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.
Ubiquilins (UBQLNs) are adaptor proteins thought to deliver ubiquitinated substrates to proteasomes. Here, we show a role for UBQLN in autophagy: enforced expression of UBQLN protects cells from starvation-induced death, whereas depletion of UBQLN renders cells more susceptible. The UBQLN protective effect requires the autophagy-related genes ATG5 and ATG7, two essential components of autophagy. The ubiquitin-associated domain of UBQLN mediates both its association with autophagosomes and its protective effect against starvation. Depletion of UBQLN delays the delivery of autophagosomes to lysosomes. This study identifies a new role for UBQLN in regulating the maturation of autophagy, expanding the involvement of ubiquitinrelated proteins in this process.
The ability to invade and grow in macrophages is necessary for Mycobacterium tuberculosis to cause disease. We have found a Mycobacterium marinum locus of two genes that is required for both invasion and intracellular survival in macrophages. The genes were designated iipA (mycobacterial invasion and intracellular persistence) and iipB. The iip mutant, which was created by insertion of a kanamycin resistance gene cassette at the 5 region of iipA, was completely avirulent to zebra fish. Expression of the M. tuberculosis orthologue of iipA, Rv1477, fully complemented the iip mutant for infectivity in vivo, as well as for invasion and intracellular persistence in macrophages. In contrast, the iipB orthologue, Rv1478, only partially complemented the iip mutant in vivo and restored invasion but not intracellular growth in macrophages. While IipA and IipB differ at their N termini, they are highly similar throughout their C-terminal NLPC_p60 domains. The p60 domain of Rv1478 is fully functional to replace that of Rv1477, suggesting that the N-terminal sequence of Rv1477 is required for full virulence in vivo and in macrophages. Further mutations demonstrated that both Arg-Gly-Asp (RGD) and Asp-Cys-Ser-Gly (DCSG) sequences in the p60 domain are required for function. The iip mutant exhibited increased susceptibility to antibiotics and lysozyme and failed to fully separate daughter cells in liquid culture, suggesting a role for iip genes in cell wall structure and function. Altogether, these studies demonstrate an essential role for a p60-containing protein, IipA, in the pathogenesis of M. marinum infection.Mycobacterium tuberculosis is an extraordinarily successful human pathogen, with 2 to 3 billion people infected worldwide (12). Its success likely reflects its complex parasitic lifestyle with sophisticated mechanisms for combating host defense. For example, M. tuberculosis inhibits acidification of the bacterium-containing phagosome and its fusion to lysosomes (6,29,33). Within the "maturation-arrested" phagosome, M. tuberculosis proliferates and ultimately kills the host cells by apoptosis and/or necrosis (11,13,22). The ability to invade and grow inside host cells is an important virulence property for pathogenic mycobacteria, and infection of macrophages by M. tuberculosis plays a key role in initiating a primary infection in the lung (29). In recent years, some progress has been made toward understanding the M. tuberculosis molecules involved both in invasion and in intracellular growth. For example, the heparin-binding hemagglutinin adhesin has been shown to mediate M. tuberculosis adherence to epithelial cells and to potentiate extrapulmonary dissemination of M. tuberculosis (23). An exported repetitive protein (Erp) of M. tuberculosis and a Mycobacterium marinum homologue of M. tuberculosis Rv3881c both are required for intracellular growth in cultured macrophages and virulence in vivo (3, 16). However, as M. tuberculosis is known to utilize multiple mechanisms for invasion (14), there must be additional myco...
Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and β-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or β form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the β-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three β-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with β-WTA, fulfill two recognition principles: binding to the β-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.
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