Necrotizing bacteria and protein targeting

Abstract

A type III-like secretion mechanism for injection of effector molecules into the cytoplasm of host cells has not yet been described for Gram-positive bacteria. These bacteria are known to secrete multiple proteins into their environments, such as those secreted by Streptococcus pyogenes, the agent responsible for strep throat and necrotizing fasciitis of soft tissues. Previous work using keratinocytes demonstrated that mutants defective in production of the hemolysin streptolysin O (SLO) lost the ability to manipulate host signaling pathways. SLO also has characteristics that would suggest it has a role in translocation. SLO is a member of a highly conserved family of pore-forming cytolysins known as cholesterol-dependent cytolysins (CDCs). Analysis of their pore size suggests that CDCs could translocate effector molecules from Gram-positive bacteria. Madden and colleagues 1xCytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in Gram-positive bacteria. Madden, J.C et al. Cell. 2001; 104: 143–152Abstract | Full Text | Full Text PDF | PubMed | Scopus (180)See all References1 recently presented evidence that a molecule of streptococcal origin with eukaryotic signal transduction properties is translocated into the keratinocyte cytosol in an SLO-dependent manner.Crossreactivity with a commercially available anti-streptolysin O antiserum revealed the presence of a 52-kDa streptococcal protein in infected keratinocytes, which was not present in SLO-deficient mutants or uninfected host cells. Using cytoplasmic fractions prepared from host cells under conditions that lyse keratinocytes but not bacteria, the 52-kDa protein was found to be translocated in an SLO-dependent manner. Other host cell types were analyzed and similar results were obtained. A streptococcal NAD-glycohydrolase (SPN)-deficient mutant did not translocate the 52-kDa protein into host cells however, indicating that this particular protein was derived from the bacteria and was not of host origin. Co-infection with bacteria that were deficient in either SLO or SPN did not rescue protein transport.These observations suggest that SPN enters the cytosol through the SLO pore. The SPN protein is biochemically active in the host cell. The presence of the microfilament inhibitor cytochalasin D did not alter enzymatic activity in wild-type cells, indicating that the presence of SPN activity was not the result of endocytosis or bacterial invasion. Furthermore, SPN activity was dependent on SLO and was not attributed to an endogenous NAD-glycohydrolase. To demonstrate that SPN and SLO work in a synergistic manner, a cytotoxic response assay was used where keratinocytes were infected with either the wild-type strain, the SLO-mutant or the SPN-mutant. At various times, samples were removed and stained with the fluorescent probes calcein AM (activated by cellular esterases) and ethidium homodimer-1 (EtH-1) (which stains the nucleus). Loss of staining with calcein AM and positive staining with EtH-1 would be indicative of membrane damage. By 3h, ∼35% of the wild-type cells have lost their ability to exclude EtH-1. At all time points, infection with SLO-deficient mutants resembled SPN-deficient mutants in terms of the number of cells damaged and number of cells that remained adherent. Both mutants had reduced levels of permeability compared with the wild-type cell. These data are consistent with a model where SLO is required for the translocation of SPN into the cytosol of the host cell.With these data and previous studies of protein targeting in Gram-positive bacteria, a working model of protein translocation suggests that effector molecules are exported across the bacterial cellular membrane and cell wall via the generalized secretion pathway and translocated into the target cell using a CDC. Other than in Listeria, little is known about the function of CDCs in pathogenesis. These interesting and novel findings set the stage for analysis of function of virulence factors secreted from Gram-positive pathogens.