Hospitalization of the elderly for invasive pneumococcal disease is frequently accompanied by the occurrence of an adverse cardiac event; these are primarily new or worsened heart failure and cardiac arrhythmia. Herein, we describe previously unrecognized microscopic lesions (microlesions) formed within the myocardium of mice, rhesus macaques, and humans during bacteremic Streptococcus pneumoniae infection. In mice, invasive pneumococcal disease (IPD) severity correlated with levels of serum troponin, a marker for cardiac damage, the development of aberrant cardiac electrophysiology, and the number and size of cardiac microlesions. Microlesions were prominent in the ventricles, vacuolar in appearance with extracellular pneumococci, and remarkable due to the absence of infiltrating immune cells. The pore-forming toxin pneumolysin was required for microlesion formation but Interleukin-1β was not detected at the microlesion site ruling out pneumolysin-mediated pyroptosis as a cause of cell death. Antibiotic treatment resulted in maturing of the lesions over one week with robust immune cell infiltration and collagen deposition suggestive of long-term cardiac scarring. Bacterial translocation into the heart tissue required the pneumococcal adhesin CbpA and the host ligands Laminin receptor (LR) and Platelet-activating factor receptor. Immunization of mice with a fusion construct of CbpA or the LR binding domain of CbpA with the pneumolysin toxoid L460D protected against microlesion formation. We conclude that microlesion formation may contribute to the acute and long-term adverse cardiac events seen in humans with IPD.

Staphylococcus aureus trigger inflammation through inflammasome activation and recruitment of neutrophils, responses critical for pathogen clearance but associated with substantial tissue damage. We postulated that necroptosis, cell death mediated by the RIPK1/RIPK3/MLKL pathway, would function to limit pathological inflammation. In models of skin infection or sepsis, Mlkl−/− mice had high bacterial loads, inability to limit IL.1b production and excessive inflammation. Similarly, mice treated with RIPK1 or RIPK3 inhibitors had increased bacterial loads in a model of sepsis. Ripk3−/− mice exhibited increased staphylococcal clearance and decreased inflammation in skin and systemic infection, due to direct effects of RIPK3 on IL-1b activation and apoptosis. In contrast to Casp1/4 −/− mice with defective S. aureus killing, the poor outcomes of Mlkl−/− mice could not be attributed to impaired phagocytic function. We conclude that necroptotic cell death limits the pathological inflammation induced by S. aureus.

Skin is the most common site of Staphylococcus aureus infection. While most of these infections are self-limited, recurrent infections are common. Keratinocytes and recruited immune cells participate in skin defense against infection. We postulated that S. aureus is able to adapt to the milieu within human keratinocytes to avoid keratinocyte-mediated clearance. From a collection of S. aureus isolated from chronically infected patients with atopic dermatitis, we noted 22% had an agr mutant-like phenotype. Using several models of human skin infection, we demonstrate that toxin-deficient, agr mutants of methicillin-resistant S. aureus (MRSA) USA300 are able to persist within keratinocytes by stimulating autophagy and evading caspase-1 and inflammasome activation. MRSA infection induced keratinocyte autophagy, as evidenced by galectin-8 and LC3 accumulation. Autophagy promoted the degradation of inflammasome components and facilitated staphylococcal survival. The recovery of more than 58% agr or RNAIII mutants (P < 0.0001) of an inoculum of wild-type (WT) MRSA from within wortmannin-treated keratinocytes compared to control keratinocytes reflected the survival advantage for mutants no longer expressing agr-dependent toxins. Our results illustrate the dynamic interplay between S. aureus and keratinocytes that can result in the selection of mutants that have adapted specifically to evade keratinocyte-mediated clearance mechanisms.

Streptococcus pneumoniae is the leading cause of communityacquired pneumonia and sepsis, with adult hospitalization linked to approximately 19% incidence of an adverse cardiac event (e.g., heart failure, arrhythmia, infarction). Herein, we review the specific host-pathogen interactions that contribute to cardiac dysfunction during invasive pneumococcal disease: (1) cell wall-mediated inhibition of cardiomyocyte contractility; (2) the new observation that S. pneumoniae is capable of translocation into the myocardium and within the heart, forming discrete, nonpurulent, microscopic lesions that are filled with pneumococci; and (3) the bacterial virulence determinants, pneumolysin and hydrogen peroxide, that are most likely responsible for cardiomyocyte cell death. Pneumococcal invasion of heart tissue is dependent on the bacterial adhesin choline-binding protein A that binds to laminin receptor on vascular endothelial cells and binding of phosphorylcholine residues on pneumococcal cell wall to platelet-activating factor receptor. These are the same interactions responsible for pneumococcal translocation across the blood-brain barrier during the development of meningitis. We discuss these interactions and how their neutralization, either with antibody or therapeutic agents that modulate platelet-activating factor receptor expression, may confer protection against cardiac damage and meningitis. Considerable collagen deposition was observed in hearts of mice that had recovered from invasive pneumococcal disease. We discuss the possibility that cardiac scar formation after severe pneumococcal infection may explain why individuals who are hospitalized for pneumonia are at greater risk for sudden death up to 1 year after infection.

Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a polymorphic fungus, are common constituents of the microbiome as well as increasingly problematic causes of infections. Interestingly, we previously showed that these two species antagonize each other’s virulence and that E. faecalis inhibition of C. albicans was specifically mediated by EntV. EntV is a bacteriocin encoded by the entV (ef1097) locus that reduces C. albicans virulence and biofilm formation by inhibiting hyphal morphogenesis. In this report, we studied the posttranslational modifications necessary for EntV antifungal activity. First, we show that the E. faecalis secreted enzyme gelatinase (GelE) is responsible for cleaving EntV into its 68-amino-acid, active form and that this process does not require the serine protease SprE. Furthermore, we demonstrate that a disulfide bond that forms within EntV is necessary for antifungal activity. Abrogating this bond by chemical treatment or genetic modification rendered EntV inactive against C. albicans. Moreover, we identified the likely catalyst of this disulfide bond, a previously uncharacterized thioredoxin within the E. faecalis genome called DsbA. Loss of DsbA, or disruption of its redox-active cysteines, resulted in loss of EntV antifungal activity. Finally, we show that disulfide bond formation is not a prerequisite for cleavage; EntV cleavage proceeded normally in the absence of DsbA. In conclusion, we present a model in which following secretion, EntV undergoes disulfide bond formation by DsbA and cleavage by GelE in order to generate a peptide capable of inhibiting C. albicans. IMPORTANCE Enterococcus faecalis and Candida albicans are among the most important and problematic pathobionts, organisms that normally are harmless commensals but can cause dangerous infections in immunocompromised hosts. In fact, both organisms are listed by the Centers for Disease Control and Prevention as serious global public health threats stemming from the increased prevalence of antimicrobial resistance. The rise in antifungal resistance is of particular concern considering the small arsenal of currently available therapeutics. EntV is a peptide with antifungal properties, and it, or a similar compound, could be developed into a therapeutic alternative, either alone or in combination with existing agents. However, to do so requires understanding what properties of EntV are necessary for its antifungal activity. In this work, we studied the posttranslational processing of EntV and what modifications are necessary for inhibition of C. albicans in order to fill this gap in knowledge.