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Pathogenic Diversity among Chlamydia trachomatis Ocular Strains in Nonhuman Primates Is Affected by Subtle Genomic Variations

  1. Laszlo Kari1,
  2. William M. Whitmire1,
  3. John H. Carlson1,
  4. Deborah D. Crane1,
  5. Nathalie Reveneau1,a,
  6. David E. Nelson2,
  7. David C. W. Mabey3,
  8. Robin L. Bailey3,
  9. Martin J. Holland3,4,
  10. Grant McClarty5 and
  11. Harlan D. Caldwell1
  1. 1Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana
  2. 2Department of Biology, Indiana University, Bloomington
  3. 3Clinical Research Unit, Infectious Tropical Disease Department, London School of Hygiene and Tropical Medicine, London, United Kingdom
  4. 4Viral Diseases Programme, MRC Laboratories, Fajara, Banjul, The Gambia, West Africa
  5. 5Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
  1. Reprints or correspondence: Dr. Harlan D. Caldwell, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, National Institutes of Health, 903 S. 4th St., Hamilton, MT 59840 (hcaldwell{at}niaid.nih.gov).

  • ↵a Present affiliation: Sanofi Pasteur, Ltd., Toronto, Ontario, Canada.

 
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Abstract

Chlamydia trachomatis is the etiological agent of trachoma, the leading cause of preventable blindness. Trachoma presents distinct clinical syndromes ranging from mild and self-limiting to severe inflammatory disease. The underlying host and pathogen factors responsible for these diverse clinical outcomes are unclear. To assess the role played by pathogen variation in disease outcome, we analyzed the genomes of 4 trachoma strains representative of the 3 major trachoma serotypes, using microarray-based comparative genome sequencing. Outside of ompA, trachoma strains differed primarily in a very small subset of genes (n = 22). These subtle genetic variations were manifested in profound differences in virulence as measured by in vitro growth rate, burst size, plaque morphology, and interferon-γ sensitivity but most importantly in virulence as shown by ocular infection of non-humanprimates. Ourfindings are the first to identify genes that correlate with differences in pathogenicityamong trachoma strains.

Blinding trachoma is caused by the obligatory intracellular prokaryotic pathogen Chlamydia trachomatis. Despite its long history, trachoma remains the world's leading cause of preventable blindness [1]. Trachoma has been recognized as 1 of the 7 major neglected diseases of the 21st century [2]. Although trachoma has largely disappeared from developed nations, it remains endemic in sub-Saharan Africa, parts of Asia, and the Middle East. Trachoma is a progressive disease characterized by multiple stages that differ in severity among infected individuals. In areas of hyperendemicity, the majority of the population is infected by preschool age. Infections in these children typically present as self-limiting acute follicular conjunctivitis. However, a significant minority of the infected individuals progress to a chronic inflammatory disease, leading to conjunctival scarring and trachomatous blindness [3–6].

Variability in trachoma progression and severity among individuals in similar environments suggest that host genetic factors influence outcome. Various studies have addressed this question by analyzing an array of host factors, including HLA haplotypes [7, 8], gene polymorphisms in interferon (IFN)—γ, interleukin-10, tumor necrosis factor—α, and matrix metalloproteinase 9 [9–11]. Despite these efforts, as of today no single polymorphic human allele can fully explain the diversity in disease outcome. Although it remains possible that host factors contribute collectively to disease severity, current data suggest that they fall short of providing a complete explanation.

Trachoma is caused by serotypes A, B, Ba, and C. The C. trachomatis major outer membrane protein (MOMP), encoded by ompA [12], is the immunodominant surface antigen that differentiates all C. trachomatis isolates into serogroups and serotypes [5, 6, 13]. A limited number of studies have addressed the possibility that chlamydial factors could be responsible for the disparity in clinical trachoma outcomes. Most of these have addressed the role played by polymorphisms in ompA. Although ompA is variable among trachoma serovars [14] and genetic diversity of ompA has been associated with a higher prevalence of infection [15], neither MOMP immunotyping [16, 17] nor genotyping [18] of clinical isolates has revealed a compelling association between MOMP variation and disease severity.

To investigate whether strain-variable chlamydial factors might influence the severity of trachoma, we sequenced and compared the genomes of 3 trachoma reference strains (serotypes A, B, and C), and a recent serotype A clinical isolate. Comparative genomics revealed only a small subset of variable genes among all reference trachoma strains, genes that also varied in a limited number of recent clinical trachoma isolates. We found that 2 serovar A strains differing in these alleles exhibited a marked difference in pathogenicity in cell culture and, most importantly, in a nonhuman-primate ocular-infection model. These findings indicate that a limited number of variable chlamydial genes may contribute to the pathogenesis of trachoma and that polymorphisms among these genes might contribute to the known variability in the severity of trachoma observed in areas of endemicity.

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Materials and Methods

Chlamydial strains and propagation. Chlamydial strains included A/HAR-13 (from a 4-year-old trachoma patient; Saudi Arabia), A2497 (from a 2.5-year-old with intense active trachoma [TF/TI]; Rombo District, Kilimanjaro Region, Tanzania; 2001), B/HAR-36 (from a 12-month-old boy with trachoma; TR II follicles and papillae; Saudi Arabia), and C/TW-3/OT (conjunctiva; Taiwan). Strains were cultured in HeLa 229 (ATCC) monolayers, and elementary bodies (EBs) were purified by density gradient centrifugation [12]. C. trachomatis A/HAR-13 and A2497 were plaque cloned in McCoy (ATCC) monolayers [19]. The 8 clinical isolates were collected in trachoma-endemic areas of The Gambia (isolates D2–4, D2–13, and D2-30) and Tanzania (isolates 363, 12023, 15048, 18843, and 25256) between 2000 and 2003.

Genome sequencing. C. trachomatis A/HAR-13, A2497, B/HAR-36, and C/TW-3/OT genomic DNA was purified from EBs [20]. Ten micrograms of genomic DNA was subjected to genomic mutation mapping and resequencing (Nimblegen Systems). Single-nucleotide polymorphisms (SNPs) and locations of unidentified mutations were identified by comparative genome sequencing on custom-made oligonucleotide microarrays [21]. Open reading frames (ORFs) containing unidentified mutations, nonsense mutations, or >5 nonsynonymous SNPs were amplified by polymerase chain reaction (PCR) and then capillary sequenced. Gene polymorphisms were analyzed by comparing ORFs in the 4 genomes by means of ClustalW and BLAST. The nucleotide sequences of the highly polymorphic genes for strain A2497, B/HAR-36, and C/TW-3 have been deposited in GenBank (accession numbers EU121594-EU121633).

Sequencing of highly polymorphic genes from clinical isolates. DNA from 8 serovar A clinical isolates was extracted from infected HeLa 229 cells by use of 0.5 mol/L NaOH and 0.1 mol/L Tris (pH 8.0) [22]. Highly polymorphic genes were amplified by PCR and capillary sequenced.

One-step growth curves and plaque characterization. Chlamydiae were plated onto 24-well HeLa 229 monolayers at an MOI of 0.5. At different intervals after infection, chlamydiae were harvested and replated onto HeLa 229 monolayers for determination of inclusion-forming units. Burst size was calculated as the ratio of the highest recoverable number of inclusion-forming units to the number of infected cells. Plaquing of A2497 and A/HAR-13 was done using McCoy cell monolayers [19]. Kinetics of plaque formation was determined after neutral red staining at 5, 7, 9, 11, 13, and 15 days after infection. Plaque size was measured from photographs of stained monolayers.

Growth inhibition by IFN-γ. HeLa 229 cells were infected with either A2497 or A/HAR-13 at an MOI of 1. After infection, cells were fed with Dulbecco's modified Eagle medium (DMEM-10) containing 4 mg/L tryptophan and 0, 0.25, 0.5, 1, 2, 4, 8, 16, or 32 ng/mL IFN-γ (R&D Systems). All infected cells were harvested and titrated for recoverable inclusion-forming units when active mature inclusions were visible in untreated monolayers.

Infection of monkeys. Male cynomolgus monkeys (Macaca fascicularis) were used. Six monkeys were infected with 104 ifu of A2497 or A/HAR-13 by direct instillation onto the lower and upper conjunctival surfaces of both eyes. Swabs of the upper and lower conjunctival surfaces were collected to evaluate infection rates by culture as described above. Monkeys were scored for hyperemia and follicle formation of the upper conjunctival surfaces in both eyes. Hyperemia was scored as follows: 0, no hyperemia; 1, mild hyperemia; and 2, severe hyperemia. Follicles were scored as follows: 0, no follicles; 1, one to three follicles; 2, four to ten follicles, 3, more than ten follicles; and 4, follicles too numerous to count. The scores recorded for upper conjunctival surfaces of both eyes were added for each animal and termed the “clinical response score.” The highest possible clinical response score for an animal was 12. The work was conducted in full compliance with the guidelines given in the Guide for Care and Use of Laboratory Animals [23] as well as with all applicable federal laws and regulations. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care.

Statistical analysis. Statistical analysis of the clinical response scores and recoverable inclusion-forming units in all experiments were done by Student's t test (2-tailed distribution; 2-sample unequal variance), and differences were considered significant at P < .05.

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Results

Comparative Genomic Analysis

We compared the genomes of 4 trachoma strains to determine the extent of genomic polymorphism. The strains included isolates from all 3 major trachoma ompA serotypes, A, B, and C, collected from different geographic regions: A/HAR-13 (Saudi Arabia), A2497 (Tanzania), B/HAR-36 (Saudi Arabia), and C/TW-3 (Taiwan). The genome sequences of strains A2497, B/HAR-36, and C/TW-3 were determined by microarray-based comparative genome sequencing, with the A/HAR-13 genome [20] as the reference sequence. This technology is not limited to the discovery of SNPs but also provides accurate locations of any other type of mutations; hence, after PCR amplification and capillary sequence confirmation, a full and accurate genomic sequence can be obtained. All trachoma genome ORFs were compared with the corresponding ORFs of the other 3 genomes. Any ORF exhibiting >6 amino acid differences in any of these comparisons was considered polymorphic.

The 4 genomes exhibited remarkable similarity. Most genes previously described as being highly divergent between a C. trachomatis genital and a trachoma strain [20, 24] were unaffected by mutations or changed minimally. No novel genes were identified in any of the newly sequenced genomes. Gene polymorphisms in the trpRBA operon and the toxin region of ocular isolates has been described previously; however, all ocular isolates lack the predicted glycosyltransferase and YopT active-site motifs in the chlamydial cytotoxin and a functional trpRBA operon [22, 25]. Excluding the highly polymorphic ompA (MOMP) and the dysfunctional toxin and trpRBA operon, most of the mutations localized to 22 genes (table 1). Two types of polymorphic genes were identified: truncated genes and highly polymorphic genes. Eight genes were truncated by frame-shift or nonsense mutations, most of them hypothetical proteins. Fourteen genes were highly polymorphic, exhibiting numerous nonsynonymous SNPs or large insertions/deletions. These genes were CTA0050 (hctB), CTA0156 (early endosomal antigen 1 [EEA1]), CTA0164 (phospholipase D [PLD]), CTA0165 (PLD), CTA0166 (PLD), CTA0498 (translocated actin-recruiting phosphoprotein [TARP]), CTA0675, CTA0739 (tsf), CTA0740 (rpsB), CTA0743 (pbpB), CTA0747 (sufD), CTA0892 (tyrP2), CTA0947, and CTA0948 (figure 1A). We sequenced these genes from 8 recent serovar A clinical isolates obtained from hyperendemic-trachoma regions in The Gambia and Tanzania. The results showed that the majority of the same hypervariable genes found for the trachoma reference serovars also varied in these more recent clinical isolates (figure 1B), providing a more cognate argument that these genes might be important to trachoma pathogenesis. To test this hypothesis, we next performed a comprehensive pathogenic analysis of the 2 fully sequenced serovar A strains (A/HAR-13 and A2497).

Figure 1
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Figure 1

Genomic analysis of trachoma strains. A, Fully sequenced trachoma strains A, B, and C. Each colored circle represents the genome of a sequenced strain. Highly polymorphic genes, relative to A/HAR-13 [20], and their locations in the chromosome are indicated by black bars within each genome circle. Corresponding gene names are shown inside the circles. B, Genotypes of the 14 highly polymorphic loci in the 4 fully sequenced and 8 recent clinical isolates (ompA is also included in the analysis). Yellow indicates A/HAR-13 genotype; red, A2497 genotype (if different from A/HAR-13); green, other genotype(s). The first 2 rows show the 2 serovar A strains, the second 2 rows show serovar B and serovar C reference strains, and the last 5 rows show the genotypes of the 8 recent clinical isolates. Some of the clinical isolates have identical genotypes in these 14 loci and thus are grouped in the same row. EEA1, early endosomal antigen 1; TARP, translocated actin-recruiting phosphoprotein.

Pathogenicity of Trachoma Strains

In vitro growth and plaque morphology. A/HAR-13 and A2497 were selected for these studies because they have the same ompA genotype, so any observed differences should be independent of MOMP, and they have the most extensive polymorphisms in 16 of the 22 variable loci (table 1). Strains were initially compared for growth efficiency and plaque morphology (figure 2). A/HAR-13 and A2497 exhibited marked differences in growth rate and burst size by 1-step-growth analysis in human epithelial cells (figure 2A). The number of recoverable inclusionforming units was 10-fold higher for strain A2497 at each time point 24 h or more after infection (P < .05, Student's t test; n = 3), and burst size (inclusion-forming units per infected cells) was ∼8-fold greater (55:7) for A2497 than for A/HAR/13. Of particular interest was the dramatic difference in plaque phenotype between the 2 strains (figure 2B), because plaquing characteristics are commonly associated with virulence and attenuated properties of intracellular pathogens [26]. A2497 formed uniform, large, clear plaques that were first distinguishable 5 days after infection, whereas A/HAR-13 produced small, diffuse plaques that were undetectable until 11 days after infection.

Figure 2
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Figure 2

Difference in growth kinetics and plaque morphology between Chlamydia trachomatis serovar A strains A2397 and A/HAR-13. A, One-step growth curve of A/HAR-13 and A2497. Cells were infected, samples were harvested at different time points, and the no. of recoverable inclusion-forming units was determined. B, Plaque morphology. A/HAR-13 and A2497 exhibit small, diffuse (average size, 86 μm) and large, clear plaques (average size, 340 μm), respectively. Arrows indicate individual plaques. A typical plaque is shown at higher magnification for each strain.

Growth inhibition by IFN-γ. IFN-γ plays an important role in immune defense against C. trachomatis infection [27–30]. Therefore, the sensitivity of both trachoma strains to the inhibitory effects of IFN-γ was studied. Strains A2497 and A/HAR-13 differed dramatically in their sensitivity to IFN-γ (figure 3). The growth of A/HAR-13 was completely inhibited when infected cells were treated with IFN-γ concentrations of 2 ng/mL or higher. In contrast, A2497 exhibited significant resistance to IFN-γ even at the highest concentration tested (32 ng/mL).

Figure 3
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Figure 3

Interferon (IFN)—γ sensitivity of serovar A strains. HeLa cells were infected, and different concentrations of IFN-γ (0–32 ng/mL) were added to the cells at the time of infection. Samples were harvested when mature inclusions were visible in the absence of IFN-γ, and the no. of recoverable inclusion-forming units was determined.

Infectivity for nonhuman primates. We compared the infectivity of the 2 trachoma strains in a nonhuman primate model of ocular infection that has been extensively used for the study of trachoma infection and disease pathogenesis [31, 32]. The eyes of Macaca fascicularis cynomolgus monkeys were infected with A/HAR13 or A2497, and the course of infection was monitored after infection, by culture and ocular clinical response. The upper tarsal conjunctiva was evaluated and scored for hyperemia and follicle formation. A clinical response score of 0 indicates no disease, and a score of 12 indicates maximal disease. Symptoms were clinically evident by 1–3 weeks after infection and were maximal in all 6 monkeys between weeks 3 and 5 (figure 4). Importantly, the clinical response score between groups differed markedly. Between weeks 4 and 10, the differences in disease between the A2497 and A/HAR-13 groups were highly significant (P = .001 to P = .021, Student's t test; n = 3), with the exception of week 7 (P = .057). At later time points, the disease subsided in all animals, although the average clinical response score for the A2497 group remained consistently greater than that in the A/HAR-13 group.

Figure 4
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Figure 4

Virulence of Chlamydia trachomatis serovar A strains in nonhuman primates. Clinical response and shedding of organisms in eyes of cynomolgus monkeys infected with A2497 (left) and A/HAR-13 (right) are plotted individually for each animal. The clinical response score (gray area) was based on the hyperemia and follicular formation of the upper conjunctiva of both eyes; 0 indicates no disease, and 12 indicates maximum disease. Nos. of recoverable inclusion-forming units from swabs were also determined weekly and plotted for each monkey (lines). Between weeks 4 and 10, clinical response scores were significantly different (P = .001 to P = .021, Student's t test; n = 3) between the 2 groups, except at week 7 (P = .057). This time period is indicated with rectangles on the graphs. Shedding of organisms differed significantly during peak shedding periods, indicated by asterisks (P = .027, Student's t test; n = 6). The unique identifiers (Rocky Mountain Laboratories [RML] nos.) of the primates are shown above each graph.

We also measured chlamydial shedding from the infected conjunctivae. Consistent with disease results, there was a significant difference in chlamydial burden between the 2 groups (figure 4). The A2497-infected monkeys shed significantly more infectious organisms (>10-fold) than the A/HAR-13—infected monkeys during the peak shedding periods (P = .027, Student's t test, n = 6). The peak shedding period in all A2497-infected monkeys was at the first week after infection. In contrast, peak shedding in A/HAR-13—infected monkeys was consistently delayed to the second week after the infectious challenge. Conjunctival shedding declined less rapidly in the A2497 animals, and 2 of 3 monkeys (RML134 and RML126) continued to shed moderately high levels of chlamydiae over a 2-month period. The greater infectious burden and prolonged shedding mirrored the enhanced disease score in the A2497 group.

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Discussion

The pathogenesis of trachoma remains poorly understood. Defining the factors that dictate progression of chronic ocular inflammation and scarring would facilitate the control of this most important blinding disease. Both environmental and host factors contribute to blinding disease [7, 9–11, 33], although no single risk factor appears decisive. There have been no studies of whether differences in trachoma strains might contribute to more severe disease. The aim of the present study was to investigate this possibility. To that end, we have performed a comprehensive genomic analysis of the major trachoma serotypes and found them to differ in a very small number of genes. Importantly, we experimentally tested whether these subtle genetic differences resulted in different pathogenic properties of trachoma isolates. We provide compelling in vitro and in vivo pathogenic analysis demonstrating that there is a direct relationship between polymorphisms in this subset of genes and virulence properties of trachoma strains. Thus, it is possible that, in addition to host and environmental factors, differences in virulence among trachoma strains may also contribute to disease severity.

We sequenced and compared the genomes of 4 trachoma strains. These analyses revealed marked similarity in all trachoma genomes. However, a small subset of genes, representing ∼2% of the total genome, is polymorphic among these trachoma strains. Importantly, we demonstrated that most highly polymorphic genes also vary in recent isolates. Interestingly, previous studies have also shown that some of these genes exhibit similar polymorphism in C. trachomatis genital strains [34, 35]. To determine whether these polymorphic alleles are associated with strain pathogenic differences, we compared the virulence of 2 serovar A strains, so that any differences observed would be independent of ompA (MOMP). Most notable was our finding that these strains exhibited marked differences in virulence in amacaquetrachoma infection model, the only model relevant to the human disease. The observed strain differences in virulence for macaques were consistent with our in vitro findings. The greater infection burden and prolonged shedding period of A2497 monkeys agrees with the faster in vitro growth rate and larger burst size of this strain (figure 2). Similarly, the delayed clearance patterns of A2497-infected monkeys might also be related to this strain's faster growth rate and/or increased resistance to the inhibitory effects of IFN-γ (figure 3). Collectively, our results are the first to provide evidence that genetic variation among trachoma strains affects the pathogenesis of trachoma.

Nine highly polymorphic genes have different genotypes between A2497 and A/HAR-13 (table 1), and 6 of these can be associated with the distinct phenotypic properties. Four genes— hctB, CTA0156 (EEA1), tsf, and rpsB—may affect growth rate. Histone-like protein (hctB) of C. trachomatis can bind DNA and may condense DNA during the transition from reticulate body (RB) to EB [36]. Thus, mutations in hctB might affect the efficiency or rate of transition from noninfectious RB to infectious EB at the end of the developmental cycle. CTA0156 is a homologue of the eukaryotic EEA1. Although uncharacterized functionally in chlamydiae, CTA0156 has been implicated in the recruitment of nutrient-rich endocytic vesicles to the chlamydial inclusion [37]. Thus, mutations in CTA0156 could affect the ability of chlamydiae to compete for limiting intracellular nutrient pools. Protein translation elongation factor Ts (tsf; CTA0739) and 30S ribosomal protein S2 (rpsB; CTA0740) are involved in protein synthesis [38, 39], and their mutations might also affect growth rate.

Table 1
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Table 1

Polymorphic genes in A2497, B/HAR-36, and C/TW-3, compared with A/HAR-13.

Two highly polymorphic genes, tyrP2 and sufD, have putative functional antagonistic properties relevant to the welldocumented inhibitory effect that IFN-γ has on chlamydial growth and development that involves tryptophan and iron acquisition [27, 29, 40]. C. trachomatis has 2 copies of tyrP, a tryptophan and tyrosine transporter. The second copy in A/HAR-13 has 8 nonsynonymous SNPs and a 10-bp deletion, truncating the predicted protein by 84 aa, which may explain the greater sensitivity of this strain to the inhibitory action of IFN-γ. The protein associated with adenosine trisphosphate—binding cassette transporter (sufD; CTA0747) is part of a 4-gene cluster (CTA0745–CTA0748), and all 4 are homologous with members of the E. coli suf operon (sufBCDS). In E. coli, the suf operon is activated by iron starvation [41], and it has been proposed to be a virulence factor in other pathogens via its involvement in iron acquisition [42]. Variability in either tyrP or the suf operon—or both—may explain the differential sensitivity of these strains to IFN-γ in vitro (figure 3). However, pretreatment of cultures with IFN-γ 24 h before infection negated this difference between the strains (data not shown). Therefore, the differences in sensitivity might result from the distinct growth characteristics of the strains, as has been suggested previously for other chlamydial strains [43]. Regardless of the explanation, the different IFN-γ sensitivities are likely important to evading host defense during natural infections.

Three members of the PLD family (CTA0164–CTA0166) and TARP (CTA0498) are also highly polymorphic among trachoma strains. Although these genes are not polymorphic between A/HAR-13 and A2497 (with the exception of the truncated CTA0166 in A2497), putative functions have been associated with them that might affect pathogenesis. All C. trachomatis strains encode multiple copies of PLD genes in the plasticity zone of their genomes that are absent in Chlamydophila caviae, Chlamydia pneumoniae, and parachlamydiae. Inhibition of PLDs by primary alcohols prevents the recovery of C. trachomatis from IFN-γ—induced persistence [44]. TARP is a type 3 secreted effector [45], is present in all pathogenic Chlamydia species, and is highly variable among C. trachomatis strains [20]. TARP has been implicated in chlamydial entry into host cells through a parasite-directed remodeling of the actin cytoskeleton [45, 46]. Hence, polymorphisms in the PLDs and TARP might also affect the pathogenicity of trachoma strains.

A possible caveat of our study is that A2497 and A/HAR-13 were isolated decades apart. A2497 is a recent clinical isolate, and A/HAR-13 is a reference strain isolated in the 1950s. We cannot exclude the possibility that A/HAR-13 has accumulated the observed polymorphisms in vitro, although various lines of evidence suggest that this is not the case. Probably the most important selective pressure organisms have to face in vitro is selection against a slower growth rate. Because the most important in vitro phenotypic characteristic of A/HAR-13 is its slow growth rate, it is unlikely that a mutant with a slower growth rate has been able to outcompete faster-growing genetic variants within the population. Rather, we think that the long passage history of A/HAR-13 without any evidence of reversion to a faster growth rate suggests that this phenotype is multifactorial and not just a result of a single base pair insertion/deletion or minimal SNPs. Furthermore, by analyzing recent clinical isolates we have shown that the most polymorphic alleles of A/HAR-13 still exist naturally in Africa. For example, the remarkable conservation of elongation factor Ts (CTA0739), in which 46 of the 47 synonymous SNPs are conserved between recent clinical isolates and A/HAR-13, suggests that the A/HAR-13 genome has not been significantly affected by the propagation in vitro.

The polymorphism in trachoma strains described here suggests that strains of different pathogenic potential may simultaneously exist in geographically restricted host populations. Thus, these polymorphisms, independently or collectively with host and environmental risk factors, may contribute to the different clinical outcomes that are characteristic of blinding trachoma. It remains unclear which genotype might be associated with more severe disease. A priori, one might assume that fastergrowing IFN-γ—resistant organisms that were more pathogenic in nonhuman primates would be more virulent in natural infection. However, persistent infection is thought to be an important factor in the pathogenesis of trachoma. In fact, persistent infections might be more greatly facilitated by less virulent genotypes that have evolved specifically with the human host.

The findings of our study suggest that a multilocus sequencetyping strategy, using the genomic markers identified here, could identify genetic differences associated with trachoma severity in hyperendemic-trachoma populations. Because chlamydiae lack a tractable genetic system, we are unable to analyze individually the functions of the polymorphic genes. However, this difficulty might be circumvented by correlating the different genotypes with clinical descriptions for a large number of field isolates. This approach could further reduce the number of genomic markers and identify alleles strongly correlated with more or less severe clinical outcomes. Such analysis might identify the subset of patients at higher risk of developing trachomatous blindness and thus dictate future strategies for targeted intervention. Achieving these goals would greatly enhance our ability to understand the pathogenesis of trachoma and perhaps control blinding trachoma.

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Acknowledgments

We thank the Rocky Mountain Veterinary Branch of Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (RML/NIAID); the Genomics Unit of the RML Research Technologies Section, RML/NIAID; Anita Mora for assistance in graphic art; and Kelly Matteson for manuscript formatting.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health.

  • Received May 16, 2007.
  • Accepted September 18, 2007.
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