Study On The Role Of Streptococcus Pyogenes Htra In Protein Secrection

Streptococcus pyogenes (GAS) is a gram-positive coccus, as a bacterial pathogen S.pyogenes responsible for a wide variety of human diseases, ranging from mild suppurative infections of the skin (impetigo) and throat (pharyngitis), to life-threatening invasive infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. Serious immune sequelae, including acute rheumatic fever and glomerulonephritis, may also develop upon repeated GAS infection. GAS is the most common serogroup that can cause purulent infection. Secreted virulences and invasive enzyme arethe main factors of GAS pathogenicity, two important secreted virulence factors of S.pyogenes are the cysteine protease SpeB and the hemolysin streptolysin S (SLS). Some researchers believe that GAS has only one transport system called "Exportal", a unique micro-domain of GAS membrane, specialized for protein secretion and prossessing. HtrA is localized to the Exportal secretory micro-domain and is reportedly essential for the maturation of SpeB and hemolysin.In this research, we constructed the htrA mutant of transmembrane domain deletion (SPD14); the htrA gene deletion complemented strain (SPC15) and the htr A gene deletion-insertion mutation made by replacing an internal fragment with the spectinomycin resistance gene aad9. The SpeB proteolytic activity of wild-type (SPXL1), htr A mutant of transmembrane domain deletion (SPD14), htrA insertion mutant SP128and htrA gene complemented strain SPC15was determined by measurement of zones of clearance on casein agar plates medium, we found that SPI28delay the mature of SpeB, while SPD14decrease the secretion of SpeB. The research showed that the htrA gene transmembrane domain mutation and htr A gene insertion mutation decreased the activities of SpeB and homolysin. To analysis the secretion of SpeB, SDS-PAGE electrophoresis is used, the result showed that, compared to the wild-type strain SPXL1, htr A insertion mutant SPI28and htr A gene complemented strain SPC15, htrA transmembrane domain deletion affect the secretion and mature of SpeB.The alkaline phosphatase activity experiment showed that htrA may affect the activity of PhoZ, compared to SPZ35, PhoZ activity of SPZ7and SPZ36are15%and106%of SPXLl.To test the function of htr A in the adherence of GAS cells on human umbilical vein endothelial cell (huvec), SPD14was compared to its wild-typeSPXLl as well as to SP128. In comparison to the wild-type, SPD14demonstrated about90.4%reduction in adherence, while SP128bound to huvec is almost the same as the wild-type. Consider the deletion of transmembrane damain can affect the secretion of protein, we speculate that the deletion of transmembrane damain may reduce the adhesion factors of SPD14.In this research, we found that htr A affect the hemolysin and SpeB protease activity of GAS, htr A transmembrane domains affect the secretion of the SpeB, extracellular alkaline phosphatase activity, as well as adherence to host cell. We speculate that htrA transmembrane domains may involve in secretion of protein, it may be the composition of "ExPortal" micro-domain.