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Neutrophils kill invading pathogens by AMPs, including cathelicidins, ROS, and NETs. The human pathogen Staphylococcus aureus exhibits enhanced resistance to neutrophil AMPs, including the murine cathelicidin CRAMP, in part, as a result of alanylation of teichoic acids by the dlt operon. In this study, we took advantage of the hypersusceptible phenotype of S. aureus DeltadltA against cationic AMPs to study the impact of the murine cathelicidin CRAMP on staphylococcal killing and to identify its key site of action in murine neutrophils. We demonstrate that CRAMP remained intracellular during PMN exudation from blood and was secreted upon PMA stimulation. We show first evidence that CRAMP was recruited to phagolysosomes in infected neutrophils and exhibited intracellular activity against S. aureus. Later in infection, neutrophils produced NETs, and immunofluorescence revealed association of CRAMP with S. aureus in NETs, which similarly killed S. aureus wt and DeltadltA, indicating that CRAMP activity was reduced when associated with NETs. Indeed, the presence of DNA reduced the antimicrobial activity of CRAMP, and CRAMP localization in response to S. aureus was independent of the NADPH oxidase, whereas killing was partially dependent on a functional NADPH oxidase. Our study indicates that neutrophils use CRAMP in a timed and locally coordinated manner in defense against S. aureus.

Staphylococcus epidermidis is an opportunistic bacterium whose infections often involve the formation of a biofilm on implanted biomaterials. In S. epidermidis, the exopolysaccharide facilitating bacterial adherence in a biofilm is polysaccharide intercellular adhesin (PIA), whose synthesis requires the enzymes encoded within the intercellular adhesin operon (icaADBC). In vitro, the formation of S. epidermidis biofilms is enhanced by conditions that repress tricarboxylic acid (TCA) cycle activity, such as growth in a medium containing glucose. In many Gram-positive bacteria, repression of TCA cycle genes in response to glucose is accomplished by catabolite control protein A (CcpA). CcpA is a member of the GalR-LacI repressor family that mediates carbon catabolite repression, leading us to hypothesize that catabolite control of S. epidermidis biofilm formation is indirectly regulated by CcpA-dependent repression of the TCA cycle. To test this hypothesis, ccpA deletion mutants were constructed in strain 1457 and 1457-acnA and the effects on TCA cycle activity, biofilm formation and virulence were assessed. As anticipated, deletion of ccpA derepressed TCA cycle activity and inhibited biofilm formation; however, ccpA deletion had only a modest effect on icaADBC transcription. Surprisingly, deletion of ccpA in strain 1457-acnA, a strain whose TCA cycle is inactive and where icaADBC transcription is derepressed, strongly inhibited icaADBC transcription. These observations demonstrate that CcpA is a positive effector of biofilm formation and icaADBC transcription and a repressor of TCA cycle activity.

Staphylococcus aureus infection elicits through its mature lipoproteins an innate immune response by TLR2–MyD88 signaling, which improves bacterial clearing and disease outcome. The role of dendritic cells (DCs) and T cells in this immune activation and the function of T and B cells in defense against S. aureus infection remain unclear. Therefore, we first evaluated DC and T cell activation after infection with S. aureus wild type (WT) and its isogenic mutant, which is deficient in lipoprotein maturation, in vitro. Lipoproteins in viable S. aureus contributed via TLR2–MyD88 to activation of DCs, which promoted the release of IFN-γ and IL-17 in CD4+ T cells. This strong effect was independent of superantigens and MHC class II. We next evaluated the function of T cells and their cytokines IFN-γ and IL-17 in infection in vivo. Six days after systemic murine infection IFN-γ, IL-17, and IL-10 production in total spleen cells were MyD88-dependent and their levels increased until day 21. The comparison of CD3−/−, Rag2−/−, and C57BL/6 mice after infection revealed that IFN-γ and IL-17 originated from T cells and IL-10 originated from innate immune cells. Furthermore, vaccination of mice to activate T and B cells did not improve eradication of S. aureus from organs. In conclusion, S. aureus enhances DC activation via TLR2–MyD88 and thereby promotes TH1 and TH17 cell differentiation. However, neither T cells and their MyD88-regulated products, IFN-γ and IL-17, nor B cells affected bacterial clearing from organs and disease outcome.

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