Capsule deletion via a λ-Red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH 78578
© Huang et al.; licensee BioMed Central Ltd. 2014
Received: 1 August 2013
Accepted: 31 December 2013
Published: 8 January 2014
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.
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.
These results are expected to provide insight into the interaction between capsule biosynthesis, biofilm formation, and fimbriae expression in this organism.
Klebsiella pneumoniae is a Gram-negative bacterium that is a member of the family Enterobacteriaceae and is closely related phylogenetically to the genera Escherichia, Salmonella, Shigella, and Yersinia. Medically, K. pneumoniae causes a wide range of diseases worldwide such pneumonia, urinary tract infections, and surgical wound infections that primarily afflict immunocompromised patients in hospital settings and long-term care facilities. The number of community-acquired infections caused by K. pneumoniae, however, has increased worldwide over the past several decades. For example, invasive forms of the disease characterized by bacteremic liver abscesses or endophthalmitis that are contracted in the community are endemic in Asia [1–3], especially in Taiwan [4–7], and reports of their occurrence are now emerging in other parts of the world [8, 9]. Infections caused by K. pneumoniae can be difficult to treat since many clinical isolates possess an extensive repertoire of antibiotic resistance genes. Ominously, some strains have now been isolated from different parts of the world that harbor New Delhi metallo-β-lactamase 1 (NDM-1) [10, 11], a gene that confers resistance to carbapenem antibiotics, the last-line treatment option against most K. pneumoniae infections. Compounding the medical threat is the paucity of new antibiotics that are being developed against multi-drug resistant Gram-negative bacteria such as K. pneumoniae.
Despite its close relationship to other enterobacteria, one notable difference between K. pneumoniae and other members of this family is the presence of an extremely thick, hypermucoviscous, extracellular polysaccharide capsule that surrounds this bacterium. At least 77 distinct capsular serotypes have been reported to date , but virulent strains have been predominantly associated with the K1 and K2 serotypes, in particular K1 [12–15]. The genes responsible for biosynthesis of the capsule are normally located in a cluster that is 21 to 30 kb in length and comprise 16 to 25 ORFs . The capsule is believed to be a major virulence determinant by protecting K. pneumoniae from phagocytosis [17–19] and destruction by antimicrobial peptides . Furthermore, the capsule is thought to play a crucial role in biofilm formation, which allows the organism to colonize indwelling medical devices and better survive hostile conditions such as detergents aimed at removing the biofilms, since mutants of K. pneumoniae strain LM21 with disruptions in different genes involved in capsule biosynthesis produce less biofilm . Consistent with these findings, a separate signature-tagged mutagenesis study also identified a mutation in ORF12 of the K. pneumoniae strain 43816 K2 capsule gene cluster that resulted in less biofilm formation . On the other hand, a third study found that a non-encapsulated derivative of K. pneumoniae strain C105 produced greater amounts of biofilm than the parental strain, and correlated this observation to the expression of type 1 fimbriae . A fourth study found that type 3 fimbriae promoted biofilm formation in a strain that still possessed its capsule .
These findings in aggregate highlight the complex interaction between capsule biosynthesis, biofilm formation, and fimbriae expression. Against this backdrop, we investigated the relationship among encapsulation, biofilm formation, and fimbriae expression in K. pneumoniae strain MGH 78578. Significantly, we found that a non-encapsulated mutant produced larger amounts of biofilm as has been reported , but in contrast also found that no fimbriae was required for this phenotype.
Materials and methods
Strains and primers
List of strains and plasmids used in this study
K. pneumoniae strains
Clinical isolate from a pneumonia patient. Parental (wild-type) strain for gene deletion.
In-frame deletion of wzabc operon (KPN_02510-KPN_02512)
In-frame deletion of capsule biosynthesis cluster ranging from ugd (KPN_02493) to galF (KPN_02515)
E. coli strains
Competent cells for general cloning
A p15A replicon plasmid containing an arabinose-inducible λ-Red recombinase and chloramphenicol resistance selection marker
General vector containing ampicillin resistance used during sub-cloning to make pACBSR-Hyg
Vector containing a promoter-less hygromycin resistance gene
Same as pEXP5-CT, but with the ampicillin resistance marker replaced with a hygromycin resistance marker
Plasmid bearing a heat-shock inducible FLP recombinase
Template for amplification of the apramycin resistance cassette
Same as pACBSR, but with the chloramphenicol resistance marker replaced with a hygromycin resistance marker
Same as pCP20, but with the pSC101 replicon and chloramphenicol resistance marker of pCP20 replaced with the p15A replicon and hygromycin resistance marker from pACBSR-Hyg
All experiments were conducted using bacteria grown in LB, low salt LB, or glucose M9 media. Low salt LB consisted of (per liter) 5 g yeast extract, 10 g tryptone, and 5 g NaCl, and was adjusted to pH 8.0 with NaOH. Glucose M9 was composed of the following chemicals (per liter): 6.8 g Na2HPO4; 3 g KH2PO4; 0.5 g NaCl; 1 g NH4Cl; 100 μM CaCl2; 2 mM MgSO4; 2 g dextrose. Stock solutions of CaCl2, MgSO4, and dextrose were filter sterilized through Millipore ExpressPlus 0.22 μm membranes (Millipore, Billerica, MA) and subsequently added individually to autoclaved solutions containing the first four chemicals. Hygromycin (Sigma-Aldrich, St. Louis, MO) and apramycin (Research Products International, Mt. Prospect, IL) were added to the media to final concentrations of 100 μg/ml and 50 μg/ml, respectively, as necessary. Low salt LB and hygromycin are always used together; all other steps use standard LB.
Construction of knockout mutants
The capsule surrounding K. pneumoniae MGH 78578 belongs to the K52 serotype, which has a hexasaccharide repeating unit composed of two rhamnose, one glucose, one glucuronic acid, and two galactose sugars . Measurements of the amount of capsule surrounding wild-type K. pneumoniae MGH 78578 and all the mutants were based on the protocol of Domenico, et al..
For transcriptome profiling, a high-density oligonucleotide tiling array consisting of 379,528 50-mer probes spaced 30 bp apart across the whole K. pneumoniae MGH 78578 genome was custom-designed by NimbleGen (Roche). Total RNA from OD600 ~0.5 cultures were hybridized to the arrays according to the protocol of Qiu, et al. . The normalized probe level information was transformed into expression level data for each gene using the Genbank annotation for K. pneumoniae MGH 78578 (accession number PRJNA57619). Genes were deemed differentially expressed between the two mutants and the wild-type if there was a 2-fold or greater change and they had a p-value less than 0.05. The expression profiling datasets of K. pneumoniae wild-type and capsule deletion mutant (Δcps) have been deposited in the Gene Expression Omnibus (GEO) database and assigned the accession number GSE40011. Three biological replicates of the wild-type, Δcps, and Δwzabc strains were used to generate the array data.
Transmission electron microscopy imaging
The samples were negatively stained with 1% aqueous uranyl acetate and examined on an FEI Tecnai G2 Sphera transmission electron microscope at 200 keV. Images were recorded on a Gatan Ultrascan UHS CCD camera.
Results and discussion
Deletion of the entire capsule biosynthesis cluster and the export subsystem leads to phenotypic defects in the two mutants
We developed a gene knockout procedure for K. pneumoniae that is based on the widely-used E. coli λ-Red recombinase system. The independent development of a similar protocol was recently reported . The method developed here requires two selection markers, one (apramycin resistance) is used to replace the target gene and the other (hygromycin resistance) selects for two plasmids used at different times to mediate homologous recombination and excision of the apramycin resistance cassette.
We examined how different homology lengths affect transformation efficiency by deleting the same locus in K. pneumoniae using cassettes that contained 39-bp, 60-bp, and 700-bp homology to the target region. This target region was the three genes (wza, wzb, and wzc) that function in capsule export. A total of one microgram of each of the three PCR products was electroporated into the host. We recovered 26, 46, and 255 apramycin-resistant transformants using the 39-bp, 60-bp, and 700-bp homology lengths, resulting in transformation efficiencies of 1.3 × 10-8, 2.5 × 10-8, and 1.1 × 10-7, respectively. To confirm these mutant candidates, we next examined 24 colonies from each group by PCR. None of the 24 colonies from the 39-bp group was a correct knockout mutant, whereas two colonies from the 60-bp group and ten colonies from the 700-bp group were correct (data not shown). Although longer homology lengths resulted in higher transformation efficiency, these results implied that replacement cassettes containing 60-bp homology were sufficient to create knockout mutants. The construction of such cassettes requires DNA oligos that are only 80–85 nucleotides long, which can be readily purchased from commercial vendors. The use of knockout cassettes bearing longer homology arms would require an additional cloning step. For these reasons, we constructed all knockout mutants in this study using 60-bp homology.
The quantity of uronic acid that can be extracted from cells provides an indirect measure of the amount of extracellular capsule [34–37]. When compared to wild-type levels, the quantity of uronic acid in the Δcps and Δwzabc mutants declined to 0.29 ± 0.17 and 0.38 ± 0.04 fold change, respectively (P < 7.5 × 10-4 for both, Student’s t-test) (Figure 2B). As a negative control, we performed the same assay side-by-side using blank media only. Absorbance measurements for these control samples were consistently below the detection threshold of our spectrophotometer.
We next measured the sensitivity of the wild-type, Δcps, and Δwzabc strains to oxidative stress and the antimicrobial peptide polymyxin B since the absence of capsule was expected to increase sensitivity to these two stresses. Both measurements were carried out using a disc diffusion assay in which hydrogen peroxide or polymyxin B was impregnated into the disc and then placed onto agar plates covered by a lawn of each strain. The diameter of the inhibition zone of the wild-type and Δwzabc mutant to hydrogen peroxide was 22.3 mm in contrast to 26.7 mm for the Δcps mutant (P < 0.001, Student’s t-test) (Figure 2C). Similarly, the antimicrobial peptide polymyxin B assay produced a diameter of 19.0 mm each time when three independent tests on Δcps were performed, but only 14 mm on wild-type and Δwzabc (Figure 2C).
Expression profiling of the Δcps and Δwzabc deletion mutants reveals interplay between capsule biosynthesis and phenotypic defects
List of the 20 genes most significantly up- and down-regulated in the Δcps mutant relative to wild-type levels
Expression level (wild-type)
Expression level (Δcps)
Lipoprotein, osmotically inducible
Periplasmic repressor CpxP
Putative stress-response protein
Putative polysaccharide deacetylase
Outer membrane protein PgaA
Putative sulfate transporter
Putative carbonic anhydrase
Copper exporting ATPase
methylthioribose kinase MtnK
Colicin I receptor
Bifunctional chorismate mutase/prephenate dehydratase
Putative fimbrial usher protein
Putative pili assembly chaperone
Carbon starvation protein
Eighty-nine genes were down-regulated more than two-fold in the Δcps mutant and fell within several groups: 17 plasmid-borne genes from pKPN3 and pKPN4, 13 metabolic genes, 11 tRNA genes, and eight genes associated with type 1 and type 3 fimbriae. Of the eight fimbriae-associated genes, five were located within the same cluster (KPN_03274 – KPN_03278) (Additional file 3). Genbank currently does not associate this set of genes with a specific type of fimbriae; however, each one is 100% homologous at the amino acid level to the mrk JFDCB cluster from K. pneumoniae NTUH-2044 that encodes type 3 fimbriae. We therefore assume that this set of five genes encodes type 3 fimbriae in K. pneumoniae MGH 78578 as well. The other three (fimA, fimI, and fimC; KPN_03287 – KPN_03289) (Additional file 3) are located within a cluster whose gene product is annotated to be type 1 fimbriae.
Fimbriae biosynthesis is abolished in Δcps but not Δwzabc
Up-regulation of the pga operon appears to be a more important factor in biofilm formation than down-regulation of the fimbriae-associated operons
In this study, we observed a complex interplay among encapsulation, biofilm formation, and fimbriae expression in Klebsiella pneumoniae MGH 78578. This strain exhibited increased biofilm formation despite the absence of capsule, an effect likely stemming from strong up-regulation of the pga operon. In contrast, other strains with different deletions within the capsule biosynthesis cluster have been reported to produce less biofilm, not more [21, 22, 40]. It is not known, however, how expression levels of the pga operon might have changed in these other mutants. In addition, type 1 fimbriae have been reported to play an important role in biofilm formation in a non-encapsulated K. pneumoniae mutant , but no fimbriae was necessary for increased biofilm formation in the non-encapsulated mutant we generated (Figure 3). Taken together, these observations suggest that overlapping regulatory mechanisms likely act to regulate these three features in K. pneumoniae in a strain-specific manner.
This study was funded from a grant from the National Health Research Institutes of Taiwan through PH-099-SP-10. We acknowledge the use of the UCSD Cryo-Electron Microscopy Facility which is supported by NIH grants to Dr. Timothy S. Baker and a gift from the Agouron Institute to UCSD. We thank Jay Sung-Joong Hong and Yu Qiu for helpful discussion on microarray experiments. We thank Nicole Fong and Valerie Khaw for technical assistance. We thank Dr. Frederick R. Blattner at the University of Wisconsin-Madison for kindly sharing the pACBSR plasmid and Dr. Carton W. Chen at National Yang-Ming University for kindly sharing the pIJ773 plasmid.
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