Integrated experimental and computational analyses of Klebsiella pneumoniae ClpK: metal ion modulation, vaccine targeting, and interaction networks.

Abstract

Klebsiella pneumoniae is a Gram-negative opportunistic pathogen which has been classified by the World Health Organization as a critical priority organism underscoring the need for the development of new therapeutics and alternative interventions. The genetic diversity and horizontal transmission of antimicrobial resistance determinants in this pathogen demonstrate its capacity for rapid adaptation and persistence thus posing a significant global health challenge. To address the challenges caused by this pathogen, this thesis focused on the structural and functional characterisation of ClpK ATPase, together with complementary vaccine-based approaches aimed at disrupting its survival and stress response pathways. ClpK is a thermotolerant caseinolytic ATPase implicated in protein quality control and stress tolerance. Biophysical and enzymatic characterization showed that ClpK is predominantly α-helical and moderately thermally stable, with ATP binding conferring conformational stabilization. Divalent cations modulated ClpK activity, with Mg²⁺ enhancing structural stability and Mn²⁺ promoting conformational flexibility and enzymatic activity. These findings provide critical insights into the metal ion-dependant regulation of ClpK and highlight its potential as a drug target. To exploit metal ion dependant regulation of this protein, and in pursuit of novel compounds and drugbinding mechanisms targeting this protein, structure-based virtual screening, ADMET analyses and molecular dynamics simulations were used to identify natural and synthetic inhibitors of ClpK. Among the screened drugs, ADEP3 and sclerotiamide derivatives exhibited the most favourable binding energetics and stable interactions under varying ionic conditions, highlighting their potential as ClpK inhibitors. Targeting ClpK alone may not be sufficient to fully disrupt the pathogen’s adaptive capacity; therefore, exploring its protein–protein interaction network provides an additional strategy for interfering with stress-regulated proteostasis mechanisms. Given the role of ClpK in bacterial stress response and protein homeostasis, identifying its interacting partners offers valuable insight into how this ATPase integrates within broader cellular survival pathways. Therefore, mapping its protein– protein interaction partners provide an additional avenue for interfering with stressregulated proteostasis mechanisms to disrupt the pathogen’s adaptive mechanisms. Computational analyses were employed to predict high-confidence ClpK interaction partners involved in chaperone and stress response pathways. This study predicted several chaperones and stress response proteins as potential interacting partners for ClpK. This was in line with the role of ClpK in protein homeostasis. Molecular docking and dynamics simulations confirmed the stability of the predicted interactions, providing a basis for understanding the role of ClpK in bacterial stress response pathways. These findings expand the understanding of ClpK’s role beyond its intrinsic ATPase activity, positioning it as part of a broader proteostasis network and reinforcing its potential as a multifaceted therapeutic target. As part of a multi-tiered strategy to combat K. pnuemoniae, a reverse vaccinology pipeline was applied to six outer membrane proteins of this pathogen, yielding seven multiepitope vaccine constructs. Five constructs demonstrated stable binding to TLR2 and TLR4 receptors, high predicted antigenicity, and global epitope coverage of ~75%. Codon optimization and high codon adaptation index values further supported the feasibility for expression in Escherichia coli. A comprehensive exploration of ClpK as a multifunctional therapeutic target was undertaken using combined computational structural and biophysical approaches. The study characterised metal ion dependent regulation of ClpK, identified several promising inhibitors, mapped its interaction network with key chaperones and stress response proteins providing a mechanistic insight into ClpK’s role in bacterial proteostasis and highlight its significance in stress adaptation and survival. Furthermore, the integration of a reverse vaccinology pipeline led to the rational design of multiepitope vaccine constructs, thereby advancing preventive intervention strategies against K. pnuemoniae. Together, these complementary approaches bridge molecular understanding with translational application, contributing to the development of next-generation therapeutic and prophylactic solutions targeting this WHO-designated critical priority pathogen.