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Infections caused by Staphylococcus aureus are a leading cause of mortality. Treating infections caused by S. aureus is difficult due to resistance against most traditional antibiotics, including β-lactams. We previously reported the presence of mutations in gdpP among S. aureus strains that were obtained by serial passaging in β-lactam drugs. Similar mutations have recently been reported in natural S. aureus isolates that are either nonsusceptible or resistant to β-lactam antibiotics. gdpP codes for a phosphodiesterase that cleaves cyclic-di-AMP (CDA), a newly discovered second messenger. In this study, we sought to identify the role of gdpP in β-lactam resistance in S. aureus. Our results showed that gdpP-associated mutations caused loss of phosphodiesterase function, leading to increased CDA accumulation in the bacterial cytosol. Deletion of gdpP led to an enhanced ability of the bacteria to withstand a β-lactam challenge (2 to 3 log increase in bacterial CFU) by promoting tolerance without enhancing MICs of β-lactam antibiotics. Our results demonstrated that increased drug tolerance due to loss of GdpP function can provide a selective advantage in acquisition of high-level β-lactam resistance. Loss of GdpP function thus increases tolerance to β-lactams that can lead to its therapy failure and can permit β-lactam resistance to occur more readily.

Penicillin binding protein 4 (PBP4) can provide high-level β-lactam resistance in Staphylococcus aureus A series of missense and promoter mutations associated with pbp4 were detected in strains that displayed high-level resistance. We show here that the missense mutations facilitate the β-lactam resistance mediated by PBP4 and the promoter mutations lead to overexpression of pbp4 Our results also suggest a cooperative interplay among PBPs for β-lactam resistance.

Autosomal dominant hyper IgE syndrome (AD-HIES), or Job's syndrome, is a primary immune deficiency caused by dominant-negative mutations in STAT3. Recurrent Staphylococcus aureus skin abscesses are a defining feature of this syndrome. A widely held hypothesis that defects in peripheral Th17 differentiation confer this susceptibility has never been directly evaluated. To assess the cutaneous immune response in AD-HIES, we induced suction blisters in healthy volunteers (HVs) and patients with AD-HIES and then challenged the wound with lethally irradiated bacteria. We show that cutaneous production of IL-17A and IL-17F was normal in patients with AD-HIES. Overproduction of TNF- [alpha] differentiated the responses in ADHIES from HVs. This was associated with reduced IL-10 family signaling in blister- infiltrating cells and defective epithelial cell function. Mouse models of AD-HIES recapitulated these aberrant epithelial responses to S. aureus and involved defective epithelial-to-mesenchymal transition (EMT) rather than a failure of bacterial killing. Defective responses in mouse models of AD-HIES and primary keratinocyte cultures from patients with AD-HIES could be reversed by TNF-[alpha] blockade and by drugs with reported modulatory effects on EMT. Our results identify these as potential therapeutic approaches in patients with ADHIES suffering S. aureus infections., Introduction Autosomal dominant hyper IgE syndrome (AD-HIES), also known as Job's syndrome, is a primary immune deficiency caused by dominant-negative loss-of-function mutations in the STAT3 gene. Recurrent Staphylococcus aureus skin [...]

TLRs sense various microbial products. Their function has been best characterized in DCs and macrophages, where they act as important mediators of innate immunity. TLR4 is also expressed on [CD4.sup.+] T cells, but its physiological function on these cells remains unknown. Here, we have shown that TLR4 triggering on [CD4.sup.+] T cells affects their phenotype and their ability to provoke intestinal inflammation. In a model of spontaneous colitis, [Il10.sup.[-/-]] [Tlr4.sup.[-/-]] mice displayed accelerated development of disease, with signs of overt colitis as early as 8 weeks of age, when compared with [Il10.sup.[-/-]] and [Il10.sup.[-/-]][Tlr9.sup.[-/-]] mice, which did not develop colitis by 8 months. Similar results were obtained in a second model of colitis in which transfer of naive [Il10.sup.[-/-]] [Tlr4.sup.[-/-]] [CD4.sup.4] T cells into [Rag1.sup.[-/-]]recipients sufficient for both IL-10 and TLR4 induced more aggressive colitis than the transfer of naive [Il10.sup.[-/-]] [CD4.sup.+] T cells. Mechanistically, LPS stimulation of TLR4-bearing [CD4.sup.+] T cells inhibited ERK1/2 activation upon subsequent TCR stimulation via the induction of MAPK phosphatase 3 (MKP-3). Our data therefore reveal a tonic inhibitory role for TLR4 signaling on subsequent TCR-dependent [CD4.sup.+] T cell responses., Introduction IL-10 is an antiinflammatory cytokine that regulates T cell proliferation and functions (1). [Il10.sup.[-/-]] mice develop spontaneous chronic enterocolitis with mucosal infiltration of lymphocytes, macrophages, and neutrophils (2) similarly [...]

Most modern research into the immune effects of breast milk has focused on the impacts of immunoglobulin or oligosaccharide content. However, immediately prior to parturition, the cell populations of breast milk become selectively enriched for CD8+ T cells of an effector memory subtype. Despite this observation that the cellular content of breast milk contains a distinct leukocyte population when compared to peripheral blood, the physiologic role of these CD8+ effector memory cells is unknown. Research encompassing animal models and humans has demonstrated that leukocytes are capable of transferring antigen‐specific immunity even when lysed, dialyzed to enrich for fractions less than 10 kDa, and orally administered. Our previous work built upon these reports to elucidate several aspects of this dialyzable leukocyte extract (DLE) activity: only DLE from T effector memory CD8+ cells was capable of transferring antigen‐specific immunity; the DLE activity was TCRβ dependent; dendritic cells (DCs) were the cellular target of DLE; and DLE enhanced immune activity in epithelial challenge models via induction of IL‐6 from DCs. Herein, we reveal that breast milk dialysate activates similar cytokine and genetic pathways as DLE taken from peripheral blood and murine spleens through TCRβ‐ and CD8‐dependent mechanisms. These findings suggest that the CD8+ memory T cells enriched in breast milk, even after potential lysis in the infant gut, may represent a mechanism for passive transfer of cellular immunity from mother to child. [ABSTRACT FROM AUTHOR]