A Naturally Occurring Novel Allele of Escherichia coli Outer Membrane Protein A Reduces Sensitivity to Bacteriophage

A novel Escherichia coli outer membrane protein A (OmpA) wasdiscovered through a proteomic investigation of cell surfaceproteins. DNA polymorphisms were localized to regions encodingthe protein's surface-exposed loops which are known phage receptorsites. Bacteriophage sensitivity testing indicated an associationbetween bacteriophage resistance and isolates having the novelompA allele.

INTRODUCTION

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Introduction

Outer membrane protein A (OmpA) is a major, two-domain, heat-modifiablemembrane protein in bacteria. The N-terminal domain is comprisedof antiparallel ß-strands that cross the membraneeight times, producing four large surface-exposed hydrophilicloops and three short periplasmic turns. The C terminus, locatedin the periplasm, is connected to the outer membrane via interactionswith peptidoglycan (6). It has been proposed that OmpA is involvedin the structural integrity of the outer membrane (1, 2). OmpAalso acts as a phage and colicin receptor (1, 3, 7), and a numberof ompA mutants with alterations near residues 25, 70, and 110have been found to be resistant to bacteriophage (6, 7). Theresidues involved in phage resistance occur in the large surface-exposedloops of the protein, the same loops that act as phage receptors.

Outer membrane proteins similar to OmpA have been identifiedin 17 species of gram-negative bacteria (1). Similarities inthe structure of OmpA and the high degree of similarity withinthe nucleotide and amino acid sequences of several enteric speciesindicate a high degree of evolutionary conservation. Further,a comparison of five closely related genera have shown thatthe ß-strands are highly conserved, whereas the surface-exposedloops are highly variable (9).

An investigation to identify differences in the outer membraneproteins of Escherichia coli from different animal sources resultedin the identification of a novel ompA allele (ompA2). Here wedescribe the genetic characteristics of the novel ompA allele,its frequency in isolates from a range of vertebrate hosts,and an evolutionary advantage of organisms possessing the novelallele.

Outer membrane protein identification and characterization.

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Outer membrane protein...

Outer membrane proteins from three vertebrate E. coli isolates(H474 from a human, TA024 from a Tasmanian devil, and B194 froma varied honeyeater) were isolated using a carbonate extractionmethod combined with two-dimensional gel electrophoresis (8).Differential display of protein profiles from each of the threeisolates showed a distinct shift in the isoelectric point ofan integral outer membrane protein for isolate B194. Matrix-assistedlaser desorption ionization-time of flight mass spectrometrywas used to obtain mass fingerprints for each protein spot (8).Peptide analysis using appropriate databases (Profound and TrEMBL)indicated that the proteins were most similar to the E. coliOmpA protein.

Nucleotide analysis of the ompA allele from isolates characterizedusing proteomics revealed two sequences (ompA1 and ompA2). ABLASTN search identified ompA1 as being the ompA sequence ofE. coli (GenBank accession no. OMPAECOLI). The ompA2 allelewas distinct from previously described ompA genes, althoughit was most similar (approximately 97%) to Shigella flexneri(GenBank accession no. AY305875). Sequencing across the variableregions of a further 14 E. coli isolates indicated the novelompA2 allele to be present in human and marsupial isolates (Table1).

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TABLE 1. Type of ompA allele present in E. coli isolates from humans and Australian vertebrates for which sequence data were obtained

Translation and alignment of the OmpA amino acid sequences showedthat the surface-exposed loops 2 and 3 were the most variable,and single-amino-acid changes were identified in loops 1 and4 (Fig. 1). The percentages of identity of the E. coli ompA2allele to the described sequences for E. coli and S. flexneriwere 96.9% and 99.6%, respectively, and a single amino acidchange between the E. coli ompA2 allele and S. flexneri withinß-strand 5 at residue 93 was observed.

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FIG. 1. Translation of the E. coli ompA1 and ompA2 alleles from three E. coli isolates H474 (human), TA024 (Tasmanian devil), and B194 (bird) and alignment to the E. coli and S. flexneri ompA sequences (GenBank accession no. ECOMPA and AY305875, respectively). Variations occur within the exposed loops of the protein (shaded gray). A single amino acid change was identified external to a loop region in isolate TA024 (shaded black).

Frequency of the OmpA variant in vertebrate hosts.

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Frequency of the ompa...

The frequency of the ompA2 allele was determined by screening524 E. coli isolates selected from a collection of greater than1,300 isolates sampled from a variety of sources throughoutAustralia. Human clinical and fecal isolates were chosen, inaddition to fecal isolates from nondomesticated Australian mammals.These strains were previously screened for mitomycin C-induciblecolicin production and lysogeny (4). PCR primers were designedto specifically amplify a region of the ompA2 sequence (OmpAVF1[5'-GGCTAACGTACCTGGTGGCGCA-3'] and OmpAVR1 [5'-CGACGATCCGGAGCCAGGCA-3']).E. coli isolates were identified as having the ompA2 alleleby the presence of a 550-bp product using electrophoresis.

The novel ompA2 allele was identified in 43% of E. coli strainsscreened using the ompA2-specific PCR. The frequency of theompA2 variant was dependent on the source of the strain (humanversus animal) and the strain's E. coli Reference Collection(ECOR) group membership (2) (nominal logistic regression, likelihoodratio test, source, ${chi}$ ²₁ = 2.39, P = 0.122; ECOR group, ${chi}$ ²₃ = 18.7,P = 0.003; source x ECOR interaction, ${chi}$ ²₃ = 9.9, P = 0.02). Thefrequency of the ompA2 allele was 45% in the human isolatesand did not vary significantly among the four ECOR groups. Infecal isolates from nondomesticated mammals, the frequency was38%, and significant differences were observed in the frequenciesof the ompA2 variant among the four ECOR groups (Fig. 2). Further,of the 524 strains examined, those strains with the ompA2 allelewere significantly less likely to be lysogenic (3.2%) than strainswith the ompA1 allele (16.8%) (likelihood ratio test, ${chi}$ ²₁ = 19.1,P = <0.001).

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FIG. 2. Frequency of Escherichia coli strains carrying the ompA2 allele with respect to source of strain and ECOR group membership of the strains.

Phage sensitivity of the novel allele.

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Phage sensitivity of the...

We hypothesized that the high frequency of the ompA2 allelein E. coli isolates conferred a selective advantage to the organism.The majority of sequence variation in the ompA2 allele occurswithin loops 2 and 3, the same regions in which laboratory-engineeredmutations induced resistance to bacteriophage (6).

To determine whether the ompA2 allele reduced sensitivity tobacteriophage, 52 ompA1 and 52 ompA2 isolates (13 from eachof the four ECOR groups) were randomly selected (5). Lawns ofthe selected isolates were spotted with phage extracts (25 µl)prepared from 24 bacteriophage-positive E. coli strains (4).Natural E. coli populations having the ompA2 allele were lesssensitive to lysis by bacteriophage than strains with the ompA1allele were. This screening indicated that 19.2% of the ompA2strains were sensitive to one or more of the 24 phages, while39.2% of ompA1 strains were sensitive (likelihood ratio test, ${chi}$ ²₁ = 5.05, P = 0.025). Although the phages used in this studywere not identified, the use of 24 different phages from infectedE. coli isolates would have increased the chances of screeningmultiple phage types.

Past studies have demonstrated that the exposed-loop regionsof the protein function as phage receptors and that inducedmutations in these regions alter sensitivity to bacteriophage(6, 7). We have identified a naturally occurring ompA allelethat is associated with increased resistance to bacteriophageover typical E. coli OmpA expression. To confirm this hypothesis,the sensitivity of E. coli carrying the ompA2 allele to bacteriophageknown to use OmpA as the receptor needs to be tested. Furtherinvestigations will aid in understanding the functions of theexposed loops of the OmpA protein.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

Sequences from this study have been entered in GenBank underaccession numbers AY682204, AY682205, and AY682206.

ACKNOWLEDGMENTS

Funding for this work was provided by Sydney Water Corporation,a Macquarie University Collaborative Research grant, and theNational Capital Authority.

We acknowledge Raj Shanker for assistance in instigating thisresearch. The proteomics aspect of this work was conducted throughdirect access to the Australian Proteome Analysis Facility (APAF),Sydney, Australia. Thank you to the APAF staff for their assistance,especially Martin Larsen for mass spectrometry analysis. Wethank Peter Cox and Mark Angles from the Sydney Water Corporationfor assistance during the project and review of the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Biological Sciences, Division of Environmental and Life Sciences, Macquarie University, North Ryde, NSW 2109, Australia. Phone: 612 9850 6974. Fax: 612 9850 8253. E-mail: mpower{at}els.mq.edu.au.

Published ahead of print on 15 September 2006.

REFERENCES

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