Characterization of Klebsiella pneumoniae type 1 fimbriae by detection of phase variation during colonization and infection and impact on virulence

Abstract

Klebsiella pneumoniae is recognized as an important gram-negative opportunistic pathogen. The ability of bacteria to adhere to host structures is considered essential for the development of infections; however, few studies have examined the influence of adhesion factors on K. pneumoniae virulence. In this study, we cloned and characterized the type 1 fimbria gene cluster of a clinical K. pneumoniae isolate. Although this cluster was not identical to the Escherichia coli type 1 fimbria gene cluster, an overall high degree of structural resemblance was demonstrated. Unique to the K. pneumoniae fim gene cluster is the fimK gene, whose product contains an EAL domain, suggesting that it has a role in regulation of fimbrial expression. Like expression of type 1 fimbriae in E. coli, expression of type 1 fimbriae in K. pneumoniae was found to be phase variable, and an invertible DNA element (fim switch) was characterized. An isogenic type 1 fimbria mutant was constructed and used to evaluate the influence of type 1 fimbriae in different infection models. Type 1 fimbriae did not influence the ability of K. pneumoniae to colonize the intestine or infect the lungs, but they were determined to be a significant virulence factor in K. pneumoniae urinary tract infection. By use of a PCR-based assay, the orientation of the fim switch during colonization and infection was investigated and was found to be all "off" in the intestine and lungs but all "on" in the urinary tract. Our results suggest that during colonization and infection, there is pronounced selective pressure in different host environments for selection of either the type 1 fimbriated or nonfimbriated phenotype of K. pneumoniae.

FIG. 1.

(A) Genetic organization of the fim region in wild-type strain C3091 and the constructed Δfim mutant. Primers used for mutant construction and for verification of correct allelic exchange are indicated. (B) Verification of correct allelic exchange in the Δfim mutant by PCR analysis using primers flanking the type 1 fimbria gene cluster in combination with primers specific for the kanamycin resistance-encoding cassette. Lane M contained DNA molecular size markers. Lanes a, primers Upfim and Dwfim; lanes b, primers Upfim and K1; lanes c, primers K2 and Dwfim. Wt, wild type.

FIG. 2.

Transmission electron micrographs of (A) E. coli HB101 and (B) E. coli HB101 transformed with pCAS624 encoding K. pneumoniae type 1 fimbriae.

FIG. 3.

Genetic organization of the type 1 fimbria gene cluster (fim) in K. pneumoniae C3091. The levels of amino acid identity to the E. coli homologues are indicated for the individual genes. The fimK gene is unique to the K. pneumoniae fim gene cluster.

FIG. 4.

Characterization of the K. pneumoniae fim phase switch. (A) Type 1 fimbria expression in K. pneumoniae is regulated by an invertible DNA element (phase switch) containing the promoter (P) of the major fimbrial subunit-encoding gene, fimA. The orientation of the phase switch, “on” or “off,” can be determined by PCR amplification of the switch region, followed by digestion with HinfI. Due to the asymmetric location of the HinfI cleavage site within the invertible element, different fragments are obtained depending on the orientation of the phase switch. IR, inverted repeat. (B) Detection of the fim phase switch orientation in cultures grown in LB broth (lane B) or on LB agar plates (lane AP). Lane M contained DNA molecular size markers.

FIG. 5.

Orientation of the fim phase switch in four K. pneumoniae strains after overnight culture in either static broth or shaking broth or on agar plates. Lane M contained DNA molecular size markers. Lanes a, strain C3091; lanes b, strain C2918; lanes c, strain C4712; lanes d, strain 747.

FIG. 6.

Colonization of the intestine by wild-type strain C3091 and the type 1 fimbria mutant fed simultaneously to three mice. Means and standard errors of the means are shown. The symbol for day 0 indicates the size of the inoculum. Wt, wild type.

FIG. 7.

Comparison of the abilities of wild-type strain C3091 and the type 1 fimbria mutant to infect the lungs and disseminate to the spleen and liver. Mice were intranasally inoculated with equal numbers of the wild type and the type 1 fimbria mutant. The competitive index (CI) was determined by dividing the ratio of wild-type bacteria to mutant bacteria recovered from infected organs by the ratio of wild-type bacteria to mutant bacteria in the inoculum. Wt, wild type.

FIG. 8.

Comparison of the urovirulence of wild-type strain C3091 and the urovirulence of the type 1 fimbria mutant. Mice were inoculated with equal numbers of the wild type and the type 1 fimbria mutant. The competitive index (CI) was determined by dividing the ratio of wild-type bacteria to mutant bacteria recovered from infected organs by the ratio of wild-type bacteria to mutant bacteria in the inoculum. (B) Comparison of the urovirulence of the type 1 fimbria mutant complemented with plasmid pCAS704 containing the fim gene cluster and the urovirulence of the wild-type strain transformed with the cloning vector. Outliers indicate that only the wild-type strain containing pEpiFOS-5 or the complemented mutant was detected in the infected tissue. Wt, wild type.

FIG. 9.

Detection of the fim phase switch orientation in the inoculum suspension (Ino) and K. pneumoniae C3091 cells infecting the colons, bladders, and lungs of mice. Lane M contained DNA molecular size markers.