The Mutation in wbaP cps Gene Cluster Selected by Phage-Borne Depolymerase Abolishes Capsule Production and Diminishes the Virulence of Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.

Susceptible bacteria survive antibiotic treatment in the mammalian gastrointestinal tract without evolving resistance

“…Bacterial capsular deletion mutants have been shown to be internalized by macrophages and bladder epithelial cells at a higher rate than wildtype counterparts. 50,65,66 To test whether perturbations in capsule production in our clinical E. coli strain could play a role in colonocyte invasion, we generated a wbaP deletion mutant of the clinical strain PEc and performed intestinal epithelial cell invasion assays using cultured HT-29 human colonocytes 67 . We were able to culture significantly larger numbers of PEc wbaP deletion mutants (Δ wbaP ) from colonocyte cells compared to colonocytes co-incubated with PEc ( Figure 5A ).…”

Klebsiella LPS O1-antigen prevents complement-mediated killing by inhibiting C9 polymerization

“…However, in general, resistance is mostly linked to hyperencapsulation, especially with K-types K1 or K2 51,52 . It has been speculated that O-antigen might in uence retention of capsule polysaccharides 53 , but the role of capsule is thought to be of bigger importance eliciting resistance to opsonophagocytic killing 41 .…”

“…Besides the O-antigen, the capsule has been described to have a role in serum resistance 40 . Finally, we wanted to determine if the capsule also plays a role, we created a capsule knockout by deleting the gene wbaP 41,42 . In contrast to the O1-antigen, deletion of capsule in strain KpO1_1 had no effect on serum survival ( g. S6).…”

Characterization of a Lytic Bacteriophage and Demonstration of Its Combined Lytic Effect with a K2 Depolymerase on the Hypervirulent Klebsiella pneumoniae Strain 52145

“…Testing other phages in combination with B1dep is scheduled in the future. On the other hand, the activity of phage 13 and 731 on 52145 supported the fact that, by capsule degradation, phages embarking depolymerases that act on different capsule serotypes may gain higher lytic spectrum by reaching previously masked irreversible outer membrane receptors, enabled by independent, recombinant depolymerases [ 107 ]. This also suggests that irreversible receptor specificity of particular phages is independent of capsule specificity and capsule depolymerase arsenal.…”