Consequences of reduction of Klebsiella pneumoniae capsule expression on interactions of this bacterium with epithelial cells

Abstract

Most Klebsiella pneumoniae clinical isolates are fully encapsulated and adhere in vitro to intestinal cell lines with an aggregative pattern. In this study, the influence of the capsule on interactions with epithelial cells was investigated by creating an isogenic mutant defective in the synthesis of the capsule. Determination of the uronic acid content of bacterial extracts confirmed that the mutant did not produce capsular polysaccharides whereas, with the wild-type strain, the level of encapsulation was growth phase dependent and reached a maximum during the lag and early log phases. Assays performed with different epithelial cell lines, Int-407, A-549, and HEp-2, showed that the capsule-defective mutant demonstrated greater adhesion than did the wild-type strain and that the aggregative pattern was maintained, indicating that the capsule was not related to the adhesion phenotype. In contrast, when the mucus-producing HT-29-MTX cells were used, the encapsulated wild-type strain adhered more strongly than did the capsule-defective mutant. No invasion properties were observed with any of the capsular phenotypes or cell lines used. The K. pneumoniae adhesin CF29K was detected by Western blot analysis and enzyme-linked immunosorbent assay on the surface of transconjugants obtained after transfer of a conjugative plasmid harboring the CF29K-encoding genes into both the wild-type and the capsule-defective strains. The amounts of adhesin detected were greater in the capsule-defective background strain than in the wild-type encapsulated strain and were associated with an increase in the level of adhesion to Caco-2 cells. Moreover, RNA slot blot experiments showed that transcription of the adhesin-encoding gene was markedly increased in the capsule-defective mutant compared to the wild-type encapsulated background. These results suggest (i) that the capsule plays an active role during the initial steps of the pathogenesis by interacting with mucus-producing cells but is subsequently not required for the adhesin-related interaction with the epithelial cell surface and (ii) that the expression of the adhesin is modulated by the presence of a capsule at a transcriptional level.

FIG. 1

Construction of the capsule-defective mutant K. pneumoniae LM21(cps). (A) A 2,560-bp DNA fragment was amplified from K. pneumoniae LM21 chromosomal DNA by PCR and ligated into the BamHI site of pUC18 to obtain pS1-3. An internal BclI 509-bp fragment was deleted and replaced by the Kn^r cassette, and the construct was cloned into the BamHI site of pLD55. B′, BamHI-BclI junctions. (B) Allelic exchange mutagenesis. After introducing pSFB1-3ΔKn into K. pneumoniae LM21, a single recombination event leads to the formation of a cointegrate (Kn^r Tet^r) and a double recombination event leads to the mutated copy (Kn^r) after loss of the suicide vector (Tet^r).

FIG. 2

Electrophoretic analysis of the amplified products from the K. pneumoniae LM21 wild-type strain (lane A) and the isogenic mutant strain LM21(cps) (lane B) with primers kcps1 and kcps3. The 2,560- and 3,300-bp fragments (arrows) are indicated. Lane M contains the 1-kb molecular size ladder (Gibco BRL, Cergy-Pontoise, France).

FIG. 3

Aspects of the colonies of K. pneumoniae LM21 (A) and its capsule-defective mutant strain LM21(cps) (B) after overnight culture at 37°C on D.W. agar plates.

FIG. 4

Plots of capsule quantities related to the growth phase of wild-type K. pneumoniae LM21 (A) and mutant K. pneumoniae LM21(cps) (B). Growth curves were determined by measuring the number of CFU (solid triangles). Capsule was quantified by measuring the quantity of uronic acid per 10⁶ CFU (open squares). Values for uronic acid contents are the mean of measurements made in triplicate.

FIG. 5

Adherence assays performed with Int-407, A-549, HEp-2 and HT-29-MTX 10⁻⁶ cell lines with the wild-type K. pneumoniae LM21 (solid bars) and the capsule-defective mutant LM21(cps) (hatched bars). The results are expressed in CFU per monolayer. The data are the mean of measurements made in triplicate.

FIG. 6

SDS-PAGE (A) and Western blot (B) of bacterial surface proteins from K. pneumoniae CF504 (lane 1), E. coli transconjugant CF604 (lane 2), K. pneumoniae CH067 (lane 3), and K. pneumoniae CH009 (lane 4). Molecular weight marker positions are shown for carbonic anhydrase (30,000) and ovalbumin (43,000).

FIG. 7

Anti-CF29K antibody-binding analysis of the whole bacteria: K. pneumoniae CH067 and CH009 (A) and K. pneumoniae CF504 with its transconjugant E. coli CF604 (B). The abilities of the bacteria from different backgrounds to bind the anti-CF29K antibodies were measured by ELISA. Microtiter plates precoated with anti-CF29K antibodies were incubated with different quantities of the whole K. pneumoniae CH067 (open squares), K. pneumoniae CH009 (solid squares), K. pneumoniae CF504 (solid triangles), or E. coli CF604 (open triangles). To detect bound bacteria, wells were incubated with biotinylated anti-CS31A antiserum; streptavidin-alkaline phosphatase and an appropriate substrate were added, and the optical density at 405 nm was monitored. The optical density measured at 405 nm from the negative control (no bacteria added) was equal to 0.130.

FIG. 8

Regulation of cf29A transcription by the presence of capsule. For the RNA slot-blot analysis: 1 or 5 μg of RNA isolated from K. pneumoniae CH067 (encapsulated) and CH009 (nonencapsulated) was hybridized with a cf29A-specific probe (left). The same blot was probed with an rrnB7 probe as a standard for the total amount loaded onto the membrane (right).