Infectivity Acts asIn VivoSelection for Maintenance of the Chlamydial Cryptic Plasmid

Abstract

Chlamydia trachomatis contains a conserved ϳ7.5-kb plasmid. Loss of the plasmid results in reduced glycogen accumulation, failure to activate TLR2, and reduced infectivity. We hypothesized that reduced infectivity functions as a means of selection for plasmid maintenance. We directly examined the biological significance of the reduced infectivity associated with plasmid deficiency by determining the relative fitness of plasmid-deficient CM972 versus that of wild-type C. muridarum Nigg in mixed inocula in vitro and in vivo. C. muridarum Nigg rapidly out-competed its plasmid-cured derivative CM972 in vitro but was not competitive with CM3.1, a derivative of CM972 that has reverted to a normal infectivity phenotype. C. muridarum Nigg also effectively competed with CM972 during lower and upper genital tract infection in the mouse, demonstrating that strong selective pressure for plasmid maintenance occurs during infection. The severity of oviduct inflammation and dilatation resulting from these mixed infections correlated directly with the amount of C. muridarum Nigg in the initial inoculum, confirming the role of the plasmid in virulence. Genetic characterization of CM972 and CM3.1 revealed no additional mutations (other than loss of the plasmid) to account for the reduced infectivity of CM972 and detected a single base substitution in TC_0236 in CM3.1 that may be responsible for its restored infectivity. These data demonstrate that a chlamydial strain that differs genetically from its wild-type parent only with respect to the lack of the chlamydial plasmid is unable to compete in vitro and in vivo, likely explaining the rarity of plasmid-deficient isolates in nature.

Plasmid deficiency in urogenital isolates ofChlamydia trachomatisreduces infectivity and virulence in a mouse model

“…glycogen accumulation) and in controlling the expression of certain genes on the Chlamydia chromosome. Overall, studies of infection with both wild‐type and plasmid‐free C. trachomatis and C. muridarum strains in mouse urogenital infection indicate that the plasmid modulates infectivity and may be essential for pathogenic immune responses (O'Connell et al ., 2007; Carlson et al ., 2008; Olivares‐Zavaleta et al ., 2010; Russell et al ., 2011). It is all but certain that either C. muridarum plasmid gene products or products of plasmid responsive loci on the bacterial chromosome engage Toll‐like receptor‐2 (TLR‐2) causing deleterious responses in mouse urogenital infections with C. muridarum (O'Connell et al ., 2011).…”

“…It is not possible to predict whether any of these SNPs contributed directly to the differences we have observed in the mouse model. However, our observations are quite consistent with the aforementioned in vivo observations of plasmid‐free strains in human disease (Bjartling et al ., 2009; Harris et al ., 2012) and in other animal models (O'Connell et al ., 2007; Carlson et al ., 2008; Olivares‐Zavaleta et al ., 2010; Russell et al ., 2011). Thus, we feel the most logical inference is to attribute the differences seen to presence and function of the plasmid.…”

“…Thus, one should be mindful of these host and pathogen differences when interpreting our data. Nonetheless, it is pleasing that our findings largely support and agree with those of others investigating the chlamydial plasmid in mouse urogenital tract infections with LGV strains either C. muridarum or C. trachomatis (O'Connell et al ., 2007; Carlson et al ., 2008; Olivares‐Zavaleta et al ., 2010; Russell et al ., 2011). In the context of what has already been reported by others regarding the chlamydial plasmid and in vivo fitness, our results add to the growing body of evidence that in many hosts and at specific anatomical sites, the plasmid plays a role in modulating infectivity and virulence, but this role is not universal.…”

Toll-Like Receptor 2 Activation by Chlamydia trachomatis Is Plasmid Dependent, and Plasmid-Responsive Chromosomal Loci Are Coordinately Regulated in Response to Glucose Limitation by C. trachomatis but Not by C. muridarum

“…This may reflect procedural differences in the in vitro assays performed, e.g., determination of plaquing efficiency versus plating efficiency (6). Most recently, we observed that coculture competition assays in vitro are more reflective of in vivo differences than differences in plaquing efficiency (35) and could facilitate further analysis of 25667R's infectivity. An additional confounding factor is the differences in cell attachment/entry associated with strains of the C. trachomatis LGV serogroup, including L2/434/Bu and 25667R (11), that might mask or override plasmid-associated infectivity differences.…”

“…Although we were unable to investigate the contribution of glycogen to infectivity in CTD153 because we did not recover spontaneous "revertants" of the infectivity defect, we were also unable to demonstrate a role for glycogen in infectivity in C. muridarum, because plasmid-deficient CM3.1, which displays wild-type infectivity in vitro, had a normal course of infection when inoculated intravaginally into mice and established infection in the oviducts at levels that were not significantly different from the parental Nigg strain (29) despite its inability to accumulate glycogen. Recently, genetic characterization of both C. muridarum strains CM972 and CM3.1 via comparative genome hybridization revealed no additional mutations other than loss of the plasmid to account for the phenotypic changes in CM972, and a single base substitution in TC0236 was detected in CM3.1 that may be responsible for restored infectivity (35).…”