scholarly journals Haemophilus ducreyi Hfq Contributes to Virulence Gene Regulation as Cells Enter Stationary Phase

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Dharanesh Gangaiah ◽  
Maria Labandeira-Rey ◽  
Xinjun Zhang ◽  
Kate R. Fortney ◽  
Sheila Ellinger ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Stationary Phase ◽  
Virulence Gene ◽  
Haemophilus Ducreyi ◽  
Alternative Sigma Factor ◽  
Gram Negative Bacteria ◽  
Major Contributor ◽  
Virulence Determinants ◽  
Content Type ◽  
Virulence Gene Regulation

ABSTRACTTo adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes ofHaemophilus ducreyi, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog ofhfq. Insertional inactivation ofhfqaltered the expression of ~16% of theH. ducreyigenes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in thehfqinactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation ofhfqdownregulated genes in theflp-tadandlspB-lspA2operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarkedhfqdeletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion ofhfqdownregulated Flp1 and impaired the ability ofH. ducreyito form microcolonies, downregulated DsrA and renderedH. ducreyiserum susceptible, and downregulated LspB and LspA2, which allowH. ducreyito resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor ofH. ducreyistationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog.IMPORTANCEPathogenic bacteria encounter a wide range of stresses in their hosts, including nutrient limitation; the ability to sense and respond to such stresses is crucial for bacterial pathogens to successfully establish an infection. Gram-negative bacteria frequently utilize the alternative sigma factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, includingHaemophilus ducreyi, which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required forH. ducreyito infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.

scholarly journals Phasevarion-Regulated Virulence in the Emerging Pediatric Pathogen Kingella kingae

2017 ◽  
Vol 85 (12) ◽  
Author(s):  
Yogitha N. Srikhanta ◽  
Ka Yee Fung ◽  
Georgina L. Pollock ◽  
Vicki Bennett-Wood ◽  
Benjamin P. Howden ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Heat Shock ◽  
Virulence Gene ◽  
Dna Methyltransferases ◽  
Epigenetic Mechanism ◽  
Phase Variable ◽  
Kingella Kingae ◽  
Content Type ◽  
Associated Proteins ◽  
Virulence Gene Regulation

ABSTRACT Kingella kingae is a common etiological agent of pediatric osteoarticular infections. While current research has expanded our understanding of K. kingae pathogenesis, there is a paucity of knowledge about host-pathogen interactions and virulence gene regulation. Many host-adapted bacterial pathogens contain phase variable DNA methyltransferases (mod genes), which can control expression of a regulon of genes (phasevarion) through differential methylation of the genome. Here, we identify a phase variable type III mod gene in K. kingae, suggesting that phasevarions operate in this pathogen. Phylogenetic studies revealed that there are two active modK alleles in K. kingae. Proteomic analysis of secreted and surface-associated proteins, quantitative PCR, and a heat shock assay comparing the wild-type modK1 ON (i.e., in frame for expression) strain to a modK1 OFF (i.e., out of frame) strain revealed three virulence-associated genes under ModK1 control. These include the K. kingae toxin rtxA and the heat shock genes groEL and dnaK. Cytokine expression analysis showed that the interleukin-8 (IL-8), IL-1β, and tumor necrosis factor responses of THP-1 macrophages were lower in the modK1 ON strain than in the modK1::kan mutant. This suggests that the ModK1 phasevarion influences the host inflammatory response and provides the first evidence of this phase variable epigenetic mechanism of gene regulation in K. kingae.

scholarly journals DksA and (p)ppGpp Have Unique and Overlapping Contributions to Haemophilus ducreyi Pathogenesis in Humans

2015 ◽  
Vol 83 (8) ◽  
pp. 3281-3292 ◽  
Author(s):  
Concerta L. Holley ◽  
Xinjun Zhang ◽  
Kate R. Fortney ◽  
Sheila Ellinger ◽  
Paula Johnson ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Stringent Response ◽  
Open Reading Frames ◽  
Haemophilus Ducreyi ◽  
Maximal Effect ◽  
Virulence Determinants ◽  
Bacterial Survival ◽  
Altered Expression ◽  
Content Type ◽  
Human Foreskin Fibroblasts

The (p)ppGpp-mediated stringent response is important for bacterial survival in nutrient limiting conditions. For maximal effect, (p)ppGpp interacts with the cofactor DksA, which stabilizes (p)ppGpp's interaction with RNA polymerase. We previously demonstrated that (p)ppGpp was required for the virulence ofHaemophilus ducreyiin humans. Here, we constructed anH. ducreyidksAmutant and showed it was also partially attenuated for pustule formation in human volunteers. To understand the roles of (p)ppGpp and DksA in gene regulation inH. ducreyi, we defined genes potentially altered by (p)ppGpp and DksA deficiency using transcriptome sequencing (RNA-seq). In bacteria collected at stationary phase, lack of (p)ppGpp and DksA altered expression of 28% and 17% ofH. ducreyiopen reading frames, respectively, including genes involved in transcription, translation, and metabolism. There was significant overlap in genes differentially expressed in the (p)ppGpp mutant relative to thedksAmutant. Loss of (p)ppGpp or DksA resulted in the dysregulation of several known virulence determinants. Deletion ofdksAdownregulatedlspBand rendered the organism less resistant to phagocytosis and increased its sensitivity to oxidative stress. Both mutants had reduced ability to attach to human foreskin fibroblasts; the defect correlated with reduced expression of the Flp adhesin proteins in the (p)ppGpp mutant but not in thedksAmutant, suggesting that DksA regulates the expression of an unknown cofactor(s) required for Flp-mediated adherence. We conclude that both (p)ppGpp and DksA serve as major regulators ofH. ducreyigene expression in stationary phase and have both overlapping and unique contributions to pathogenesis.

scholarly journals MgrA Activates Staphylococcal Capsule via SigA-Dependent Promoter

2020 ◽  
Vol 203 (2) ◽  
pp. e00495-20
Author(s):  
Mei G. Lei ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Gene Regulation ◽  
Virulence Factor ◽  
Virulence Gene ◽  
Major Effect ◽  
Mobility Shift ◽  
Content Type ◽  
Regulatory Pathways ◽  
Mobility Shift Assay ◽  
Virulence Gene Regulation

ABSTRACTStaphylococcus aureus capsule polysaccharide is an important antiphagocytic virulence factor. The cap genes are regulated at the promoter element (Pcap) upstream of the cap operon. Pcap, which consists of a dominant SigB-dependent promoter and a weaker upstream SigA-dependent promoter, is activated by global regulator MgrA. How MgrA activates capsule is unclear. Here, we showed that MgrA directly bound to the Pcap region and affected the SigA-dependent promoter. Interestingly, an electrophoretic mobility shift assay showed that MgrA bound to a large region of Pcap, mainly downstream of the SigA-dependent promoter. We further showed that the ArlRS two-component system and the Agr quorum sensing system activated capsule primarily through MgrA in the early growth phases.IMPORTANCE The virulence of Staphylococcus aureus depends on the expression of various virulence factors, which is governed by a complex regulatory network. We have been using capsule as a model virulence factor to study virulence gene regulation in S. aureus. MgrA is one of the regulators of capsule and has a major effect on capsule production. However, how MgrA regulates capsule genes is not understood. In this study, we were able to define the mechanism involving MgrA regulation of capsule. In addition, we also delineated the role of MgrA in capsule regulatory pathways involving the key virulence regulators Agr and Arl. This study further advances our understanding of virulence gene regulation in S. aureus, an important human pathogen.

scholarly journals RNA G-Quadruplex Structures Mediate Gene Regulation in Bacteria

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaolong Shao ◽  
Weitong Zhang ◽  
Mubarak Ishaq Umar ◽  
Hei Yuen Wong ◽  
Zijing Seng ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Cell Envelope ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Content Type ◽  
Cellular Processes ◽  
Wide Range ◽  
G Quadruplex ◽  
Eukaryotic Organisms

ABSTRACT Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both in vitro and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species. IMPORTANCE G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.

scholarly journals The Cpx System Regulates Virulence Gene Expression in Vibrio cholerae

2015 ◽  
Vol 83 (6) ◽  
pp. 2396-2408 ◽  
Author(s):  
Nicole Acosta ◽  
Stefan Pukatzki ◽  
Tracy L. Raivio
Keyword(s):  
Vibrio Cholerae ◽  
Response Regulator ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Receptor Protein ◽  
Virulence Determinants ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Envelope Stress ◽  
El Tor

Bacteria possess signal transduction pathways capable of sensing and responding to a wide variety of signals. The Cpx envelope stress response, composed of the sensor histidine kinase CpxA and the response regulator CpxR, senses and mediates adaptation to insults to the bacterial envelope. The Cpx response has been implicated in the regulation of a number of envelope-localized virulence determinants across bacterial species. Here, we show that activation of the Cpx pathway inVibrio choleraeEl Tor strain C6706 leads to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT) and the toxin-coregulated pilus (TCP). Our results indicate that this occurs through the repression of production of the ToxT regulator and an additional upstream transcription factor, TcpP. The effect of the Cpx response on CT and TCP expression is mostly abrogated in a cyclic AMP receptor protein (CRP) mutant, although expression of thecrpgene is unaltered. Since TcpP production is controlled by CRP, our data suggest a model whereby the Cpx response affects CRP function, which leads to diminished TcpP, ToxT, CT, and TCP production.

scholarly journals MroQ Is a Novel Abi-Domain Protein That Influences Virulence Gene Expression in Staphylococcus aureus via Modulation of Agr Activity

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Stephanie Marroquin ◽  
Brittney Gimza ◽  
Brooke Tomlinson ◽  
Michelle Stein ◽  
Andrew Frey ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Gene ◽  
Transcriptional Analysis ◽  
Data Sets ◽  
Virulence Determinants ◽  
Sequencing Data ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Function Mechanism ◽  
Family Protein

ABSTRACT Numerous factors have, to date, been identified as playing a role in the regulation of Agr activity in Staphylococcus aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigated the product of SAUSA300_1984 (termed MroQ), a transmembrane Abi-domain/M79 protease-family protein, as a novel effector of this system. Using a USA300 mroQ mutant, we observed a drastic reduction in proteolysis, hemolysis, and pigmentation that was fully complementable. This appears to result from diminished agr activity, as transcriptional analysis revealed significant decreases in expression of both RNAII and RNAIII in the mroQ mutant. Such effects appear to be direct, rather than indirect, as known agr effectors demonstrated limited alterations in their activity upon mroQ disruption. A comparison of RNA sequencing data sets for both mroQ and agr mutants revealed a profound overlap in their regulomes, with the majority of factors affected being known virulence determinants. Importantly, the preponderance of alterations in expression were more striking in the agr mutant, indicating that MroQ is necessary, but not sufficient, for Agr function. Mechanism profiling revealed that putative residues for metalloprotease activity within MroQ are required for its Agr-controlling effect; however, this was not wielded at the level of AgrD processing. Virulence assessment demonstrated that both mroQ and agr mutants exhibited increased formation of renal abscesses but decreased skin abscess formation alongside diminished dermonecrosis. Collectively, we present the characterization of a novel agr effector in S. aureus which would appear to be a direct regulator, potentially functioning via interaction with the AgrC histidine kinase.

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