Molecular variations in Klebsiella pneumoniae and Escherichia coli FimH affect function and pathogenesis in the urinary tract - PubMed (original) (raw)

Molecular variations in Klebsiella pneumoniae and Escherichia coli FimH affect function and pathogenesis in the urinary tract

David A Rosen et al. Infect Immun. 2008 Jul.

Abstract

Type 1 pili mediate binding, invasion, and biofilm formation of uropathogenic Escherichia coli (UPEC) in the host urothelium during urinary tract infection (UTI) via the adhesin FimH. In this study, we characterized the molecular basis of functional differences between FimH of the UPEC isolate UTI89 and the Klebsiella pneumoniae cystitis isolate TOP52. Type 1 pili characteristically mediate mannose-sensitive hemagglutination of guinea pig erythrocytes. Although the adhesin domain of K. pneumoniae TOP52 FimH (FimH(52)) is highly homologous to that of E. coli, with an identical mannose binding pocket and surrounding hydrophobic ridge, it lacks the ability to agglutinate guinea pig erythrocytes. In addition, FimH-dependent biofilm formation in K. pneumoniae is inhibited by heptyl mannose, but not methyl mannose, suggesting the need for contacts outside of the mannose binding pocket. The binding specificity differences observed for FimH(52) resulted in significant functional differences seen in the pathogenesis of K. pneumoniae UTI compared to E. coli UTI. Infections in a murine model of UTI demonstrated that although the K. pneumoniae strain TOP52 required FimH(52) for invasion and IBC formation in the bladder, FimH(52) was not essential for early colonization. This work reveals that a limited amount of sequence variation between the FimH of E. coli and K. pneumoniae results in significant differences in function and ability to colonize the urinary tract.

PubMed Disclaimer

Figures

FIG. 1.

FimH52 and FimH89 are highly conserved in sequence and structure. (A) The FimH amino acid sequences of E. coli UTI89 and K. pneumoniae TOP52 are shown. Residues known to interact with mannose (orange) and form the hydrophobic ridge (green) are fully conserved. The purple line denotes sequence of the AD, the blue line denotes sequence of the PD, and the yellow line denotes the short linker region. Amino acid numbers refer to the mature protein without signal sequence. (B) Structure of E. coli FimH (yellow) (from the J96 strain FimC-H complex; PDB identification no. 1KLF) bound to mannose (red) overlaid with a threaded model of K. pneumoniae TOP52 FimH. Side chains of TOP52 amino acids that vary from the UTI89 sequence are shown as blue sticks. (Inset) Space-filling model of a view into the mannose binding pocket (same colors described above). This shows that all residues in direct contact with the mannose moiety and those that form the hydrophobic ridge are fully conserved between E. coli UTI89 and K. pneumoniae TOP52.

FIG. 2.

The fim operon of UTI89 Δ_fimH_ is primarily in the phase-off orientation. Phase assays of the fimS invertible promoter region of the fim operon were done for E. coli UTI89, UTI89 Δ_fimH_, UTI89 Δ_fimH_/pBAD, UTI89 Δ_fimH_/p_fimH_89, and UTI89 Δ_fimH_/p_fimH_52. UTI89 was largely phase on, while the UTI89 Δ_fimH_ strains were all primarily phase off despite complementation.

FIG. 3.

fimH constructs in UTI89 Δ_fimH_ are expressed in some bacteria and localized at the tips of pili. Immunoelectron microscopy using an anti-FimH antibody was performed against UTI89 Δ_fimH_/p_fimH_89 (A), UTI89 Δ_fimH_/pAD89PD52 (B), UTI89 Δ_fimH_/pAD52PD89 (C), and UTI89 Δ_fimH_/p_fimH_52 (D). While the majority of bacteria did not appear to be expressing type 1 pili, piliated bacteria could be found in all four samples. Piliated bacteria displayed FimH immmunostaining at the distal tips of pili.

FIG. 4.

Heptyl mannose, but not methyl mannose, inhibits FimH-dependent biofilm formation of K. pneumoniae TOP52/p_fimX_. A 48-h biofilm assay was used to quantify biofilms produced by E. coli UTI89 and K. pneumoniae TOP52 strains in the presence of no mannose, 1 mM methyl mannose, 100 mM methyl mannose, or 1 mM heptyl mannose. TOP52 forms a FimH-dependent biofilm with induced expression of type 1 pili via the E. coli recombinase, fimX. This biofilm formation is inhibited by heptyl mannose but not high concentrations of methyl mannose. UTI89 forms robust biofilm without mannose but is inhibited by heptyl mannose or high concentrations of methyl mannose. Error bars represent standard deviations.

FIG. 5.

FimH of K. pneumoniae TOP52 is required for invasion, IBC formation, and persistence but not colonization in the murine model of UTI. Female C3H/HeN mice were inoculated with 107 E. coli UTI89 (▴), UTI89 Δ_fimH_ (▵), K. pneumoniae TOP52 (•), or TOP52 Δ_fimH_ (○) by transurethral inoculation. For organ titers, bladders (A) and kidneys (B) were harvested at various time points postinfection and CFU were calculated. Titer data are combined from three independent experiments. For ex vivo gentamicin protection assays, bladders were harvested at 1 h postinfection and luminal (C) and intracellular (D) populations of bacteria were quantified. IBCs were quantified (E) after visualization by LacZ staining at 6 h postinoculation. Short bars represent geometric means of each group, and horizontal dotted lines represent limits of detection. Significant P values, as calculated using the Mann-Whitney U test, are displayed.

FIG. 6.

Complementation of UTI89 Δ_fimH_ with p_fimH_89 but not p_fimH_52 leads to increased bacterial burden in the murine model of UTI. Female C3H/HeN mice were inoculated with 107 cells of the UTI89 Δ_fimH_/pBAD vector control, UTI89 Δ_fimH_/p_fimH_89, or UTI89 Δ_fimH_/p_fimH_52 by transurethral inoculation. Ex vivo gentamicin protection assays were performed in which bladders were harvested at 1 h postinfection and luminal (A) and intracellular (B) populations of bacteria were quantified. For organ titers, bladders (C) and kidneys (D) were harvested at 6 h postinfection and CFU were enumerated. Short bars represent geometric means of each group, and horizontal dotted lines represent limits of detection. Significant P values, as calculated using the Mann-Whitney U test, are displayed.

References

1. Anderson, G. G., J. J. Palermo, J. D. Schilling, R. Roth, J. Heuser, and S. J. Hultgren. 2003. Intracellular bacterial biofilm-like pods in urinary tract infections. Science 301105-107. -PubMed
1. Bahrani-Mougeot, F. K., E. L. Buckles, C. V. Lockatell, J. R. Hebel, D. E. Johnson, C. M. Tang, and M. S. Donnenberg. 2002. Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract. Mol. Microbiol. 451079-1093. -PubMed
1. Barnhart, M. M., F. G. Sauer, J. S. Pinkner, and S. J. Hultgren. 2003. Chaperone-subunit-usher interactions required for donor strand exchange during bacterial pilus assembly. J. Bacteriol. 1852723-2730. -PMC -PubMed
1. Bennett-Lovsey, R. M., A. D. Herbert, M. J. Sternberg, and L. A. Kelley. 2008. Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre. Proteins 70611-625. -PubMed
1. Bouckaert, J., J. Berglund, M. Schembri, E. De Genst, L. Cools, M. Wuhrer, C. S. Hung, J. Pinkner, R. Slattegard, A. Zavialov, D. Choudhury, S. Langermann, S. J. Hultgren, L. Wyns, P. Klemm, S. Oscarson, S. D. Knight, and H. De Greve. 2005. Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin. Mol. Microbiol. 55441-455. -PubMed

Molecular variations in Klebsiella pneumoniae and Escherichia coli FimH affect function and pathogenesis in the urinary tract - PubMed (original) (raw)