Deciphering the diversity, structure, and function of cycle-inhibiting factor in Burkholderia pseudomallei...

ObjectiveThis study aimed to characterize the genetic diversity, structural variation, and functional role of the cycle inhibiting factor (Cif) in Burkholderia pseudomallei, with a particular focus on its involvement in neuronal infections.MethodsWe analyzed the cif gene (bpss1385) from 1294 clinical isolates of B. pseudomallei using phylogenetic analysis and structural modeling to identify Cif variant types. Functional characterization of selected variants was performed using plaque formation assays in SH-SY5Y neuroblastoma cells. Additionally, proteomic profiling was conducted to assess differential host protein expression in SH-SY5Y cells infected with the B. pseudomallei strain K96243 versus a cif-deleted mutant.ResultsThe cif gene was present in 57.7% of clinical isolates, revealing 18 distinct variant types. The wild-type variant (bpss1385) was the most prevalent and shared high sequence and structural similarity with most other variants. However, VT4, VT6, and VT15 displayed notable structural divergence, with VT4 exhibiting the most pronounced alterations, particularly in substrate-binding and catalytic regions. Although VT4 produced a similar number of plaques as the wild type, the plaque size was significantly smaller, suggesting reduced intracellular activity or attenuated virulence. Most other variants retained structural and functional similarity to the wild type. Proteomic analysis identified 52 differentially expressed proteins upon cif deletion, implicating Cif in regulating neuronal cell processes, including mRNA metabolism and cytoskeletal organization.ConclusionOur findings highlight substantial structural variation among B. pseudomallei Cif variants, with VT4 emerging as the most structurally distinct. Despite overall conservation in infection efficiency, VT4's reduced plaque size suggests functional consequences of its structural changes. Along with proteomic evidence of host pathway disruption, these results underscore the role of Cif in modulating host neuronal pathways and support its potential as a therapeutic target in neurological melioidosis.