Capsule impedes adhesion to and invasion of epithelial cells by Klebsiella pneumoniae

Abstract

The adhesion of K21a, K26, K36, and K50 capsulated Klebsiella strains to ileocecal (HCT-8) and bladder (T24) epithelial cell lines was significantly lower than that of their corresponding spontaneous noncapsulated variants K21a/3, K26/1, K36/3, and K50/3, respectively. Internalization of the bacteria by both epithelial cell lines was also significantly reduced. Similarly, a capsule-switched derivative, K2(K36), that exhibited a morphologically larger K36 capsule and formed more capsular material invaded the ileocecal epithelial cell line poorly compared to the corresponding K2 parent strain. None of the capsulated strains exhibited significant mannose-sensitive type 1 fimbriae, whereas two of the noncapsulated variants K21a/3 and K50/3 exhibited potent mannose-sensitive hemagglutinating activity. Although hemagglutinating activity that could be attributed to mannose-resistant Klebsiella type 3 fimbriae was weak in all strains, in several cases the encapsulated parent strains exhibited lower titers than their corresponding noncapsulated variants. Although the level of adhesion to the ileocecal cells is not different from adhesion to bladder cells, bacterial internalization by bladder cells was significantly lower than internalization by ileocecal cells, suggesting that bladder cells lack components required for the internalization of Klebsiella.

FIG. 1

Immunoelectron micrographs of K2 (A), its capsule-switched derivative K2(K36) (C), and the parent strain K36 (B) grown on agar supplemented with 1% lactose and labeled with rabbit anti-K36 capsule antibody. Note that the anti-K36 serum labeled only the K36 parent strain and the derivative K2(K36).

FIG. 1

Immunoelectron micrographs of K2 (A), its capsule-switched derivative K2(K36) (C), and the parent strain K36 (B) grown on agar supplemented with 1% lactose and labeled with rabbit anti-K36 capsule antibody. Note that the anti-K36 serum labeled only the K36 parent strain and the derivative K2(K36).

FIG. 2

Capsule formation, cellular adhesion, and internalization of the K2 and K36 parent strains and their capsule-switched derivative. Confluent monolayers of HCT-8 cells were exposed to K2 (closed box) and K36 (striped box) capsulated parent strains and their derivative K2(K36) (open box). The amount of glucuronic acid (micrograms per 10⁹ bacteria), percent adhesion to, and percent internalization into the cell lines were estimated as described in Materials and Methods. Data represent mean values and standard deviations of at least quadruplicate experiments for each strain. Mean values for the K2 and K36 parent strains were significantly higher than those for K2(K36) (P > 0.01) for both the adhesion and internalization assays.

FIG. 3

Internalization of noncapsulated variants of Klebsiella into HCT-8 cells. Relative invasion represents the percent increase in invasion of three noncapsulated variants (open boxes) from each of the indicated Klebsiella serotypes compared to the corresponding capsulated parent strains (=100%; closed boxes). The hatched box is the relative invasion of the K21a capsulated revertant strain derived from a noncapsulated variant strain. Percents invasion of the capsulated parent strains were 0.75, 2.94, 4.55, 3.58, and 2.74 for K26, K36, K50, K2, and K21a, respectively. All values for the noncapsulated variants are significantly higher (P < 0.01) than those for the corresponding capsulated parent strains. Data shown represent mean values and standard deviations.

FIG. 4

Intracellular survival of capsulated parent strain K50 and its noncapsulated variant K50/3. Monolayers of HCT-8 cells were infected with the indicated K. pneumoniae strains. After invasion, the medium was replaced with fresh medium containing 10 μg of gentamicin ml, and the monolayers were lysed at the indicated times to determine the number of the intracellular bacteria expressed as a percentage of inoculum. The asterisk denotes a value significantly different from that obtained after 2 h (P < 0.01). Data shown represent mean values and standard deviations.