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.

Keywords: HMV; RmpA; RmpC; capsule; hypermucoviscous; hypervirulent.

FIG 1

RmpD is required for HMV. Following transformation of the ΔrmpAC mutant with pRmpA, pRmpC, or pRmpA-C, manC expression (A) and mucoviscosity (B) were assayed as described in Materials and Methods. (C) Schematic of the rmp locus (top) and operon structure (bottom). Reverse transcription-PCR (RT-PCR) was performed using primers positioned near the 5′ or 3′ end of the three genes to amplify DNA fragments (blue lines) spanning the intergenic regions. Products were separated on a 1% agarose gel. PCR templates: RT+, products from cDNA synthesis reaction; RT−, cDNA synthesis without reverse transcriptase; no temp, PCR with no cDNA added; gDNA, genomic DNA (positive control). (D) Effect on mucoviscosity of trans expression of pRmpD in WT, ΔrmpA, ΔrmpC, and ΔrmpAC strains. One-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test was performed using WT with vector as the reference. ns, not significant; ****, P < 0.0001; ***, P < 0.001; *, P ≤ 0.05. Data were obtained after a 6-h induction of plasmid-borne rmp genes.

FIG 2

Analysis of ΔrmpD strain indicates RmpD but not RmpA is required for HMV. manC-gfp expression (A) and mucoviscosity (B) were measured in WT and ΔrmpD strains with the indicated plasmids as described in Materials and Methods. Data were obtained after a 6-h induction of plasmid-borne rmp genes. One-way ANOVA with Dunnett’s posttest was used to determine significance using WT with vector as the reference. ns, not significant; ****, P < 0.0001.

FIG 3

rmpD encodes a protein that is conserved among hypervirulent K. pneumoniae. (A) rmpD genes from several hvKp strains were identified using Geneious Prime, cloned, and expressed in either WT or ΔrmpD strains, which were assayed for mucoviscosity. One-way ANOVA with Tukey’s posttest was used to determine significance using the WT or the ΔrmpD mutant with vector as the reference. ****, P < 0.0001. (B) Western blot analysis of whole-cell extracts from WT carrying pRmpD-2×FLAG probed with α-FLAG antibody. (C) RmpD from known hvKp strains. c, chromosomal copy; p, plasmid copy; orange and blue boxes, residues conserved in chromosomal and plasmid copies, respectively; gray boxes, nonconserved residues; *, fully conserved residues; red residues, positively charged side chains, Black lines indicate predicted transmembrane domain regions: residues 3 to 23 predicted by Geneious Prime and 13 to 28 predicted by Phyre2. Accession numbers for these sequences are in the Text S1 in the supplemental material.

FIG 4

No strong correlation between capsule levels and HMV. Mucoviscosity assay (A) and uronic acid assay (B) of WT, ΔrmpD, ΔmanC, and ΔwcaJ strains with vector (pMWO-078) or pRmpD. Data were obtained after a 6-h induction of plasmid-borne rmp genes as described in Materials and Methods. One-way ANOVA with Tukey’s posttest was used to determine significance to obtain all pairwise comparisons. ns, not significant; ****, P < 0.0001.

FIG 5

The rmpD mutant is encapsulated. Bacteria expressing gfp were stained with India ink and imaged at ×1,000 magnification. (India ink) Exopolysaccharide is visualized by a clearing zone (ink exclusion) around the bacteria. (GFP) Fluorescence images indicating the presence of the bacteria. Background shading varies due to uneven liquid distribution under the coverslip.

FIG 6

Low capsule levels are sufficient for hyper-HMV. Mucoviscosity assay (A) and uronic acid assay (B) of WT and regulatory mutants (ΔrcsB, ΔkvrA, and ΔkvrB) with vector (pMWO-078) or pRmpD. Data were obtained after a 6-h induction of plasmid-borne rmpD as described in Materials and Methods. One-way ANOVA with Tukey’s posttest was used to determine significance to obtain all pairwise comparisons. ns, not significant; ****, P < 0.0001.

FIG 7

rmpD contributes to immune evasion and virulence. Adherence (A) and uptake (B) of bacteria were determined as described in Materials and Methods, using cytochalasin D to prevent phagocytosis (A) and gentamicin to kill extracellular bacteria (B). WT, ΔrmpD, and ΔmanC strains carrying either the vector (pMWO-078) or pRmpD were tested. Two-tailed Student’s t test was used to determine significance. ns, not significant; ***, P = 0.0001; ****, P < 0.0001. C57BL/6J mice were inoculated with 2 × 10⁴ CFU of the indicated strains; lungs (C) and spleens (D) were harvested at 24 and 72 hpi for bacterial enumeration. Each circle represents one mouse, solid lines indicate median values, and dotted lines represent the limit of detection. Mann-Whitney test was applied to determine significance. ns, not significant; **, P < 0.05.