Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator.

ABSTRACT Control of the virulence regulator/sensor kinase (CovRS) two-component system (TCS) serves as a model for investigating the impact of signaling pathways on the pathogenesis of Gram-positive bacteria. However, the molecular mechanisms by which CovR, an OmpR/PhoB family response regulator, controls virulence gene expression are poorly defined, partly due to the labile nature of its aspartate phosphorylation site. To better understand the regulatory effect of phosphorylated CovR, we generated the phosphorylation site mutant strain 10870-CovR-D53E, which we predicted to have a constitutive CovR phosphorylation phenotype. Interestingly, this strain showed CovR activity only for a subset of the CovR regulon, which allowed for classification of CovR-influenced genes into D53E-regulated and D53E-nonregulated groups. Inspection of the promoter sequences of genes belonging to each group revealed distinct promoter architectures with respect to the location and number of putative CovR-binding sites. Electrophoretic mobility shift analysis demonstrated that recombinant CovR-D53E protein retains its ability to bind promoter DNA from both CovR-D53E-regulated and -nonregulated groups, implying that factors other than mere DNA binding are crucial for gene regulation. In fact, we found that CovR-D53E is incapable of dimerization, a process thought to be critical to OmpR/PhoB family regulator function. Thus, our global analysis of CovR-D53E indicates dimerization-dependent and dimerization-independent modes of CovR-mediated repression, thereby establishing distinct mechanisms by which this critical regulator coordinates virulence gene expression. IMPORTANCE Streptococcus pyogenes causes a wide variety of diseases, ranging from superficial skin and throat infections to life-threatening invasive infections. To establish these various disease manifestations, Streptococcus pyogenes requires tightly coordinated production of its virulence factor repertoire. Here, the response regulator CovR plays a crucial role. As an OmpR/PhoB family member, CovR is activated by phosphorylation on a conserved aspartate residue, leading to protein dimerization and subsequent binding to operator sites. Our transcriptome analysis using the monomeric phosphorylation mimic mutant CovR-D53E broadens this general notion by revealing dimerization-independent repression of a subset of CovR-regulated genes. Combined with promoter analyses, these data suggest distinct mechanisms of CovR transcriptional control, which allow for differential expression of virulence genes in response to environmental cues.

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Listeria monocytogenes σB Modulates PrfA-Mediated Virulence Factor Expression

Infection and Immunity ◽

10.1128/iai.01205-08 ◽

2009 ◽

Vol 77 (5) ◽

pp. 2113-2124 ◽

Cited By ~ 66

Author(s):

Juliane Ollinger ◽

Barbara Bowen ◽

Martin Wiedmann ◽

Kathryn J. Boor ◽

Teresa M. Bergholz

Keyword(s):

Gene Expression ◽

Listeria Monocytogenes ◽

Transcriptional Activation ◽

Virulence Gene ◽

Transcript Profiling ◽

Virulence Gene Expression ◽

Qrt Pcr ◽

Genome Wide

ABSTRACT Listeria monocytogenes σB and positive regulatory factor A (PrfA) are pleiotropic transcriptional regulators that coregulate a subset of virulence genes. A positive regulatory role for σB in prfA transcription has been well established; therefore, observations of increased virulence gene expression and hemolytic activity in a ΔsigB strain initially appeared paradoxical. To test the hypothesis that L. monocytogenes σB contributes to a regulatory network critical for appropriate repression as well as induction of virulence gene expression, genome-wide transcript profiling and follow-up quantitative reverse transcriptase PCR (qRT-PCR), reporter fusion, and phenotypic experiments were conducted using L. monocytogenes prfA*, prfA* ΔsigB, ΔprfA, and ΔprfA ΔsigB strains. Genome-wide transcript profiling and qRT-PCR showed that in the presence of active PrfA (PrfA*), σB is responsible for reduced expression of the PrfA regulon. σB-dependent modulation of PrfA regulon expression reduced the cytotoxic effects of a PrfA* strain in HepG2 cells, highlighting the functional importance of regulatory interactions between PrfA and σB. The emerging model of the role of σB in regulating overall PrfA activity includes a switch from transcriptional activation at the P2 prfA promoter (e.g., in extracellular bacteria when PrfA activity is low) to posttranscriptional downregulation of PrfA regulon expression (e.g., in intracellular bacteria when PrfA activity is high).

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The response regulator ResD modulates virulence gene expression in response to carbohydrates in Listeria monocytogenes

Molecular Microbiology ◽

10.1111/j.1365-2958.2006.05328.x ◽

2006 ◽

Vol 61 (6) ◽

pp. 1622-1635 ◽

Cited By ~ 46

Author(s):

Marianne H. Larsen ◽

Birgitte H. Kallipolitis ◽

Janne K. Christiansen ◽

John E. Olsen ◽

Hanne Ingmer

Keyword(s):

Gene Expression ◽

Listeria Monocytogenes ◽

Response Regulator ◽

Virulence Gene ◽

Virulence Gene Expression

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The Global Regulator CodY Regulates Toxin Gene Expression in Bacillus anthracis and Is Required for Full Virulence

Infection and Immunity ◽

10.1128/iai.00716-09 ◽

2009 ◽

Vol 77 (10) ◽

pp. 4437-4445 ◽

Cited By ~ 60

Author(s):

Willem van Schaik ◽

Alice Château ◽

Marie-Agnès Dillies ◽

Jean-Yves Coppée ◽

Abraham L. Sonenshein ◽

...

Keyword(s):

Gene Expression ◽

Bacillus Anthracis ◽

Mutant Strain ◽

Virulence Gene ◽

Etiologic Agent ◽

Toxin Gene ◽

Promoter Regions ◽

Virulence Gene Expression ◽

Toxin Gene Expression ◽

Virulence Regulator

ABSTRACT In gram-positive bacteria, CodY is an important regulator of genes whose expression changes upon nutrient limitation and acts as a repressor of virulence gene expression in some pathogenic species. Here, we report the role of CodY in Bacillus anthracis, the etiologic agent of anthrax. Disruption of codY completely abolished virulence in a toxinogenic, noncapsulated strain, indicating that the activity of CodY is required for full virulence of B. anthracis. Global transcriptome analysis of a codY mutant and the parental strain revealed extensive differences. These differences could reflect direct control for some genes, as suggested by the presence of CodY binding sequences in their promoter regions, or indirect effects via the CodY-dependent control of other regulatory proteins or metabolic rearrangements in the codY mutant strain. The differences included reduced expression of the anthrax toxin genes in the mutant strain, which was confirmed by lacZ reporter fusions and immunoblotting. The accumulation of the global virulence regulator AtxA protein was strongly reduced in the mutant strain. However, in agreement with the microarray data, expression of atxA, as measured using an atxA-lacZ transcriptional fusion and by assaying atxA mRNA, was not significantly affected in the codY mutant. An atxA-lacZ translational fusion was also unaffected. Overexpression of atxA restored toxin component synthesis in the codY mutant strain. These results suggest that CodY controls toxin gene expression by regulating AtxA accumulation posttranslationally.

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Listeria monocytogenes σB Regulates Stress Response and Virulence Functions

Journal of Bacteriology ◽

10.1128/jb.185.19.5722-5734.2003 ◽

2003 ◽

Vol 185 (19) ◽

pp. 5722-5734 ◽

Cited By ~ 246

Author(s):

Mark J. Kazmierczak ◽

Sharon C. Mithoe ◽

Kathryn J. Boor ◽

Martin Wiedmann

Keyword(s):

Gene Expression ◽

Listeria Monocytogenes ◽

Stress Response ◽

Consensus Sequence ◽

Virulence Gene ◽

Wild Type ◽

Promoter Regions ◽

Virulence Gene Expression ◽

Stress Response Genes ◽

In The Wild

ABSTRACT While the stress-responsive alternative sigma factor σB has been identified in different species of Bacillus, Listeria, and Staphylococcus, theσ B regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify σB-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidateσ B-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted σB-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and ΔsigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significantσ B-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting σB-dependent expression, 54 were preceded by a sequence resembling the σB promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the σB-dependent nature of a subset of eight selected promoter regions. Notably, theσ B-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, σB also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest thatσ B contributes to L. monocytogenes gene expression during infection.

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The Fatty Acid Regulator FadR Influences the Expression of the Virulence Cascade in the El Tor Biotype of Vibrio cholerae by Modulating the Levels of ToxT via Two Different Mechanisms

Journal of Bacteriology ◽

10.1128/jb.00762-16 ◽

2017 ◽

Vol 199 (7) ◽

Cited By ~ 8

Author(s):

Gabriela Kovacikova ◽

Wei Lin ◽

Ronald K. Taylor ◽

Karen Skorupski

Keyword(s):

Gene Expression ◽

Vibrio Cholerae ◽

Virulence Gene ◽

Enteric Bacteria ◽

Posttranslational Regulation ◽

Content Type ◽

Virulence Gene Expression ◽

Expression Of Genes ◽

El Tor ◽

Virulence Regulator

ABSTRACT FadR is a master regulator of fatty acid (FA) metabolism that coordinates the pathways of FA degradation and biosynthesis in enteric bacteria. We show here that a ΔfadR mutation in the El Tor biotype of Vibrio cholerae prevents the expression of the virulence cascade by influencing both the transcription and the posttranslational regulation of the master virulence regulator ToxT. FadR is a transcriptional regulator that represses the expression of genes involved in FA degradation, activates the expression of genes involved in unsaturated FA (UFA) biosynthesis, and also activates the expression of two operons involved in saturated FA (SFA) biosynthesis. Since FadR does not bind directly to the toxT promoter, we determined whether the regulation of any of its target genes indirectly influenced ToxT. This was accomplished by individually inserting a double point mutation into the FadR-binding site in the promoter of each target gene, thereby preventing their activation or repression. Although preventing FadR-mediated activation of fabA, which encodes the enzyme that carries out the first step in UFA biosynthesis, did not significantly influence either the transcription or the translation of ToxT, it reduced its levels and prevented virulence gene expression. In the mutant strain unable to carry out FadR-mediated activation of fabA, expressing fabA ectopically restored the levels of ToxT and virulence gene expression. Taken together, the results presented here indicate that V. cholerae FadR influences the virulence cascade in the El Tor biotype by modulating the levels of ToxT via two different mechanisms. IMPORTANCE Fatty acids (FAs) play important roles in membrane lipid homeostasis and energy metabolism in all organisms. In Vibrio cholerae, the causative agent of the acute intestinal disease cholera, they also influence virulence by binding into an N-terminal pocket of the master virulence regulator, ToxT, and modulating its activity. FadR is a transcription factor that coordinately controls the pathways of FA degradation and biosynthesis in enteric bacteria. This study identifies a new link between FA metabolism and virulence in the El Tor biotype by showing that FadR influences both the transcription and posttranslational regulation of the master virulence regulator ToxT by two distinct mechanisms.

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