wbp-encoded LPS O-antigen architecture as a prognostic and therapeutic target in Pseudomonas aeruginosa keratitis.

Abstract

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) keratitis can progress rapidly to vision-threatening disease, even with intensive therapy. Virulence-associated genes are key determinants of ocular-surface pathogenesis. We therefore sought to develop a composite wbp-exo genotyping framework for risk stratification and to guide wbp-dependent, LPS-directed, levofloxacin-polymyxin B (LVX-POL) combination therapy for high-risk corneal infections. METHODS: A well-characterised clinical P. aeruginosa keratitis cohort was integrated with whole-genome sequencing. Based on comprehensive virulence-gene identification and annotation, the relationship between strain-level genetic features and clinical prognosis was analysed. The differences between WBP1 strains and WBP2 strains in adhesion, invasion, and biofilm formation in corneal epithelial cells were further evaluated. To establish biological plausibility, wbp genotypes were correlated with LPS O-antigen electrophoretic profiles and in vivo corneal inflammatory phenotypes in murine infection, including the observation of leucocyte recruitment and cytokine responses. To further confirm the key role of wbp gene status and LPS O-antigen in pathogenicity, wbpL knockout and reconstitution strains were constructed. Their appearances in vitro and in vivo were evaluated. Finally, a mechanistic rationale for an LPS-directed LVX-POL regimen was tested in a high-risk WBP1 P. aeruginosa murine keratitis. FINDINGS: Whole-genome sequencing was performed on 46 clinical P. aeruginosa isolates and identified an average of 332 virulence- and fitness-associated genes per strain. The exo and wbp gene families were significantly associated with patient prognosis. A fusion model (AUC = 0.86) outperformed single-gene-family models (EXO: 0.66; WBP: 0.72) for predicting clinical outcomes. Intact wbp cassettes were enriched in poor-outcome isolates, and electrophoretic LPS profiles indicated that WBP1 strains produce highly polymerised O-antigen associated with sustained neutrophil recruitment and cytokine production. Murine experiments further implicated wbp genes in clinical pathogenesis, showing stronger immune responses and higher expression of TLR4, MyD88, TRAF6, p65, p-p65, IL-6, TNF-α, and IL-1β throughout the inflammatory course. After knocking out wbpL gene, the WBP1 strain got stronger in biofilm formation and adhesion but weaker in inflammation and ocular surface survival. In the high-risk WBP1 P. aeruginosa keratitis model, LVX-POL combinations achieved complete ulcer resolution and markedly improved stromal infiltration and hypopyon, outperforming LVX monotherapy. INTERPRETATION: The wbp gene family was identified as a key genetic factor that contributes to the LPS O-antigen structure, inflammatory intensity, bacterial ocular surface survival and poor prognosis in P. aeruginosa keratitis. A WBP1-targeted, LPS-directed LVX-POL regimen was proposed as a mechanistically informed option for high-risk strains. FUNDING: This research was supported by Beijing Public Health High-level Talent Training Program (Phase III-03-14), Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project (2026ZD01909300) and Beijing Natural Science Foundation "QiYan" Undergraduate Research Fund (QY26496).