The Small Protein RmpD Drives Hypermucoviscosity in Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae has a remarkable ability to cause a wide range of human diseases. It is divided into two broad classes: classical strains that are a notable problem in health care settings due to multidrug resistance, and hypervirulent (hv) strains that are historically drug sensitive but able to establish disease in immunocompetent hosts. Alarmingly, there has been an increased frequency of clinical isolates that have both drug resistance and hv-associated genes. One such gene, rmpA, encodes a transcriptional regulator required for maximal capsule (cps) gene expression and confers hypermucoviscosity (HMV). This link has resulted in the assumption that HMV is caused by elevated capsule production. However, we recently reported a new cps regulator, RmpC, and ΔrmpC mutants have reduced cps expression but retain HMV, suggesting that capsule production and HMV may be separable traits. Here, we report the identification of a small protein, RmpD, that is essential for HMV but does not impact capsule. RmpD is 58 residues with a putative N-terminal transmembrane domain and highly positively charged C-terminal half, and it is conserved among other hv K. pneumoniae strains. Expression of rmpD in trans complements both ΔrmpD and ΔrmpA mutants for HMV, suggesting that RmpD is the key driver of this phenotype. The rmpD gene is located between rmpA and rmpC, within an operon regulated by RmpA. These data, combined with our previous work, suggest a model in which the RmpA-associated phenotypes are largely due to RmpA activating the expression of rmpD to produce HMV and rmpC to stimulate cps expression. IMPORTANCE Capsule is a critical virulence factor in Klebsiella pneumoniae, in both antibiotic-resistant classical strains and hypervirulent strains. Hypervirulent strains usually have a hypermucoviscosity (HMV) phenotype that contributes to their heightened virulence capacity, but the production of HMV is not understood. The transcriptional regulator RmpA is required for HMV and also activates capsule gene expression, leading to the assumption that HMV is caused by hyperproduction of capsule. We have identified a new gene (rmpD) required for HMV but not for capsule production. This distinction between HMV and capsule production will promote a better understanding of the mechanisms of hypervirulence, which is in great need given the alarming increase in clinical isolates with both drug resistance and hypervirulence traits.

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

“…K . pneumoniae hmv can also be quantified in liquid cultures by sedimentation since hypermucoviscous cells are retained in the supernatant after centrifugation, while non-mucoviscous cells fully sediment to form a tight pellet [ 27 , 31 ]. This objective assay is more quantitative and reproducible than the string test.…”

“…pneumoniae literature despite early studies suggesting that hmv may not only be due to overproduction of CPS [ 24 , 25 ]. More recently, discordant changes in CPS production and hmv have been shown at the phenotypic and genotypic levels [ 3 , 26 , 27 ]. Some examples include the rmpC mutant in strain KPPR1S, which exhibits reduced CPS production, but retains full hmv; and the rmpD mutant in strain KPPR1S, which synthesizes WT levels of CPS, but is non-mucoviscous [ 26 , 27 ].…”

“…More recently, discordant changes in CPS production and hmv have been shown at the phenotypic and genotypic levels [ 3 , 26 , 27 ]. Some examples include the rmpC mutant in strain KPPR1S, which exhibits reduced CPS production, but retains full hmv; and the rmpD mutant in strain KPPR1S, which synthesizes WT levels of CPS, but is non-mucoviscous [ 26 , 27 ]. In fact, recent data have shown that rmpA , rmpD , and rmpC form a single operon, where RmpA auto-regulates the operon, rmpD expression increases hmv independently of capsule biosynthesis, and rmpC expression increases capsule biosynthesis without impacting hmv [ 27 ].…”

“…Some examples include the rmpC mutant in strain KPPR1S, which exhibits reduced CPS production, but retains full hmv; and the rmpD mutant in strain KPPR1S, which synthesizes WT levels of CPS, but is non-mucoviscous [ 26 , 27 ]. In fact, recent data have shown that rmpA , rmpD , and rmpC form a single operon, where RmpA auto-regulates the operon, rmpD expression increases hmv independently of capsule biosynthesis, and rmpC expression increases capsule biosynthesis without impacting hmv [ 27 ]. The mounting evidence that hmv and CPS overproduction has been conflated into a single characteristic of hvKp spotlights our limited understanding of K .…”

Identification of a Chromosomal Integrated DNA Fragment Containing the rmpA2 and iucABCDiutA Virulence Genes in Klebsiella pneumoniae

“…Chromosomal rmpA has been described in K. pneumoniae strain NTUH-K2044 previously, which was located in ICEKp1 together with the iroBCDN gene cluster ( 15 , 16 ). Recent studies suggested that rmpA mainly activates the expression of rmpD and rmpC to produce the hypermucoviscous phenotype and stimulate capsular expression, respectively ( 17 , 18 ), while strain 16HN-263 did not harbor the newly identified rmpC and rmpD genes, which may result in the negative string test. The virulence-associated genes of strain 16HN-263 were located on a 100-kbp insertion fragment flanked by IS 26 mobile elements (Tn 7074 ) ( 19 ) ( Fig.…”