Capsule and fimbria interaction in Klebsiella pneumoniae

Abstract

The capsular polysaccharide and type 1 fimbriae are two of the major surface-located virulence properties associated with the pathogenesis of Klebsiella pneumoniae. The capsule is an elaborate polysaccharide matrix that encases the entire cell surface and provides resistance against many host defense mechanisms. In contrast, type 1 fimbriae are thin adhesive thread-like surface organelles that can extend beyond the capsular matrix and mediate d-mannose-sensitive adhesion to host epithelial cells. These fimbriae are archetypical and consist of a major building block protein (FimA) that comprises the bulk of the organelle and a tip-located adhesin (FimH). It is assumed that the extended major-subunit protein structure permits the FimH adhesin to function independently of the presence of a capsule. In this study, we have employed a defined set of K. pneumoniae capsulated and noncapsulated strains to show that the function of type 1 fimbriae is actually impeded by the concomitant expression of a polysaccharide capsule. Capsule expression had significant effects on two parameters commonly used to define FimH function, namely, yeast cell agglutination and biofilm formation. Our data suggest that this effect is not due to transcriptional/translational changes in fimbrial gene/protein expression but rather the result of direct physical interference. This was further demonstrated by the fact that we could restore fimbrial function by inhibiting capsule synthesis. It remains to be determined whether the expression of these very different surface components occurs simply via random events of phase variation or in a coordinated manner in response to specific environmental cues.

FIG. 1.

Yeast cell agglutination profile of K. pneumoniae strains C105 (grey bars) and C105NCV (black bars) after induction of FimB expression. Bacterial cells were mixed with a 5% suspension of yeast cells on a glass slide, and the time until agglutination occurred was recorded. The assay is a direct measurement of functional FimH expression. The growth rates of the two strains during the course of the experiment were identical. Data from a single experiment are presented; the experiment was repeated three times, and the results were essentially the same.

FIG. 2.

In vitro recombination assay for monitoring of the influence of the capsule on FimB-mediated switching. (A) Map of the phase switch region in both on and off orientations. Indicated are the positions of the primers used in the PCR amplification and the sizes of the DNA fragments resulting from BsaBI digestion. IRL, inverted repeat left; IRR, inverted repeat right. (B) Acrylamide gel electrophoresis showing the amounts of on and off DNA fragments obtained after BsaBI digestion. Samples were taken at regular time intervals after induction of FimB expression, and the switch orientation was monitored. On fragments (204 and 446) and off fragments (322 and 328) are indicated. A DNA size marker (100 bp to 1,200 bp, increasing by regular 100-bp increments) is shown in the middle of the gel.

FIG. 3.

Western blot (A) and receptor blot (B) of total whole-cell lysates of recombinant cells reacted with either type 1 fimbrial antisera or α-d-mannosylated BSA, respectively. Lanes 1, C105 containing pPKL4; lanes 2, C105NCV containing pPKL4; lanes 3, MS428 containing pPKL4 (positive control); lanes 4, MS428 containing pBR322 (negative control). The FimH protein is indicated. Equal amounts of FimH were detected in both capsulated and noncapsulated K. pneumoniae cells (lanes 1 and 2).

FIG. 4.

Transmission electron micrographs of K. pneumoniae strains expressing different combinations of capsule and type 1 fimbriae. (A) C105 (capsule⁺); (B) C105 plus pPKL4 (capsule⁺ fimbriae⁺); (C) C105NCV (capsule⁻); (D) C105NCV plus pPKL4 (capsule⁻ fimbriae⁺). Scanning of multiple cells by TEM did not reveal any significant differences in the numbers of fimbriae between C105 and C105NCV (A versus C) or between C105(pPKL4) and C105NCV(pPKL4) (B versus D).

FIG. 5.

Biofilm formation by the capsule-producing K. pneumoniae strain C105 containing either pJKS60 (FimB⁺) or pBAD/Myc-HisA (control) and the capsule-negative strain C105NCV containing either pJKS60 (FimB⁺) or pBAD/Myc-HisA (control). FimB-promoted type 1 fimbria expression promoted an enhanced biofilm formation phenotype only in the absence of any capsular material. The strains were grown under hydrodynamic conditions in LB media containing 0.2% arabinose on polystyrene microtiter plates. Adhered cells were stained with 0.1% crystal violet, and the absorbance was measured at 600 nm. Shown are the averages of readings from three experiments (plus standard deviations).