Signature-Tagged Mutagenesis in a Chicken Infection Model Leads to the Identification of a Novel Avian Pathogenic Escherichia coli Fimbrial Adhesin

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

Visualization of novel virulence activities of the Xanthomonas type III effectors AvrBs1, AvrBs3 and AvrBs4.

International audienceXanthomonas campestris pv. vesicatoria secretes at least 20 effector proteins through the type III secretion system directly into plant cells. In this study, we uncovered virulence activities of the effector proteins AvrBs1, AvrBs3 and AvrBs4 using Agrobacterium-mediated transient expression of the corresponding genes in Nicotiana benthamiana, followed by microscopic analyses. We showed that, in addition to the nuclear-localized AvrBs3, the effector AvrBs1, which localizes to the plant cell cytoplasm, also induces a morphological change in mesophyll cells. Comparative analyses revealed that avrBs3-expressing plant cells contain highly active nuclei. Furthermore, plant cells expressing avrBs3 or avrBs1 show a decrease in the starch content in chloroplasts and an increased number of vesicles, indicating an enlargement of the central vacuole and the cell wall. Both AvrBs1 and AvrBs3 cause an increased ion efflux when expressed in N. benthamiana. By contrast, expression of the avrBs3 homologue avrBs4 leads to large catalase crystals in peroxisomes, suggesting a possible virulence function of AvrBs4 in the suppression of the plant defence responses. Taken together, our data show that microscopic inspection can uncover subtle and novel virulence activities of type III effector proteins

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

<p>Differences between IMT5155 and IMT5155Δ<i>yqi</i> were statistically significant with a <i>p</i><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 IMT5155Δ<i>yqi</i> were statistically significant with a <i>p</i><0.02 (n = 6). Strain IMT11327 is the negative control (B).</p

Physical map showing genomic organization of the 4,975 bp <i>yqi</i> adhesin gene cluster in APEC strain IMT5155.

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

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

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

<p>Differences between IMT5155 and IMT5155Δ<i>yqi</i> were statistically significant with a <i>p</i><0.005 at 1.5 h and <i>p</i><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 IMT5155Δ<i>yqi</i> and IMT5155Δ<i>yqi</i> (pDSK602:<i>yqi</i>) was significant with a <i>p</i><0.04 (B).</p

Score values for severity of organ lesions ± standard deviation in respiratory and other organs.

<p>Infection with IMT5155, IMT5155Δ<i>yqi</i> and IMT11327 at infection dose 10⁹ CFU. Differences in organ scores between IMT5155 and IMT5155Δ<i>yqi</i> and between IMT5155 and negative control IMT11327 were statistically significant for air sacs, lungs and liver with a <i>p</i><0.05^*.</p

Expression of the ExPEC adhesin I (<i>yqi</i>) gene cluster <i>in vitro</i>.

<p>Electron micrographs show negatively stained afimbriate strain <i>E. coli</i> AAEC189 (pKESK:<i>yqi</i>_4975_XB) over-expressed with the <i>yqi</i> adhesin gene cluster at a magnification of 45,000x, 65,000x and 100,000x (A–C), negative control afimbriate strain <i>E. coli</i> AAEC189 (D) and wild type fimbriated <i>E. coli</i> strain IMT5155 (E). The arrows indicate the location of the fimbriae.</p

Phylogenetic analysis of ExPEC adhesin I (<i>yqi</i>).

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

Oligonucleotide primers used in this study.

<p>Oligonucleotide primers used in this study.</p