H-NS enhances phage resistance in Klebsiella pneumoniae by suppressing quorum sensing

Abstract

The molecular mechanisms underlying Klebsiella pneumoniae (KP) resistance to phages have not been fully elucidated, especially those involving the quorum-sensing (QS) system and the global regulator H-NS. In this work, we investigate the relationship between H-NS and QS in phage resistance by detecting transcriptional variation under phage pressure with RT-qPCR. LuxS and lsr operon knockout strains are generated via CRISPR editing, and H-NS-overexpressing mutants are constructed using plasmid-based overexpression. We also determine phage susceptibility by measuring the efficiency of plating (EOP). The capsular uronic acid content and phage adsorption efficiency are evaluated. The results show that phage pressure strongly downregulates luxS and lsr operon gene expressions but upregulates the transcription of H-NS. Knockout of either luxS/lsr operon genes or overexpression of H-NS results in diminished phage sensitivity and the downregulation of CPS synthase genes. However, these changes do not reduce the capsular uronic acid content or affect phage adsorption rates, suggesting that H-NS-mediated resistance is independent of capsular modulation. In addition, this H-NS-QS regulatory link is conserved between HvKP and CRKP. H-NS enhances phage resistance by suppressing QS through a mechanism independent of capsular polysaccharide modulation, as evidenced by unaltered phage adsorption and statistically insignificant changes in capsular uronic acid. This provides a novel explanation for a resistance mechanism involving intracellular defenses (e.g., abortive infection) and provides possible avenues for improving phage therapy targeting multidrug-resistant K. pneumoniae.

1. Introduction

Klebsiella pneumoniae is a major pathogen of hospital-acquired infections, causing various diseases, including pneumonia, sepsis, and intra-abdominal infections. The global spread of carbapenem-resistant K. pneumoniae (CRKP) and highly virulent K. pneumoniae (HvKP) has added great challenges to clinical treatment [1,2]. Phage therapy is considered a new means to effectively combat multidrug-resistant bacterial infections because of its advantages of selective killing and less pan-resistance [3,4]. Bacteria have evolved various mechanisms to resist phage infection, such as CRISPR-Cas system-mediated immune defense against phages [5], adsorption blockade due to mutations in receptor proteins [6], and abortive infection [7]. Therefore, bacterial resistance regulatory mechanisms are very diverse, which hinders the effectiveness of phage therapy, and further exploration of bacterial resistance regulatory networks is needed for the effective treatment of bacterial infections.

Quorum sensing (QS) is a control system that consists of sensing population size and population coordination among bacteria through small signaling molecules, which are involved in regulating biofilm production, pathogenesis-related gene expression, and metabolic response [8]. In recent years, QS has also been reported to be involved in regulating the interactions between bacteria and phages; e.g., LasI/LasR can regulate phage infection by affecting the expression of its outer membrane proteins in Pseudomonas aeruginosa [9]. In addition, the QS signaling molecules (CAI-1 and AI-2) of Vibrio cholerae can also affect host survival by directly regulating phage lysis-related decisions [10]. However, reports on whether the QS system of KP is involved in the regulation of phage resistance have not yet been reported in the literature, and the molecular mechanism of phage resistance is still unknown.

Histone-like nucleoid structuring protein (H-NS) is a highly conserved global transcriptional regulator in gram-negative bacteria that regulates chromosome structure and gene expression by binding to DNA and plays a central role in the environmental stress response (e.g., osmotic pressure changes and acidic environments) [11]. Recent studies have shown that H-NS can limit bacterial adaptive evolution by inhibiting the lateral transfer of virulence island genes [12], but its functional association with the QS system and its role in phage resistance have not been explored. On the basis of these findings, the present study proposes the scientific hypothesis that H-NS may mediate phage resistance in KPs by modulating key pathways of the QS system and that this mechanism is generalizable across different clinical strains.