To enhance the research capabilities of investigators interested in Staphylococcus aureus, the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) isolate USA300. To demonstrate the utility of this library for large-scale screening of phenotypic alterations, we spotted the library on indicator plates to assess hemolytic potential, protease production, pigmentation, and mannitol utilization. As expected, we identified many genes known to function in these processes, thus validating the utility of this approach. Importantly, we also identified genes not previously associated with these phenotypes. In total, 71 mutants displayed differential hemolysis activities, the majority of which were not previously known to influence hemolysin production. Furthermore, 62 mutants were defective in protease activity, with only 14 previously demonstrated to be involved in the production of extracellular proteases. In addition, 38 mutations affected pigment formation, while only 7 influenced mannitol fermentation, underscoring the sensitivity of this approach to identify rare phenotypes. Finally, 579 open reading frames were not interrupted by a transposon, thus providing potentially new essential gene targets for subsequent antibacterial discovery. Overall, the Nebraska Transposon Mutant Library represents a valuable new resource for the research community that should greatly enhance investigations of this important human pathogen.

Microbial virulence is a complex and often multifactorial phenotype, intricately linked to a pathogen's evolutionary trajectory. Toxicity, the ability to destroy host cell membranes, and adhesion, the ability to adhere to human tissues, are the major virulence factors of many bacterial pathogens, including Staphylococcus aureus. Here, we assayed the toxicity and adhesiveness of 90 MRSA (methicillin resistant S. aureus) isolates and found that while there was remarkably little variation in adhesion, toxicity varied by over an order of magnitude between isolates, suggesting different evolutionary selection pressures acting on these two traits. We performed a genome-wide association study (GWAS) and identified a large number of loci, as well as a putative network of epistatically interacting loci, that significantly associated with toxicity. Despite this apparent complexity in toxicity regulation, a predictive model based on a set of significant single nucleotide polymorphisms (SNPs) and insertion and deletions events (indels) showed a high degree of accuracy in predicting an isolate's toxicity solely from the genetic signature at these sites. Our results thus highlight the potential of using sequence data to determine clinically relevant parameters and have further implications for understanding the microbial virulence of this opportunistic pathogen.

Phagocyte oxidative burst is the primary source of lethal lung injury during influenza and MRSA coinfection.

Influenza and bacterial coinfection is a significant cause of hospitalization and death in humans during influenza epidemics and pandemics. However, the fundamental protective and pathogenic mechanisms involved in this complex virus-host-bacterium interaction remain incompletely understood. In this study, we have developed mild to lethal influenza and coinfection models for comparative analyses of disease pathogenesis. Specifically, wild-type and IL-1R type 1-deficient ( ) mice were infected with influenza virus and then superchallenged with noninvasive serotype 14 (Spn14) or serotype 19A (Spn19A). The coinfections were followed by comparative analyses of inflammatory responses and animal protection. We found that resident alveolar macrophages are efficient in the clearance of both pneumococcal serotypes in the absence of influenza infection; in contrast, they are essential for airway control of Spn14 infection but not Spn19A infection. In agreement, TNF-α and neutrophils play a compensatory protective role in secondary bacterial infection associated with Spn19A; however, the essential requirement for alveolar macrophage-mediated clearance significantly enhances the virulence of Spn14 during postinfluenza pneumococcal infection. Furthermore, we show that, although IL-1 signaling is not required for host defense against pneumococcal infection alone, it is essential for sustaining antibacterial immunity during postinfluenza pneumococcal infection, as evidenced by significantly aggravated bacterial burden and animal mortality in mice. Mechanistically, we show that through preventing alveolar macrophage depletion, inflammatory cytokine IL-1 signaling is critically involved in host resistance to influenza and pneumococcal coinfection.

The incidence of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in otherwise healthy individuals is increasing. To investigate the mechanism underlying the epidemiological success of predominant community-associated (CA)-MRSA strains, we examined their fitness traits during the initial interaction between bacteria and the host occurring in the lower airway. Using a mouse respiratory infection model, we show that clinical isolates often responsible for community-associated infections are highly resistant to clearance from healthy airways, while S. aureus strains not as prevalent or traditionally associated with hospital-associated infections are relatively susceptible. Mechanistically, the competitive fitness of S. aureus is a result of both agr-dependent and -independent resistance to innate bacterial killing. Furthermore, we show that rather than evasion from neutrophil-dependent bactericidal process, the observed S. aureus fitness in the lower airways is due to its intrinsic resistance to resident alveolar macrophage-mediated intracellular killing. Importantly, we demonstrate that the virulence determinants responsible for bacterial persistence in immune-competent mice are dispensable in mice with predisposing conditions such as influenza infection. Together, these novel findings of the improved competence of predominant CA-MRSA strains to survive innate killing in healthy hosts, particularly at the very beginning stage of infection, provide a unique insight into their epidemiological success.

The death and lysis of a subpopulation of Staphylococcus aureus cells during biofilm development benefit the whole bacterial population through the release of an important component of the biofilm matrix, extracellular DNA. Previously, we have demonstrated that these processes are affected by the gene products of the cidABC operon, the expression of which is controlled by the LysR-type transcriptional regulator, CidR. In this study, we characterized cis- and trans-acting elements essential for the induction of the cidABC operon. In addition to a CidR-binding site located within the cidABC promoter region, sequence analysis revealed the presence of a putative catabolite responsive element (cre box), suggestive of the involvement of the catabolite control protein A (CcpA) in the regulation of cidABC expression. This was confirmed using electrophoretic mobility shift assays and real-time reverse transcriptase PCR analysis demonstrating the direct positive control of cidABC transcription by the master regulator of carbon metabolism. Furthermore, the importance of CcpA and the identified cre site for the induction of the cidABC operon was demonstrated by examining the expression of PcidABC-lacZ reporter fusions in various mutant strains in which the genes involved in carbon metabolism and carbon catabolite repression were disrupted. Together the results of this study demonstrate the necessity of both transcriptional regulators, CidR and CcpA, for the induction of the cidABC operon and reveal the complexity of molecular interactions controlling its expression. IMPORTANCE This work focuses on the characterization of cis- and trans-acting elements essential for the induction of the cidABC operon in S. aureus. The results of this study are the first to demonstrate the synergistic control of cidABC expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full induction of cidABC expression depends on the metabolic state of bacteria and requires both CidR and CcpA. Together, these findings delineate regulatory control of cidABC expression under different metabolic conditions and provide important new insights into our understanding of cell death mechanisms during biofilm development in S. aureus.

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. SOCS1−/− mice die within three weeks postnatally due to IFN-γ-induced hyperinflammation. Since it is well established that IFN-γ is dispensable for protection against influenza infection, we generated SOCS1−/−IFN-γ−/− mice to determine whether SOCS1 regulates antiviral immunity in vivo. Here we show that SOCS1−/−IFN-γ−/− mice exhibited significantly enhanced resistance to influenza infection, as evidenced by improved viral clearance, attenuated acute lung damage, and consequently increased survival rates compared to either IFN-γ−/− or WT animals. Enhanced viral clearance in SOCS1−/−IFN-γ−/− mice coincided with a rapid onset of adaptive immune responses during acute infection, while their reduced lung injury was associated with decreased inflammatory cell infiltration at the resolution phase of infection. We further determined the contribution of SOCS1-deficient T cells to antiviral immunity. Anti-CD4 antibody treatment of SOCS1−/−IFN-γ−/− mice had no significant effect on their enhanced resistance to influenza infection, while CD8+ splenocytes from SOCS1−/−IFN-γ−/− mice were sufficient to rescue RAG1−/− animals from an otherwise lethal infection. Surprisingly, despite their markedly reduced viral burdens, RAG1−/− mice reconstituted with SOCS1−/−IFN-γ−/− adaptive immune cells failed to ameliorate influenza-induced lung injury. In conclusion, in the absence of IFN-γ, the cytoplasmic protein SOCS1 not only inhibits adaptive antiviral immune responses but also exacerbates inflammatory lung damage. Importantly, these detrimental effects of SOCS1 are conveyed through discrete cell populations. Specifically, while SOCS1 expression in adaptive immune cells is sufficient to inhibit antiviral immunity, SOCS1 in innate/stromal cells is responsible for aggravated lung injury.

Postinfluenza methicillin-resistant Staphylococcus aureus (MRSA) infection can quickly develop into severe, necrotizing pneumonia, causing over 50% mortality despite antibiotic treatments. In this study, we investigated the efficacy of antibiotic therapies and the impact of S. aureus alpha-toxin in a model of lethal influenza virus and MRSA coinfection. We demonstrate that antibiotics primarily attenuate alpha-toxin-induced acute lethality, even though both alpha-toxin-dependent and -independent mechanisms significantly contribute to animal mortality after coinfection. Furthermore, we found that the protein synthesis-suppressing antibiotic linezolid has an advantageous therapeutic effect on alpha-toxin-induced lung damage, as measured by protein leak and lactate dehydrogenase (LDH) activity. Importantly, using a Panton-Valentine leucocidin (PVL)-negative MRSA isolate from patient sputum, we show that linezolid therapy significantly improves animal survival from postinfluenza MRSA pneumonia compared with vancomycin treatment. Rather than improved viral or bacterial control, this advantageous therapeutic effect is associated with a significantly attenuated proinflammatory cytokine response and acute lung damage in linezolid-treated mice. Together, our findings not only establish a critical role of alpha-toxin in the extreme mortality of secondary MRSA pneumonia after influenza but also provide support for the possibility that linezolid could be a more effective treatment than vancomycin to improve disease outcomes.