Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

Abstract

Background: Serotyping of O-(lipopolysaccharide) and H-(flagellar) antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies.

Results: The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) was determined and both were found 99.3% (1043 of 1050 nucleotides) identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella.

Conclusion: The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strains which were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of different molecular weigh are functionally expressed and coassembled in the same flagellar filament in E. coli.

Figure 1

HhaI digested fliC PCR products obtained with primers fliC-1 and fliC-2 from E. coli reference strains for 53 different expressed H-types as indicated at the right side. Similarity of restriction fragment patterns was calculated with BioNumerics software and is indicated by the dendrogram on the left side. The flagellar antigens of strains encoding H-types H3, H17, H35, H36, H44, H47, H53, H54 and H55 are not encoded by fliC but by other genes (flkA, fllA, flmA and others) in the corresponding E. coli strains and the fliC HhaI patterns obtained from these strains do therefore not correspond to their H-serotypes [21, 25, 26, 27].

Figure 2

Alignment of the deduced FliC (flagellin) sequences from E. coli strains representing the flagellar antigen H4 and its genetic variants: U9-41 (accession BAA85081); C107-54 (accession CAE53943), E1541-68 (accession CAD60547); P12b (accession CAD56695); and P7d (accession CAE53942).

Figure 3

Electrophoretic separation of restriction enzyme digested fliC PCR products (primers fliC-1 and fliC-2) of E. coli strains U9-41 and P12b on 2% agarose: Lanes: 1+8= molecular weight standard; 2 = U9-41 (HpaII); 3 = P12b (HpaII); 4 = U9-41 (undigested); 5 = P12b (undigested); 6 = U9-41 (MboI); 7 = P12b (MboI). Sizes of restriction fragments are listed in Table 2.

Figure 4

(A) Low power micrograph of E. coli strain TPE1978 cells showing the density of the bacterial samples used for indirect IEM and the presentation of flagella (bar length = 1 μm) (B) IEM of strain TPE1978 flagella after incubation with rabbit flagellar H48 antiserum (1:1000) and detection of bound antibody by anti-rabbit-IgG- 10 nm gold (1:20), bar length = 100 nm. (C) Strain JM109 flagella after incubation with rabbit flagellar H48 antiserum and detection of bound antibody by anti-rabbit-IgG- 10 nm gold. (D) Strain TPE1978 flagella after incubation with rabbit flagellar H4 antiserum (1:1000) and detection of bound antibody by anti-rabbit-IgG- 5 nm gold. (E) Strain JM109 flagella after incubation with rabbit flagellar H4 antiserum and detection of bound antibody by anti-rabbit-IgG- 5 nm gold. (F) Double-labeling IEM of strain TPE1978 after sequential incubations with rabbit flagellar H4 antiserum and anti-rabbit-IgG 5 nm gold, followed by rabbit flagellar H48 antiserum detected by anti-rabbit-IgG- 10 nm gold. Both, 5 nm and 10 nm gold markers are bound at comparable amounts over all flagella present on the bacteria. (G) Double-labeling IEM of strain JM109 after sequential incubations with rabbit flagellar H4 antiserum and anti-rabbit-IgG- 5 nm gold followed by rabbit flagellar H48 antiserum and anti-rabbit-IgG- 10 nm gold. Only H48 specific (10 nm) gold particles are bound to the flagella of JM109.