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YhjC is a novel transcriptional regulator required for Shigella flexneri virulence.

ABSTRACT: Shigella is an intracellular pathogen that primarily infects the human colon and causes shigellosis. Shigella virulence relies largely on the type III secretion system (T3SS) and secreted effectors. VirF, the master Shigella virulence regulator, is essential for the expression of T3SS-related genes. In this study, we found that YhjC, a LysR-type transcriptional regulator, is required for Shigella virulence through activating the transcription of virF. Pathogenicity of the yhjC mutant, including colonization in the colons of guinea pigs as well as its ability for host cell adhesion and invasion, was significantly lowered. Expression levels of virF and nearly all VirF-dependent genes were downregulated by yhjC deletion, indicating that YhjC can activate virF transcription. Electrophoretic mobility shift assay analysis demonstrated that YhjC could bind directly to the virF promoter region. Therefore, YhjC is a novel virulence regulator that positively regulates the virF expression and promotes Shigella virulence. Additionally, genome-wide expression analysis identified the presence of other genes in the large virulence plasmid and a genome exhibiting differential expression in response to yhjC deletion, with 169 downregulated and 99 upregulated genes, indicating that YhjC also functioned as a global regulatory factor.

SUBMITTER: Li W

PROVIDER: S-EPMC8218686 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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YhjC is a novel transcriptional regulator required for Shigella flexneri virulence.

Li Wanwu W Jiang Lingyan L Liu Xiaoqian X Guo Rui R Ma Shuai S Wang Jingting J Ma Shuangshuang S Li Shujie S Li Huiying H

Virulence 20211201 1

Shigella is an intracellular pathogen that primarily infects the human colon and causes shigellosis. Shigella virulence relies largely on the type III secretion system (T3SS) and secreted effectors. VirF, the master Shigella virulence regulator, is essential for the expression of T3SS-related genes. In this study, we found that YhjC, a LysR-type transcriptional regulator, is required for Shigella virulence through activating the transcription of virF. Pathogeni ...[more]

PMID: 34152261

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Project description:Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli, and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-GMP (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. c-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri. In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae, in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation and reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes. IMPORTANCE The intracellular human pathogen Shigella causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance among Shigella species is on the rise. Here, we explored how the widely conserved c-di-GMP bacterial signaling system alters Shigella behaviors associated with pathogenesis. We found that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in Shigella's ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.

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.

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YhjC is a novel transcriptional regulator required for Shigella flexneri virulence.

Publications

YhjC is a novel transcriptional regulator required for Shigella flexneri virulence.

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