Arginine Regulates the Mucoid Phenotype of Hypervirulent Klebsiella pneumoniae

Abstract

Hypervirulent Klebsiella pneumoniae is associated with severe community-acquired infections. Hypervirulent K. pneumoniae colonies typically exhibit a mucoid phenotype. K. pneumoniae mucoidy is influenced by a complex combination of environmental factors and genetic mechanisms. Mucoidy results from altered capsular polysaccharide chain length, yet the specific environmental cues regulating this phenotype and their impact on pathogenesis remain unclear. This study demonstrates that casamino acids enhance the mucoidy phenotype but do not affect total capsular polysaccharide levels. Through targeted screening of each amino acid present in casamino acids, we identified that arginine is necessary and sufficient to stimulate the mucoid phenotype without altering capsule abundance. Furthermore, arginine activates the rmpADC promoter, increasing rmpD transcript levels, which in turn modulates capsular polysaccharide chain length and diversity. The arginine regulator, ArgR, plays a pivotal role in this regulatory cascade since deleting argR decreases mucoidy and increases capsular polysaccharide chain length diversity. Additionally, the ∆argR mutant displays increased macrophage association and has a substantial competitive defect in the lungs of mice, suggesting a link between arginine-dependent gene regulation, immune evasion and in vivo fitness. We discovered that arginine-dependent regulation of mucoidy is conserved in four additional hypervirulent K. pneumoniae isolates likely via a conserved ARG binding box present in rmp promoters. Our findings support a model in which arginine activates ArgR and increases mucoidy in hypervirulent K. pneumoniae. As a result, it is possible that arginine-dependent regulation of mucoidy allows hypervirulent K. pneumoniae to adapt the cell surface across different niches. This study underscores the significance of arginine as a regulatory signal in bacterial virulence.

Keywords: Klebsiella pneumoniae; amino acids; arginine; capsular polysaccharide; capsule; hypermucoviscosity; hypervirulence; mucoidy.

Figure 1.

Specific nutrients dissociate CPS from mucoidy. Wild type K. pneumoniae strain KPPR1 was cultured in LB or low-iron M9 minimal medium with either 0.4% glucose (Glc), 1% casamino acids (CAA) or both as nutrient sources. (A) Mucoidy was determined by quantifying the supernatant OD₆₀₀ after sedimenting 0.5 OD₆₀₀ unit of culture at 1,000 x g for 5 min. (B) Uronic acid content of crude CPS extracts were quantified and normalized to the OD₆₀₀ of the overnight culture. Data presented are the mean, and error bars represent the standard error of the mean. Statistical significance was determined using a one-way ANOVA with a Tukey post-test. The statistics displayed above the data bars represent values relative to the LB medium. The additional statistics connected by lines serve as direct comparisons to highlight differences between M9 media conditions. # p < 0.0001. Experiments were performed ≥3 independent times, in triplicate.

Figure 2.

Arginine and phenylalanine are necessary for KPPR1 to regulate mucoidy. Wild type K. pneumoniae strain, KPPR1, was cultured in (A, B) low-iron M9 minimal medium with 20 mM sodium pyruvate and all 18 amino acids (M9+All) or individual amino acid absent. In (C, D) KPPR1 was cultured in M9+All or M9 medium without arginine, phenylalanine, or both absent. (A, C) Mucoidy was determined by quantifying the supernatant OD₆₀₀ after sedimenting 0.5 OD₆₀₀ unit of culture at 1,000 x g for 5 min. Uronic acid content of crude CPS extracts were quantified and (B) normalized to the OD₆₀₀ of the overnight culture or (D) normalized to 10⁹ CFUs. Data presented are the mean, and error bars represent the standard error of the mean. Statistical significance was determined using a one-way ANOVA and Dunnett post-test compared to M9+All. * p < 0.05; ** p < 0.01; *** p < 0.001; # p < 0.0001. Experiments were performed ≥3 independent times, in triplicate.

Figure 3.

Arginine restores mucoidy in KPPR1 and ArgR is required for mucoidy regulation. (A,B) KPPR1 was cultured overnight in low-iron M9 medium supplemented with 18 amino acids (All), 0.2% glycerol (Glycerol), or increasing concentrations of arginine (0.025%–0.2%) and 0.2% glycerol. Additionally, KPPR1, astA::kan, argG::kan, and ∆argR mutants were cultured overnight in M9 minimal media under the following conditions: with 18 amino acids (All), without arginine (-Arg), with 0.2% glycerol (Glycerol), or with 0.2% glycerol and 0.2% arginine (Glycerol+Arg). (A,C) Mucoidy was determined for each of the conditions by sedimenting 0.5 OD₆₀₀ unit of culture at 1,000 x g for 5 min and then measuring the supernatant OD600. (B,D) The total capsule abundance was determined by measuring the uronic acid content of crude CPS extracts and normalized to 10⁹ CFUs. Data presented are the mean, and error bars represent the standard error of the mean. In panels (C,D) the cross indicates no growth in a condition. (A,B) Statistical significance was determined using one-way ANOVA with a Dunnett post-test. (C,D) Statistical significance was determined using two-way ANOVA with a Tukey post-test. Experiments were performed ≥3 independent times, in triplicate ** p < 0.01; # p < 0.0001.

Figure 4.

Arginine regulates mucoidy by upregulating rmpD transcription in an ArgR-dependent manner. KPPR1 and the ∆argR mutant were cultured in different amino acid conditions (M9+All, M9-Arg, M9+Glycerol, M9+Glycerol+Arg). (A) Promoter activity was measured by GFP expression under the control of the rmp operon. RNA was isolated from mid-log cultures of KPPR1 in the four media conditions or from ∆argR in M9+All. (B, C) The relative abundance of rmpD in the different conditions was determined by qRT-PCR and normalized to gap2 transcript abundance. (B) Relative rmpD transcript levels in KPPR1 cultured in M9-Arg or M9+Glycerol+Arg were compared to M9+All or M9+Glycerol, respectively, while (C) the ∆argR mutant cultured in M9+All transcript levels were compared to wildtype, KPPR1. Data presented are the mean, and error bars represent the standard error of the mean. Statistical significance was determined in panel A with a two-way ANOVA with Tukey’s post-test to compare wild-type KPPR1 to the ∆argR mutant and comparing wildtype-KPPR1 in the four conditions. In panels B and C significance was determined using a student’s t-test to determine if either group were significantly different than 1. * p<0.05; ** p < 0.01; *** p < 0.001; # p < 0.0001. Experiments were performed ≥3 independent times, in triplicate.

Figure 5.

Arginine and ArgR regulate mucoidy by upregulating rmpD transcription and decreasing capsule Chain diversity. KPPR1 and the ∆argR mutant were cultured in different amino acid conditions (M9+All, M9-Arg, M9+Glycerol, M9+Glycerol+Arg) or (M9+All), respectively. (A) Capsular polysaccharides (CPS) were separated on a 4–15% SDS-PAGE gel and stained with alcian blue and silver stain. Three distinct polysaccharide types emerged: diverse mid- to high-molecular-weight chains (Type A), uniform high-molecular-weight chains (Type B), and diffuse ultra-high-molecular-weight chains (Type C). Data presented are the mean, and error bars represent the standard error of the mean. Representative images are shown in panel A with the quantification being shown in panels B and C. Statistical significance was determined in panel B using a two-way ANOVA with Tukey’s post-test was used to compare specific groups, while panel H a one-way ANOVA with a Dunnett’s post-test was used. * p < 0.05; ** p < 0.01. Experiments were performed ≥3 independent times, in triplicate.

Figure 6.

ArgR blocks bacterial association with immortalized bone marrow-derived macrophages (iBMDMs) and promotes competition fitness in the lung. The strains KPPR1 and ∆argR were transformed with either the empty vector (EV) or argR (PargR) or rmpD (PrmpD). The EV backbone is pACYC184Δtet. (A) Wildtype and the ∆argR mutant carrying the vectors were cultured overnight in M9+All and mucoidy was determined by a sedimentation assay (0.5 OD₆₀₀ unit centrifuged at 1,000 x g for 5 min). Macrophages (BEI NR-9456) were incubated with KPPR1 wildtype or ∆argR for 2 hours (MOI = 10). (B) Macrophages were washed with PBS 3x, then lysed with 0.2% TritonX100. CFUs were enumerated and normalized to input CFUs for association. Data presented (A, B) are the mean, and error bars represent the standard error of the mean. (C) KPPR1 EV and ∆argR carrying EV or PargR were cultured overnight and 1×10⁶ CFU of 1:1 mixture of WT_EV and ∆argR_EV or ∆argR_PargR .were administered retropharyngeally to C57BL/6 mice. The log₁₀ competitive index at 24 h post infection is shown relative to 1.0 for individual mice with bars representing the median and interquartile range. Statistical significance was determined in panel A with a one-way ANOVA and Dunnett’s post-test, while in panel B a paired t-test was used. In panel C, an unpaired t-test was used to determine statistical significance. *** p < 0.001; # p < 0.0001. Experiments were performed ≥2 independent times, with each dot representing an individual mouse

Figure 7.

Multiple HvKp strains regulate mucoidy in response to arginine and encode an ARG binding box in the rmp promoter. One hypervirulent K. pneumoniae (hvKp) laboratory strain (NTUH-K2044) and three hvKp clinical isolates (Kp4289, Kp4585, and Kp6557) were cultured in M9+All or M9-Arg. (A) Mucoidy was assessed by quantifying the supernatant OD₆₀₀ after sedimenting 0.5 OD₆₀₀ unit of culture at 1,000 x g for 5 min. (B) A BPROM-predicted ARG binding box from the rmp promoter of five HvKp strains was aligned with ClustalW. The yellow highlighted nucleotides represent highly conserved nucleotides in the ARG box with stars representing conservation of the nucleotide across all five strains. (C). KPPR1 carrying either the P_rmp-GFP reporter plasmid or a scrambled ARG box (P_rmp∆ARG-GFP) was cultured in different amino acid conditions (M9+All, M9-Arg, M9+Glycerol, M9+Glycerol+Arg). Promoter activity was measured by GFP expression under the control of the rmp operon. Data presented are the mean, and error bars represent the standard error of the mean. Statistical significance was determined using an unpaired t-test. ** p < 0.01; # p < 0.0001. Experiments were performed >3 independent times, in triplicate.

Figure 8.

Model of K. pneumoniae regulation of mucoidy in response to arginine. When cultured in the presence of the semi-essential amino acid, arginine, K. pneumoniae increases mucoidy without altering the total capsule abundance. When arginine is brought into the cell, the arginine regulator, ArgR, will bind with arginine and become active. The activated ArgR-arginine can act as a transcriptional regulator by binding to ARG boxes. The arginine-ArgR complex activates the rmpADC promoter in an ARG-box dependent manner and upregulates rmpD, the small protein known to regulate mucoidy. The RmpD protein then goes on to interact with the Wzc protein, a tyrosine kinase that regulates high-level capsule polymerization. RmpD-Wzc interactions decrease ‘Type A’ and increase ‘Type B’ polysaccharide chains, which decreases CPS diversity and presents as the mucoid phenotype. Created with Biorender.com