Abstract
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; 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 (log
2
ratio ≥ 1,
p
2
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
.