The study described in this report shows that mutations in a single genetic locus (CT135) are sufficient to change the in vivo virulence of the human C. trachomatis urogenital strain D/UW-3/CX in the female genital tract of C3H/HeJ mice. Our findings support the conclusion that the D-LC strain but not the D-EC strain produced a naturally ascending infection in the female genital tract and evoked a chronic mononuclear inflammatory response, resulting in salpingitis. To our knowledge, this is the first demonstration of a naturally ascending infection in female mice with accompanying upper genital tract sequelae achieved using a C. trachomatis human STI isolate. We were successful in identifying the virulent human strain and mapping the virulence to CT135 by performing an in vivo infection screen that utilized a large cohort of mice infected with our laboratory reference strain, D/UW-3/CX, and de novo genome sequencing of the reisolated strains. The ID50s of D-EC and D-LC were similar, a finding consistent with the conclusion that CT135 is not critical for chlamydial colonization of urogenital epithelial cells. Strain virulence differences were discernible postcolonization and were characterized by significantly higher infectious burdens, durations of infection, and ascending infections, resulting in salpingitis. Our finding that CT135 mutations do not discriminate pathobiological differences in in vitro assays of infectivity, plaque morphology, or resistance to IFN-γ strongly support the conclusion that the function of CT135 is restricted to host-pathogen interactions within the in vivo infection environment.
Analysis of CT135 from our serovar D reference strain by CEL I digestion revealed numerous CT135 polymorphisms suggesting a mixture of phenotypes differing in virulence. The heterogeneity in CT135 was proven by direct sequencing from plaque-cloned isolates, also revealing that all genotypes had disruptions in CT135. Analysis of the 15 C. trachomatis reference strains by CEL I digestion showed that CT135 polymorphisms were not unique to serovar D. There was considerable CT135 diversity in most serovars, suggesting that similar disrupting mutations occur in these strains. The exception was the lack of CT135 polymorphisms in serovars K, L1, L2, and L3, the significance of which is unknown. Interestingly, sequencing of CT135 from clinical samples that had undergone minimal in vitro passages in cell culture showed no CT135 disruptions by either capillary sequencing or CEL I digestion. These findings suggest that most reference strains have lost their intact CT135, probably due to negative selection pressure, while there is an in vivo positive selection for intact CT135 in clinical isolates. A caveat, however, is that the clinical diagnosis associated with these clinical samples is not known. Therefore, future epidemiological genotyping studies of CT135 should minimally incorporate comparisons of symptomatic and asymptomatic subjects to more fully appreciate a potential role for CT135 in the pathogenesis of human infection.
CT135 encodes a hypothetical protein unique to the Chlamydiaceae
. All sequenced C. trachomatis
genomes and the C. muridarum
genome contain a CT135 ortholog located immediately downstream from CT134, a gene that also encodes a hypothetical protein with orthologs in other Chlamydiaceae
. The CT134 and CT135 ORFs are on the same DNA strand and are separated by only 58 bp in the C. trachomatis
) and C. muridarum
) genomes, suggesting that these genes might form a bicistronic operon with a related function (20
). Consistent with this theory, it was shown that in C. trachomatis
-infected human epithelial cells, both genes were coordinately upregulated at 3 h p.i. (3
); in contrast, the adjacent genes CT133 and CT136 exhibited upregulation at 1 and 8 h p.i., respectively. However, in Chlamydophila
species, the intergenic regions between CT134 and CT135 are larger than what would be predicted for a bicistronic operon. The conservation and organization of these genes, particularly among human C. trachomatis
isolates, suggest that they could be common virulence factors that play important roles in the pathogenesis of chlamydial infection and disease. In support of the importance of CT135 in chlamydial pathogenesis, others have reported similar frameshift mutations in this gene in strains differing in in vitro
or in vivo
pathogenic properties (6
). Unlike our findings, however, those studies identified mutations in multiple other genetic loci, making it impossible to associate a single gene with a pathogenic phenotype. Nevertheless, those studies collectively support our conclusion that CT135 is an important chlamydial virulence factor. Our CT135 sequence results are confounding, given that both the D-EC and D-LC strains contain mutations predicted to disrupt the ORF as initially annotated (26
). Given the distinct in vivo
phenotypic differences between the strains, it is likely that D-LC CT135 encodes a protein with enhanced virulence properties. Another possible explanation is that CT135 is an antivirulence factor and that it is functional in D-EC but not in D-LC (14
). However, the fact that all clinical samples examined in this study possessed intact CT135 makes the second possibility less likely.
A fundamental question is how does CT135 function in the pathogenesis of in vivo
chlamydial infection? The difference in virulence between the two strains was the ability of D-LC to sustain infections with greater organism burdens and infection durations and, importantly, to generate ascending infection of the upper reproductive tract. It is possible that a single metabolic, structural, or secreted protein could exhibit this phenotypic property, an argument supported by the critical role described for the C. trachomatis
tryptophan synthase gene in the pathogenesis of human genital but not ocular infections (5
). It is also possible that CT135 might be a regulatory gene that affects the expression of multiple genes in response to in vivo
environmental stimuli that are important to in vivo
growth or survival. Interestingly, transcriptional profiling studies have shown that the levels of expression of CT135 and its putative bicistronic partner, CT134, are increased 2.7-fold when C. trachomatis
is grown in IFN-γ-treated human epithelial cells (2
). Under this growth condition, C. trachomatis
exhibits differential gene expression that has been postulated to be part of a stress response stimulon. Our results did not show a difference in the susceptibilities of D-EC and D-LC strains to growth inhibition by IFN-γ in cultured mouse cells. However, the effects of IFN-γ in this in vitro
environment may not be sufficiently selective or hostile to differentiate the strains.
Our findings could have important implications for the understanding of human disease and the utilization of the murine model for studying human infection and immunity. CT135 genotyping of C. trachomatis-infected humans might be useful for identifying individuals at greater risk for complicated postinfection sequelae. This will require sequencing of CT135 from patients with asymptomatic, symptomatic, or complicated infections. Also, identifying additional C. trachomatis strains from other serovars causing STIs that demonstrate an enhanced murine virulence phenotype would significantly improve the mouse genital tract small animal model to study the pathophysiology and immunity of human urogenital infection.
In summary, we have identified a single genetic locus that significantly changes the in vivo pathogenicity of a human clinical isolate for the female mouse genital tract. Future studies will focus on determining whether CT135 plays a similar role in other C. trachomatis serovars for the murine genital tract and defining the role of CT135 in the pathogenicity of human infection and disease.