The KP1_4563 gene is regulated by the cAMP receptor protein and controls type 3 fimbrial function in Klebsiella pneumoniae NTUH-K2044

Abstract

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen that can adhere to host cells or extracellular matrix via type 1 and type 3 fimbriae. KP1_4563 is a gene encoding a hypothetical protein in K. pneumoniae NTUH-K2044. KP1_4563 is located between the type 1 and type 3 fimbrial gene clusters and is likely associated with fimbrial function given its putative conserved domains of unknown function (DUF1471). Cyclic AMP receptor protein (CRP) regulates virulence-related gene expression and is a crucial transcriptional regulator in many bacteria. The predicted DNA recognition motif of CRP is present in the KP1_4563 promoter region. This study aimed to investigate the function of KP1_4563 in fimbriae and its transcriptional regulation mechanism by CRP. We generated Kp-Δ4563 mutant and complementation strains. We utilized phenotype and adhesion assays to evaluate the role of KP1_4563 in fimbriae. We conducted quantitative RT-PCR (qRT-PCR), LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays to study the transcriptional regulation of KP1_4563 gene by CRP. We found that KP1_4563 negatively regulates the function of type 3 fimbriae. Compared with NTUH-K2044, the absence of KP1_4563 enhanced the ability of Kp-Δ4563 to adhere to A549 cells. CRP negatively regulates KP1_4563 by directly binding to its promoter region. KP1_4563 plays an important role in type 3 fimbrial function. This novel insight will assist in the development of strategies for preventing K. pneumoniae infection.

Fig 1. Phenotype and adhesion assays of KP1_4563.

(A) Hemagglutination assays. Mannose-resistant hemagglutination (MRHA) assays were performed with human erythrocytes. The results are expressed as the minimum bacterial density (CFU/ml) required to cause a visible agglutination reaction. Values represent the mean of three independent experiments, and the error bars represent standard deviation. P values were calculated by one-way ANOVA and Tukey HSD post hoc comparisons. (B) Mannan-binding assay. Mean values and standard deviation of six technical replicates are showed. P values were calculated by one-way ANOVA and LSD post hoc comparisons. (C) Bacterial adhesion assays. Data are the means of measurements made in technical triplicates. Error bars represent the standard deviation. P values were calculated by one-way ANOVA and LSD post hoc comparisons. Significant differences are indicated by * for P<0.05 or ** for P<0.01.

Fig 2. Transcriptional regulation of KP1_4563 by CRP.

(A) Quantitative RT-PCR (qRT-PCR). Transcriptional expression of KP1_4563 in WT and Kp-Δcrp. The results are expressed as the percentage of WT expression. Data are presented as the mean of at least three technical replicates (mean ± standard deviation). Statistical significance was analyzed by independent samples t- test. Significant difference is indicated by * for P<0.05. (B) LacZ fusion assay. The putative promoter region of KP1_4563 was cloned into the lacZ transcriptional fusion placZ15 plasmid and then introduced into CCW01 or CCW01Δcrp to determine promoter activity. The results are expressed as β-galactosidase activity (Miller units) in the cellular extracts. Statistical significance was analyzed by independent samples t-test. Significant difference is indicated by * for P<0.05. (C) EMSA. The radioactively labeled putative promoter region of KP1_4563 was incubated with increasing amounts of purified His–CRP protein with cAMP and was then subjected to 4% (w/v) native polyacrylamide gels electrophoresis. The interaction between His–CRP and the promoter region of KP1_4563 formed a DNA–CRP complex, which produced a retarded DNA band with decreased mobility. (D) DNase I footprinting. A labeled coding or non-coding DNA fragment was incubated with increasing amounts of His–CRP (lanes 1, 2, 3, and 4 represent 0, 8.5, 16.9, and 25.4 pmol of purified His–CRP protein, respectively) with cAMP and was then subjected to 8 M urea-6% (w/v) polyacrylamide gels electrophoresis. The footprint region is indicated by vertical bars with positions, and the negative numbers indicate the nucleotide positions upstream of the KP1_4563 gene start codon ATG where the A in the ATG start codon refers to position 1.