Faculty Bibliography

Staphylococcus aureus is a global health concern, resulting in significant disease burden in both hospital and community settings. To establish infection, the bacteria must contend with a multitude of host defense mechanisms, including "nutritional immunity", in which nutrients are sequestered away from invading pathogens. Importantly, S. aureus requires iron for growth during infection, which it acquires through the lysis of erythrocytes (hemolysis). HlgAB, a secreted bi-component pore forming toxin, contributes to the ability of S. aureus to lyse erythrocytes to release heme iron. HlgAB consists of two subunits, the S-subunit HlgA and the F-subunit HlgB. Prior work has shown that the hemolytic activity of HlgAB is dependent on the binding of HlgA to the host receptor Duffy Antigen Receptor for Chemokines (DARC). Here we show that HlgB binds the surface of erythrocytes independently of DARC or HlgA. Our comparative genomic analysis reveals high conservation of hlgA and hlgB genes across S. aureus lineages. By performing structure-function studies, we identified a series of loops within the rim domain of HlgB that are required for the binding of HlgB to erythrocytes and erythrocyte lysis by HlgAB. The importance of HlgB-mediated host targeting was validated in a tissue culture model of S. aureus-mediated lysis of primary human erythrocytes, in an in vivo murine model of intoxication, and during in vivo systemic infection. Altogether, these findings expand our mechanistic insights into how S. aureus overcomes nutritional immunity, and the role of HlgB in S. aureus pathophysiology.

We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the Φ11 prophage (mΦ11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present report shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding a known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an mΦ11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPA's role in biofilm formation. Collectively, these data reveal a critical mechanism-epigenetic regulation of staphylococcal gene expression-by which phage can regulate virulence to drive adaptive leaps by S. aureus.

Pathogens have evolved to be highly adapted to their natural host. Community-associated methicillin-resistant Staphylococcus aureus USA300, for instance, is a lineage responsible for the epidemic of skin and soft tissue infections (SSTIs) in humans. Owing to its human tropism, mechanisms that enabled the rise of USA300 as a major skin pathogen remain incompletely defined. By leveraging a rodent-adapted strain of S. aureus, we developed a natural model of SSTIs. We found that LukMF', a pore-forming leukocidin homolog to the human-specific LukSF-PV toxin, drives skin pathology in mice. LukMF' lyses neutrophils via the chemokine receptor CCR1, which in turn fuels inflammatory pathology and microbial survival within the infectious nidus. Ablation of CCR1, depletion of neutrophils, or vaccination with LukMF' all protected mice from skin pathology. Thus, these data support epidemiological studies linking leukocidins with human SSTIs and highlight the power of natural models to unearth potential targets to curtail infections.

In the human pathogen Staphylococcus aureus, the two-component regulatory system SaeRS contributes to the expression of numerous virulence factors essential for pathogenesis. The kinase and phosphatase activities of SaeS are stimulated by several host and physiological signals, resulting in increased phosphorylation of the transcription factor SaeR and increased transcriptional activity of regulated promoters. It was recently demonstrated that the accumulation of fatty acids negatively impacts SaeS activity, decreasing titers of phosphorylated SaeP and transcriptional output. Triclosan is an effective antimicrobial that has been integrated as an ingredient in a variety of healthcare and consumer products. The chlorinated compound is recalcitrant to natural or biological transformations, resulting in environmental accumulation. At low concentrations, triclosan is a bacteriostatic inhibitor of enoyl-acetyl carrier protein reductase (FabI) of the type II fatty acid synthesis system (FASII), which is necessary for the elongation and synthesis of fatty acids. Herein, we demonstrate that the treatment of S. aureus with a growth-permissive concentration of triclosan alters the titers of cell-associated fatty acids and thereby functions as an activator of SaeRS. Triclosan-dependent activation of SaeRS subsequently resulted in increased transcription and expression of genes that code for virulence factors. These phenotypes are chemically reversed by the exogenous addition of oleic acid, which inactivates SaeRS, and genetically reversed by crippling the FakAB fatty acid kinase system, which generates phosphorylated fatty acids for incorporation into phospholipids. These findings present implications for the widespread use of triclosan as an antimicrobial agent in household products and its role as a persistent environmental pollutant.

The T300A substitution in ATG16L1 associated with Crohn's disease impairs autophagy, yet up to 50% of humans are heterozygous for this allele. Here, we demonstrate that heterozygosity for the analogous substitution in mice (Atg16L1^T316A), but not homozygosity, protects against lethal Salmonella enterica Typhimurium infection. One copy of Atg16L1^T316A was sufficient to enhance cytokine production through inflammasome activation, which was necessary for protection. In contrast, two copies of Atg16L1^T316A inhibited the autophagy-related process of LC3-associated phagocytosis (LAP) and increased susceptibility. Macrophages from human donors heterozygous for ATG16L1^T300A displayed elevated inflammasome activation while homozygosity impaired LAP, similar to mice. These results clarify how the T300A substitution impacts ATG16L1 function and suggest it can be beneficial to heterozygous carriers, providing an explanation for its prevalence within the human population.

Staphylococcus aureus is a major cause of bacterial infection-related deaths. Increasing antimicrobial resistance highlights the urgent need for effective preventative strategies. Antibody-mediated opsonophagocytosis, the key mechanism for protection against S. aureus, is disabled by critical virulence factors such as Staphylococcal protein A (SpA) and leukocidin AB (LukAB). In our study, we combined genetically detoxified vaccine candidates SpA* and LukAB RARPR-33 with a T_H1 adjuvant aiming to restore host antibody functionality. To evaluate these vaccine candidates, we developed both surgical site infection (SSI) and superficial wound infection (SWI) models in minipigs. Our results showed a significant reduction in bacterial load and systemic dissemination in the SSI model, while skin infection severity was markedly decreased after intradermal immunization in the SWI model. This study introduces a novel S. aureus vaccine strategy by targeting immune evasion factors SpA and LukAB, utilizing potent T_H1 adjuvants, and employing minipig challenge models.

Gastrointestinal (GI) colonization by methicillin-resistant Staphylococcus aureus (MRSA) is associated with a high risk of transmission and invasive disease in vulnerable populations. The immune and microbial factors that permit GI colonization remain unknown. Male sex is correlated with enhanced Staphylococcus aureus nasal carriage, skin and soft tissue infections, and bacterial sepsis. Here, we established a mouse model of sexual dimorphism during GI colonization by MRSA. Our results show that in contrast to male mice that were susceptible to persistent colonization, female mice rapidly cleared MRSA from the GI tract following oral inoculation in a manner dependent on the gut microbiota. This colonization resistance displayed by female mice was mediated by an increase in IL-17A+ CD4+ T cells (Th17) and dependent on neutrophils. Ovariectomy of female mice increased MRSA burden, but gonadal female mice that have the Y chromosome retained enhanced Th17 responses and colonization resistance. Our study reveals a novel intersection between sex and gut microbiota underlying colonization resistance against a major widespread pathogen.

Pathogenic Enterobacter species are of increasing clinical concern due to the multidrug-resistant nature of these bacteria, including resistance to carbapenem antibiotics. Our understanding of Enterobacter virulence is limited, hindering the development of new prophylactics and therapeutics targeting infections caused by Enterobacter species. In this study, we assessed the virulence of contemporary clinical Enterobacter hormaechei isolates in a mouse model of intraperitoneal infection and used comparative genomics to identify genes promoting virulence. Through mutagenesis and complementation studies, we found two porin-encoding genes, ompC and ompD, to be required for E. hormaechei virulence. These porins imported clinically relevant carbapenems into the bacteria, and thus loss of OmpC and OmpD desensitized E. hormaechei to the antibiotics. Our genomic analyses suggest porin-related genes are frequently mutated in E. hormaechei, perhaps due to the selective pressure of antibiotic therapy during infection. Despite the importance of OmpC and OmpD during infection of immunocompetent hosts, we found the two porins to be dispensable for virulence in a neutropenic mouse model. Moreover, porin loss provided a fitness advantage during carbapenem treatment in an ex vivo human whole blood model of bacteremia. Our data provide experimental evidence of pathogenic Enterobacter species gaining antibiotic resistance via loss of porins and argue antibiotic therapy during infection of immunocompromised patients is a conducive environment for the selection of porin mutations enhancing the multidrug-resistant profile of these pathogens.

BACKGROUND:Bloodstream infections (BSI) are associated with high mortality rates, particularly when caused by resistant pathogens. Reducing the delay in diagnosis and initiation of appropriate treatment is crucial for improving clinical outcomes. The implementation of polymerase chain reaction (PCR) tests in the diagnostic process offers a promising approach to achieving quicker identification of pathogens, thereby potentially reducing mortality associated with BSI. METHODS:BSI, for which diagnostic protocol has been unchanged. RESULTS:(VRE) BSI and 384 with MSSA BSI. The mean 30-day mortality risk difference in the period post-intervention estimated in our difference-in-differences model was -6.03 per 100 (95% CI: -10.35 to -1.7), with event study plots suggesting minimal deviation from parallel trends in the pre-treatment period. CONCLUSIONS:Findings suggest that introduction of BCID2 PCR testing for enterococcal bloodstream infections (BSI) may be associated with a reduction in mortality, however, interpretation of the effects must be approached with caution given the relative imprecision of estimates. Further research with larger samples is essential to establish a definitive conclusion on the impact of rapid PCR testing on mortality in BSI. This is an innovative approach using causal methods to evaluate interventions aimed at the improvement of infection control and antimicrobial treatment strategies.

The antimicrobial activity of histones was discovered in the 1940s, but their mechanism of action is not fully known. Here we show that methicillin-resistant Staphylococcus aureus (MRSA) is susceptible to histone H1 (H1), even in the presence of divalent cations and serum. Through selective evolution and a genome-wide screen of a transposon library, as well as physiological and pharmacological experiments, we elucidated how H1 kills MRSA. We show that H1 first binds to wall teichoic acids with high affinity. Once bound, H1 requires a potentiated membrane and a metabolically active bacterium to permeabilize the membrane and enter the cell. Upon entry, H1 accumulates intracellularly, in close association with the bacterial DNA. Of note, anti-H1 antibodies inhibit neutrophil extracellular trap killing of MRSA. Moreover, H1 colocalizes with bacterial DNA in abscess samples of MRSA-infected patients, suggesting a role for H1 in combating MRSA in vivo.