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. 2015 May;83(5):1881-92.
doi: 10.1128/IAI.03158-14. Epub 2015 Feb 23.

In vitro passage selects for Chlamydia muridarum with enhanced infectivity in cultured cells but attenuated pathogenicity in mouse upper genital tract

Chaoqun Chen  1 , Zhou Zhou  1 , Turner Conrad  2 , Zhangsheng Yang  2 , Jin Dai  2 , Zhongyu Li  3 , Yimou Wu  4 , Guangming Zhong  5
Affiliations

In vitro passage selects for Chlamydia muridarum with enhanced infectivity in cultured cells but attenuated pathogenicity in mouse upper genital tract

Chaoqun Chen et al. Infect Immun. 2015 May.

Abstract

Although modern Chlamydia muridarum has been passaged for decades, there are no reports on the consequences of serial passage with strong selection pressure on its fitness. In order to explore the potential for Pasteurian selection to induce genomic and phenotypic perturbations to C. muridarum, a starter population was passaged in cultured cells for 28 generations without standard infection assistance. The resultant population, designated CMG28, displays markedly reduced in vitro dependence on centrifugation for infection and low incidence and severity of upper genital tract pathology following intravaginal inoculation into mice compared to the parental C. muridarum population, CMG0. Deep sequencing of CMG0 and CMG28 revealed novel protein variants in the hypothetical genes TC0237 (Q117E) and TC0668 (G322R). In vitro attachment assays of isogenic plaque clone pairs with mutations in either TC0237 and TC0668 or only TC0237 reveal that TC0237(Q117E) is solely responsible for enhanced adherence to host cells. Paradoxically, double mutants, but not TC0237(Q117E) single mutants, display severely attenuated in vivo pathogenicity. These findings implicate TC0237 and TC0668 as novel genetic factors involved in chlamydial attachment and pathogenicity, respectively, and show that serial passage under selection pressure remains an effective tool for studying Chlamydia pathogenicity.

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Figures

FIG 1
FIG 1
Attachment of CMG0 and CMG28 populations and plaque-purified clones to cultured HeLa cells. The same numbers of CMG0 and CMG28 organisms, as listed along the x axis, were inoculated onto HeLa cell monolayers under unassisted-infection conditions. The infected cells were washed and processed for immunofluorescence detection of the remaining chlamydial organisms 1 h after incubation at 4°C (A) or 6 h (a) or 22 h (b) after incubation at 37°C (B). The cell-associated chlamydial organisms or inclusions were counted and expressed as the number of chlamydial organisms or inclusions per cell, as shown along the y axis. The 37°C incubation conditions were applied only to the CMG0 and CMG28 population cultures (B). All experiments were repeated three times. **, P < 0.01 (Kruskal-Wallis test).
FIG 2
FIG 2
Induction of hydrosalpinx in mice by CMG0 and CMG28 populations and plaque-purified clones. C3H/HeJ mice were intravaginally infected with 2 × 105 IFU of the 8 C. muridarum organisms, including the CMG0 (a) and CMG28 (b) population pair (n = 13/group) and three plaque-purified isogenic clone pairs CMG0.1.1 (n = 8) (c) and CMG28.38.1 (n = 8) (d), CMG0.21.3 (n = 8) (e) and CMG28.12.3 (n = 5) (f), and CMG0.10.1 (n = 5) (g) and CMG28.54.1 (n = 5) (h). The mice were sacrificed 60 days after infection for observation of hydrosalpinx. One representative image of whole genital tract from each group of mice is presented in the left columns, with vagina on the left and oviduct/ovary on the right. The areas covering the oviduct/ovary portions were magnified and are shown on the right side of the corresponding images of whole genital tracts. Hydrosalpinges are indicated by white arrows, and hydrosalpinx severity scores are indicated by white numbers. The hydrosalpinx incidence rates and severity scores are listed below the corresponding images. Mice with hydrosalpinx in either or both oviducts were considered positive for hydrosalpinx. The severities of both hydrosalpinges from a given mouse were scored separately and added together for the severity score assigned to that particular mouse. Fisher's exact test was used for comparing incidence rates (&, P < 0.05; &&, P < 0.01) and a Wilcoxon rank sum test was used for comparing severity scores (*, P < 0.05; **, P < 0.01) between the CMG0 and CMG28 organisms. Note that all CMG28 organisms induced more significant hydrosalpinges than did their CMG0 counterparts, with the exception of the CMG0.1.1 and CMG28.38.1 pair.
FIG 3
FIG 3
Microscopic observation of oviduct dilation induced by CMG0 and CMG28 populations and plaque-purified clones. The oviduct tissues of mice infected with the four pairs of CMG0 and CMG28 organisms were harvested and subjected to H&E staining for microscopic evaluation of oviduct dilation. (A) Representative images for each group were taken under a 4× objective lens from mice infected with CMG0 (a) and CMG28 (b) populations and from the three pairs of plaque-purified clone-infected mice (c to h), as indicated at the top of each image. Uterine horn (UH), normal oviduct, and ovary tissues are marked. Dilated oviducts are indicated with white lines with arrowheads at both ends. Bar, 0.5 mm. (B) Severity of lumenal dilation was scored as described in Materials and Methods and is listed along the y axis. The four pairs of C. muridarum organisms are listed along the x axis. Note that all CMG28 organisms induced more significant oviduct lumenal dilation than did their CMG0 counterparts, with the exception of the CMG0.1.1 and CMG28.38.1 pair. *, P < 0.05; **, P < 0.01 (Wilcoxon rank sum test).
FIG 4
FIG 4
Shedding of live chlamydial organisms from mouse lower genital tract following infection with CMG0 and CMG28 populations and plaque-purified clones. C3H/HeJ mice were intravaginally infected with four pairs of C. muridarum CMG0 and CMG28 organisms, as described in the legend of Fig. 2 and as indicated above the corresponding plots. On different days after infection, as shown along the x axis, vaginal swabs were taken for titration of live organisms on HeLa cell monolayers. The live organisms recovered from each swab are expressed as log10 IFU along the y axis in panels a, c, e, and g. The percentage of mice remaining positive for shedding of live organisms at each time point is plotted along the y axis in panels b, d, f, and h. Note that there is no significant difference in live-organism shedding from the lower genital tracts of mice infected with CMG0 and those infected with CMG28 organisms.
FIG 5
FIG 5
Recovery of live chlamydial organisms from mouse genital tract tissues following infection with CMG0 and CMG28 populations. C3H/HeJ mice intravaginally infected with CMG0 (n = 5) or CMG28 (n = 5) population organisms were sacrificed on day 14 after infection. Vaginal swabs were taken prior to mouse sacrifice. The entire genital tract tissue was harvested from each mouse and divided into lower genital tract (LGT) vagina/cervix (VC) and upper genital tract (UGT) uterus/uterine horn (UH) and oviduct/ovary (OV) sections, as listed along the x axis. Each tissue section was homogenized for titration of live C. muridarum organisms. Vaginal swabs were similarly titrated for live organisms. Log10 IFU values were used to calculate means and standard deviations for each group, as displayed along the y axis. Note that the numbers of live organisms recovered from mice infected with CMG0 and those infected with CMG28 were similar, without any significant difference (Kruskal-Wallis test).
FIG 6
FIG 6
Oviduct inflammatory infiltration induced by CMG0 and CMG28 populations and plaque-purified clones. Oviduct tissues from mice infected with the CMG0 and CMG28 populations and plaque-purified clones, as described in the legend of Fig. 2, were subjected to H&E staining, as described in the legend of Fig. 3, for evaluation of inflammatory histopathology. (A) Representative images from each group taken under 10× (left) and 100× (right) objective lenses. White rectangles in the 10× objective lens images indicate the same areas from which the right images were taken under a 100× objective lens. (B) The severity of inflammatory infiltration in oviduct tissue was scored as described in Materials and Methods and is listed along the y axis. The four pairs of C. muridarum organisms are listed along the x axis. Note that all CMG28 organisms induced more significant oviduct inflammatory infiltration than that induced by their CMG0 counterparts, with the exception of the CMG0.1.1 and CMG28.38.1 pair. *, P < 0.05; **, P < 0.01 (Wilcoxon rank sum test).
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