Signature-tagged mutagenesis in a chicken infection model leads to the identification of a novel avian pathogenic Escherichia coli fimbrial adhesin

Abstract

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was successfully expressed in vitro. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial-like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the 153 intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the B2 group of the EcoR classification and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its significant role during APEC infection in chickens.

Figure 1. Graphic illustrating growth of transposon mutant EA7F9 in competition with IMT5155 and separately in Luria Bertani (LB) growth medium.

A competition index (CI) in vitro was calculated at a time point of 4 h.

Figure 2. Physical map showing genomic organization of the 4,975 bp yqi adhesin gene cluster in APEC strain IMT5155.

A hypothetical protein preceeds the putative outer membrane usher protein, followed by the putative chaperone and finally the putative adhesin.

Figure 3. Bacterial adhesion to chicken fibroblast cells 1.5 h and 3 h after infection with an MOI = 100.

Differences between IMT5155 and IMT5155Δyqi were statistically significant with a p<0.005 at 1.5 h and p<0.05 at 3 h (A). Bacterial adhesion to polarized Madin Darby canine kidney (MDCK-1) cells 3 h after infection with an MOI = 100. The difference between IMT5155Δyqi and IMT5155Δyqi (pDSK602:yqi) was significant with a p<0.04 (B).

Figure 4. Bacterial colonization of the chicken lungs 24 h after intra-tracheal infection with 10⁶ CFU of bacteria.

Differences between IMT5155 and IMT5155Δyqi were statistically significant with a p<0.05 (n = 6). Strain IMT11327 is the negative control (A). Bacterial colonization of the chicken lungs 24 h after intra-tracheal infection with 10⁹ CFU of bacteria. Differences between IMT5155 and IMT5155Δyqi were statistically significant with a p<0.02 (n = 6). Strain IMT11327 is the negative control (B).

Figure 5. Bacterial re-isolation of IMT5155, IMT5155Δyqi and IMT11327 from the lungs, spleen, liver, heart, kidneys and brain 24 h after intra-tracheal infection with 10⁹ CFU of bacteria (n = 6).

Absence of columns indicates that no bacteria were isolated from the organ.

Figure 6. Expression of the ExPEC adhesin I (yqi) gene cluster in vitro.

Electron micrographs show negatively stained afimbriate strain E. coli AAEC189 (pKESK:yqi_4975_XB) over-expressed with the yqi adhesin gene cluster at a magnification of 45,000x, 65,000x and 100,000x (A–C), negative control afimbriate strain E. coli AAEC189 (D) and wild type fimbriated E. coli strain IMT5155 (E). The arrows indicate the location of the fimbriae.

Figure 7. Phylogenetic analysis of ExPEC adhesin I (yqi).

Maximum Parsimony tree shows distances between yqi gene sequences among strains belonging to different sequence types (ST: Sequence type; STC: Sequence type complex).