Role Of The ATP-dependent Proteases In Stress Tolerance And Virulence Of Streptococcus Suis Serotype 2

Streptococcus suis(SS)is one of the predominant zoonotic pathogens responsible for numerous diseases,including septicemia,meningitis,and consequently death in swine and humans.It is also a major threat to public health through zoonotic transmission and is responsible to the swine industry worldwide for severe economic loss.Based on the difference in antigenicity to capsular polysaccharide(CPSs),SS are classified serologically into 33 serotypes(1-31,33,and 1/2).Among the 33 serotypes,serotype 2 shows the highest prevalence worldwide with the powerful pathogenicity.To date,over 100 putative virulence factors have been revealed to be involved in SS2 virulence,of which at least 37 have been claimed to be "critical" virulence factors.Unfortunately,the pathogenic mechanism of SS2 infection is still not well understood.Proteases help to maintain cellular homeostasis by clearing short-lived regulatory proteins as well as misfolded and damaged proteins during protein quality control mechanism.Many bacterial species in prokaryotes contain four distinct types of ATP-dependent proteases Clp proteases(ClpAP,ClpBP,ClpXP,and ClpP),single-chain AAA(FtsH),ClpYQ(also known as HsIUV)and the Lon family proteases(Lon).Stress tolerance,pathogenicity,expression,and regulation of three ATP-dependent proteases in ClpX,ClpP and FtsH were studied in this thesis.1 ClpP is required for the stress tolerance and virulence in Streptococcus suis serotype 2Streptococcus suis has received increasing attention for its involvement in severe infections in pigs and humans;however,their pathogenesis remains unclear.ClpP,one subunit of the ATP-dependent caseinolytic protease Clp,plays key roles in bacterial adaptation to various environmental stresses.In this study,a clpP deletion mutant was constructed and characterized from SS2(ZY05719)strain.The mutant strain ?clpP displayed significant sensitivity to acidic pH and H2O2-induced oxidative stress.ClpP were required for the optimal growth during heat stress.An in vitro experiment on RAW264.7 macrophage cells showed a significant decrease(25%)of anti-phagocytosis in ?clpP compared to the wild-type strain.The inactivation of ClpP significantly decreased the adherence of SS2 to HEp-2 cells at 43.5%compared to the wild-type strain.Mouse infection assays verified the abilities of ?clpP to colonize the tissues,where ?clpP were significantly attenuated compared to the wild-type strain.These virulence-related phenotypes were partially recoverable by genetic complementation.Furthermore,the deletion of clpP caused a significant decrease in the expression of tpx,and apuA compared with the wild-type strain,suggesting that these genes may be regulated by ClpP as downstream response factors to facilitate the bacterial tolerance against various environmental stresses.Taken together,these results suggest that ClpP play important roles in stress tolerance for achieving the full virulence of S.suis serotype 2 during infection.2 Role of ClpX in SS2 stress tolerance and virulenceIn this study,we evaluated the potential role of ClpX ATP-dependent caseinolytic protease in the stress tolerance and pathogenesis of Streptococcus suis serotype 2.The clpX was deleted to generate mutant(?clpX).The ?c1pX strains showed reduced growth significantly under acidic pH and oxidative stress induced by H2O2 compared to the wild-type strain.However,ClpX were required for the optimal growth during cold stress.Moreover,the ?clpX reduced the anti-phagocytic abilities of SS2 to RAW264.7 macrophage cells at 28%in comparision with that of the wild-type strain.The mouse infection assays demonstrated that ClpX inactivation caused not only fewer clinical symptoms and low mortality but also severe attenuation in bacterial colonization.These virulence-related phenotypes were partially recoverable by genetic complementation.The deletion of clpX also resulted in a significant decrease in the expression of sodA,and tpx,genes,compared with the wild-type strain,which suggested that these genes could be regulated by ClpX as downstream response factors to facilitate bacterial tolerance to diverse environmental stresses.3 The roles of filamentation temperature sensitive H(FtsH)in the survival of SS2 under stresses and the virulence in BALB/c miceFtsH,the membrane-bound ATP-dependent metalloprotease plays a critical role in resistance to diverse stressors in the Gram-positive bacteria.The ftsH was deleted to generate isogenic of mutant.The S.suis FtsH gene was heat resistance at 42?,since its'expression was upregulated during high temperature and combined heat and osmotic shock.The ?ftsH leads to pleiotropic defects including,sensitivity to NaCl,H2O2,acidic pH,and reduced biofilm formation,whereas it was restored when the ftsH gene was complemented.The ?ftsH showed a significant decrease in the anti-phagocytosis at 57%in RAW264.7 macrophage cells and also a decrease in adhesion at 31.3%in HEp-2 cells compared to the wild-type strain.Moreover,the virulence of wild-type strains was significantly higher than that of the ?ftsH strains in zebrafish infection experiment.Mouse infection experiment further revealed that the loss of ftsH reduced colonization in blood,brain,lung and spleen compared to wild-type strain.4 The CtsR regulated the expression of clpP in SS2Stress proteins play a monumental role in virulence,the regulation of stress response in gram-positive bacteria is still unknown.In this study,a virulent S.suis serotype 2(SS2)strain ZY05719 was explored to construct ctsR deletion mutants and their complementation strains.The ?ctsR significantly attenuated the virulence of SS2 in both infection models of BALB/c mice and zebrafish.The transcriptional level of clpP in the ?ctsR was strongly upregulated,suggesting that clpP of SS2 is controlled by CtsR repressor protein.Direct CtsR-dependent regulation was demonstrated by EMSA assays using recombinant CtsR and the DNA probe containing clpP promoter region.The purified CtsR protein of S.suis specifically binds to the regulatory regions of the clpP.Our results demonstrate a CtsR regulon controlling heat shock genes of class ? clpP in this SS2 strain.