Journal articles on the topic 'H-NS-mediated gene'

ABSTRACT The bacterial nucleoid-associated protein H-NS, which preferentially targets and silences A+T-rich genes, binds the ubiquitous reporter gene gfp and dramatically reduces local transcription. We have redesigned gfp to reduce H-NS-mediated transcription silencing and simultaneously improve translation in vivo without altering the amino acid sequence of the GFP protein.

Swarming motility is ultimately mediated by the proton-powered lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf genes is tightly regulated by a number of environmental conditions and regulatory factors. The nucleoid-associated DNA-binding protein H-NS is a small and abundant protein that is widely distributed in bacteria, and H-NS-like protein-dependent expression of laf genes has been identified in Vibrio cholerae and V. parahaemolyticus. The data presented here show that H-NS acts as a repressor of the swarming motility in V. parahaemolyticus. A single σ28-dependent promoter was detected for lafA encoding the flagellin of the lateral flagella, and its activity was directly repressed by H-NS. Thus, H-NS represses swarming motility by directly acting on lafA. Briefly, this work revealed a novel function for H-NS as a repressor of the expression of lafA and swarming motility in V. parahaemolyticus.

ABSTRACT H-NS regulates the flagellar master operon (flhDC) and thus is necessary for flagellation of Escherichia coli. However, the molecular mechanism of its regulation has remained unknown. Genetic screening of a transposon insertion abolishing the H-NS effect revealed a previously unidentified gene, namedhdfR, encoding a LysR family protein. Binding of purified HdfR to the flhDC promoter was demonstrated by a DNA mobility shift assay, indicating that HdfR is a transcriptional regulator for the flagellar master operon. Furthermore, the expression of the hdfR gene was shown to be negatively regulated by H-NS.

ABSTRACT H-NS is an abundant nucleoid-associated protein involved in the maintenance of chromosomal architecture in bacteria. H-NS also has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. In this study we demonstrate a role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon ofVibrio cholerae. Deletion of hns resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin-coregulated pilus, and the ToxT virulence gene regulatory protein. For the cholera toxin- and ToxT-encoding genes, elevated expression in an hns mutant was found to occur in the absence of the cognate activator proteins, suggesting that H-NS functions directly at these promoters to decrease gene expression. Deletion analysis of the region upstream of toxT suggests that an extensive region located far upstream of the transcriptional start site is required for complete H-NS-mediated repression of gene expression. These data indicate that H-NS negatively influences multiple levels of gene expression within the V. choleraevirulence cascade and raise the possibility that the transcriptional activator proteins in the ToxR regulon function to counteract the repressive effects of H-NS at the various promoters as well as to recruit RNA polymerase.

ABSTRACT IS1, the smallest active transposable element in bacteria, encodes a transposase that promotes inter- and intramolecular transposition. Host-encoded factors, e.g., histone-like proteins HU and integration host factor (IHF), are involved in the transposition reactions of some bacterial transposable elements. Host factors involved in the IS1 transposition reaction, however, are not known. We show that a plasmid with an IS1 derivative that efficiently produces transposase did not generate miniplasmids, the products of intramolecular transposition, in mutants deficient in a nucleoid-associated DNA-binding protein, H-NS, but did generate them in mutants deficient in histone-like proteins HU, IHF, Fis, and StpA. Nor did IS1 transpose intermolecularly to the target plasmid in the H-NS-deficient mutant. The hns mutation did not affect transcription from the indigenous promoter of IS1 for the expression of the transposase gene. These findings show that transpositional recombination mediated by IS1 requires H-NS but does not require the HU, IHF, Fis, or StpA protein in vivo. Gel retardation assays of restriction fragments of IS1-carrying plasmid DNA showed that no sites were bound preferentially by H-NS within the IS1 sequence. The central domain of H-NS, which is involved in dimerization and/or oligomerization of the H-NS protein, was important for the intramolecular transposition of IS1, but the N- and C-terminal domains, which are involved in the repression of certain genes and DNA binding, respectively, were not. The SOS response induced by the IS1 transposase was absent in the H-NS-deficient mutant strain but was present in the wild-type strain. We discuss the possibility that H-NS promotes the formation of an active IS1 DNA-transposase complex in which the IS1 ends are cleaved to initiate transpositional recombination through interaction with IS1 transposase.

ABSTRACT We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated inShigella, being functional at 30°C and nonfunctional at 37 to 40°C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40°C postirradiation, shows up to 30-fold higher survival than when incubated at 30°C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40°C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, inShigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40°C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40°C. These K-12 strains were originally selected fromE. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.

Coordination of the expression of Salmonella enterica invasion genes on Salmonella pathogenicity island 1 (SPI1) depends on a complex circuit involving several regulators that converge on expression of the hilA gene, which encodes a transcriptional activator (HilA) that modulates expression of the SPI1 virulence genes. Two of the global regulators that influence hilA expression are the nucleoid-associated proteins Hha and H-NS. They interact and form a complex that modulates gene expression. A chromosomal transcriptional fusion was constructed to assess the effects of these modulators on hilA transcription under several environmental conditions as well as at different stages of growth. The results obtained showed that these proteins play a role in silencing hilA expression at both low temperature and low osmolarity, irrespective of the growth phase. H-NS accounts for the main repressor activity. At high temperature and osmolarity, H-NS-mediated silencing completely ceases when cells enter the stationary phase, and hilA expression is induced. Mutants lacking IHF did not induce hilA in cells entering the stationary phase, and this lack of induction was dependent on the presence of H-NS. Band-shift assays and in vitro transcription data showed that for hilA induction under certain growth conditions, IHF is required to alleviate H-NS-mediated silencing.

ABSTRACT The histone-like protein H-NS is a major component of the bacterial nucleoid and plays a crucial role in global gene regulation of enteric bacteria. It is known that the expression of a variety of genes is repressed by H-NS, and mutations in hns result in various phenotypes, but the role of H-NS in the drug resistance of Escherichia coli has not been known. Here we present data showing that H-NS contributes to multidrug resistance by regulating the expression of multidrug exporter genes. Deletion of the hns gene from the ΔacrAB mutant increased levels of resistance against antibiotics, antiseptics, dyes, and detergents. Decreased accumulation of ethidium bromide and rhodamine 6G in the hns mutant compared to that in the parental strain was observed, suggesting the increased expression of some drug exporter(s) in this mutant. The increased drug resistance and decreased drug accumulation caused by the hns deletion were completely suppressed by deletion of the multifunctional outer membrane channel gene tolC. At least eight drug exporter systems require TolC for their functions. Among these, increased expression of acrEF, mdtEF, and emrKY was observed in the Δhns strain by quantitative real-time reverse transcription-PCR analysis. The Δhns-mediated multidrug resistance pattern is quite similar to that caused by overproduction of the AcrEF exporter. Deletion of the acrEF gene greatly suppressed the level of Δhns-mediated multidrug resistance. However, this strain still retained resistance to some compounds. The remainder of the multidrug resistance pattern was similar to that conferred by overproduction of the MdtEF exporter. Double deletion of the mdtEF and acrEF genes completely suppressed Δhns-mediated multidrug resistance, indicating that Δhns-mediated multidrug resistance is due to derepression of the acrEF and mdtEF drug exporter genes.

ABSTRACT The nucleoid-associated proteins H-NS and StpA inEscherichia coli bind DNA as oligomers and are implicated in gene regulatory systems. There is evidence for both homomeric and heteromeric H-NS–StpA complexes. The two proteins show differential turnover, and StpA was previously found to be subject to protease-mediated degradation by the Lon protease. We investigated which regions of the H-NS protein are able to prevent degradation of StpA. A set of truncated H-NS derivatives was tested for their ability to mediate StpA stability and to form heteromers in vitro. The data indicate that H-NS interacts with StpA at two regions and that the presence of at least one of the H-NS regions is necessary for StpA stability. Our results also suggest that a proteolytically stable form of StpA, StpAF21C, forms dimers, whereas wild-type StpA in the absence of H-NS predominantly forms tetramers or oligomers, which are more susceptible to proteolysis.

Proteus mirabilis, a cause of catheter-associated urinary tract infection, relies on several virulence factors to colonize the urinary tract. Among these, urease contributes to the development of urinary stones resulting from the increase in local pH due to urease-mediated hydrolysis of urea to NH3 and CO2. UreR, an AraC-like transcriptional activator, activates transcription of the genes encoding the urease subunits and accessory proteins (ureDABCEFG) in the presence of urea. UreR also initiates transcription of its own gene in a urea-inducible manner by binding to the intergenic region between ureR and ureD. The intergenic region contains poly(A) tracts that appear to be the target of H-NS. It has been shown that Escherichia coli and P. mirabilis H-NS acts to repress transcription of ureR in an E. coli model system. It was hypothesized that H-NS represses urease gene expression in the absence of UreR and urea by binding to the intergenic region. To demonstrate this the P. mirabilis hns gene was cloned and the 15·6 kDa H-NS was overexpressed and purified as a myc-His tail fusion. Using a gel shift assay, purified H-NS-myc-His bound preferentially to a 609 bp DNA fragment containing the entire ureR-ureD intergenic region. H-NS and UreR were able to displace each other from the ureR-ureD intergenic region. Circular permutation analysis revealed that the intergenic region is bent. Moreover, H-NS recognizes this curvature, binds the DNA fragment and induces further bending of the DNA as shown by a circular ligation assay. The effects of H-NS, urea and temperature (25 vs 37 °C) on urease expression were shown in E. coli containing an hns knockout and P. mirabilis where expression was increased at 37 °C. Increased transcription from pureR was seen in the E. coli hns knockout when temperature was increased from 25 to 37 °C. These findings suggest H-NS and UreR differentially regulate urease in a negative and positive manner, respectively.

ABSTRACT RovA is a MarR/SlyA-type regulator that mediates the transcription of inv in Yersinia enterocolitica and Y. pseudotuberculosis. In Y. pseudotuberculosis, rovA transcription is controlled primarily by H-NS and RovA, which bind to similar regions within the rovA promoter. At 37°C, rovA transcription is repressed by H-NS. Transcription of rovA results when RovA relieves H-NS-mediated repression. The region of the rovA promoter that H-NS and RovA bind is not conserved in the Y. enterocolitica promoter. Using green fluorescent protein reporters, we determined that the Y. enterocolitica rovA (rovA Yent ) promoter is weaker than the Y. pseudotuberculosis promoter. However, despite the missing H-NS/RovA binding site in the rovA Yent promoter, H-NS and RovA are still involved in the regulation of rovA Yent . DNA binding studies suggest that H-NS and RovA bind with a higher affinity to the Y. pseudotuberculosis/Y. pestis rovA (rovA Ypstb/Ypestis ) promoter than to the rovA Yent promoter. Furthermore, H-NS appears to bind to two regions in a cooperative fashion within the rovA Yent promoter that is not observed with the rovA Ypstb/Ypestis promoter. Finally, using a transposon mutagenesis approach, we identified a new positive regulator of rovA in Y. enterocolitica, LeuO. In Escherichia coli, LeuO regulates gene expression via changes in levels of RpoS and H-NS, but LeuO-mediated regulation of rovA Yent appears to be independent of either of these two proteins. Together, these data demonstrate that while the rovA regulatory factors are conserved in Yersinia, divergence of Y. enterocolitica and Y. pseudotuberculosis/Y. pestis during evolution has resulted in modifications in the mechanisms that are responsible for controlling rovA transcription.

ABSTRACT The Escherichia coli hlyE gene (also known as clyA or sheA) codes for a novel pore-forming toxin. Previous work has shown that the global transcription factors FNR and CRP positively regulate hlyE expression by binding at the same site. Here in vivo transcription studies reveal that FNR occupies the hlyE promoter more frequently than CRP, providing a mechanism for the moderate upregulation of hlyE expression in response to two distinct environmental signals (oxygen and glucose starvation). It has been reported that H-NS interacts with two large regions of the hlyE promoter (PhlyE), one upstream of the −35 element and one downstream of the −10 element. Here we identify two high-affinity H-NS sites, H-NS I, located at the 3′ end of the extended upstream footprint, and H-NS II, located at the 5′ end of the extended downstream footprint. It is suggested that these high-affinity sites initiate the progressive formation of higher order complexes, allowing a range of H-NS-mediated regulatory effects at PhlyE. Finally, the identification of a SlyA binding site that overlaps the H-NS I site in PhlyE suggests a mechanism to explain how SlyA overproduction enhances hlyE expression by antagonizing the negative effects of H-NS.

ABSTRACT The VirB protein of Shigella flexneri is a positive regulator of the major virulence operons of this enteroinvasive intracellular pathogen. VirB resembles no other transcription factor but is strongly homologous to plasmid partition proteins. We found that the binding of the VirB protein to the promoter region of the icsB virulence gene induced hypersensitivity to cleavage by DNase I over a region to which the H-NS repressor protein binds and completely abolished the protection of this sequence from DNase I by H-NS. In the absence of H-NS, the VirB protein had no additive effect on the ability of the icsB promoter to form an open transcription complex, indicating that VirB is not involved in the recruitment of RNA polymerase to the promoter or in open complex formation. Similarly, VirB did not stimulate promoter function in an in vitro transcription assay but acted as an antagonist of H-NS-mediated repression. A sequence located upstream of the icsB promoter and related to cis-acting elements involved in plasmid partitioning was required for promoter derepression by VirB. Alterations to one heptameric motif within this DNA sequence attenuated VirB binding and derepression of icsB transcription.

The marine bacterium Vibrio parahaemolyticus, a major cause of food-borne gastroenteritis, employs a type VI secretion system 1 (T6SS1), a recently discovered protein secretion system, to combat competing bacteria. Environmental signals such as temperature, salinity, cell density and surface sensing, as well as the quorum-sensing master regulator OpaR, were previously reported to regulate T6SS1 activity and expression. In this work, we set out to identify additional transcription regulators that control the tightly regulated T6SS1 activity. To this end, we determined the effect of deletions in several known virulence regulators and in two regulators encoded within the T6SS1 gene cluster on expression and secretion of the core T6SS component Hcp1 and on T6SS1-mediated anti-bacterial activity. We report that VP1391 and VP1407, transcriptional regulators encoded within the T6SS1 gene cluster, are essential for T6SS1 activity. Moreover, we found that H-NS, a bacterial histone-like nucleoid structuring protein, which mediates transcription silencing of horizontally acquired genes, serves as a repressor of T6SS1. We also show that activation of surface sensing and high salt conditions alleviate the H-NS-mediated repression. Our results shed light on the complex network of environmental signals and transcription regulators that govern the tight regulation over T6SS1 activity.

The type III secretion system (T3SS) is an appendage used by many bacterial pathogens, such as pathogenic Yersinia, to subvert host defenses. However, because the T3SS is energetically costly and immunogenic, it must be tightly regulated in response to environmental cues to enable survival in the host. Here we show that expression of the Yersinia Ysc T3SS master regulator, LcrF, is orchestrated by the opposing activities of the repressive H-NS/YmoA histone-like protein complex and induction by the iron and oxygen-regulated IscR transcription factor. While deletion of iscR or ymoA has been shown to decrease and increase LcrF expression and type III secretion, respectively, the role of H-NS in this system has not been definitively established because hns is an essential gene in Yersinia. Using CRISPRi knockdown of hns, we show that hns depletion causes derepression of lcrF. Furthermore, we find that while YmoA is dispensable for H-NS binding to the lcrF promoter, YmoA binding to H-NS is important for H-NS repressive activity. We bioinformatically identified three H-NS binding regions within the lcrF promoter and demonstrate binding of H-NS to these sites in vivo using chromatin immunoprecipitation. Using promoter truncation and binding site mutation analysis, we show that two of these H-NS binding regions are important for H-NS/YmoA-mediated repression of the lcrF promoter. Surprisingly, we find that IscR is dispensable for lcrF transcription in the absence of H-NS/YmoA. Indeed, IscR-dependent regulation of LcrF and type III secretion in response to changes in oxygen, such as those Yersinia is predicted to experience during host infection, only occurs in the presence of an H-NS/YmoA complex. These data suggest that, in the presence of host tissue cues that drive sufficient IscR expression, IscR can act as a roadblock to H-NS/YmoA-dependent repression of RNA polymerase at the lcrF promoter to turn on T3SS expression.

ABSTRACT DNA adenine methylase (Dam−) mutants of Salmonella enterica serovar Typhimurium contain reduced levels of FinP RNA encoded on the virulence plasmid. Dam methylation appears to regulate finP transcription, rather than FinP RNA stability or turnover. The finP promoter includes canonical −10 and −35 modules and depends on the σ70 factor. Regulation of finP transcription by Dam methylation does not require DNA sequences upstream from the −35 module, indicating that Dam acts at the promoter itself or downstream. Unexpectedly, a GATC site overlapping with the −10 module is likewise dispensable for Dam-mediated regulation. These observations indicate that Dam methylation regulates finP transcription indirectly and suggest the involvement of a host factor(s) responsive to the Dam methylation state of the cell. We provide evidence that one such factor is the nucleoid protein H-NS, which acts as a repressor of finP transcription in a Dam− background. H-NS also restrains transcription of the overlapping traJ gene, albeit in a Dam-independent fashion. Hence, the decreased FinP RNA content found in Dam− hosts of S. enterica appears to result from H-NS-mediated repression of finP transcription.

Second messenger nucleotides, such as guanosine penta- or tetra-phosphate, commonly referred to as (p)ppGpp, are powerful signaling molecules, used by all bacteria to fine-tune cellular metabolism in response to nutrient availability. Indeed, under nutritional starvation, accumulation of (p)ppGpp reduces cell growth, inhibits stable RNAs synthesis, and selectively up- or down- regulates the expression of a large number of genes. Here, we show that the E. colihns promoter responds to intracellular level of (p)ppGpp. hns encodes the DNA binding protein H-NS, one of the major components of bacterial nucleoid. Currently, H-NS is viewed as a global regulator of transcription in an environment-dependent mode. Combining results from relA (ppGpp synthetase) and spoT (ppGpp synthetase/hydrolase) null mutants with those from an inducible plasmid encoded RelA system, we have found that hns expression is inversely correlated with the intracellular concentration of (p)ppGpp, particularly in exponential phase of growth. Furthermore, we have reproduced in an in vitro system the observed in vivo (p)ppGpp-mediated transcriptional repression of hns promoter. Electrophoretic mobility shift assays clearly demonstrated that this unusual nucleotide negatively affects the stability of RNA polymerase-hns promoter complex. Hence, these findings demonstrate that the hns promoter is subjected to an RNA polymerase-mediated down-regulation by increased intracellular levels of (p)ppGpp.

ABSTRACT Bile induces pleiotropic responses that affect production of virulence factors, motility, and other phenotypes in the enteric pathogen Vibrio cholerae. Since bile is a heterogeneous mixture, crude bile was fractionated, and the components that mediate virulence gene repression and enhancement of motility were identified by nuclear magnetic resonance, gas chromatography (GC), and GC-mass spectrometry analyses. The unsaturated fatty acids detected in bile, arachidonic, linoleic, and oleic acids, drastically repressed expression of the ctxAB and tcpA genes, which encode cholera toxin and the major subunit of the toxin-coregulated pilus, respectively. The unsaturated fatty acid-dependent repression was due to silencing of ctxAB and tcpA expression by the histone-like nucleoid-structuring protein H-NS, even in the presence of the transcriptional activator ToxT. Unsaturated fatty acids also enhanced motility of V. cholerae due to increased expression of flrA, the first gene of a regulatory cascade that controls motility. H-NS had no role in the fatty acid-mediated enhancement of motility. It is likely that the ToxR/ToxT system that negatively regulates motility is rendered nonfunctional in the presence of unsaturated fatty acids, leading to an increase in motility. Motility and flrA expression were also increased in the presence of cholesterol, another component of bile, in an H-NS- and ToxR/ToxT-independent manner.

ABSTRACT The gene cluster gspCDEFGHIJKLM codes for various structural components of the type II secretion pathway which is responsible for the secretion of heat-labile enterotoxin by enterotoxigenic Escherichia coli (ETEC). In this work, we used a variety of molecular approaches to elucidate the transcriptional organization of the ETEC type II secretion system and to unravel the mechanisms by which the expression of these genes is controlled. We showed that the gspCDEFGHIJKLM cluster and three other upstream genes, yghJ, pppA, and yghG, are cotranscribed and that a promoter located in the upstream region of yghJ plays a major role in the expression of this 14-gene transcriptional unit. Transcription of the yghJ promoter was repressed 168-fold upon a temperature downshift from 37°C to 22°C. This temperature-induced repression was mediated by the global regulatory proteins H-NS and StpA. Deletion mutagenesis showed that the promoter region encompassing positions −321 to +301 relative to the start site of transcription of yghJ was required for full repression. The yghJ promoter region is predicted to be highly curved and bound H-NS or StpA directly. The binding of H-NS or StpA blocked transcription initiation by inhibiting promoter open complex formation. Unraveling the mechanisms of regulation of type II secretion by ETEC enhances our understanding of the pathogenesis of ETEC and other pathogenic varieties of E. coli.

Hepatic cytokine gene expression is independently stimulated by circulating microbial products and reductions in the cellular O2 supply. Although these stimuli occur sequentially after gram-negative bacteremia, it is unknown whether their interplay augments production of interleukin (IL)-1 by the liver. We studied the effects of intraportal Escherichia coli (EC) bacteremia and secondary constant-flow hypoxia (Po2, approximately 46 Torr for 30 min) on IL-1 alpha and IL-1 beta gene expression in ex situ buffer-perfused rat livers over 180 min (n = 67). At t = 0, normoxic EC and normal saline (NS) controls received 10(9) live EC serotype 055:B5 and 0.9% NaCl, respectively; in livers subjected to EC+hypoxia-reoxygenation (H/R) and NS+H/R, hypoxia began 0.5 h after EC or NS and was followed by 120 min of reoxygenation. Portal and hepatic venous perfusates were serially analyzed for bacterial colony-forming units, O2 uptake, and aspartate aminotransferase. At 60 min (peak hypoxia) and 180 min, cDNAs for IL-1 alpha and IL-1 beta were hybridized to whole liver RNA, and IL-1 beta protein levels in venous perfusates were assessed. Intrahepatic levels of reduced glutathione (GSH) were measured as an index of oxidative stress. Compared with normoxic EC, IL-1 alpha transcripts decreased at 180 min in EC+H/R livers (P < 0.0001) as did IL-1 beta mRNA (P < 0.05), despite similar EC clearance, GSH levels, posthypoxic O2 uptake, and aspartate aminotransferase release. Hepatic secretion of IL-1 beta likewise fell in EC+H/R vs. EC controls (P < 0.005). Prostaglandin H synthase-2 (COX-2) message accumulation was not enhanced by H/R, and indomethacin did not reverse H/R-mediated suppression of IL-1 production. In contrast, H/R-related falls in EC-induced IL-1S expression were reversed by allopurinol or catalase. Thus brief hypoxic stress of the liver causing neither GSH depletion nor functional impairment downregulates postbacteremic IL-1 expression by a mechanism involving O2 radicals but not cyclooxygenase metabolites.

ABSTRACT Curli fibers could be described as a virulence factor able to confer adherence properties to both abiotic and eukaryotic surfaces. The ability to adapt rapidly to changing environmental conditions through signal transduction pathways is crucial for the growth and pathogenicity of bacteria. OmpR was shown to activate csgD expression, resulting in curli production. The CpxR regulator was shown to negatively affect curli gene expression when binding to its recognition site that overlaps the csgD OmpR-binding site. This study was undertaken to clarify how the interplay between the two regulatory proteins, OmpR and CpxR, can affect the transcription of the curli gene in response to variation of the medium osmolarity. Band-shift assays with purified CpxR proteins indicate that CpxR binds to the csgD promoter region at multiple sites that are ideally positioned to explain the csg repression activity of CpxR. To understand the physiological meaning of this in vitro molecular phenomenon, we analyzed the effects of an osmolarity shift on the two-component pathway CpxA/CpxR. We establish here that the Cpx pathway is activated at both transcriptional and posttranscriptional levels in response to a high osmolarity medium and that CpxR represses csgD expression in high-salt-content medium, resulting in low curli production. However, csgD repression in response to high sucrose content is not mediated by CpxR but by the global regulatory protein H-NS. Therefore, multiple systems (EnvZ/OmpR, Cpx, Rcs, and H-NS) appear to be involved in sensing environmental osmolarity, leading to sophisticated regulation of the curli genes.

ABSTRACT Two Escherichia coli genes, expressed from multicopy plasmids, are shown to cause partial induction of prophage λ inrecA mutant lysogens. One is rcsA, which specifies a positive transcriptional regulator of the cpsgenes, which are involved in capsular polysaccharide synthesis. The other is dsrA, which specifies an 85-nucleotide RNA that relieves repression of the rcsA gene by histone-like protein H-NS. Genetic contexts known to increase Cps expression also cause RecA-independent λ induction: the rcsC137 mutation, which causes constitutive Cps expression, and the lon andrcsA3 mutations, which stabilize RcsA. Lambdoid phages 21, φ80, and 434 are also induced by RcsA and DsrA overexpression inrecA lysogens. Excess λ cI repressor specifically blocks λ induction, suggesting that induction involves repressor inactivation rather than repressor bypass. RcsA-mediated induction requires RcsB, the known effector of the cps operon, whereas DsrA-mediated induction is RcsB independent in stationary phase, pointing to the existence of yet another RecA-independent pathway of prophage induction.

ABSTRACT Transcription of the K+ transport operonkdp in Escherichia coli is induced during K+-limited growth by the action of a dual-component phosphorelay regulatory system comprised of a sensor kinase (integral membrane protein), KdpD, and a DNA-binding response regulator (cytoplasmic protein), KdpE. In this study, we screened for newdke (named dke for decreased kdpexpression) mutations (in loci other than kdpDE) that led to substantially decreased kdp expression. Onedke mutation was shown to be in hns, encoding the nucleoid protein H-NS. Another dke mutation was mapped to trxB (encoding thioredoxin reductase), and an equivalent reduction in kdp expression was demonstrated also fortrxA mutants that are deficient in thioredoxin 1. Exogenously provided dithiothreitol rescued the kdpexpression defect in trxB but not trxA mutants. Neither trxB nor trxA affected gene regulation mediated by another dual-component system tested, EnvZ-OmpR. Mutations in genes dsbC and dsbD did not affectkdp expression, suggesting that the trx effects on kdp are not mediated by alterations in protein disulfide bond status in the periplasm. Reduced kdp expression was observed even in a trxB strain that harbored a variant KdpD polypeptide bearing no Cys residues. A trxB hns double mutant was even more severely affected for kdp expression than either single mutant. The dke mutations themselves had no effect on strength of the signal controlling kdpexpression, and constitutive mutations in kdpDE were epistatic to hns and trxB. These results indicate that perturbations in cytoplasmic thiol oxidation status and in levels of the H-NS protein exert additive effects, direct or indirect, at a step(s) upstream of KdpD in the signal transduction pathway, which significantly influence the magnitude of KdpD kinase activity obtained for a given strength of the inducing signal forkdp transcription.

ABSTRACTAraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenicEscherichia coli(EPEC), enterotoxigenicE. coli, enteroaggregativeE. coli, andCitrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, ofC. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target,sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression ofsefAby binding to a region upstream of thesefApromoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22.

We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE2, and PGI2. The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by NG-monomethyl-l-arginine (l-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by l-NMMA. Muscle contractility stimulated by 3 μM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with l-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.

Bradykinin (BK) is released in the brain during injury and inflammation. Activation of endothelial BK receptors produces acute dilatation of cerebral arterioles that is mediated by reactive oxygen species (ROS). ROS can also modulate gene expression, including expression of the inducible isoform of cyclooxygenase (COX-2). We hypothesized that exposure of the brain to BK would produce acute dilatation, which would be followed by a delayed dilatation mediated by COX-2. To test this hypothesis in anesthetized rats, BK was placed twice in cranial windows for 7 min, after which the windows were flushed to remove residual BK. The two BK exposures were separated by 30 min. Each BK exposure produced acute dilatation of cerebral arterioles, after which diameter rapidly returned to baseline. Over the subsequent 4.5 h after the second BK exposure, arterioles dilated 48 ± 8%. Treatment of the cranial window with NS-398, a selective COX-2 inhibitor, or dexamethasone, significantly attenuated the delayed dilatation. Aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, did not alter the delayed dilatation. Cotreatment of cranial windows with BK, superoxide dismutase, and catalase also prevented the delayed dilatation. In separate experiments, exposure of the cortical surface to BK upregulated leptomeningeal expression of COX-2 mRNA. Our results suggest that acute, time-limited exposure of the brain to BK produces delayed dilatation of cerebral arterioles dependent on expression and activity of COX-2.

ABSTRACTEnteropathogenic (EPEC) and enterohemorrhagic (EHEC)Escherichia coliare clinically important diarrheagenic pathogens that adhere to the intestinal epithelial surface. TheE. colicommon pili (ECP), or meningitis-associated and temperature-regulated (MAT) fimbriae, are ubiquitous among both commensal and pathogenicE. colistrains and play a role as colonization factors by promoting the interaction between bacteria and host epithelial cells and favoring interbacterial interactions in biofilm communities. The first gene of theecpoperon encodes EcpR (also known as MatA), a proposed regulatory protein containing a LuxR-like C-terminal helix-turn-helix (HTH) DNA-binding motif. In this work, we analyzed the transcriptional regulation of theecpgenes and the role of EcpR as a transcriptional regulator. EHEC and EPECecpRmutants produce less ECP, while plasmids expressing EcpR increase considerably the expression of EcpA and production of ECP. Theecpgenes are transcribed as an operon from a promoter located 121 bp upstream of the start codon ofecpR. EcpR positively regulates this promoter by binding to two TTCCT boxes distantly located upstream of theecppromoter, thus enhancing expression of downstreamecpgenes, leading to ECP production. EcpR mutants in the putative HTH DNA-binding domain are no longer able to activateecpexpression or bind to the TTCCT boxes. EcpR-mediated activation is aided by integration host factor (IHF), which is essential for counteracting the repression exerted by histone-like nucleoid-structuring protein (H-NS) on theecppromoter. This work demonstrates evidence about the interplay between a novel member of a diverse family of regulatory proteins and global regulators in the regulation of a fimbrial operon.

ABSTRACT Shiga toxigenic Escherichia coli (STEC) strains are important human pathogens which are capable of causing diarrhea, hemorrhagic colitis, and the potentially fatal hemolytic-uremic syndrome (HUS). An important virulence trait of certain STEC strains, such as those belonging to serogroup O157, is the capacity to produce attaching and effacing (A/E) lesions on enterocytes, a property encoded by the locus for enterocyte effacement (LEE). LEE contains theeae gene, which encodes intimin, an outer membrane protein which mediates the intimate attachment of bacteria to the host epithelial cell surface, and eae is routinely used as a marker for LEE-positive STEC strains. However, the O157:H−STEC strain 95SF2 carries eae but did not produce A/E lesions on HEp-2 cells, as judged by a fluorescent actin staining assay. In this assay, 95SF2 adhered poorly to the HEp-2 cells, and those that did bind exhibited abnormal cell division. In contrast, the O157:H7 STEC strain EDL933 adhered strongly and produced typical A/E lesions. We have demonstrated that 95SF2 carries a defective LEE regulatory gene, ler, with a single base change with respect to that published for ler of EDL933, resulting in an Ile57-to-Thr substitution. Ler shows homology to H-NS-like regulators, which are modulators of transcription, and the mutation occurs in a domain implicated in oligomerization. 95SF2 was able to adhere and produce A/E lesions on HEp-2 cells when EDL933ler was expressed from a multicopy plasmid. Conversely, introduction of a plasmid carrying 95SF2 ler into EDL933 abolished adherence and capacity to form A/E lesions. Studies witheae deletion derivatives of 95SF2 and EDL933 demonstrated that the ler-mediated adherence to HEp-2 cells is largely independent of intimin. We have also demonstrated that EDL933ler, but not 95SF2 ler, increases the level of intimin in O157 STEC.

We determined the functional implications of calcium-sensing receptor (CaR)-dependent, Gq- and Gi-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells and tumor necrosis factor (TNF)-α gene transcription that cause expression of cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of Gq, Gi, protein kinase C (PKC), or calcineurin (CaN) activities abolished CaR-mediated COX-2 expression and PGE2 synthesis. We tested the hypothesis that these pathways contribute to the effects of CaR activation on ion transport in mTAL cells. Ouabain-sensitive O2 consumption, an in vitro correlate of ion transport in the mTAL, was inhibited by ∼70% in cells treated for 6 h with extracellular Ca2+ (1.2 mM), an effect prevented in mTAL cells transiently transfected with a dominant negative CaR overexpression construct (R796W), indicating that the effect was initiated by stimulation of the CaR. Pretreatment with the COX-2-selective inhibitor, NS-398 (1 μM), reversed CaR-activated decreases in ouabain-sensitive O2 consumption by ∼60%, but did not alter basal levels of ouabain-sensitive O2 consumption. Similarly, inhibition of either Gq, Gi, PKC, or CaN, which are components of the mechanism associated with CaR-stimulated COX-2-derived PGE2 synthesis, reversed the inhibitory effects of CaR on O2 consumption without affecting basal O2 consumption. Our findings identified signaling elements required for CaR-mediated TNF production that are integral components regulating mTAL function via a mechanism involving COX-2 expression and PGE2 production.

Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors. To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell’s exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for β-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 × 107pfu/ml were sufficient for expression in 50% of the ∼1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel–GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by −17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function.

ppGpp is an intracellular sensor that, in response to different types of stress, coordinates the rearrangement of the gene expression pattern of bacteria to promote adaptation and survival to new environmental conditions. First described to modulate metabolic adaptive responses, ppGpp modulates the expression of genes belonging to very diverse functional categories. In Escherichia coli, ppGpp regulates the expression of cellular factors that are important during urinary tract infections. Here, we characterize the role of this alarmone in the regulation of the hlyCABDII operon of the UPEC isolate J96, encoding the toxin α-hemolysin that induces cytotoxicity during infection of bladder epithelial cells. ppGpp is required for the expression of the α-hemolysin encoded in hlyCABDII by stimulating its transcriptional expression. Prototrophy suppressor mutations in a ppGpp-deficient strain restore the α-hemolysin expression from this operon to wild-type levels, confirming the requirement of ppGpp for its expression. ppGpp stimulates hlyCABDII expression independently of RpoS, RfaH, Zur, and H-NS. The expression of hlyCABDII is promoted at 37 °C and at low osmolarity. ppGpp is required for the thermoregulation but not for the osmoregulation of the hlyCABDII operon. Studies in both commensal and UPEC isolates demonstrate that no UPEC specific factor is strictly required for the ppGpp-mediated regulation described. Our data further support the role of ppGpp participating in the coordinated regulation of the expression of bacterial factors required during infection.

ABSTRACTPseudomonas aeruginosais a Gram-negative bacterium that is ubiquitous in the environment, and it is an opportunistic pathogen that can infect a variety of hosts, including humans. During the process of infection,P. aeruginosacoordinates the expression of numerous virulence factors through the production of multiple cell-to-cell signaling molecules. The production of these signaling molecules is linked through a regulatory network, with the signalN-(3-oxododecanoyl) homoserine lactone and its receptor LasR controlling the induction of a second acyl-homoserine lactone signal and thePseudomonasquinolone signal (PQS). LasR-mediated control of PQS occurs partly by activating the transcription ofpqsR, a gene that encodes the PQS receptor and is necessary for PQS production. We show that LasR interacts with a single binding site in thepqsRpromoter region and that it does not influence the transcription of the divergently transcribed gene,nadA. Using DNA affinity chromatography, we identified additional proteins that interact with thepqsR-nadAintergenic region. These include the H-NS family members MvaT and MvaU, and CysB, a transcriptional regulator that controls sulfur uptake and cysteine biosynthesis. We show that CysB interacts with thepqsRpromoter and that CysB repressespqsRtranscription and PQS production. Additionally, we provide evidence that CysB can interfere with the activation ofpqsRtranscription by LasR. However, as seen with other CysB-regulated genes,pqsRexpression was not differentially regulated in response to cysteine levels. These findings demonstrate a novel role for CysB in influencing cell-to-cell signal production byP. aeruginosa.IMPORTANCEThe production of PQS and other 4-hydroxy-2-alkylquinolone (HAQs) compounds is a key component of theP. aeruginosacell-to-cell signaling network, impacts multiple physiological functions, and is required for virulence. PqsR directly regulates the genes necessary for HAQ production, but little is known about the regulation ofpqsR. We identified CysB as a novel regulator ofpqsRand PQS production, but, unlike other CysB-controlled genes, it does not appear to regulatepqsRin response to cysteine. This implies that CysB functions as both a cysteine-responsive and cysteine-unresponsive regulator inP. aeruginosa.

Abstract Activation of T cells leading to graft-vs-host disease (GVHD) requires two signaling events: the Ag-specific signal generated through the engagement of the TCR/CD3 complex with antigenic peptide fragments presented by MHC molecules on APCs and the second signal provided through additional costimulatory ligands. T cells have preferential costimulatory requirements depending on their state of activation-induced maturation. In the present study, we investigated the role of the receptor-ligand pair VLA-4 (alpha 4 beta 1) and VCAM-1 in allogeneic T cell responses in vitro and in vivo. Anti-VCAM-1 mAb effectively inhibited mixed lymphocyte culture (MLC) across several major MHC barriers and secondary MLC across minor histocompatibility Ags (95.5% and 90.0% inhibition, respectively). In contrast, anti-VLA-4 mAb inhibited a CD8(+)-mediated primed CTL response in vitro by 100%, yet had little effect on proliferative responses. In the B10.D2/nSnJ--&gt;BALB/c (both H-2d) system of GVHD, BALB/c received anti-VCAM-1, or anti-VLA-4 or controls NS-1 or Y13-259 for the first 5 wk after transplant. Anti-VLA-4 mAb delayed the onset of GVHD, but failed to reduce incidence, severity or GVHD-related mortality. In contrast, anti-VCAM-1 reduced the incidence of GVHD from 100% (18/18) in control animals to 53.3% (8/15) (p &lt; 0.01) on day 70 post-transplant and significantly decreased GVHD-related mortality. Sixty percent (9/15) of anti-VCAM-1 recipients survived more than 180 days after transplant. Long-term engraftment of allogeneic bone marrow was documented in all transplanted mice by PCR analysis of a microsatellite region in the IL-1 beta gene.

Abstract Histone deacetylase (HDAC) inhibitors reduce experimental acute graft-versus-host disease (GVHD) and recent work by us and others suggest that HDAC inhibitors regulate dendritic cell (DC) function. However, the critical cellular and molecular mechanisms underpinning these observations are not known. We investigated the mechanisms by utilizing two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and ITF 2357. Pretreatment of murine bone marrow (BM) and human peripheral blood mononuclear cell derived DCs with either HDAC inhibtors and stimulated with TLR ligands such as LPS caused a significant reduction in the secretion of TNF-α compared to the untreated controls (P< 0.01). Pre-treatment also significantly reduced the DC mediated in vitro and in vivo stimulation of allogeneic T cell proliferation (P<0.05). SAHA and ITF 2357 increased expression of indoleamine 2, 3-dioxygenase (IDO) at both mRNA and protein levels. Blockade of IDO induction with specific small interfering RNA (siRNA) in the wild type (WT) DCs and those derived from IDO deficient (IDO−/−) animals confirmed a functional role for IDO in the HDAC inhibitor mediated regulation TNFα secretion and allo-T cell proliferation. DNA-protein interaction analysis with ChIP assay demonstrated that both acetylated histone(H) 4 and STAT3 bound to murine IDO promoter. Using TESS DNA soft-wear analysis we found two potential STAT3 binding Gamma Activated Sites (GAS sites) in the IDO promoter and it was recently reported that acetylation of STAT3 is sufficient for its activation (Yuan, et al. Science 2005). We therefore sought to determine whether direct acetylation of STAT3 by the HDAC inhibitors is critical for the induction of IDO in DCs. SAHA or ITF2357 treatment induced acetylation, activation and dimerization of STAT-3 as determined by protein-protein interaction studies. Co-culture studies with JSI-124, an inhibitor of STAT3 signaling, demonstrated that STAT3 is critical for induction of IDO by the HDAC inhibitors. Functional relevance was confirmed by the lack of HDAC inhibitor induced suppression of DC function when co-treated with JSI-124. We next cloned 1500bp DNA fragment upstream of mouse IDO gene start codon and attached it to luciferase gene and peformed mutagenesis studies to evaluate for luciferase activity. Deletion of the GAS regions or treatment with JSI-124 impaired luciferase activity of the IDO promoter constructs demonstrating that STAT3 is necessary and sufficient for transcriptional induction of IDO by the HDAC inhibitors. To specifically address the in vivo relevance of IDO induction by HDAC inhibitors in only the host type DCs, we generated [B6 → B6] and [IDO−/−B6 → B6] BM chimeras and utilized them in a well characterized [BALB/c B6] mouse model of acute GVHD. [B6 → B6] and [IDO−/− B6 → B6] animals received 800 Gy on day −1 and were treated orally with of 50mg/kg of ITF 2357 or diluent on days −1 to +2. Mice were transplanted on day 0 with 3 x 106 T cells and 5 x 106 BM from either syngeneic B6 or allogeneic BALB/c donors. Treatment with ITF 2357 resulted in significantly better survival in the allogeneic [B6 → B6] animals (80% vs. 40%, P < 0.02) but did not confer any survival benefit to the [IDO−/− B6 → B6] animals when compared with diluent treated recipients [20% vs. 30%, P = NS]. Our data thus demonstrate a novel molecular pathway in modulation of GVHD through a STAT3 dependent induction of IDO in the host DCs.

Salvianolic acid B (Sal B) is an effective treatment agent for ischemic disease in China. However, Sal B’s effects on peripheral arterial disease (PAD) and its mechanism remains poorly understood. Macrophage polarization plays a crucial role in PAD. Nevertheless, treatment modalities that increase the population of anti-inflammatory (M2) macrophages are limited. This study aimed to explore the protective effects of Sal B on limb perfusion and investigate the mechanism of Sal B-induced macrophage polarization. C57BL/6 male mice (6 weeks) were randomized into control, Model + NS, and Model + Sal B groups (n = 5). Then, we established a hind limb ischemia mouse model to assess the Sal B’s role (15 mg/kg/d) in PAD. We quantified the blood perfusion via laser speckle contrast imaging (LSCI) and measured the capillary density and muscle edema with CD31 and H&E staining. The Sal B-induced macrophage polarization was confirmed by qPCR and ELISA. The results showed that the Sal B group exhibited a significant improvement in the blood perfusion, capillary density, muscle edema, and M2 markers gene expressions. Cell migration and tube formation were promoted in the endothelial cells stimulated with a culture supernatant from Sal B-treated macrophages. In contrast, endothelial functions improved by Sal B-treated macrophages were impaired in groups treated with SIRT1 and PI3K inhibitors. These findings provide evidence for Sal B’s protective role in PAD and demonstrate the enhancement of macrophage polarization via the SIRT1/PI3K/AKT pathway.

Abstract Studies in vitro, and in vivo using animal models show that leukocytes play a key role in vasoocclusion and clinical research suggests that high leukocyte counts correlate with mortality, stroke and acute chest syndrome in sickle cell disease (SCD). Lungs are particularly vulnerable to vaso-occlusive events because of their anatomic features in SCD. Transgenic mice expressing exclusively human sickle hemoglobin (SS) are well-established models for the study of vascular inflammation. Previous studies have shown that systemic LPS challenge causes exaggerated inflammation, including increased serum and broncoalveolar lavage (BAL) TNF-α and IL-1 cytokines and sVCAM-1 in sickle mice. The aim of this study was to examine the contribution of acute airway inflammation in SCD using SS mice and the role of chemokines and matrix metalloproteinases (MMPs) in this process. Acute lung inflammation and injury were induced by intranasal administration of lipopolysaccharide (LPS, 50 μl of 250 μg/ml) in control (C57BL/6) and SS mice. The vehicle mice group received a similar volume of sterile PBS. BAL was performed 4 h after LPS challenge. qRT-PCR analysis was used to examine gene expression and ELISA protein production. The intranasal administration of LPS to mice triggered a huge influx of leukocytes (neutrophils, NS) in BAL of control and SS mice compared with the respective vehicle groups, but this influx was greater in SS mice, when compared with control mice (1.4 ± 0.06 vs 0.66 ± 0.12 WBCx106/BAL); p=0.0006, 1.06 ± 0.1 vs 0.40 ±0.12 NSx106/BAL; p=0.004, respectively). At baseline levels, KC and MIP-2 chemokines (functional homologues of human IL-8 in mice) are higher in BAL fluid of SS mice compared to control mice (186.6 ± 14.1 pg/ml vs 14.1 ± 5.8 pg/ml; 41.2 ± 7.9 pg/ml vs 11.4 ± 7.3 pg/ml, p=, respectively). Corresponding with influx of NS, lung lavage levels of KC and MIP-2 were significantly higher in SS BALF compared to control mice (2491 ± 454 pg/ml vs 798.1 vs 98.2 pg/ml; 1726 ± 307 pg/ml vs 887.3 ± 149.5 pg/ml, respectively). Enhanced levels of TNF-α were also observed at baseline and after LPS instillation compared to those of the control mice (20.8 ± 8.8 pg/ml vs 2.5 ± 1.6 pg/ml; 4250 ± 636 pg/ml vs 1585 ± 263 pg/ml, respectively). Instillation of LPS markedly increased KC, TNF-α, MMP-8, MMP-9 and TIMP-1 mRNA levels in the lungs of control and SS mice compared to animals that received PBS instead of LPS (Control, KC: 0.19 ± 0.047 vs 0.01 ± 0.005; TNF-α: 0.30 ± 0.07 vs 0.01 ± 0.002; MMP-8: 0.2 ± 0.06 vs 0.016 ± 0.004; MMP-9: 0.22 ± 0.03 vs 0.08 ± 0.01; TIMP-1: 0.32 ± 0.06 vs 0.09 ± 0.03); (SS, KC: 0.42 ± 0.1 vs 0.039 ± 0.02; TNF-α: 0.23 ± 0.025 vs 0.02 ± 0.007; MMP-8: 0.42 ± 0.06 vs 0.06 ± 0.03; MMP-9: 0.49 ± 0.11 vs 0.11 ± 0.05; TIMP-1: 0.49 ± 0.11 vs 0.09 ± 0.03). However, the LPS-induced KC, MMP-8 and MMP-9 expression was significantly higher in SS mice lung compared than that of the control group (p&lt;0.05). Lung MMP-2, MMP-12 and TIMP-2 gene expressions were similar in the PBS and LPS groups and were not significantly different between SS and control mice. Our results indicate that chemokines and MMPs are critically involved in the recruitment of neutrophils to the lung following LPS challenge, and suggest that these inflammatory mediators may play a role in the development of pulmonary diseases in SCD. The findings from this study provide further support to the claim that a proinflammatory state is present in SCD and have important implications for the pathophysiology of lung injury in SCD.

Myelofibrosis (MF) is characterized by progressive bone marrow (BM) fibrosis resulting from aberrant megakaryopoeisis and expression of pro-inflammatory cytokines. These two processes, heavily influenced by bromodomain and extraterminal domain (BET)-mediated gene regulation, lead to myeloproliferation and cytopenias. CPI-0610 is a potent, selective and unique BET inhibitor under investigation in MF patients as monotherapy or in combination with ruxolitinib in the MANIFEST trial (NCT02158858). In ruxolitinib naïve and experienced MF patients, treatment with CPI-0610 monotherapy or in combination with ruxolitinib not only reduced spleen volume and symptoms, but also improved hemoglobin levels and overcame transfusion dependency in a subset of patients. To evaluate the effects of CPI-0610 on BM biology, correlative analyses were conducted using patient samples collected from the MANIFEST trial. Pre-treatment and post-treatment BM biopsy samples were available from 78 evaluable patients, with reticulin staining conducted and evaluated by local pathologists available for 34 pre-treatment and 24-week post-treatment pairs of BM biopsy samples. Improvement in reticulin staining of at least one grade was observed in 32% (11/34) of patients. Central pathology review of reticulin staining is ongoing and will be presented. Additional bone marrow biopsy pairs collected pre-treatment and 24-week post-treatment for exploratory histopathological assessments were available from a total of 23 unselected MANIFEST patients at the time of the abstract submission. To assess changes in erythroid and megakaryocytic lineages, H&E and immunohistochemistry (IHC) staining of CD71 and CD61 were conducted centrally. Semi-quantitative analysis revealed an overt increase in erythroid lineage formation in 61% (14/23) of patients. Patients with CD71 improvement in BM IHC generally showed a greater increase in median hemoglobin levels than those without improvement. Tight clusters of megakaryocytes (MK), characteristic in MF BM, were observed at baseline in all cases and were decreased/absent after treatment in 52% (12/23) patients. In addition, decreased MK number to &lt;5/high powered field was also observed in 39% (9/23) of patients. Overall improvement in MK histotopography was observed in 65% of patients (15/23). Central histopathology review with IHC staining, including reticulin staining, will be conducted on additional samples and will be available for presentation at the conference. To further dissect the underlying mechanism of these relative improvements in bone marrow composition and histotopography induced by CPI-0610, CD34+ hematopoietic stem cells were isolated from peripheral blood collected from multiple MF patients at baseline to evaluate the impact of CPI-0610 on MK and erythroid differentiation in vitro. When CD34+ cells from MF patients were treated with CPI-0610 in erythroid differentiation conditions in the presence of SCF, IL3 and EPO, a dose-dependent increase in more mature erythroid cell populations was observed. However, under the same conditions, ruxolitinib suppressed erythroid differentiation, which is consistent with ruxolitinib inhibition of EPO signaling and the anemia caused by ruxolitinib treatment in MF patients. Suppressive effects of ruxolitinib on erythroid differentiation were partially rescued by CPI-0610 in a dose-dependent manner. CPI-0610 treatment of CD34+ cells from MF patients in MK differentiating conditions in the presence of SCF, IL6, IL9 and TPO resulted in a dose-dependent decrease in proliferation of CD34+ cells and an increase in the ratio of late MK (CD34-/CD41a+/CD42b+) and early MK (CD34+/CD41a+/CD42b+). This CPI-0610-induced dose-dependent maturation of aberrant immature MK cells may play a role in reducing MF disease manifestations. Molecular studies to further our understanding of the underlying mechanisms of CPI-0610 treatment are ongoing. Taken together, these paired BM biopsy and in vitro myeloid maturation results demonstrated an effect of CPI-0610 in promoting erythroid and MK differentiation. These results may partially explain CPI-0610's clinical effects in MF patients, including rising hemoglobin, reduced transfusion dependency and reduction in spleen volume and symptoms. Disclosures Mertz: Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Keller:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Meyer:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Zavidij:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Cui:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Verstovsek:Roche: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Sierra Oncology: Consultancy, Research Funding; Protagonist Therapeutics: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding; Novartis: Consultancy, Research Funding; Blueprint Medicines Corp: Research Funding; Celgene: Consultancy, Research Funding; CTI Biopharma Corp: Research Funding; PharmaEssentia: Research Funding; Incyte Corporation: Consultancy, Research Funding; ItalPharma: Research Funding; NS Pharma: Research Funding. Gupta:Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Incyte: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Talpaz:IMAGO: Consultancy; Takeda: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Constellation Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Harrison:Promedior: Honoraria; AOP Orphan Pharmaceuticals: Honoraria; Roche: Honoraria; Incyte Corporation: Speakers Bureau; Gilead Sciences: Honoraria, Speakers Bureau; CTI Biopharma Corp: Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Janssen: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Sierra Oncology: Honoraria. Mead:CTI: Consultancy; Gilead: Consultancy; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; Celgene/BMS: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding; Abbvie: Consultancy. Mesa:Novartis: Consultancy; CTI BioPharma: Research Funding; Bristol Myers Squibb: Research Funding; Sierra Oncology: Consultancy; Genentech: Research Funding; Samus Therapeutics: Research Funding; LaJolla Pharmaceutical Company: Consultancy; Incyte: Research Funding; Promedior: Research Funding; AbbVie: Research Funding. Mascarenhas:Celgene, Prelude, Galecto, Promedior, Geron, Constellation, and Incyte: Consultancy; Incyte, Kartos, Roche, Promedior, Merck, Merus, Arog, CTI Biopharma, Janssen, and PharmaEssentia: Other: Research funding (institution). Senderowicz:Constellation Pharmaceuticals: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months; Puma Biotechnology: Membership on an entity's Board of Directors or advisory committees. Colak:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Shao:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Luptakova:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Humphrey:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Wang:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Trojer:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

The pandemic of COVID-19 has afflicted every individual and has initiated a cascade of directly or indirectly involved events in precipitating mental health issues. The human species is a wanderer and hunter-gatherer by nature, and physical social distancing and nationwide lockdown have confined an individual to physical isolation. The present review article was conceived to address psychosocial and other issues and their aetiology related to the current pandemic of COVID-19. The elderly age group has most suffered the wrath of SARS-CoV-2, and social isolation as a preventive measure may further induce mental health issues. Animal model studies have demonstrated an inappropriate interacting endogenous neurotransmitter milieu of dopamine, serotonin, glutamate, and opioids, induced by social isolation that could probably lead to observable phenomena of deviant psychosocial behavior. Conflicting and manipulated information related to COVID-19 on social media has also been recognized as a global threat. Psychological stress during the current pandemic in frontline health care workers, migrant workers, children, and adolescents is also a serious concern. Mental health issues in the current situation could also be induced by being quarantined, uncertainty in business, jobs, economy, hampered academic activities, increased screen time on social media, and domestic violence incidences. The gravity of mental health issues associated with the pandemic of COVID-19 should be identified at the earliest. Mental health organization dedicated to current and future pandemics should be established along with Government policies addressing psychological issues to prevent and treat mental health issues need to be developed. References World Health Organization (WHO) Coronavirus Disease (COVID-19) Dashboard. Available at: https://covid19.who.int/ [Accessed on 23 August 2020] Sim K, Chua HC. The psychological impact of SARS: a matter of heart and mind. CMAJ. 2004; 170:811e2. https://doi.org/10.1503/cmaj.1032003. Wu P, Fang Y, Guan Z, Fan B, Kong J, Yao Z, et al. The psychological impact of the SARS epidemic on hospital employees in China: exposure, risk perception, and altruistic acceptance of risk. Can J Psychiatr. 2009; 54:302e11. https://doi.org/10.1177/070674370905400504. Brooks SK, Webster RK, Smith LE, Woodland L, Wessely S, Greenberg N, et al. The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020; 395:912e20. https://doi.org/10.1016/S0140-6736(20)30460-8. Robertson E, Hershenfield K, Grace SL, Stewart DE. The psychosocial effects of being quarantined following exposure to SARS: a qualitative study of Toronto health care workers. Can J Psychiatr. 2004; 49:403e7. https://doi.org/10.1177/070674370404900612. Barbisch D, Koenig KL, Shih FY. Is there a case for quarantine? Perspectives from SARS to Ebola. Disaster Med Public Health Prep. 2015; 9:547e53. https://doi.org/10.1017/dmp.2015.38. Jeong H, Yim HW, Song YJ, Ki M, Min JA, Cho J, et al. Mental health status of people isolated due to Middle East Respiratory Syndrome. Epidemiol Health. 2016;38: e2016048. https://doi.org/10.4178/epih.e2016048. Liu X, Kakade M, Fuller CJ, Fan B, Fang Y, Kong J, et al. Depression after exposure to stressful events: lessons learned from the severe acute respiratory syndrome epidemic. Compr Psychiatr. 2012; 53:15e23. https://doi.org/10.1016/j.comppsych.2011.02.003 Chadda RK, Deb KS. Indian family systems, collectivistic society and psychotherapy. Indian J Psychiatry. 2013;55: S299‑ https://dx.doi.org/10.4103%2F0019-5545.105555. Grover S, Sahoo S, Mehra A, Avasthi A, Tripathi A, Subramanyan A, et al. Psychological impact of COVID‑19 lockdown: An online survey from India. Indian J Psychiatry. 2020; 62:354-62. https://doi.org/ 10.4103/psychiatry.IndianJPsychiatry _427_20. Hawkley LC, Cacioppo JT. Loneliness matters: a theoretical and empirical review of consequences and mechanisms. Ann Behav Med. 2010; 40: 218–27. https://dx.doi.org/10.1007%2Fs12160-010-9210-8. Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet. 2020;395(10223):507-13. https://doi.org/10.1016/S0140-6736(20)30211-7. Bhandari S, Sharma R, Singh Shaktawat A, Banerjee S, Patel B, Tak A, et al. COVID-19 related mortality profile at a tertiary care centre: a descriptive study. Scr Med. 2020;51(2):69-73. https://doi.org/10.5937/scriptamed51-27126. Baumeister RF, Leary MR. The need to belong: desire for interpersonal attachments as a fundamental human motivation. Psychol Bull. 1995; 117: 497–529. https://doi.org/10.1037/0033-2909.117.3.497. Caspi A, Harrington H, Moffitt TE, Milne BJ, Poulton R. Socially isolated children 20 years later: risk of cardiovascular disease. Arch Pediatr Adolesc Med. 2006; 160(8):805-11. https://doi.org/10.1001/archpedi.160.8.805. Eaker ED, Pinsky J, Castelli WP. Myocardial infarction and coronary death among women: psychosocial predictors from a 20-year follow-up of women in the Framingham Study. Am J Epidemiol. 1992; 135(8):854-64. https://doi.org/10.1093/oxfordjournals.aje.a116381. Luo Y, Hawkley LC, Waite LJ, Cacioppo JT. Loneliness, health, and mortality in old age: a national longitudinal study. Soc Sci Med. 2012 Mar; 74(6):907-14. https://dx.doi.org/10.1016%2Fj.socscimed.2011.11.028. Olsen RB, Olsen J, Gunner-Svensson F, Waldstrøm B. Social networks and longevity. A 14-year follow-up study among elderly in Denmark. Soc Sci Med. 1991; 33(10):1189-95. https://doi.org/10.1016/0277-9536(91)90235-5. Patterson AC, Veenstra G. Loneliness and risk of mortality: a longitudinal investigation in Alameda County, California. Soc Sci Med. 2010; 71(1):181-6. https://doi.org/10.1016/j.socscimed.2010.03.024. Savikko N, Routassalo P, Tilvis RS, Strandberg TE, Pitkalla KH. Predictors and subjective causes of loneliness in an aged population. Arch Gerontol Geriatrics. 2005; 41:3;223-33. https://doi.org/10.1016/j.archger.2005.03.002. Health Advisory for Elderly Population of India during COVID19. Available at: https://www.mohfw.gov.in/pdf/AdvisoryforElderlyPopulation.pdf [Accessed on 13 August 2020]. Dicks D, Myers R, Kling A. Uncus and amygdala lesions: effects on social behavior in the free-ranging rhesus monkey. Science. 1969; 165:69–71. https://doi.org/10.1126/science.165.3888.69. Kanai R, Bahrami B, Duchaine B, Janik A, Banissy MJ, Rees G. Brain structure links loneliness to social perception. Curr Biol. 2012; 22(20):1975-9. https://dx.doi.org/10.1016%2Fj.cub.2012.08.045. Bender AR, Daugherty A, Raz N. Vascular risk moderates associations between hippocampal subfield volumes and memory. J Cogn Neurosci. 2013; 25:1851–62. https://doi.org/10.1162/jocn_a_00435. Raz N. Diabetes: brain, mind, insulin–what is normal and do we need to know? Nat Rev Endocrinol. 2011; 7:636–7. https://doi.org/10.1038/nrendo.2011.149. Colcombe SJ, Erickson KI, Naftali R, Andrew GW, Cohen NJ, McAuley E, et al. Aerobic fitness reduces brain tissue loss in aging humans. J Gerontol A Biol Sci Med Sci. 2003; 58:176–80. https://doi.org/10.1093/gerona/58.2.m176. Maass A, Düzel S, Goerke M, Becke A, Sobieray U, Neumann K, et al. Vascular hippocampal plasticity after aerobic exercise in older adults. Mol Psychiatry. 2015; 20, 585–93. https://doi.org/10.1038/mp.2014.114. Wilson RS, Krueger KR, Arnold SE, Schneider JA, Kelly JF, Barnes LL, et al. Loneliness and Risk of Alzheimer Disease. Arch Gen Psychiatry. 2007;64(2):234–240. https://doi.org/10.1001/archpsyc.64.2.234. Kogan JH, Frankland PW, Silva AJ. Long-term memory underlying hippocampus-dependent social recognition in mice. Hippocampus. 2000;10(1):47-56. https://doi.org/10.1002/(sici)1098-1063(2000)10:1%3C47::aid-hipo5%3E3.0.co;2-6. Yorgason JT, España RA, Konstantopoulos JK, Weiner JL, Jones SR. Enduring increases in anxiety-like behavior and rapid nucleus accumbens dopamine signaling in socially isolated rats. Eur J Neurosci. 2013;37(6):1022-31. https://doi.org/10.1111/ejn.12113. Bledsoe AC, Oliver KM, Scholl JL, Forster GL. Anxiety states induced by post-weaning social isolation are mediated by CRF receptors in the dorsal raphe nucleus. Brain Res Bull. 2011;85(3-4):117-22. https://dx.doi.org/10.1016%2Fj.brainresbull.2011.03.003. Lukkes JL, Engelman GH, Zelin NS, Hale MW, Lowry CA. Post-weaning social isolation of female rats, anxiety-related behavior, and serotonergic systems. Brain Res. 2012; 1443:1-17. https://dx.doi.org/10.1016%2Fj.brainres.2012.01.005. Ago Y, Araki R, Tanaka T, Sasaga A, Nishiyama S, Takuma K, et al. Role of social encounter-induced activation of prefrontal serotonergic systems in the abnormal behaviors of isolation-reared mice. Neuropsychopharmacology. 2013; 38(8):1535-47. https://doi.org/10.1038/npp.2013.52. Veenema AH. Early life stress, the development of aggression and neuroendocrine and neurobiological correlates: what can we learn from animal models? Front Neuroendocrinol. 2009;30(4):497-518. https://doi.org/10.1016/j.yfrne.2009.03.003. Zhao X, Sun L, Jia H, Meng Q, Wu S, Li N, et al. Isolation rearing induces social and emotional function abnormalities and alters glutamate and neurodevelopment-related gene expression in rats. Prog Neuropsychopharmacol Biol Psychiatry. 2009;33(7):1173-1177. https://doi.org/10.1016/j.pnpbp.2009.06.016. Sciolino NR, Bortolato M, Eisenstein SA, Fu J, Oveisi F, Hohmann AG, et al. Social isolation and chronic handling alter endocannabinoid signaling and behavioral reactivity to context in adult rats. Neuroscience. 2010;168(2):371-86. https://dx.doi.org/10.1016%2Fj.neuroscience.2010.04.007. Ghasemi M, Phillips C, Trillo L, De Miguel Z, Das D, Salehi A. The role of NMDA receptors in the pathophysiology and treatment of mood disorders. Neurosci Biobehav Rev. 2014; 47:336-358. https://doi.org/10.1016/j.neubiorev.2014.08.017. Olivenza R, Moro MA, Lizasoain I, Lorenzo P, Fernández AP, Rodrigo J, et al. Chronic stress induces the expression of inducible nitric oxide synthase in rat brain cortex. J Neurochem. 2000;74(2):785-791. https://doi.org/10.1046/j.1471-4159.2000.740785.x. Maeng S, Zarate CA Jr, Du J, Schloesser RJ, McCammon J, Chen G, et al. Cellular mechanisms underlying the antidepressant effects of ketamine: role of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors. Biol Psychiatry. 2008;63(4):349-352. https://doi.org/10.1016/j.biopsych.2007.05.028. Kalia LV, Kalia SK, Salter MW. NMDA receptors in clinical neurology: excitatory times ahead. Lancet Neurol. 2008;7(8):742-755. https://dx.doi.org/10.1016%2FS1474-4422(08)70165-0. Waxman EA, Lynch DR. N-methyl-D-aspartate Receptor Subtypes: Multiple Roles in Excitotoxicity and Neurological Disease. The Neuroscientist. 2005; 11(1), 37–49. https://doi.org/10.1177/1073858404269012. Hermes G, Li N, Duman C, Duman R. Post-weaning chronic social isolation produces profound behavioral dysregulation with decreases in prefrontal cortex synaptic-associated protein expression in female rats. Physiol Behav. 2011;104(2):354-9. https://dx.doi.org/10.1016%2Fj.physbeh.2010.12.019. Sestito RS, Trindade LB, de Souza RG, Kerbauy LN, Iyomasa MM, Rosa ML. Effect of isolation rearing on the expression of AMPA glutamate receptors in the hippocampal formation. J Psychopharmacol. 2011;25(12):1720-1729. https://doi.org/10.1177/0269881110385595. Toua C, Brand L, Möller M, Emsley RA, Harvey BH. The effects of sub-chronic clozapine and haloperidol administration on isolation rearing induced changes in frontal cortical N-methyl-D-aspartate and D1 receptor binding in rats. Neuroscience. 2010;165(2):492-499. https://doi.org/10.1016/j.neuroscience.2009.10.039. Alò R, Avolio E, Mele M, Storino F, Canonaco A, Carelli A et al. Excitatory/inhibitory equilibrium of the central amygdala nucleus gates anti-depressive and anxiolytic states in the hamster. Pharmacol Biochem Behav. 2014; 118:79-86. https://doi.org/10.1016/j.pbb.2014.01.007. St JP, Petkov VV. Changes in 5-HT1 receptors in different brain structures of rats with isolation syndrome. General pharmacology. 1990;21(2):223-5. https://doi.org/10.1016/0306-3623(90)90905-2. Miachon S, Rochet T, Mathian B, Barbagli B, Claustrat B. Long-term isolation of Wistar rats alters brain monoamine turnover, blood corticosterone, and ACTH. Brain Res Bull. 1993;32(6):611-614. https://doi.org/10.1016/0361-9230(93)90162-5. Van den Berg CL, Van Ree JM, Spruijt BM, Kitchen I. Effects of juvenile isolation and morphine treatment on social interactions and opioid receptors in adult rats: behavioural and autoradiographic studies. Eur J Neurosci. 1999;11(9):3023-3032. https://doi.org/10.1046/j.1460-9568.1999.00717.x. Vanderschuren LJ, Stein EA, Wiegant VM, Van Ree JM. Social play alters regional brain opioid receptor binding in juvenile rats. Brain Res. 1995;680(1-2):148-156. https://doi.org/10.1016/0006-8993(95)00256-p. Moles A, Kieffer BL, D'Amato FR. Deficit in attachment behavior in mice lacking the mu-opioid receptor gene. Science. 2004;304(5679):1983-1986. https://doi.org/10.1126/science.1095943. Panksepp J, Herman BH, Vilberg T, Bishop P, DeEskinazi FG. Endogenous opioids and social behavior. Neurosci Biobehav Rev. 1980;4(4):473-487. https://doi.org/10.1016/0149-7634(80)90036-6. Gong JP, Onaivi ES, Ishiguro H, Liu Q, Tagliaferro PA, Brusco A, et al. Cannabinoid CB2 receptors: immunohistochemical localization in rat brain. Brain Res. 2006;1071(1):10-23. https://doi.org/10.1016/j.brainres.2005.11.035. Breivogel CS, Sim-Selley LJ. Basic neuroanatomy and neuropharmacology of cannabinoids. Int Rev Psychiatry 2009; 21:2:113-121. https://doi.org/10.1080/09540260902782760. Haj-Mirzaian A, Amini-Khoei H, Haj-Mirzaian A, Amiri S, Ghesmati M, Zahir M, et al. Activation of cannabinoid receptors elicits antidepressant-like effects in a mouse model of social isolation stress. Brain Res Bull. 2017; 130:200-210. https://doi.org/10.1016/j.brainresbull.2017.01.018. Banach M, Piskorska B, Czuczwar SJ, Borowicz KK. Nitric Oxide, Epileptic Seizures, and Action of Antiepileptic Drugs. CNS & Neurological Disorders - Drug Targets 2011;10: 808. https://doi.org/10.2174/187152711798072347. Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012;33(7):829-37, 837a-837d. https://dx.doi.org/10.1093%2Feurheartj%2Fehr304. Hu Y, Wu D, Luo C, Zhu L, Zhang J, Wu H, et al. Hippocampal nitric oxide contributes to sex difference in affective behaviors. PNAS. 2012, 109 (35) 14224-14229. https://doi.org/10.1073/pnas.1207461109. Khan MI, Ostadhadi S, Zolfaghari S, Mehr SE, Hassanzadeh G, Dehpour, A et al. The involvement of NMDA receptor/NO/cGMP pathway in the antidepressant like effects of baclofen in mouse force swimming test. Neuroscience Letters. 2016; 612:52-61. https://doi.org/10.1016/j.neulet.2015.12.006. Matsumoto K, Puia G, Dong E, Pinna G. GABAA receptor neurotransmission dysfunction in a mouse model of social isolation-induced stress: Possible insights into a non-serotonergic mechanism of action of SSRIs in mood and anxiety disorders. Stress. 2007; 10:1:3-12. https://doi.org/10.1080/10253890701200997. Zlatković J, Filipović D. Chronic social isolation induces NF-κB activation and upregulation of iNOS protein expression in rat prefrontal cortex. Neurochem Int. 2013;63(3):172-179. https://doi.org/10.1016/j.neuint.2013.06.002. Haj-Mirzaian A, Amiri S, Kordjazy N, Momeny M, Razmi A, Balaei MR, et al. Lithium attenuated the depressant and anxiogenic effect of juvenile social stress through mitigating the negative impact of interlukin-1β and nitric oxide on hypothalamic-pituitary-adrenal axis function. Neuroscience. 2016; 315:271-285. https://doi.org/10.1016/j.neuroscience.2015.12.024. Larson HJ. The biggest pandemic risk? Viral misinformation. Nature 2018; 562:309. https://doi.org/10.1038/d41586-018-07034-4. Zarocostas J. How to fight an infodemic. Lancet 2020; 395:676. https://doi.org/10.1016/S0140-6736(20)30461-X. World Health Organization, 2019. Ebola Virus Disease – Democratic Republic of the Congo. Geneva, Switzerland: WHO. Available at: https://www.who.int/csr/don/28-november-2019-ebola-drc/en/ [Accessed on August 8, 2020] Times of India. Covid-19: doctors gone to collect samples attacked in Indore. Available at: https://timesofindia.indiatimes.com/videos/news/covid-19-doctors-goneto- collect-samples-attacked-in-indore/videoshow/74942153.cms; 2020 [Accessed on August 8, 2020]. Withnall A. Coronavirus: why India has had to pass new law against attacks on healthcare workers. The Independent. April 23, 2020. Semple K. “Afraid to be a nurse”: health workers under attack. The New York Times. 2020 Apr 27. The Economist. Health workers become unexpected targets during COVID-19. The Economist. May 11, 2020. Turan B, Budhwani H, Fazeli PL, Browning WR, Raper JL, Mugavero MJ, et al. How does stigma affect people living with HIV? The mediating roles of internalized and anticipated HIV stigma in the effects of perceived community stigma on health and psychosocial outcomes. AIDS Behav. 2017; 21: 283–291. https://doi.org/10.1007/s10461-016-1451-5. James PB, Wardle J, Steel A, Adams J. An assessment of Ebola-related stigma and its association with informal healthcare utilisation among Ebola survivors in Sierra Leone: a cross sectional study. BMC Public Health. 2020; 20: 182. https://doi.org/10.1186/s12889-020-8279-7. Aljazeera, 2020. Iran: Over 700 Dead after Drinking Alcohol to Cure Coronavirus. Aljazeera. Available at: https://www.aljazeera.com/ news/2020/04/iran-700-dead-drinking-alcohol-cure-coronavirus200427163529629.html. (Accessed June 4, 2020) Delirrad M, Mohammadi AB, 2020. New methanol poisoning outbreaks in Iran following COVID-19 pandemic. Alcohol Alcohol. 55: 347–348. https://doi.org/10.1093/alcalc/agaa036. Hassanian-Moghaddam H, Zamani N, Kolahi A-A, McDonald R, Hovda KE. Double trouble: methanol outbreak in the wake of the COVID-19 pandemic in Iran-a cross-sectional assessment. Crit Care. 2020; 24: 402. https://doi.org/10.1186/s13054-020-03140-w. Soltaninejad K. Methanol Mass Poisoning Outbreak: A Consequence of COVID-19 Pandemic and Misleading Messages on Social Media. Int J Occup Environ Med. 2020;11(3):148-150. https://dx.doi.org/10.34172%2Fijoem.2020.1983. Islam MS, Sarkar T, Khan SH, Kamal AM, Hasan SMM, Kabir A, et al. COVID-19–Related Infodemic and Its Impact on Public Health: A Global Social Media Analysis. Am J Trop Med Hyg. 2020; 00(0):1–9. https://doi.org/10.4269/ajtmh.20-0812. Hawryluck L, Gold W, Robinson S, Pogorski S, Galea S, Styra R. SARS control and psychological effects of quarantine, Toronto, Canada. Emerg Infect Dis. 2004;10(7):1206–1212. https://dx.doi.org/10.3201%2Feid1007.030703. Lee S, Chan LYY, Chau AAM, Kwok KPS, Kleinman A. The experience of SARS-related stigma at Amoy Gardens. Soc Sci Med. 2005; 61(9): 2038-2046. https://doi.org/10.1016/j.socscimed.2005.04.010. Yoon MK Kim SY Ko HS Lee MS. System effectiveness of detection, brief intervention and refer to treatment for the people with post-traumatic emotional distress by MERS: a case report of community-based proactive intervention in South Korea. Int J Ment Health Syst. 2016; 10: 51. https://doi.org/10.1186/s13033-016-0083-5. Reynolds DL, Garay JR, Deamond SL, Moran MK, Gold W, Styra R. Understanding, compliance and psychological impact of the SARS quarantine experience. Epidemiol Infect. 2008; 136: 997-1007. https://dx.doi.org/10.1017%2FS0950268807009156. Marjanovic Z, Greenglass ER, Coffey S. The relevance of psychosocial variables and working conditions in predicting nurses' coping strategies during the SARS crisis: an online questionnaire survey. Int J Nurs Stud. 2007; 44(6): 991-998. https://doi.org/10.1016/j.ijnurstu.2006.02.012. Bai Y, Lin C-C, Lin C-Y, Chen J-Y, Chue C-M, Chou P. Survey of stress reactions among health care workers involved with the SARS outbreak. Psychiatr Serv. 2004; 55: 1055-1057. https://doi.org/10.1176/appi.ps.55.9.1055. Ministry of Health and Family Welfare. Available at: https://www.mohfw.gov.in/pdf/Guidelinesforhomequarantine.pdf [Accessed on 25 August 2020]. Ministry of Health and Family Welfare. Available at: https://www.mohfw.gov.in/pdf/RevisedguidelinesforHomeIsolationofverymildpresymptomaticCOVID19cases10May2020.pdf [Accessed on 25 August 2020]. Ministry of Health and Family Welfare. Available at: https://www.mohfw.gov.in/pdf/AdvisoryformanagingHealthcareworkersworkinginCOVIDandNonCOVIDareasofthehospital.pdf (Accessed on 25 August 2020). Ministry of Health and Family Welfare. Available at: https://www.mohfw.gov.in/pdf/RevisedguidelinesforInternationalArrivals02082020.pdf [Accessed on 25 August 2020]. Cost of the lockdown? Over 10% of GDP loss for 18 states. Available at: https://timesofindia.indiatimes.com/india/cost-of-the-lockdown-over-10-of-gdp-loss-for-18-states/articleshow/76028826.cms [Accessed on 21 August 2020]. Jorda O, Singh SR, Taylor AM. Longer-Run Economic Consequences of Pandemics. Federal Reserve Bank of San Francisco Working Paper. 2020-09. https://doi.org/10.24148/wp2020-09. Firdaus G. Mental well‑being of migrants in urban center of India: Analyzing the role of social environment. Indian J Psychiatry. 2017; 59:164‑ https://doi.org/10.4103/psychiatry.indianjpsychiatry_272_15. National Crime Record Bureau. Annual Crime in India Report. New Delhi, India: Ministry of Home Affairs; 2018. 198 migrant workers killed in road accidents during lockdown: Report. Available at: https://www.hindustantimes.com/india-news/198-migrant-workers-killed-in-road-accidents-during-lockdown-report/story-hTWzAWMYn0kyycKw1dyKqL.html [Accessed on 25 August 2020]. Qiu H, Wu J, Hong L, Luo Y, Song Q, Chen D. Clinical and epidemiological features of 36 children with coronavirus disease 2019 (COVID-19) in Zhejiang, China: an observational cohort study. Lancet Infect Dis. 2020; 20:689-96. https://doi.org/10.1016/S1473-3099(20)30198-5. Dalton L, Rapa E, Stein A. Protecting the psychological health of through effective communication about COVID-19. Lancet Child Adolesc Health. 2020;4(5):346-347. https://doi.org/10.1016/S2352-4642(20)30097-3. Centre for Disease Control. Helping Children Cope with Emergencies. Available at: https://www.cdc.gov/childrenindisasters/helping-children-cope.html [Accessed on 25 August 2020]. Liu JJ, Bao Y, Huang X, Shi J, Lu L. Mental health considerations for children quarantined because of COVID-19. Lancet Child & Adolesc Health. 2020; 4(5):347-349. https://doi.org/10.1016/S2352-4642(20)30096-1. Sprang G, Silman M. Posttraumatic Stress Disorder in Parents and Youth After Health-Related Disasters. Disaster Med Public Health Prep. 2013;7(1):105-110. https://doi.org/10.1017/dmp.2013.22. Rehman U, Shahnawaz MG, Khan NH, Kharshiing KD, Khursheed M, Gupta K, et al. Depression, Anxiety and Stress Among Indians in Times of Covid-19 Lockdown. Community Ment Health J. 2020:1-7. https://doi.org/10.1007/s10597-020-00664-x. Cao W, Fang Z, Hou, Han M, Xu X, Dong J, et al. The psychological impact of the COVID-19 epidemic on college students in China. Psychiatry Research. 2020; 287:112934. https://doi.org/10.1016/j.psychres.2020.112934. Wang C, Zhao H. The Impact of COVID-19 on Anxiety in Chinese University Students. Front Psychol. 2020; 11:1168. https://dx.doi.org/10.3389%2Ffpsyg.2020.01168. Kang L, Li Y, Hu S, Chen M, Yang C, Yang BX, et al. The mental health of medical workers in Wuhan, China dealing with the 2019 novel coronavirus. Lancet Psychiatry 2020;7(3): e14. https://doi.org/10.1016/s2215-0366(20)30047-x. Lai J, Ma S, Wang Y, Cai Z, Hu J, Wei N, et al. Factors associated with mental health outcomes among health care workers exposed to coronavirus disease 2019. JAMA Netw Open 2020;3(3): e203976. https://doi.org/10.1001/jamanetworkopen.2020.3976. Lancee WJ, Maunder RG, Goldbloom DS, Coauthors for the Impact of SARS Study. Prevalence of psychiatric disorders among Toronto hospital workers one to two years after the SARS outbreak. Psychiatr Serv. 2008;59(1):91-95. https://dx.doi.org/10.1176%2Fps.2008.59.1.91. Tam CWC, Pang EPF, Lam LCW, Chiu HFK. Severe acute respiratory syndrome (SARS) in Hongkong in 2003: Stress and psychological impact among frontline healthcare workers. Psychol Med. 2004;34 (7):1197-1204. https://doi.org/10.1017/s0033291704002247. Lee SM, Kang WS, Cho A-R, Kim T, Park JK. Psychological impact of the 2015 MERS outbreak on hospital workers and quarantined hemodialysis patients. Compr Psychiatry. 2018; 87:123-127. https://dx.doi.org/10.1016%2Fj.comppsych.2018.10.003. Koh D, Meng KL, Chia SE, Ko SM, Qian F, Ng V, et al. Risk perception and impact of severe acute respiratory syndrome (SARS) on work and personal lives of healthcare workers in Singapore: What can we learn? Med Care. 2005;43(7):676-682. https://doi.org/10.1097/01.mlr.0000167181.36730.cc. Verma S, Mythily S, Chan YH, Deslypere JP, Teo EK, Chong SA. Post-SARS psychological morbidity and stigma among general practitioners and traditional Chinese medicine practitioners in Singapore. Ann Acad Med Singap. 2004; 33(6):743e8. Yeung J, Gupta S. Doctors evicted from their homes in India as fear spreads amid coronavirus lockdown. CNN World. 2020. Available at: https://edition.cnn.com/2020/03/25/asia/india-coronavirus-doctors-discrimination-intl-hnk/index.html. [Accessed on 24 August 2020] Violence Against Women and Girls: the Shadow Pandemic. UN Women. 2020. May 3, 2020. Available at: https://www.unwomen.org/en/news/stories/2020/4/statement-ed-phumzile-violence-against-women-during-pandemic. [Accessed on 24 August 2020]. Gearhart S, Patron MP, Hammond TA, Goldberg DW, Klein A, Horney JA. The impact of natural disasters on domestic violence: an analysis of reports of simple assault in Florida (1999–2007). Violence Gend. 2018;5(2):87–92. https://doi.org/10.1089/vio.2017.0077. Sahoo S, Rani S, Parveen S, Pal Singh A, Mehra A, Chakrabarti S, et al. Self-harm and COVID-19 pandemic: An emerging concern – A report of 2 cases from India. Asian J Psychiatr 2020; 51:102104. https://dx.doi.org/10.1016%2Fj.ajp.2020.102104. Ghosh A, Khitiz MT, Pandiyan S, Roub F, Grover S. Multiple suicide attempts in an individual with opioid dependence: Unintended harm of lockdown during the COVID-19 outbreak? Indian J Psychiatry 2020; [In Press]. The Economic Times. 11 Coronavirus suspects flee from a hospital in Maharashtra. March 16 2020. Available at: https://economictimes.indiatimes.com/news/politics-and-nation/11-coronavirus-suspects-flee-from-a-hospital-in-maharashtra/videoshow/74644936.cms?from=mdr. [Accessed on 23 August 2020]. Xiang Y, Yang Y, Li W, Zhang L, Zhang Q, Cheung T, et al. Timely mental health care for the 2019 novel coronavirus outbreak is urgently needed. The Lancet Psychiatry 2020;(3):228–229. https://doi.org/10.1016/S2215-0366(20)30046-8. Van Bortel T, Basnayake A, Wurie F, Jambai M, Koroma A, Muana A, et al. Psychosocial effects of an Ebola outbreak at individual, community and international levels. Bull World Health Organ. 2016;94(3):210–214. https://dx.doi.org/10.2471%2FBLT.15.158543. Kumar A, Nayar KR. COVID 19 and its mental health consequences. Journal of Mental Health. 2020; ahead of print:1-2. https://doi.org/10.1080/09638237.2020.1757052. Gupta R, Grover S, Basu A, Krishnan V, Tripathi A, Subramanyam A, et al. Changes in sleep pattern and sleep quality during COVID-19 lockdown. Indian J Psychiatry. 2020; 62(4):370-8. https://doi.org/10.4103/psychiatry.indianjpsychiatry_523_20. Duan L, Zhu G. Psychological interventions for people affected by the COVID-19 epidemic. Lancet Psychiatry. 2020;7(4): P300-302. https://doi.org/10.1016/S2215-0366(20)30073-0. Dubey S, Biswas P, Ghosh R, Chatterjee S, Dubey MJ, Chatterjee S et al. Psychosocial impact of COVID-19. Diabetes Metab Syndr. 2020; 14(5): 779–788. https://dx.doi.org/10.1016%2Fj.dsx.2020.05.035. Wright R. The world's largest coronavirus lockdown is having a dramatic impact on pollution in India. CNN World; 2020. Available at: https://edition.cnn.com/2020/03/31/asia/coronavirus-lockdown-impact-pollution-india-intl-hnk/index.html. [Accessed on 23 August 2020] Foster O. ‘Lockdown made me Realise What’s Important’: Meet the Families Reconnecting Remotely. The Guardian; 2020. Available at: https://www.theguardian.com/keep-connected/2020/apr/23/lockdown-made-me-realise-whats-important-meet-the-families-reconnecting-remotely. (Accessed on 23 August 2020) Bilefsky D, Yeginsu C. Of ‘Covidivorces’ and ‘Coronababies’: Life During a Lockdown. N. Y. Times; 2020. Available at: https://www.nytimes.com/2020/03/27/world/coronavirus-lockdown-relationships.html [Accessed on 23 August 2020]

Salmonella Typhimurium utilizes a type three secretion system (T3SS) encoded on the Salmonella pathogenicity island 1 (SPI1) to invade intestinal epithelial cells and induce inflammatory diarrhea. HilA activates expression of the T3SS structural genes. Expression of hilA is controlled by the transcription factors HilD, HilC and RtsA, which act in a complex feed-forward regulatory loop. The nucleoid-associated protein H-NS is a xenogenic silencer that has a major effect on SPI1 expression. In this work, we use genetic techniques to show that disruptions of the chromosomal region surrounding hilD have a cis-effect on H-NS-mediated repression of the hilD promoter; this effect occurs asymmetrically over ∼4 kb spanning the prgH-hilD intergenic region. CAT cassettes inserted at various positions in this region are also silenced in relation to the proximity to the hilD promoter. We identify a putative H-NS nucleation site, mutation of which results in de-repression of the locus. Furthermore, we genetically show that HilD abrogates H-NS-mediated silencing to activate the hilD promoter. In contrast, H-NS-mediated repression of the hilA promoter, downstream of hilD , is through its control of HilD, which directly activates hilA transcription. Likewise, activation of the prgH promoter, although in a region silenced by H-NS, is strictly dependent on HilA. In summary, we propose a model in which H-NS nucleates within the hilD promoter region to polymerize and exert its repressive effect. Thus, H-NS-mediated repression of SPI1 is primarily through control of hilD expression, with HilD capable of overcoming H-NS to autoactivate. IMPORTANCE The foodborne pathogen Salmonella relies on a type III secretion system to invade intestinal epithelial cells and initiate infection. This system was acquired through horizontal gene transfer, essentially creating the Salmonella genus. Expression of this critical virulence factor is controlled by a complex regulatory network. The nucleoid protein H-NS is a global repressor of horizontally acquired genomic loci. Here we identify the critical site of H-NS regulation in this system and show that alterations to the DNA over a surprisingly large region affect this regulation, providing important information regarding the mechanism of H-NS action.

The DNA-binding protein H-NS is a pleiotropic gene regulator in gram-negative bacteria. Through its capacity to sense temperature and other environmental factors, H-NS allows pathogens like Salmonella to adapt their gene expression to their presence inside or outside warm-blooded hosts. To investigate how this sensing mechanism may have evolved to fit different bacterial lifestyles, we compared H-NS orthologs from bacteria that infect humans, plants, and insects, and from bacteria that live on a deep-sea hypothermal vent. The combination of biophysical characterization, high-resolution proton-less NMR spectroscopy and molecular simulations revealed, at an atomistic level, how the same general mechanism was adapted to specific habitats and lifestyles. In particular, we demonstrate how environment-sensing characteristics arise from specifically positioned intra- or intermolecular electrostatic interactions. Our integrative approach clarified the exact modus operandi for H-NS–mediated environmental sensing and suggests that this sensing mechanism resulted from the exaptation of an ancestral protein feature.

Bacteria frequently need to adapt to altered environmental conditions. Adaptation requires changes in gene expression, often mediated by global regulators of transcription. The nucleoid-associated protein H-NS is a key global regulator in Gram-negative bacteria and is believed to be a crucial player in bacterial chromatin organization via its DNA-bridging activity. H-NS activity in vivo is modulated by physico-chemical factors (osmolarity, pH, temperature) and interaction partners. Mechanistically, it is unclear how functional modulation of H-NS by such factors is achieved. Here, we show that a diverse spectrum of H-NS modulators alter the DNA-bridging activity of H-NS. Changes in monovalent and divalent ion concentrations drive an abrupt switch between a bridging and non-bridging DNA-binding mode. Similarly, synergistic and antagonistic co-regulators modulate the DNA-bridging efficiency. Structural studies suggest a conserved mechanism: H-NS switches between a ‘closed’ and an ‘open’, bridging competent, conformation driven by environmental cues and interaction partners.

ABSTRACT A multifunctional autoprocessing repeats-in-toxin (MARTX) toxin plays an essential role in the virulence of many pathogens, including a fulminating human pathogen Vibrio vulnificus. H-NS and HlyU repress and derepress expression of the MARTX toxin gene rtxA in V. vulnificus, respectively. However, little is known about other regulatory proteins and environmental signals involved in rtxA regulation. In this study, we found that a leucine-responsive regulatory protein (Lrp) activates rtxA by binding directly and specifically to the rtxA promoter, PrtxA. Phased hypersensitivity resulting from DNase I cleavage of the PrtxA regulatory region suggests that Lrp probably induces DNA bending in PrtxA. Lrp activates PrtxA independently of H-NS and HlyU, and leucine inhibits Lrp binding to PrtxA and reduces the Lrp-mediated activation. Furthermore, a cyclic AMP receptor protein (CRP) represses PrtxA, and exogenous glucose relieves the CRP-mediated repression. Biochemical and mutational analyses demonstrated that CRP binds directly and specifically to the upstream region of PrtxA, which presumably alters the DNA conformation in PrtxA and thus represses rtxA. Moreover, CRP represses expression of lrp and hlyU by binding directly to their upstream regions, forming coherent feed-forward loops with Lrp and HlyU. In conclusion, expression of rtxA is controlled by a regulatory network comprising CRP, Lrp, H-NS, and HlyU in response to changes in host environmental signals such as leucine and glucose. This collaborative regulation enables the elaborate expression of rtxA, thereby enhancing the fitness and pathogenesis of V. vulnificus during the course of infection. IMPORTANCE A MARTX toxin, RtxA, is an essential virulence factor of many pathogens, including Vibrio species. H-NS and HlyU repress and derepress, respectively, rtxA expression of a life-threatening pathogen, Vibrio vulnificus. We found that Lrp directly activates rtxA independently of H-NS and HlyU, and leucine inhibits the Lrp-mediated activation of rtxA. Furthermore, we demonstrated that CRP represses rtxA but derepresses in the presence of exogenous glucose. CRP represses rtxA not only directly by binding to upstream of rtxA but also indirectly by repressing lrp and hlyU. This is the first report of a regulatory network comprising CRP, Lrp, H-NS, and HlyU, which coordinates the rtxA expression in response to environmental signals such as leucine and glucose during infection. This elaborate regulatory network will enhance the fitness of V. vulnificus and contribute to its successful infection within the host.

ABSTRACT Shigella species, the causal agents of bacillary dysentery, use a type III secretion system (T3SS) to inject two waves of virulence proteins, known as effectors, into the colonic epithelium to subvert host cell machinery. Prior to host cell contact and secretion of the first wave of T3SS effectors, OspD1, an effector and antiactivator protein, prevents premature production of the second wave of effectors. Despite this important role, regulation of the ospD1 gene is not well understood. While ospD1 belongs to the large regulon of VirB, a transcriptional antisilencing protein that counters silencing mediated by the histone-like nucleoid structuring protein H-NS, it remains unclear if VirB directly or indirectly regulates ospD1. Additionally, it is not known if ospD1 is regulated by H-NS. Here, we identify the primary ospD1 transcription start site (+1) and show that the ospD1 promoter is remotely regulated by both VirB and H-NS. Our findings demonstrate that VirB regulation of ospD1 requires at least one of the two newly identified VirB regulatory sites, centered at −978 and −1270 relative to the ospD1 +1. Intriguingly, one of these sites lies on a 193-bp sequence found in three conserved locations on the large virulence plasmids of Shigella. The region required for H-NS-dependent silencing of ospD1 lies between −1120 and −820 relative to the ospD1 +1. Thus, our study provides further evidence that cis-acting regulatory sequences for transcriptional antisilencers and silencers, such as VirB and H-NS, can lie far upstream of the canonical bacterial promoter region (i.e., −250 to +1). IMPORTANCE Transcriptional silencing and antisilencing mechanisms regulate virulence gene expression in many important bacterial pathogens. In Shigella species, plasmid-borne virulence genes, such as those encoding the type III secretion system (T3SS), are silenced by the histone-like nucleoid structuring protein H-NS and antisilenced by VirB. Previous work at the plasmid-borne icsP locus revealed that VirB binds to a remotely located cis-acting regulatory site to relieve transcriptional silencing mediated by H-NS. Here, we characterize a second example of remote VirB antisilencing at ospD1, which encodes a T3SS antiactivator and effector. Our study highlights that remote transcriptional silencing and antisilencing occur more frequently in Shigella than previously thought, and it raises the possibility that long-range transcriptional regulation in bacteria is commonplace.

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) poses a challenging threat to public health. Based on the analysis of tet(X4)-positive plasmids in the NCBI database, we found that the IncX1-type plasmid is one of the most common vectors for spreading tet(X4) gene, but the mechanisms by which these plasmids adapt to host bacteria and maintain the persistence of antibiotic resistance genes (ARGs) remain unclear. Herein, we investigated the underlying mechanisms of how host bacteria modulate the fitness cost of IncX1 plasmids carrying tet(X4) gene. Interestingly, we found that the tet(X4)-bearing IncX1 plasmids encoding H-NS protein imposed low or no fitness cost in Escherichia coli and Klebsiella pneumoniae; instead, they partially promoted the virulence and biofilm formation in host bacteria. Regression analysis revealed that the expression of hns gene in plasmids was positively linked to the relative fitness of host bacteria. Furthermore, when pCE2::hns was introduced, the fitness of tet(X4)-positive IncX1 plasmid pRF55-1 without hns gene was significantly improved, indicating that hns mediates the improvement of fitness. Finally, we showed that the expression of hns gene is negatively correlated with the expression of tet(X4) gene, suggesting that the regulatory effect of H-NS on adaptability may be attributed to its inhibitory effect on the expression of ARGs. Together, our findings suggest the important role of plasmid-encoded H-NS protein in modulating the fitness of tet(X4)-bearing IncX1 plasmids, which shed new insight into the dissemination of tet(X4) gene in a biological environment.

Abstract Bacterial adaptation is largely shaped by horizontal gene transfer, xenogeneic silencing mediated by lineage-specific DNA bridgers (H-NS, Lsr2, MvaT and Rok), and various anti-silencing mechanisms. No xenogeneic silencing DNA bridger is known for α-proteobacteria, from which mitochondria evolved. By investigating α-proteobacterium Sinorhizobium fredii, a facultative legume microsymbiont, here we report the conserved zinc-finger bearing MucR as a novel xenogeneic silencing DNA bridger. Self-association mediated by its N-terminal domain (NTD) is required for DNA–MucR–DNA bridging complex formation, maximizing MucR stability, transcriptional silencing, and efficient symbiosis in legume nodules. Essential roles of NTD, CTD (C-terminal DNA-binding domain), or full-length MucR in symbiosis can be replaced by non-homologous NTD, CTD, or full-length protein of H-NS from γ-proteobacterium Escherichia coli, while NTD rather than CTD of Lsr2 from Gram-positive Mycobacterium tuberculosis can replace the corresponding domain of MucR in symbiosis. Chromatin immunoprecipitation sequencing reveals similar recruitment profiles of H-NS, MucR and various functional chimeric xenogeneic silencers across the multipartite genome of S. fredii, i.e. preferring AT-rich genomic islands and symbiosis plasmid with key symbiosis genes as shared targets. Collectively, the convergently evolved DNA bridger MucR predisposed α-proteobacteria to integrate AT-rich foreign DNA including symbiosis genes, horizontal transfer of which is strongly selected in nature.