Regulation of Klebsiella pneumoniae mucoidy by the bacterial tyrosine kinase Wzc

Abstract

Klebsiella pneumoniae is a frequent nosocomial pathogen associated with urinary tract infections (UTI), pneumonia, and septicemia. Two lineages of K. pneumoniae have emerged that challenge the treatment of these infections. One lineage includes hypervirulent strains, causing invasive community-acquired infections, and the other encompasses carbapenem-resistant isolates. Non-mucoid, carbapenem-resistant isolates are most frequently isolated from UTI. Therefore, we hypothesized that environmental conditions may drive K. pneumoniae adaptation to the urinary tract. We found that two rmp-positive, hypervirulent strains and two rmp-negative clinical UTI isolates all suppressed mucoidy when cultured in urine compared to rich LB medium without altering capsular polysaccharide (CPS) abundance. We then performed a transposon screen to identify genetic factors through which urine suppresses mucoidy. Due to variations within transposon hits, we performed whole genome sequencing on our hits and found 100% association between secondary mutations in Wzc and increased mucoidy. Wzc is an inner membrane tyrosine kinase that regulates CPS assembly. When Wzc mutations are encoded chromosomally we observed both increased mucoidy and increased cell-free extracellular polysaccharide (EPS). However, when Wzc mutations are expressed in trans we observed increased mucoidy without impacting CPS or cell-free EPS abundance. Functionally, one periplasmic mutation increased Wzc autophosphorylation, while four active site mutations reduced Wzc autophosphorylation. These four active site mutations are also sufficient to increase mucoidy in one of the rmp-negative clinical UTI isolates without affecting EPS abundance. Combined, these data implicate mutation-driven modulation of Wzc activity as a global, rmp-independent mechanism for increasing K. pneumoniae mucoidy, without altering EPS uronic acid content. Wzc activity may represent the lynchpin that coordinates both capsule biosynthesis and mucoidy, explaining why these two phenotypes have been historically intertwined.

Adaptation to novel spatially-structured environments is driven by the capsule and alters virulence-associated traits

“…S6A). This is also in line with recent research showing that some wzc mutations in trans lead to HMV 61 and it further underlines the complex regulatory pathway leading to the hypermucoviscous phenotype 43,44 . It will be important to test whether the mechanisms of adaptation and the advantage of the capsule are similar in more complex environments, for instance, in mixed bacterial communities.…”

BY-kinases: Protein tyrosine kinases like no other

“…A possible role of mutations localized within key conformationally labile regions could be to impart fitness in pathogenic species by altering the stabilities of the OS and CS, thereby affecting the relative lifetimes of Σ Μ and Σ Ο and allowing rapid adaption to environmental cues. It has been shown that Wzc mutations enable drug-resistant strains of Klebsiella pneumoniae to adapt to the urinary tract by regulating mucoidy ( 67 ). G569, which lies on α4 (rigid body motions of α4 distinguish the CS and OS; Fig.…”

“…2 ). Furthermore, an insertion at the C-terminal end of α2 (at position 526) was also found to enhance mucoidy without reducing kinase activity ( 67 ). It is notable that this insertion coincides with the region modified to generate the G 4 -construct that stabilizes the CS in E. coli Wzc ( 22 ), as discussed previously ( Fig.…”

Characterization of Klebsiella pneumoniae Extracellular Polysaccharides

“…Hypermucoviscosity is caused by changes in capsular polysaccharide chain length (Khadka et al., 2023; Ovchinnikova et al., 2023). Mucoviscosity was conventionally measured by the ‘string test’.…”

“…So far, more than 80 capsular types have been identified in the K. pneumoniae complex (Pan et al., 2015). Additionally, hypermucoviscosity is a unique virulence feature of K. pneumoniae , characterized by sticky bacterial colonies and associated with changes in capsule polymer chain length (Khadka et al., 2023; Ovchinnikova et al., 2023). The capsule protects K. pneumoniae from opsonophagocytosis and complement‐mediated killing, while hypermucoviscosity is often associated with increased dissemination in the host (Domenico et al., 1994; Mike et al., 2021).…”