# Differential contributions of ClpX and ClpP to pulmonary virulence in classical and hypervirulent Klebsiella pneumoniae

## TL;DR

This study shows that ClpX is a key protein in the virulence and antibiotic resistance of both classical and hypervirulent Klebsiella pneumoniae strains, making it a potential therapeutic target.

## Contribution

The study identifies ClpX as a conserved virulence determinant in K. pneumoniae, linking it to capsule production, pili regulation, and antimicrobial susceptibility.

## Key findings

- Loss of ClpX impairs infection in both classical and hypervirulent K. pneumoniae.
- ClpX regulates capsule production in hypervirulent strains and pili in both pathotypes.
- Loss of ClpX or ClpP increases antibiotic susceptibility across both K. pneumoniae pathotypes.

## Abstract

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen and a common cause of antibiotic-resistant infections including neonatal sepsis and hospital-acquired pneumonia. K. pneumoniae strains can be categorized into two pathotypes: classical K. pneumoniae (cKp), which often causes nosocomial infections, and hypervirulent K. pneumoniae (hvKp), which can cause severe disease in healthy hosts. New therapies are urgently needed for these infections, and caseinolytic proteins have emerged as promising therapeutic targets in other bacterial pathogens. ClpX and ClpP have been implicated in bacterial protein homeostasis, regulation of virulence, and antimicrobial susceptibility in other species, but their specific roles in K. pneumoniae pathogenesis have yet to be defined. Here, we investigate the contribution of K. pneumoniae ClpX and ClpP to lung infection, virulence factor regulation, and antibiotic susceptibility. In a murine pneumonia model, loss of ClpX impairs infection of both hvKp and cKp. Loss of ClpX results in decreased capsule production in hvKp and enhances type 1 pilus production in both pathotypes. In hvKp, loss of ClpX increases type 3 pili, while in cKp, increased type 3 piliation is observed with loss of ClpP. Across both pathotypes, loss of ClpX or ClpP increases susceptibility to a range of antibiotics. These data identify ClpX as critical to K. pneumoniae virulence and antimicrobial susceptibility. By connecting ClpX to capsule production, pili regulation, and in vivo virulence, this work highlights a conserved putative therapeutic target that may enable adjunctive strategies to enhance antibiotic efficacy or attenuate the severity of K. pneumoniae infection.

Klebsiella pneumoniae is a leading cause of antibiotic-resistant and hospital-acquired infections. The emergence of highly virulent strains of K. pneumoniae capable of causing severe disease is of utmost concern. Here, we investigate two specific caseinolytic proteins, ClpX and ClpP, produced by both classical and hypervirulent strains of K. pneumoniae and their role in K. pneumoniae lung infection. We show that ClpX is a key regulator of virulence factors including bacterial pili and capsule; it is essential for murine pulmonary fitness across both classical and hypervirulent pathotypes. Furthermore, loss of ClpX increases susceptibility to multiple antibiotics, indicating a role in both protein homeostasis and pathogenicity. These findings suggest ClpX is a conserved virulence determinant in multiple strains of K. pneumoniae and highlight its potential as a therapeutic target to enhance antibiotic efficacy or mitigate disease severity.