The Function Of The Novel LPXTG Surface-Anchored Protein (SasX) In Staphylococcus Aureus And The Exploration Of Its Potential As A Target Of Vaccine

Staphylococcus aureus is a commensal opportunistic pathogen, which can be frequently isolated from hospital and community environment. As one of the most important pathogen causing hospital-associated infections, S.aureus can cause both superficial and invasive, even life-threatening, infections such as pneumonia, endocarditis, and sepsis. On the other hand, S.aureus colonizes about 30% of the human population. It will migrate to the appropriate site when skin damage or hypoimmunity happen to the host. Since the superbug MRS A emerged in the 1960s, antibiotic treatment became more and more ineffective owing to the rapid development of antibiotic-resistant strains. We need new antibiotic or vaccine to fight against these high resistant bugs. Exploration of the novel targets of S.aureus has become a new hotspot in the scientific field.The surface of S.aureus is covered with varies surface proteins that are covalently anchored to the peptidoglycan in the cell wall. So, they are named as cell wall-anchored proteins. In recent years, several researchers reported these cell wall-anchored proteins can play important roles in both the colonization and infection process. For these proteins are expressed on the surface of S.aureus and are always determined as virulence factors, they are always good candidates of targets in investigation of new antibiotics and vaccine.ST239 is the international health care-assoicated MRS A lineage prevalent in the hospitals of Asia. To investigate the genetic basis for this clone adapts to survive in the health care environment, in 2010, HA-MRSA ST239 （TW20） genome was completely sequenced. SATW20₂1850 in the prophage ψSPβ-like of TW20 encodes an LPXTG motif surface-anchored protein that does not have orthologs in any of the genomes of the other sequenced S.aureus strains currently available. But this protein is highly similar with SesI, a virulence determinant in Staphylococcus epidermidis RP62A. So we named this novel protein SasX （Staphylococcus aureus surface protein X）. To determine whether this novel protein is an important virulence or not for HA-MRSA ST239 persistent in the hospital setting, we carried out studies on several aspects, such as the epidermiology of sasX in China, the role of SasX in colonization and infection of S.aureus, and the possibility of it to be a target for new vaccine in S.aureus. The aim of the study is to elucidate the molecular mechanism of SasX in promoting the persistent infection and spreading of MRS A ST239 in the hospital environment and to explorate the novel target for investigation of antibiotics and vaccine.Part â… . Distribution of the novel surface-anchored protein-encoding gene sasX in the Staphylococcus aureus strainsST239 is the international health care-associated MRS A lineage prevalent in the hospitals of Asia. The genome sequence of the ST239 strain contained a novel gene of unknown function （SATW20₂1850）. This new gene does not have any similarity to other proteins found in the NCBI databases. However, the protein encoded by this gene showed high similarity in the amino level with SesI, a virulence determinant of S.epidermidis RP62A. We named the novel gene sasX. We conjectured that sasX might play a role in the persistence of ST239 in hospital infection. In this part, we first used the method of molecular epidermiology to analyze the contribution of sasX in S.aureus strains. A total of 807 S.aureus strains were randomly collected from three teaching hospitals （Shanghai Huashan hospital, Shandong Provincial hospital, and the first affiliated hospital of Wenzhou Medical College） in China from the year of 2003 to the year of 2011. As part of a population-based community prevalence study,170 S.aureus strains were collected from more than 1500 healthy volunteers in Wenzhou and Shanghai. Compared to hospital isolated strains which have a high frequency of sasX positive strains, msX was only found in 0.59% of the S.aureus strains from the nasal swabs of healthy individuals without any healthcare association. The sasX gene was first found in MRSA ST239 strains. The frequency of sasX in MRSA ST239 increased in recent years. The sasX gene was horizontally transferred between different S.aureus clonal types in the hospital environment, such as ST5 and ST88. The sasX gene was located in ψSPβ-like prophage in all of the clones analyzed. Most sasX-carrying S.aureus were MRSA strains, from 2004 to the year of 2010, the percentage of sasX positive MRSA was increased from 26.4% to 50.8%, but the percentage of sasX positive MSSA was only about 10%. Antibiotic resistance of sasX positive strains was higher than that of sasX negative strains. This indicated that sasX might have something to do with the resistance to antibiotic. The distribution of SasX is different to the other surface anchored proteins. This might indicate that the SasX protein is a virulence factor of S.aureus or at least a marker of invasive capacity. And, the presence of sasX gene is related to antibiotic resistance. For better understanding the function of this novel gene, further studies will be carried out in the following parts.Part â…¡. The novel surface-anchored protein SasX promotes biofilm formation and is a virulence factor of S. aureusMRSA ST239 HS sasX gene mutant and complement were obtained by gene knock-out and complement methods using temperature sensitive plasmid pKOR â…  and pRBsasX expressing plasmid, respectively. Semiquantitative biofilm assay was used for detection of the biofilm formation of wild type and mutant strains. The sasX mutant strain showed a very clear reduction of biofilm formation compared to wild type and complement, while there was no significant difference of biofilm formation between wild type and complement. Primary attachment assays demonstrated that comparing to wild type, there was a significant reduction of initial accumulative phases of biofilm development in mutant strain, but there was no difference between wild type and complement. To investigate the function of SasX in the pathogenicity ofS. aureus, the mice abcess model and the pneumonia model were constructed. In the mice abcess model, both sasX wild type and mutant could cause abscess in the skin of mice, while no abscess were forming in the PBS control group. But on the same time point, the wild type group produced significantly larger abscesses compared to mutant group. In the mice pneumonia model, the results of the ratio of wet lung weight/body weight, the level of TNF-a and pathological analysis of the lung tissue revealed that SasX played an important role in lung infection. The immune evasion ability of bacteria also contributes to the successful persistence of infection. The neutrophil phagocytosis rate of wild type strain was significantly lower than the sasX mutant strain. And, the wild type with sasXgene also showed the ability to cause a cell lysis of neutrophil.Part â…¢. The novel surface-anchored protein SasX promotes aggregation and colonization of S. aureusThe role of SasX played in the biofilm formation indicated that SasX might have functions in promoting the adherence and aggregation ability of S. aureus. Examination by the optical microscopy showed that the mutant strain showed a reduction in cell aggregation when compared to the wild type strain, while the complement had no difference with wild type in aggregation. The adherence ability of S. aureus to the human nasal epithelial cells has an important role in colonization, speading and infection of S. aureus. By adherence assay which was used for detection of the adherence ability of wild type and mutant to human nasal epithelial cells and blocking experiments which detected the ability of the purified recombinant SasX protein in blocking the adherence of S. aureus to human nasal epithelial cells, we investigated the influence SasX excercised on colonization of S.aureus. The sasX mutant strain showed a significant reduction of adherence to human nasal epithelial cells compared to wild type, and the complement strain showed a very clear increasement of adherence to human nasal epithelial cells compared to wild type strain. Preincubation of nasal epithelial cells with the purified recombinant SasX protein inhibited S.aureus binding significantly. As an in vivo experiment of adherence to nasal, the mice nasal colonization model showed that the sasX mutant strain has reduced adherence ability when compared to the wild type strain. The competition assay of colonization also showed that the wild type strain with sasX gene has much stronger adherence ability when compared to the mutant one. In conclusion, SasX had an influence in aggregation of S.aureus and its adherence to human nasal epithelial cells. By acquiring sasX, S.aureus colonized more easily to the susceptible sites of the host, and thus caused infection.Part â…£. The initial exploration of SasX to serve as a novel target of vaccine towards S.aureusThe results of the functional experiments of SasX revealed that the novel surface anchored protein SasX is an important virulence factor of S.aureus. In this part, we intended to investigate passive and active immunization strategies using SasX as a target to control MRSA colonization and infection. Mice immunized with the purified SasX protein could generated the specific immune response, the highest specific antibody titer could be detected about one week after the first boosting immunization with incomplete adjuvant. The dominated subtype antibodies specific to SasX protein in the first week after immunization belonged to IgG2a and IgG2b. After the first boosting immunization, IgGl increased significantly and became the most dominated one. The in vivo mouse models of skin abscess, pneumonia and bacteremia model confirmed:The purified SasX protein immunized mice could produce specific antibody, the antibody has significant protective immunity. The inflammation and tissue injury or infection mortality were significantly reduced in the group immunized with purified SasX protein. Active immunization with purified SasX protein has better anti-infective effects or immune effects than passive immunization with SasX antibody. This study identified purified SasX protein could generated the specific immune response and the antibody has significant protective immunity. SasX protein might be a promising target for therapeutic interference.