The N-terminal domain is required for cell surface localisation of VapA, a member of the Vap family of Rhodococcus equi virulence proteins.
Authors: Miranda-CasoLuengo Raúl, Yerlikaya Zeynep, Luo Haixia, Cheng Cheng, Blanco Alfonso, Haas Albert, Meijer Wim G
Journal: PloS one
Summary
Rhodococcus equi pneumonia remains a significant cause of foal mortality globally, with virulent strains harbouring an 80–85 kb plasmid encoding six virulence-associated proteins (Vaps); VapA is the predominant virulence factor, localising to the bacterial cell surface to establish an intracellular replication niche, whilst VapC, VapD and VapE are secreted into the host cytoplasm. Miranda-CasoLuengo and colleagues used an unmarked vapA deletion strain as a genetic background to systematically express Strep-tagged Vap-derivatives and assess their subcellular localisation by flow cytometry, demonstrating that VapA is the only surface-localised Vap among those encoded on the virulence plasmid. Through construction of chimeric fusion proteins in which the N-terminal domain of VapD was swapped with VapA's N-terminus, the team provided direct experimental evidence that VapA's N-terminal domain (N-domain) is both necessary and sufficient for cell surface localisation—a distinction from the other Vaps despite their high sequence identity in the C-terminal region. The researchers further showed that VapA's N-terminus is exposed to the extracellular environment using an N-terminally tagged construct, and since VapA lacks the characteristic lipobox motif typical of lipid-anchored proteins, they propose that surface retention is mediated by direct interactions between the N-domain and cell wall components. These findings refine our understanding of how R. equi manipulates host cell biology and may inform development of targeted immunotherapies or novel antimicrobials that disrupt VapA-mediated pathogenesis in affected foals.
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Practical Takeaways
- •Understanding VapA's cell surface localization mechanism may inform development of targeted therapeutics or vaccines against R. equi pneumonia in foals, since VapA creates the intracellular niche essential for bacterial survival
- •The finding that soluble recombinant VapA can functionally complement native VapA suggests potential for novel immunotherapeutic or antimicrobial approaches in clinical practice
Key Findings
- •VapA is the only cell surface virulence-associated protein (Vap) encoded by the R. equi virulence plasmid, while VapC, VapD, and VapE are secreted
- •The N-terminal domain of VapA is essential for cell surface localization, as demonstrated by domain-swap experiments where VapD N-domain substitution abolished surface targeting
- •The N-terminus of VapA is exposed to the extracellular environment and likely mediates cell surface localization through interactions with cell surface components rather than lipid anchoring