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Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection.
Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.
Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens.
Staphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins.