Proteomic identification of Acanthamoeba castellanii excretory-secretory proteins associated with Klebsiella pneumoniae capsule enlargement: a candidate role for C-type lysozyme.

BACKGROUND

Acanthamoeba castellanii and Klebsiella pneumoniae are environmental commensals that co-exist in aquatic habitats. Our previous studies demonstrated that A. castellanii genotype T4 (AcT4) induces capsule enlargement and hypermucoviscosity in K. pneumoniae through small excretory-secretory proteins (ESPs; <10 kDa) that activate the Rcs two-component system, upregulating capsule biosynthesis genes independently of virulence plasmids. However, the molecular identity of these active ESPs remained undefined.

RESULTS

To identify candidate proteins, we performed label-free quantitative proteomics (LC-MS/MS) on conditioned medium from AcT4 co-cultured with K. pneumoniae (Ac/Kp) versus AcT4 alone (Ac only; n = 3 per group). A total of 391 A. castellanii proteins were identified; seven showed significantly increased extracellular abundance in Ac/Kp medium (log2 ratio ≥ 1, p < 0.05), and 98 showed decreased abundance. Functional enrichment analysis revealed that increased-abundance proteins were enriched in transport, signaling/regulation, redox activity, ion/metal binding, and chaperone/stress response categories. Among these, a C-type lysozyme/α-lactalbumin superfamily protein (UniProt L8GZI4; 14.7 kDa; log2 ratio = 5.19) was identified as a priority candidate, as lysozyme-mediated peptidoglycan hydrolysis generates muropeptides-known activators of the Rcs phosphorelay.

CONCLUSIONS

This comparative secretome analysis provides the first proteomic catalogue of AcT4 extracellular proteins during co-culture with K. pneumoniae and identifies C-type lysozyme as a biologically plausible candidate mediating Rcs-dependent capsule induction. These findings lay the groundwork for future validation of AcT4-derived ESPs in protozoa-bacteria interactions and their potential role in the environmental emergence of hypervirulent K. pneumoniae.