| Brain abscess(BA)is a serious fatal central nervous system infectious disease that can be caused by many bacteria.Staphylococcus aureus(S.aureus)is an important pathogen,and its isolation and culture rate in brain abscess patients ranks the second.S.aureus is a common opportunistic pathogen colonized in the human body,and its colonization significantly increases the risk of invasive infection in the host.S.aureus causes disease mainly by expressing and releasing a variety of virulence factors.S.aureus can produce up to fifty virulence factors,including three categories:toxins,adhesins and immune evasion factors.In different host environments,S.aureus may utilize different virulence factors for pathogenesis.So far,the key virulence factors in brain abscess formation by S.aureus have not been elucidated.Screening and identification of related virulence factors is not only important for elucidating the specific mechanism of S.aureus-induced brain abscess formation,but also have important practical significance for the prevention and effective treatment of brain abscess.S.aureus can invade directly through the adjacent parts of the brain(such as the ear,nasal cavity),or spread through blood dissemination of systemic infection,destroy and cross the blood-brain barrier,getting access to the skull and causing fatal central nervous system(central nervous system,CNS)infections(such as meningitis,brain abscesses,etc.).In this study,we constructed murine ear colonization,bacteremia,and BA models to mimic the pathogenic process of S.aureus BA.Real-time quantitative PCR(RT-qPCR)was used to screen the candidate virulence genes of S.aureus-induced brain abscess.Gene knockout mutants were constructed to further identify the key virulence determinants contributing to BA formation through S.aureus infection.Finally,monoclonal antibodies were applied to block the function of certain factors in S.aureus-caused BA to further evaluate the key virulence factors.The main research contents and results of this dissertation are as follows:1.Establishment of S.aureus ear colonization,bacteremia and BA mouse modelsThe mouse models of ear colonization,bacteremia and brain abscess were established with S.aureus Newman strain and XQ strain as experimental strains respectively.2.Screening for the key virulence determinants of S.aureus in BA formation by RT-qPCR2.1 Total RNA extraction,reverse transcription and quality control of S.aureus ear colonization,bacteremia,and BA samplesThe ear swabs,blood and brain tissue samples of the model animals were collected,and the total RNA of the mixed samples of S.aureus and host cells was extracted and reverse transcribed into cDNA.The quality control DNA was used to exclude PCR amplification inhibitors in cDNA samples.2.2 Screening for the key virulence determinants of S.aureus in BA formation via SYBR~?Green RT-qPCRApplying SYBR~?Green RT-qPCR and 16S rRNA gene as internal reference,the expression of 42 virulence genes in Newnan strain and XQ strain in brain abscess model and in vitro culture medium,ear colonization and bacteremia status were compared.The results showed that the expression level of S.aureus virulence factors in vivo(brain abscess)was significantly different from that in vitro(TSB or RPMI).Compared with the ear colonization state,most of the S.aureus genes up-regulated expressions in brain abscess(clfA and essA of Newnan strain,except icaD of XQ strain).Compared with the bacteremia state,nine genes(isdA,isdB,isdC,sasF,sdrC,hla,pvlF,aur and spa)of S.aureus in brain abscess were up-regulated in at least one strain.2.3 Further screening for the key virulence determinants of S.aureus in BA formation via TaqMan~?RT-qPCRBased on the high specificity of TaqMan~?probe,23 virulence genes were further selected(including 9 genes up-regulated in brain abscess relative to bacteremia,and 14genes significantly up-regulated at least one strain in brain abscess compared with ear colonization).The candidate genes were further screened by TaqMan~?RT-qPCR to compare the expression differences of these virulence factors in brain abscess,ear colonization and bacteremia.It was found that 5 virulence genes(isdB,sasF,hla,pvlF and spa)in the brain abscess compared with the ear colonization state,of which were significantly up-regulated in both strains.The remaining 18 genes,except adsA,were significantly up-regulated in at least one strain.Compared with the bacteremia state,13genes increased transcription in brain abscess.Among them,6 virulence genes(isdA,isdB,isdC,sasF,sdrC and adsA)were sinificantly up-regulated in at least one strain.These results suggest that these virulence factors may play key roles in the formation of brain abscess by S.aureus.Through the progressive screening by SYBR~?Green and TaqMan~?RT-qPCR,11candidate genes for brain abscess formation by S.aureus were selected,including:i)Five genes(isdB,sasF,hla,pvlF and spa)whose expression levels were significantly upregulated in two strains in BA compared with those in ear colonization,and the lgt gene that was significantly upregulated in XQ strain in BA compared with that in ear colonization;ii)Three genes(isdA,isdC and aur)that were upregulated in both strains and two genes(sdrC and adsA)that were upregulated in Newman strain in BA compared with those in the bacteremia.3.Identification of key virulence determinants for S.aureus BA formationGene deletion and antibody blocking experiments were performed to identify the key virulence determinants in S.aureus for BA formation among the 11 candidate genes.3.1 Construction and identification of single and double gene knockout strainsThe knockout strains were constructed in strain Newman.Markerless knockout was carried out based on the principle of homologous recombination.As results,8 out of the 11genes(isdA,isdC,lgt,hla,spa,sdrC,aur,and sasF)were successfully deleted,and 3(isdB,pvlF and adsA)failed to be deleted,which may own to the technological problems.In addition,three double-gene knockout strains(Newman-Δhla/Δspa,-ΔsdrC/Δaur,and-ΔsdrC/ΔsasF)were also constructed.All mutant strains were identified by PCR amplification and DNA sequencing.3.2 Evaluation of the pathogenic effects of gene knockout mutants or antibody blocking of certain factors in murine BA modelMice were separately infected with 8 single gene knockout mutants and 3 double-gene knockout strains,and their abilities in causing BA were tested and compared with the wild-type Newman.As a result,Newman-ΔisdA,-ΔisdC,-Δlgt,-Δhla,-Δspa,and-Δhla/Δspa mutant strains significantly decreased their bacterial loads and the abscess volumes in BAs.The remaining mutants were not differed compared with the wild-type strain.These results indicate that 5 genes(isdA,isdC,lgt,hla,and spa)are key virulence determinants for S.aureus-induced BA.To further evaluate the roles of hla and spa in S.aureus-caused BA,the antibody blocking experiment was performed using monoclonal antibodies(mAbs)against SpA and Hla in BA model.As a result,the BA bacterial loads and volumes were significantly decreased in the mice of antibody-treated group compared with those in mice of the control group.The pathological section of BA and three-dimensional reconstruction of the whole brain showed that the antibody blocking significantly inhibited S.aureus BA formation,further indicates that Spa and Hla are the key virulence factors for BA formation by S.aureus.3.3 Effects of the key virulence determinants for BA formation in S.aureus ear colonization and bacteremiaTo determine the specificity of the screened virulence determinants in S.aureus BA formation,the pathogenic effects of gene knockout mutants and wild-type Newman were compared in mouse models of S.aureus ear colonization and bacteremia.As results,the bacterial burdens of Newman-ΔisdA,-ΔisdC,-Δlgt,-Δhla,-Δspa,and-Δhla/Δspa mutant strains in the mouse ear colonization were negligibly differed from those of the wild-type Newman.In mouse bacteremia model,no difference was observed in the bacterial burden between wild-type Newman and its isogenic mutants except that of Newman-Δlgt 1hr post-infection.These results further indicate that isdA,isdC,lgt,hla,and spa play roles in S.aureus-induced BA,and have limited functions in S.aureus ear colonization and early bacteremia.In conclusion,we constructed murine models of S.aureus ear colonization,bacteremia and brain abscess,targeted 11 candidate genes by SYBR~?Green and TaqMan~?RT-qPCR,then gene knockout,pathogenicity evaluation,and antibody blocking experiment were used to screen and identify the key virulence determinants for BA formation through S.aureus infection,and 5 genes(isdA,isdC,lgt,hla and spa)that contributing to S.aureus BA were screened and confirmed.Our data will help to understand the mechanism underlying S.aureus-caused BA,and provide potential targets for the development of new strategies to treat BA caused by S.aureus infections. |
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