Characterization of the ospZ promoter in Shigella flexneri and its regulation by VirB and H-NS.
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ABSTRACT:
SUBMITTER: Basta DW
PROVIDER: S-EPMC3676053 | biostudies-literature | 2013 Jun
REPOSITORIES: biostudies-literature
Publications
Characterization of the ospZ promoter in Shigella flexneri and its regulation by VirB and H-NS.
Basta David W DW Pew Krystle L KL Immak Joy A JA Park Hiromichi S HS Picker Michael A MA Wigley Amanda F AF Hensley Christopher T CT Pearson Jaclyn S JS Hartland Elizabeth L EL Wing Helen J HJ
Journal of bacteriology 20130329 11
OspZ is an effector protein of the type III secretion system in Shigella spp. that downregulates the human inflammatory response during bacterial infection. The ospZ gene is located on the large virulence plasmid of Shigella. Many genes on this plasmid are transcriptionally repressed by the nucleoid structuring protein H-NS and derepressed by VirB, a DNA-binding protein that displays homology to the plasmid partitioning proteins ParB and SopB. In this study, we characterized the ospZ promoter an ...[more]
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Project description:Shigella species, the causal agents of bacillary dysentery, use a type III secretion system (T3SS) to inject two waves of virulence proteins, known as effectors, into the colonic epithelium to subvert host cell machinery. Prior to host cell contact and secretion of the first wave of T3SS effectors, OspD1, an effector and antiactivator protein, prevents premature production of the second wave of effectors. Despite this important role, regulation of the ospD1 gene is not well understood. While ospD1 belongs to the large regulon of VirB, a transcriptional antisilencing protein that counters silencing mediated by the histone-like nucleoid structuring protein H-NS, it remains unclear if VirB directly or indirectly regulates ospD1 Additionally, it is not known if ospD1 is regulated by H-NS. Here, we identify the primary ospD1 transcription start site (+1) and show that the ospD1 promoter is remotely regulated by both VirB and H-NS. Our findings demonstrate that VirB regulation of ospD1 requires at least one of the two newly identified VirB regulatory sites, centered at -978 and -1270 relative to the ospD1 +1. Intriguingly, one of these sites lies on a 193-bp sequence found in three conserved locations on the large virulence plasmids of Shigella The region required for H-NS-dependent silencing of ospD1 lies between -1120 and -820 relative to the ospD1 +1. Thus, our study provides further evidence that cis-acting regulatory sequences for transcriptional antisilencers and silencers, such as VirB and H-NS, can lie far upstream of the canonical bacterial promoter region (i.e., -250 to +1).IMPORTANCE Transcriptional silencing and antisilencing mechanisms regulate virulence gene expression in many important bacterial pathogens. In Shigella species, plasmid-borne virulence genes, such as those encoding the type III secretion system (T3SS), are silenced by the histone-like nucleoid structuring protein H-NS and antisilenced by VirB. Previous work at the plasmid-borne icsP locus revealed that VirB binds to a remotely located cis-acting regulatory site to relieve transcriptional silencing mediated by H-NS. Here, we characterize a second example of remote VirB antisilencing at ospD1, which encodes a T3SS antiactivator and effector. Our study highlights that remote transcriptional silencing and antisilencing occur more frequently in Shigella than previously thought, and it raises the possibility that long-range transcriptional regulation in bacteria is commonplace.
| S-EPMC7186461 | biostudies-literature
Project description:The virF gene of Shigella, responsible for triggering the virulence cascade in this pathogenic bacterium, is transcriptionally repressed by the nucleoid-associated protein H-NS. The primary binding sites of H-NS within the promoter region of virF have been detected here by footprinting experiments in the presence of H-NS or its monomeric DNA-binding domain (H-NSctd), which displays the same specificity as intact H-NS. Of the 14 short DNA fragments identified, 10 overlap sequences similar to the H-NS binding motif. The 'fast', 'intermediate' and 'slow' H-NS binding events leading to the formation of the nucleoprotein complex responsible for transcription repression have been determined by time-resolved hydroxyl radical footprinting experiments in the presence of full-length H-NS. We demonstrate that this process is completed in ≤1 s and H-NS protections occur simultaneously on site I and site II of the virF promoter. Furthermore, all 'fast' protections have been identified in regions containing predicted H-NS binding motifs, in agreement with the hypothesis that H-NS nucleoprotein complex assembles from a few nucleation sites containing high-affinity binding sequences. Finally, data are presented showing that the 22-bp fragment corresponding to one of the HNS binding sites deviates from canonical B-DNA structure at three TpA steps.
| S-EPMC4245942 | biostudies-literature
Project description:Quorum-sensing systems regulate the expression of virulence factors in a wide variety of plant and animal pathogens, including members of the Enterobacteriaceae. Studies of Shigella virulence gene expression have demonstrated that maximal expression of genes encoding the type III secretion system and its substrates and maximal activity of this virulence organelle occur at high cell density. In these studies, we demonstrate that the expression of ipa, mxi, and spa invasion operons is maximal in stationary-phase bacteria and that conditioned media derived from stationary-phase cultures enhance the expression of these loci. In contrast, expression of virB, a transcription factor essential for the expression of invasion loci, peaks in late log phase; accordingly, virB expression is enhanced by a signal(s) present in conditioned media derived from late-log-phase cultures. Autoinducer 2 (AI-2), a quorum signaling molecule active in late log phase, was synthesized by Shigella species and enteroinvasive Escherichia coli and shown to be responsible for the observed peak of virB expression. However, AI-2 does not influence invasion operon expression and is not required for Shigella virulence, as mutants deficient in AI-2 synthesis are fully virulent. The implications of these findings with regard to both virB and invasion operon expression and the evolution of circuitries governing virulence gene expression are discussed.
| S-EPMC97850 | biostudies-literature
Project description:The transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneri and enteroinvasive Escherichia coli (EIEC) is induced at 37 degreesC and repressed at 30 degreesC. In this work, we report that the O135: K-:H- EIEC strain HN280 and S. flexneri SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the apy gene. In addition, the S. flexneri strains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the phoN-Sf gene. Both apy and phoN-Sf are pINV-carried loci whose contribution to the pathogenicity of enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both the apy and the phoN-Sf genes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of virB transcription) expressed the apy gene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Deltahns deletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of virB transcription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb-) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of virF or virB and by the presence of the intact apy locus or intact apy and phoN-Sf loci were detected among Crb- mutants of HN280 and SFZM53, respectively. While all Crb- apy+ mutants of HN280 failed to produce apyrase, Crb- apy+ phoN-Sf+ mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying cloned virF (pMYSH6504) or virB (pBN1) into Crb- mutants of HN280 and SFZM53 lacking virF or virB, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes, apy is another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of enteroinvasive bacteria.
| S-EPMC108614 | biostudies-literature
Project description:Shigella flexneri is a leading cause of diarrheal disease in children under five in developing countries. There is currently no licensed vaccine and broad spectrum protection requires coverage of multiple serotypes. The live attenuated vaccines CVD 1213 and CVD 1215 were derived from two prominent S. flexneri serotypes: S. flexneri 3a and S. flexneri 6. To provide broad-spectrum immunity, they could be combined with CVD 1208S, a S. flexneri 2a strain that demonstrated promising results in phase I and II clinical trials. Each strain contains a mutation in the guaBA operon. These vaccine candidates were tested in vitro and in vivo and were found to be auxotrophic for guanine and defective in intracellular replication, but capable of inducing cytokine production from both epithelial cells and macrophages. Both strains were attenuated for virulence in the guinea pig Serény test and induced robust serotype-specific antibody responses following immunization. Each strain induced homologous serotype protection against challenge and a mixed inoculum of the three S. flexneri vaccines conferred protection against all three virulent wild-type strains. These data support the use of CVD 1213, CVD 1215 and CVD 1208S in a multivalent vaccine to confer broad protection against disease caused by Shigella flexneri.
| S-EPMC5985478 | biostudies-literature
Project description:The O antigen of the Shigella flexneri lipopolysaccharide (LPS) is an important virulence determinant and immunogen. We have isolated S. flexneri mutants which produce a semi-rough LPS by using an O-antigen-specific phage, Sf6c. Western immunoblotting was used to show that the LPS produced by the semi-rough mutants contained only one O-antigen repeat unit. Thus, the mutants are deficient in production of the O-antigen polymerase and were termed rfc mutants. Complementation experiments were used to locate the rfc adjacent to the rfb genes on plasmid clones previously isolated and containing this region (D. F. Macpherson, R. Morona, D. W. Beger, K.-C. Cheah, and P. A. Manning, Mol. Microbiol 5:1491-1499, 1991). A combination of deletions and subcloning analysis located the rfc gene as spanning a 2-kb region. Insertion of a kanamycin resistance cartridge into a SalI site in this region inactivated the rfc gene. The DNA sequence of the rfc region was determined. An open reading frame spanning the SalI site was identified and encodes a protein with a predicted molecular mass of 43.7 kDa. The predicted protein is highly hydrophobic and showed little sequence homology with any other protein. Comparison of its hydropathy plot with that of other Rfc proteins from Salmonella enterica (typhimurium) and Salmonella enterica (muenchen) revealed that the profiles were similar and that the proteins have 12 or more potential membrane-spanning segments. A comparison of the S. flexneri rfc gene and protein product with other rfc and rfc-like proteins revealed that they have a similarly low percentage of G + C content and have similar codon usage, and all have a high percentage of rare codons. An attempt to identify the S. flexneri Rfc protein was unsuccessful, although proteins encoded upstream and downstream of the rfc gene could be identified. Examination of the distribution of rare or minor codons in the rfc gene revealed that it has several minor codons within the first 25 amino acids. This is in contrast to the upstream gene rfbG, which also has a high percentage of rare codons but whose gene product could be detected. The positioning of the rare codons in the rfc gene may restrict translation and suggests that minor isoaccepting tRNA species may be involved in translational regulation of rfc expression. The low percentage of G + C content of rfc genes may be a consequence of the selection pressure to maintain this form of control.
| S-EPMC205111 | biostudies-other
Project description:The rfb region of Shigella flexneri encodes the proteins required to synthesize the O-antigen component of its cell surface lipopolysaccharides (LPS). We have previously reported that a region adjacent to rfb was involved in regulating the length distribution of the O-antigen polysaccharide chains (D. F. Macpherson et al., Mol. Microbiol. 5:1491-1499, 1991). The gene responsible has been identified in Escherichia coli O75 (called rol [R. A. Batchelor et al., J. Bacteriol. 173:5699-5704, 1991]) and in E. coli O111 and Salmonella enterica serovar typhimurium strain LT2 (called cld [D. A. Bastin et al., Mol. Microbiol. 5:2223-2231, 1991]). Through a combination of subcloning, deletion, and transposon insertion analysis, we have identified a gene adjacent to the S. flexneri rfb region which encodes a protein of 36 kDa responsible for the length distribution of O-antigen chains in LPS as seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels. DNA sequence analysis identified an open reading frame (ORF) corresponding to the rol gene. The corresponding protein was almost identical in sequence to the Rol protein of E. coli O75 and was highly homologous to the functionally identical Cld proteins of E. coli O111 and S. enterica serovar typhimurium LT2. These proteins, together with ORF o349 adjacent to rfe, had almost identical hydropathy plots which predict membrane-spanning segments at the amino- and carboxy-terminal ends and a hydrophilic central region. We isolated a number of TnphoA insertions which inactivated the rol gene, and the fusion end points were determined. The PhoA+ Rol::PhoA fusion proteins had PhoA fused within the large hydrophilic central domain of Rol. These proteins were located in the whole-membrane fraction, and extraction with Triton X-100 indicated a cytoplasmic membrane location. This finding was supported by sucrose density gradient fractionation of the whole-cell membranes and of E. coli maxicells expressing L-[35S]methionine-labelled Rol protein. Hence, we interpret these data to indicate that the Rol protein is anchored into the cytoplasmic membrane via its amino- and carboxy-terminal ends but that the majority of the protein is located in the periplasmic space. To confirm that rol is responsible for the effects on O-antigen chain length observed with the cloned rfb genes in E. coli K-12, it was mutated in S. flexneri by insertion of a kanamycin resistance cartridge. The resulting strains produced LPS with O antigens of nonmodal chain length, thereby confirming the function of the rol gene product. We propose a model for the function of Rol protein in which it acts as a type of molecular chaperone to facilitate the interaction of the O-antigen ligase (RfaL) with the O-antigen polymerase (Rfc) and polymerized, acyl carrier lipid-linked, O-antigen chains. Analysis of the DNA sequence of the region identified a number of ORFs corresponding to the well-known gnd and hisIE genes. The rol gene was located immediately downstream of two ORFs with sequence similarity to the gene encoding UDPglucose dehydrogenase (HasB) of Streptococcus pyogenes. The ORFs arise because of a deletion or frameshift mutation within the gene we have termed udg (for UDPglucose dehydrogenase).
| S-EPMC176702 | biostudies-other
Project description:BACKGROUND: Shigella bacteria cause dysentery, which remains a significant threat to public health. Shigella flexneri is the most common species in both developing and developed countries. Five Shigella genomes have been sequenced, revealing dynamic and diverse features. To investigate the intra-species diversity of S. flexneri genomes further, we have sequenced the complete genome of S. flexneri 5b strain 8401 (abbreviated Sf8401) and compared it with S. flexneri 2a (Sf301). RESULTS: The Sf8401 chromosome is 4.5-Mb in size, a little smaller than that of Sf301, mainly because the former lacks the SHI-1 pathogenicity island (PAI). Compared with Sf301, there are 6 inversions and one translocation in Sf8401, which are probably mediated by insertion sequences (IS). There are clear differences in the known PAIs between these two genomes. The bacteriophage SfV segment remaining in SHI-O of Sf8401 is clearly larger than the remnants of bacteriophage SfII in Sf301. SHI-1 is absent from Sf8401 but a specific related protein is found next to the pheV locus. SHI-2 is involved in one intra-replichore inversion near the origin of replication, which may change the expression of iut/iuc genes. Moreover, genes related to the glycine-betaine biosynthesis pathway are present only in Sf8401 among the known Shigella genomes. CONCLUSION: Our data show that the two S. flexneri genomes are very similar, which suggests a high level of structural and functional conservation between the two serotypes. The differences reflect different selection pressures during evolution. The ancestor of S. flexneri probably acquired SHI-1 and SHI-2 before SHI-O was integrated and the serotypes diverged. SHI-1 was subsequently deleted from the S. flexneri 5b genome by recombination, but stabilized in the S. flexneri 2a genome. These events may have contributed to the differences in pathogenicity and epidemicity between the two serotypes of S. flexneri.
| S-EPMC1550401 | biostudies-literature
Project description:VirB is a key regulator of genes located on the large virulence plasmid (pINV) in the bacterial pathogen Shigella flexneri VirB is unusual; it is not related to other transcriptional regulators, instead, it belongs to a family of proteins that primarily function in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer. To do this, a GFP-VirB fusion that retains the regulatory activity of VirB and yet, does not undergo significant protein degradation in S. flexneri, was used. Surprisingly, discrete fluorescent foci were observed in live wild-type S. flexneri cells and an isogenic virB mutant using fluorescence microscopy. In contrast, foci were rarely observed (<10%) in pINV-cured cells or in cells expressing a GFP-VirB fusion carrying amino acid substitutions in the VirB DNA binding domain. Finally, the 25 bp VirB-binding site was demonstrated to be sufficient and necessary for GFP-VirB focus formation using a set of small surrogate plasmids. Combined, these data demonstrate that the VirB:DNA interactions required for the transcriptional anti-silencing activity of VirB on pINV are a prerequisite for the subcellular localization of VirB in the bacterial cytoplasm. The significance of these findings, in light of the anti-silencing activity of VirB, is discussed.ImportanceThis study reveals the subcellular localization of VirB, a key transcriptional regulator of virulence genes found on the large virulence plasmid (pINV) in Shigella. Fluorescent signals generated by an active GFP-VirB fusion form 2, 3, or 4 discrete foci in the bacterial cytoplasm, predominantly at the quarter cell position. These signals are completely dependent upon VirB interacting with its DNA binding site found either on the virulence plasmid or an engineered surrogate. Our findings: 1) provide novel insight into VirB:pINV interactions, 2) suggest that VirB may have utility as a DNA marker, and 3) raise questions about how and why this anti-silencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm.
| S-EPMC8117518 | biostudies-literature
Project description:The glutamate-dependent acid-resistance system is the most effective acid tolerance pathway in Shigella, allowing survival in extremely acidic environments. However, the regulation of this system in Shigella remains elusive. In the current study, we identified significant differences in the levels of glutamate decarboxylase between three Shigella flexneri strains with different levels of acid resistance using blue native-polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF)/sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results showed that the degree of acid resistance and the levels of GadA/B were significantly lower in strain 2457T compared with two other S. flexneri strains. It has been reported that plasmid pSf-R27 is expressed in strain 2457T but not in the other 142 sequenced S. flexneri isolates. pSf-R27 encodes protein Sfh, which belongs to a family of histone-like nucleoid-structuring (H-NS) proteins that participate in the transcriptional control of glutamate-dependent acid resistance, implicating pSf-R27 in the lower acid resistance of strain 2457T. Transformation of pSf-R27 or sfh alone into strain 301 resulted in decreased expression of GadA/B in the recombinant strains. Thus, we confirmed that H-NS family protein Sfh, bound to the gadA/B regulatory region and regulates the expression of glutamate decarboxylase at the transcriptional level. We also examined the acid tolerance of the wild-type and recombinant strains using flow cytometry and determined that the acid tolerance of S. flexneri is closely related to the expression of GadA/B. These findings further our understanding of the acid tolerance of S. flexneri, especially via the glutamate-dependent pathway.
| S-EPMC5633597 | biostudies-literature