Auswahl der wissenschaftlichen Literatur zum Thema „WhiB6“

A strain of <i>Kytococcus sedentarius</i> was isolated from a dehumidifier operating in a university lecture theatre. Genome analysis and phenotypic characterisation showed that this strain, <i>K. sedentarius</i> MBB13, was a moderately halotolerant aerobe with a branched aerobic electron transport chain and genes that could contribute to erythromycin resistance. The major compatible solute was glycine betaine, with ectoine and proline being deployed at higher osmolarities. Actinobacteria possess multiple WhiB-like (Wbl) regulatory proteins, and <i>K. sedentarius</i> MBB13 has four (WhiB1, WhiB2, WhiB3, and WhiB7). Wbls are iron-sulfur proteins that regulate gene expression through interactions with RNA polymerase sigma factors and/or other regulatory proteins. Bacterial two-hybrid analyses suggested that WhiB1 and WhiB2, but not WhiB3 and WhiB7, interact with the C-terminal domain of the major sigma factor, σ^A; no interaction was detected between any of the Wbl proteins and the only alternative sigma factors, σ^B, σ^H, or σ^J. The interaction between σ^A and WhiB1 or WhiB2 was disrupted in a heterologous system under growth conditions that produce nitric oxide and the iron-sulfur clusters of the isolated WhiB1 and WhiB2 proteins reacted with nitric oxide. Thus, <i>K. sedentarius</i> strain exhibits the major phenotypic characteristics of the type strain and a comprehensive examination of the interactions between its four Wbl proteins and four sigma factors suggested that the Wbl proteins all operate through interaction with σ^A.

ABSTRACT The seven Mycobacterium tuberculosis whiB-like genes encode small proteins postulated to be transcriptional regulators. A systematic real-time reverse transcription-PCR analysis following exposure to antibiotics and a variety of growth and in vitro stress conditions indicates differential, and in some cases dramatic, transcription modulations for the different M. tuberculosis whiB family members. This information together with biochemical analyses of the whiB1 to whiB7 gene products will be important for understanding the biology of this novel family of proteins in mycobacteria and related actinomycetes.

ESX (ESAT-6 system) export systems play diverse roles across mycobacterial species. Interestingly, genetic disruption of ESX systems in different species does not result in an accumulation of protein substrates in the mycobacterial cell. However, the mechanisms underlying this observation are elusive. We hypothesized that the levels of ESX substrates were regulated by a feedback-control mechanism, linking the levels of substrates to the secretory status of ESX systems. To test this hypothesis, we used a combination of genetic, transcriptomic, and proteomic approaches to define export-dependent mechanisms regulating the levels of ESX-1 substrates inMycobacterium marinum. WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that, in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of thewhiB6gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected inM. marinumstrains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb1, a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of thewhiB6gene. Finally, we found that the loss of WhiB6 resulted in a virulentM. marinumstrain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates inM. marinumare fine-tuned by negative feedback control, linking the expression of thewhiB6gene to the presence, not the functionality, of the ESX-1 membrane complex.

WhiB is a transcription regulator which has been reported to be involved in the regulation of cell morphogenesis, cell division, antibiotic resistance, stress, etc., in several members of the family Actinomycetes . The present study describes functional characterization of a WhiB family protein, WhiB1 (protein ID: WP_065632651.1), from Gordonia sp. IITR100. We demonstrate that WhiB1 affects chromosome segregation and cell morphology in recombinant Escherichia coli , Gordonia sp. IITR100 as well as in Rhodococcus erythropolis . Multiple sequence alignment suggests that WhiB1 is a conserved protein among members of the family Actinomycetes . It has been reported that overexpression of WhiB1 leads to repression of the biodesulfurization operon in recombinant E. coli , Gordonia sp. IITR100 and R. erythropolis . A WhiB1-mut containing a point mutation Q116A in the DNA binding domain of WhiB1 led to partial alleviation of repression of the biodesulfurization operon. We show for the first time that the WhiB family protein WhiB1 is also involved in repression of the biodesulfurization operon by directly binding to the dsz promoter DNA.

ABSTRACT A growing body of evidence suggests that the WhiB-like proteins exclusive to the GC-rich actinomycete genera play significant roles in pathogenesis and cell division. Each of these proteins contains four invariant cysteine residues and a conserved helix-turn-helix motif. whmD, the Mycobacterium smegmatis homologue of Streptomyces coelicolor whiB, is essential in M. smegmatis, and the conditionally complemented mutant M. smegmatis 628-53 undergoes filamentation under nonpermissive conditions. To identify residues critical to WhmD function, we developed a cotransformation-based assay to screen for alleles that complement the filamentation phenotype of M. smegmatis 628-53 following inducer withdrawal. Mycobacterium tuberculosis whiB2 and S. coelicolor whiB complemented the defect in M. smegmatis 628-53, indicating that these genes are true functional orthologues of whmD. Deletion analysis suggested that the N-terminal 67 and C-terminal 12 amino acid residues are dispensable for activity. Site-directed mutagenesis indicated that three of the four conserved cysteine residues (C90, C93, and C99) and a conserved aspartate (D71) are essential. Mutations in a predicted loop glycine (G111) and an unstructured leucine (L116) were poorly tolerated. The region essential for WhmD activity encompasses 6 of the 10 residues conserved in all seven M. tuberculosis WhiBs, as well as in most members of the WhiB family identified thus far. WhmD structure was found to be sensitive to the presence of a reducing agent, suggesting that the cysteine residues are involved in coordinating a metal ion. Iron-specific staining strongly suggested that WhmD contains a bound iron atom. With this information, we have now begun to comprehend the functional significance of the conserved sequence and structural elements in this novel family of proteins.

The wbl (whiB-like) genes encode putative transcription factors unique to actinomycetes. This study characterized the promoter element of one of the seven wbl genes of Mycobacterium tuberculosis, whiB1 (Rv3219c). The results reveal that whiB1 is transcribed by a class I-type cAMP receptor protein (CRP)-dependent promoter, harbouring a CRP-binding site positioned at −58.5 with respect to its transcription start point. In vivo promoter activity analysis and electrophoretic mobility shift assays suggest that the expression of whiB1 is indeed regulated by cAMP-dependent binding of CRPM (encoded by the M. tuberculosis gene Rv3676) to the whiB1 5′ untranslated region (5′UTR). β-Galactosidase gene fusion analysis revealed induction of the whiB1 promoter in M. tuberculosis on addition of exogenous dibutyric cAMP (a diffusible cAMP analogue) only when an intact CRP-binding site was present. These results indicate that M. tuberculosis whiB1 transcription is regulated in part by cAMP levels via direct binding of cAMP-activated CRPM to a consensus CRP-binding site in the whiB1 5′UTR.

Abstract WhiB1 is a monomeric iron–sulfur cluster-containing transcription factor in the WhiB-like family that is widely distributed in actinobacteria including the notoriously persistent pathogen Mycobacterium tuberculosis (M. tuberculosis). WhiB1 plays multiple roles in regulating cell growth and responding to nitric oxide stress in M. tuberculosis, but its underlying mechanism is unclear. Here we report a 1.85 Å-resolution crystal structure of the [4Fe–4S] cluster-bound (holo-) WhiB1 in complex with the C-terminal domain of the σ70-family primary sigma factor σA of M. tuberculosis containing the conserved region 4 (σA4). Region 4 of the σ70-family primary sigma factors is commonly used by transcription factors for gene activation, and holo-WhiB1 has been proposed to activate gene expression via binding to σA4. The complex structure, however, unexpectedly reveals that the interaction between WhiB1 and σA4 is dominated by hydrophobic residues in the [4Fe–4S] cluster binding pocket, distinct from previously characterized canonical σ704-bound transcription activators. Furthermore, we show that holo-WhiB1 represses transcription by interaction with σA4in vitro and that WhiB1 must interact with σA4 to perform its essential role in supporting cell growth in vivo. Together, these results demonstrate that holo-WhiB1 regulates gene expression by a non-canonical mechanism relative to well-characterized σA4-dependent transcription activators.

ABSTRACTThe proteins belonging to the WhiB superfamily are small global transcriptional regulators typical of actinomycetes. In this paper, we characterize the role of WhiB5, aMycobacterium tuberculosisprotein belonging to this superfamily. A null mutant was constructed inM. tuberculosisH37Rv and was shown to be attenuated during both progressive and chronic mouse infections. Mice infected with the mutant had smaller bacillary burdens in the lungs but a larger inflammatory response, suggesting a role of WhiB5 in immunomodulation. Most interestingly, thewhiB5mutant was not able to resume growth after reactivation from chronic infection, suggesting that WhiB5 controls the expression of genes involved in this process. The mutant was also more sensitive than the wild-type parental strain toS-nitrosoglutathione (GSNO) and was less metabolically active following prolonged starvation, underscoring the importance of GSNO and starvation in development and maintenance of chronic infection. DNA microarray analysis identified 58 genes whose expression is influenced by WhiB5, includingsigM, encoding an alternative sigma factor, and genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4.

La tuberculose (TB) est, hors COVID, la première cause de décès lié à un agent infectieux. La détermination rapide du profil de résistance complet de Mycobacterium tuberculosis (Mtb) est essentielle pour démarrer un traitement adapté dans les cas de TB multirésistante (MDR) et ainsi limiter l’acquisition de résistances supplémentaires, augmenter les chances de succès thérapeutique et réduire la transmission de ces souches. Il existe 9 lignées principales au sein du complexe M. tuberculosis, dont 2 sont largement répandues dans le monde (L2 et L4). Au sein de la lignée 2, le clone W148 (ou complexe clonal CC 100-32) a diffusé récemment en Europe et en Asie. Cette diffusion pourrait s'expliquer par des mutations spécifiques au sein du génome.En premier lieu, nous avons étudié la technologie Deeplex Myc-TB pour détecter rapidement le génotype et la résistance des souches cliniques de Mtb MDR. Cette technologie, basée sur une PCR multiplex et un séquençage haut débit, détermine à la fois l’espèce (hsp65), le génotype (spoligotype) et la résistance à 13 antituberculeux de première et seconde ligne (18 cibles). Le travail d’évaluation a inclus 112 prélèvements et 94 souches adressés au Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux (CNR MyRMA). Nous avons observé que le test Deeplex Myc-TB fonctionne sur prélèvement positif à l’examen microscopique et sur souche. Les profils de résistance obtenus sont concordants à 85,4% avec les résultats de la méthode phénotypique de référence. L'utilisation de Deeplex Myc-TB fournit des résultats en une dizaine de jours, soit environ 6 semaines avant ceux de l’antibiogramme phénotypique. Deeplex Myc-TB permet donc d’adapter l’antibiothérapie bien avant que ce qui était fait jusqu’à présent, pour le bénéfice des patients. Nous avons ainsi pu valider cet outil maintenant utilisé en routine au CNR MyRMA. Nous avons ensuite étudié les mutations spécifiques du CC 100-32 MDR. L’analyse du génome complet de 36 souches reçues au CNR MyRMA a permis d’identifier 30 mutations non synonymes et petites délétions spécifiques. Parmi elles, nous avons choisi d’étudier celles présentes dans kdpD et whiB6, car des données de la littérature semblent montrer un impact de ces gènes sur la virulence de la souche. KdpDE est un système à deux composants régulant l’expression du système de transport du potassium inductible KdpFABC. La mutation présente dans le complexe CC 100-32 MDR est une délétion de 2 nucléotides à la fin du gène kdpD entraînant une protéine de fusion KdpDE. Nous avons donc construit un tel mutant chez Mtb H37Rv en supprimant les deux gènes kdpD et kdpE avant de le complémenter avec la forme sauvage ou mutée de kdpDE. Nous n’avons pas observé de différence de croissance in vitro des différentes souches, y compris en l’absence de potassium, suggérant que KdpDE n’est pas essentiel pour le fitness de la bactérie en présence et absence de potassium. L’étude de l’impact de la délétion sur l’activité transcriptionnelle et la virulence est en cours. D’autre part, WhiB6 est un facteur de transcription qui régule le système ESX-1, nécessaire à la virulence. Des travaux du laboratoire ont permis d'obtenir le mutant ∆whiB6 et les souches complémentées WT et mutée (T51P) et leur ré-analyse indique que la souche mutée T51P entraîne une production d’ESAT-6 et de cytokines pro-inflammatoires plus faible que la souche sauvage ; nous réalisons actuellement une analyse du transcriptome. Les premiers résultats obtenus semblent indiquer que la mutation T51P dans WhiB6 entraîne moins de virulence et d’inflammation. Ce travail a permis de valider un outil maintenant indispensable au diagnostic des TB MDR en routine au CNR MyRMA et d’étudier la diffusion d’un clone MDR à travers les fonctions de deux facteurs de transcription impliqués dans la virulence
Tuberculosis (TB) is, excluding COVID, the leading cause of death linked to an infectious agent. Rapid determination of the full resistance profile of Mycobacterium tuberculosis (Mtb) is essential to initiate appropriate treatment of multidrug resistant (MDR) cases, thereby limiting the acquisition of additional resistance, increasing the chances of therapeutic success and reducing transmission of these strains. There are 9 main lineages within the M. tuberculosis complex, 2 of which are widespread throughout the world (L2 and L4). Within lineage 2, the clone W148 (or clonal complex CC 100-32) has recently spread to Europe and Asia. This spread could be explained by specific mutations within the genome.First, we investigated the Deeplex Myc-TB tool for rapid detection of genotype and resistance in clinical Mtb MDR strains. The Deeplex Myc-TB technology, based on multiplex PCR and high-throughput sequencing, determines species (hsp65), genotype (spoligotype) and resistance to 13 first- and second-line anti-tuberculosis drugs (18 targets). Our evaluation included 112 samples and 94 strains sent to the Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux (CNR MyRMA). We have observed that the Deeplex Myc-TB test is efficient on microscopically positive samples and strains. The resistance profiles obtained are 85.4% concordant with the results of the phenotypic reference method. The use of Deeplex Myc-TB provides results within ten days, and around 6 weeks before those of the phenotypic antibiogram. Deeplex Myc-TB can therefore be used to adapt antibiotic therapy much earlier than was previously possible, to the patient benefit. We were thus able to validate this tool, which is now routinely used at CNR MyRMA. We next studied mutations specific to CC 100-32 MDR. Genome-wide analysis of 36 strains received at CNR MyRMA identified 30 non-synonymous mutations and small deletions specific to CC 100-32 MDR strains. Among these, we chose to study mutations present in kdpD and whiB6, as data in the literature indicated an impact of these genes on the virulence of the strain. On the one hand, KdpDE is a two-component system regulating expression of the inducible potassium transport system KdpFABC. The mutation present in the CC 100-32 MDR complex is a 2-nucleotides deletion at the end of the kdpD gene, resulting in a KdpDE fusion protein. We therefore constructed such a mutant in Mtb H37Rv by deleting both the kdpD and kdpE genes before complementing it with the wild-type or mutated form of kdpDE. We observed no difference in the in vitro growth of the different strains, even in the absence of potassium, suggesting that KdpDE is not essential for bacterial fitness in presence or absence of potassium. The impact of the deletion on transcriptional activity and virulence is currently being studied. On the other hand, WhiB6 is a transcription factor that regulates the ESX-1 system, necessary for virulence. Work from the laboratory generated a ∆whiB6 mutant and the complemented WT and mutated (T51P) strains and their re-analysis indicated that T51P mutant strain produces less ESAT-6 and pro-inflammatory cytokines than the wild-type strain. Our transcriptome analysis of these strains is currently underway. Initial results suggest that the T51P mutation in WhiB6 results in less virulence and inflammation. This work has validated a tool that is now essential for routine diagnosis of MDR TB at CNR MyRMA, and to study the spread of an MDR clone through the function of two transcription factors involved in virulence

Mycobacterium tuberculosis is a major human pathogen, infecting approximately one third of the world's population. The majority of infected individuals are asymptomatic, and in these cases M. tuberculosis is present in a non-replicating persistent state. Mycobacterium tuberculosis is capable of emerging from this persistent state and causing active disease. Extensive gene regulation facilitates the entry into and emergence from the persistent state, and understanding this reprogramming of genes may help provide novel methods of treatment. The cAMP receptor protein Rv3676 is a global gene regulator in M. tuberculosis that is required for virulence, regulating ~100 genes including whiB1. The whiB1 gene encodes a member of the Wbl family of proteins, which are iron-sulphur cluster-containing transcription factors associated with a role in developmental processes in the actinomycetes. WhiB1, one of seven Wbl proteins encoded by M. tuberculosis, was the focus of this study. The whiB1 gene was identified as being essential for M. tuberculosis survival. The WhiB1 protein incorporated a [4Fe-4S] cluster, ligated by four essential cysteine residues, which was stable in the presence of oxygen but reacted rapidly with nitric oxide in a multiphasic reaction forming an unusual octa-nitrosylated cluster. The reduced and oxidised apo-WhiB1 bound both the whiB1 and groEL2 promoter regions, but the [4Fe-4S] containing form of WhiB1 (holo-WhiB1) was unable to bind DNA. Treatment of holo-WhiB1 with nitric oxide led to restoration of DNA-binding. The DNA-binding capabilities of WhiB1 were found to be, in part, due to specific amino acid residues in the helix-turn-helix region of the protein. Transcription of whiB1 is negatively-regulated by apo-WhiB1 in both the presence and absence of Rv3676, which has been identified as activating whiB1 expression. Thus whiB1 expression is regulated by two important infection signals viz. nitric oxide and cAMP, suggesting a role for WhiB1 in tuberculosis pathogenesis.

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, continues to be the world’s deadliest human bacterial pathogen. Current treatments are notoriously limited, lengthy, and becoming increasingly ineffective due to drug-resistant mutant strains. WhiB7, a putative transcriptional regulator, is an essential component of intrinsic antibiotic resistance in Mtb. Unique to Actinobacteria, multiple paralogous WhiB-like proteins have diverse roles in physiology, but little is known about their mode of action or regulation. To investigate WhiB7, a combination of in vitro run-off, two-hybrid assays, protein pull-down experiments, and genetic approaches was used. WhiB7 was characterized as an auto-regulatory, redox-sensitive transcriptional activator, providing the first biochemical proof that a WhiB-like protein directly promotes transcription. WhiB7’s antibiotic resistance function was dependent on three regions: an iron-sulfur cluster binding region likely required for stability; a middle region for binding to the vegetative sigma factor SigA; and a C-terminal DNA-binding region. Mutations disrupting any one of the regions led to an inability of WhiB7 to activate resistance. These experimental constraints were combined with protein modelling techniques to visualize the WhiB7:SigA:DNA complex which may serve as a platform for the design of inhibitors. Additionally, a GFP reporter was constructed to monitor whiB7 induction, and was used to screen our custom library of almost 600 bioactive compounds including the majority of clinical antibiotics. Expression was induced by compounds having diverse structures and targets, which did not correlate with drug susceptibility of the whiB7 mutant. Antibiotic-induced transcription was synergistically increased by the reductant dithiothreitol, an effect mirrored by a whiB7-dependent shift to a highly reduced intracellular condition reflected by the reduced:oxidized mycothiol ratio. Amino acid metabolism also contributed to WhiB7-mediated intrinsic resistance. To gain insights into whether other genetic loci contribute to WhiB7-mediated antibiotic resistance, a transposon library of Mycobacterium smegmatis was screened for WhiB7-like drug susceptibility or resistance. These studies revealed a putative aspartate aminotransferase (MSMEG_4060) that contributed to whiB7 repression and a pair of adjacent hypothetical genes (MSMEG_3637/3638) that contributed to whiB7 induction. Continued characterization of WhiB7 may serve as a paradigm for other WhiB-like proteins and lead to novel and desperately needed therapies for tuberculosis.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

Intrinsic resistance of the intracellular pathogen Mycobacterium tuberculosis is one of the main reasons that the disease tuberculosis is difficult to treat and why it remains as one of the world’s most prevalent and dangerous infectious diseases. The intrinsic resistance regulator WhiB7 controls a regulon that contains many genes predicted to have physiological functions including aspartate aminotransferases, aspB and aspC. Multi-drug susceptibility was observed in an aspC mutant and an aspB constitutive expression strain. The expression of aspC was positively regulated by WhiB7 while expression of aspB downregulated whiB7 expression. The fitness of Mycobacterium smegmatis was affected negatively by oxaloacetate and positively by α-ketoglutarate, substrates of aspartate aminotransferase, which then altered the growth inhibition by antibiotics. Recombinant AspB and AspC both catalyze measurable transamination of aspartate and α-ketoglutarate. AspC plays an important role in mycobacteria physiology as deletion of this gene caused many growth deficiencies. Furthermore, the physiological role of AspC extended beyond amino acid intermediary metabolism to redox homeostasis and oxidative stress detoxification. These results revealed a link between intrinsic antibiotic resistance and metabolism mediated through AspB and AspC. Since antibiotic resistance in mycobacterium is a complex function of its physiology, it is important to screen for tuberculosis drugs under growth conditions that resemble those found in vivo.

Tuberculosis is a re-emerging global health problem. Bacille Calmette-Guerin (BCG), the available vaccine against the disease, is only effective short term and is associated with adverse reactions clinically. The development of new effective vaccines will require an understanding of virulence, immunogenic factors and the beneficial immune responses induced in the human host. My thesis investigates phoP and whiB3, two genes associated with virulence and immunogenicity in Mycobacterium tuberculosis. Study of PhoP in a natural phoP mutant, BCG-Prague, and in the clinically safe BCG-Japan, shows that over-expression of PhoP increases the immunogenicity of these vaccine strains. In addition, I found that WhiB3 impacts carbon metabolism in BCG-Birkhaug and BCG-Sweden, although the effect of this on virulence in vivo is still unclear. The characterization of genes involved in virulence and immunogenicity allows us to develop novel approaches for improving the efficacy of BCG, which has important implications for future TB vaccine development.