Discovery of an antivirulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin-1 production.

Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets virulence of S. aureus, and it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS.

Vaginal community state types (CSTs) alter environmental cues and production of the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1).

Menstrual toxic shock syndrome (mTSS) is a rare but life-threatening disease associated with the use of high-absorbency tampons. The production of the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) superantigen is involved in nearly all cases of mTSS and is tightly controlled by regulators responding to the environment. In the prototypic mTSS strain S. aureus MN8, the major repressor of TSST-1 is the carbon catabolite protein A (CcpA), which responds to glucose concentrations in the vaginal tract. Healthy vaginal Lactobacillus species also depend on glucose for both growth and acidification of the vaginal environment through lactic acid production. We hypothesized that interactions between the vaginal microbiota [herein referred to as community state types (CSTs)] and S. aureus MN8 depend on environmental cues and that these interactions subsequently affect TSST-1 production. Using S. aureus MN8 ΔccpA growing in various glucose concentrations, we demonstrate that the supernatants from different CSTs grown in vaginally defined medium (VDM) could significantly decrease tst expression. When co-culturing CST species with MN8 ∆ccpA, we show that Lactobacillus jensenii completely inhibits TSST-1 production in conditions mimicking healthy menstruation or mTSS. Finally, we show that growing S. aureus in "unhealthy" or "transitional" CST supernatants results in higher interleukin 2 (IL-2) production from T cells. These findings suggest that dysbiotic CSTs may encourage TSST-1 production in the vaginal tract and further indicate that the CSTs are likely important for the protection from mTSS.IMPORTANCEIn this study, we investigate the impact of the vaginal microbiota against Staphylococcus aureus in conditions mimicking the vaginal environment at various stages of the menstrual cycle. We demonstrate that Lactobacillus jensenii can inhibit toxic shock syndrome toxin-1 (TSST-1) production, suggesting the potential for probiotic activity in treating and preventing menstrual toxic shock syndrome (mTSS). On the other side of the spectrum, "unhealthy" or "transient" bacteria such as Gardnerella vaginalis and Lactobacillus iners support more TSST-1 production by S. aureus, suggesting that community state types are important in the development of mTSS. This study sets forward a model for examining contact-independent interactions between pathogenic bacteria and the vaginal microbiota. It also demonstrates the necessity of replicating the environment when studying one as dynamic as the vagina.

Novel insights into the immune response to bacterial T cell superantigens.

Bacterial T cell superantigens (SAgs) are a family of microbial exotoxins that function to activate large numbers of T cells simultaneously. SAgs activate T cells by direct binding and crosslinking of the lateral regions of MHC class II molecules on antigen-presenting cells with T cell receptors (TCRs) on T cells; these interactions alter the normal TCR-peptide-MHC class II architecture to activate T cells in a manner that is independent of the antigen specificity of the TCR. SAgs have well-recognized, central roles in human diseases such as toxic shock syndrome and scarlet fever through their quantitative effects on the T cell response; in addition, numerous other consequences of SAg-driven T cell activation are now being recognized, including direct roles in the pathogenesis of endocarditis, bloodstream infections, skin disease and pharyngitis. In this Review, we summarize the expanding family of bacterial SAgs and how these toxins can engage highly diverse adaptive immune receptors. We highlight recent findings regarding how SAg-driven manipulation of the adaptive immune response may operate in multiple human diseases, as well as contributing to the biology and life cycle of SAg-producing bacterial pathogens.

Interplay between Staphylococcus aureus and the vaginal microbiota.

Staphylococcus aureus is a proficient colonizer and opportunistic pathogen which can lead to vaginal dysbiosis, aerobic vaginitis, or life-threatening menstrual toxic shock syndrome. Here we explore the complex but underappreciated interactions that S. aureus may impose on the vaginal environment leading to additional disease outcomes.

The fadXDEBA locus of Staphylococcus aureus is required for metabolism of exogenous palmitic acid and in vivo growth.

In Staphylococcus aureus, genes that should confer the capacity to metabolize fatty acids by ß-oxidation occur in the fadXDEBA locus, but their function has not been elucidated. Previously, incorporation into phospholipid through the fatty acid kinase FakA pathway was thought to be the only option available for S. aureus to metabolize exogenous saturated fatty acids. We now find that in S. aureus USA300, a fadX::lux reporter was repressed by glucose and induced by palmitic acid but not stearic acid, while in USA300ΔfakA basal expression was significantly elevated, and enhanced in response to both fatty acids. When cultures were supplemented with palmitic acid, palmitoyl-CoA representing the first metabolite in the ß-oxidation pathway was detected in USA300, but not in a fadXDEBA deletion mutant USA300Δfad, which relative to USA300 exhibited increased incorporation of palmitic acid into phospholipid accompanied by a rapid loss of viability. USA300Δfad also exhibited significantly reduced viability in a murine tissue abscess infection model. Our data are consistent with FakA-mediated incorporation of fatty acids into phospholipid as a preferred pathway for metabolism of exogenous fatty acids, while the fad locus is critical for metabolism of palmitic acid, which is the most abundant free fatty acid in human plasma.

Glucose Mediates Niche-Specific Repression of Staphylococcus aureus Toxic Shock Syndrome Toxin-1 through the Activity of CcpA in the Vaginal Environment.

Staphylococcus aureus chronically colonizes up to 30% of the human population on the skin or mucous membranes, including the nasal tract or vaginal canal. While colonization is often benign, this bacterium also has the capability to cause serious infections. Menstrual toxic shock syndrome (mTSS) is a serious toxinosis associated with improper use of tampons, which can induce an environment that is favorable to the production of the superantigen known as toxic shock syndrome toxin-1 (TSST-1). To better understand environmental signaling that influences TSST-1 production, we analyzed expression in the prototype mTSS strain S. aureus MN8. Using transcriptional and protein-based analysis in two niche-related media, we observed that TSST-1 expression was significantly higher in synthetic nasal medium (SNM) than in vaginally defined medium (VDM). One major divergence in medium composition was high glucose concentration in VDM. The glucose-dependent virulence regulator gene ccpA was deleted in MN8, and, compared with wild-type MN8, we observed increased TSST-1 expression in the ΔccpA mutant when grown in VDM, suggesting that TSST-1 is repressed by catabolite control protein A (CcpA) in the vaginal environment. We were able to relieve CcpA-mediated repression by modifying the glucose level in vaginal conditions, confirming that changes in nutritional conditions contribute to the overexpression of TSST-1 that can lead to mTSS. We also compared CcpA-mediated repression to other key regulators of tst, finding that CcpA regulation is dominant compared to other characterized regulatory mechanisms. This study underlines the importance of environmental signaling for S. aureus pathogenesis in the context of mTSS. IMPORTANCE Menstrual toxic shock syndrome (mTSS) is caused by strains of Staphylococcus aureus that overproduce a toxin known as toxic shock syndrome toxin-1 (TSST-1). This work studied how glucose levels in a model vaginal environment could influence the amount of TSST-1 that is produced by S. aureus. We found that high levels of glucose repress TSST-1 production, and this is done by a regulatory protein called catabolite control protein A (CcpA). The research also demonstrated that, compared with other regulatory proteins, the CcpA regulator appears to be the most important for maintaining low levels of TSST-1 in the vaginal environment, and this information helps to understand how changes in the vaginal environmental can lead to mTSS.

Identification of Crp as a novel regulator of the Std fimbrial expression in Salmonella.

The Salmonella enterica serovar Typhi genome contains 14 putative fimbrial systems. The Std fimbriae belong to the chaperone-usher family and its regulation has not been investigated in S. Typhi. Several regulators of Std were previously identified in the closely related serovar Typhimurium. We hypothesize that regulators of S. Typhimurium may be shared with S. Typhi, but that several other regulators remain to be discovered. Here, we describe the role of more than 50 different candidate regulators on std expression. Three types of regulators were investigated: known regulators in S. Typhimurium, in silico predicted regulators and virulence/metabolic regulators. Expression of std was determined in the regulator mutants and compared with the wild-type strain. Overall, 21 regulator mutations affect std promoter expression. The role of Crp, a newly identified factor for std expression, was further investigated. Crp acted as an activator of std expression on a distal region of the std promoter region. Together, our results demonstrate the major influence of Crp as a novel transcriptional factor on std promoter expression and later production of Std fimbriae in Salmonella.

New Roles for Two-Component System Response Regulators of Salmonella enterica Serovar Typhi during Host Cell Interactions.

In order to survive external stresses, bacteria need to adapt quickly to changes in their environment. One adaptive mechanism is to coordinate and alter their gene expression by using two-component systems (TCS). TCS are composed of a sensor kinase that activates a transcriptional response regulator by phosphorylation. TCS are involved in motility, virulence, nutrient acquisition, and envelope stress in many bacteria. The pathogenic bacteria Salmonella enterica serovar Typhi (S. Typhi) possess 30 TCSs, is specific to humans, and causes typhoid fever. Here, we have individually deleted each of the 30 response regulators. We have determined their role during interaction with host cells (epithelial cells and macrophages). Deletion of most of the systems (24 out of 30) resulted in a significant change during infection. We have identified 32 new phenotypes associated with TCS of S. Typhi. Some previously known phenotypes associated with TCSs in Salmonella were also confirmed. We have also uncovered phenotypic divergence between Salmonella serovars, as distinct phenotypes between S. Typhi and S. Typhimurium were identified for cpxR. This finding highlights the importance of specifically studying S. Typhi to understand its pathogenesis mechanisms and to develop strategies to potentially reduce typhoid infections.

Functional Analysis of the Chaperone-Usher Fimbrial Gene Clusters of Salmonella enterica serovar Typhi.

The human-specific pathogen Salmonella enterica serovar Typhi causes typhoid, a major public health issue in developing countries. Several aspects of its pathogenesis are still poorly understood. S. Typhi possesses 14 fimbrial gene clusters including 12 chaperone-usher fimbriae (stg, sth, bcf, fim, saf, sef, sta, stb, stc, std, ste, and tcf). These fimbriae are weakly expressed in laboratory conditions and only a few are actually characterized. In this study, expression of all S. Typhi chaperone-usher fimbriae and their potential roles in pathogenesis such as interaction with host cells, motility, or biofilm formation were assessed. All S. Typhi fimbriae were better expressed in minimal broth. Each system was overexpressed and only the fimbrial gene clusters without pseudogenes demonstrated a putative major subunits of about 17 kDa on SDS-PAGE. Six of these (Fim, Saf, Sta, Stb, Std, and Tcf) also show extracellular structure by electron microscopy. The impact of fimbrial deletion in a wild-type strain or addition of each individual fimbrial system to an S. Typhi afimbrial strain were tested for interactions with host cells, biofilm formation and motility. Several fimbriae modified bacterial interactions with human cells (THP-1 and INT-407) and biofilm formation. However, only Fim fimbriae had a deleterious effect on motility when overexpressed. Overall, chaperone-usher fimbriae seem to be an important part of the balance between the different steps (motility, adhesion, host invasion and persistence) of S. Typhi pathogenesis.

Biology and Assembly of the Bacterial Envelope.

All free-living bacterial cells are delimited and protected by an envelope of high complexity. This physiological barrier is essential for bacterial survival and assures multiple functions. The molecular assembly of the different envelope components into a functional structure represents a tremendous biological challenge and is of high interest for fundamental sciences. The study of bacterial envelope assembly has also been fostered by the need for novel classes of antibacterial agents to fight the problematic of bacterial resistance to antibiotics. This chapter focuses on the two most intensively studied classes of bacterial envelopes that belong to the phyla Firmicutes and Proteobacteria. The envelope of Firmicutes typically has one membrane and is defined as being monoderm whereas the envelope of Proteobacteria contains two distinct membranes and is referred to as being diderm. In this chapter, we will first discuss the multiple roles of the bacterial envelope and clarify the nomenclature used to describe the different types of envelopes. We will then define the architecture and composition of the envelopes of Firmicutes and Proteobacteria while outlining their similarities and differences. We will further cover the extensive progress made in the field of bacterial envelope assembly over the last decades, using Bacillus subtilis and Escherichia coli as model systems for the study of the monoderm and diderm bacterial envelopes, respectively. We will detail our current understanding of how molecular machines assure the secretion, insertion and folding of the envelope proteins as well as the assembly of the glycosidic components of the envelope. Finally, we will highlight the topics that are still under investigation, and that will surely lead to important discoveries in the near future.

Response to variable light intensity in photoacclimated algae and cyanobacteria exposed to atrazine.

Atrazine is frequently detected in freshwater ecosystems exposed to agricultural waste waters and runoffs worldwide and it can affect non-target organisms (mainly photoautotrophic) and modify community structure. Meanwhile, light environment is known to vary between aquatic ecosystems, but also before and during the exposure to atrazine and these variations may modify the sensitivity to atrazine of photoautotroph organisms. In this study, 10 species of phytoplankton (chlorophytes, baccilariophytes and cyanophytes) acclimated to low or high light intensities were exposed to atrazine and light of different intensities to compare their combined effect. Our data showed that chlorophytes and baccilariophytes were more resistant to atrazine compared to cyanophytes for all light conditions. Atrazine was found to inhibit Φ'(M), Ψ(0), P(M) and non-photochemical quenching for all species indicating an effect on electron transport, primary production and photoregulation processes. These data also indicate a higher sensitivity of Ψ(0) (average Ψ(0)-EC(50) of 91 ± 11 nM or 19.6 ± 0.9 µgL(-1)) compared to Φ'(M) (average Φ'(M)-EC(50) of 217 ± 19 nM or 46.8 ± 4.1 µgL(-1)) and suggest that photoregulation processes activated in presence of light decrease the effect of atrazine. We also showed that increasing light intensity decreased Φ'(M)-EC(50) in both low (except baccilariophytes) and high light acclimated conditions. Despite this similarity, most species acclimated to high light were found to have higher or similar Φ'(M)-EC(50) compared to low light acclimated cells and thus, were less sensitive to atrazine in low light and high light environments. We concluded that an increase in the plastoquinone pool induced by acclimation to high light decreased the sensitivity to atrazine in phytoplankton and we hypothesized that the effect observed was the result of a dilution of atrazine toxicity through increased binding site availability (quinones) combined with increased photoregulation processes capacity.