Vaccine antigen identification against dangerous Gram-positive ESKAPE pathogens

Sadones, Océane (2024): Vaccine antigen identification against dangerous Gram-positive ESKAPE pathogens. Dissertation, LMU München: Medizinische Fakultät

Abstract

Enterococcus faecium and Staphylococcus aureus, the Gram-positive pathogens of the ESKAPE group, are known to represent a great threat to human health, especially due to their presence in the healthcare setting where they often present with high virulence and multiple resistances to antibiotics. Together, S. aureus and E. faecium account for 45% of annual antimicrobial resistance related-deaths in the US. In this study, we combined several techniques for antigen discovery in an effort to reduce the number of identified proteins to only promising candidates. We used a subtractive proteome analysis that we combined with a false positive analysis of peptides obtained by trypsin shaving. Briefly, the subtractive proteome analysis was conducted on proteins extractions obtained by lysostaphin digestion, SDS boiling or sonication that were run through SDS-PAGE in triplicates. Two of the gels were blotted onto a membrane and detected with either pooled human sera previously tested for the presence of opsonic antibodies, or with the same sera previously depleted of S. aureus-specific antibodies. Bands identified with the pooled sera but not with the depleted one were reported to the remaining gel and matching bands were excised for protein identification by mass spectrometry. The false positive analysis was performed using trypsin shaving: bacteria were incubated with or without trypsin in a hypotonic solution and the resulting supernatant was run through mass spectrometry, leading to a list of proteins enriched by the treatment with the enzyme. Combination of the results obtained with both techniques formed a list of 40 potential antigens which was narrowed down to 10 candidates after removal of already described antigens, cytoplasmic and non-immunogenic proteins. We picked five of them: the DUF5011 domain-containing Fe/B12 transporter, and Fe(3+) dicitrate ABC transporter; and two having an amino acid sequence really close to two already discovered antigens in enterococci: PrsA and AdcAau. Cross-opsonic effect was investigated and showed that antibodies raised against the two enterococcal antigens can mediate the killing of S. aureus. We showed that our selected proteins are able to elicit specific and opsonic antibodies against the S. aureus strain MW2. We also demonstrate that PrsA and AdcAau can cross-bind to their homologs and cross-opsonize several S. aureus, E. faecium and E. faecalis strains. These findings show that the novel experimental approach for antigen discovery can lead to the identification of promising candidates that can induce the production of opsonic antibodies. We showed that three of the investigated candidates could mediate the killing of S. aureus. Also, two of the identified proteins are antigens that could be considered for vaccine formulation against both Gram-positive ESKAPE pathogens.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: ESKAPE, Staphylococcus aureus, Enterococcus, vaccine, antigens, antibodies
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 22. Oktober 2024
• 1. Bericht­erstatter:in: Hübner, Johannes