Complete deletion of the Chlamydia muridarum putative cytotoxin locus reveals contributions during invasion in tissue culture and oviduct pathology during murine genital tract infection (Mouse data)

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Figure 5. C57Bl/6 female mice were infected intravaginally with 105 IFU of C. muridarum WT, Δtox, or toxRep. A) IFU enumerated from vaginal swabs collected at days 3-28 post infection. Data presented as means +/- standard deviation from five mice/group/time point. Statistical significance was assessed by two-way RM ANOVA followed by Tukey’s multiple comparisons test; p < 0.05 for WT vs. other strains at day 3 and 7. B) Ascension at day 7 post intravaginal infection as measured by qPCR on upper genital tract homogenates. Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple comparisons test and found to be not significant. C) Frequency of hydrosalpinx at 51 days post intravaginal infection, pooled from two independent experiments..Figure 6. C57Bl/6 female mice were infected transcervically with 106 IFU of C. muridarum WT, Δtox or toxRep. At three days (A) and seven days (B) post-infection, bacterial burden in the upper genital tract was quantified by qPCR. Data are plotted as mean +/- SEM. Data in B are pooled from two independent experiments. Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple comparisons test; ns: not significant. Figure 7. Seven days after transcervical infection of C57Bl/6 mice with 106 IFU of C. muridarum WT, Δtox or toxRep, female genital tracts were excised and histopathology was assessed on randomized, anonymized slides. A-C) Pathology scores and representative images of H&E stained oviduct (OD, A), ovary (OV, B), and uterus (UT, C). Figure 8. Fifty-one days after transcervical infection of C57Bl/6 mice with 106 IFU of C. muridarum WT, Δtox or toxRep, female genital tracts were assessed for gross pathology. A) Representative images of excised upper genital tracts; asterisk indicates hydrosalpinx. B) Frequency of hydrosalpinx pooled from two independent experiments. Statistical significance was assessed by Fisher’s exact test; **, P = 0.0078, *, P = 0.0209, ns: not significant. Figure 9. Fifty-one days after transcervical infection of C57Bl/6 mice with 106 IFU of C. muridarum WT, Δtox or toxRep, female genital tracts were excised and histopathology was assessed on randomized, anonymized slides. A) Oviduct (OD) pathology scores. B) Representative images of H&E-stained oviduct and ovary. C) Ovary (OV) pathology scores. D) Pathology scores and representative images of H&E-stained uterus (UT). Two images of uterus are shown from each infection condition to represent the within-group variability of horn dilation that was present after infection with all three bacterial strains. Table S2. WT, tox and cis-tox were subjected to whole genome sequencing and variant analysis was done using C. muridarum Nigg AE002160.2

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Mice and genital tract infections. Intravaginal and transcervical infections were performed with density gradient purified EBs. Intravaginal infections were performed by clearing the vaginal vault with a calcium alginate tipped swab then instilling 105 IFU of C. muridarum WT, Δtox, or toxRep into the lower genital tract. Transcervical infections were performed using a nonsurgical embryo transfer device (ParaTechs) to instill 106 IFU of C. muridarum WT, Δtox, or toxRep directly into the upper genital tract. Infected mice were monitored for the duration of the study. Quantification of bacterial burden in vivo. Vaginal swabs were collected at 3-28 days post infection. To measure upper genital tract bacterial burden, qPCR was performed on total DNA isolated from homogenized upper genital tracts using a DNeasy Blood and Tissue Kit (Qiagen). Chlamydia 16S DNA and mouse GAPDH DNA was amplified and quantitated on an AriaMX Real-Time PCR System (Agilent) using specific primer pairs and probes (IDT or Applied Biosystems). The ratio of bacterial/mouse DNA in homogenates was calculated using standard curves generated from known amounts of purified DNA.Assessment of upper genital tract pathology. Gross pathology phenotypes (e.g. hydrosalpinx, uterine horn dilation) were evaluated in situ upon necropsy. Excised tissues were fixed in 10% formalin and processed by the Rodent Histopathology Core Facility at Harvard Medical School where they were embedded in paraffin, sectioned to 4-5 μm, then stained with H&E prior to being assessed for histopathology. Each tissue was assigned a severity score based on the prevalence of pathology including fibrosis, edema, epithelial/membrane thickening and/or degeneration, luminal/tissue cellular infiltration, and increased vascularity. Left and right uterine horn, oviduct, and ovary were scored separately. Uterus pathology scores range from 0 (no pathology); 1 (mild/rare pathology, less than 1/3 of tissue affected); 2 (moderate/multifocal pathology, between 1/3–2/3 of tissue affected); to 3 (severe/coalescing pathology, greater than 2/3 of tissue affected). Oviduct and ovary scores ranged from 0 (no pathology), 1 (less than ½ the tissue affected), to 2 (more than ½ of the tissue affected.) For chronic infection when oviduct edema can be extreme (hydrosalpinx visible by eye), histopathology was scored using an expanded 0-4 scale in the manner of Chen et al., 2014 (14) ranging from 0 (no pathology); 1 (less than ½ of the tissue affected), 2 (more than ½ of the tissue affected, though dilation is smaller than the ovary on the same side), 3 (more than ½ of the tissue affected with dilation equal to the ovary size on the same side) and 4 (more than ½ of the tissue affected with confluent dilation larger than the ovary on the same side).

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