Identification of protective and broadly conserved vaccine antigens from the genome of extraintestinal pathogenic Escherichia coli

Abstract

Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli. We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains.

Fig. 1.

Comparative genome analysis between IHE3034 and other ExPEC strains. Comparison between IHE3034 ORFs with the nonpathogenic E. coli strain MG1655 (external gray) and other seven ExPEC strains. Nineteen genomic islands (black boxes in the outside, identified by roman numerals), specific to ExPEC strains, and not present in the nonpathogenic strain MG1655 are shown. The green circles represent UPEC strains, the light blue is a human K1 strain, and the red is an avian K1 strain.

Fig. 2.

Passive and active immunization using the antigen encoded by ECOK1_3385. Mice (10 per group) were actively (A and C) or passively (B and D) immunized with recombinant ECOK1_3385 (⧫), heat-inactivated IHE3034 (△) and saline solution with Freund’s adjuvant (□). Following challenge with the strain IHE3034 survival of mice was followed for 4 days (A and B). Bacterial counts in the blood (C and D) were determined at 20 h by counting the colony forming units (CFUs) in the blood detected by 10-fold serial dilutions and plating method. Bacteremia mean values in each group were determined assuming that mice, dead before 20 h, had the maximum level of CFU causing mouse death.

Fig. 3.

MSTree analysis of ECOK1_3385 distribution in a collection of E. coli isolates. The MSTree was constructed by a graphical tool, implemented as part of Bionumerics V4.6 (Applied Maths Belgium), that links allele designations within an MLST database. ST complexes are defined as containing at least three STs, differing in no more than one allele to their nearest neighbor. The MSTree displays the quantitative relationships between STs (given as numbers) and ST complexes (given as underlined numbers), measured as the number of shared alleles, by lines of different thickness and type. (A) Distribution of different E. coli groups (ExPEC, extraintestinal pathogenic E. coli; InPEC, intestinal pathogenic E. coli; commensals) in the phylogenetic background of 573 strains. The groups have been classified not only on the basis of their clinical history but also according to extensive virulence typing. (B) Distribution of ECOK1_3385 in the phylogenetic background of 573 strains.

Fig. 4.

T2SS is necessary for the secretion of the antigen encoded by ECOK1_3385. Map of the genomic region encoding ECOK1_3385 and the adjacent T2SS in strains IHE3034 and MG1655. The figure shows that the T2SS is truncated in the strain MG1655. Presence of the antigen in the total cell lysates and culture supernatants of strains, MG1655 (lane 1), W3110 (lane 2), CFT073 (lane 3), 536 (lane 4), IHE3034 (lane 5), IHE3034ΔECOK1_3385 (lane 6), IHE3034ΔECOK1_3381 (lane 7), IHE3034ΔECOK1_3380 (lane 8), and IHE3034ΔECOK1_3374 (lane 9). Antigen ECOK1_3385 is present in the total cell lysates of all strains with the exception of lanes 3 and 6 that contain strain CFT073 and the knockout mutant IHE3034ΔECOK1_3385 used as negative controls. The antigen is secreted only in strains 536 and IHE3034 that have an intact T2SS (lanes 4 and 5, respectively). Deletion of T2SS components such as ECOK1_3381 (component D), ECOK1_3380 (component E), and ECOK1_3374 (component K) leads to the abrogation of its secretion but not expression (lanes 7, 8, and 9, respectively).