Differential contributions of ClpX and ClpP to pulmonary virulence in classical and hypervirulent <i>Klebsiella pneumoniae</i>

<i>Klebsiella pneumoniae</i> is an opportunistic Gram-negative pathogen and a common cause of antibiotic-resistant infections including neonatal sepsis and hospital-acquired pneumonia. <i>K. pneumoniae</i> strains can be categorized into two pathotypes: classical <i>K. pneumoniae</i> (cKp), which often causes nosocomial infections, and hypervirulent <i>K. pneumoniae</i> (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 <i>K. pneumoniae</i> pathogenesis have yet to be defined. Here, we investigate the contribution of <i>K. pneumoniae</i> 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 <i>K. pneumoniae</i> virulence and antimicrobial susceptibility. By connecting ClpX to capsule production, pili regulation, and <i>in vivo</i> virulence, this work highlights a conserved putative therapeutic target that may enable adjunctive strategies to enhance antibiotic efficacy or attenuate the severity of <i>K. pneumoniae</i> infection.IMPORTANCE<i>Klebsiella pneumoniae</i> is a leading cause of antibiotic-resistant and hospital-acquired infections. The emergence of highly virulent strains of <i>K. pneumoniae</i> 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 <i>K. pneumoniae</i> and their role in <i>K. pneumoniae</i> 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 <i>K. pneumoniae</i> and highlight its potential as a therapeutic target to enhance antibiotic efficacy or mitigate disease severity.