Molecular characterization and adhesive properties of CF29K, an adhesin of Klebsiella pneumoniae strains involved in nosocomial infections

Abstract

We previously described a CS31A-related protein, CF29K, expressed by Klebsiella pneumoniae strains involved in nosocomial infections. In this study, we cloned and sequenced cf29A, the structural gene of the CF29K protein, and showed that CF29K is an antigenic subtype of CS31A. The CF29K protein was found to be identical to the CS31A-L protein on the basis of biochemical and immunological properties. In contrast, the CS31A-H protein presented a different apparent molecular mass during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a different limited degradation pattern with endopeptidase V8, and a specific conformational epitope. We cloned and sequenced the CS31A-L structural gene and confirmed that CF29K and CS31A-L are identical, but their major subunits differ from ClpG (the CS31A-H subtype major subunit) by one amino acid at position 89 of the mature protein, which is a lysine in CF29K instead of the asparagine in ClpG. Site-directed mutagenesis experiments demonstrated that the biochemical and immunological differences between CS31A-H and CF29K or CS31A-L were dependent only on the amino acid at position 89 of the mature protein. To study the adhesive properties of CS31A-H and CF29K in the same Escherichia coli reference strain, we performed transcomplementation experiments with the cloned CS31A major-subunit structural genes or cloned cf29A gene and the clp accessory genes of the CS31A operon. We showed that CS31A-L, CF29K, and CS31A-H were involved in adhesion to Caco-2 and Int-407 cells but not to HEp-2 cells. Nevertheless, K. pneumoniae strains and corresponding E. coli transconjugants producing CF29K adhered to cultured Caco-2, Int-407, and HEp-2 cells, indicating the expression of another R-plasmid-encoded adhesin that mediated adhesion to HEp-2 cells. The carbohydrate part of the eucaryotic receptor of CF29K and CS31A-H adhesins was investigated by adhesion inhibition experiments with Int-407 cells. Although CS31A and CF29K belong to the K88 adhesin family, the receptor does not contain N-acetyl-D-galactosamine residues but contains Nacetylneuraminic acid and N-acetyl-D-glucosamine. Klebsiella pneumoniae strains resistant to multiple antibiotics are involved in nosocomial infections in intensive care units (7, 28). They are isolated from patients with urinary tract and respiratory infections, bacteremia, septicemia, and suppurative lesions. The origins of nosocomial strains have been studied, and epidemiologic investigations have shown the reservoir for K. pneumoniae strains to be the gastrointestinal tracts of patients (7, 37). The carriage of K. pneumoniae strains may be the result of bacterial adhesion to the intestinal epithelial cells of the host. Adherence-mediating pili are often involved in the adhesion process. K. pneumoniae can express type 1 and type 3 pili. Type 1 pili are involved in mannose-sensitive hemagglutination of various erythrocytes and adhesion to ciliated hamster tracheal cells and rat bladder epithelial cells (11, 12). Type 3 pili have been found to mediate adhesio...

Klebsiella pneumoniaedisassembles host microtubules in lung epithelial cells

“…We found that the ATCC K6 reference strain (ATCC700603) and the NCTC K1 reference strain (NCTC204) also disassembled microtubules ( Figure S6a K. pneumoniae expresses virulence factors that include the capsular polysaccharide, outer membrane porins, and those secreted through a T6SS. Because all of these have been correlated to disease progression (Cortés et al, 2002;Fung et al, 2012;Majumdar et al, 2015;March et al, 2011;Martino et al, 1995;Siu et al, 2012;Tsai et al, 2011), we examined if any of those factors caused microtubule severing. Using mutated bacteria, we infected A549 cells for 6 hr and saw that all still caused microtubule disassembly indicating that none of those components were responsible for the microtubule severing phenotype (Figure S7a…”

“…Unfortunately, only a few virulence factors have been characterised from these microbes. These include the capsular polysaccharide, outer membrane porins, and the type VI secretion system (T6SS), which show decreased virulence in bacteria mutated in their components March et al, 2011;Martino et al, 1995;Rosen et al, 2008;Tsai et al, 2011). The spread of K. pneumoniae is significant in hospital settings as these bacteria can form biofilms on contaminated hospital equipment (Schroll, Barken, Krogfelt, & Struve, 2010;Yinnon, Butnaru, Raveh, Jerassy, & Rudensky, 1996).…”

Adhesive properties and antibiotic resistance of Klebsiella, Enterobacter, and Serratia clinical isolates involved in nosocomial infections

“…A nonfimbrial adhesin, termed CF29K, is involved in the diffuse phenotype. The cf29 operon is located on a conjugative plasmid together with genes encoding other virulence factors (i.e., aerobactin) and resistance to antibiotics (i.e., the CAZ-1/TEM-5 extended-spectrum ␤-lactamase-encoding gene) (5,7).…”

Protein Haemaglutinin Outer Membran Protein (OMP) 35 kDa sebagai Protein Adhesin Proteus mirabilis pada Vesika Urinaria Kelinci

“…Preparat diamati dengan mikroskop pembesaran 1000 kali dan dihitung jumlah bakteri yang menempel pada sel epitel. Indeks adhesi adalah jumlah rerata bakteri yang menempal pada epitel, dihitung untuk setiap pengamatan terhadap 100 epitel ( Martino et al, 1995).…”