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Journal of Bacteriology, December 2007, p. 9145-9149, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.00722-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genetic Diversity of the Gene Cluster Encoding Longus, a Type IV Pilus of Enterotoxigenic Escherichia coli{triangledown} ,{dagger}

Oscar G. Gomez-Duarte,1* Sujay Chattopadhyay,2 Scott J. Weissman,3 Jorge A. Giron,4 James B. Kaper,5 and Evgeni V. Sokurenko2*

Division of Infectious Diseases, Department of Pediatrics, University of Iowa Children's Hospital, Iowa City, Iowa,1 Department of Microbiology, University of Washington, Seattle, Washington,2 Division of Infectious Disease, Immunology, and Rheumatology, Department of Pediatrics, University of Washington, Seattle, Washington,3 Department of Immunobiology, University of Arizona, Tucson, Arizona,4 Department of Microbiology, University of Maryland, Baltimore, Maryland5

Received 7 May 2007/ Accepted 20 September 2007


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ABSTRACT
 
Enterotoxigenic Escherichia coli (ETEC) strains produce a type IV pilus named Longus. We identified a 16-gene cluster involved in the biosynthesis of Longus that has 57 to 95% identity at the protein level to CFA/III, another type IV pilus of ETEC. Alleles of the Longus structural subunit gene lngA demonstrate a diversity of 12 to 19% at the protein level with strong positive selection for point replacements and horizontal transfer.


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TEXT
 
Enterotoxigenic Escherichia coli (ETEC) is an important cause of infant diarrhea in developing countries (5, 18), a leading cause of traveler's diarrhea (1, 14), and a re-emergent diarrheal pathogen in the United States (2, 35). One of the ETEC putative colonization factors is Longus, a type IV pilus (T4P) composed of a 22-kDa major structural subunit designated LngA (11, 13), which is estimated to be encoded or expressed by 10 to 35% of ETEC strains (12, 15, 20, 21). Antibodies reacting with LngA were found in stool from patients with ETEC infections (22). LngA shares homology with the major subunit of various T4Ps (9, 13), including the toxin-coregulated pilin of Vibrio cholerae (29) and the bundle-forming pilin of enteropathogenic E. coli (10). The highest homology (79%) is shared by LngA with CofA, the major subunit of another T4P of ETEC—CFA/III pili (13, 16, 27). Here we identify the genes involved in the assembly and regulation of Longus and describe their genetic and evolutionary variability.

Three plasmid libraries were constructed by partial restriction endonuclease digestion of the Longus-encoding virulence plasmid from ETEC strain E9034A. Plasmids were screened by PCR with lngA-derived primers and then other lng-specific primers (the plasmids used in this study are listed in Table 1). The Longus gene cluster obtained from ETEC strain E9034A is 14 kb in length and contains 16 open reading frames. Fourteen genes share considerable homology, as well as cluster topology, with CFA/III genes and are thus designated, in homology with the cof cluster, lngR, lngS, lngT, lngA, lngB, lngC, lngD, lngE, lngF, lngG, lngH, lngI, lngJ, and lngP (Fig. 1). Gene length, homology to known proteins, and putative functions are presented for each gene in Table 2. Two open reading frames, lngX1 and lngX2, were identified that have no homology to CFA/III pilin genes.


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  		TABLE 1. Host strains and plasmids used or generated in this study


Figure 1
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  		FIG. 1. Homology of Longus and CFA/III gene clusters. Arrows indicate the direction of transcription. Genes in black correspond to major structural subunit genes.


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  		TABLE 2. Biosynthetic Longus genes, putative functions, gene homologues, and allelic variations

We screened a collection of ETEC strains by PCR with a previously described lngA-specific primer pair (13) and a cofA-specific primer pair based on the published cofA sequence (28). The bacterial strains used in this study belong to different serotypes and were collected at different time points and in different regions of the world. Among the 56 ETEC strains studied, we identified two (4%) cofA-positive strains and 21 (38%) lngA-positive strains (see Table S1 in the supplemental material). We also screened the collection with specific primers based on other gene sequences of the Longus or CFA/III cluster (R, S, T, B, C, E, F, G, H, I, J, and P). The lngA-positive strains tested positive only with Longus-derived primers, while the cofA-positive strains tested positive only with cof-derived primers.

The lngA genes from the different ETEC strains were sequenced, multiple sequence alignments were performed with ClustalX 1.83 (30), and then PAUP* 4.0b (26) was used to construct maximum-likelihood-based phylogenetic trees. The lngA alleles segregated into three distinctive phylogenetic groups defined as groups 1, 2, and 3 (Fig. 2a). Primary (the most basal) nodes of groups 1 and 2 differed in 103 nucleotides (19%), leading to 22 amino acid changes (12%). Groups 1 and 2 differed from group 3 in 142 (26%) and 125 (23%) nucleotides, respectively, each leading to 34 (19%) amino acid changes. Alleles of the cofA gene in the two cofA-positive strains were also highly divergent from each other, with 137 nucleotide differences (24.8%) leading to 46 residue changes (25%). Although both allelic variants of cofA were more closely related to each other than to any of the lngA variants, the phylogenetic separation was not distinct (Fig. 2a). For example, the lngA group 1 and group 3 variants are phylogenetically more distant from each other than the lngA group 3 and cofA group 2 variants, obscuring evolutionary distinction of the Longus and CFA/III fimbrial structures.


Figure 2
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  		FIG. 2. Maximum-likelihood phylograms of Longus sequences. (a) Unrooted phylogram of lngA sequences. lngA sequences from 21 ETEC strains along with cofA sequences from CFA/III ETEC strains were included. (b) Phylogram of concatenated nonstructural genes in the Longus (based on a subset of six strains) and CFA/III gene clusters. The intra-cof (260-1/E2528C/M403-C1) comparison of nonstructural genes is based on 4 out of 13 loci (cofC, -E, -H, and -P). (c) Phylogram of concatenated adk and fumC genes from 21 ETEC strains. The branch lengths refer to numbers of nucleotide differences. For panels A and B, two different length scales are used. The symbol # indicates strains in which the non-lngA genes were sequenced (see panel B). Homologous lngA allelic groups are denoted by underlined (group 1), bold (group 2), and plain (group 3) text. Strains from which sequences were derived are represented by three-letter designations. lngA group 1 strains: E9034A, L1A; 01117-5, L1B; B2C, L1C; M104, L1D; M145-C2, L1E; M424-C1, L1F; M526-C6B, L1G; P307, L1H; M408-C1, L1I. lngA group 2 strains: 10159-a, L2A; 11381a, L2B, 2108-2, L2C; B7A, L2D; G1026, L2E; M445-C1, L2F; M452-C1, L2G; M626-C, L2H; M633-C1, L2I; MP215-1, L2J; BR5, L2K. lngA group 3 strain ECOR27, L3A. cofA group 1 strains: 2528C, C1A; 260-1, C1B. cofA group 2 strain M403-C1, C2A.

Within-group analysis of lngA sequences identified three distinct allelic variants within group 1 and three variants within group 2 that differed from each other by three to seven nucleotides (Fig. 2a). However, all 15 within-group mutations resulted in amino acid substitutions, indicating that these point replacements accumulated under strong positive selection. While immune escape may drive the accumulation of these point mutations, the changes may also be functionally adaptive for ETEC isolates by modifying some functional (e.g., putative adhesive) properties of Longus fimbriae, as shown for mannose- and di-galactose-specific fimbrial adhesins of uropathogenic E. coli (24, 25, 32), bfpA of EPEC (3), decay-accelerating factor-specific Dr adhesins of uropathogenic and diarrheal E. coli (6, 17, 31), and type 1 fimbrial adhesin of Salmonella enterica serovar Typhimurium (4).

Alignment of predicted LngA protein sequences (182 amino acids long) revealed that the between-group diversity of the protein is clustered in three regions, amino acids 56 to 77, 104 to 121, and 148 to 182 (see Fig. S1 in the supplemental material). Most of the within-group point replacements are located in these diversity regions as well. Unlike the amino acid diversity, the diversity of silent changes across lngA does not exhibit distinct clustering (see Fig. S1 in the supplemental material). The distribution of amino acid polymorphisms between the LngA and CofA protein sequences (as well as between the two CofA groups) shows less distinct clustering but reflects the general pattern of diversity seen among the LngA groups. Both LngA and CofA represent major surface subunits and thus antigens expected to be under strong selection for structural diversification. In particular, surface epitope regions would be prone to accumulate extensive structural changes while regions that are critical for proper tertiary structure or fimbrial morphology and/or function would remain conserved.

Unlike the structural subunit genes, nonstructural Longus genes are highly conserved (determined for the entire gene cluster in a subset of six strains), with more than 98% identity among homologs at the DNA (Fig. 2b) and protein (data not shown) levels. Homologs of nonstructural genes of the two CFA/III clusters (determined for cofC, -E, -H, and -P) were also conserved (99% identical to each other, on average), and after concatenation, they formed a distinct outgroup relative to the Longus genes (Fig. 2b), which is in contrast to the lngA/cofA tree (Fig. 2a). This indicates that while Longus and CFA/III pili are evolutionarily related, the cof and lng genes are paralogous in nature; i.e., they belong to evolutionarily distinct types of fimbriae.

To assess phylogenetic congruence among different lng loci, we performed an incongruence length difference test (8) with PAUP* 4.0b (26) that compared the sum of the branch lengths of a given pair of trees with the sum of the branch-lengths obtained through 1,000 random partitionings of the original data sets. Individual phylograms for the nonstructural genes were congruent with each other, providing no evidence of recombinational shuffling of alleles among different clusters (not shown). In contrast, a gene tree of concatenated nonstructural genes (Fig. 2b) was not congruent with the tree of corresponding lngA alleles (P = 0.001), suggesting that the major structural subunit gene is moving horizontally among the fimbrial gene clusters.

To determine the populational and intergenomic dynamics of the Longus gene clusters, we also sequenced two housekeeping loci—adk and fumC—in different ETEC strains. Housekeeping genes constitute the "backbone" of the bacterial chromosome, subject to recombinational events only infrequently in the species E. coli, providing the basis for phylogenetic and clonal grouping of E. coli strains (34). Both loci demonstrated high levels (98.1% for fumC and 99.4% for adk) of identity at the DNA level (Fig. 2c) that are within the range shown for housekeeping genes of E. coli at the species level (23). No congruence was detected between the concatenated adk/fumC tree and the lngA groups (P = 0.001), indicating frequent horizontal transfer of the lngA genes among ETEC strains. The lngA alleles have shown incongruence even with the major phylogenetic clusters of E. coli—ECOR groups A and B1—to which the vast majority of the ETEC strains studied belong (determined by the Clermont method [7]; see Table S1 in the supplemental material), indicating gene movement among distant clones as well. At the same time, the trees of adk/fumC and nonstructural lng genes were congruent with each other (the incongruence length difference test P value equals 1) for the same sample set, indicating that the nonstructural genes are significantly less prone to transfer than lngA. Thus, it appears that the main mechanism for horizontal transfer of lngA among ETEC strains involves single-gene recombination rather than transfer of the entire Longus-carrying plasmid.

Sequence analysis of the fimbrial genes also provides insight into the evolutionary history of Longus. It appears that the highly conserved nonstructural lng genes evolved in a manner similar to that of housekeeping genes. It involved accumulation of point mutations (rather than recombination) and purifying selection against amino acid replacement changes, the latter based on the low ratio of nonsynonymous changes (Dn as determined according to reference 35 by DnaSP software) to synonymous, silent changes (Ds) seen in most of the genes (Table 2). Silent changes are considered to be functionally neutral and accumulate randomly over time, reflecting the molecular clock. We used two different molecular clock rates, 3E-8 changes/year (16) and 6E-9 changes/year (33). Thus, on the basis of nonstructural gene diversity, Longus and CFA/III fimbriae were acquired by E. coli around 0.73 to 3.64 and 0.97 to 4.83 million years ago, respectively (16, 33), i.e., probably at some time after E. coli speciation, which is estimated to have occurred around 5 to 6 million years ago (19).

The synonymous and nonsynonymous diversity of lngA and cofA alleles is approximately 10-fold higher than that of corresponding nonstructural genes (Table 2). The high rates of both nonsynonymous and synonymous changes in the major subunit genes could be due to a high rate of either nonhomologous intragenic recombination (gene shuffling) or point mutations in the structural genes comparative to the nonstructural ones. Alternatively, this could be explained by parallel evolution of the structural allelic variants for extended periods of evolutionary time, with the lngA (and cofA) gene groups starting to diverge at the approximate time of the divergence of the Longus and CFA/III gene clusters from each other 20 to 100 million years ago, i.e., well before E. coli speciation. We propose that lng and cof clusters were acquired by E. coli in separate unique events after the time of speciation, with this evolutionary bottleneck explaining both the distinct phylogeny and low diversity of the lng and cof nonstructural genes. Afterwards, there were continuous horizontal-transfer events acting only on the major subunit genes lngA and cofA, both between and within species, driven by strong pressure for antigenic diversity.

Despite the antigenic diversity of lngA and cofA, the present study indicates that there is significant structural conservation between the group variants, allowing for likely cross-reactivity among them and thus the development of a Longus-CFA/III immunoprotective multicomponent vaccine against ETEC diarrheal disease.

Nucleotide sequence accession number. Newly described DNA sequences reported in this study were deposited in the GenBank database. The entire Longus cluster DNA sequence was assigned accession number EF595770. The cofA and lngA gene DNA sequences were assigned accession numbers EU107087 to EU107107.


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ACKNOWLEDGMENTS
 
This work was supported in part by NIH grant GM60731 to E.V.S., grants AI21657 and DK58957 to J.B.K., a K08 award to S.J.W., and a K12 award to O.G.G.-D. It was also supported by grant 1892-2007 from the Children's Miracle Network—University of Iowa Children's Hospital to O.G.G.-D.

We are grateful to Steve L. Moseley for providing E. coli strains for this study. We thank Jing Bai for technical assistance with DNA experiments.


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FOOTNOTES
 
* Corresponding author. Mailing address for Oscar G. Gomez-Duarte: Division of Pediatric Infectious Diseases, University of Iowa Children's Hospital, 200 Hawkins Drive, Iowa City, IA 52242. Phone: (319) 356-2270. Fax: (319) 356-4855. E-mail: oscar-gomez{at}uiowa.edu. Mailing address for Evgeni V. Sokurenko: Department of Microbiology, University of Washington, Seattle, WA 98195-7242. Phone: (206) 685-2162. Fax: (206) 543-8297. E-mail: evs{at}u.washington.edu Back

{triangledown} Published ahead of print on 19 October 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, December 2007, p. 9145-9149, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.00722-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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