Streptococcus pyogenes TrxSR Two-Component System Regulates Biofilm Production in Acidic Environments

Bacteria form biofilms for their protection against environmental stress and produce virulence factors within the biofilm. Biofilm formation in acidified environments is regulated by a two-component system, as shown by studies on isogenic mutants of the sensor protein of the two-component regulatory system in Streptococcus pyogenes . In this study, we found that the LiaS histidine kinase sensor mediates biofilm production and pilus expression in an acidified environment through glucose fermentation. The liaS isogenic mutant produced biofilms in a culture acidified by hydrochloric acid but not glucose, suggesting that the acidified environment is sensed by another protein. In addition, the trxS isogenic mutant could not produce biofilms or activate the mga promoter in an acidified environment. Mass spectrometry analysis showed that TrxS regulates M Protein, consistent with the transcriptional regulation of emm , which encodes M protein. Our results demonstrate that biofilm production during environmental acidification is directly under the control of TrxS.

Staphylococcus aureus secreted lipases do not inhibit innate immune killing mechanisms

Background: Staphylococcus aureus causes an array of diseases in both humans and livestock. Pathogenesis is mediated by a plethora of proteins secreted by S. aureus, many of which remain incompletely characterised. For example, S. aureus abundantly secretes two isoforms of the enzyme lipase into the extracellular milieu, where they scavenge upon polymeric triglycerides. It has previously been suggested that lipases may interfere with the function of innate immune cells, such as macrophages and neutrophils, but the impact of lipases on phagocytic killing mechanisms remains unknown. Methods: We employed the epidemic S. aureus clone USA300 strain LAC and its lipase deficient isogenic mutant, along with recombinant lipase proteins, in in vitro experimental infection assays. To determine if lipases can inhibit innate immune killing mechanisms, the bactericidal activity of whole blood, human neutrophils, and macrophages was analysed. In addition, gentamycin protection assays were carried out to examine the influence of lipases on S. aureus innate immune cell escape. Results: There were no differences in the survival of S. aureus USA300 LAC wild type and its lipase-deficient isogenic mutant after incubation with human whole blood or neutrophils. Furthermore, there was no detectable lipase-dependent effect on phagocytosis, intracellular survival, or escape from both human primary and immortalised cell line macrophages, even upon supplementation with exogenous recombinant lipases. Conclusions: S. aureus lipases do not inhibit bacterial killing mechanisms of human macrophages, neutrophils, or whole blood. These findings broaden our understanding of the interaction of S. aureus with the innate immune system.

Functional insights into the high-molecular-mass penicillin-binding proteins of Streptococcus agalactiae revealed by gene deletion and transposon mutagenesis analysis

High-molecular-mass penicillin-binding proteins (PBPs) are enzymes that catalyze the biosynthesis of bacterial cell wall peptidoglycan. The Gram-positive bacterial pathogen Streptococcus agalactiae (group B streptococcus , or GBS) produces five high-molecular-mass PBPs, namely, PBP1A, PBP1B, PBP2A, PBP2B, and PBP2X. Among these, only PBP2X is essential for cell viability, whereas the other four PBPs are individually dispensable. The biological function of the four non-essential PBPs is poorly characterized in GBS. We deleted the pbp1a , pbp1b , pbp2a , and pbp2b genes individually from a genetically well-characterized serotype V GBS strain, and studied the phenotypes of the four isogenic mutant strains. Compared to the wild-type parental strain (i) none of the pbp isogenic mutant strains had a significant growth defect in THY rich medium, (ii) isogenic mutant strains Δ pbp1a and Δ pbp1b had significantly increased susceptibility to penicillin and ampicillin, and (iii) isogenic mutant strains Δ pbp1a and Δ pbp2b had significantly longer chain lengths. Using saturated transposon mutagenesis and transposon insertion site sequencing, we determined genes essential for the viability of wild-type GBS strain and each of the four isogenic pbp deletion mutant strains in THY rich medium. The pbp1a gene is essential for cell viability in the pbp2b deletion background. Reciprocally, pbp2b is essential in the pbp1a deletion background. Moreover, the gene encoding RodA, a peptidoglycan polymerase that works in conjunction with PBP2B, is also essential in the pbp1a deletion background. Together, our results suggest functional overlap between PBP1A and PBP2B-RodA complex in GBS cell wall peptidoglycan biosynthesis. IMPORTANCE High-molecular-mass penicillin-binding proteins (HMM-PBPs) are enzymes required for bacterial cell-wall biosynthesis. Bacterial pathogen group B streptococcus (GBS) produces five distinct HMM-PBPs. The biological functions of these proteins are not well characterized in GBS. In this study, we performed a comprehensive deletion analysis of genes encoding HMM-PBPs in GBS. We found that deleting certain PBP-encoding genes altered bacterial susceptibility to beta-lactam antibiotics, cell morphology, and the essentiality of other enzymes involved in cell-wall peptidoglycan synthesis. The results of our study shed new light on the biological functions of PBPs in GBS.

NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA. IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.

Clostridium difficile exploits a host metabolite produced during toxin-mediated infection

Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile (Cd) is responsible for a toxin-mediated colitis that causes 15,000 deaths in the U.S. yearly1, yet the molecular mechanisms by which Cd benefits from toxin-induced colitis remain understudied. Up to 21% of healthy adults are asymptomatic carriers of toxigenic Cd2, indicating that Cd can persist as part of a healthy microbiota; antibiotic-induced perturbation of the gut ecosystem is associated with transition to toxin-mediated disease. To understand how Cd metabolism adapts from a healthy gut to the inflamed conditions its toxins induce, we used RNA-seq to define the metabolic state of wild-type Cd versus an isogenic mutant lacking toxins in a mouse model. Combining bacterial and mouse genetics, we demonstrate that Cd utilizes sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation, including during Cd toxin-mediated disease. This work highlights a mechanism by which Cd can utilize a host-derived nutrient generated during toxin-induced disease by an enzyme not previously associated with infection.

Influence of streptococcal arginine deiminase on the leukocyte infiltration in murine air pouch model

Numerous pathogens express arginine deiminase, an enzyme that catalyzes the hydrolysis of L-arginine in a chain of biochemical reactions aimed at the synthesis of ATP in bacterial cells. L-arginine is a semi-essential, proteinogenic amino acid that plays an important role in regulating the functions of the immune system cells in mammals. Depletion of L-arginine may cause a weakening of the immune reaction. In order to improve the conditions of dissemination, many pathogens use a strategy of L-arginine depletion in the microenvironment of host cells. Bacterial arginine deiminase can be a pathogenicity factor aimed for dysregulating the processes of inflammation and immune response. In general, the effect of arginine deiminase on immune cells may result into disturbed production of regulatory proinflammatory molecules, such as NO, and related substances, inhibition of activation, migration and differentiation of individual leukocyte subsets. The aim of this study was to investigate the effect of arginine deiminase on the formation of inflammatory infiltrate in murine air pouch model of streptococcal infection. Materials and methods: The study was performed using S. pyogenes M49-16 expressing arginine deiminase and its isogenic mutant S. pyogenes M49-16delArcA with inactivated arginine deiminase gene. The flow cytometry analysis of the inflammatory infiltrate leukocytes subpopulation in mice infected with the original strain of S. pyogenes M49-16 and its isogenic mutant S. pyogenes M49-16delArcA at different periods of infection was performed. It was shown that the inflammation reached its peak 6 hours after streptococcal inoculation, being more pronounced in mice infected with the mutant strain. Тhis finding was affirmed by a simultaneous and more pronounced increase in the absolute numbers of all leukocyte subsets in the focus of inflammation in this group of mice when compared to mice infected with original bacterial strain. Despite the decrease in the absolute number of all leukocyte types in the inflammatory infiltrate in both groups of mice for 24 hours, this trend was more pronounced in the group of mice infected with mutant microbial strain. Comparison of the inflammatory infiltrates developing in mice infected with original versus mutant strains showed that arginine deiminase may be a pathogenicity factor leading to dysregulation of protective immune response, due to impaired migration of white blood cells to the site of infection.

NirA is an alternative nitrite reductase fromPseudomonas aeruginosawith potential as an anti-virulence target

ABSTRACTThe opportunistic pathogenPseudomonas aeruginosaproduces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, anti-virulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treatP. aeruginosainfections. To identifyP. aeruginosagenes required for virulence in multiple hosts, a random library of Tn5 mutants in PAO1-L was previously screenedin vitrofor those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we have identified a Tn5 mutant with an insertion in PA4130 showing reduced levels in a number of virulence traitsin vitro. Construction of an isogenic mutant in this gene presented similar results as those from the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models ofDrosophila melanogaster,Caenorhabditis elegansas well as decreased toxicity in human cell lines. This mutant also presented an 80% increased survival in murine acute and agar-bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulphite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this protein is a nitrite reductase operating in a siroheme and 4Fe-4S dependant manner. The preference for nitrite and the production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named as NirA.

Staphylococcus aureus secreted lipases do not inhibit innate immune killing mechanisms

Background: Staphylococcus aureus causes an array of diseases in both humans and livestock. Pathogenesis is mediated by a plethora of proteins secreted by S. aureus, many of which remain incompletely characterised. For example, S. aureus abundantly secretes two isoforms of the enzyme lipase into the extracellular milieu, where they scavenge upon polymeric triglycerides. It has previously been suggested that lipases may interfere with the function of innate immune cells, such as macrophages and neutrophils, but the impact of lipases on phagocytic killing mechanisms remains unknown. Methods: We employed the epidemic S. aureus clone USA300 strain LAC and its lipase deficient isogenic mutant, along with recombinant lipase proteins, in in vitro experimental infection assays. To determine if lipases can inhibit innate immune killing mechanisms, the bactericidal activity of whole blood, human neutrophils, and macrophages was analysed. In addition, gentamycin protection assays were carried out to examine the influence of lipases on S. aureus innate immune cell escape. Results: There were no differences in the survival of S. aureus USA300 LAC wild type and its lipase-deficient isogenic mutant after incubation with human whole blood or neutrophils. Furthermore, there was no detectable lipase-dependent effect on phagocytosis, intracellular survival, or escape from both human primary and immortalised cell line macrophages, even upon supplementation with exogenous recombinant lipases. Conclusions: S. aureus lipases do not inhibit bacterial killing mechanisms of human macrophages, neutrophils, or whole blood. These findings broaden our understanding of the interaction of S. aureus with the innate immune system.