First call EMIDA ERAnet
Year: 2011 / Consortium:
Ulrich Dobrindt (Institute of Hygiene, University of Münster, Germany) – WWU
Eliora Z. Ron (Dept. of Molecular Microbiology and Biotechnology, Tel Aviv University, Israel) – TAU
Eric Oswald (INRA UMR1225, Ecole Nationale Vétérinaire de Toulouse, France) – INRA-ENVT
Laura Serino / Mariagrazia Pizza (Novartis Vaccines and Diagnostics, Siena, Italy) - NVD
Title and acronym:
Combatting colibacillosis – genomics based approach (CombatColibacillosis)
Project coordinator: Dobrindt, Ulrich
Total provided budget: € xxxx / Duration: 36 months
Work/packages
WP title leader/ deputy leader (names and affiliations) / WP1. Comparative genomics of avian pathogenic E. coli (APEC) isolates from avian septicemia to identify candidate markers contributing to APEC virulence and fitness. U. Dobrindt (WWU) / E. Ron (TAU)
WP2. Functional characterization of ExPEC factors involved in adaptation, bacterial competition and host-bacterium interaction. E. Oswald (INRA-ENVT) / E. Ron (TAU)
WP3. Identification and testing of potential protective vaccine antigens against APEC WP. L. Serino (NVD)
Project objectives (per WP) / WP1. Characterization of APEC and commensal E. coli isolates from poultry with regard to genome content, virulence traits and phylogeny:
- Establishment of large collection of avian pathogenic E. coli and commensal E. coli isolates from poultry
- Analysis of the distribution of virulence- and fitness genes in the poultry isolates, comparison with extraintestinal pathogenic E. coli isolates from humans
- phenotypic characterization of the isolates
- molecular epidemiological analysis of the isolates
- Whole genome sequence determination for genome content analysis of the isolates
WP2. Functional characterization of ExPEC factors involved in adaptation, bacterial competition and host-bacterium interaction.
- Analysis of the expression of virulence- and fitness-associated genes under conditions which mimic infection (qPCR, transcriptomics, proteomics)
- functional characterization of virulence- or fitness-associated genes (generation of deletion mutants, overexpression of candidate genes, test in suitable in vitro and in vivo models)
WP3. Analysis of candidate genomic regions to select determinants, which encode virulence-related proteins and traits that could be used for the further development of preventive and therapeutic strategies against APEC infection.
- Identification of vaccine candidates for APEC by comparative genomics
- Recombinant expression of candidate antigens
- Test for active protection upon immunization
Results
(Please provide the main results, where possible referring to the relevant objectives and to WPs) / WP1.
- We established a well-defined collection of commensal tracheal and fecal E. coli isolates from healthy chicken raised in different European countries as well as of APEC isolates. This collection allowed to compare genome content and phylogeny of APEC with commensal E. coli isolates from healthy chicken. We provided for the first time a survey of the population structure of commensal E. coli in healthy chickens, which will be important define biomarkers for the detection and risk assessment of virulent APEC strains.
- We sequenced the genomes of 140 chicken commensal and APEC isolates
- MALDI-based analyses established a collection of well-defined human and avian pathogenic or commensal E. coli strains from the different partner countries France, Israel and Germany. The commensal isolates were also collected from hens that have been raised by different methods of animal husbandry, e.g. conventional (commercial) farming vs. organic farming.
- We analyzed the clonal distribution of characteristic virulence- and fitness associated genes among human and avian pathogenic E. coli by PCR-based screening and by whole genome shotguns sequencing. According to MLST and whole genome-based phylogeny, we demonstrated that commensal and APEC isolates can usually be distinguished based on their phylogeny and allocation to clonal lineages, whereas the detection of known virulence-associated genes of extraintestinal pathogenic E. coli (ExPEC) did not unequivocally allow the discrimination of APEC and chicken commensal isolates, most likely due to their frequent localization on mobile genetic elements and the fact that the reservoir of ExPEC/APEC is the gut microbiota but maybe for chicken also the trachaea. The intestinal microbiota is also the reservoir of human ExPEC, which are not truly commensal E. coli.
- We performed antibiotic susceptibility testing by agar diffusion assays and found out that in contrast to virulence-associated genes, animal husbandry (commercial farming vs. organic farming) had a significant impact on the presence of antibiotic resistance-associated genes and antibiotic resistance phenotypes was significantly affected by different forms of animal husbandry (commercial farming vs. organic farming)
- Plasmid MLST and incompatibility group typing allowed an initial characterization of the variability and composition of the plasmidome of E. coli isolates from poultry. Several so far undiscovered pMLST clones and Inc types could be described in the commensal isolates. According to Inc types, the plasmidome of chicken commensals from an organic farm displayed a greater diversity than the plasmidome of E. coli isolates from hens from commercial farming.
WP2.
- We worked on the functional characterization of APEC virulence-/ and fitness determinants, e.g. the contribution of iron uptake systems, the ETT-2 type 3 secretion system and the Bor/Iss proteins on serum resistance of E. coli isolates. Additionally, the importance of HlyF for OMV production was investigated as well as the impact of the SslE mucinase for intestinal fitness and colonization. For this purpose, we applied structural and functional analyses of the corresponding determinants coding for virulence- and fitness traits of interest.
- We generated deletion mutants of candidate virulence or fitness genes and compared the virulence- and fitness phenotypes of the wildtype strains with those of the isogenic deletion mutants in vitro as well as in suitable in vivo intestinal colonization and/or infection models. Simultaneously, candidate genes were overexpressed and their effect on virulence- or fitness-associated phenotypes was assessed relative to the wild type strain.
- We compared the expression of these determinants in media and under conditions mimicking host infection using qRT-PCR and reporter gene fusions or by Western blot analysis
- We demonstrated that HlyF, which is expressed by the most virulent APEC strains, regulates production of outer membrane vesicles (OMVs). HlyF expression resulted in increased release of cytotoxins, such as ClyA and CDT. Deletion of hlyF led to reduced persistence in blood and attenuation in a chicken model of colibacillosis. OMVs exhibit remarkable potential for immunomodulation of immune responses and can be used for the delivery of bacteria-specific antigens or unrelated antigens incorporated into the vesicle structure.
- The function of mucinase SslE was further studied in a physiologically relevant model of mucus-expressing HT29-MTX intestinal epithelial cells. E. coli strain IHE3034 expressing SslE variant I was shown to have an impaired association rate to mucus-secreting HT29-MTX cells relative to its isogenic sslE deletion mutant. These differences in the adhesive capacity were abolished upon removal of the mucus layer. SslE seems to promote tight adhesion of the bacteria to the mucus layer, but also the surface accessibility of HT290-MTX cells, because it renders the mucus layer looser and easily removable, thus allowing to penetrate the mucus layer.
- Whole genome sequence determination, annotation and comparative analysis of one O78 and one O2 APEC isolate with publicly available genome sequences of other APEC and human ExPEC isolates; we identified several potential virulence factors, conserved in all the colisepticemic strains examined, and determined their contribution to growth in serum, which is a requirement for septicemia. Based on our findings, specific vaccines or drugs can be developed against these critical virulence factors to combat avian colisepticemia.
- We determined the differential effects of iron uptake systems on growth under the iron-limiting conditions in APEC O78; iron limitation is a critical factor for bacterial survival in serum. Our study defined an iron uptake system (yersiniabactin), which is essential under low-iron conditions and thus represents a promising vaccine or therapeutic target.
- We described the contribution of the ETT-2 variant of T3SS expression to serum resistance of E. coli.
- We characterized two iss alleles in the APEC O78 genome with differential effect on serum resistance
- The usability of candidate markers for improved and facilitated diagnostics, e.g. by MALDI-TOF analysis was tested. Whole-cell MALDI measurements demonstrated that some commensal and APEC variants cannot be distinguished based on protein expression profiles. However, some of the commensal clones could be easily discriminated from other clones based on whole- cell MALDI tests.
- We systematically assessed bacterial transcriptomes by RNA-seq in response to growth under in vivo-like conditions, e.g. upon growth in blood serum or under iron depletion or in serum in the presence of subinhibitory concentrations of relevant antibiotics used for the treatment of septicaemia.
WP3.
- Based on the comparative genomic analyses, we identified novel candidates for diagnostic proteins and vaccine targets
- The corresponding determinants were subcloned, heterologously expressed in suitable E. coli production strain and purified.
- The purified proteins were tested for their efficacy as protective antigens against APEC infection
- One vaccine candidate provided (cross-)protection against E. coli infection in mice. Protection was also tested in a newly established murine model of intestinal colonization. The intranasal immunization of mice led to significantly reduced intestinal colonization of enterotoxigenic E. coli (ETEC) strain GL53. Expression of the corresponding gene was demonstrated upon intestinal colonization using a promoter::luciferase reporter fusion, which was expressed in ETEC strain GL53 and monitored by in vivo in mice using imaging system technology (IVIS).
Strength and Weaknesses / A particular strength of our project was the collaboration between partners from several European countries working on different aspects of APEC pathogenomics. The partners have complementary expertise, i.e., an extensive experience in microbiology, veterinary medicine, avian pathology, molecular genetics, cell biology, bioinformatics, and vaccine development. Our joint efforts to establish a collection of well-characterized commensal chicken isolates from different European countries represented one important central aspect of our activities and allowed to quickly collect a large number of isolates from different countries. Furthermore, the comparative genomic analysis of APEC genomes relied on the successful cooperation of different partners with complementary expertise and included the sharing of genome sequence information, e.g. between partners 1, 2 and 4, as well as the functional characterization of virulence-/fitness determinants in vitro and in vivo, and the tests of protection due to vaccination with purified protective antigens in experimental models of infection. Short term visits between members of different partner laboratories (partners 3 and 4) were organized to learn new techniques and to perform experiments.
We believe that one missing expertise was an avian pathologist. Having one in the group would have enabled to run a series of animal experiments that could improve the data
If this project had more time (and budget) there was a good chance that we could have brought it to the point of developing a novel vaccine. We did obtain extensive data that could lead to this end.
Further steps and emerging gaps / Further steps required to establish an improved APEC vaccine would include the efficiency assessment of the protective antigens against APEC infection and maybe the transfer of results from our project on OMV expression to improve the delivery of our novels antigens.
Conclusions
(Please include here also all references to published papers and other dissemination activities) / Conclusions:
The establishment of a well-defined collection of E. coli isolates from healthy and diseased chicken raised in different European countries allowed us to compare genome content and phylogeny of APEC with commensal E. coli isolates from healthy chicken. We provided for the first time a survey of the population structure of commensal E. coli in healthy chickens, which will be important to assess the pathogenic potential of APEC and to define biomarkers for the detection and risk assessment of virulent APEC strains. Genome sequencing and comparative analysis APEC and commensal E. coli isolates from poultry led to the identification of several potential virulence factors, which are conserved in all the colisepticemic strains examined. Based on these findings, specific vaccines or drugs can be developed against these critical virulence factors to combat avian colisepticemia. We successfully characterized the expression and function of important virulence- and fitness-associated genes of APEC: The detailed investigation of the contribution of iron uptake systems to growth under the iron-limiting conditions in APEC O78 corroborated that iron limitation is a critical factor for bacterial survival in serum. We could define an iron uptake system, which is essential under low-iron conditions and thus represents a promising vaccine or therapeutic target.
One of the virulence-associated factors that is epidemiologically linked with the most virulent APEC strains is the plasmid-encoded factor HlyF. Increased OMV release due to HlyF overexpression in clinical isolates represents a promising strategy for improved vaccine development.
Our analysis of additional APEC virulence-associated genes showed that also the ETT-2 type 3 secretion system contributes to bacterial serum resistance as well as the Iss protein.
SslE and FdeC have been functionally characterized during infection and colonization
Based on our joint work promising vaccine candidates have been identified and functionally characterized. (Cross-)protection due to vaccination with purified protective antigens against E. coli infection in mice has been confirmed. In summary, our joint efforts resulted in a significant gain of knowledge regarding the genomic variability and virulence potential of APEC that also may lead to the further development of alternative diagnostic and preventive strategies against APEC infection.
Publications:
- Huja S, Oren Y, Biran D, Meyer S, Dobrindt U, Bernhard J, Becher D, Hecker M, Sorek R, Ron EZ. (2014) Fur is the master regulator of the extraintestinal pathogenic Escherichia coli response to serum. MBio. 5:e01460-14.
- Huja S, Oren Y, Trost E, Brzuszkiewicz E, Biran D, Blom J, Goesmann A, Gottschalk G, Hacker J, Ron EZ, Dobrindt U. (2015) Genomic avenue to avian colisepticemia. MBio. 6:e01681-14.
- Moriel DG, Rosini R, Seib KL, Serino L, Pizza M, Rappuoli R. (2012) Escherichia coli: great diversity around a common core. MBio. 3:e00118-12.
- Nesta B, Spraggon G, Alteri C, Moriel DG, Rosini R, Veggi D, Smith S, Bertoldi I, Pastorello I, Ferlenghi I, Fontana MR, Frankel G, Mobley HL, Rappuoli R, Pizza M, Serino L, Soriani M. (2012) FdeC, a novel broadly conserved Escherichia coli adhesin eliciting protection against urinary tract infections. MBio 3:pii:e00010-12.