Type 3 fimbriae are adhesive organelles produced by a range of Gram-negative pathogens that cause CAUTI. Here we show that type 3 fimbriae (mrkABCD) genes from 33 CAUTI isolates representing C. freundii, C. koseri, E. coli, K. oxytoca and K. pneumoniae cluster into five well-supported clades on the basis of nucleotide sequence. Type 3 fimbriae were expressed by all of these strains as indicated by their positive MR/K agglutination. Type 3 fimbrial expression was also associated with biofilm growth in the majority of these strains. This is the first report describing the distinct grouping of type 3 fimbrial genes into phylogenetic clades at the species level, with strong evidence supporting inter-species lateral gene transfer. We also demonstrate the functional expression of type 3 fimbriae by strains of C. koseri and C. freundii.
Phylogenetic analysis with individual and concatenated mrkABCD sequences revealed five distinct clades (A-E) which were strongly supported by long internal branches. The majority of the sequences grouped in clade A, which is represented by the chromosomal mrk gene cluster from the genome sequenced K. pneumoniae strain MGH78578. Clades A and B contained mrk gene clusters from K. pneumoniae (both chromosomal and plasmid origin) and E. coli (plasmid origin). Two mrk loci have been fully sequenced from E. coli; in both cases the mrk genes are located on a conjugative plasmid (pMAS2027 and pOLA52, respectively) and flanked by transposon-like sequences [30, 40]. While the genomic location of the mrk genes in the additional seven E. coli strains identified in this study remains to be determined, the data presented here and in previous studies strongly suggests inter-genera lateral gene transfer of the mrk cluster [17, 28]. In contrast, the composition of clade E is entirely C. koseri sequences, while clades C and D are represented by a unique sequence from C freundii and K. oxytoca, respectively. The presence of cko_00966 homologs downstream of representative mrk clusters in all 5 clades strongly suggests that the ancestral mrkABCD locus was also encoded next to a cko_00966 homolog and that the clades are largely related by linear descent. Notably, the relationship determined here is not congruent with the known evolutionary relationship of Klebsiella, Citrobacter, and E. coli , supporting the occurrence of lateral gene transfer. We propose that clade A represents the K. pneumoniae lineage, with mrk regions laterally transferred to E. coli (e.g. pMAS2027 and pOLA52) and clade E represents the C. koseri lineage. Clades B, C and D, which contain mrk sequences from K. pneumoniae, E. coli, C. freundii and K. oxytoca, are clearly under-represented and additional type 3 fimbrial gene sequences are required to confirm the groupings.
Among the four genes used in the phylogenetic analysis, mrkD exhibited the highest inter-group diversity (Table 1). Thus, from the partial sequence comparisons performed in this work, the MrkD adhesin displayed greater sequence variability than the MrkA major subunit. This is inconsistent with other chaperone-usher fimbriae such as type 1 and P fimbriae, where the sequence of the adhesin (e.g. FimH, PapG) is more conserved than the major subunit protein (e.g. FimA, PapA). We note, however, that these findings require substantiation via comparison of the entire sequence of each structural subunit from multiple strains. The MrkD adhesin mediates several phenotypes, including MR/K agglutination, as well as adherence to human endothelial cells, urinary bladder cells, basement membranes and ECM proteins such as collagen IV and V [5, 31, 34, 35]. Interestingly, previous studies have demonstrated that sequence variations in the MrkD adhesin are associated with differential binding properties [42–44]. Our study demonstrates that the degree of sequence variation in MrkD might be even greater than previously predicted .
CAUTI is associated with biofilm formation on the inner surface of indwelling catheters. Thirteen independent mrk deletion mutants were generated and used to examine type 3 fimbriae associated phenotypes including MR/K agglutination and biofilm formation. All of the mrk mutants were unable to cause MR/K agglutination, confirming that this property is highly specific for type 3 fimbriae. In biofilm assays, 11/13 mrk mutants displayed a significant reduction in biofilm growth compared to their respective parent strain, demonstrating that type 3 fimbriae contribute to this phenotype across a range of different genera and species. The exceptions were C. freundii M46 and E. coli M184. C. freundii M46 failed to produce a significant biofilm in the assay conditions employed irrespective of its mrk genotype. Although this strain caused MR/K agglutination, we were also unable to detect the MrkA major subunit protein by western blot analysis. E. coli M184 showed no reduction in biofilm growth upon deletion of the mrk genes. It is likely that E. coli M184 contains additional mechanisms that promote biofilm growth and therefore deletion of the mrk genes did not result in loss of this phenotype.