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.

Infections caused by Klebsiella pneumoniae continue to be a global public health threat; the treatment of these infections is complicated by the high frequency of multidrug resistance. K. pneumoniae produces a polysaccharide capsule required for virulence. Hypervirulent isolates also have a hypermucoviscous (HMV) phenotype that increases virulence, and we recently demonstrated that a horizontally acquired gene, rmpD , is required for HMV and hypervirulence but that the identity of the polymeric product(s) in HMV isolates is uncertain.

1Klebsiella pneumoniae has a remarkable ability to cause a wide range of human diseases. It is 2 divided into two broad classes: Classical strains that are a notable problem in healthcare 3 settings due to multidrug resistance, and hypervirulent (hv) strains that are drug sensitive, but 4 able to establish disease in immunocompetent hosts. Alarmingly, there has been an increased 5 frequency of clinical isolates that have both drug resistance and hv-associated genes. One such 6 gene is rmpA that encodes a transcriptional regulator required for maximal capsule (cps) gene 7 expression and confers hypermucoviscosity (HMV). This link has resulted in the assumption that 8 HMV is caused by elevated capsule production. However, we recently reported a new cps 9 regulator, RmpC, and ∆rmpC mutants have reduced cps expression but remain HMV, suggesting 10 that capsule and HMV may be separable traits. Here, we report the identification of a small 11 protein, RmpD, that is essential for HMV, but does not impact capsule. RmpD is 58 residues 12 with a putative N-terminal transmembrane domain and highly positively charged C-terminal 13 half, and it is conserved among other hv K. pneumoniae strains. Expression of rmpD in trans 14 complements both ∆rmpD and ∆rmpA mutants for HMV, suggesting that RmpD is the key driver 15 of this phenotype. The rmpD gene is located between rmpA and rmpC, within an operon 16 regulated by RmpA. This data, combined with our previous work, suggests a model in which the 17RmpA-associated phenotypes are largely due to RmpA activating the expression of rmpD to 18 produce HMV and rmpC to stimulate cps expression.