Study On Accumulation-associated Protein (Aap) As A Vaccine Candidate Against Biofilm Formation By Staphylococcus Epidermidis

Staphylococcus epidermidis is a normal inhabitant of human skin and mucous membranes that rarely causes pyogenic infections in healthy individuals. However, during the past two decades S. epidermidis has emerged as one of the major pathogens in nosocomial infections. The primary pathogenicity trait of S. epidermidis is associated with its ability to form biofilms on surfaces of medical devices, limiting severely the efficacy of many conventional antibiotics, and biofilms may also protect the bacteria against attacks from the host defence system. In parallel, the appearance of multi-drug resistant S. epidermidis strains has increased quickly due to the increasing use of antibiotics in hospitals, which urgently require design novel vaccines against staphylococcus infections, especially in relation to biofilm development.Biofilms are bacterial communities that adhere to biological or abiotic substrata and are stabilized by adhesins, typically composed of polysaccharides, proteins, and extracellular DNA. The formation of staphylococcal biofilms involves two phases: primary adhesion followed by biofilm accumulation. Once attached to the substrata, the bacteria will proliferate, secrete and be enmeshed within adhesins, and then accumulate as multilayered cell clusters. Accumulation-associated protein (Aap) is considered one of the most important protein-based adhesins that involved in S. epidermidis biofilm formation. Implicated in both polysaccharide-based and protein-based biofilms, Aap can directly mediate intercellular adhesion. Studies has demonstrated that the intact full-length Aap may be cleaved by a protease to remove the N-terminal A domain, exposing the more distal B-repeat region, which then promotes cell-cell interaction. Antiserum against Aap showed inhibition of both proteinaceous and polysaccharide-based biofilm formation by S. epidermidis. Biofilm-inhibiting monoclonal antibodies against Aap have also been established. Aap could be considered as a vaccine candidate to prevent biofilm infections.In this study, we have investigated the key region in Aap that mediates the bacterial accumulation and biofilm formation, and studied the effects of its antibodies on bacterial biofilm formation. In addition, epitope mapping of the biofilm-affecting monoclonal antibodies against Aap was performed and the effects of the monoclonal antibodies on Aap expression, PIA biosynthesis, and eDNA release in S. epidermidis were further studied. Our studies may provide new insights into the mechanisms of staphylococcal biofilm formation and may help in developing anti-staphylococcal biofilm vaccines.ChapterⅠ. The identification of the key region in Aap that mediates bacterial accumulation and biofilm formation by S. epidermidis.It has been revealed that Aap is one of the most important adhesins mediating the intercellular adhesion of S. epidermidis, and the antibodies against Aap inhibit the biofilm formation. However, there is no report about the anti-biofilm vaccine development using Aap as the antigen to date. In order to find an appropriate region in Aap which may induce the production of anti-biofilm antibodies for further vaccine development studies, we investigated the domain structures of Aap and identified the key region in Aap that mediates bacterial accumulation and biofilm formation by S. epidermidis in the present study.According to the amino acid sequence analysis, we found that Aap contains five regions, including the N-terminal exporting signal, the A region, the B-repeat region, the collagen-like proline/glycine-rich region and a cell wall anchoring motif (LPXTG). The A region of Aap contains an N-terminal A-repeat region with 11 degenerate 16-aa repeats and a putative globular domain ("α/β"). The B-repeat region of Aap composes a few nearly identical 128-aa repeat constructs terminating in a conserved half repeat motif (79-aa). The main difference among Aap in different strains is focus on the B-repeat region. Although the B-repeat construct unit is almost identical in Aap from different strains, Aap from different strains contains variable number of the constructs in the B-repeat region. Based on the protein sequence analysis, we identified the key region in Aap that mediates bacterial accumulation and biofilm formation. By over expressing the B-repeat region of Aap from strain ATCC 12228 in Staphylococcus carnosus, it induced the biofilm formation of the biofilm negative bacteria. We conclude that the B-repeat region is the key region in mediating the biofilm formation. In addition, we further cloned, expressed, and purified the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrptl.5) in B-repeat region of Aap. We found that AapBrptl.5 formed a polymer in the presence of Zn2+, and AapBrptl.5 inhibited the biofilm formation by S. epidermidis RP62A in a dose-dependent manner. Moreover, we found that anti-AapBrptl.5 mouse immune serum also inhibited the biofilm formation by S. epidermidis RP62A. It demonstrated that AapBrptl.5 is the minimal functional unit of Aap that mediates bacterial accumulation and biofilm formation.ChapterⅡ. The identification of epitopes in Aap that induce the production of anti-biofilm antibodies.Antibodies against Aap have been reported to inhibit biofilm accumulation by S. epidermidis, indicating that Aap could serve as a vaccine candidate to prevent S. epidermidis biofilm infection. However, full-length Aap is not a safe vaccine for systemic immunization because such bacterial antigens contain many antigenic determinants and may induce hypersensitivity reactions. A peptide that induces anti-biofilm humoral immunity would be an optimal vaccine. The Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5) was found to be the basic functional unit of Aap, which is necessary to mediate bacterial accumulation, suggesting that it harbors the epitopes that would guide the development of biofilm-preventing epitope-based peptide vaccines. To locate these epitopes, monoclonal antibodies (MAbs; MAb18B6, MAb25c11, and MAb20B9) against AapBrptl.5 were prepared in the present study.Only MAb18B6 was found to possess the weak but broad-spectrum activity necessary to inhibit biofilm formation by S. epidermidis, whereas MAb25C11 and MAb20B9 enhanced the biofilm formation by some strains of S. epidermidis, including RP62A. All of the MAbs strengthened the cell aggregation of planktonic S. epidermidis. Epitope mapping of the MAbs revealed that the biofilm-inhibiting MAb18B6 recognized an identical area within all AapBrpt constructs from S. epidermidis RP62A, which was not shared by the other two MAbs. To explore the reasons for the biofilm-enhancing activities of the MAbs, extracellular polymeric substance (EPS) biosynthesis was studied, including Aap expression, extracellular DNA release, and PIA synthesis in S. epidermidis RP62A co-cultured with the MAbs, and all MAbs were found to affect EPS biosynthesis in S. epidermidis and further enhance the bacterial accumulation. We have revealed two contradictory actions of the MAbs on biofilm formation:one inhibits bacterial accumulation by binding to Aap, whereas the other enhances biofilm formation by up-regulating EPS biosynthesis. The influence of the MAbs to affect biofilm formation depends on the resultant effect of their inhibition of Aap dimerization and up-regulatiion of Aap expression and EPS biosynthesis. The key factor determining the resultant effect should be the epitope. MAb18B6 recognizes all twelve Brpt constructs in Aap thus possesses higher inhibition on Aap dimerization to combat the action of up-regulated Aap expression and EPS biosynthesis, as a result, it inhibited the biofilm formation; whereas MAb25c11 and MAb20B9 just recognize six of the AapBrpt constructs and block Aap dimerization incompletely, therefore their weak inhibition of Aap dimerization fail to overcome the action of up-regulated Aap expression and EPS biosynthesis, and then they enhanced the biofilm formation. Considering those findings, vaccine development using Aap as the antigen should pay attention to the following points:which is the optimum epitope that mediate the highest inhibition of Aap dimerization; how to reduce the effect of anti-Aap antibodies on up-regulating the bacterial Aap expression and EPS biosynthesis.These findings contribute to a better understanding of staphylococcal biofilm formation and will help to develop epitope-peptide vaccines against staphylococcal infections.