To identify putative new virulence factors of avian pathogenic Escherichia coli (APEC) strains, a genomic subtraction was performed between the APEC strain MT512 and the non-pathogenic E. coli strain of avian origin EC79. Seventeen DNA fragments were cloned that were specific for the APEC strain. Among them, nine were identified that were more frequent among pathogenic than non-pathogenic isolates in a collection of 67 avian E. coli. Chromosome or plasmid location, and the nucleotide sequence of these nine fragments were characterized. Four fragments were plasmid-located. The nucleotide sequence of two of them exhibited identity with the sequence of the RepF1B replicon of E. coli plasmids, and the amino-acid deduced sequences from the two other fragments exhibited similarity to the products of genes sitA of Salmonella Typhimurium and iroD of E. coli, which are involved in iron metabolism. Of the five chromosome-located fragments, three were predicted to encode parts of proteins that were significantly homologous to previously described proteins: TktA (transketolase) of Haemophilus influenzae, a FruA (fructokinase) homologue of Listeria innocua and Gp2 (large terminal subunit) of phage 21. The putative products of the two other chromosome-located fragments were homologous to proteins with unknown functions: Z0255 of E. coli strain EDL933 (EHEC) and RatA of Salmonella Typhimurium strain LT2. Both these chromosomal fragments, whose presence is correlated with serogroups O1 and O2 and to the virulence of APEC strains belonging to these serogroups, are good candidates for being part of novel virulence determinants of APEC. Moreover, several fragments were shown to be located close to tRNA selC, asnT or thrW, which suggests they could be part of pathogenicity islands. Six fragments that were shown to be part of whole ORFs present in the APEC strain MT 512 were also present in extra-intestinal pathogenic E. coli (ExPEC) strains of human and animal origin. Thus, the putative novel virulence factors identified in this study could be shared by ExPEC strains of different origins.
BarnesH. J.,
GrossW. B.
1997; Colibacillosis. In Diseases of Poultry pp. 131–141 Edited by
CalnekB. W.,
BarnesH. J.,
BeardC. W.,
McDougaldL. R.,
SaifY. M.
Ames, IA: Iowa State University Press;
BingenE.,
PicardB.,
BrahimiN.,
MathyS.,
DesjardinsP.,
ElionJ.,
DenamurE.
1998; Phylogenetic analysis of Escherichia coli strains causing neonatal meningitis suggests horizontal gene transfer from a predominant pool of highly virulent B2 group strains. J Infect Dis 177:642–650[CrossRef]
BonacorsiS. P.,
ClermontO.,
TinsleyC.,
Le GallI.,
BeaudoinJ. C.,
ElionJ.,
NassifX.,
BingenE.
2000; Identification of regions of the Escherichia coli chromosome specific for neonatal meningitis-associated strains. Infect Immun 68:2096–2101[CrossRef]
BoydE. F.,
HartlD. L.
1998; Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution. J Bacteriol 180:1159–1165
BréeA.,
DhoM.,
LafontJ. P.
1989; Comparative infectivity for axenic and specific-pathogen-free chickens of O2 Escherichia coli strains with or without virulence factors. Avian Dis 33:134–139[CrossRef]
BrownP. K.,
CurtissR.3rd (1996; Unique chromosomal regions associated with virulence of an avian pathogenic Escherichia coli strain. Proc Natl Acad Sci U S A 93:11149–11154[CrossRef]
CheethamB. F.,
KatzM. E.
1995; A role for bacteriophages in the evolution and transfer of bacterial virulence determinants. Mol Microbiol 18:201–208[CrossRef]
CzirokE.,
DhoM.,
HerpayM.,
GadoI.,
MilchH.
1990; Association of virulence markers with animal pathogenicity of Escherichia coli in different models. Acta Microbiol Hung 37:207–217
DhoM.,
LafontJ. P.
1982; Escherichia coli colonization of the trachea in poultry: comparison of virulent and avirulent strains in gnotoxenic chickens. Avian Dis 26:787–797[CrossRef]
DhoM.,
LafontJ. P.
1984; Adhesive properties and iron uptake ability in Escherichia coli lethal and nonlethal for chicks. Avian Dis 28:1016–1025[CrossRef]
Dho-MoulinM.,
van den BoschJ. F.,
GirardeauJ. P.,
BréeA.,
BaratT.,
LafontJ. P.
1990; Surface antigens from Escherichia coli O2 and O78 strains of avian origin. Infect Immun 58:740–745
DozoisC. M.,
DaigleF.,
CurtissR.3rd (2003; Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain. Proc Natl Acad Sci U S A 100:247–252[CrossRef]
FairbrotherJ. M.,
BroesA.,
JacquesM.,
LariviereS.
1989; Pathogenicity of Escherichia coli O115 : K“V165” strains isolated from pigs with diarrhea. Am J Vet Res 50:1029–1036
FleischmannR. D.,
AdamsM. D.,
WhiteO.
& 7 other authors; 1995; Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496–512[CrossRef]
GoulletP.,
PicardB.
1986; Highly pathogenic strains of Escherichia coli revealed by the distinct electrophoretic patterns of carboxylesterase B. J Gen Microbiol 132:1853–1858
HackerJ.,
Blum-OehlerG.,
MühldorferI., TschapeH.
1997; Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol 23:1089–1097[CrossRef]
HuangS. H.,
WassC.,
FuQ.,
PrasadaraoN. V.,
StinsM.,
KimK. S.
1995; Escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: molecular cloning and characterization of invasion gene ibe10. Infect Immun 63:4470–4475
HullR. A.,
GillR. E.,
HsuP.,
MinshewB. H.,
FalkowS.
1981; Construction and expression of recombinant plasmids encoding type 1 or d-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect Immun 33:933–938
IkeK.,
KawaharaK.,
DanbaraH.,
KumeK.
1992; Serum resistance and aerobactin iron uptake in avian Escherichia coli mediated by conjugative 100-megadalton plasmid. J Vet Med Sci 54:1091–1098[CrossRef]
JanakiramanA.,
SlauchJ. M.
2000; The putative iron transport system SitABCD encoded on SPI1 is required for full virulence of Salmonella typhimurium. Mol Microbiol 35:1146–1155[CrossRef]
JohnsonJ. R.,
OswaldE.,
O'BryanT. T.,
KuskowskiM. A.,
SpanjaardL.
2002; Phylogenetic distribution of virulence-associated genes among Escherichia coli isolates associated with neonatal bacterial meningitis in the Netherlands. J Infect Dis 185:774–784[CrossRef]
KingsleyR. A.,
HumphriesA. D.,
WeeningE. H.,
De ZoeteM. R.,
WinterS.,
PapaconstantinopouloA.,
DouganG.,
BaumlerA. J.
2003; Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype Typhimurium: identification of intestinal colonization and persistence determinants. Infect Immun 71:629–640[CrossRef]
KuhnertP.,
HackerJ.,
MühldorferI.,
BurnensA. P.,
NicoletJ.,
FreyJ.
1997; Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains. Appl Environ Microbiol 63:703–709
LacksS.,
GreenbergB.
1977; Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J Mol Biol 114:153–168[CrossRef]
MeierC.,
OelschlaegerT. A.,
MerkertH.,
KorhonenT. K.,
HackerJ.
1996; Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells. Infect Immun 64:2391–2399
OswaldE.,
De RyckeJ.,
GuillotJ. F.,
BoivinR.
1989; Cytotoxic effect of multinucleation in HeLa cell cultures associated with the presence of Vir plasmid in Escherichia coli strains. FEMS Microbiol Lett 49:95–99
ParreiraV. R.,
GylesC. L.
2003; A novel pathogenicity island integrated adjacent to the thrW tRNA gene of avian pathogenic Escherichia coli encodes a vacuolating autotransporter toxin. Infect Immun 71:5087–5096[CrossRef]
PawelzikM.,
HeesemannJ.,
HackerJ.,
OpferkuchW.
1988; Cloning and characterization of a new type of fimbria (S/F1C-related fimbria) expressed by an Escherichia coli O75 : K1 : H7 blood culture isolate. Infect Immun 56:2918–2924
Runyen-JaneckyL. J.,
ReevesS. A.,
GonzalesE. G.,
PayneS. M.
2003; Contribution of the Shigella flexneri Sit, Iuc, and Feo iron acquisition systems to iron acquisition in vitro and in cultured cells. Infect Immun 71:1919–1928[CrossRef]
SmithH. W.
1974; A search for transmissible pathogenic characters in invasive strains of Escherichia coli: the discovery of a plasmid-controlled toxin and a plasmid-controlled lethal character closely associated, or identical, with colicine V. J Gen Microbiol 83:95–111[CrossRef]
SorsaL. J.,
DufkeS.,
HeesemanJ.,
SchubertS.
2003; Characterization of an iroBCDEN gene cluster on a transmissible plasmid of uropathogenic Escherichia coli: evidence for horizontal transfer of a chromosomal virulence factor. Infect Immun 71:3285–3293[CrossRef]
StockiS. L.,
BabiukL. A.,
RawlykN. A.,
PotterA. A.,
AllanB. J.
2002; Identification of genomic differences between Escherichia coli strains pathogenic for poultry and E. coliK-12 MG1655 using suppression subtractive hybridization analysis. Microb Pathog 33:289–298[CrossRef]
WeinbergE. D.
1995; Acquisition of iron and other nutrients in vivo. In Virulence Mechanisms of Bacterial Pathogens pp. 79–93 Edited by
RothJ. A.,
BolinC. A.,
BrogdenK. A.,
MinonF. C.,
WannemuehlerM. J.
Washington, DC: American Society for Microbiology;
WelchR. A.,
BurlandV.,
PlunkettG.3rd16 other authors2002; Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc Natl Acad Sci U S A 99:17020–17024[CrossRef]
WhiteD. G.,
Dho-MoulinM.,
WilsonR. A.,
WhittamT. S.
1993; Clonal relationships and variation in virulence among Escherichia coli strains of avian origin. Microb Pathog 14:399–409[CrossRef]
WilsonK.
1987; Preparation of genomic DNA. In Current Protocols in Molecular Biology pp 2.4.1–2.4.2 Edited by
AusubelF. M.,
BrentR.,
KingstonR. E.,
SeidmanJ. G.,
SmithJ. A.,
StruhlK.
New York: Wiley;
WuW. F.,
ChristiansenS.,
FeissM.
1988; Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage λ terminase. Genetics 119:477–484
ZhouD.,
HardtW. D.,
GalanJ. E.
1999; Salmonella typhimurium encodes a putative iron transport system within the centisome 63 pathogenicity island. Infect Immun 67:1974–1981