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Published: 4 June 2021
Last updated: 18 February 2022

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1

Robertson, Kirstin P., C. Jeffrey Smith, Andrea M. Gough, and Edson R. Rocha. "Characterization of Bacteroides fragilis Hemolysins and Regulation and Synergistic Interactions of HlyA and HlyB." Infection and Immunity 74, no. 4 (April 2006): 2304–16. http://dx.doi.org/10.1128/iai.74.4.2304-2316.2006.

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ABSTRACT This study describes the presence of 10 hemolysin orthologs in the genome of the opportunistic human anaerobic pathogen Bacteroides fragilis, which is currently classified as a nonhemolytic bacterium. The hemolysins were designated HlyA through HlyI plus HlyIII. All cloned hemolysin genes were able to confer hemolytic activity to a nonhemolytic Escherichia coli strain on blood agar plates. Interestingly, HlyH was found to be present in the genome of the B. fragilis NCTC9343 strain but absent in strains 638R, YCH46, and Bacteroides thetaiotaomicron VPI-5482. The hemolysins HlyA, HlyB, and HlyIII were selected for further characterization. HlyA, HlyB, and HlyIII were cytolytic to erythrocytes on liquid hemolytic assay. When hlyA and hlyB were expressed together in a nonhemolytic E. coli strain, the strain showed enhanced hemolytic activity on blood agar plates. Further analysis revealed that HlyA and HlyB have synergistic hemolytic activity as detected by the liquid hemolytic assay. In addition, the two-component hemolysins HlyA and HlyB form a protein-protein complex in vivo as determined by bacterial two-hybrid system assay. The hlyB and hlyA genes are organized in an operon that is coordinately regulated by iron and oxygen. Northern blot hybridization analysis revealed that hlyBA were expressed as a bicistronic mRNA induced approximately 2.5-fold under low-iron conditions and repressed in iron-rich medium. The normal iron-regulated expression of hlyBA mRNA was lost in the furA mutant strain. In contrast, the hlyA gene was also expressed as a single mRNA in iron-rich medium, but its expression was reduced approximately threefold under low-iron conditions in a Fur-independent manner. This suggests that hlyA alone is regulated by an unidentified iron-dependent regulator. Moreover, the expression levels of hlyBA and hlyA were reduced about threefold following oxygen exposure and treatment with hydrogen peroxide. Taken together, these results suggest that iron and oxidative stress have an effect on the control of hlyBA and hlyA transcriptional levels. A hlyBA mutant was constructed, and its hemolytic activity was greatly diminished compared to those of the hlyIII mutant and parent strains. In addition, the hlyBA mutant had a significant modification in colony morphology and growth deficiency compared to the parent strain. The implications of these findings for the pathophysiology of B. fragilis in extraintestinal infections and competition in ecological systems for this organism are discussed.
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2

Pimenta, A. L., K. Racher, L. Jamieson, M. A. Blight, and I. B. Holland. "Mutations in HlyD, Part of the Type 1 Translocator for Hemolysin Secretion, Affect the Folding of the Secreted Toxin." Journal of Bacteriology 187, no. 21 (November 1, 2005): 7471–80. http://dx.doi.org/10.1128/jb.187.21.7471-7480.2005.

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ABSTRACT HlyD, a member of the membrane fusion protein family, is essential for the secretion of the RTX hemolytic toxin HlyA from Escherichia coli. Random point mutations affecting HlyA secretion were obtained, distributed in most periplasmic regions of the HlyD molecule. Analysis of the secretion phenotypes of different mutants allowed the identification of regions in HlyD involved in different steps of HlyA translocation. Four mutants, V349-I, T85-I, V334-I and L165-Q, were conditionally defective, a phenotype shown to be linked to the presence of inhibitory concentrations of Ca2+ in extracellular medium. Hly mutant T85-I was defective at an early stage in secretion, while mutants V334-I and L165-Q appeared to accumulate HlyA in the cell envelope, indicating a block at an intermediate step. Mutants V349-I, V334-I, and L165-Q were only partially defective in secretion, allowing significant levels of HlyA to be transported, but in the case of V349-I and L165-Q the HlyA molecules secreted showed greatly reduced hemolytic activity. Hemolysin molecules secreted from V349-I and V334-I are defective in normal folding and can be reactivated in vitro to the same levels as HlyA secreted from the wild-type translocator. Both V349-I and V334-I mutations mapped to the C-terminal lipoyl repeat motif, involved in the switching from the helical hairpin to the extended form of HlyD during assembly of the functional transport channel. These results suggest that HlyD is an integral component of the transport pathway, whose integrity is essential for the final folding of secreted HlyA into its active form.
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3

Sugamata, Yasuhiro, and Toshikazu Shiba. "Improved Secretory Production of Recombinant Proteins by Random Mutagenesis of hlyB, an Alpha-Hemolysin Transporter from Escherichia coli." Applied and Environmental Microbiology 71, no. 2 (February 2005): 656–62. http://dx.doi.org/10.1128/aem.71.2.656-662.2005.

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ABSTRACT Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA218) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 × 104 E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23°C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA218 fusion protein at 23 and 30°C but unexpectedly not at 37°C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA218 was detected only in the L448F mutant (AE104F) at 23°C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA218 fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37°C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.
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4

Stanley, Peter, Vassilis Koronakis, and Colin Hughes. "Acylation of Escherichia coli Hemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function." Microbiology and Molecular Biology Reviews 62, no. 2 (June 1, 1998): 309–33. http://dx.doi.org/10.1128/mmbr.62.2.309-333.1998.

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SUMMARY The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity. The inactive protoxin pro-HlyA is activated intracellularly by amide linkage of fatty acids to two internal lysine residues 126 amino acids apart, directed by the cosynthesized HlyC protein with acyl carrier protein as the fatty acid donor. This action distinguishes HlyC from all bacterial acyltransferases such as the lipid A, lux-specific, and nodulation acyltransferases, and from eukaryotic transferases such as N-myristoyl transferases, prenyltransferases, and thioester palmitoyltransferases. Most lipids directly attached to proteins may be classed as N-terminal amide-linked and internal ester-linked acyl groups and C-terminal ether-linked isoprenoid groups. The acylation of HlyA and related toxins does not equate to these but does appear related to a small number of eukaryotic proteins that include inflammatory cytokines and mitogenic and cholinergic receptors. While the location and structure of lipid moieties on proteins vary, there are common effects on membrane affinity and/or protein-protein interactions. Despite being acylated at two residues, HlyA does not possess a “double-anchor” motif and does not have an electrostatic switch, although its dependence on calcium binding for activity suggests that the calcium-myristoyl switch may have relevance. The acyl chains on HlyA may provide anchorage points onto the surface of the host cell lipid bilayer. These could then enhance protein-protein interactions either between HlyA and components of a host signal transduction pathway to influence cytokine production or between HlyA monomers to bring about oligomerization during pore formation.
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5

Masin, Jiri, Adriana Osickova, David Jurnecka, Nela Klimova, Humaira Khaliq, Peter Sebo, and Radim Osicka. "Retargeting from the CR3 to the LFA-1 receptor uncovers the adenylyl cyclase enzyme–translocating segment of Bordetella adenylate cyclase toxin." Journal of Biological Chemistry 295, no. 28 (May 11, 2020): 9349–65. http://dx.doi.org/10.1074/jbc.ra120.013630.

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The Bordetella adenylate cyclase toxin-hemolysin (CyaA) and the α-hemolysin (HlyA) of Escherichia coli belong to the family of cytolytic pore-forming Repeats in ToXin (RTX) cytotoxins. HlyA preferentially binds the αLβ2 integrin LFA-1 (CD11a/CD18) of leukocytes and can promiscuously bind and also permeabilize many other cells. CyaA bears an N-terminal adenylyl cyclase (AC) domain linked to a pore-forming RTX cytolysin (Hly) moiety, binds the complement receptor 3 (CR3, αMβ2, CD11b/CD18, or Mac-1) of myeloid phagocytes, penetrates their plasma membrane, and delivers the AC enzyme into the cytosol. We constructed a set of CyaA/HlyA chimeras and show that the CyaC-acylated segment and the CR3-binding RTX domain of CyaA can be functionally replaced by the HlyC-acylated segment and the much shorter RTX domain of HlyA. Instead of binding CR3, a CyaA1-710/HlyA411-1024 chimera bound the LFA-1 receptor and effectively delivered AC into Jurkat T cells. At high chimera concentrations (25 nm), the interaction with LFA-1 was not required for CyaA1-710/HlyA411-1024 binding to CHO cells. However, interaction with the LFA-1 receptor strongly enhanced the specific capacity of the bound CyaA1-710/HlyA411-1024 chimera to penetrate cells and deliver the AC enzyme into their cytosol. Hence, interaction of the acylated segment and/or the RTX domain of HlyA with LFA-1 promoted a productive membrane interaction of the chimera. These results help delimit residues 400–710 of CyaA as an “AC translocon” sufficient for translocation of the AC polypeptide across the plasma membrane of target cells.
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6

Russo, Thomas A., Zhengdong Wang, Bruce A. Davidson, Stacy A. Genagon, Janet M. Beanan, Ruth Olson, Bruce A. Holm, Paul R. Knight, Patricia R. Chess, and Robert H. Notter. "Surfactant dysfunction and lung injury due to theE. colivirulence factor hemolysin in a rat pneumonia model." American Journal of Physiology-Lung Cellular and Molecular Physiology 292, no. 3 (March 2007): L632—L643. http://dx.doi.org/10.1152/ajplung.00326.2006.

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This study tests the hypothesis that the virulence factor hemolysin (Hly) expressed by extraintestinal pathogenic Escherichia coli contributes to surfactant dysfunction and lung injury in a rat model of gram-negative pneumonia. Rats were instilled intratracheally with CP9 (wild type, Hly-positive), CP9 hlyA (Hly-minus), CP9 /pEK50 (supraphysiological Hly), or purified LPS. At 6 h postinfection, rats given CP9 had a decreased percentage content of large surfactant aggregates in cell-free bronchoalveolar lavage (BAL), decreased large aggregate surface activity, decreased PaO2/FiO2ratio, increased BAL albumin/protein levels, and increased histological evidence of lung injury compared with rats given CP9 hlyA or LPS. In addition, rats given CP9/ pEK50 or CP9 had decreased large aggregate surface activity, decreased PaO2/FiO2ratios, and increased BAL albumin/protein levels at 2 h postinfection compared with rats given CP9 hlyA. The severity of permeability lung injury based on albumin/protein levels in BAL at 2 h was ordered as CP9 /pEK50 > CP9 > CP9 hlyA > normal saline controls. Total lung titers of bacteria were increased at 6 h in rats given CP9 vs. CP9 hlyA, but bacterial titers were not significantly different at 2 h, indicating that increased surfactant dysfunction and lung injury were associated with Hly as opposed to bacterial numbers per se. Further studies in vitro showed that CP9 could directly lyse transformed pulmonary epithelial cells (H441 cells) but that indirect lysis of H441 cells secondary to Hly-induced neutrophil lysis did not occur. Together, these data demonstrate that Hly is an important direct mediator of surfactant dysfunction and lung injury in gram-negative pneumonia.
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7

Wang, Changying, Qianqian Li, Junqiang Lv, Xuan Sun, Yang Cao, Kaiyuan Yu, Chunhui Miao, Zhi-Song Zhang, Zhi Yao, and Quan Wang. "Alpha-hemolysin of uropathogenic Escherichia coli induces GM-CSF-mediated acute kidney injury." Mucosal Immunology 13, no. 1 (November 12, 2019): 22–33. http://dx.doi.org/10.1038/s41385-019-0225-6.

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Abstract Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), inducing acute pyelonephritis and may result in permanent renal scarring and failure. Alpha-hemolysin (HlyA), a key UPEC toxin, causes serious tissue damage; however, the mechanism through which HlyA induces kidney injury remains unclear. In the present study, granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by renal epithelial cells was upregulated by HlyA in vitro and in vivo, which induced M1 macrophage accumulation in kidney, and ADAM10 was found involved in HlyA-induced GM-CSF. Macrophage elimination or GM-CSF neutralization protected against acute kidney injury in mice, and increased GM-CSF was detected in urine of patients infected by hlyA-positive UPEC. In addition, HlyA was found to promote UPEC invasion into renal epithelial cells by interacting with Nectin-2 in vitro. However, HlyA did not affect bacterial titers during acute kidney infections, and HlyA-induced invasion did not contribute to GM-CSF upregulation in vitro, which indicate that HlyA-induced GM-CSF is independent of bacteria invasion. The role of GM-CSF in HlyA-mediated kidney injury may lead to novel strategies to treat acute pyelonephritis.
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8

Zaitseva, J., S. Jenewein, C. Oswald, T. Jumpertz, I. B. Holland, and L. Schmitt. "A molecular understanding of the catalytic cycle of the nucleotide-binding domain of the ABC transporter HlyB." Biochemical Society Transactions 33, no. 5 (October 26, 2005): 990–95. http://dx.doi.org/10.1042/bst0330990.

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The ABC transporter (ATP-binding-cassette transporter) HlyB (haemolysin B) is the central element of a type I secretion machinery, dedicated to the secretion of the toxin HlyA in Escherichia coli. In addition to the ABC transporter, two other indispensable elements are necessary for the secretion of the toxin across two membranes in a single step: the transenvelope protein HlyD and the outer membrane protein TolC. Despite the fact that the hydrolysis of ATP by HlyB fuels secretion of HlyA, the essential features of the underlying transport mechanism remain an enigma. Similar to all other ABC transporters, ranging from bacteria to man, HlyB is composed of two NBDs (nucleotide-binding domains) and two transmembrane domains. Here we summarize our detailed biochemical, biophysical and structural studies aimed at an understanding of the molecular principles of how ATP-hydrolysis is coupled to energy transduction, including the conformational changes occurring during the catalytic cycle, leading to substrate transport. We have obtained individual crystal structures for each single ground state of the catalytic cycle. From these and other biochemical and mutational studies, we shall provide a detailed molecular picture of the steps governing intramolecular communication and the utilization of chemical energy, due to ATP hydrolysis, in relation to resulting structural changes within the NBD. These data will be summarized in a general model to explain how these molecular machines achieve translocation of molecules across biological membranes.
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9

Jumpertz, Thorsten, Christian Chervaux, Kathleen Racher, Maria Zouhair, Mark A. Blight, I. Barry Holland, and Lutz Schmitt. "Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin." Microbiology 156, no. 8 (August 1, 2010): 2495–505. http://dx.doi.org/10.1099/mic.0.038562-0.

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Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of β-lactamase when the latter was fused to a C-terminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the β-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.
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10

Reimann, Sven, Gereon Poschmann, Kerstin Kanonenberg, Kai Stühler, Sander H. J. Smits, and Lutz Schmitt. "Interdomain regulation of the ATPase activity of the ABC transporter haemolysin B from Escherichia coli." Biochemical Journal 473, no. 16 (August 11, 2016): 2471–83. http://dx.doi.org/10.1042/bcj20160154.

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Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli. HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we describe the purification and biochemical characterization of detergent-solubilized HlyB in the presence of its transport substrate. Our results exhibit a positive co-operativity in ATP hydrolysis. We characterized further the influence of the CLD on kinetic parameters by using an HlyB variant lacking the CLD (HlyB∆CLD). The biochemical parameters of HlyB∆CLD revealed an increased basal maximum velocity but no change in substrate-binding affinity in comparison with full-length HlyB. We also assigned a distinct interaction of the CLD and a transport substrate (HlyA1), leading to an inhibition of HlyB hydrolytic activity at low HlyA1 concentrations. At higher HlyA1 concentrations, we observed a stimulation of the hydrolytic activities of both HlyB and HlyB∆CLD, which was completely independent of the interaction of HlyA1 with the CLD. Notably, all observed effects on ATPase activity, which were also analysed in detail by mass spectrometry, were independent of the HlyA1 secretion signal. These results assign an interdomain regulatory role for the CLD modulating the hydrolytic activity of HlyB.
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11

Smith, Yarery C., Susan B. Rasmussen, Kerian K. Grande, Richard M. Conran, and Alison D. O'Brien. "Hemolysin of Uropathogenic Escherichia coli Evokes Extensive Shedding of the Uroepithelium and Hemorrhage in Bladder Tissue within the First 24 Hours after Intraurethral Inoculation of Mice." Infection and Immunity 76, no. 7 (April 28, 2008): 2978–90. http://dx.doi.org/10.1128/iai.00075-08.

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ABSTRACT Many uropathogenic Escherichia coli (UPEC) strains produce both hemolysin (Hly) and cytotoxic necrotizing factor type 1 (CNF1), and the loci for these toxins are often linked. The conclusion that Hly and CNF1 contribute to urovirulence is supported by the results of epidemiological studies associating the severity of urinary tract infections (UTIs) with toxin production by UPEC isolates. Additionally, we previously reported that mouse bladders and rat prostates infected with UPEC strain CP9 exhibit a more profound inflammatory response than the organs from animals challenged with CP9cnf 1 and that CNF1 decreases the antimicrobial activities of polymorphonuclear leukocytes. More recently, we created an Hly mutant, CP9ΔhlyA 1::cat, and showed that it was less hemolytic and destructive for cultured bladder cells than CP9 was. Here we evaluated the relative effects of mutations in hlyA 1 or cnf 1 alone or together on the pathogenicity of CP9 in a mouse model of ascending UTI. To do this, we constructed an hlyA 1-complemented clone of CP9ΔhlyA 1::cat and an hlyA 1 cnf 1 CP9 double mutant. We found that Hly had no influence on bacterial colonization of the bladder or kidneys in single or mixed infections with the wild type and CP9ΔhlyA 1::cat but that it did provoke sloughing of the uroepithelium and bladder hemorrhage within the first 24 h after challenge. Finally, we confirmed that CNF1 expression induces bladder inflammation and, in particular, as shown in this study, submucosal edema. From these data, we speculate that Hly and CNF1 may be largely responsible for the signs and symptoms of cystitis in humans infected with toxigenic UPEC.
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12

Wiles, Travis J., Bijaya K. Dhakal, Danelle S. Eto, and Matthew A. Mulvey. "Inactivation of Host Akt/Protein Kinase B Signaling by Bacterial Pore-forming Toxins." Molecular Biology of the Cell 19, no. 4 (April 2008): 1427–38. http://dx.doi.org/10.1091/mbc.e07-07-0638.

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Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTIs), and they have the capacity to induce the death and exfoliation of target uroepithelial cells. This process can be facilitated by the pore-forming toxin α-hemolysin (HlyA), which is expressed and secreted by many UPEC isolates. Here, we demonstrate that HlyA can potently inhibit activation of Akt (protein kinase B), a key regulator of host cell survival, inflammatory responses, proliferation, and metabolism. HlyA ablates Akt activation via an extracellular calcium-dependent, potassium-independent process requiring HlyA insertion into the host plasma membrane and subsequent pore formation. Inhibitor studies indicate that Akt inactivation by HlyA involves aberrant stimulation of host protein phosphatases. We found that two other bacterial pore-forming toxins (aerolysin from Aeromonas species and α-toxin from Staphylococcus aureus) can also markedly attenuate Akt activation in a dose-dependent manner. These data suggest a novel mechanism by which sublytic concentrations of HlyA and other pore-forming toxins can modulate host cell survival and inflammatory pathways during the course of a bacterial infection.
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13

Strack, Karen, Natalia Lauri, Sabina María Maté, Andrés Saralegui, Carlos Muñoz-Garay, Pablo J. Schwarzbaum, and Vanesa Herlax. "Induction of erythrocyte microvesicles by Escherichia Coli Alpha hemolysin." Biochemical Journal 476, no. 22 (November 21, 2019): 3455–73. http://dx.doi.org/10.1042/bcj20190546.

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Alpha hemolysin (HlyA) is the major virulence factor of uropathogenic Escherichia coli (UPEC) strains. Once in circulation, a low concentration of the toxin induces an increase in intracellular calcium that activates calpains — which proteolyse cytoskeleton proteins — and also favours the exposure of phosphatidylserine (PS) in the outer leaflet of erythrocyte membranes. All these events are considered part of eryptosis, as well as the delivery of microvesicles (MVs). Within this context, we studied the delivery of MVs by erythrocytes treated with sublytic concentrations of HlyA and demonstrated that HlyA-treated erythrocytes secrete MVs of diameter ∼200 nm containing HlyA and PS by a mechanism involving an increment of intracellular calcium concentration and purinergic receptor activation. Despite the presence of toxin in their membrane, HlyA-MVs are not hemolytically active and do not induce ATP release in untreated erythrocytes, thus suggesting that the delivery of HlyA-MVs might act as a protective mechanism on the part of erythrocytes that removes the toxin from the membrane to prevent the spread of infection. Although erythrocytes have been found to eliminate denatured hemoglobin and several membrane proteins by shedding MVs, the present work has revealed for the first time that an exogenous protein, such as a toxin, is eliminated by this process. This finding sheds light on the mechanism of action of the toxin and serves to further elucidate the consequences of UPEC infection in patients exhibiting HlyA-related diseases.
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14

Gupta, Prateek, and Kelvin H. Lee. "Silent mutations result in HlyA hypersecretion by reducing intracellular HlyA protein aggregates." Biotechnology and Bioengineering 101, no. 5 (December 1, 2008): 967–74. http://dx.doi.org/10.1002/bit.21979.

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15

Schulz, Emanuel, Michael Schumann, Martina Schneemann, Violaine Dony, Anja Fromm, Oliver Nagel, Jörg-Dieter Schulzke, and Roland Bücker. "Escherichia coli Alpha-Hemolysin HlyA Induces Host Cell Polarity Changes, Epithelial Barrier Dysfunction and Cell Detachment in Human Colon Carcinoma Caco-2 Cell Model via PTEN-Dependent Dysregulation of Cell Junctions." Toxins 13, no. 8 (July 26, 2021): 520. http://dx.doi.org/10.3390/toxins13080520.

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Escherichia coli (E. coli) of the B2 phylotype reside in human and animal intestines. The bacteria possess pathogenicity factors such as α-hemolysin (HlyA) that can induce intestinal epithelial leaks. We addressed the questions which host cell processes were dysregulated by E. coli HlyA that can potentiate intestinal diseases. The colon carcinoma cell line Caco-2 was infected by HlyA+ E. coli. Cell polarity regulation was analyzed by live cell imaging for the phosphatidylinositol-4,5-bisphosphate (PIP2) abundance. In Caco-2 monolayers, transepithelial electrical resistance was measured for characterization of barrier function. Cell proliferation and separation were assessed microscopically. Epithelial regulation and cell signaling were analyzed by RNA-Seq and Ingenuity Pathway Analysis (IPA). Our main findings from E. coli HlyA toxinogenicity in the colon carcinoma cell line are that (i) PIP2 at the membrane decrease, (ii) PTEN (phosphatase and tensin homolog) inhibition leads to cell polarity changes, (iii) epithelial leakiness follows these polarity changes by disruption of cell junctions and (iv) epithelial cell detachment increases. HlyA affected pathways, e.g., the PTEN and metastasis signaling, were identified by RNA-Seq bioinformatics calculations in IPA. In conclusion, HlyA affects cell polarity, thereby inducing epithelial barrier dysfunction due to defective tight junctions and focal leak induction as an exemplary mechanism for leaky gut.
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16

Dutta, Somnath, Budhaditya Mazumdar, Kalyan K. Banerjee, and Amar N. Ghosh. "Three-Dimensional Structure of Different Functional Forms of the Vibrio cholerae Hemolysin Oligomer: a Cryo-Electron Microscopic Study." Journal of Bacteriology 192, no. 1 (October 23, 2009): 169–78. http://dx.doi.org/10.1128/jb.00930-09.

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ABSTRACT Vibrio cholerae hemolysin (HlyA) is a 65-kDa water-soluble pore-forming toxin that causes lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. The HlyA monomer self-assembles on the target cell surface to the more stable β-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa β-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an ∼1,000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, we determined three-dimensional structures of 65- and 50-kDa HlyA oligomers, using cryo-electron microscopy and single-particle methods. Our study clearly shows that the HlyA oligomer has sevenfold symmetry but that the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like, and ring-like domains. The bowl-like domain is coupled with the ring-like domain, and seven side openings are present just beneath the ring-like domain. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore size as well as in shape of the molecules and channel. These structural differences may be relevant to the striking difference in efficiencies of functional channel formation by the two toxin forms.
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17

Grimminger, F., H. Wahn, H. J. Kramer, J. Stevens, K. Mayer, D. Walmrath, and W. Seeger. "Differential influence of arachidonic vs. eicosapentaenoic acid on experimental pulmonary hypertension." American Journal of Physiology-Heart and Circulatory Physiology 268, no. 6 (June 1, 1995): H2252—H2259. http://dx.doi.org/10.1152/ajpheart.1995.268.6.h2252.

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The impact of the 2- and 3-series prostanoid precursors arachidonic acid (AA) and eicosapentaenoic acid (EPA) on experimental pulmonary hypertension was investigated. The model of buffer-perfused rabbit lungs was stimulated by infusion of Escherichia coli hemolysin (HlyA), which is known to provoke sustained thromboxane (Tx)-mediated pulmonary hypertension. Release of di- and trienoic Tx into the recirculating perfusate was quantified by a post-high-performance liquid chromatography enzyme-linked immunosorbent assay technique. HlyA at 0.08 hemolytic unit/ml caused a sustained rise in pulmonary arterial pressure (PAP; maximum increase 14 +/- 2 mmHg) accompanied by progressive TxB2 liberation (maximum perfusate concn 33 +/- 4 pg/ml, baseline < 2 pg/ml). Between 5 and 30 nM, AA provoked a transient monophasic rise in PAP (maximum pressor response 1.5-15 mmHg) and concomitant TxB2 release (peak concn 2-30 pg/ml). Simultaneous administration of HlyA and AA exhibited additive effects with regard to mediator release and pressor responses. EPA at 200-2,000 nM caused a transient rise in PAP similar to that provoked by 5-30 nM AA (maximum pressor response 3-18 mmHg). This was accompanied by liberation of TxB2 (peak concn 16 +/- 5 and 28 +/- 4 pg/ml after 1,000 and 2,000 nM EPA) and TxB3 (peak concn 9 +/- 4 and 30 +/- 3 pg/ml). Combined application of HlyA and EPA resulted in approximate addition of the TxB2 release reaction to each single compound, and TxB3 liberation more than doubled (maximum concn 59 +/- 12 pg/ml). The pressor responses to HlyA-EPA (200-2,000 nM) did not, however, surpass those to HlyA-AA (5-30 nM).(ABSTRACT TRUNCATED AT 250 WORDS)
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18

Ha, Thi Quyen. "Analysis of gene encoding haemolysin A of Vibrio cholerae isolated in Vietnam." Journal of Vietnamese Environment 9, no. 4 (August 8, 2018): 202–6. http://dx.doi.org/10.13141/jve.vol9.no4.pp202-206.

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Vibrio cholerae is the cholera causing agent, divided into two biotypes, including the classical biotype and ElTor biotype. Both of these biotypes caused cholera epidemics in the world. The classical biotype caused 6th cholera pandemic (from 1921 to 1961), and ElTor biotype caused 7th cholera pandemic (from 1961 to the 70s). Haemolysin A, a hemolytic protein of V. cholerae ElT or biotype, is encoded by the hlyA gene. This gene is often used for analyzing genetic relationship between strains in the same species or between species in the same Vibrio genus. Results of analyzing nucleotide and amino acid sequences of hlyA gene of V. cholerae strain causing cholera in Vietnam (named hlyA.VN) showed that: the hlyA.VN gene sequence was similar to the hlyA gene sequences of V. cholerae strains of the 6th and 7th cholera epidemics. The hlyA gene of the 6th cholera epidemic strain was deficient in 11 nuleotides (this deficiency leading to the loss of 4 amino acids in the haemolysin A protein) comparing to hlyA.VN gene and hlyA gene of the 7th cholera epidemic strain. The results of genetic distance analysis as well as phylogenetic tree construction also confirmed V. cholerae causing cholera in Vietnam was closely relationship to the strains causing cholera pandemics in the world. It is great significance for the surveillance of molecular epidemiology to prevent cholera effectively. Vibrio cholerae là tác nhân gây bệnh tả, được chia thành hai typ sinh học, đó là typ sinh học cổ điển và typ sinh học ElTor. Cả hai typ này đã từng gây ra các đại dịch tả trên thế giới. Typ sinh học cổ điển đã từng gây ra đại dịch tả lần thứ 6 (từ năm 1921 đến 1961), còn typ sinh học ElTor đã từng gây ra đại dịch tả lần thứ 7 (từ 1961 đến những năm 70). Haemolysin A, một protein có chức năng làm tan máu của V. cholerae typ sinh học ElTor, được mã hóa bởi gen hlyA. Gene này thường được sử dụng cho các phân tích quan hệ di truyền giữa các chủng trong cùng một loài V. cholerae hay giữa các loài trong cùng một chi Vibrio. Kết quả phân tích trình tự nucleotide và axit amin gen hlyA của chủng V. cholerae gâybệnh ở Việt Nam (hlyA.VN) cho thấy: trình tự gen hlyA.VN có sự tương đồng lớn với trình tự gen hlyA của chủng gây đại dịch tả 6 và 7. Gen hlyA của chủng gây đại dịch tả 6 bị thiếu hụt 11 nuleotide (sự thiếu hụt này dẫn tới sự mất đi 4 axit amin trong phân tử haemolysin A) so với gen hlyA.VN và gene hlyA của chủng gây đại dịch tả 7. Kết quả phân tích khoảng cách di truyền cũng như xây dựng cây phát sinh chủng loại cũng đã khẳng định: chủng gây bệnh ở Việt Nam có quan hệ rất gần với các chủng gây đại dịch tả trên thế giới. Nhận định này có ý nghĩa rất lớn đối với công tác giám sát dịch tễ học phân tử để ngăn chặn bệnh tả hiệu quả.
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19

Huntley, James S., Venugopal Sathyamoorthy, Robert H. Hall, and Andrew C. Hall. "Membrane attack induced by HlyA, a pore-forming toxin of Vibrio cholerae." Human & Experimental Toxicology 16, no. 2 (February 1997): 101–5. http://dx.doi.org/10.1177/096032719701600205.

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Determining the activity of purified toxins has generally provided the basis for establishing their role in the host- pathogen relationship. The bacterial genus Vibrio pro duces a number of exotoxins in addition to cholera toxin, including haemolysin A (HIyA; Vibrio cholerae) and thermostable direct haemolysin (TDH; Vibrio parahae molyticus) , both of which possess membrane-targeting cytolytic activity. The action of HlyA has been analyzed using protocols previously applied to TDH: lysis and flux experiments on human erythrocytes showed that HlyA similarly causes lysis after cell swelling (by colloid osmosis) due to an elevation of cation permeability. However, kinetic measurements of flux, haemolysis and cation selectivity showed that HlyA and TDH form pores with distinct and characteristic features.
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20

Rose, F., L. Kiss, F. Grimminger, K. Mayer, U. Grandel, W. Seeger, E. Bieniek, and U. Sibelius. "E. colihemolysin-induced lipid mediator metabolism in alveolar macrophages: impact of eicosapentaenoic acid." American Journal of Physiology-Lung Cellular and Molecular Physiology 279, no. 1 (July 1, 2000): L100—L109. http://dx.doi.org/10.1152/ajplung.2000.279.1.l100.

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Escherichia coli hemolysin (HlyA) is a prototype of a large family of pore-forming proteinaceous exotoxins that have been implicated in the pathogenetic sequelae of severe infection and sepsis, including development of acute lung injury. In the present study in rabbit alveolar macrophages (AMs), subcytolytic concentrations of purified HlyA evoked rapid synthesis of platelet-activating factor, with quantities approaching those in response to maximum calcium ionophore challenge. In parallel, large quantities of leukotriene (LT) B4and 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) were liberated from HlyA-exposed AMs depending on exogenous arachidonic acid (AA) supply. Coadministration of eicosapentaenoic acid (EPA) dose dependently suppressed generation of the proinflammatory lipoxygenase products LTB4and 5-, 8-, 9-, and 12-HETE in parallel with the appearance of the corresponding EPA-derived metabolites LTB5and 5-, 8-, 9-, and 12–hydroxyeicosapentaenoic acid (HEPE). At equimolar concentrations, EPA turned out to be the preferred substrate over AA for these AM lipoxygenase pathways, with the sum of LTB5and 5-, 8-, 9-, and 12-HEPE surpassing the sum of LTB4and 5-, 8-, 9-, and 12-HETE by >80-fold. In contrast, coadminstration of EPA did not significantly reduce HlyA-elicited generation of the anti-inflammatory AA lipoxygenase product 15-HETE. We conclude that AMs are sensitive target cells for HlyA attack, resulting in marked proinflammatory lipid mediator synthesis. In the presence of EPA, lipoxygenase product formation is shifted from a pro- to an anti-inflammatory profile.
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21

Walmrath, D., H. A. Ghofrani, S. Rosseau, H. Schütte, A. Cramer, W. Kaddus, F. Grimminger, S. Bhakdi, and W. Seeger. "Endotoxin "priming" potentiates lung vascular abnormalities in response to Escherichia coli hemolysin: an example of synergism between endo- and exotoxin." Journal of Experimental Medicine 180, no. 4 (October 1, 1994): 1437–43. http://dx.doi.org/10.1084/jem.180.4.1437.

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The pore-forming hemolysin of Escherichia coli (HlyA), an important virulence factor in extraintestinal E. coli infections, causes thromboxane generation and related vasoconstriction in perfused rabbit lungs (Seeger, W., H. Walter, N. Suttorp, M. Muhly, and S. Bhakdi. 1989. J. Clin. Invest. 84:220). We investigated the influence of pulmonary vascular "priming" with endotoxin on the responsiveness of the lung to a low-dose HlyA challenge. Rabbit lungs were perfused with Krebs Henseleit buffer containing 0.1-100 ng/ml Salmonella abortus equii lipopolysaccharide (LPS) for 60-180 min. This treatment caused protracted release of tumor necrosis factor into the recirculating medium, but did not induce significant alterations of pulmonary hemodynamics and fluid balance. At a dose of 1 ng/ml, HlyA elicited only moderate thromboxane release (&lt; 200 pg/ml) and pulmonary artery pressure increase (&lt; or = 6 mmHg) in control lungs. Acceleration and potentiation of both the metabolic and vasoconstrictor response occurred in lungs primed with LPS. This priming effect displayed dose (threshold integral of 0.1-1 ng/ml LPS) and time dependencies (threshold integral of 60-90 min LPS incubation). Maximum thromboxane release and pulmonary artery pressure increase surpassed the responses to HlyA in nonprimed lungs by more than 15-fold. Cyclooxygenase inhibition and thromboxane-receptor antagonism blocked these effects. These data demonstrate that LPS priming synergizes with HlyA challenge to provoke vascular abnormalities that are possibly relevant to the pathogenesis of organ failure in severe local and systemic infections.
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22

Johnson, James R., Hank A. Lockman, Krista Owens, Srdjan Jelacic, and Phillip I. Tarr. "High-Frequency Secondary Mutations after Suicide-Driven Allelic Exchange Mutagenesis in Extraintestinal Pathogenic Escherichia coli." Journal of Bacteriology 185, no. 17 (September 1, 2003): 5301–5. http://dx.doi.org/10.1128/jb.185.17.5301-5305.2003.

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ABSTRACT Frequent unintended secondary mutations occurred in extraintestinal pathogenic Escherichia coli strains CP9, CFT073, and RS218 during suicide plasmid-mediated, putatively specific deletions of hlyA, papG allele III, and iha. Pulsed-field gel electrophoresis and PCR analyses demonstrated genomic alterations and/or unintended loss of defined virulence genes (papG, the F7-2 papA allele, iutA, sat, hlyD, and cnf). Caution is warranted when attributing the observed phenotypic changes to the intended mutation.
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23

Valadbeigi, Hassan, Elham Esmaeeli, Sobhan Ghafourian, Abbas Maleki, and Nourkhoda Sadeghifard. "Molecular Analysis of Uropathogenic E.coli Isolates from Urinary Tract Infections." Infectious Disorders - Drug Targets 19, no. 3 (October 4, 2019): 322–26. http://dx.doi.org/10.2174/1871526518666181113091322.

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Introduction: The aim of the current study was to investigate the prevalence of virulence genes in uropathogenic Escherichia coli (UPEC) isolates in Ilam. Materials and Methods: For this purpose, a total of 80 UPEC isolates were collected for patients with UTIs during a 6 months period. The multiplex polymerase chain reaction (multiplex PCR) was used to detect the papEF, fimH, iucD, hlyA, fyuA, and ompT genes. Results: The prevalence of fimH, papEF, iucD, fyuA, hlyA, hlyA, and ompT genes were 87.5%, 47.5%, 60%, 67.5%, 27.5%, 47.5% and 71.2%, respectively. Among all of the isolates, 27 profiles were obtained. Conclusion: Our findings demonstrated that the most prevalence was found for fimH, and different distribution of virulence genes suggested different ability of pathogenicity.
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24

Onlen, Cansu, Nizami Duran, Suphi Bayraktar, Emrah Ay, and Burçin Ozer. "The frequency of shiga-like toxin (stx1 and stx2) and EHEC-hlyA in food by multiplex PCR." Revista Romana de Medicina de Laborator 25, no. 4 (October 26, 2017): 317–26. http://dx.doi.org/10.1515/rrlm-2017-0025.

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Abstract Aim: The aim of the present study was to determine the frequency of shiga-like toxin (stx1 and stx2) and drug resistance profiles food-borne Escherichia coli O157:H7 in Hatay province, Turkey. Methods: The presence of the virulence genes (stx1, stx2, hlyA) in a total of 150 E.coli isolates were studied with multiplex PCR. Results: A total of 327 salad samples were analyzed. E. coli O157:H7 was detected in 150 (45.8 %) out of 327 analyzed samples. Of these 150 isolates, the presence of hly-A gene was detected in 32 (21.3%) E.coli isolates. A total of five (15.6%) isolates in this 32 hlyA positive isolates had stx2 gene, two (6.3%) of them had stx1 gene and one (3.1%) of the isolates was found to be positive for both stx1 and stx2 genes. It was found that all E.coli O157:H7 isolates were resistant to erythromycin. While the highest rate of antibiotic resistance was observed for ampicillin (68.8%), no antibiotic resistance against cefuroxime, ciprofloxacin and cephaperasone was identified. Conclusions: The results obtained in our province showed that E.coli strains isolated from salad samples were found to have some important virulence genes such as stx1, stx2, and hlyA. The stx2 frequency was found to be higher than stx1 frequency. Also, it was observed that there was not any significant correlation between drug resistance profiles and presence of toxin genes in E.coli O157:H7 strains. As a result, increasing frequency of STEC O157 serotype among foodborne pathogens is a growing public health problem.
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25

Tsou, Amy M., and Jun Zhu. "Quorum Sensing Negatively Regulates Hemolysin Transcriptionally and Posttranslationally in Vibrio cholerae." Infection and Immunity 78, no. 1 (October 26, 2009): 461–67. http://dx.doi.org/10.1128/iai.00590-09.

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ABSTRACT Recent work has shown that in addition to cholera toxin (CT) and the toxin-coregulated pilus (TCP), other cytotoxic proteins in Vibrio cholerae also cause disease symptoms, and this is particularly evident in strains lacking CT. One such protein is the hemolysin encoded by hlyA. Here we show that, like CT and TCP, HlyA is repressed by the quorum-sensing-regulated transcription factor HapR. This repression occurs on two levels: one at the transcriptional level that is independent of the metalloprotease HapA and one at the posttranslational level that is mediated by HapA. The transcriptional regulation is significantly more apparent on solid media than in liquid cultures. This is the first time that hemolysis has been shown to be directly regulated by quorum sensing in V. cholerae, and it is interesting that, like other virulence factors, HlyA is also repressed by HapR, which is expressed late in infection.
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26

Thomas, Sabrina, Sander H. J. Smits, and Lutz Schmitt. "A simple in vitro acylation assay based on optimized HlyA and HlyC purification." Analytical Biochemistry 464 (November 2014): 17–23. http://dx.doi.org/10.1016/j.ab.2014.07.001.

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Rivas, Amable J., Miguel Balado, Manuel L. Lemos, and Carlos R. Osorio. "Synergistic and Additive Effects of Chromosomal and Plasmid-Encoded Hemolysins Contribute to Hemolysis and Virulence in Photobacterium damselae subsp. damselae." Infection and Immunity 81, no. 9 (June 24, 2013): 3287–99. http://dx.doi.org/10.1128/iai.00155-13.

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ABSTRACTPhotobacterium damselaesubsp.damselaecauses infections and fatal disease in marine animals and in humans. Highly hemolytic strains produce damselysin (Dly) and plasmid-encoded HlyA (HlyApl). These hemolysins are encoded by plasmid pPHDD1 and contribute to hemolysis and virulence for fish and mice. In this study, we report that all the hemolytic strains produce a hitherto uncharacterized chromosome-encoded HlyA (HlyAch). Hemolysis was completely abolished in a singlehlyAchmutant of a plasmidless strain and in adly hlyAplhlyAchtriple mutant. We found that Dly, HlyApl, and HlyAchare needed for full hemolytic values in strains harboring pPHDD1, and these values are the result of the additive effects between HlyApland HlyAch, on the one hand, and of the synergistic effect of Dly with HlyApland HlyAch, on the other hand. Interestingly, Dly-producing strains produced synergistic effects with strains lacking Dly production but secreting HlyA, constituting a case of the CAMP (Christie,Atkins, andMunch-Petersen) reaction. Environmental factors such as iron starvation and salt concentration were found to regulate the expression of the three hemolysins. We found that the contributions, in terms of the individual and combined effects, of the three hemolysins to hemolysis and virulence varied depending on the animal species tested. While Dly and HlyAplwere found to be main contributors in the virulence for mice, we observed that the contribution of hemolysins to virulence for fish was mainly based on the synergistic effects between Dly and either of the two HlyA hemolysins rather than on their individual effects.
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Fagan, Peter K., Michael A. Hornitzky, Karl A. Bettelheim, and Steven P. Djordjevic. "Detection of Shiga-Like Toxin (stx1 andstx2), Intimin (eaeA), and Enterohemorrhagic Escherichia coli (EHEC) Hemolysin (EHEC hlyA) Genes in Animal Feces by Multiplex PCR." Applied and Environmental Microbiology 65, no. 2 (February 1, 1999): 868–72. http://dx.doi.org/10.1128/aem.65.2.868-872.1999.

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ABSTRACT A multiplex PCR was developed for the rapid detection of genes encoding Shiga toxins 1 and 2 (stx1 andstx2 ), intimin (eaeA), and enterohemolysin A (hlyA) in 444 fecal samples derived from healthy and clinically affected cattle, sheep, pigs, and goats. The method involved non-solvent-based extraction of nucleic acid from an aliquot of an overnight culture of feces in EC (modified) broth. The detection limit of the assay for both fecal samples and pure cultures was between 18 and 37 genome equivalents. stx1 and hlyA were the most commonly encountered virulence factors.
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29

Nagamatsu, Kanna, Thomas J. Hannan, Randi L. Guest, Maria Kostakioti, Maria Hadjifrangiskou, Jana Binkley, Karen Dodson, Tracy L. Raivio, and Scott J. Hultgren. "Dysregulation ofEscherichia coliα-hemolysin expression alters the course of acute and persistent urinary tract infection." Proceedings of the National Academy of Sciences 112, no. 8 (February 9, 2015): E871—E880. http://dx.doi.org/10.1073/pnas.1500374112.

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Urinary tract infections (UTIs) are among the most common bacterial infections, causing considerable morbidity in females. Infection is highly recurrent despite appropriate antibiotic treatment. UropathogenicEscherichia coli(UPEC), the most common causative agent of UTIs, invades bladder epithelial cells (BECs) and develops into clonal intracellular bacterial communities (IBCs). Upon maturation, IBCs disperse, with bacteria spreading to neighboring BECs to repeat this cycle. This process allows UPEC to gain a foothold in the face of innate defense mechanisms, including micturition, epithelial exfoliation, and the influx of polymorphonuclear leukocytes. Here, we investigated the mechanism and dynamics of urothelial exfoliation in the early acute stages of infection. We show that UPEC α-hemolysin (HlyA) induces Caspase-1/Caspase-4–dependent inflammatory cell death in human urothelial cells, and we demonstrate that the response regulator (CpxR)-sensor kinase (CpxA) two-component system (CpxRA), which regulates virulence gene expression in response to environmental signals, is critical for fine-tuning HlyA cytotoxicity. Deletion of thecpxRtranscriptional response regulator derepresseshlyAexpression, leading to enhanced Caspase-1/Caspase-4– and NOD-like receptor family, pyrin domain containing 3-dependent inflammatory cell death in human urothelial cells. In vivo, overexpression of HlyA during acute bladder infection induces more rapid and extensive exfoliation and reduced bladder bacterial burdens. Bladder fitness is restored fully by inhibition of Caspase-1 and Caspase-11, the murine homolog of Caspase-4. Thus, we have discovered that fine-tuning of HlyA expression by the CpxRA system is critical for enhancing UPEC fitness in the urinary bladder. These results have significant implications for our understanding of how UPEC establishes persistent colonization.
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30

Yousif, Maitham Ghaly, and Ataa Khalil Al-shamari. "PHYLOGENETIC CHARATERIZATION OF LISTERIA MONOCYTOGENES ISOLATED FROM DIFFERENT SOURCES IN IRAQ." Asian Journal of Pharmaceutical and Clinical Research 11, no. 2 (February 1, 2018): 289. http://dx.doi.org/10.22159/ajpcr.2018.v11i2.22633.

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Objective: The main goal of the current study was to isolate and detect the phylogenetic characterization of Listeria monocytogenes isolate from clinical and non-clinical specimens.Methods: L. monocytogenes was isolated from 353 samples including: (94) Vaginal swabs, (81) piece of placenta, (51) frozen chicken, (69) soft cheese, and (58) frozen red meat samples using the Association of Official Agricultural Chemists method. Polymerase chain reaction (PCR) was performed for the detection of virulence gene hlyA followed by DNA sequence analysis for this gene.Results: A total of 13 isolates of L. monocytogenes were isolated from 2 (2.1%) vaginal swabs, 4 (4.9%) piece placenta, 2 (3.9%) frozen chicken, 3 (4.3%) frozen soft cheese, and 2 (3.4%) frozen red meat samples, and hlyA gene was detected in 100% of L. monocytogenes isolates.Conclusion: All the isolates tested positive for hlyA gene, so this is important role in the study of the L. monocytogenes infection and its pathogenicity. The phylogenetic analysis showed a clear convergence in the L. monocytogenes isolates from chicken, red meat, and soft cheese 70%, while there is a marked difference in isolates of L. monocytogenes isolated from placenta and vaginal swabs.
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31

Wang, Jui-Hsing, Guo-Cheng He, Yao-Ting Huang, and Po-Yu Liu. "Comparative Genomics Reveals Pathogenicity-Related Loci in Shewanella algae." Canadian Journal of Infectious Diseases and Medical Microbiology 2020 (March 30, 2020): 1–10. http://dx.doi.org/10.1155/2020/9205197.

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Shewanella algae is an emerging marine zoonotic pathogen and accounts for considerable mortality and morbidity in compromised hosts. However, there is scarce literature related to the understanding of the genetic background of virulence determinants in S. algae. In this study, we aim to determine the occurrence of common virulence genes in S. algae using whole-genome sequence and comparative genomic analysis. Comparative genomics reveals putative-virulence genes related to bile resistance, chemotaxis, hemolysis, and motility. We detected the existence of hlyA, hlyD, and hlyIII involved in hemolysis. We also found chemotaxis gene cluster cheYZA operon and cheW gene. The results provide insights into the genetic basis underlying pathogenicity in S. algae.
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32

BABACAN, Orkun. "Antibiotic susceptibility and phylogenetic analyses for the origins and serotypes of Listeriamonocytogenes strains isolated from ice cream and cream cakes." TURKISH JOURNAL OF VETERINARY AND ANIMAL SCIENCES 44, no. 5 (October 27, 2020): 1100–1109. http://dx.doi.org/10.3906/vet-2003-116.

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Listeria monocytogenes is a zoonotic bacterium which also infects humans. The aim of this study was to isolate this organism from cream cakes and ice cream and obtain 16S rRNA and hlyA gene sequences from isolates in order to perform phylogenetic analyses. Serotypes and antibiotic susceptibility were also determined. The cream cake and ice cream samples were examined for L. monocytogenes according to ISO 11290-1 and using the mini Vidas LMO 2 kit procedure. Antibiotic susceptibilities were investigated using the disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) standards. The Sanger DNA sequencing method for phylogenetic analysis was used for L. monocytogenes isolates. A total of 16 (n =128, 12.50%) L. monocytogenes strains, 9 (12.16%) from cream cake samples and 7 (12.96%) from ice cream samples, were isolated. This was the first investigation involving sequencing of L. monocytogenes isolated from cream cakes and ice creams in Turkey. All isolates were susceptible to sulfamethoxazole/trimethoprim, tetracycline, streptomycin, gentamicin, meropenem, and erythromycin. The numbers of isolates resistant to sulbactam/ampicillin, penicillin G, chloramphenicol, and ampicillin were 16, 2, 1, and 1, respectively. Moreover, 3 isolates showed intermediate resistance to amikacin and 2 to ciprofloxacin. The hlyA gene sequences of 11 of the L. monocytogenesisolates isolated from milk were closely related to the hlyA gene sequences of the GenBank reference strains. The comparison of the 16s rRNA gene sequences of the L. monocytogenes strains with the GenBank reference serotypes identified 1 isolate as serotype 1/2c, 1 as serotype 1/2a, and 1 as serotype 4. Nucleic acid sequencing is useful for identification of L. monocytogenes. The 16S rRNAand hlyA sequences can be used to determine the origin and relationship between L. monocytogenesisolates, as well as the serotype.
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33

BARKOCY-GALLAGHER, GENEVIEVE A., TERRANCE M. ARTHUR, MILDRED RIVERA-BETANCOURT, XIANGWU NOU, STEVEN D. SHACKELFORD, TOMMY L. WHEELER, and MOHAMMAD KOOHMARAIE. "Characterization of O157:H7 and Other Escherichia coli Isolates Recovered from Cattle Hides, Feces, and Carcasses†." Journal of Food Protection 67, no. 5 (May 1, 2004): 993–98. http://dx.doi.org/10.4315/0362-028x-67.5.993.

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In a previous study, the seasonal prevalence was reported for stx+ Escherichia coli O157:H7 in feces and on hides and carcasses of cattle at processing. Overall, 1,697 O157:H7 isolates have now been characterized for the incidence of (i) eaeO157, hlyA, stx1, and stx2 in the recovered isolates and (ii) presumptive rough and presumptive nonmotile isolates. Seven O157:H7 isolates (0.4%) lacked stx genes, although they carried eae and hlyA. All but one of the isolates carried both eae and hlyA. Approximately two-thirds of the isolates (64% when one isolate per sample was considered) carried both stx1 and stx2. E. coli O157:H7 cells that harbored both stx1 and stx2 were more often recovered from hides in the fall (79% of the fall hide isolates) and winter (84% of the winter hide isolates) than in the spring (53%) and summer (59%). Isolates recovered from preevisceration carcasses showed a similar but not statistically significant trend. Twenty-three of the 25 O157:H7 isolates carrying stx1 but not stx2 were recovered during summer. Fifteen presumptive rough and 117 presumptive nonmotile stx+ O157:H7 isolates were recovered. Ten (67%) of the presumptive rough isolates were recovered during summer. Ninety-five of the presumptive nonmotile isolates (81%) were recovered during fall. Forty-eight percent of the false-positive isolates (175 of 363) tentatively identified as O157:H7 were O157+ H7− and lacked eaeO157, hlyA, and stx. These data suggest that in beef processing samples (i) there are minor seasonal variations in the prevalence of stx genes among E. coli O157:H7 isolates, (ii) presumptive rough and presumptive nonmotile stx+ O157:H7 isolates are present, (iii) E. coli O157:H7 isolates lacking stx genes may be rare, and (iv) O157+ H7− isolates lacking stx genes can result in many false-positive results.
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34

Osickova, Adriana, Humaira Khaliq, Jiri Masin, David Jurnecka, Anna Sukova, Radovan Fiser, Jana Holubova, Ondrej Stanek, Peter Sebo, and Radim Osicka. "Acyltransferase-mediated selection of the length of the fatty acyl chain and of the acylation site governs activation of bacterial RTX toxins." Journal of Biological Chemistry 295, no. 28 (May 27, 2020): 9268–80. http://dx.doi.org/10.1074/jbc.ra120.014122.

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In a wide range of organisms, from bacteria to humans, numerous proteins have to be posttranslationally acylated to become biologically active. Bacterial repeats in toxin (RTX) cytolysins form a prominent group of proteins that are synthesized as inactive protoxins and undergo posttranslational acylation on ε-amino groups of two internal conserved lysine residues by co-expressed toxin-activating acyltransferases. Here, we investigated how the chemical nature, position, and number of bound acyl chains govern the activities of Bordetella pertussis adenylate cyclase toxin (CyaA), Escherichia coli α-hemolysin (HlyA), and Kingella kingae cytotoxin (RtxA). We found that the three protoxins are acylated in the same E. coli cell background by each of the CyaC, HlyC, and RtxC acyltransferases. We also noted that the acyltransferase selects from the bacterial pool of acyl–acyl carrier proteins (ACPs) an acyl chain of a specific length for covalent linkage to the protoxin. The acyltransferase also selects whether both or only one of two conserved lysine residues of the protoxin will be posttranslationally acylated. Functional assays revealed that RtxA has to be modified by 14-carbon fatty acyl chains to be biologically active, that HlyA remains active also when modified by 16-carbon acyl chains, and that CyaA is activated exclusively by 16-carbon acyl chains. These results suggest that the RTX toxin molecules are structurally adapted to the length of the acyl chains used for modification of their acylated lysine residue in the second, more conserved acylation site.
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35

RODRÍGUEZ-CALLEJA, JOSE M., ISABEL GARCÍA-LÓPEZ, MARÍA-LUISA GARCÍA-LÓPEZ, JESÚS A. SANTOS, and ANDRÉS OTERO. "Rabbit Meat as a Source of Bacterial Foodborne Pathogens." Journal of Food Protection 69, no. 5 (May 1, 2006): 1106–12. http://dx.doi.org/10.4315/0362-028x-69.5.1106.

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Even though worldwide production of rabbit meat is &gt;1,000,000 tons, little information is available for rabbit meat microbiology. This study provides data on the prevalence of Salmonella, Escherichia coli O157:H7, Yersinia enterocolitica, Listeria spp., motile Aeromonas spp., and Staphylococcus aureus on rabbit meat. A total of 24 rabbit carcasses from two abattoirs and 27 rabbit meat packages from supermarket displays were examined. In addition to culturing methods, associated virulence genes were investigated by PCR in suspect isolates and samples. Neither Salmonella nor E. coli O157:H7 was detected. All samples were negative for virulence-associated invA, stx1, and stx2 genes. At one abattoir, two carcasses (3.9%) carried Y. enterocolitica yst−, and two were positive for the yst gene, although viable Y. enterocolitica cells were not recovered from these samples. Seven samples (13.7%) were contaminated with Listeria. Of them, three were positive for hly and iap genes (Listeria monocytogenes hly+/iap+), two carried Listeria seeligeri, one carried Listeria ivanovii, and one carried Listeria innocua. For detectable motile Aeromonas spp. (average count, 1.77 ± 0.62 log CFU/g), the contamination rate was 35.3%, although ca. 90% of the samples were positive for the aerA and/or hlyA genes. The majority of aeromonad isolates were Aeromonas hydrophila aerA+/hlyA+. Aeromonas caviae, Aeromonas popoffii, Aeromonas schubertii, and the two biovars of Aeromonas veronii were also isolated. The prevalence of S. aureus contamination (average count, 1.37 ± 0.79 log CFU/g) was 52.9%. Among 27 S. aureus isolates, two harbored genes for staphylococcal enterotoxin B (seb), and two harbored genes for staphylococcal enterotoxin C (sec). The remaining isolates were negative for sea, seb, sec, sed, and see.
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36

Oropeza-Wekerle, R. L., W. Speth, B. Imhof, I. Gentschev, and W. Goebel. "Translocation and compartmentalization of Escherichia coli hemolysin (HlyA)." Journal of Bacteriology 172, no. 7 (1990): 3711–17. http://dx.doi.org/10.1128/jb.172.7.3711-3717.1990.

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37

Hall, R. H., and B. S. Drasar. "Vibrio cholerae HlyA hemolysin is processed by proteolysis." Infection and Immunity 58, no. 10 (1990): 3375–79. http://dx.doi.org/10.1128/iai.58.10.3375-3379.1990.

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38

Körkoca, Hanifi, Yusuf Alan, Sedat Bozari, Mustafa Berktas, and Yaşar Goz. "Detection of putative virulence genes in Aeromonas isolates from humans and animals." Journal of Infection in Developing Countries 8, no. 11 (November 13, 2014): 1398–406. http://dx.doi.org/10.3855/jidc.4879.

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Introduction: Aeromonas are food- and water-borne bacteria that are considered to be zoonotic human pathogens. This study aimed to investigate the presence of genes associated with virulence in human and animal Aeromonas isolates and the potential role of animal isolates with regards to human Aeromonas infections. Methodology: The presence of aerA, hlyA, alt, ast, laf, ascF-G, stx1 and stx2 putative virulence genes in 40 human and animal Aeromonas isolates (16 human and 24 animal isolates) were examined by polymerase chain reaction (PCR). DNA fragments of expected sizes were purified and sequenced. BLAST in the NCBI was used to verify any amplified products. Results: PCR screening showed that hlyA, alt, and laf genes were determined at ratios of 6.25%, 50%, and 6.25%, respectively, in human isolates. The ratios of hlyA, alt, ascF-G, laf, stx2, and stx1 genes in animal isolates were 58.3%, 20.83%, 33.3%, 20.83%, 8.33%, and 4.17%, respectively. Neither aerA nor ast genes were detected in any isolates. Any one of eight putative virulence genes was not detected in seven human and eight animal isolates in the study. Conclusions: The current study is the first to investigate the presence of the virulence gene in gull Aeromonas isolates. The manifestation of the presence of the virulence gene and gene combinations was considerable, especially in fish and gull isolates when compared with clinical human isolates. The current study demonstrates the potential importance of fish and gulls in terms of human Aeromonas infections.
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39

Byun, Roy, Liam D. H. Elbourne, Ruiting Lan, and Peter R. Reeves. "Evolutionary Relationships of Pathogenic Clones of Vibrio cholerae by Sequence Analysis of Four Housekeeping Genes." Infection and Immunity 67, no. 3 (March 1, 1999): 1116–24. http://dx.doi.org/10.1128/iai.67.3.1116-1124.1999.

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ABSTRACT Studies of the Vibrio cholerae population, using molecular typing techniques, have shown the existence of several pathogenic clones, mainly sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones. However, the relationship of the pathogenic clones to environmental V. cholerae isolates remains unclear. A previous study to determine the phylogeny of V. cholerae by sequencing the asd (aspartate semialdehyde dehydrogenase) gene of V. cholerae showed that the sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones had very differentasd sequences which fell into separate lineages in theV. cholerae population. As gene trees drawn from a single gene may not reflect the true topology of the population, we sequenced the mdh (malate dehydrogenase) and hlyA(hemolysin A) genes from representatives of environmental and clinical isolates of V. cholerae and found that the mdhand hlyA sequences from the three pathogenic clones were identical, except for the previously reported 11-bp deletion inhlyA in the sixth-pandemic clone. Identical sequences were obtained, despite average nucleotide differences in the mdhand hlyA genes of 1.52 and 3.25%, respectively, among all the isolates, suggesting that the three pathogenic clones are closely related. To extend these observations, segments of the recAand dnaE genes were sequenced from a selection of the pathogenic isolates, where the sequences were either identical or substantially different between the clones. The results show that the three pathogenic clones are very closely related and that there has been a high level of recombination in their evolution.
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40

Kędrak-Jabłońska, Agnieszka, Sylwia Budniak, Anna Szczawińska, Monika Reksa, Marek Krupa, and Krzysztof Szulowski. "Application of SYBR Green I and TaqMan probe-based real-time PCRs for the identification of Listeria spp. and Listeria monocytogenes." Bulletin of the Veterinary Institute in Pulawy 59, no. 4 (December 1, 2015): 489–94. http://dx.doi.org/10.1515/bvip-2015-0073.

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Abstract The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes. Five strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of the tests. QuantiTect SYBR Green PCR and QuantiTect Probe PCR kits were selected for the study. In the first stage of the study, SYBR Green I real-time PCRs were performed under several methods, the first one allowing detection of the 23S rDNA gene and the remainder based on the amplification of the hlyA gene. In the next part, three varied in method TaqMan probe-based real-time PCRs allowing confirmation of strains belonging to Listeria spp. and L. monocytogenes were conducted. The observation of amplification curves in real-time PCR methods enabled the detection of both genes, and these methods demonstrated a significant sensitivity and high specificity. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes, which confirmed the tested strains as Listeria spp. and L. monocytogenes respectively. Isolates of other microbial species did not yield real-time PCR products.
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41

Williams, S. G., and P. A. Manning. "Transcription of the Vibrio cholerae haemolysin gene, hlyA, and cloning of a positive regulatory locus, hlyU." Molecular Microbiology 5, no. 8 (August 1991): 2031–38. http://dx.doi.org/10.1111/j.1365-2958.1991.tb00825.x.

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42

Stanley, Peter, Vassilis Koronakis, Kim Hardie, and Colin Hughes. "Independent interaction of the acyltransferase HlyC with two maturation domains of the Escherichia coli toxin HlyA." Molecular Microbiology 20, no. 4 (May 1996): 813–22. http://dx.doi.org/10.1111/j.1365-2958.1996.tb02519.x.

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43

Guzmán-Verri, C., F. García, and S. Arvidson. "Incomplete activation of Escherichia coli hemolysin (HlyA) due to mutations in the 3' region of hlyC." Journal of bacteriology 179, no. 18 (1997): 5959–62. http://dx.doi.org/10.1128/jb.179.18.5959-5962.1997.

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44

Vazquez, Romina F., Sabina M. Maté, Laura S. Bakás, Marisa M. Fernández, Emilio L. Malchiodi, and Vanesa S. Herlax. "Novel evidence for the specific interaction between cholesterol and α-haemolysin of Escherichia coli." Biochemical Journal 458, no. 3 (February 28, 2014): 481–89. http://dx.doi.org/10.1042/bj20131432.

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The present study shows, for the first time, the interaction of HlyA with cholesterol. This interaction seems to favour a conformational state of the protein that allows its correct insertion into the membrane and its further oligomerization to form pores.
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45

Bücker, Roland, Emanuel Schulz, Stephanie Wiegand, Britta Siegmund, Hanno Troeger, and Joerg D. Schulzke. "Mo1940 E. Coli α-Hemolysin (HlyA) Mediates Colonic Inflammation." Gastroenterology 146, no. 5 (May 2014): S—697. http://dx.doi.org/10.1016/s0016-5085(14)62530-0.

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46

Williams, S. G., L. T. Varcoe, S. R. Attridge, and P. A. Manning. "Vibrio cholerae Hcp, a secreted protein coregulated with HlyA." Infection and immunity 64, no. 1 (1996): 283–89. http://dx.doi.org/10.1128/iai.64.1.283-289.1996.

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47

WAGNER, Wilma, Michael KUHN, and Werner GOEBEL. "Active and Inactive Forms of Hemolysin (HlyA) fromEscherichia coli." Biological Chemistry Hoppe-Seyler 369, no. 1 (January 1988): 39–46. http://dx.doi.org/10.1515/bchm3.1988.369.1.39.

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48

Senior, B. W. "The production of HlyA toxin by Proteus penneri strains." Journal of Medical Microbiology 39, no. 4 (October 1, 1993): 282–89. http://dx.doi.org/10.1099/00222615-39-4-282.

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49

Hou, Hong Man, Yu Na Cui, Gong Liang Zhang, and Hao Yu Wang. "Detection of Listeria monocytogenes in Foods by SYBR Green I Melting Curve." Advanced Materials Research 781-784 (September 2013): 1771–75. http://dx.doi.org/10.4028/www.scientific.net/amr.781-784.1771.

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Two pairs of specific primers were composited based on the hlyA gene of L.monocytogenes and the 16sRNA gene of the genus Listeria. Optimal reaction system and condition of the SYBR GreenⅠfluorescent PCR were established, respectively, which could detect two specific genes. The reaction solution was verified by the method of gel electrophoresis. The results showed that two target fragments possessed different sizes and Tm valves which were amplfied corresponding two separate peaks. The standard curve based on the L.monocytogenes hlyA gene was established and the correlation coefficient was 0.996. The minimal detection limit was 89CFU/mL. Moreover, suspicious strain isolated from white clams was tested by using this method so as to study the feasibility. The positive result was evaluated with the National Standard Methods (GB 4789.30-2010). The verified result indicated that the positive result of SYBR GreenⅠfluorescent PCR matched with the GB 4789.30-2010 exactly.
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50

Christensen, Mette, Nanna Johnsen, Marianne Skals, Aimi Hamilton, Peter Rubak, Anne-Mette Hvas, and Helle Praetorius. "Prevention of P2 Receptor-Dependent Thrombocyte Activation by Pore-Forming Bacterial Toxins Improves Outcome in A Murine Model of Urosepsis." International Journal of Molecular Sciences 21, no. 16 (August 6, 2020): 5652. http://dx.doi.org/10.3390/ijms21165652.

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Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes in the major organs. Development of a thrombocytopenic state is associated with a poorer outcome of sepsis. Uropathogenic Escherichia coli frequently produce the pore-forming, virulence factor α-haemolysin (HlyA), of which the biological effects are mediated by ATP release and subsequent activation of P2 receptors. Thus, we speculated that inhibition of thrombocyte P2Y1 and P2Y12 receptors might ameliorate the septic response to HlyA-producing E. coli. The study combined in vitro measurements of toxin-induced thrombocyte activation assessed as increased membrane abundance of P-selectin, fibronectin and CD63 and data from in vivo murine model of sepsis-induced by HlyA-producing E. coli under infusion of P2Y1 and P2Y12 antagonists. Our data show that the P2Y1 receptor antagonist almost abolishes thrombocyte activation by pore-forming bacterial toxins. Inhibition of P2Y1, by constant infusion of MRS2500, markedly increased the survival in mice with induced sepsis. Moreover, MRS2500 partially prevented the sepsis-induced depletion of circulating thrombocytes and dampened the sepsis-associated increase in proinflammatory cytokines. In contrast, P2Y12 receptor inhibition had only a marginal effect in vivo and in vitro. Taken together, inhibition of the P2Y1 receptor gives a subtle dampening of the thrombocyte activation and the cytokine response to bacteraemia, which may explain the improved survival observed by P2Y1 receptor antagonists.
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