A Smooth-Type, Phage-Resistant Klebsiella pneumoniae Mutant Strain Reveals that OmpC Is Indispensable for Infection by Phage GH-K3

Abstract

Bacteriophage can be used as an alternative or complementary therapy to antibiotics for treating multidrug-resistant bacterial infections. However, the rapid emergence of resistant host variants during phage treatment has limited its therapeutic applications. In this study, a potential phage-resistant mechanism of Klebsiella pneumoniae was revealed. After phage GH-K3 treatment, a smooth-type colony, named K7RB, was obtained from the K. pneumoniae K7 culture. Treatment with IO4− and/or proteinase K indicated that polysaccharides of K7 played an important role in phage recruitment, and protein receptors on K7 were essential for effective infection by GH-K3. Differences in protein expression between K7 and K7RB were quantitatively analyzed by liquid chromatography-tandem mass spectrometry. Among differentially expressed proteins, OmpC, OmpN, KPN_02430, and OmpF were downregulated significantly in K7RB. trans-Complementation of OmpC in K7RB conferred rapid adsorption and sensitivity to GH-K3. In contrast, a single-base deletion mutation of ompC in K7, which resulted in OmpC silencing, led to lower adsorption efficiency and resistance to GH-K3. These assays proved that OmpC is the key receptor-binding protein for GH-K3. In addition, the native K. pneumoniae strains KPP14, KPP27, and KPP36 showed low or no sensitivity to GH-K3. However, these strains became more sensitive to GH-K3 after their native receptors were replaced by OmpC of K7, suggesting that OmpCK7 was the most suitable receptor for GH-K3. This study revealed that K7RB became resistant to GH-K3 due to gene mutation of ompC and that OmpC of K7 is essential for effective infection by GH-K3. IMPORTANCE With increased incidence of multidrug-resistant (MDR) bacterial strains, phages have regained attention as promising potential antibacterial agents. However, the rapid emergence of resistant variants during phage treatment has limited the therapeutic applications of phage. According to our trans-complementation, ompC mutation, and phage adsorption efficiency assays, we identified OmpC as the key receptor-binding protein (RBP) for phage GH-K3, which is essential for effective infection. This study revealed that the phage secondary receptor of K. pneumoniae, OmpC, is the essential RBP not only for phage infecting Gram-negative bacteria, such as Escherichia coli and Salmonella, but also for K. pneumoniae.

Global Trends in Proteome Remodeling of the Outer Membrane Modulate Antimicrobial Permeability in Klebsiella pneumoniae

“…Another possibility, and not mutually exclusive, is that selection for variation in OmpK36, OmpK37, and OmpK38 is being driven by microbial factors, such as bacteriophages or colicins (74,75). Bacteriophages such as GH-K3 that use OmpK36 as a receptor would produce the selective pressure for the evolution of phage-resistant ompK36 mutants (74), which would select for a collateral drug resistance phenotype given the MIC data in our study. Given prospects for phage therapy to treat CRE infections, the evolutionary drivers on outer membrane protein remodeling in K. pneumoniae and other CRE pathogens need further attention.…”

“…One possibility is host factors involved in driving antigenic variation for protection against antibodies and/or complement, as OmpK36 has been shown to be targeted by components of the complement pathway (71)(72)(73). Another possibility, and not mutually exclusive, is that selection for variation in OmpK36, OmpK37, and OmpK38 is being driven by microbial factors, such as bacteriophages or colicins (74,75). Bacteriophages such as GH-K3 that use OmpK36 as a receptor would produce the selective pressure for the evolution of phage-resistant ompK36 mutants (74), which would select for a collateral drug resistance phenotype given the MIC data in our study.…”

Three Capsular Polysaccharide Synthesis-Related Glucosyltransferases, GT-1, GT-2 and WcaJ, Are Associated With Virulence and Phage Sensitivity of Klebsiella pneumoniae

“…Phage GH-K3 and its host strain K. pneumoniae K7 were previously isolated and maintained in our laboratory (Gu et al, 2012). A rough-type, phage-resistant K. pneumoniae mutant strain was isolated and named as K7R R in a previous study (Cai et al, 2018).…”

“…Especially, the abortion of adsorption is the most common bacteriophage resistance mechanism (Silva et al, 2016). Similar to other Gram-negative bacteria, the first and second phage adsorption receptors based on polysaccharides and outer membrane proteins of K. pneumoniae have been described (Cai et al, 2018). During the interaction of phage and host strain, majority of the anti-phage mutant colonies became small-rough type due to loss of cell-surface polysaccharides.…”

Fitness Trade-Offs Resulting from Bacteriophage Resistance Potentiate Synergistic Antibacterial Strategies

“…Alongside LPS, early studies of coliphage adsorption to E. coli K-12 identified the maltose transporter LamB (75) and OmpC (49), an outer membrane porin, as primary receptors during phage infection. OmpC recognition by phages appears to be conserved in other enteric bacteria such as the nosocomial pathogen Klebsiella pneumoniae (76). In addition, the outer membrane protein OmpF is also an important phage receptor of enteric bacteria (77).…”