3.1. Genetic Divergence of Incs between C. trachomatis Strains
To identify putative Incs within C. trachomatis
L2/434/Bu, a computational approach was designed to identify proteins that contained a hydrophobic domain of greater than 40 amino acids or two transmembrane domains of 20–30 amino acids separated by a small loop region [16
]. Each protein identified was analyzed using Kyte and Doolittle plot analysis to verify the presence of the characteristic bilobed hydrophobic domain [23
]. A list of predicted Inc proteins is provided in . The Incs predicted by our computational method provided assemblages similar to those previously compiled [16
]. Corresponding Incs from other C. trachomatis
strains where complete genomes were available were downloaded and analyzed for evolutionary distance using MEGA4 (). As an internal control for comparative purposes, the polymorphic outer membrane proteins (pmpA-I
) were also analyzed using nucleotide sequences obtained from the available genomes. The mean genetic distances obtained for C. trachomatis
Incs ranged from 0.001 (CT789) to 0.017 (CT116) with Incs CT115, CT116, CT223, and CT229 being the most divergent. Many of the Incs appeared genetically conserved in that they exhibited very little divergence (). The genetic divergence seen among the pmp
s was similar to what was previously described. PmpE, pmpF
, and pmpH
contained the most polymorphisms (mean genetic distances of 0.025, 0.065, and 0.014 resp.) [32
]. These data suggest that despite being highly conserved, some Incs may be evolving at different rates, equivalent to the more divergent of the pmp
Open reading frames with predicted Inc-like domainsa.
Figure 1 Genetic divergence of incs and pmps within C. trachomatis. Mean genetic distance within each predicted inc and pmp genes based on the average p-distance was determined from a pairwise comparison between all possible sequences for the same gene. Error (more ...)
Although distinct diseases and tissue tropisms are associated with different C. trachomatis
serovars, the genomes examined to date exhibit a high degree of synteny and greater than 99% sequence identity [33
]. The overall conservation seen between genomes indicates that there are relatively few loci involved in tissue tropism or that small polymorphisms in certain genes may greatly impact the infection process between serovars. Currently, there are few genetic loci that have been linked to clinical phenotype or tissue tropism within C. trachomatis.
These include members of the Pmps, pmpB, pmpC, pmpF, pmpG, pmpH
], and hctB
]. Here we observed an overall conservation of Incs within different strains of C. trachomatis
although four Incs (CT115, CT116, CT223, and CT229) appeared to be more divergent than others.
3.2. Phylogenetic Analysis of Incs within C. trachomatis
The variations in genetic divergence seen within predicted Incs of C. trachomatis suggested that certain Incs may be under different selective evolutionary pressures. Phylogenetic reconstructions of unrooted trees were performed for each Inc using existing genome sequences from all available serovars. The most divergent Incs, CT115, CT116, CT223, and CT229, exhibited clustering into clinical groupings with CT116 showing a separate clade for LGV strains while CT115, CT223, and CT229 exhibited separate clusters for genital, ocular, and LGV strains (Figures and ). Incs CT214, CT383, CT618, and CT195 also demonstrated phylogenetic clustering according to clinical groupings although they showed less divergence based on genetic distance than Incs CT115, CT116, CT223, and CT229 (). It was also possible to identify Incs that displayed partial clustering according to clinical groupings in that there were either separate clusters identified for ocular isolates or LGV isolates but not both. The most phylogenetically divergent clinical cluster was the LGV isolates. Separate LGV clusters could be identified for 35 Incs (, ). Separate ocular clusters were identified for six Incs (, ). Another trend that was evident was the separate clustering of serovar E isolates forming a separate clade from the ocular, LGV, and the other urogenital isolates. Finally, there were Incs that did not appear to segregate according to clinical disease ().
Figure 2 Phylogenetic reconstructions of C. trachomatis Incs displaying tissue tropism clusters. Evolutionary history of Incs was inferred with the Neighbor-Joining method using the bootstrap test with 1000 replicates with the percentage of replicate trees associated (more ...)
Phylogenetic clustering of Incs in C. trachomatis.
The differences in phylogenetic clustering of Incs suggest that certain Incs may be evolving at different rates than others. Most of the Incs exhibited some clustering according to disease groupings. Seven Incs produced separate clusters for urogenital, LGV, and ocular strains, 35 Incs produced separate LGV clades, and 6 Incs produced separate ocular clades. These findings suggest the possibility that specific Incs may be evolving towards different infection strategies for different host tissues. A microarray analysis of niche specific genes previously identified four Incs, CT116, CT223, CT288, and CT618 as LGV specific [45
] which were also identified in our study. However, no other Incs in that study were correlated with tissue tropism or clinical grouping.
The greater phylogenetic divergence of those Incs producing separate LGV clades suggests that Incs within LGV strains may be undergoing evolutionary divergence at rates greater than those of other clinical groupings. This divergence has been noted with other phylogenetically defined tissue tropic genes including tarP
] and the pmp
]. Phylogenetic analysis of tarP
indicated that the LGV isolates were the first to diverge to produce a distinct clade containing L1-L3 isolates [37
]. A similar divergence of LGV strains was seen for the pmp
]. This suggests that the evolutionary trend seen with the Incs in which LGV isolates appear more evolutionarily distinct coincides with other characterized genetic loci. It may be that LGV isolates in general show the greatest evolutionary divergence and that this divergence may not be limited to genes predicted to contribute to tissue tropism. The infections caused by LGV isolates differ from those caused by the ocular and urogenital serovars in that they are able to replicate within macrophages and cause a more invasive, systemic disease than the infections of mucosal epithelium caused by the ocular and urogenital strains [46
]. The differences in host selective pressures may be driving the evolutionary differences seen within the LGV Incs as well as other loci.
3.3. Conservation of Incs between Chlamydiae Species
The same computational approach used for C. trachomatis
was implemented to predict Incs for C. caviae
, C. felis
, C. muridarum,
and C. pneumoniae
(). All predicted Incs were cross-referenced to the other genomes using PSI-BLAST in attempts to identify divergent homologues in other species. Certain predicted Incs appear more than once in due to potential similarity to more than one identified homologue. C. pneumoniae
was predicted to contain the most Incs (92) with C. felis
and C. caviae
possessing 69 and 79, respectively. C. trachomatis
and C. muridarum
contained the fewest number of Incs within this comparison consisting of 55 and 54, respectively. Again, the predictions were highly overlapping but not identical to previous predictions [16
We were able to define a core subset of 23 Incs for which homologues could be identified in all five Chlamydiae species. C. trachomatis and C. muridarum were found to be the most related sharing 49 Inc homologues but also showed differences with each species containing unique Incs: 6 for C. trachomatis and 5 for C. muridarum (, ). The three remaining species, C. felis, C. caviae, and C. pneumoniae, also appeared very similar in that they shared a core of 47 Inc homologues. A pairwise comparison of these latter three species also identified shared Incs between any two given species with C. felis and C. caviae sharing a minimum 16 Incs, C. caviae and C. pneumoniae sharing 8, and C. felis and C. pneumoniae not sharing any outside of the core Incs (, ). Incs that appeared to be expanded in that there were more than one homologue per genome were only counted once in the Venn diagram. Genomic comparisons between all five species identified a core family of 23 conserved Incs for which Inc homologues are present in all species (CT005, CT006, CT058, CT134, CT179, CT195, CT232, CT233, CT288, CT324, CT365, CT383, CT440, CT449, CT483, CT484, CT565, CT616, CT618, CT642, CT728, CT788, and CT850) (, ). The analysis also identified Incs that were unique to each species ().
Figure 3 Venn diagram analysis of shared Inc homologues across Chlamydiae species. Two Venn diagrams are depicted showing shared Incs between C. trachomatis and C. muridarum (a) and between C. felis, C. caviae, and C. pneumoniae (b). Homologues that lacked an (more ...)
Generally, if an Inc was identified in one species, then its homologues in other species were also identified as Incs, although some exceptions were noted (). C. pneumoniae CP0481 was identified as an Inc based on the presence of a bilobed hydrophobic domain whereas its homologues in C. caviae (CCA00586) and C. felis (CF0422) lack the characteristic bilobed hydrophobic domain. C. pneumoniae also encodes CP0667 which lacks a bilobed hydrophobic domain but contains homologues to CP0667 that do contain the hydrophobic domain (CP0387, CP0388, and CP0390; ). Homologues to CP0667 in all four other chlamydial species also contain the characteristic bilobed hydrophobic domain (). C. trachomatis contained two genes which lacked a discernible bilobed hydrophobic domain; however the homologues of these genes in other chlamydial species are predicted to contain the characteristic hydrophobic bilobed domain (). As such, it appears that most Inc homologues maintain the bilobed hydrophobic domain.
Comparison of Incs between species provided an opportunity to evaluate the overall topology and conservation of Incs. Each Inc and its corresponding homologues identified were evaluated for the presence or absence of the characteristic bilobed hydrophobic domain. It was found that homologues of most Incs also contained a bilobed hydrophobic domain and that those homologues were also identified as Incs within this study suggesting that an Inc in one species has a corresponding homologue that is also an Inc in another species. Interestingly, the overall hydrophobic topology of the homologues was maintained despite a great degree of sequence divergence (an overall conserved identity of 27.3% for CT483 and 17.2% for CT850 at the amino acid level). illustrates two examples of Inc homologues that were identified in all five species, CT850 (a) and CT483 (b). Not only the presence of the bilobed hydrophobic domain but also its location was conserved suggesting that the bilobed hydrophobic domain is an integral part of an Inc protein.
Figure 4 Hydropathy plot analysis and conservation of core Incs. Incs CT850 (a) and CT483 (b) from C. trachomatis, C. muridarum, C. pneumoniae, C. felis, and C. caviae were visualized using Kyte and Doolittle hydropathy plots. Regions of the bilobed hydrophobic (more ...)
Examination of all chlamydial species for which sequenced genomes were available indicates that the predicted Incs within each species represent a significant fraction of the genome. Overall, a great diversity of Incs was identified, not only those which are shared between species but also those which appeared to be unique to each individual species. It is possible that Incs present in only one species may play a role in infection that is unique to that species. There were also 23 Incs identified in all five species. These Incs represent core Incs that may be involved in conserved interactions with the host cell.
Very few of the known Incs have had functions assigned. IncA, first identified in C. caviae
], is required for the homotypic fusion of inclusions in cells multiply infected with C. trachomatis
] and when transfected into host cells blocks C. caviae
]. IncA appears to be nonessential for C. trachomatis
survival and multiplication since clinical isolates of C. trachomatis
lacking IncA have been isolated from patients [49
]. IncA has been shown to have structural similarities to SNARE (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors) proteins, a class of membrane proteins that control the specificity of vesicle fusion [50
]. IncA has been shown to interact with the SNARE proteins Vamp3, Vamp7, and Vamp8 although depletion of these three SNAREs by siRNA had no deleterious impact on chlamydial growth [50
]. A number of Rab-family GTPases are recruited to the chlamydial inclusion membrane in a species-dependent manner [51
] and it appears that certain Inc proteins may play a role in specific Rab recruitment to the inclusion membrane. C. trachomatis
CT229 mediates recruitment of Rab 4 [52
] and C. pneumoniae
Cpn585 displays affinity for Rab 1, Rab 10, and Rab 11 [53
]. Other Incs known to recruit host proteins include IncG, which recruits the adaptor molecule 14-3-3β
in a species-specific fashion [13
]. Although a few Inc functions and interactions with host components have been identified, they are for the most part restricted to unique chlamydial species. One possibility is that the function of the majority of Incs and a reason for their duplication and diversity may not necessarily involve specific interactions with the host cell but be related to their unique hydrophobic structure and potential roles in the structural integrity of the inclusion membrane.
3.4. Genetic Expansion of Incs within Different Chlamydiae Strains
The C. trachomatis and C. muridarum genomes contained operons or clusters of Incs in which there was little sequence similarity between Inc open reading frames (CT115-119; CT222-229, ). Although these Incs cluster together in the genome, they appear to have arisen independently of each other or may represent expansion of an ancestral inc that diverged at a rapid rate. In contrast, C. felis, C. pneumonia, and C. caviae each contained Inc operons in which there were intracluster similarities suggesting gene expansion within these genomes (, ). Additionally, orthologous Incs could be located outside of operons in other regions of the genome. Multiple examples of duplicated Incs could be identified in C. felis, C. pneumonia, and C. caviae however none were identified in C. trachomatis or C. muridarum (). C. felis contained three examples of Inc expansion, one of which is illustrated in . The operon containing Incs CF0449-CF0451 had intracluster similarity but only a single identifiable homologue in either C. caviae (CCA00557) or C. pneumoniae (CP0730) (). C. caviae contained three genetic expansions of Incs, two of which are depicted in Figures and . One Inc cluster (CCA00633–CCA00639) contained three non-Inc genes within the operon and was homologues to only one Inc in C. felis (CF0574) or C. pneumoniae (CP0401) (). The second Inc expansion (CCA00425, CCA00426, CCA00221, and CCA00398) corresponded to an Inc expansion in C. pneumoniae (CP0397-CP0390; CP0667), where one homologue, CP0667, did not contain an identifiable bilobed hydrophobic domain (). C. pneumoniae not only contained the greatest number of Incs but also exhibited the most Inc expansions (eight) of the species examined, one of which is depicted in .
Figure 5 Examples of Inc expansion in C. caviae, C. felis, and C. pneumoniae. Three loci are depicted showing Inc expansion in C. felis (a), C. caviae (b), and C. caviae and C. pneumoniae (b and c). *designates that the predicted gene products lack the bilobed (more ...)
The advent of genome sequencing and comparative genomics has led to the recognition that there has been genetic expansion of genes from C. pneumoniae
], C. caviae
] but not C. felis
. Initial comparisons between C. pneumoniae
and C. trachomatis
genomes showed a high degree of sequence conservation and synteny but also noted a remarkable increase in the number of predicted Pmp genes. C. pneumoniae
encodes for 21
Pmps while C. trachomatis
encodes for only 9 [54
]. The amplification of the Pmp family of proteins suggests that either C. pneumoniae
contains mechanisms that enhance genetic expansion and diversity or, alternatively, the unique biology of C. pneumoniae
may have selected for these expansions. Similarly, we were able to identify Incs that have been expanded in C. pneumoniae, C. caviae,
and C. felis
. The genetic expansion of CT058 homologues was one of the most prominent in our study with C. caviae
and C. pneumoniae
each having four homologues. The regions flanking these genes also show a high degree of conservation [55
]. Incs unique to C. pneumoniae
appear to have undergone genetic expansion. C. pneumoniae
lacks an IncA homologue but encodes a closely related set of Inc paralogues that are predicted to contain an IncA domain [57
]. This group of paralogous genes has previously been referred to as the CP1054 family and each of these is shown to contain the characteristic bilobed hydrophobic domain characteristic of Incs. The reason for the level of genetic expansion seen in C. pneumoniae, C. caviae,
and C. felis
is uncertain but may represent a level of redundancy that is advantageous for these species with regards to infection or transmission that is not necessary for C. trachomatis
or C. muridarum
While the analyses here are focused upon the human and veterinary pathogens that comprise the genus Chlamydia
, members of the so-called environmental chlamydia of the family Parachlamydiaceae
also express proteins displaying the characteristic bilobed hydrophobic structure of chlamydial inclusion membrane proteins. The Parachlamydiaceae
are obligate intracellular symbiotes of amoebae believed to be ancestral to the pathogenic Chlamydia
and contain several of the virulence factors expressed in Chlamydia
. The genomes of the environmental chlamydiae are larger; that of Protochlamydia amoebophila
is approximately 2.41 Mbp [58
] versus that of C. trachomatis
serovar D which is 1.04 Mbp [34
]. Despite the much larger genome, the number of putative Incs in P. amoebophila
is only twenty-three [59
]. Of these, only three (pc0156, pc0184, and pc1857) bear significant similarity to chlamydial Incs and the Incs to which they are most closely related are among the core Incs identified here in Chlamydia spp.
Like the chlamydial inclusion membrane proteins, the functions of the Incs from P. amoebophila
are largely unknown although five of these have been confirmed as localized to the inclusion membrane [59
], and thus the predictive value of the bilobed hydrophobic domain appears to be viable in this family as well.