Characterization of Stg fimbriae from an avian pathogenic Escherichia coli O78:K80 strain and assessment of their contribution to colonization of the chicken respiratory tract

Abstract

In a previous study, ecs-3, a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 strain chi7122, was found to be expressed in vivo in infected chicken tissues. The region encompassing ecs-3 carries a fimbrial gene cluster that is a putative ortholog of the stg fimbrial gene cluster of Salmonella enterica serovar Typhi. This APEC fimbrial gene cluster, which we have termed stg, is a member of a distinct group of related fimbriae that are located in the glmS-pstS intergenic region of certain E. coli and S. enterica strains. Under the control of the pBAD promoter, the production of Stg fimbriae was demonstrated by Western blotting and immunogold electron microscopy with E. coli K-12. Transcriptional fusions suggest that stg expression is influenced by the carbohydrate source and decreased by the addition of iron and that Fur plays a role in the regulation of stg expression. stg sequences were associated with APEC O78 isolates, and stg was phylogenetically distributed among E. coli reference strains and clinical isolates from human urinary tract infections. Stg fimbriae contributed to the adherence of a nonfimbriated E. coli K-12 strain to avian lung sections and human epithelial cells in vitro. Coinfection experiments with APEC strain chi7122 and an isogenic Deltastg mutant demonstrated that compared to the wild-type parent, the Deltastg mutant was less able to colonize air sacs, equally able to colonize lungs, and able to more effectively colonize tracheas of infected chickens. Stg fimbriae, together with other adhesins, may therefore contribute to the colonization of avian respiratory tissues by certain APEC strains.

FIG. 1.

(A) The glmS-pstS intergenic region of APEC strain χ7122. Black arrows indicate the four ORFs of the stg gene cluster. Numbers indicate the predicted number of amino acids. White arrows indicate corresponding genes present in E. coli K-12. Gray arrows indicate positions of primers used for cloning the stg encoding region (glmS-F and pstS-R) and for screening the presence of stg sequences among isolates (stgC-F and stgC-R). The small white rectangle corresponds to the region amplified for screening the presence of stg sequences among isolates. The dashed lines indicate the 5′ and 3′ products amplified to assess the presence of a full-length stg gene cluster among isolates. (B) Phylogram based on Clustal analysis of proteins sharing highest identities/similarities with the predicted stgA gene product. The APEC StgA protein is highlighted within a box. Fimbrial proteins from gene clusters inserted in the yhjW-yhjX region, which includes LpfA of serovar Typhimurium, cluster together and belong to the LP fimbrial group. Fimbrial proteins from gene clusters inserted in the glmS-pstS region, including StgA from E. coli and serovar Typhi, comprise a distinct cluster and belong to the Stg fimbrial group. Entries from top to bottom correspond to GenBank accession numbers P43660, C86029, AAO22843, AAO52822, P22595, AAL18161, AAN45247, CAD03135, AAG58930, and CAE14452. The scale indicates percent difference in similarity. E. tarda, Edwardsiella tarda; P. luminescens, Photorhabus luminescens; S. flexneri, Shigella flexneri; S. marcescens, Serratia marcescens.

FIG. 2.

(A and B) Immunodetection of Stg fimbriae and immunogold labeling with anti-StgA antiserum and observation by transmission electron microscopy. Bars = 200 nm. Strain ORN172(pIJ39) was grown overnight on TSA plates at 37°C without arabinose (A) or with 0.05% arabinose (B). Arrows indicate some of the StgA-labeled gold particles associated with fimbrial structures. (C) Immunoblot of fimbrial extracts obtained from ORN172(pIJ39) cells grown under the same conditions as those described above. Samples were migrated on an SDS-15% polyacrylamide gel electrophoresis gel and incubated with anti-StgA-specific antiserum. Lane 1, extract from cells grown without arabinose; lane 2, extract from cells grown with 0.05% arabinose. Molecular mass markers are indicated to the right. A band reacting with StgA-antiserum, at an apparent molecular mass of 23 kDa, was detected only in cells induced with arabinose.

FIG. 3.

(A) β-Galactosidase activity expressed from the PstgA::lacZ fusion in APEC strain QT891 grown overnight at 37°C on M9 minimal medium plates supplemented with different carbohydrates or other carbon sources. (B) Effect of iron on β-galactosidase activity expressed from the PstgA::lacZ fusion in APEC strain QT891 and isogenic Δfur strain QT865. M9, M9-glucose. Error bars indicate standard deviations.

FIG. 4.

Bacterial adherence assays using human UM-UC-3 kidney and INT407 intestinal epithelial cell lines. The percentage of the initial bacterial inoculum associated with epithelial cells after 90 min of incubation is indicated. Bacteria containing stg genes (pIJ2) were significantly more adherent (P < 0.001) to both cell lines than was the negative control strain ORN103(pCR-XL-TOPO). Error bars indicate standard deviations.

FIG. 5.

Adherence of Stg-positive strain ORN103(pIJ2) (A) and Stg-negative control strain ORN103(pCR-XL-TOPO) (B) to chicken lung sections. Bacteria were grown on TSA, and 50 μl of a 5 × 10⁹ bacterial suspension was added to the tissue sections. Slides were stained using a Diff-Quick stain set.

FIG. 6.

Comparative colonization of respiratory tissues 6 days following coinfection of chickens with APEC strain χ7122 and isogenic ΔstgABCD::kan derivative QT302. Bars represent the mean proportion (as a percentage) of the Δstg mutant compared to the total CFU of bacteria recovered. Error bars indicate standard errors of the means. Asterisks indicate significant differences in the proportions of Δstg mutant and wild-type APEC χ7122 recovered from tissues compared to the input ratio. P values obtained using the Mann-Whitney test are indicated. The dashed line indicates the initial input ratio of the inoculum of the Δstg mutant. n = number of tissue samples from which either strain could be recovered from 12 analyzed tissues.