Jeffrey S. Kavanaugh

Diabetic wounds are often infected with microbes, which perpetuate inflammation, and stall wound healing. The bacterium Group B Streptococcus (GBS) is frequently isolated from diabetic wounds; however, little is known about how GBS adapts to survive in this niche. Previously, we found that GBS acquires stable mutations in the major two-component system CovRS during murine diabetic wound infection that result in increased pigmentation. Here, we further characterize these pigmented variants and determine the consequences on GBS survival. Using a murine model of wound infection, we find that covRS mutants arise specifically in diabetic hosts and are selected for across multiple GBS backgrounds. Whole genome sequencing of pigmented isolates revealed mutations in both covR and covS , with most isolates having a single nucleotide insertion or deletion in the covR promoter region. Phenotypic analysis of murine-acquired mutants reveals enhanced traits associated with virulence, including increased hemolytic activity, host cell cytotoxicity, and elevated nuclease activity. While our previous and current study indicated that engineered covR deletion mutants do not exhibit increased survival in the diabetic wound, we observe that a pigmented isolate survived better than wild-type during co-infection with Enterococcus faecalis , another frequently isolated wound pathogen. Finally, we find that depletion of neutrophils reduces the frequency of covRS mutant variants that arise in the population. Our work highlights the emergence of covRS mutations in GBS, and the consequences of these variations are associated with enhanced virulence and competitive fitness, underscoring the importance of these regulatory changes in the context of diabetic wounds.

Staphylococcus aureus, the most frequent cause of skin infections, is more common in men than women and selectively colonizes the skin during inflammation. Yet, the specific cues that drive infection in these settings remain unclear. Here we show that the host androgens testosterone and dihydrotestosterone promote S. aureus pathogenesis and skin infection. Without the secretion of these hormones, skin infection in vivo is limited. Testosterone activates S. aureus virulence in a concentration dependent manner through stimulation of the agr quorum sensing system, with the capacity to circumvent other inhibitory signals in the environment. Taken together, our work defines a previously uncharacterized inter-kingdom signal between the skin and the opportunistic pathogen S. aureus and identifies the mechanism of sex-dependent differences in S. aureus skin infection. One-Sentence Summary: Testosterone promotes S. aureus pathogenesis through activation of the agr quorum sensing system.

Neutrophils, polymorphonuclear leukocytes (PMN), play a critical role in the innate immune response to Staphylococcus aureus, a pathogen that continues to be associated with significant morbidity and mortality. Neutrophil extracellular trap (NET) formation is involved in ensnaring and killing of S. aureus, but this host–pathogen interaction also leads to host tissue damage. Importantly, NET components including neutrophil proteases are under consideration as therapeutic targets in a variety of disease processes. Although S. aureus lipoproteins are recognized to activate cells via TLRs, specific mechanisms of interaction with neutrophils are poorly delineated. We hypothesized that a lipoprotein-containing cell membrane preparation from methicillin-resistant S. aureus (MRSA-CMP) would elicit PMN activation, including NET formation. We investigated MRSA-CMP–elicited NET formation, regulated elastase release, and IL-8 production in human neutrophils. We studied PMN from healthy donors with or without a common single-nucleotide polymorphism in TLR1, previously demonstrated to impact TLR2/1 signaling, and used cell membrane preparation from both wild-type methicillin-resistant S. aureus and a mutant lacking palmitoylated lipoproteins (lgt). MRSA-CMP elicited NET formation, elastase release, and IL-8 production in a lipoprotein-dependent manner. TLR2/1 signaling was involved in NET formation and IL-8 production, but not elastase release, suggesting that MRSA-CMP–elicited elastase release is not mediated by triacylated lipoproteins. MRSA-CMP also primed neutrophils for enhanced NET formation in response to a subsequent stimulus. MRSA-CMP–elicited NET formation did not require Nox2-derived reactive oxygen species and was partially dependent on the activity of peptidyl arginine deiminase (PAD). In conclusion, lipoproteins from S. aureus mediate NET formation via TLR2/1 with clear implications for patients with sepsis. Visual Abstract Key Points S. aureus lipoproteins drive NET formation and involve TLR2/1 and PAD enzymes. NET formation can be primed by lipoprotein-containing S. aureus cell membrane.

Implant-associated Staphylococcus aureus infections are difficult to treat because of biofilm formation. Bacteria in a biofilm are often insensitive to antibiotics and host immunity. Monoclonal antibodies (mAbs) could provide an alternative approach to improve the diagnosis and/or treatment of biofilm-related infections. Here we show that mAbs targeting common surface components of S. aureus can recognize clinically relevant biofilm types. We identify two groups of antibodies: one group that uniquely binds S. aureus in biofilm state and one that recognizes S. aureus in both biofilm and planktonic state. In a mouse model, we show that mAb 4497 (recognizing wall teichoic acid (WTA)) specifically localizes to biofilm-infected implants. In conclusion, we demonstrate the capacity of several human mAbs to detect S. aureus biofilms in vitro and in vivo . This is an important first step to develop mAbs for imaging or treating S. aureus biofilms.

Human hemoglobin produced in the Escherichiacoli coexpression system of Hernan et al. [(1992) Biochemistry31, 8619−8628] has been transformed into a functionally homogeneous protein whose properties closely approximate those of normal hemoglobin A. Both of the α and β chains of this hemoglobin contain a valine−methionine substitution at position 1 in order to accommodate the difference in specificity of the protein-processing enzymes of procaryotes. Despite extensive purification, functional homogeneity of the E.coli expressed hemoglobin was achieved only by the complete disassembly of the hemoglobin into its component α and β globins and their reassembly in the presence of hemin. The kinetics of CO combination and the thermodynamics of O₂ binding and cooperativity of the reassembled αV1M−βV1M hemoglobin closely approximate those of HbA. The α globin obtained from the E.coli expressed hemoglobin was also combined with normal human β chains and hemin to form the αV1M variant. The α+M variant of HbA, in which the normal N-terminal valine of the α chains is preceded by a methionine residue, was prepared by the same procedure. The kinetics of the reactions of CO with the αV1M and α+M variants are similar to those for HbA. The equilibria of oxygen binding to αV1M and HbA are similar whereas α+M exhibits a significantly higher oxygen affinity. The three-dimensional structures of αV1M and α+M offer an explanation for the latter affinity difference. Although the structures of αV1M and HbA, which have been determined by X-ray crystallography, are virtually indistinguishable except at the N-terminal residues, that of α+M indicates the displacement of a solvent molecule, possibly a chloride ion, from arginine 141α. Such an alteration in an anion binding site could result in increased oxygen affinity.