Epigenetic Control of Virulence Gene Expression in Pseudomonas aeruginosa by a LysR-Type Transcription Regulator

Phenotypic variation within an isogenic bacterial population is thought to ensure the survival of a subset of cells in adverse conditions. The opportunistic pathogen Pseudomonas aeruginosa variably expresses several phenotypes, including antibiotic resistance, biofilm formation, and the production of CupA fimbriae. Here we describe a previously unidentified bistable switch in P. aeruginosa. This switch controls the expression of a diverse set of genes, including aprA, which encodes the secreted virulence factor alkaline protease. We present evidence that bistable expression of PA2432, herein named bexR (bistable expression regulator), which encodes a LysR-type transcription regulator, controls this switch. In particular, using DNA microarrays, quantitative RT–PCR analysis, chromatin immunoprecipitation, and reporter gene fusions, we identify genes directly under the control of BexR and show that these genes are bistably expressed. Furthermore, we show that bexR is itself bistably expressed and positively autoregulated. Finally, using single-cell analyses of a GFP reporter fusion, we present evidence that positive autoregulation of bexR is necessary for bistable expression of the BexR regulon. Our findings suggest that a positive feedback loop involving a LysR-type transcription regulator serves as the basis for an epigenetic switch that controls virulence gene expression in P. aeruginosa

Identification of Group A Streptococcus Genes Directly Regulated by CsrRS and Novel Intermediate Regulators

Adaptation of group A Streptococcus (GAS) to its human host is mediated by two-component systems that transduce external stimuli to regulate bacterial physiology. Among such systems, CsrRS (also known as CovRS) is the most extensively characterized for its role in regulating ∼10% of the GAS genome, including several virulence genes. Here, we show that extracellular magnesium and the human antimicrobial peptide LL-37 have opposing effects on the phosphorylation of the response regulator CsrR by the receptor kinase CsrS. Genetic inactivation of CsrS phosphatase or kinase activity, respectively, had similar but more pronounced effects on CsrR phosphorylation compared to growth in magnesium or LL-37. These changes in CsrR phosphorylation were correlated with the repression or activation of CsrR-regulated genes as assessed by NanoString analysis. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) revealed CsrR occupancy at CsrRS-regulated promoters and lower-affinity associations at many other locations on the GAS chromosome. Because ChIP-seq did not detect CsrR occupancy at promoters associated with some CsrR-regulated genes, we investigated whether these genes might be controlled indirectly by intermediate regulators whose expression is modulated by CsrR. Transcriptional profiling of mutant strains deficient in the expression of either of two previously uncharacterized transcription regulators in the CsrR regulon indicated that one or both proteins participated in the regulation of 22 of the 42 CsrR-regulated promoters for which no CsrR association was detected by ChIP-seq. Taken together, these results illuminate CsrRS-mediated regulation of GAS gene expression through modulation of CsrR phosphorylation, CsrR association with regulated promoters, and the control of intermediate transcription regulators. Importance: Group A Streptococcus (GAS) is an important public health threat as a cause of sore throat, skin infections, life-threatening invasive infections, and the postinfectious complications of acute rheumatic fever, a leading cause of acquired heart disease. This work characterizes CsrRS, a GAS system for the detection of environmental signals that enables adaptation of the bacteria for survival in the human throat by regulating the production of products that allow the bacteria to resist clearance by the human immune system. CsrRS consists of two proteins: CsrS, which is on the bacterial surface to detect specific stimuli, and CsrR, which receives signals from CsrS and, in response, represses or activates the expression of genes coding for proteins that enhance bacterial survival. Some of the genes regulated by CsrR encode proteins that are themselves regulators of gene expression, thereby creating a regulatory cascade

Improved transformation efficiency of group A Streptococcus by inactivation of a type I restriction modification system

Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in electrotransformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes

Twin RNA Polymerase–Associated Proteins Control Virulence Gene Expression in Francisella tularensis

The MglA protein is the only known regulator of virulence gene expression in Francisella tularensis, yet it is unclear how it functions. F. tularensis also contains an MglA-like protein called SspA. Here, we show that MglA and SspA cooperate with one another to control virulence gene expression in F. tularensis. Using a directed proteomic approach, we show that both MglA and SspA associate with RNA polymerase (RNAP) in F. tularensis, and that SspA is required for MglA to associate with RNAP. Furthermore, bacterial two-hybrid and biochemical assays indicate that MglA and SspA interact with one another directly. Finally, through genome-wide expression analyses, we demonstrate that MglA and SspA regulate the same set of genes. Our results suggest that a complex involving both MglA and SspA associates with RNAP to positively control virulence gene expression in F. tularensis. The F. tularensis genome is unusual in that it contains two genes encoding different α subunits of RNAP, and we show here that these two α subunits are incorporated into RNAP. Thus, as well as identifying SspA as a second critical regulator of virulence gene expression in F. tularensis, our findings provide a framework for understanding the mechanistic basis for virulence gene control in a bacterium whose transcription apparatus is unique

Deep sequencing analyses expands the Pseudomonas aeruginosa AmpR regulon to include small RNA-mediated regulation of iron acquisition, heat shock and oxidative stress response

Pathogenicity of Pseudomonas aeruginosa, a major cause of many acute and chronic human infections, is determined by tightly regulated expression of multiple virulence factors. Quorum sensing (QS) controls expression of many of these pathogenic determinants. Previous microarray studies have shown that the AmpC β-lactamase regulator AmpR, a member of the LysR family of transcription factors, also controls non-β-lactam resistance and multiple virulence mechanisms. Using RNA-Seq and complementary assays, this study further expands the AmpR regulon to include diverse processes such as oxidative stress, heat shock and iron uptake. Importantly, AmpR affects many of these phenotypes, in part, by regulating expression of non-coding RNAs such as rgP32, asRgsA, asPrrF1 and rgRsmZ. AmpR positively regulates expression of the major QS regulators LasR, RhlR and MvfR, and genes of the Pseudomonas quinolone system. Chromatin immunoprecipitation (ChIP)-Seq and ChIP–quantitative real-time polymerase chain reaction studies show that AmpR binds to the ampC promoter both in the absence and presence of β-lactams. In addition, AmpR directly binds the lasR promoter, encoding the QS master regulator. Comparison of the AmpR-binding sequences from the transcriptome and ChIP-Seq analyses identified an AT-rich consensus-binding motif. This study further attests to the role of AmpR in regulating virulence and physiological processes in P. aeruginosa

Twin RNA Polymerase–associated Proteins Control Virulence Gene Expression in Francisella tularensis

The MglA protein is the only known regulator of virulence gene expression in Francisella tularensis, yet it is unclear how it functions. F. tularensis also contains an MglA-like protein called SspA. Here, we show that MglA and SspA cooperate with one another to control virulence gene expression in F. tularensis. Using a directed proteomic approach, we show that both MglA and SspA associate with RNA polymerase (RNAP) in F. tularensis, and that SspA is required for MglA to associate with RNAP. Furthermore, bacterial two-hybrid and biochemical assays indicate that MglA and SspA interact with one another directly. Finally, through genome-wide expression analyses, we demonstrate that MglA and SspA regulate the same set of genes. Our results suggest that a complex involving both MglA and SspA associates with RNAP to positively control virulence gene expression in F. tularensis. The F. tularensis genome is unusual in that it contains two genes encoding different α subunits of RNAP, and we show here that these two α subunits are incorporated into RNAP. Thus, as well as identifying SspA as a second critical regulator of virulence gene expression in F. tularensis, our findings provide a framework for understanding the mechanistic basis for virulence gene control in a bacterium whose transcription apparatus is unique

Control of a Programmed Cell Death Pathway in Pseudomonas aeruginosa by an Antiterminator

In Pseudomonas aeruginosa the alp system encodes a programmed cell death pathway that is switched on in a subset of cells in response to DNA damage and is linked to the virulence of the organism. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified target locus, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream. AlpA thus functions in a mechanistically unusual manner to control the expression of virulence genes in this opportunistic pathogen

A Self-Lysis Pathway that Enhances the Virulence of a Pathogenic Bacterium

In mammalian cells, programmed cell death (PCD) plays important roles in development, in the removal of damaged cells, and in fighting bacterial infections. Although widespread among multicellular organisms, there are relatively few documented instances of PCD in bacteria. Here we describe a potential PCD pathway in Pseudomonas aeruginosa that enhances the ability of the bacterium to cause disease in a lung infection model. Activation of the system can occur in a subset of cells in response to DNA damage through cleavage of an essential transcription regulator we call AlpR. Cleavage of AlpR triggers a cell lysis program through de-repression of the alpA gene, which encodes a positive regulator that activates expression of the alpBCDE lysis cassette. Although this is lethal to the individual cell in which it occurs, we find it benefits the population as a whole during infection of a mammalian host. Thus, host and pathogen each may use PCD as a survival-promoting strategy. We suggest that activation of the Alp cell lysis pathway is a disease-enhancing response to bacterial DNA damage inflicted by the host immune system

Anr and Its Activation by PlcH Activity in Pseudomonas aeruginosa Host Colonization and Virulence

Pseudomonas aeruginosa hemolytic phospholipase C (PlcH) degrades phosphatidylcholine (PC), an abundant lipid in cell membranes and lung surfactant. A ΔplcHR mutant, known to be defective in virulence in animal models, was less able to colonize epithelial cell monolayers and was defective in biofilm formation on plastic when grown in lung surfactant. Microarray analyses found that strains defective in PlcH production had lower levels of Anr-regulated transcripts than the wild type. PC degradation stimulated the Anr regulon in an Anr-dependent manner under conditions where Anr activity was submaximal because of the presence of oxygen. Two PC catabolites, choline and glycine betaine (GB), were sufficient to stimulate Anr activity, and their catabolism was required for Anr activation. The addition of choline or GB to glucose-containing medium did not alter Anr protein levels, growth rates, or respiratory activity, and Anr activation could not be attributed to the osmoprotectant functions of GB. The Δanr mutant was defective in virulence in a mouse pneumonia model. Several lines of evidence indicate that Anr is important for the colonization of biotic and abiotic surfaces in both P. aeruginosa PAO1 and PA14 and that increases in Anr activity resulted in enhanced biofilm formation. Our data suggest that PlcH activity promotes Anr activity in oxic environments and that Anr activity contributes to virulence, even in the acute infection phase, where low oxygen tensions are not expected. This finding highlights the relationships among in vivo bacterial metabolism, the activity of the oxygen-sensitive regulator Anr, and virulence

Tn-Seq reveals hidden complexity in the utilization of host-derived glutathione in \u3cem\u3eFrancisella tularensis\u3c/em\u3e

Host-derived glutathione (GSH) is an essential source of cysteine for the intracellular pathogen Francisella tularensis. In a comprehensive transposon insertion sequencing screen, we identified several F. tularensis genes that play central and previously unappreciated roles in the utilization of GSH during the growth of the bacterium in macrophages. We show that one of these, a gene we named dptA, encodes a proton-dependent oligopeptide transporter that enables growth of the organism on the dipeptide Cys-Gly, a key breakdown product of GSH generated by the enzyme γ-glutamyltranspeptidase (GGT). Although GGT was thought to be the principal enzyme involved in GSH breakdown in F. tularensis, our screen identified a second enzyme, referred to as ChaC, that is also involved in the utilization of exogenous GSH. However, unlike GGT and DptA, we show that the importance of ChaC in supporting intramacrophage growth extends beyond cysteine acquisition. Taken together, our findings provide a compendium of F. tularensis genes required for intracellular growth and identify new players in the metabolism of GSH that could be attractive targets for therapeutic intervention