Harnessing antifungal immunity in pursuit of a Staphylococcus aureus vaccine strategy.

Staphylococcus aureus (S. aureus) is one of the most common bacterial infections worldwide, and antibiotic resistant strains such as Methicillin-Resistant S. aureus (MRSA) are a major threat and burden to public health. MRSA not only infects immunocompromised patients but also healthy individuals and has rapidly spread from the healthcare setting to the outside community. However, all vaccines tested in clinical trials to date have failed. Immunocompromised individuals such as patients with HIV or decreased levels of CD4+ T cells are highly susceptible to S. aureus infections, and they are also at increased risk of developing fungal infections. We therefore wondered whether stimulation of antifungal immunity might promote the type of immune responses needed for effective host defense against S. aureus. Here we show that vaccination of mice with a fungal ß-glucan particle (GP) loaded with S. aureus antigens provides protective immunity to S. aureus. We generated glucan particles loaded with the four S. aureus proteins ClfA, IsdA, MntC, and SdrE, creating the 4X-SA-GP vaccine. Vaccination of mice with three doses of 4X-SA-GP promoted protection in a systemic model of S. aureus infection with a significant reduction in the bacterial burden in the spleen and kidneys. 4X-SA-GP vaccination induced antigen-specific Th1 and Th17 CD4+ T cell and antibody responses and provided long-term protection. This work suggests that the GP vaccine system has potential as a novel approach to developing vaccines for S. aureus.

YpdA, a putative bacillithiol disulfide reductase, contributes to cellular redox homeostasis and virulence in Staphylococcus aureus.

The intracellular redox environment of Staphylococcus aureus is mainly buffered by bacillithiol (BSH), a low molecular weight thiol. The identity of enzymes responsible for the recycling of oxidized bacillithiol disulfide (BSSB) to the reduced form (BSH) remains elusive. We examined YpdA, a putative bacillithiol reductase, for its role in maintaining intracellular redox homeostasis. The ypdA mutant showed increased levels of BSSB and a lower bacillithiol redox ratio vs. the isogenic parent, indicating a higher level of oxidative stress within the bacterial cytosol. We showed that YpdA consumed NAD(P)H; and YpdA protein levels were augmented in response to stress. Wild type strains overexpressing YpdA showed increased tolerance to oxidants and electrophilic agents. Importantly, YpdA overexpression in the parental strain caused an increase in BSH levels accompanied by a decrease in BSSB concentration in the presence of stress, resulting in an increase in bacillithiol redox ratio vs. the vector control. Additionally, the ypdA mutant exhibited decreased survival in human neutrophils (PMNs) as compared with the parent, while YpdA overexpression protected the resulting strain from oxidative stress in vitro and from killing by human neutrophils ex vivo. Taken together, these data present a new role for YpdA in S. aureus physiology and virulence through the bacillithiol system.

Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection.

Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγnull (NSG) mice engrafted with human CD34+ umbilical cord blood cells. These "humanized" NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL+ versus isogenic PVL- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL+ S. aureus but not PVL- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.

Importance of bacillithiol in the oxidative stress response of Staphylococcus aureus.

In Staphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed mutants with the deletion of bshA (sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, and fosB (sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains, including clinical isolates. Mutation of fosB or bshA caused a 16- to 60-fold reduction in fosfomycin resistance in these S. aureus strains. High-pressure liquid chromatography analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present across S. aureus strains. Deletion of fosB led to a decrease in BSH levels. The fosB and bshA mutants of strain COL and a USA300 isolate, upon further characterization, were found to be sensitive to H2O2 and exhibited decreased NADPH levels compared with those in the isogenic parents. Microarray analyses of COL and the isogenic bshA mutant revealed increased expression of genes involved in staphyloxanthin synthesis in the bshA mutant relative to that in COL under thiol stress conditions. However, the bshA mutant of COL demonstrated decreased survival compared to that of the parent in human whole-blood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival of S. aureus under oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independently of its capability to produce staphyloxanthin.

The impact of CodY on virulence determinant production in community-associated methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is a leading human pathogen of both hospital and community-associated diseases worldwide. This organism causes a wealth of infections within the human host as a result of the vast arsenal of toxins encoded within its genome. Previous transcriptomic studies have shown that toxin production in S. aureus can be strongly impacted by the negative regulator CodY. CodY acts by directly, and indirectly (via Agr), repressing toxin production during times of plentiful nutrition. In this study, we use iTRAQ-based proteomics for the first time to study virulence determinant production in S. aureus, so as to correlate transcriptional observations with actual changes in protein synthesis. Using a codY mutant in the epidemic CA-MRSA clone USA300 we demonstrate that deletion of this transcription factor results in a major upregulation of toxin synthesis in both post-exponential and stationary growth. Specifically, we observe hyper-production of secreted proteases, leukocidins and hemolysins in both growth phases in the USA300 codY mutant. Our findings demonstrate the power of mass spectrometry-based quantitative proteomics for studying toxin production in S. aureus, and the importance of CodY to this central process in disease causation and infection.

NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus.

Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.

Propionibacterium acnes-induced immunopathology correlates with health and disease association.

Genomic studies revealed the existence of health- and acne-associated P. acnes strains and suggested novel approaches for broadening understanding of acne vulgaris. However, clinical association of P. acnes with disease or health has yet to be corroborated experimentally. Current animal models of acne do not closely mimic human disease and have unclear translational value. We have developed a murine model of acne by combining P. acnes inoculation with topical application of a synthetic human sebum. We showed that human sebum promoted persistence of intradermally injected P. acnes with little loss of viability after 1 week and permitted use of more physiologic inoculums. Application of acne-associated P. acnes RT4/5 strains led to development of moderate to severe skin pathology compared with application of health-associated type II P. acnes strains (RT2/6). RT4/5 P. acnes strains uniformly induced higher levels of KC (IL-8), IL-1α, IL-1ß, and IL-6 in vitro and in vivo compared with type II P. acnes strains. Overall, our data provide immunopathologic corroboration of health and disease association of clinical P. acnes strains and inform on a platform to query putative virulence factors uncovered by genomic studies.

RAS and ROS-A Story of Pseudomonas Survival.

Some pathogens block generation of reactive oxygen species to evade neutrophil killing, but how that is accomplished is poorly understood. In this issue of Cell Host & Microbe, Vareechon et al. (2017) describe ADP-ribosylation of Ras as a strategy to inhibit assembly of neutrophil NADPH oxidase.

O-Acetylation of Peptidoglycan Limits Helper T Cell Priming and Permits Staphylococcus aureus Reinfection.

Humans do not usually develop effective immunity to Staphylococcus aureus reinfection. Using a murine model that mimics human infection, we show that lack of protective immunity to S. aureus systemic reinfection is associated with robust interleukin-10 (IL-10) production and impaired protective Th17 responses. In dendritic cell co-culture assays, priming with S. aureus promotes robust T cell proliferation, but limits Th cells polarization and production of IL-1ß and other cytokines important for Th1 and Th17 differentiation. We show that O-acetylation of peptidoglycan, a mechanism utilized by S. aureus to block bacterial cell wall breakdown, limits the induction of pro-inflammatory signals required for optimal Th17 polarization. IL-10 deficiency in mice restores protective immunity to S. aureus infection, and adjuvancy with a staphylococcal peptidoglycan O-acetyltransferase mutant reduces IL-10, increases IL-1ß, and promotes development of IL-17-dependent, Th cell-transferable protective immunity. Overall, our study suggests a mechanism whereby S. aureus modulates cytokines critical for induction of protective Th17 immunity.

Group B Streptococcus Evades Host Immunity by Degrading Hyaluronan.

In response to tissue injury, hyaluronan (HA) polymers are cleaved by host hyaluronidases, generating small fragments that ligate Toll-like receptors (TLRs) to elicit inflammatory responses. Pathogenic bacteria such as group B Streptococcus (GBS) express and secrete hyaluronidases as a mechanism for tissue invasion, but it is not known how this activity relates to immune detection of HA. We found that bacterial hyaluronidases secreted by GBS and other Gram-positive pathogens degrade pro-inflammatory HA fragments to their component disaccharides. In addition, HA disaccharides block TLR2/4 signaling elicited by both host-derived HA fragments and other TLR2/4 ligands, including lipopolysaccharide. Application of GBS hyaluronidase or HA disaccharides reduced pulmonary pathology and pro-inflammatory cytokine levels in an acute lung injury model. We conclude that breakdown of host-generated pro-inflammatory HA fragments to disaccharides allows bacterial pathogens to evade immune detection and could be exploited as a strategy to treat inflammatory diseases.

Mannitol utilisation is required for protection of Staphylococcus aureus from human skin antimicrobial fatty acids.

Mannitol (Mtl) fermentation, with the subsequent production of acid, is a species signature of Staphylococcus aureus, and discriminates it from most other members of the genus. Inactivation of the gene mtlD, encoding Mtl-1-P dehydrogenase was found to markedly reduce survival in the presence of the antimicrobial fatty acid, linoleic acid. We demonstrate that the sugar alcohol has a potentiating action for this membrane-acting antimicrobial. Analysis of cellular metabolites revealed that, during exponential growth, the mtlD mutant accumulated high levels of Mtl and Mtl-P. The latter metabolite was not detected in its isogenic parent strain or a deletion mutant of the entire mtlABFD operon. In addition, the mtlD mutant strain exhibited a decreased MIC for H2O2, however virulence was unaffected in a model of septic arthritis.

Extracellular proteases are key mediators of Staphylococcus aureus virulence via the global modulation of virulence-determinant stability.

Staphylococcus aureus is a highly virulent and successful pathogen that causes a diverse array of diseases. Recently, an increase of severe infections in healthy subjects has been observed, caused by community-associated methicillin-resistant S. aureus (CA-MRSA). The reason for enhanced CA-MRSA virulence is unclear; however, work suggests that it results from hypersecretion of agr-regulated toxins, including secreted proteases. In this study, we explore the contribution of exo-proteases to CA-MRSA pathogenesis using a mutant lacking all 10 enzymes. We show that they are required for growth in peptide-rich environments, serum, in the presence of antimicrobial peptides (AMPs), and in human blood. We also reveal that extracellular proteases are important for resisting phagocytosis by human leukocytes. Using murine infection models, we reveal contrasting roles for the proteases in morbidity and mortality. Upon exo-protease deletion, we observed decreases in abscess formation, and impairment during organ invasion. In contrast, we observed hypervirulence of the protease-null strain in the context of mortality. This dichotomy is explained by proteomic analyses, which demonstrates exo-proteases to be key mediators of virulence-determinant stability. Specifically, increased abundance of both secreted (e.g. α-toxin, Psms, LukAB, LukE, PVL, Sbi, γ-hemolysin) and surface-associated (e.g. ClfA+B, FnbA+B, IsdA, Spa) proteins was observed upon protease deletion. Collectively, our findings provide a unique insight into the progression of CA-MRSA infections, and the role of secreted proteolytic enzymes.