The Nucleoid-Associated Protein Fis Represses Type 3 Fimbriae to Modulate Biofilm and Adherence Formation in Klebsiella pneumoniae

Abstract

The nucleoid-associated protein Fis functions as a global regulator that influences various cellular processes in Gram-negative bacteria. In this study, we examined the role of Fis in the transcriptional regulation of type 3 fimbriae in Klebsiella pneumoniae, a notable opportunistic pathogen associated with hospital-acquired infections. Our transcriptional analyses revealed that deleting the fis gene caused a significant upregulation of mrkA and mrkH, the genes responsible for the structure and regulation of type 3 fimbriae, respectively. Additionally, phenotypic assays demonstrated that the Δfis mutant exhibited enhanced biofilm formation and greater adherence to A549 lung epithelial cells compared to the wild-type strain. These effects were restored to wild-type levels in the cis-complemented strain. Electrophoretic mobility shift assays confirmed that Fis directly binds to the regulatory regions upstream of both mrkA and mrkH, indicating that repression occurs through direct interaction with the promoter. In summary, our findings show that Fis acts as a transcriptional repressor of mrkA and mrkH, thereby negatively regulating the expression of type 3 fimbriae, biofilm formation, and adherence. This study highlights Fis as a direct regulator of fimbrial expression and biofilm development in K. pneumoniae, deepening our understanding of its virulence regulatory network.

Keywords: Fis; Klebsiella pneumoniae; adherence; biofilm; mrkA; mrkH; type 3 fimbriae.

Figure 1

Growth curves of K. pneumoniae WT, Δfis, and Δfis::fis strains. Cultures were grown in TSB at 37 °C for 8 h, and the optical density at 600 nm (OD₆₀₀) was measured at hourly intervals. Data represent the mean ± standard deviation from three independent biological replicates. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test, comparing to the growth of the WT strain (* p < 0.05).

Figure 2

Regulation of mrkA and mrkH type 3 fimbrial gene expression by the nucleoid-associated protein Fis. RT-qPCR analysis of (A) mrkA and (B) mrkH expression, and measurement of CAT activity from transcriptional fusions to the cat reporter gene (C) pKK-mrkA-cat and (D) pKK-mrkH-cat in K. pneumoniae WT, Δfis, and Δfis::fis strains. Data represent the mean values ± standard deviation from at least two independent biological replicates. Statistical significance was determined relative to the WT strain using one-way ANOVA followed by Tukey’s post hoc test (** p < 0.01; *** p < 0.001; ns, not significant).

Figure 3

Effect of Fis on biofilm formation and adherence to epithelial cells in K. pneumoniae. (A) Quantitative biofilm assay performed in polystyrene microtiter plates showing biofilm biomass of WT, Δfis, and Δfis::fis strains. (B) Representative crystal violet-stained biofilms formed by the indicated strains in 96-well plates. The blank corresponds to TSB medium without bacterial inoculation and was included as a negative control. (C) Adherence of the same strains to A549 lung epithelial cells. The Δfis mutant exhibited significantly increased biofilm formation and adherence compared with the WT strain, whereas complementation (Δfis::fis) restored these phenotypes to WT levels. Data represent mean values ± standard deviation from three independent biological experiments. Statistical significance was determined relative to the WT strain using one-way ANOVA followed by Tukey’s post hoc test (*** p < 0.001; ns, not significant).

Figure 4

Direct interaction of Fis with the mrkA and mrkH promoter regions was demonstrated by electrophoretic mobility shift assays (EMSA). Increasing concentrations of purified Fis-His₆ recombinant protein were incubated with DNA fragments containing the promoter regions of (A) mrkA and (B) mrkH. A progressive, concentration-dependent retardation in DNA mobility was observed for both fragments, indicating the formation of Fis–DNA complexes. As a negative control, a DNA fragment corresponding to the fbpA coding region, unrelated to type 3 fimbrial regulation, was included in the same assays, showing no detectable shift. Arrows indicate free DNA, or Fis/DNA complexes, stained with ethidium bromide.

Figure 5

In silico analysis of the regulatory regions of mrkA and mrkH. (A) Predicted Fis-binding motifs identified within the upstream regulatory region of mrkA. (B) Predicted Fis-binding motifs identified within the upstream regulatory region of mrkH. Putative Fis-binding sites are enclosed in boxes; nucleotides matching the consensus sequence are shown in bold, whereas non-conserved positions are indicated in bold red. The experimentally determined promoter regions for each gene are also shown. (C) Alignment of the predicted Fis-binding motifs from the K. pneumoniae mrkA and mrkH regulatory regions with the consensus Fis-binding sequence from E. coli. Nucleotides that differ from the E. coli consensus are highlighted in bold red.