Protection elicited by chlamydial pre-infection lasts until at least day 9 pci.

<p>(A) Mice were super-infected with 10⁶ IFU <i>C</i>. <i>muridarum</i> on day 0 pci then with 5 x 10³ PFU HSV-2 on day 9 pci (Cm-9D-H) and swabbed every three days until day 27 pci. (B) Morbidity and mortality resulting from HSV-2 was monitored daily and the percent survival between experimental groups was compared using the log rank statistic. Significant (p<0.05) difference from Cm singly-infected control is indicated by an asterisk (*). The survival curve includes data combined from 3 separate experiments with n = 24 for each group. (C) Chlamydial shedding was determined by chlamydial titer assay and is reported as average IFU/mouse +/- SEM. (D) Average chlamydial shedding at day 9 pci (indicated by bars) and individual mouse chlamydial shedding (segregated according to survival status) are shown; n = 8 for both Cm (circles) and Cm-9D-H (triangles). (E) HSV-2 recovery was determined by plaque assay and is reported as average PFU/mouse +/- SEM. (F) Average HSV-2 recovery at day 12 pci (indicated by bars) and individual mouse HSV-2 recovery (segregated according to survival status) are shown; n = 8 for both HSV-2 (circles) and Cm-3D-H (triangles). Survivors and non-survivors are indicated by S and NS, respectively. The data in panels C-F are representative of 3 independent experiments. Differences in pathogen shedding/recovery between groups were determined with the paired Student’s t-test with p<0.05 considered significant, as indicated by an asterisk (*).</p

Protection from HSV-2-induced disease requires viable chlamydiae.

<p>(A) Mice were super-infected with 10⁶ UV-irradiated Cm (UVCm) then with 5 x 10³ PFU HSV-2 on day 3 pci (UVCm-3D-H). As controls, mice were singly-infected with UV-irradiated Cm (UVCm) on day 0 or with HSV-2 on day 3 pci (HSV-2). Vaginal swabbing was performed every 3 days until day 21 pci. (B) Morbidity and mortality resulting from HSV-2 was monitored daily and the percent survival between experimental groups was compared using the log rank statistic. Significant (p<0.05) difference from Cm singly-infected control is indicated by an asterisk (*). The survival curve depicts data combined from 3 separate experiments with n = 24 for UVCm and n = 36 for both the HSV-2 and UVCm-3D-H groups. Note that 100% survival was also observed in mock, Cm, and super-infected Cm-3D-H control groups that were included within this experiment (not shown). (C) Chlamydial shedding was determined by chlamydial titer assay and is reported as average IFU/mouse +/- SEM. (D) Average chlamydial shedding at day 3 pci (indicated by bars) and individual mouse chlamydial shedding (segregated according to survival status) are shown; n = 8 for Cm (circles), and n = 12 for both Cm-3D-H (diamonds) and UVCm-3D-H (triangles). (E) HSV-2 recovery was determined by plaque assay and is reported as average PFU/mouse +/- SEM. (F) Average HSV-2 recovery at day 6 pci (indicated by bars) and individual mouse HSV-2 recovery (segregated according to survival status) are shown; n = 8 for HSV-2 (circles) and n = 12 for UVCm-3D-H (triangles). Survivors and non-survivors are indicated by S and NS, respectively. Differences in pathogen shedding/recovery between groups in panels C-F were determined with the paired Student’s t-test with p<0.05 considered significant and are representative of 3 independent experiments.</p

Chlamydial pre-infection protects from challenge with a 100% lethal dose of HSV-2.

<p>(A) Female BALB/c mice were singly-infected with either 10⁵ PFU HSV-2 (H5) or with 10⁶ IFU <i>C</i>. <i>muridarum</i> (Cm). Super-infected mice were infected with Cm on day 0 pci and the high inoculum of HSV-2 on day 3 pci (Cm-3D-H5). Morbidity and mortality resulting from HSV-2 was monitored daily and the percent survival between experimental groups was compared using the log rank statistic. Significant (p<0.05) difference between HSV-2 singly-infected controls and super-infected group is indicated by an asterisk (*). The survival curve depicts data combined from 2 separate experiments with n = 16 for Cm and n = 20 for both the H5 and Cm-3D-H5 groups. (B) Chlamydial shedding was determined by chlamydial titer assay and is reported as average IFU/mouse +/- SEM. (C) Average chlamydial shedding at day 3 pci (indicated by bars) and individual mouse chlamydial shedding (segregated according to survival status) are shown; n = 8 for Cm (circles) and n = 12 for Cm-3D-H (triangles). (D) HSV-2 recovery was determined by plaque assay and is reported as average PFU/mouse +/- SEM. (E) Average HSV-2 recovery at day 6 pci (indicated by bars) and individual mouse HSV-2 recovery (segregated according to survival status) are shown; n = 12 for both HSV-2 (circles) and Cm-3D-H (triangles). (C and E) Survivor and non-survivor mice are indicated by S and NS, respectively. The data in panels C-F are representative of 2 independent experiments. Differences in pathogen shedding/recovery between groups were determined with the paired Student’s t-test with p<0.05 considered significant, as indicated by an asterisk (*).</p

Protection elicited by chlamydial pre-infection is lost by day 27 pci.

<p>(A) Mice were super-infected with 10⁶ IFU <i>C</i>. <i>muridarum</i> on day 0 pci then with 5 x 10³ HSV-2 on day 27 pci (Cm-27D-H) and swabbed every 3 days until day 45 pci. (B) Morbidity and mortality resulting from HSV-2 was monitored daily and the percent survival between experimental groups was compared using the log rank statistic. Significant (p<0.05) difference from HSV-2 singly-infected control is indicated by an asterisk (*).The survival curve contains data combined from 2 separate experiments with n = 16 for each group. (C) Chlamydial shedding was determined by chlamydial titer assay and is reported as average IFU/mouse +/- SEM. (D) Average chlamydial shedding at day 27 pci (indicated by bars) and individual mouse chlamydial shedding (segregated according to survival status) are shown; n = 8 for both Cm (circles) and Cm-9D-H (triangles). (E) HSV-2 recovery was determined by plaque assay and is reported as average PFU/mouse +/- SEM. (F) Average HSV-2 recovery at day 30 pci (indicated by bars) and individual mouse HSV-2 shedding (segregated according to survival status) are shown; n = 8 for both HSV-2 (circles) and Cm-3D-H (triangles). Survivors and non-survivors are indicated by S and NS, respectively. The data in panels C-F are representative of 2 independent experiments. Differences in pathogen shedding/recovery between groups were determined with the paired Student’s t-test with p<0.05 considered significant, as indicated by an asterisk (*).</p

Active chlamydial infection is required to protect from HSV-2-induced disease.

<p>(A) Mice were infected with 10⁶ IFU <i>C</i>. <i>muridarum</i> on day 0 pci and then treated with 200 mg/kg azithromycin (Az) via oral gavage on day 6 pci to cure the chlamydial infection. On day 9 pci, the mice were super-infected with 5 x 10³ PFU HSV-2. As a control, mice were treated with Az on day 6 pci then singly-infected with HSV-2 on day 9 pci. Pathogen shedding was determined from swab samples performed every 3 days until day 27 pci. (B) Morbidity and mortality resulting from HSV-2 was monitored daily and the percent survival between experimental groups was compared using the log rank statistic. Significant (p<0.05) difference from Cm singly-infected control is indicated by an asterisk (*). The survival curve depicts data combined from 2 separate experiments with n = 16 for each group. (C) Chlamydial shedding was determined by chlamydial titer assay and is reported as average IFU/mouse +/- SEM. (D) Average chlamydial shedding at day 9 pci (indicated by bars) and individual mouse chlamydial shedding (segregated according to survival status) are shown; n = 8 for Cm (circles), Cm-9D-H (diamonds) and Cm-Az-9D-H (triangles). (E) HSV-2 recovery was determined by plaque assay and is reported as average PFU/mouse +/- SEM. (F) Average HSV-2 recovery at day 12 pci (indicated by bars) and individual mouse HSV-2 shedding (segregated according to survival status) are shown; n = 8 for both Az-9D-H (circles) and Cm-Az-9D-H (triangles). Survivors and non-survivors are indicated by S and NS, respectively. Differences in pathogen shedding/recovery between groups in panels C-F were determined with the paired Student’s t-test with p<0.05 considered significant and are representative of 2 independent experiments.</p

Nectin-1 is not required for male mouse rectal chlamydial infection.

<p>Male mice were rectally infected with 1x10⁶ IFU <i>C</i>. <i>muridarum</i> as described in the methods. A) Swab samples every 3 days from day 3 to 24 pi were used in chlamydial titer assays to determine chlamydial shedding. N = 16 and n = 18 for nectin-1^+/+ and nectin-1^-/- groups, respectively. Shedding is reported as the average IFU/mouse +/- SEM at each day pi. Combined data from two independent experiments are shown. B) Immunohistochemical staining of male wild type colon tissue at 430x (left panels) and 630x (right panels). Yellow arrows indicate chlamydial inclusions. Colon tissue was harvested from <i>C</i>. <i>muridarum</i> rectally infected mice at day 24 pi; n = 3. Two representative inclusions are depicted from one wild type mouse. Data represent a single experiment.</p

Nectin-1^-/- female mice have fewer detectable chlamydial inclusions in cervical tissue.

<p>A) Immunohistochemical staining of nectin-1^+/+ (upper panels) and nectin-1^-/- (lower panels) <i>C</i>. <i>muridarum</i> infected cervical tissue harvested day 6 pi; n = 5 for each group. Yellow arrows indicate chlamydial inclusions. All mouse cervical samples were stained and cervical samples shown are from two individual mice per experimental group. Data are representative of two independent experiments.</p

Host nectin-1 is required for chlamydial shedding in intravaginally infected mice.

<p>A) Example of genotypic characterization of nectin-1 heterozygous mice (lanes 1 and 2), nectin-1^+/+ mice (lanes 3 and 4), and nectin-1^-/- mice (lanes 5 and 6). Molecular size ladders are represented by lanes labeled “L”. Nectin-1^+/+ mice exhibit a single band at 639bp, nectin-1^-/- mice exhibit a single band at 459bp and heterozygotes exhibit bands at 639bp and 459bp. B and C) Mice were infected with either 1 x 10³ IFU (B) or 1 x 10⁶ IFU <i>C</i>. <i>muridarum</i> (C) on day 0. Swab samples from days 3 through 21 pi were used in chlamydial titer assays to determine chlamydial shedding. For panel B, n = 10 for the nectin-1^+/+ group and n = 13 for the nectin-1^-/- group. For panel C, n = 19 per group. Shedding data are reported as the average IFU/mouse +/- SEM at each day pi. Shedding data are depicted as the combined data from 2 independent experiments each. Differences in shedding between groups at each day post shedding were determined with the unpaired Student’s t-test with p<0.05 considered significant, as indicated by an asterisk (*).</p

Nectin-1^-/- female mice have fewer detectable chlamydiae in the lower genital tract.

<p>A) Day 3 pi PCR semi-quantification of chlamydial genomes using 16s DNA normalized to host β-actin. Each group n = 4 and data are representative of 2 independent experiments. B) Day 3 pi PCR semi-quantification of chlamydial viability determined by amplification of chlamydial 16s rRNA normalized to chlamydial 16s DNA and host β-actin. A single data set was analyzed and n = 4 per group. Panels A and B are reported as average integrated intensity +/-SEM. Differences between groups were determined with the unpaired Student’s t-test with p<0.05 considered significant, as indicated by an asterisk (*). Non-significant comparisons are designated NS. C) Representative gel electrophoresis of chlamydial 16s DNA, chlamydial pre-16s RNA, and host β-actin PCR bands from one nectin-1^+/+ and one nectin-1^-/- female mouse.</p