c-di-AMP-Regulated K+ Importer KtrAB Affects Biofilm Formation, Stress Response, and SpeB Expression in Streptococcus pyogenes.

The second messenger cyclic di-AMP (c-di-AMP) controls biofilm formation, stress response, and virulence in Streptococcus pyogenes The deletion of the c-di-AMP synthase gene, dacA, results in pleiotropic effects including reduced expression of the secreted protease SpeB. Here, we report a role for K+ transport in c-di-AMP-mediated SpeB expression. The deletion of ktrB in the ΔdacA mutant restores SpeB expression. KtrB is a subunit of the K+ transport system KtrAB that forms a putative high-affinity K+ importer. KtrB forms a membrane K+ channel, and KtrA acts as a cytosolic gating protein that controls the transport capacity of the system by binding ligands including c-di-AMP. SpeB induction in the ΔdacA mutant by K+ specific ionophore treatment also supports the importance of cellular K+ balance in SpeB production. The ΔdacA ΔktrB double deletion mutant not only produces wild-type levels of SpeB but also partially or fully reverts the defective ΔdacA phenotypes of biofilm formation and stress responses, suggesting that many ΔdacA phenotypes are due to cellular K+ imbalance. However, the null pathogenicity of the ΔdacA mutant in a murine subcutaneous infection model is not restored by ktrB deletion, suggesting that c-di-AMP controls not only cellular K+ balance but also other metabolic and/or virulence pathways. The deletion of other putative K+ importer genes, kup and kimA, does not phenocopy the deletion of ktrB regarding SpeB induction in the ΔdacA mutant, suggesting that KtrAB is the primary K+ importer that is responsible for controlling cellular K+ levels under laboratory growth conditions.

Glutathione Synthesis Contributes to Virulence of Streptococcus agalactiae in a Murine Model of Sepsis.

Streptococcus agalactiae, a leading cause of sepsis and meningitis in neonates, utilizes multiple virulence factors to survive and thrive within the human host during an infection. Unique among the pathogenic streptococci, S. agalactiae uses a bifunctional enzyme encoded by a single gene (gshAB) to synthesize glutathione (GSH), a major antioxidant in most aerobic organisms. Since S. agalactiae can also import GSH, similar to all other pathogenic streptococcal species, the contribution of GSH synthesis to the pathogenesis of S. agalactiae disease is not known. In the present study, gshAB deletion mutants were generated in strains representing three of the most prevalent clinical serotypes of S. agalactiae and were compared against isogenic wild-type and gshAB knock-in strains. When cultured in vitro in a chemically defined medium under nonstress conditions, each mutant and its corresponding wild type had comparable growth rates, generation times, and growth yields. However, gshAB deletion mutants were found to be more sensitive than wild-type or gshAB knock-in strains to killing and growth inhibition by several different reactive oxygen species. Furthermore, deletion of gshAB in S. agalactiae strain COH1 significantly attenuated virulence compared to the wild-type or gshAB knock-in strains in a mouse model of sepsis. Taken together, these data establish that GSH is a virulence factor important for resistance to oxidative stress and that de novo GSH synthesis plays a crucial role in S. agalactiae pathogenesis and further suggest that the inhibition of GSH synthesis may provide an opportunity for the development of novel therapies targeting S. agalactiae disease.IMPORTANCE Approximately 10 to 30% of women are naturally and asymptomatically colonized by Streptococcus agalactiae However, transmission of S. agalactiae from mother to newborn during vaginal birth is a leading cause of neonatal meningitis. Although colonized mothers who are at risk for transmission to the newborn are treated with antibiotics prior to delivery, S. agalactiae is becoming increasingly resistant to current antibiotic therapies, and new treatments are needed. This research reveals a critical stress resistance pathway, glutathione synthesis, that is utilized by S. agalactiae and contributes to its pathogenesis. Understanding the role of this unique bifunctional glutathione synthesis enzyme in S. agalactiae during sepsis may help elucidate why S. agalactiae produces such an abundance of glutathione compared to other bacteria.

The Second Messenger c-di-AMP Regulates Diverse Cellular Pathways Involved in Stress Response, Biofilm Formation, Cell Wall Homeostasis, SpeB Expression, and Virulence in Streptococcus pyogenes.

Cyclic di-AMP (c-di-AMP) is a recently discovered second messenger in bacteria. The cellular level of c-di-AMP in Streptococcus pyogenes is predicted to be controlled by the synthase DacA and two putative phosphodiesterases, GdpP and Pde2. To investigate the role of c-di-AMP in S. pyogenes, we generated null mutants in each of these proteins by gene deletion. Unlike those in other Gram-positive pathogens such as Staphylococcus aureus and Listeria monocytogenes, DacA in S. pyogenes was not essential for growth in rich media. The DacA null mutant presented a growth defect that manifested through an increased lag time, produced no detectable biofilm, and displayed increased susceptibility toward environmental stressors such as high salt, low pH, reactive oxygen radicals, and cell wall-targeting antibiotics, suggesting that c-di-AMP plays significant roles in crucial cellular processes involved in stress management. The Pde2 null mutant exhibited a lower growth rate and increased biofilm formation, and interestingly, these phenotypes were distinct from those of the null mutant of GdpP, suggesting that Pde2 and GdpP play distinctive roles in c-di-AMP signaling. DacA and Pde2 were critical to the production of the virulence factor SpeB and to the overall virulence of S. pyogenes, as both DacA and Pde2 null mutants were highly attenuated in a mouse model of subcutaneous infection. Collectively, these results show that c-di-AMP is an important global regulator and is required for a proper response to stress and for virulence in S. pyogenes, suggesting that its signaling pathway could be an attractive antivirulence drug target against S. pyogenes infections.

Genetics of Group A Streptococci.

Streptococcus pyogenes (group A streptococcus) is remarkable in terms of the large number of diseases it can cause in humans and for the large number of streptococcal factors that have been identified as potential virulence determinants for these diseases. A challenge is to link the function of potential virulence factors to the pathogenesis of specific diseases. An exciting advance has been the development of sophisticated genetic systems for the construction of loss-of-function, conditional, hypomorphic, and gain-of-function mutations in targeted S. pyogenes genes that can be used to test specific hypotheses regarding these genes in pathogenesis. This will facilitate a mechanistic understanding of how a specific gene function contributes to the pathogenesis of each streptococcal disease. Since the first S. pyogenes genome was completed in 2001, hundreds of complete and draft genome sequences have been deposited. We now know that the average S. pyogenes genome is approximately 1.85 Mb and encodes ∼1,800 genes and that the function of most of those genes in pathogenesis remains to be elucidated. However, advances in the development of a variety of genetic tools for manipulation of the S. pyogenes genome now provide a platform for the interrogation of gene/phenotype relationships for individual S. pyogenes diseases, which may lead to the development of more sophisticated and targeted therapeutic interventions. This article presents an overview of these genetic tools, including the methods of genetic modification and their applications.

c-di-AMP: An Essential Molecule in the Signaling Pathways that Regulate the Viability and Virulence of Gram-Positive Bacteria.

Signal transduction pathways enable organisms to monitor their external environment and adjust gene regulation to appropriately modify their cellular processes. Second messenger nucleotides including cyclic adenosine monophosphate (c-AMP), cyclic guanosine monophosphate (c-GMP), cyclic di-guanosine monophosphate (c-di-GMP), and cyclic di-adenosine monophosphate (c-di-AMP) play key roles in many signal transduction pathways used by prokaryotes and/or eukaryotes. Among the various second messenger nucleotides molecules, c-di-AMP was discovered recently and has since been shown to be involved in cell growth, survival, and regulation of virulence, primarily within Gram-positive bacteria. The cellular level of c-di-AMP is maintained by a family of c-di-AMP synthesizing enzymes, diadenylate cyclases (DACs), and degradation enzymes, phosphodiesterases (PDEs). Genetic manipulation of DACs and PDEs have demonstrated that alteration of c-di-AMP levels impacts both growth and virulence of microorganisms. Unlike other second messenger molecules, c-di-AMP is essential for growth in several bacterial species as many basic cellular functions are regulated by c-di-AMP including cell wall maintenance, potassium ion homeostasis, DNA damage repair, etc. c-di-AMP follows a typical second messenger signaling pathway, beginning with binding to receptor molecules to subsequent regulation of downstream cellular processes. While c-di-AMP binds to specific proteins that regulate pathways in bacterial cells, c-di-AMP also binds to regulatory RNA molecules that control potassium ion channel expression in Bacillus subtilis. c-di-AMP signaling also occurs in eukaryotes, as bacterially produced c-di-AMP stimulates host immune responses during infection through binding of innate immune surveillance proteins. Due to its existence in diverse microorganisms, its involvement in crucial cellular activities, and its stimulating activity in host immune responses, c-di-AMP signaling pathway has become an attractive antimicrobial drug target and therefore has been the focus of intensive study in several important pathogens.

SpxA1 and SpxA2 Act Coordinately To Fine-Tune Stress Responses and Virulence in Streptococcus pyogenes.

SpxA is a unique transcriptional regulator highly conserved among members of the phylum Firmicutes that binds RNA polymerase and can act as an antiactivator. Why some Firmicutes members have two highly similar SpxA paralogs is not understood. Here, we show that the SpxA paralogs of the pathogen Streptococcus pyogenes, SpxA1 and SpxA2, act coordinately to regulate virulence by fine-tuning toxin expression and stress resistance. Construction and analysis of mutants revealed that SpxA1- mutants were defective for growth under aerobic conditions, while SpxA2- mutants had severely attenuated responses to multiple stresses, including thermal and oxidative stresses. SpxA1- mutants had enhanced resistance to the cationic antimicrobial molecule polymyxin B, while SpxA2- mutants were more sensitive. In a murine model of soft tissue infection, a SpxA1- mutant was highly attenuated. In contrast, the highly stress-sensitive SpxA2- mutant was hypervirulent, exhibiting more extensive tissue damage and a greater bacterial burden than the wild-type strain. SpxA1- attenuation was associated with reduced expression of several toxins, including the SpeB cysteine protease. In contrast, SpxA2- hypervirulence correlated with toxin overexpression and could be suppressed to wild-type levels by deletion of speB These data show that SpxA1 and SpxA2 have opposing roles in virulence and stress resistance, suggesting that they act coordinately to fine-tune toxin expression in response to stress. SpxA2- hypervirulence also shows that stress resistance is not always essential for S. pyogenes pathogenesis in soft tissue.IMPORTANCE For many pathogens, it is generally assumed that stress resistance is essential for pathogenesis. For Streptococcus pyogenes, environmental stress is also used as a signal to alter toxin expression. The amount of stress likely informs the bacterium of the strength of the host's defense response, allowing it to adjust its toxin expression to produce the ideal amount of tissue damage, balancing between too little damage, which will result in its elimination, and too much damage, which will debilitate the host. Here we identify components of a genetic circuit involved in stress resistance and toxin expression that has a fine-tuning function in tissue damage. The circuit consists of two versions of the protein SpxA that regulate transcription and are highly similar but have opposing effects on the severity of soft tissue damage. These results will help us understand how virulence is fine-tuned in other pathogens that have two SpxA proteins.

Complete Genome Sequences of emm6 Streptococcus pyogenes JRS4 and Parental Strain D471.

We report the complete genome assemblies of the group A Streptococcus pyogenes serotype emm6 strain D471 and its streptomycin-resistant derivative JRS4. Both of these well-studied laboratory strains have been extensively characterized over the past three decades and have been instrumental in the discovery of multiple aspects of streptococcal pathogenesis.

Streptococcus pyogenes polymyxin B-resistant mutants display enhanced ExPortal integrity.

The ExPortal protein secretion organelle in Streptococcus pyogenes is an anionic phospholipid-containing membrane microdomain enriched in Sec translocons and postsecretion protein biogenesis factors. Polymyxin B binds to and disrupts ExPortal integrity, resulting in defective secretion of several toxins. To gain insight into factors that influence ExPortal organization, a genetic screen was conducted to select for spontaneous polymyxin B-resistant mutants displaying enhanced ExPortal integrity. Whole-genome resequencing of 25 resistant mutants revealed from one to four mutations per mutant genome clustered primarily within a core set of 10 gene groups. Construction of mutants with individual deletions or insertions demonstrated that 7 core genes confer resistance and enhanced ExPortal integrity through loss of function, while 3 were likely due to gain of function and/or combinatorial effects. Core resistance genes include a transcriptional regulator of lipid biosynthesis, several genes involved in nutrient acquisition, and a variety of genes involved in stress responses. Two members of the latter class also function as novel regulators of the secreted SpeB cysteine protease. Analysis of the most frequently isolated mutation, a single nucleotide deletion in a track of 9 consecutive adenine residues in pstS, encoding a component of a high-affinity Pi transporter, suggests that this sequence functions as a molecular switch to facilitate stress adaptation. Together, these data suggest the existence of a membrane stress response that promotes enhanced ExPortal integrity and resistance to cationic antimicrobial peptides.

An association between peptidoglycan synthesis and organization of the Streptococcus pyogenes ExPortal.

UNLABELLED: The ExPortal of Streptococcus pyogenes is a focal microdomain of the cytoplasmic membrane that clusters the translocons of the general secretory pathway with accessory factors to facilitate the maturation of secreted polypeptides. While it is known that the ExPortal is enriched in anionic lipids, the mechanisms that organize the ExPortal are poorly understood. In the present study, we examined the role of the cell wall in organizing and maintaining the ExPortal. Removal of the cell wall resulted in a loss of ExPortal focal integrity accompanied by the circumferential redistribution of ExPortal lipid and protein components. A similar loss occurred upon treatment with gallidermin, a nonpermeabilizing lantibiotic that targets the lipid II precursor of peptidoglycan synthesis, and this treatment disrupted the secretion of several ExPortal substrates. Furthermore, several enzymes involved in the membrane-associated steps of lipid II synthesis, including MraY and MurN, were found to localize to a single discrete focus in the membrane that was coincident with the focal location of the secretory translocons and the anionic lipid microdomain. These data suggest that the ExPortal is associated with the site of peptidoglycan precursor synthesis and that peptidoglycan biogenesis influences ExPortal organization. These data add to an emerging literature indicating that cell wall biogenesis, cell division, and protein secretion are spatially coorganized processes. IMPORTANCE: Since Gram-positive bacteria lack a periplasmic space, they lack a protected compartment to spatially coordinate interaction between newly secreted proteins and the factors required to process them. This represents a significant problem for pathogens that depend on the secretion of toxins and cell wall-associated adhesins to cause disease. Streptococci solve this dilemma by restricting secretion and processing factors to a defined region of the membrane. However, the mechanisms that promote restriction are not understood. In this study, we show that restriction of these factors in the pathogen Streptococcus pyogenes is intimately linked with the presence of the cell wall and its synthesis. Furthermore, several cell wall synthesis proteins are also restricted to the site of protein secretion. This study contributes to our understanding of how the Gram-positive cell is organized to coordinate protein secretion and biogenesis with cell wall synthesis and to the ongoing development of antibiotics that target these processes.

Complete Genome Sequence of emm Type 14 Streptococcus pyogenes Strain HSC5.

Streptococcus pyogenes causes a greater diversity of human disease than any other bacterial pathogen. Here, we present the complete genome sequence of the emm type 14 S. pyogenes strain HSC5. This strain is a robust producer of the cysteine protease SpeB and is capable of producing infection in several different animal models.

Analysis of polymorphic residues reveals distinct enzymatic and cytotoxic activities of the Streptococcus pyogenes NAD+ glycohydrolase.

The Streptococcus pyogenes NAD(+) glycohydrolase (SPN) is secreted from the bacterial cell and translocated into the host cell cytosol where it contributes to cell death. Recent studies suggest that SPN is evolving and has diverged into NAD(+) glycohydrolase-inactive variants that correlate with tissue tropism. However, the role of SPN in both cytotoxicity and niche selection are unknown. To gain insight into the forces driving the adaptation of SPN, a detailed comparison of representative glycohydrolase activity-proficient and -deficient variants was conducted. Of a total 454 amino acids, the activity-deficient variants differed at only nine highly conserved positions. Exchanging residues between variants revealed that no one single residue could account for the inability of the deficient variants to cleave the glycosidic bond of ß-NAD(+) into nicotinamide and ADP-ribose; rather, reciprocal changes at 3 specific residues were required to both abolish activity of the proficient version and restore full activity to the deficient variant. Changing any combination of 1 or 2 residues resulted in intermediate activity. However, a change to any 1 residue resulted in a significant decrease in enzyme efficiency. A similar pattern involving multiple residues was observed for comparison with a second highly conserved activity-deficient variant class. Remarkably, despite differences in glycohydrolase activity, all versions of SPN were equally cytotoxic to cultured epithelial cells. These data indicate that the glycohydrolase activity of SPN may not be the only contribution the toxin has to the pathogenesis of S. pyogenes and that both versions of SPN play an important role during infection.

The metal ion-dependent adhesion site motif of the Enterococcus faecalis EbpA pilin mediates pilus function in catheter-associated urinary tract infection.

UNLABELLED: Though the bacterial opportunist Enterococcus faecalis causes a myriad of hospital-acquired infections (HAIs), including catheter-associated urinary tract infections (CAUTIs), little is known about the virulence mechanisms that it employs. However, the endocarditis- and biofilm-associated pilus (Ebp), a member of the sortase-assembled pilus family, was shown to play a role in a mouse model of E. faecalis ascending UTI. The Ebp pilus comprises the major EbpC shaft subunit and the EbpA and EbpB minor subunits. We investigated the biogenesis and function of Ebp pili in an experimental model of CAUTI using a panel of chromosomal pilin deletion mutants. A nonpiliated pilus knockout mutant (EbpABC(-) strain) was severely attenuated compared to its isogenic parent OG1RF in experimental CAUTI. In contrast, a nonpiliated ebpC deletion mutant (EbpC(-) strain) behaved similarly to OG1RF in vivo because it expressed EbpA and EbpB. Deletion of the minor pilin gene ebpA or ebpB perturbed pilus biogenesis and led to defects in experimental CAUTI. We discovered that the function of Ebp pili in vivo depended on a predicted metal ion-dependent adhesion site (MIDAS) motif in EbpA's von Willebrand factor A domain, a common protein domain among the tip subunits of sortase-assembled pili. Thus, this study identified the Ebp pilus as a virulence factor in E. faecalis CAUTI and also defined the molecular basis of this function, critical knowledge for the rational development of targeted therapeutics. IMPORTANCE: Catheter-associated urinary tract infections (CAUTIs), one of the most common hospital-acquired infections (HAIs), present considerable treatment challenges for physicians. Inherently resistant to several classes of antibiotics and with a propensity to acquire vancomycin resistance, enterococci are particularly worrisome etiologic agents of CAUTI. A detailed understanding of the molecular basis of Enterococcus faecalis pathogenesis in CAUTI is necessary for the development of preventative and therapeutic strategies. Our results elucidated the importance of the E. faecalis Ebp pilus and its subunits for enterococcal virulence in a mouse model of CAUTI. We further showed that the metal ion-dependent adhesion site (MIDAS) motif in EbpA is necessary for Ebp function in vivo. As this motif occurs in other sortase-assembled pili, our results have implications for the molecular basis of virulence not only in E. faecalis CAUTI but also in additional infections caused by enterococci and other Gram-positive pathogens.

Listeria monocytogenes CtaP is a multifunctional cysteine transport-associated protein required for bacterial pathogenesis.

The bacterial pathogen Listeria monocytogenes survives under a myriad of conditions in the outside environment and within the human host where infections can result in severe disease. Bacterial life within the host requires the expression of genes with roles in nutrient acquisition as well as the biosynthesis of bacterial products required to support intracellular growth. A gene product identified as the substrate-binding component of a novel oligopeptide transport system (encoded by lmo0135) was recently shown to be required for L. monocytogenes virulence. Here we demonstrate that lmo0135 encodes a multifunctional protein that is associated with cysteine transport, acid resistance, bacterial membrane integrity and adherence to host cells. The lmo0135 gene product (designated CtaP, for cysteine transport associated protein) was required for bacterial growth in the presence of low concentrations of cysteine in vitro, but was not required for bacterial replication within the host cytosol. Loss of CtaP increased membrane permeability and acid sensitivity, and reduced bacterial adherence to host cells. ctaP deletion mutants were severely attenuated following intragastric and intravenous inoculation of mice. Taken together, the data presented indicate that CtaP contributes to multiple facets of L. monocytogenes physiology, growth and survival both inside and outside of animal cells.

Listeria monocytogenes - from saprophyte to intracellular pathogen.

Listeria monocytogenes is a bacterium that lives in the soil as a saprophyte but is capable of making the transition into a pathogen following its ingestion by susceptible humans or animals. Recent studies suggest that L. monocytogenes mediates its saprophyte-to-cytosolic-parasite transition through the careful modulation of the activity of a virulence regulatory protein known as PrfA, using a range of environmental cues that include available carbon sources. In this Progress article we describe the regulation of PrfA and its role in the L. monocytogenes transition from the saprophytic stage to the virulent intracellular stage.

The posttranslocation chaperone PrsA2 contributes to multiple facets of Listeria monocytogenes pathogenesis.

Listeria monocytogenes is an intracellular bacterial pathogen whose virulence depends on the regulated expression of numerous secreted bacterial factors. As for other gram-positive bacteria, many proteins secreted by L. monocytogenes are translocated across the bacterial membrane in an unfolded state to the compartment existing between the membrane and the cell wall. This compartment presents a challenging environment for protein folding due to its high density of negative charge, high concentrations of cations, and low pH. We recently identified PrsA2 as a gene product required for L. monocytogenes virulence. PrsA2 was identified based on its increased secretion by strains containing a mutationally activated form of prfA, the key regulator of L. monocytogenes virulence gene expression. The prsA2 gene product is one of at least two predicted peptidyl-prolyl cis/trans-isomerases encoded by L. monocytogenes; these proteins function as posttranslocation protein chaperones and/or foldases. In this study, we demonstrate that PrsA2 plays a unique and important role in L. monocytogenes pathogenesis by promoting the activity and stability of at least two critical secreted virulence factors: listeriolysin O (LLO) and a broad-specificity phospholipase. Loss of PrsA2 activity severely attenuated virulence in mice and impaired bacterial cell-to-cell spread in host cells. In contrast, mutants lacking prsA1 resembled wild-type bacteria with respect to intracellular growth and cell-to-cell spread as well as virulence in mice. PrsA2 is thus distinct from PrsA1 in its unique requirement for the stability and full activity of L. monocytogenes-secreted factors that contribute to host infection.

Functional impact of mutational activation on the Listeria monocytogenes central virulence regulator PrfA.

The transcriptional activator PrfA is required for the expression of virulence factors necessary for Listeria monocytogenes pathogenesis. PrfA is believed to become activated following L. monocytogenes entry into the cytosol of infected host cells, resulting in the induction of target genes whose products are required for bacterial intracellular growth and cell-to-cell spread. Several mutations have been identified that appear to lock PrfA into its highly activated cytosolic form (known as prfA* mutations). In this study PrfA and five PrfA* mutant proteins exhibiting differing degrees of activity were purified and analysed to define the influences of the mutations on distinct aspects of PrfA activity. Based on limited proteolytic digestion, conformational changes were detected for the PrfA* mutant proteins in comparison to wild-type PrfA. For all but one mutant (PrfA Y63C), the DNA binding affinity as measured by electophoretic mobility shift assay appeared to directly correlate with levels of PrfA mutational activation, such that the high-activity mutants exhibited the largest increases in DNA binding affinity and moderately activated mutants exhibited more moderate increases. Surprisingly, the ability of PrfA and PrfA* mutants to form dimers in solution appeared to inversely correlate with levels of PrfA-dependent gene expression. Based on comparisons of protein activity and structural similarities with PrfA family members Crp and CooA, the prfA* mutations modify distinct aspects of PrfA activity that include DNA binding and protein-protein interactions.

A novel prfA mutation that promotes Listeria monocytogenes cytosol entry but reduces bacterial spread and cytotoxicity.

Listeria monocytogenes is an environmental bacterium that becomes a pathogen following ingestion by a mammalian host. The transition from environmental organism to pathogen requires significant changes in gene expression, including the increased expression of gene products that contribute to bacterial growth within host cells. PrfA is an L. monocytogenes transcriptional regulator that becomes activated upon bacterial entry into mammalian cells and induces the expression of gene products required for virulence. How PrfA activation occurs is not known, however several mutations have been identified that increase PrfA activity in strains grown in vitro (prfA mutations). Here we describe a novel prfA mutation that enhances extracellular PrfA-dependent gene expression but in contrast to prfA mutants inhibits the cytosol-mediated induction of virulence genes. prfA Y154C strains entered cells and escaped from phagosomes with an efficiency similar to wild type bacteria, however the mutation prevented efficient L. monocytogenes actin polymerization and reduced spread of bacteria to adjacent cells. The prfA Y154C mutation severely attenuated bacterial virulence in mice but the mutant strains did generate target antigen specific CD8(+) effector cells. Interestingly, the prfA Y154C mutant was significantly less cytotoxic for host cells than wild type L. monocytogenes. The prfA Y154C mutant strain may therefore represent a novel attenuated strain of L. monocytogenes for antigen delivery with reduced host cell toxicity.

Identification of novel Listeria monocytogenes secreted virulence factors following mutational activation of the central virulence regulator, PrfA.

Upon bacterial entry into the cytosol of infected mammalian host cells, the central virulence regulator PrfA of Listeria monocytogenes becomes activated and induces the expression of numerous factors which contribute to bacterial pathogenesis. The mechanism or signal by which PrfA becomes activated during the course of infection has not yet been determined; however, several amino acid substitutions within PrfA (known as PrfA* mutations) that appear to lock the protein into a constitutively activated state have been identified. In this study, the PrfA activation statuses of several L. monocytogenes mutant strains were subjected to direct isogenic comparison and the mutant with the highest activity, the prfA(L140F) mutant, was identified. The prfA(L140F) strain was subsequently used as a tool to identify gene products secreted as a result of PrfA activation. By use of two-dimensional gel electrophoresis followed by liquid chromatography-electrospray ionization-tandem mass spectroscopy analyses, 15 proteins were identified as up-regulated in the prfA(L140F) secretome, while the secretion of two proteins was found to be reduced. Although some of the proteins identified were known to be subject to direct regulation by PrfA, the majority have not previously been associated with PrfA regulation and their expression or secretion may be influenced indirectly by a PrfA-dependent regulatory pathway. Plasmid insertion inactivation of the genes encoding four novel secreted products indicated that three of the four have significant roles in L. monocytogenes virulence. The use of mutationally activated prfA alleles therefore provides a useful approach towards identifying gene products that contribute to L. monocytogenes pathogenesis.