Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

Interleukin 17C (IL-17C) is a member of the IL-17 family that is selectively induced in epithelia by bacterial challenge and inflammatory stimuli. Here we show that IL-17C functioned in a unique autocrine manner, binding to a receptor complex consisting of the receptors IL-17RA and IL-17RE, which was preferentially expressed on tissue epithelial cells. IL-17C stimulated epithelial inflammatory responses, including the expression of proinflammatory cytokines, chemokines and antimicrobial peptides, which were similar to those induced by IL-17A and IL-17F. However, IL-17C was produced by distinct cellular sources, such as epithelial cells, in contrast to IL-17A, which was produced mainly by leukocytes, especially those of the T(H)17 subset of helper T cells. Whereas IL-17C promoted inflammation in an imiquimod-induced skin-inflammation model, it exerted protective functions in dextran sodium sulfate-induced colitis. Thus, IL-17C is an essential autocrine cytokine that regulates innate epithelial immune responses.

ABSTRACTThe strong restriction barrier present inStaphylococcus aureusandStaphylococcus epidermidishas limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain ofS. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiencyEscherichia colicloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates ofS. aureusandS. epidermidis. We also show that the loss of restriction (both type I and IV) in anS. aureusUSA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens.IMPORTANCEStaphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding ofStaphylococcus aureusandStaphylococcus epidermidisinfections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.

H3K27 methylation mediated by the histone methyltransferase complex PRC2 is critical for transcriptional regulation, Polycomb silencing, Drosophila segmentation, mammalian X chromosome inactivation, and cancer. PRC2-mediated H3K27 methylation can spread along the chromatin and propagate the repressive chromatin environment; thus, chromatin components that antagonize the activity of PRC2 are important for restraining Polycomb silencing. Here we report that in HeLa cells, H3 histones unmethylated at Lys-36 are mostly methylated at Lys-27, with the exception of newly synthesized H3. In addition, K27me3 rarely co-exists with K36me2 or K36me3 on the same histone H3 polypeptide. Moreover, PRC2 activity is greatly inhibited on nucleosomal substrates with preinstalled H3K36 methylation. These findings collectively identify H3K36 methylation as a chromatin component that restricts the PRC2-mediated spread of H3K27 methylation. Finally, we provide evidence that the controversial histone lysine methyltransferase Ash1, a known Trithorax group protein that antagonizes Polycomb silencing in vivo, is an H3K36-specific dimethylase, not an H3K4 methylase, further supporting the role of H3K36 methylation in antagonizing PRC2-mediated H3K27 methylation.

T-helper-17 (Th17) cells have critical roles in mucosal defense and in autoimmune disease pathogenesis 1-3. They are most abundant in the small intestine lamina propria (SILP), where their presence requires colonization of mice with microbiota 4-7. Segmented Filamentous Bacteria (SFB) are sufficient to induce Th17 cells and to promote Th17-dependent autoimmune disease in animal models 8-14. However, the specificity of Th17 cells, the mechanism of their induction by distinct bacteria, and the means by which they foster tissue-specific inflammation remain unknown. Here we show that the T cell receptor (TCR) repertoire of intestinal Th17 cells in SFB-colonized mice has minimal overlap with that of other intestinal CD4+ T cells and that most Th17 cells, but not other T cells, recognize antigens encoded by SFB. T cells with antigen receptors specific for SFB-encoded peptides differentiated into RORγt-expressing Th17 cells, even if SFB-colonized mice also harbored a strong Th1 cell inducer, Listeria monocytogenes, in their intestine. The match of T cell effector function with antigen specificity is thus determined by the type of bacteria that produce the antigen. These findings have significant implications for understanding how commensal microbiota contribute to organ-specific autoimmunity and for developing novel mucosal vaccines.