Capsule deletion via a λ-Red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH 78578

Abstract

Background: Klebsiella pneumoniae is a leading cause of hospital-acquired urinary tract infections and pneumonia worldwide, and is responsible for many cases of pyogenic liver abscess among diabetic patients in Asia. A defining characteristic of this pathogen is the presence of a thick, exterior capsule that has been reported to play a role in biofilm formation and to protect the organism from threats such antibiotics and host immune challenge.

Findings: We constructed two knockout mutants of K. pneumoniae to investigate how perturbations to capsule biosynthesis alter the cellular phenotype. In the first mutant, we deleted the entire gene cluster responsible for biosynthesis of the extracellular polysaccharide capsule. In the second mutant, we deleted the capsule export subsystem within this cluster. We find that both knockout mutants have lower amounts of capsule but produce greater amounts of biofilm. Moreover, one of the two mutants abolishes fimbriae expression as well.

Conclusions: These results are expected to provide insight into the interaction between capsule biosynthesis, biofilm formation, and fimbriae expression in this organism.

Figure 1

Construction of two capsule defective mutants, Δcps and Δwzabc. A. Genetic loci that were deleted in the two mutants ∆wzabc and ∆cps. A portion of the capsule export system within the capsule biosynthesis cluster was deleted in ∆wzabc whereas the entire biosynthesis cluster was deleted in ∆cps. The deletions in both mutants left behind an 81 bp scar region (red box). The triangles indicate the CDSs inside the gene clusters. B. Gel image confirming the deletions in both mutants. Two primer pairs (1 and 2 for ∆wzabc; 3 and 4 for ∆cps) were used to verify the deletions by PCR. The small arrows in part A denote the approximate binding location of the primers used to generate these amplicons. Pairs 1 and 3 were designed to amplify the 5′ and 3′ junctions of the wild-type sequence, respectively, while pairs 2 and 4 were designed such that either the forward or reverse primer from each pair bound inside the scar region. In this way, pairs 1 and 3 will produce a PCR amplicon if the wild-type sequence is still present, whereas pairs 2 and 4 amplify only if the correct target locus has been deleted. The amplicon sizes are: pair 1, 833 bp; pair 2, 741 bp; pair 3; 602 bp; pair 4, 428 bp.

Figure 2

Phenotypic characterization of both capsule deletion mutants. A. Growth curves for the wild-type (WT), ∆wzabc, and ∆cps strains. B. Measurements of uronic acid content in the wild-type, ∆wzabc, and ∆cps strains. Values were normalized by the optical densities of the samples when the measurements were made. C. The susceptibility of the wild-type, ∆wzabc, and ∆cps strains to hydrogen peroxide (left) and polymyxin B (right). Error bars in all three panels denote the standard deviation of three biological replicates.

Figure 3

Transmission electron microscopy images of wild-type K. pneumoniae and the Δwzabc and Δcps mutants. The wild-type and ∆wzabc are fimbriated, whereas ∆cps is not. All images were taken at 19,000X nominal magnification (27,290X CCD magnification).

Figure 4

The fold change in the amount of uronic acid (capsule) and biofilm produced by the Δcps and Δwzabc mutants relative to the wild-type (WT). Error bars represent the standard deviation of three biological replicates. The uronic acid data is identical to that found in Figure 2 but presented here as fold change.