A Klebsiella pneumoniae Regulatory Mutant Has Reduced Capsule Expression but Retains Hypermucoviscosity

Abstract

The polysaccharide capsule is an essential virulence factor for Klebsiella pneumoniae in both community-acquired hypervirulent strains as well as health care-associated classical strains that are posing significant challenges due to multidrug resistance. Capsule production is known to be transcriptionally regulated by a number of proteins, but very little is known about how these proteins collectively control capsule production. RmpA and RcsB are two known regulators of capsule gene expression, and RmpA is required for the hypermucoviscous (HMV) phenotype in hypervirulent K. pneumoniae strains. In this report, we confirmed that these regulators performed their anticipated functions in the ATCC 43816 derivative, KPPR1S: rcsB and rmpA mutants are HMV negative and have reduced capsule gene expression. We also identified a novel transcriptional regulator, RmpC, encoded by a gene near rmpA. The ΔrmpC strain has reduced capsule gene expression but retains the HMV phenotype. We further showed that a regulatory cascade exists in which KvrA and KvrB, the recently characterized MarR-like regulators, and RcsB contribute to capsule regulation through regulation of the rmpA promoter and through additional mechanisms. In a murine pneumonia model, the regulator mutants have a range of colonization defects, suggesting that they regulate virulence factors in addition to capsule. Further testing of the rmpC and rmpA mutants revealed that they have distinct and overlapping functions and provide evidence that HMV is not dependent on overproduction of capsule. This distinction will facilitate a better understanding of HMV and how it contributes to enhanced virulence of hypervirulent strains. IMPORTANCE Klebsiella pneumoniae continues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor for K. pneumoniae is the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen.

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impactKlebsiella pneumoniaefitness

“…The hmv of hvKp is generally ascribed to increased production of CPS; however, recent studies have challenged this paradigm [24–27, 30]. This paradigm shift in our understanding of K. pneumoniae hmv has been an emerging concept in recent years as genes, namely kvrA, kvrB, rmpC , and rmpD , have been identified that differentially affect hmv and CPS biosynthesis [17, 25, 26, 30]. Here, we have generated a condensed, ordered transposon library in a genetically tractable and hmv strain of K. pneumoniae , KPPR1, and used this library to query a question at the forefront of K. pneumoniae pathogenesis, namely, how do hmv and CPS biosynthesis independently and coordinately impact invasive hvKp infections?…”

“…Moreover, the ability of the bacteria to distinctly control hmv and CPS biosynthesis suggest that there are environmental conditions in which one or both properties are advantageous, emphasizing that while these two features are closely associated with hypervirulent K. pneumoniae , they likely serve distinct functions within specific environments and may actually be regulated in response to the local environment [39]. For example, the hmv low /CPS WT Δ rmpD mutant adheres to macrophage-like J774A.1 cells more than WT, while the hmv WT /CPS low Δ rmpC mutant adheres similar to WT [25, 26]. In addition, non-mucoid strains are more likely to be isolated from urine than blood [39].…”

“…More recently, discordant changes in CPS production and hmv have been shown at the phenotypic and genotypic levels [3, 25]. Some examples include the rmpC mutant in strain KPPR1S and the MagA G334A ( m ucoviscosity- a ssociated g ene A , also known as Wzy) mutant in strain NTUH-K2044, which both exhibit reduced CPS production, but remain hmv [26–29]; and the rmpD mutant in strain KPPR1S and the kvrA ( K lebsiella v irulence r egulator) mutant in the ST258 strain MKP103, which remain fully encapsulated, but are non-mucoid [25, 30]. The mounting evidence that hmv and CPS overproduction has been conflated into a single characteristic of hvKp spotlights our limited understanding of K. pneumoniae hypervirulence and points to the critical need to better understand the relationship and function of these two important features in K. pneumoniae pathogenesis and biology.…”

Hypervirulent Klebsiella pneumoniae – clinical and molecular perspectives

“…Whilst RmpA appears to coordinate capsule and HMV, RmpC only regulates capsule; an rmpC mutant synthesizes less capsule, but retains hypermucoviscosity. This suggests that capsule synthesis is not required for the HMV phenotype [121]. Indeed, early reports of RmpA and RmpB proposed that capsule overexpression was not the basis for HMV [105,115].…”

Mutation of kvrA causes OmpK35/36 porin downregulation and reduced meropenem/vaborbactam susceptibility in KPC-producing Klebsiella pneumoniae

“…DhaR is a transcriptional activator responsible for controlling the production of the metabolic enzymes glycerol dehydrogenase and dihydroxyacetone kinase (18,19). KvrA is a MarR-type transcriptional repressor with a key role in K. pneumoniae capsulation (20–22).…”