Roles Of Sortase A In The Pathogenesis Of Staphylococcus Aureus-induced Mastitis, Bacteraemia And Pneumonia

Sortase A (SrtA), a transpeptidase, anchors surface proteins with a LPXTG-motifsorting signal to the cell wall envelope. These surface proteins have numerousfunctions, including adhesion to and invasion of host cells and tissues, evasion ofimmune responses and biofilm formation. To determine the contributions of thisenzyme to the pathogenesis of Staphylococcus aureus (S. aureus) in vivo, we chose S.aureus Newman D2C as a new reference strain, which not only wascoagulase-negative but also didn‘t produce hemolysin. We could exclude the effectsof coagulase and hemolysin and focus just on the functions of SrtA. Here, wesuccessfully constructed a srtA mutant strain, ΔSrtA, by genetic recombination.Immunofluorescence assay showed that this srtA mutant failed to anchor surfaceprotein A (SpA) to cell wall peptidoglycan. Furthermore, fibronectin/fibrinogen assayrevealed that S. aureus ΔSrtA significantly decreased the ability to adhere tofibronectin and fibrinogen, respectively. We further investigated the roles of SrtA inthe pathogenesis of S. aureus-induced mastitis, bacteraemia and pneumonia in mice.The mouse model of mastitis was developed by challenging with S. aureus via themilk duct of lactating BALB/c mice. The histological and myeloperoxidase (MPO)assays showed that S. aureus ΔSrtA attenuated the inflammatory reaction in themammary tissue of mice compared with wild-type S. aureus challenged mice.Furthermore, the ELISA results showed that S. aureus ΔSrtA impaired the inductionof pro-inflammatory cytokines such as tumor necrosis factor-(TNF-),interleukin-1β (IL-1β) and interleukin-6(IL-6). Western blot analysis demonstratedthat the srtA mutant blocked the activation of nuclear factor κB (NF-κB) andmitogen-activated protein kinases (MAPKs) by attenuating the degradation andphosphorylation of signaling pathway molecules such as IκB, p65and p38. Theseresults suggest that SrtA was a key virulence factor in the pathogenesis of S. aureus-induced mastitis in mice. It appears that the srtA mutant affected theattachment of S. aureus to host cells, thus attenuating the activation of the NF-κB andMAPK signaling pathways, which regulated the expression of proinflammatorycytokines and finally decreased the susceptibility to mastitis.Mouse model of S. aureus-induced bacteraemia was obtained by injectingintravenously S. aureus into BALB/c mice. At a challenge dose of S. aureus(2×108CFUs), mice infected with both wild-type S. aureus and S. aureus ΔSrtAsurvived post infection. Microscopic analyses of kidney tissue at day5post-infectionrevealed numerous bacterial foci/abscesses in mice challenged with wild-type S.aureus, which characterized by a central core of bacteria surrounded by a robustinflammatory cell infiltration. However, the similar lesions were not observed inmice challenged with S. aureus ΔSrtA. At a higher challenge dose of S. aureus(1×109CFUs), survival analyses following intravenous injection revealed that micechallenged with wild-type S. aureus uniformly died or became moribund by day3post-infection. None of mice challenged with S. aureus ΔSrtA succumbed duringthis period and even the end time point. The white blood cells and platelets inperipheral blood were significant reduction in mice infected with either wild-type S.aureus or the srtA mutant versus uninfected control mice. The levels ofinflammatory cytokines, IL-12p70， IL-17A, IFNγ and IL-6, in serum of S. aureusΔSrtA infected mice were dramatically lower than that in mice infected withwild-type S. aureus. The results indicated that SrtA was required for abscessformation and staphylococcal persistence in host tissues. And, SrtA also might linkto inflammatory mediators but not blood cell counts which caused death of micefollowing bacteraemia.An animal model of S. aureus–induced pneumonia in adult immunocompetentC57BL/6J mice is developed that closely mimics the clinicopathological features ofhuman disease. Compared with wild-type S. aureus, histopathologic analysisrevealed extenuated hyperemia, decreased inflammatory cell infiltration and normalalveolar architecture of lung tissues following challenge with S. aureus ΔSrtA at24hours. The levels of proinflammatory cytokines, such as TNF-, IL-1β and IL-6, in the bronchoalveolar lavage fluid were significantly decreased in mice challengedwith S. aureus ΔSrtA versus wild-type S. aureus. Compared with wild-type S.aureus, significantly decreased levels in phosphorylation of IκB, p65, p38, ERK,and JNK was detected in the lungs of mice infected with S.aureus ΔSrtA by westernblot assay. These results indicated that SrtA is an important virulence factors for S.aureus-induced pneumonia.Together, deficiency of SrtA which does not anchor surface proteins like surfaceprotein A to the cell wall impairs the inflammatory response of S. aureus-inducedmastitis, bacteraemia and pneumonia in mice. Results from these studies show thatSrtA is an important potential target for S. aureus-induced mastitis, bacteraemia andpneumonia. Thus, drugs which are active at inhibiting SrtA serve as an alternative foranti-microbial therapy.