Engineered skin microbiome reduces mosquito attraction to mice.

The skin microbiome plays a pivotal role in the production of attractive cues detected by mosquitoes. Here, we leveraged recent advances in genetic engineering to significantly reduce the production of L-(+)-lactic acid as a strategy to reduce mosquito attraction to the highly prominent skin commensals Staphylococcus epidermidis and Corynebacterium amycolatum. Engraftment of these engineered bacteria onto the skin of mice reduced mosquito attraction and feeding for up to 11 uninterrupted days, which is considerably longer than the several hours of protection conferred by the leading chemical repellent N,N-diethyl-meta-toluamide. Taken together, our findings demonstrate engineering the skin microbiome to reduce attractive volatiles represents an innovative untapped strategy to reduce vector attraction, preventing bites, and pathogen transmission. These findings set the stage for new classes of long-lasting microbiome-based repellent products.

Identification of human skin microbiome odorants that manipulate mosquito landing behavior.

The resident human skin microbiome is responsible for the production of most of the human scents that are attractive to mosquitoes. Hence, engineering the human skin microbiome to synthesize less of mosquito attractants or produce repellents could potentially reduce bites and prevent the transmission of deadly mosquito-borne pathogens. In order to further characterize the human skin volatilome, we quantified the major volatiles of 39 strains of skin commensals (Staphylococci and Corynebacterium). Importantly, to validate the behavioral activity of these volatiles, we first assessed landing behavior triggered by human skin volatiles. We demonstrated that landing behavior is gated by the presence of carbon dioxide and L-(+)-lactic acid. This is similar to the combinatorial coding triggering mosquito short range attraction. Repellency behavior to selected skin volatiles and terpenes was tested in the presence of carbon dioxide and L-(+)-lactic acid. In a 2-choice landing behavior context, the skin volatiles 2- and 3-methyl butyric acids reduced mosquito landing by 62.0-81.6% and 87.1-99.6%, respectively. Similarly, the terpene geraniol was capable of reducing mosquito landing behavior by 74.9%. We also tested the potential repellency effects of terpenes in mosquitoes at short-range using a 4-port olfactometer. In these assays, geraniol reduced mosquito attraction (69-78%) to a mixture of key human kairomones carbon dioxide, L-(+)-lactic acid, and ammonia. These findings demonstrate that carbon dioxide and L-(+)-lactic acid change the valence of other skin volatiles towards mosquito landing behavior. Moreover, this study offers candidate odorants to be targeted in a novel strategy to reduce attractants or produce repellents by the human skin microbiota that may curtail mosquito bites, and subsequent mosquito-borne disease.

Volatile profiling distinguishes Streptococcus pyogenes from other respiratory streptococcal species.

Sore throat is one of the most common complaints encountered in the ambulatory clinical setting. Rapid, culture-independent diagnostic techniques that do not rely on pharyngeal swabs would be highly valuable as a point-of-care strategy to guide outpatient antibiotic treatment. Despite the promise of this approach, efforts to detect volatiles during oropharyngeal infection have yet been limited. In our research study, we sought to evaluate for specific bacterial volatile organic compounds (VOC) biomarkers in isolated cultures in vitro, in order to establish proof-of-concept prior to initial clinical studies of breath biomarkers. A particular challenge for the diagnosis of pharyngitis due to Streptococcus pyogenes is the likelihood that many metabolites may be shared by S. pyogenes and other related oropharyngeal colonizing bacterial species. Therefore, we evaluated whether sufficient metabolic differences are present, which distinguish the volatile metabolome of Group A streptococci from other streptococcal species that also colonize the respiratory mucosa, such as Streptococcus pneumoniae and Streptococcus intermedius. In this work, we identified 27 discriminatory VOCs (q-values < 0.05), composed of aldehydes, alcohols, nitrogen-containing compounds, hydrocarbons, ketones, aromatic compounds, esters, ethers, and carboxylic acid. From this group of volatiles, we identify candidate biomarkers that distinguish S. pyogenes from other species and establish highly produced VOCs that indicate the presence of S. pyogenes in vitro, supporting future breath-based diagnostic testing for streptococcal pharyngitis. IMPORTANCE Acute pharyngitis accounts for approximately 15 million ambulatory care visits in the United States. The most common and important bacterial cause of pharyngitis is Streptococcus pyogenesis, accounting for 15%-30% of pediatric pharyngitis. Distinguishing between bacterial and viral pharyngitis is key to management in US practice. The culture of a specimen obtained by a throat swab is the standard laboratory procedure for the microbiologic confirmation of pharyngitis; however, this method is time-consuming, which delays appropriate treatment. If left untreated, S. pyogenes pharyngitis may lead to local and distant complications. In this study, we characterized the volatile metabolomes of S. pyogenes and other related oropharyngeal colonizing bacterial species. We identify candidate biomarkers that distinguish S. pyogenes from other species and provide evidence to support future breath-based diagnostic testing for streptococcal pharyngitis.

Identification of human skin microbiome odorants that manipulate mosquito landing behavior.

The resident human skin microbiome is responsible for the production of most of the human scents that are attractive to mosquitoes. Hence, engineering the human skin microbiome to synthesize less of mosquito attractants or produce repellents could potentially reduce bites and prevent the transmission of deadly mosquito-borne pathogens. In order to further characterize the human skin volatilome, we quantified the major volatiles of 39 strains of skin commensals (Staphylococci and Corynebacterium). Importantly, to validate the behavioral activity of these volatiles, we first assessed landing behavior triggered by human skin bacteria volatiles. We demonstrated that this behavioral step is gated by the presence of carbon dioxide and L-(+)-lactic acid, similar to the combinatorial coding triggering short range attraction. Repellency behavior to selected skin volatiles and the geraniol terpene was tested in the presence of carbon dioxide and L-(+)-lactic acid. In a 2-choice landing behavior context, the skin volatiles 2- and 3-methyl butyric acids reduced mosquito landing by 62.0-81.6% and 87.1-99.6%, respectively. Similarly, geraniol was capable of reducing mosquito landing behavior by 74.9%. We also tested the potential repellency effects of geraniol on mosquitoes at short-range using a 4-port olfactometer. In these assays, geraniol reduced mosquito attraction (69-78%) to a mixture of key human kairomones carbon dioxide, L-(+)-lactic acid, and ammonia. These findings demonstrate that carbon dioxide and L-(+)-lactic acid changes the valence of other skin volatiles towards mosquito landing behavior. Moreover, this study offers candidate odorants to be targeted in a novel strategy to reduce attractants or produce repellents by the human skin microbiota that may curtail mosquito bites, and subsequent mosquito-borne disease.

Central carbon flux controls growth/damage balance for Streptococcus pyogenes.

Microbial pathogens balance growth against tissue damage to achieve maximum fitness. Central carbon metabolism is connected to growth, but how it influences growth/damage balance is largely unknown. Here we examined how carbon flux through the exclusively fermentative metabolism of the pathogenic lactic acid bacterium Streptococcus pyogenes impacts patterns of growth and tissue damage. Using a murine model of soft tissue infection, we systematically examined single and pair-wise mutants that constrained carbon flux through the three major pathways that S. pyogenes employs for reduction of the glycolytic intermediate pyruvate, revealing distinct disease outcomes. Its canonical lactic acid pathway (via lactate dehydrogenase) made a minimal contribution to virulence. In contrast, its two parallel pathways for mixed-acid fermentation played important, but non-overlapping roles. Anaerobic mixed acid fermentation (via pyruvate formate lyase) was required for growth in tissue, while aerobic mixed-acid pathway (via pyruvate dehydrogenase) was not required for growth, but instead regulated levels of tissue damage. Infection of macrophages in vitro revealed that pyruvate dehydrogenase was required to prevent phagolysosomal acidification, which altered expression of the immunosuppressive cytokine IL-10. Infection of IL-10 deficient mice confirmed that the ability of aerobic metabolism to regulate levels of IL-10 plays a key role in the ability of S. pyogenes to modulate levels of tissue damage. Taken together, these results show critical non-overlapping roles for anaerobic and aerobic metabolism in soft tissue infection and provide a mechanism for how oxygen and carbon flux act coordinately to regulate growth/damage balance. Therapies targeting carbon flux could be developed to mitigate tissue damage during severe S. pyogenes infection.

Engineered Skin Microbiome Reduces Mosquito Attraction to Mice.

The skin microbiome plays a pivotal role in the production of attractive cues detected by mosquitoes. Here we leveraged recent advances in genetic engineering to significantly reduce the production of L-(+)-lactic acid as a strategy to reduce mosquito attraction to the highly prominent skin commensals Staphylococcus epidermidis and Corynebacterium amycolatum . Engraftment of these engineered bacteria onto the skin of mice reduced mosquito attraction and feeding for up to 11 uninterrupted days, which is considerably longer than the several hours of protection conferred by the leading chemical repellent DEET. Taken together, our findings demonstrate engineering the skin microbiome to reduce attractive volatiles represents an innovative untapped strategy to reduce vector attraction, preventing bites, and pathogen transmission setting the stage for new classes of long-lasting microbiome-based repellent products. One-Sentence Summary: Modified microbes make skin less attractive to mosquitoes.

Rifampin resistance mutations in the rpoB gene of Enterococcus faecalis impact host macrophage cytokine production.

Antibiotic-resistant bacteria in the genus Enterococcus are a major cause of nosocomial infections and are an emergent public health concern. Similar to a number of bacterial species, resistance to the antibiotic rifampicin (RifR) in enterococci is associated with mutations in the gene encoding the ß subunit of RNA polymerase (rpoB). In Mycobacterium tuberculosis, RifRrpoB mutations alter mycobacterial surface lipid expression and are associated with an altered IL-1 cytokine response in macrophages upon infection. However, it is not clear if RifR mutations modulate host cytokine responses by other bacteria. To address this question, we utilized Enterococcus faecalis (E. faecalis). Here, we treated human monocyte-derived macrophages with heat-inactivated wild type or RifRrpoB mutants of E. faecalis and found that RifR mutations reduced IL-1ß cytokine production. However, RifR mutations elicited other potent pro- and anti-inflammatory responses, indicating that they can impact other immune pathways beyond IL-1R1 signaling. Our findings suggest that immunomodulation by mutations in rpoB may be conserved across diverse bacterial species and that subversion of IL-1R1 pathway is shared by RifR bacteria.

Breast Cancer Risk Assessment and Chemoprevention Use Among Veterans Affairs Primary Care Providers: A National Online Survey.

INTRODUCTION: Breast cancer is the most common cancer diagnosed among women and the second most common cause of cancer death among women. There are ways to reduce a woman's risk of breast cancer; however, most eligible women in the United States are neither offered personalized screening nor chemoprevention. Surveys have found that primary care providers are largely unaware of breast cancer risk assessment models or chemoprevention. This survey aims to investigate Veterans Health Administration primary care providers' comfort level, practice patterns, and knowledge of breast cancer risk assessment and chemoprevention. MATERIALS AND METHODS: An online, Research Electronic Data Capture-generated survey was distributed to VHA providers in internal medicine, family medicine, and obstetrics/gynecology. Survey domains were provider demographics, women's health experience, comfort level, practice patterns, barriers to using risk models and chemoprevention, and knowledge of chemoprevention. RESULTS: Of the 167 respondents, 33.1% used the Gail model monthly or more often and only 2.4% prescribed chemoprevention in the past 2 years. Most VHA primary care providers did not answer chemoprevention knowledge questions correctly. Designated women's health providers were more comfortable with risk assessment (P < 0.018) and chemoprevention (P < 0.011) and used both breast cancer risk models (P < 0.0045) and chemoprevention more often (P < 0.153). Reported barriers to chemoprevention were lack of education and provider time. CONCLUSIONS: VHA providers and women Veterans would benefit from a system to ensure that women at increased risk of breast cancer are identified with risk modeling and that risk reduction options, such as chemoprevention, are offered when appropriate. VHA providers requested risk reduction education, which could improve primary care provider comfort level with chemoprevention.

Predictive genomic markers of response to VEGF targeted therapy in metastatic renal cell carcinoma.

BACKGROUND: First-line treatment for metastatic renal cell carcinoma (mRCC) is rapidly changing. It currently includes VEGF targeted therapies (TT), multi-target tyrosine kinase inhibitors (TKIs), mTOR inhibitors, and immunotherapy. To optimize outcomes for individual patients, genomic markers of response to therapy are needed. Here, we aim to identify tumor-based genomic markers of response to VEGF TT to optimize treatment selection. METHODS: From an institutional database, primary tumor tissue was obtained from 79 patients with clear cell mRCC, and targeted sequencing was performed. Clinical outcomes were obtained retrospectively. Progression-free survival (PFS) on first-line VEGF TT was correlated to genomic alterations (GAs) using Kaplan-Meier methodology and Cox proportional hazard models. A composite model of significant GAs predicting PFS in the first-line setting was developed. RESULTS: Absence of VHL mutation was associated with inferior PFS on first-line VEGF TT. A trend for inferior PFS was observed with GAs in TP53 and FLT1 C/C variant. A composite model of these 3 GAs was associated with inferior PFS in a dose-dependent manner. CONCLUSION: In mRCC, a composite model of TP53 mutation, wild type VHL, and FLT1 C/C variant strongly predicted PFS on first-line VEGF TT in a dose-dependent manner. These findings require external validation.

Effect of treatment dose reductions in the setting of hand-foot syndrome on survival outcomes in patients with metastatic renal cell carcinoma treated with vascular endothelial growth factor receptor inhibitors.

Purpose Hand-foot syndrome is a common dose limiting toxicity of vascular endothelial growth factor receptor tyrosine kinase inhibitors used for treatment of patients with metastatic renal cell carcinoma. The effect of treatment dose reductions, in the context of hand-foot syndrome, on survival outcomes is reported. Methods This was a retrospective case series of patients receiving vascular endothelial growth factor receptor tyrosine kinase inhibitors from 1 January 2004 to 31 October 2013. The main outcomes were progression-free and overall survival in these patients experiencing hand-foot syndrome and undergoing treatment dose reductions. Univariate and multivariate analyses were conducted utilizing Kaplan-Meier method and COX Proportional Hazard model with landmark analyses at 2 months. Results Of the 120 patients evaluated, treatment dose reductions for any reason were required in 68 (56.7%) patients. The most common reasons for treatment dose reductions were mucositis, hand-foot syndrome, and fatigue. The median progression-free survival and overall survival were significantly longer in patients with hand-foot syndrome with or without treatment dose reductions as compared to those without hand-foot syndrome. Conclusions An improvement in survival outcomes was observed in metastatic renal cell carcinoma patients with treatment-associated hand-foot syndrome despite treatment dose reductions. These data need validation in a larger cohort to confirm the hypothesis that treatment dose reductions in the setting of hand-foot syndrome do not negatively impatient survival.

Lactobacillus reuteri induces gut intraepithelial CD4+CD8αα+ T cells.

The small intestine contains CD4+CD8αα+ double-positive intraepithelial lymphocytes (DP IELs), which originate from intestinal CD4+ T cells through down-regulation of the transcription factor Thpok and have regulatory functions. DP IELs are absent in germ-free mice, which suggests that their differentiation depends on microbial factors. We found that DP IEL numbers in mice varied in different vivaria, correlating with the presence of Lactobacillus reuteri This species induced DP IELs in germ-free mice and conventionally-raised mice lacking these cells. L. reuteri did not shape the DP-IEL-TCR (TCR, T cell receptor) repertoire but generated indole derivatives of tryptophan that activated the aryl-hydrocarbon receptor in CD4+ T cells, allowing Thpok down-regulation and differentiation into DP IELs. Thus, L. reuteri, together with a tryptophan-rich diet, can reprogram intraepithelial CD4+ T cells into immunoregulatory T cells.

Neutrophil-lymphocyte ratio as a predictive biomarker for response to high dose interleukin-2 in patients with renal cell carcinoma.

BACKGROUND: Immunotherapy with high-dose interleukin-2 (HD-IL2) results in long-term survival in some metastatic renal cell carcinoma (mRCC) patients but has significant acute toxicities. Biomarkers predicting response to therapy are needed to better select patients most likely to benefit. NLR (absolute neutrophil count (ANC)/absolute lymphocyte count (ALC)) is a prognostic and predicative biomarker in various malignancies. The goal was to determine whether NLR can predict response to HD-IL2 in this setting. METHODS: Patients with clear cell mRCC treated with HD-IL2 were identified from an institutional database from 2003-2012. Baseline variables for the assessment of IMDC risk criteria, and neutrophil and lymphocyte count, were collected. Best response criteria were based on RECIST 1.0. Wilcoxon rank-sum test was used to evaluate the association of continuous baseline variables with disease control. NLR was stratified by ≤4 or >4. Progression free survival (PFS) and overall survival (OS) were estimated with the Kaplan-Meier method and Cox proportional hazard models assessed associations of NLR with survival. RESULTS: In 71 eligible patients, median NLR in those with an objective response (n = 14, 20%) was 2.3 vs 3.4 in those without (n = 57, 80%, p = 0.02). NLR ≤4 was associated with improved progression free and overall survival. After adjustment for IMDC risk criteria, NLR remained a significant predictor of OS (ANC/ALC ≤4 vs >4, HR 0.41, 95% CI 1.09-5.46, p = 0.03; ANC/ALC continuous variable per unit change in NLR, HR 1.08, 95% CI 1.01-1.14, p = 0.03). CONCLUSIONS: In this discovery set, NLR predicts overall survival in patients treated with HD-IL2 in mRCC, and may allow better patient selection in this setting. Data needs validation in an independent cohort.

Temporal and Racial Differences Associated with Atopic Dermatitis Staphylococcusaureus and Encoded Virulence Factors.

Atopic dermatitis (AD) is an inflammatory skin condition strongly associated with Staphylococcus aureus colonization and infection. S. aureus strains shift in populations in ~10-year intervals depending on virulence factors. Shifts in S. aureus virulence factors may in part explain the racial differences observed in the levels of prevalence and severity of AD. AD S. aureus isolates collected from 2011 to 2014 (103 isolates) and in 2008 (100 isolates) were examined for the prevalence of genes encoding superantigens (SAgs). The strains from 2011 to 2014 were obtained from AD patients as a part of the National Institute of Allergy and Infectious Diseases (NIAID) Atopic Dermatitis Research Network (ADRN). The prevalence of SAg genes was investigated temporally and racially. The enterotoxin gene cluster (EGC) was more prevalent in the 2011-2014 AD isolates than in the 2008 AD isolates. The prevalences of virulence factor genes were similar in European American (EA) and Mexican American (MA) patients but differed in 6 of 22 SAg genes between EA and African American (AA) or MA and AA isolates; notably, AA isolates lacked tstH, the gene encoding toxic shock syndrome toxin 1 (TSST-1). The presence of tstH and sel-p (enterotoxin-like P) was associated with decreased clinical severity and increased blood eosinophils, respectively. The EGC is becoming more prevalent, consistent with the previously observed 10 years of cycling of S. aureus strains. Race-specific S. aureus selection may account for differences in virulence factor profiles. The lack of TSST-1-positive (TSST-1+) AD S. aureus in AA is consistent with the lack of AAs acquiring TSST-1-associated menstrual toxic shock syndrome (TSS). IMPORTANCE Monitoring pathogen emergence provides insight into how pathogens adapt in the human population. Secreted virulence factors, important contributors to infections, may differ in a manner dependent on the strain and host. Temporal changes of Staphylococcus aureus toxigenic potential, for example, in encoding toxic shock syndrome toxin 1 (TSST-1), contributed to an epidemic of TSS with significant health impact. This study monitored changes in atopic dermatitis (AD) S. aureus isolates and demonstrated both temporal and host infection differences according to host race based on secreted superantigen potential. The current temporal increase in enterotoxin gene cluster superantigen prevalence and lack of the gene encoding TSST-1 in AAs predict differences in infection types and presentations.

Conditional survival of metastatic renal cell carcinoma patients treated with high-dose interleukin-2.

Conditional survival (CS) is a clinically useful prediction measure which adjusts a patient's prognosis based on their duration of survival since initiation of therapy. CS has been described in numerous malignancies, and recently described in patients with metastatic renal cell carcinoma (mRCC) who received vascular endothelial growth factor tyrosine kinase inhibitor (VEGFTKI) therapy. However, CS has been not reported in the context of mRCC treated with high-dose interleukin-2 therapy (HDIL-2). A total of 176 patients with histologically confirmed metastatic clear cell RCC (mccRCC) treated with HDIL-2 at the University of Utah Huntsman Cancer Institute from 1988-2012 were evaluated. Using the Heng/IMDC model, they were stratified by performance status and prognostic risk groups. Two-year CS was defined as the probability of surviving an additional two years from initiation of HDIL-2 to 18 months after the start of HDIL-2 at three-month intervals. The median overall survival (OS) was 19.9 months. Stratifying patients into favourable (n = 35; 20%), intermediate (n = 110; 63%), and poor (n = 31; 18%) prognostic groups resulted in median OS of 47.5 (HR 0.57, 95% CI 0.35-0.88, p = 0.0106 versus intermediate), 19.6 (HR 0.33, 95% CI 0.10-0.33, p < 0.0001 versus poor), and 8.8 (HR 5.34, 95% CI 3.00-9.62, p < 0.0001 versus favourable) months respectively. Two-year overall CS increased from 43% at therapy initiation to 100% at 18 months. These results have significant ramifications in prognostication. Furthermore, it is important when counseling patients with mccRCC who have completed treatment with HDIL-2 and are in active follow-up.

Extension of overall survival beyond objective responses in patients with metastatic renal cell carcinoma treated with high-dose interleukin-2.

PURPOSE: In metastatic renal cell carcinoma (mRCC), survival benefit associated with objective response rates of 16-20 % with high-dose interleukin-2 (HDIL-2) is well established and discussed. Based on recently emerged data on efficacy of cancer immunotherapy, we hypothesized that the survival benefit with HDIL-2 extends beyond those achieving objective responses, i.e., to those who achieve stable disease as the best response to treatment. MATERIALS AND METHODS: All sequential treatment naïve mRCC patients treated with HDIL-2 at the University of Utah (1988-2013) and University of Michigan (1997-2013) were included. Best responses on treatment were associated with survival outcomes using log-rank and COX regression with a landmark analysis at 2 months. RESULTS: 391 patients (75 % male; median age 55 years) were included and belonged to the following prognostic risk categories: 20 % good, 64 % intermediate, and 15 % poor. Best responses on treatment were complete response (9 %), partial response (10 %), stable disease (32 %), progressive disease (42 %), and not evaluable for response (7 %). No significant differences in progression-free survival (HR 0.74, 95 % CI 0.48-1.1, p = 0.14) or overall survival (HR 0.66, 95 % CI 0.39-1.09, p = 0.11) were observed between patients achieving partial response versus stable disease. Significant differences in progression-free survival (HR 0.13, 95 % CI 0.09-0.22, p < 0.0001) and overall survival (HR 0.33, 95 % CI 0.23-0.48, p < 0.0001) were observed between patients achieving stable disease compared to those with progressive disease and who were not evaluable. CONCLUSIONS: Survival benefit with HDIL-2 is achieved in ~50 % patients and extends beyond those achieving objective responses.

The Staphylococcus aureus Global Regulator MgrA Modulates Clumping and Virulence by Controlling Surface Protein Expression.

Staphylococcus aureus is a human commensal and opportunistic pathogen that causes devastating infections in a wide range of locations within the body. One of the defining characteristics of S. aureus is its ability to form clumps in the presence of soluble fibrinogen, which likely has a protective benefit and facilitates adhesion to host tissue. We have previously shown that the ArlRS two-component regulatory system controls clumping, in part by repressing production of the large surface protein Ebh. In this work we show that ArlRS does not directly regulate Ebh, but instead ArlRS activates expression of the global regulator MgrA. Strains lacking mgrA fail to clump in the presence of fibrinogen, and clumping can be restored to an arlRS mutant by overexpressing either arlRS or mgrA, indicating that ArlRS and MgrA constitute a regulatory pathway. We used RNA-seq to show that MgrA represses ebh, as well as seven cell wall-associated proteins (SraP, Spa, FnbB, SasG, SasC, FmtB, and SdrD). EMSA analysis showed that MgrA directly represses expression of ebh and sraP. Clumping can be restored to an mgrA mutant by deleting the genes for Ebh, SraP and SasG, suggesting that increased expression of these proteins blocks clumping by steric hindrance. We show that mgrA mutants are less virulent in a rabbit model of endocarditis, and virulence can be partially restored by deleting the genes for the surface proteins ebh, sraP, and sasG. While mgrA mutants are unable to clump, they are known to have enhanced biofilm capacity. We demonstrate that this increase in biofilm formation is partially due to up-regulation of SasG, a surface protein known to promote intercellular interactions. These results confirm that ArlRS and MgrA constitute a regulatory cascade, and that they control expression of a number of genes important for virulence, including those for eight large surface proteins.

Novel Tissue Level Effects of the Staphylococcus aureus Enterotoxin Gene Cluster Are Essential for Infective Endocarditis.

BACKGROUND: Superantigens are indispensable virulence factors for Staphylococcus aureus in disease causation. Superantigens stimulate massive immune cell activation, leading to toxic shock syndrome (TSS) and contributing to other illnesses. However, superantigens differ in their capacities to induce body-wide effects. For many, their production, at least as tested in vitro, is not high enough to reach the circulation, or the proteins are not efficient in crossing epithelial and endothelial barriers, thus remaining within tissues or localized on mucosal surfaces where they exert only local effects. In this study, we address the role of TSS toxin-1 (TSST-1) and most importantly the enterotoxin gene cluster (egc) in infective endocarditis and sepsis, gaining insights into the body-wide versus local effects of superantigens. METHODS: We examined S. aureus TSST-1 gene (tstH) and egc deletion strains in the rabbit model of infective endocarditis and sepsis. Importantly, we also assessed the ability of commercial human intravenous immunoglobulin (IVIG) plus vancomycin to alter the course of infective endocarditis and sepsis. RESULTS: TSST-1 contributed to infective endocarditis vegetations and lethal sepsis, while superantigens of the egc, a cluster with uncharacterized functions in S. aureus infections, promoted vegetation formation in infective endocarditis. IVIG plus vancomycin prevented lethality and stroke development in infective endocarditis and sepsis. CONCLUSIONS: Our studies support the local tissue effects of egc superantigens for establishment and progression of infective endocarditis providing evidence for their role in life-threatening illnesses. In contrast, TSST-1 contributes to both infective endocarditis and lethal sepsis. IVIG may be a useful adjunct therapy for infective endocarditis and sepsis.

Staphylococcus aureus ß-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

ß-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of ß-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N ß-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. ß-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of ß-toxin and suggests ß-toxin functions importantly in infective endocarditis through both of its mechanisms of action.