Construction and characterization of mutations within the Klebsiella mrkD1P gene that affect binding to collagen type V

Abstract

The fimbria-associated MrkD1P protein mediates adherence of type 3 fimbriate strains of Klebsiella pneumoniae to collagen type V. Currently, three different MrkD adhesins have been described in Klebsiella species, and each possesses a distinctive binding pattern. Therefore, the binding abilities of mutants possessing defined mutations within the mrkD1P gene were examined in order to determine whether specific regions of the adhesin molecule were responsible for collagen binding. Both site-directed and chemically induced mutations were constructed within mrkD1P, and the ability of the gene products to be incorporated into fimbrial appendages or bind to collagen was determined. Binding to type V collagen was not associated solely with one particular region of the MrkD1P protein, and two classes of nonadhesive mutants were isolated. In one class of mutants, the MrkD adhesin was not assembled into the fimbrial shaft, whereas in the second class of mutants, the adhesin was associated with fimbriae but did not bind to collagen. Both hemagglutinating and collagen-binding activities were associated with the MrkD1P molecule, since P pili and type 3 fimbriae carrying adhesive MrkD proteins exhibited identical binding properties.

FIG. 1

Hydropathicity profile of MrkD_1P. The numbers at the bottom of the plot indicate the length, in amino acids, of the polypeptide. The arrows above the plot indicate the sites of the mutations characterized in this study.

FIG. 2

Western immunoblots of purified type 3 fimbriae from K. pneumoniae IApc35 transformants. The gels were probed with anti-type 3 fimbria-specific serum to detect the MrkA major subunit, stripped, and subsequently probed with the anti-MrkD_1P serum. Lanes 1, 2, and 3 include fimbriae from transformants possessing pTS68, pTS71, and pTS72, respectively, and were overloaded with fimbrial proteins in order to demonstrate the absence of MrkD_1P in these samples. Lanes 4 and 5 include fimbriae from pTS76 and pTS78.