Nucleotide and phylogenetic analyses of the Chlamydia trachomatis ompA gene indicates it is a hotspot for mutation

Chlamydia trachomatis is an obligate intracellular bacterium that is the most frequently reported bacterial sexually transmitted disease in the United States [1], as well as the leading cause of infectious blindness worldwide [2]. Strains of C. trachomatis are differentiated into serovars based on the serospecificity of the major outer membrane protein (MOMP) that constitutes over 60% of all the surface-exposed proteins [3] and functions as a porin [4]. Serovars A-C primarily infect ocular tissue, serovars D-K typically infect urogenital tissue, and serovars L1-L3 infect lymphatic tissue. There appears to be a very high level of nucleotide identity across the genomes of the serovars [5, 6] as even the most divergent genomes are over 99% identical in sequence [7]. Additionally, genomic data has demonstrated that the phylogenetic pattern of the serovars correlates with tissue tropism [8–10].

In contrast to the low level of nucleotide variation observed in most of the genome, the ompA gene that encodes MOMP is characterized by a very high level of variability as nearly 30% of the coding sequence exhibits polymorphisms [11]. For comparison, the porB gene in C. trachomatis also encodes a surface-exposed porin that elicits neutralizing antibodies [12, 13], but it only varies at 1% of its nucleotide sites [8]. MOMP has four variable domains that encode the surface-exposed epitopes and contain the amino acid substitutions that define serospecificity; these are separated by five membrane-spanning constant domains that are considerably more conserved than the variable domains, but are nevertheless more polymorphic than the majority of the genes in the genome. Phylogenetic characterization of the ompA gene exhibits a pattern of strongly supported groupings that differ markedly from the tissue tropism [11, 14]: serovars A, C, H, I, J, K, and L3 comprise one group; F and G a second; D, E, and L1 a third; and B and L2 a fourth (see Figure 1A).

The discordance between the ompA gene tree and trees from other C. trachomatis loci is considered to be due to intra-species recombination [8, 9, 15–17]. Recombination is thought to be rare in C. trachomatis due to the limited opportunity other bacteria, plasmids, or exogenous DNA have to enter a chlamydial inclusion [18], but genomic sequence data indicate that intra-species recombination has occurred in 8 genes in the genome, including ompA, and may have occurred in as many as 47 additional genes [17]. There is evidence that recombination has acted on portions of the ompA gene to create mosaic MOMP structures [19–22], as well as on the entire ompA gene in order to replace the MOMP of an isolate [8, 9, 16]. Further support that chlamydia has the means and mechanisms to acquire and stably incorporate exogenous DNA is the presence of a genomic island containing a tetracycline-resistance gene found in the chromosome of several swine isolates of Chlamydia suis[23]. In addition to recombination, diversity generated in MOMP can be attributed to the positive selection of non-synonymous substitutions that lead to beneficial amino acid replacements, especially in a gene that encodes an antigenic outer membrane protein.

It is currently unknown, however, if the increase in nucleotide diversity in ompA is solely due to recombination and positive selection. In order to establish if there are additional factors involved in the generation of ompA diversity, we examined the nucleotide and phylogenetic associations of the five constant and four variable domains of the ompA gene, as these regions are under different immune and functional constraints. We also evaluated the relationships of the loci surrounding the ompA gene. These results demonstrate that the synonymous nucleotide diversity generated in the ompA gene cannot be explained by recombination and positive selection alone, and indicate that this region is prone to increased mutations compared to other regions in the genome. The data also show that the increased nucleotide substitution rate and the lack of phylogenetic associations in the regions proximal to the ompA gene are characteristic motifs of gene conversion.

Examination of the ompA gene of C. trachomatis and its surrounding loci demonstrate an increase in nucleotide substitutions and differing phylogenetic histories compared to other regions of the genome. The increase in nucleotide substitution rates and the lack of any phylogenetic congruence in the regions flanking ompA are characteristic motifs of gene conversion. Gene conversion in bacteria is a non-reciprocal recombinatorial process in which one gene replaces another through strand invasion/displacement and repair via the recA-D proteins [27]. Such a process is common in bacteria; it occurs in at least 17 different genera [27], including Chlamydia[17, 28, 29]. Typically, gene conversion can serve one of two functions in the genome. First, it can promote diversification of antigenic outer membrane genes, as has been reported in species of Anaplasma[30], Bartonella[31], Borrelia[32, 33], Mycoplasma[34], Neisseria[35, 36], and Treponema[34]. Second, it can reduce nucleotide variation in functional genes by homogenizing homologs within a genome; there is evidence that all bacterial genomes with multiple-copy rDNA undergo gene conversion, which maintains the uniformity and the low-level of nucleotide diversity observed in the ribosomal gene sequences (concerted evolution) [37]. Both models, however, also lead to an increase in nucleotide diversity and indel events in the flanking regions [30, 38, 39]. This is what we observed in the ompA gene, supporting gene conversion of the ompA gene region.

Gene conversion alone does not explain the increase in polymorphisms within the ompA variable domains. An increase in the rate of amino acid replacements in the variable domains of the ompA gene compared to the constant domains is expected, as these regions are surface-exposed and are the main antigenic targets of the host immune system. However, the 3.8-fold increase in the rate of synonymous change in the variable domains relative to the constant domains is notable, especially considering that the constant domains contain more synonymous changes than most other genes in the genome. Though synonymous changes will hitchhike with the selection of favorable non-synonymous changes, this selection should occur for any polymorphism across the entire gene and not just in the variable domains. Nevertheless, in the ompA gene, the rate of synonymous change is clearly not evenly distributed. This disparity in the distribution of synonymous changes is not due to recombination of the variable domain because the phylogenetic trees for each constant and variable domain, in addition to the trees constructed from either synonymous or non-synonymous sites, are all congruent with each other and with the ompA gene. There appears to be some factor that increases the propensity of mutations in the variable regions (i.e. a mutational hotspot) that would explain the observations; a mutational hotspot has previously been demonstrated to occur in the trpA gene of C. trachomatis[40, 41]. The high mutation rate observed in the synonymous sites in the variable domains compared to the constant domains appears to be evolutionarily conserved: the divergence analysis from the C. muridarum MoPn isolate indicates an equivalent level of synonymous change between the variable domains, the constant domains, and the neighboring genes.

It is not known what mechanism(s) promote the mutational increase in ompA and its neighboring loci relative to the rest of the genome. Codon bias causes a greater level of synonymous change in some genes as a way to increase transcriptional efficiency [42], but this would not occur in distinct segments of a single gene. Mutational bias associated with chromosomal location at the replication terminus [43] is also not a factor due to the fact that the ompA gene is only 60 kb from the origin of replication. One possible explanation is that a several-kilobase genomic region surrounding ompA has a unique secondary structure, which predisposes the region to increased mutations and decreased access to DNA repair machinery. While the whole region would be subject to increased mutations, more mutations could become fixed in the ompA gene if it had fewer functional constraints than the neighboring loci. In addition, the level of ompA expression may play a role: because ompA is one of the most highly expressed genes in C. trachomatis[44], it spends more time than other genes being single stranded and therefore may have a higher propensity of acquiring mutations in this state [45, 46]. Mutations are more likely to occur in the non-template strand because it is single-stranded and not protected by the replication bubble, and is therefore more prone to DNA damage and deamination that can lead to nucleotide replacements, insertions, or deletions [47–50]. Coupled with the potential translesion activity of DNA polymerase I [51] and positive selection, these mutations in ompA could become permanent.

The discordant phylogenetic associations of the loci neighboring the ompA gene, along with the increased rate of nucleotide diversity in these regions, can be ascribed to recombination of the ompA gene via gene conversion. In addition, the high synonymous substitution rate within the variable domains of ompA appears to be the result of an unidentified influence that generates an increase in mutations within the gene or genomic region. Together, the increased mutation rate in the ompA gene acts in concert with positive selection and recombination to promote the high degree of variability observed in MOMP in order to evade immune detection.