Conserved FimK Truncation Coincides with Increased Expression of Type 3 Fimbriae and Cultured Bladder Epithelial Cell Association in Klebsiella quasipneumoniae

Abstract

Klebsiella spp. commonly cause both uncomplicated urinary tract infection (UTI) and recurrent UTI (rUTI). Klebsiella quasipneumoniae, a relatively newly defined species of Klebsiella, has been shown to be metabolically distinct from Klebsiella pneumoniae, but its type 1 and type 3 fimbriae have not been studied. K. pneumoniae uses both type 1 and type 3 fimbriae to attach to host epithelial cells. The type 1 fimbrial operon is well conserved between Escherichia coli and K. pneumoniae apart from fimK, which is unique to Klebsiella spp. FimK contains an N-terminal DNA binding domain and a C-terminal phosphodiesterase (PDE) domain that has been hypothesized to cross-regulate type 3 fimbriae expression via modulation of cellular levels of cyclic di-GMP. Here, we find that a conserved premature stop codon in K. quasipneumoniae fimK results in truncation of the C-terminal PDE domain and that K quasipneumoniae strain KqPF9 cultured bladder epithelial cell association and invasion are dependent on type 3 but not type 1 fimbriae. Further, we show that basal expression of both type 1 and type 3 fimbrial operons as well as cultured bladder epithelial cell association is elevated in KqPF9 relative to uropathogenic K. pneumoniae TOP52. Finally, we show that complementation of KqPF9ΔfimK with the TOP52 fimK allele reduced type 3 fimbrial expression and cultured bladder epithelial cell attachment. Taken together these data suggest that the C-terminal PDE of FimK can modulate type 3 fimbrial expression in K. pneumoniae and its absence in K. quasipneumoniae may lead to a loss of type 3 fimbrial cross-regulation. IMPORTANCE K. quasipneumoniae is often indicated as the cause of opportunistic infections, including urinary tract infection, which affects >50% of women worldwide. However, the virulence factors of K. quasipneumoniae remain uninvestigated. Prior to this work, K. quasipneumoniae and K. pneumoniae had only been distinguished phenotypically by metabolic differences. This work contributes to the understanding of K. quasipneumoniae by evaluating the contribution of type 1 and type 3 fimbriae, which are critical colonization factors encoded by all Klebsiella spp., to K. quasipneumoniae bladder epithelial cell attachment in vitro. We observe clear differences in bladder epithelial cell attachment and regulation of type 3 fimbriae between uropathogenic K. pneumoniae and K. quasipneumoniae that coincide with a structural difference in the fimbrial regulatory gene fimK.

Keywords: Klebsiella; Klebsiella pneumoniae; Klebsiella quasipneumoniae; fimbriae; urinary tract infection; urothelium; virulence regulation.

FIG 1

K. quasipneumoniae association and invasion to bladder epithelial cells is mannose insensitive. (A) Illustration of Klebsiella type 1 and type 3 fimbriae. FimA and FimH, MrkA, and MrkD represent the major structural subunit and adhesin for type1 and 3 fimbriae, respectively. FimF and FimG represent minor structural subunits of type 1 fimbriae. (B) Schematic of the fim and mrk operons encoding type 1 and type 3 fimbriae of K. quasipneumoniae isolate PF9 (KqPF9), respectively. Illustrations were made using Biorender.com. (C and D) 5637 bladder epithelial cell association and invasion in the absence (−DM) and presence (+DM) of 2.5% d-mannose. Uropathogenic E. coli strain UTI89 was used as mannose-sensitive control, and K. pneumoniae type strain MGH78578 was used as mannose-insensitive control. Bladder epithelial cell association of PBS control was evaluated as 100%, and the effect of mannose was determined relative to respective PBS control. Experiments were performed in biological and technical triplicate. The error bars indicate standard error of mean. Significance was evaluated by two-tailed, paired Student's t test. **, P < 0.01 and ****, P < 0.0005.

FIG 2

K. quasipneumoniae association to cultured bladder epithelial cells is dependent on type 3 fimbriae. (A) Representative electron micrographs of KqPF9 and isogenic KqPF9ΔfimA, KqPF9ΔmrkA, and KqPF9ΔfimAΔmrkA mutant and complement strains. fimgc Indicates the fim gene cluster (fimAICDFGHK), and mrkgc indicates the mrk gene cluster (mrkABCDF). Arrows point to fimbriae. Scale bar = 500 nm. (B and C) Cell association of KqPF9 and isogenic type 1 and type 3 fimbriae mutants and mutants expressing respective complementing plasmids to 5637 bladder epithelial cells. The cell association of respective mutants were evaluated relative to KqPF9. The experiments were performed in biological and technical triplicate. The error bars indicate standard error of mean. Significance was evaluated using one-way ANOVA with Dunnett’s multiple comparisons post hoc. *, P < 0.05; ***, P < 0.005; and ****, P < 0.0005.

FIG 3

Expression of type 1 and type 3 fimbriae as well as bladder epithelial cell attachment and biofilm formation is elevated in KqPF9 relative to TOP52. (A and B) qRT-PCR analysis of type 1 fimbriae (fimA) and type 3 fimbriae (mrkA) expression in TOP52 and KqPF9 isolates. The expression values of fimA and mrkA were normalized to rho, and the fold change was determined relative to TOP52. (C) Percent association KqPF9 with 5637 bladder epithelial cells relative to TOP52. (D) Static biofilm formation of TOP52 and KqPF9. Percent KqPF9 biofilm formation was determined relative to TOP52. All experiments were performed at least in biological and technical triplicate. All error bars indicate standard error of mean and two-tailed, paired Student's t test was used to evaluate significance. **, P < 0.01; ***, P < 0.005; and ****, P < 0.0005.

FIG 4

The FimK C-terminal PDE is truncated in K. quasipneumonaie. (A) Comparison of the fim operon encoding type 1 fimbriae among the Enterobacteriaceae, E. coli, K. pneumoniae, and K. quasipneumoniae. E. coli does not encode fimK, and K. quasipneumoniae encodes a truncated fimK encompassing only the putative helix-turn-helix (HTH) domain with a premature stop codon truncating the EAL phosphodiesterase domain. Schematic made using Biorender.com. (B) Amino acid alignment of FimK sequences of K. pneumoniae and K. quasipneumoniae strains deposited in NCBI database is shown with respective sequence IDs. The start and end positions indicate the beginning and end of the FimK amino acid sequence. Mismatches are indicated in red. (C) A magnification of the alignment in B encompassing the region between amino acids 185 and 261 showing the truncation of K. quasipneumoniae FimK at position 218.

FIG 5

The role of mrkH and mrkJ in type 3 fimbriae expression is conserved in KqPF9. (A) Schematic of regulation of the mrk operon by transcriptional regulator MrkH, phosphodiesterase MrkJ, and cyclic di-GMP in K. pneumoniae. (B to D) qRT-PCR analysis of mrkH (B), mrkJ (C), and type 3 fimbriae (mrkA) (D) expression in KqPF9ΔmrkH and ΔmrkJ mutant and complement strains. Expression of each gene was normalized to rho, and the fold change is expressed relative to wild type. (E) Cell association of KqPF9ΔmrkH and ΔmrkJ mutant and complemented strains to 5637 bladder epithelial cells. The cell association of respective mutant strains was evaluated relative to wild-type KqPF9. (F) Static biofilm formation of KqPF9 along with isogenic mutants of respective genes, and strains expressing appropriate complementing plasmids. The biofilm formation of the mutant strains was determined relative to KqPF9. All experiments were performed in at least in biological and technical triplicate. The error bars indicate standard error of mean. Statistical testing was performed by one-way ANOVA analysis with Dunnett’s multiple-comparison test. **, P < 0.01; ***, P < 0.005; and ****, P < 0.0005.

FIG 6

TOP52 fimK but not KpPF9 fimK reduces type 3 fimbrial expression and bladder epithelial cell attachment. (A and B) qRT-PCR analysis of mrkH and type 3 fimbriae (mrkA) gene transcription in wild-type TOP52 and respective TOP52ΔfimK mutant and complemented strains. The expression of each gene was normalized to rho, and the fold change is relative to wild type. (C) 5637 bladder epithelial cell association of TOP52 and respective TOP52ΔfimK and complemented strains. qRT-PCR evaluating the expression of mrkH and mrkA in wild-type KqPF9 and respective (D and E) KqPF9ΔfimK mutant and complement strains. rho Was used for normalization, and expression is relative to wild type. (F) 5637 bladder epithelial cell association of KqPF9 and KqPF9ΔfimK mutant and complemented strains. All experiments were performed at least in biological and technical triplicate. The error bars indicate standard error of mean. One-way ANOVA with Dunnett’s multiple-comparison test was used to evaluate significance. **, P < 0.01; ***, P < 0.005; and ****, P < 0.0005.