New insights into the bacterial fitness-associated mechanisms revealed by the characterization of large plasmids of an avian pathogenic E. coli

Abstract

Extra-intestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health, with illness causing substantial economic loss. APEC strain χ7122 (O78∶K80∶H9), containing three large plasmids [pChi7122-1 (IncFIB/FIIA-FIC), pChi7122-2 (IncFII), and pChi7122-3 (IncI(2))]; and a small plasmid pChi7122-4 (ColE2-like), has been used for many years as a model strain to study the molecular mechanisms of ExPEC pathogenicity and zoonotic potential. We previously sequenced and characterized the plasmid pChi7122-1 and determined its importance in systemic APEC infection; however the roles of the other pChi7122 plasmids were still ambiguous. Herein we present the sequence of the remaining pChi7122 plasmids, confirming that pChi7122-2 and pChi7122-3 encode an ABC iron transport system (eitABCD) and a putative type IV fimbriae respectively, whereas pChi7122-4 is a cryptic plasmid. New features were also identified, including a gene cluster on pChi7122-2 that is not present in other E. coli strains but is found in Salmonella serovars and is predicted to encode the sugars catabolic pathways. In vitro evaluation of the APEC χ7122 derivative strains with the three large plasmids, either individually or in combinations, provided new insights into the role of plasmids in biofilm formation, bile and acid tolerance, and the interaction of E. coli strains with 3-D cultures of intestinal epithelial cells. In this study, we show that the nature and combinations of plasmids, as well as the background of the host strains, have an effect on these phenomena. Our data reveal new insights into the role of extra-chromosomal sequences in fitness and diversity of ExPEC in their phenotypes.

Figure 1. Circular representation of pChi7122-2 (A), pChi7122-3 (B), and pChi7122-4 (C).

The different rings represent (from outer to inner) CDS which are color coded by functional group (ring 1 and 2), deviation from average G+C content (ring 3), and GC skew [(G−C)/(G+C); ring 4]. Colors represent the following: red, virulence-associated; green, plasmid transfer; yellow, plasmid replication; grey other functions; brown, hypothetical protein.

Figure 2. Comparison of physical and genetic maps for eitABCD and sugar pathway regions.

The eitABCD region from pChi7122-2 was compared to its equivalent in pHK01 (HM355591.1), pEG356 (FN594520.1), pAPEC-O2-ColV (NC_007675.1), pAA (FN554767.1), and pAPEC-O1-ColBM (NC_009837.1) (A); and sugars pathways genome region pChiA pChiOTDR in pChi7122-2 to its equivalent found in the genomes of S. Enteritidis (AM933172.1), S. Gallinarum (AM933173.1), S. Weltevreden (FR775220.1) and S. Agona (CP001138.1) respectively (B).

Figure 3. Plasmid pChi7122-2 putative sugar utilization pathway.

Illustration of the proposed biochemical pathway for putative sugar utilization encoded by the operon pChiOTDR (A), the predicted 3D model and function of each enzyme are presented; and diagrammatic representation of the genetic organization of the putative sugar utilization operon pChiA pChiOTDR (B). The two promoter regions identified are indicated with arrows. Bioinformatic analysis indicated that pChiR may act as a transcriptional regulator of pChiOTDR genes. The promoter region contains independent CRP binding boxes indicated in red. The promoter elements for pChiOTDR (P_pchi) located in the positive DNA strand are underlined. The promoter elements for pChiA (P_pChiA) in the negative strand are in bold and red without underlining.

Figure 4. Comparative of association and invasion of strains with 3-D INT-407 cells.

Mean of Log CFU/ml and standard deviation of bacteria association and invasion with cells (A) Representative confocal laser scanning micrographs showing association, and invasion of 3-D INT-407 cells with χ7122 and derivatives (B). Bacteria are marked in green, while the blue and red colors represent cell nuclei labeled with DAPI and F-actin cytoskeleton labeled with phalloidin, respectively; and ZO-1 staining of non-infected and infected 3-D INT-407 with plasmid-derivative strains (C). Non-infected 3-D INT-407 aggregates (a) or 3-D INT-407 aggregates infected for 2 h with wild type (χ7122) (b), ΔpChi7122-1,2,3 (χ7368) (c), pChi7122-1 (χ7394) (d), pChi7122-2 (χ7392) (e), pChi7122-3 (χ7367) (f), pChi7122-1,2 (χ7561) (g), pChi7122-1,3 (χ7562) (h), and pChi7122-2,3 (χ7274) (I). The ZO-1 antigen is marked in green, while the blue color represents cell nuclei labeled with DAPI. Images are presented with (indicated as “2”) and without (indicated as “1”) DAPI labeling for clarity purposes. Images are based on 400× magnifications. Arrows indicate the bacteria stained in green; Abbreviations used are: pChi7122-1,2,3 = pChi7122-1, pChi7122-2, and pChi7122-3; pChi7122-1,2 = pChi7122-1 and pChi7122-2; pChi7122-1,3 = pChi7122-1 and pChi7122-3; pChi7122-2-3 = pChi7122-2 and pChi7122-3.

Figure 5. Bile and acid tolerance of strains.

Bile sensitivity assay for E. coli K-12 and derivatives, no-plasmids (χ6092), pChi7122-1 (χ7346), pChi7122-2 (χ7347), and pChi7122-3 (χ7348). Five-microliters of serial ten-fold dilutions of each strain were spotted on both LB and LB +1% (w/v) DOC agar plates. The approximate numbers of bacteria present in each dilution are indicated on the right side of the plate (A). Percent acid survival of wild-type derivative strains in acid shock for 18 hours (B). Abbreviations used are: pChi7122-1,2,3 = pChi7122-1, pChi7122-2, and pChi7122-3; pChi7122-1,2 = pChi7122-1 and pChi7122-2; pChi7122-1,3 = pChi7122-1 and pChi7122-3; pChi7122-2-3 = pChi7122-2 and pChi7122-3.

Figure 6. Effect of plasmids, O-LPS, and temperature on biofilm formation.

Biofilm formation of different strains were compared at different temperatures: χ7122 and its plasmid derivatives strains: No-plasmids (χ7368), pChi7122-1 (χ7394), pChi7122-2 (χ7392), pChi7122-3 (χ7367), pChi7122-1,2 (χ7561), pChi7122-1-3 (χ7562), and pChi7122-2,3 (χ7274) (A), E. coli K-12 (χ6092) and its derivatives: pChi7122-1 (χ7346), pChi7122-2 (χ7347), and pChi7122-3 (χ7348) (B), and χ7122 and its LPS derivative strains : rough mutant O78(-) (χ7145), smooth strains with either O1-LPS (O1) (χ7193) or O111-LPS (O111) (χ7167) (C) at either 30°C, 37°C, or 42°C.