The Type I Interferon Receptor Is Not Required for Protection in the Chlamydia Muridarum and HSV-2 Murine Super-Infection Model

Chlamydia trachomatis/HSV-2 vaginal co-infections are seen clinically, suggesting that these sexually transmitted pathogens may interact. We previously established an intravaginal Chlamydia muridarum/HSV-2 super-infection model and observed that chlamydial pre-infection protects mice from a subsequent lethal HSV-2 challenge. However, the mechanism of protection remains unknown. The type I interferon, IFN-β, binds to the type I interferon receptor (IFNR), elicits a host cellular antiviral response and inhibits HSV replication in vitro and in vivo. Previous studies have demonstrated that C. muridarum infection stimulates genital tract (GT) IFN-β production; therefore, we hypothesized that chlamydial pre-infection protects mice from HSV-2 challenge via the IFN-β/IFNR-induced antiviral response. To test this prediction, we quantified IFN-β levels in vaginal swab samples. Detection of IFN-β in C. muridarum singly infected, but not in mock-infected animals, prompted the use of the super-infection model in IFNR knockout (IFNR-/-) mice. We observed that C. muridarum pre-infection reduces HSV-2-induced mortality by 40% in wild-type mice and by 60% IFNR-/-mice. Severity of HSV-2 disease symptoms and viral shedding was also similarly reduced by C. muridarum pre-infection. These data indicate that, while chlamydial infection induces GT production of IFN-β, type I IFN-induced antiviral responses are likely not required for the observed protective effect

Host Nectin-1 Promotes Chlamydial Infection in the Female Mouse Genital Tract, But is Not Required for Infection in a Novel Male Murine Rectal Infection Model

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen, but more than 70% of patients fail to seek treatment due to the asymptomatic nature of these infections. Women suffer from numerous complications from chronic chlamydial infections, which include pelvic inflammatory disease and infertility. We previously demonstrated in culture that host cell nectin-1 knockdown significantly reduced chlamydial titers and inclusion size. Here, we sought to determine whether nectin-1 was required for chlamydial development in vivo by intravaginally infecting nectin-1-/- mice with Chlamydia muridarum and monitoring chlamydial shedding by chlamydial titer assay. We observed a significant reduction in chlamydial shedding in female nectin-1-/- mice compared to nectin-1+/+ control mice, an observation that was confirmed by PCR. Immunohistochemical staining in mouse cervical tissue confirmed that there are fewer chlamydial inclusions in Chlamydia-infected nectin-1-/- mice. Notably, anorectal chlamydial infections are becoming a substantial health burden, though little is known regarding the pathogenesis of these infections. We therefore established a novel male murine model of rectal chlamydial infection, which we used to determine whether nectin-1 is required for anorectal chlamydial infection in male mice. In contrast to the data from vaginal infection, no difference in rectal chlamydial shedding was observed when male nectin-1+/+ and nectin-1-/- mice were compared. Through the use of these two models, we have demonstrated that nectin-1 promotes chlamydial infection in the female genital tract but does not appear to contribute to rectal infection in male mice. These models could be used to further characterize tissue and sex related differences in chlamydial infection

Chlamydial Pre-Infection Protects From Subsequent Herpes Simplex Virus-2 Challenge in a Murine Vaginal Super-Infection Model

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chlamydia trachomatis and Herpes Simplex Virus-2 (HSV-2) genital tract co-infections have been reported in humans and studied in vitro but the clinical consequences are unknown. Limited epidemiologic evidence suggests that these co-infections could be more severe than single infections of either pathogen, but the host-pathogen interactions during co-infection remain uncharacterized. To determine whether disease progression and/or pathogen shedding differs between singly-infected and super-infected animals, we developed an in vivo super-infection model in which female BALB/c mice were vaginally infected with Chlamydia muridarum (Cm) followed later by HSV-2. Pre-infection with Chlamydia 3 or 9 days prior to HSV-2 super-infection conferred significant protection from HSV-2-induced neurologic disease and significantly reduced viral recovery compared to HSV-2 singlyinfected controls. Neither protection from mortality nor reduced viral recovery were observed when mice were i) super-infected with HSV-2 on day 27 post Cm; ii) infected with UV-irradiated Cm and super-infected with HSV-2; or iii) azithromycin-treated prior to HSV-2 super-infection. Therefore, protection from HSV-2-induced disease requires active infection with viable chlamydiae and is not observed after chlamydial shedding ceases, either naturally or due to antibiotic treatment. Thus, Chlamydia-induced protection is transient and requires the continued presence of chlamydiae or their components. These data demonstrate that chlamydial pre-infection can alter progression of subsequent HSV-2 infection, with implications for HSV-2 transmission from co-infected humans

Host Nectin-1 Promotes Chlamydial Infection in the Female Mouse Genital Tract, but Is Not Required for Infection in a Novel Male Murine Rectal Infection Model.

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen, but more than 70% of patients fail to seek treatment due to the asymptomatic nature of these infections. Women suffer from numerous complications from chronic chlamydial infections, which include pelvic inflammatory disease and infertility. We previously demonstrated in culture that host cell nectin-1 knockdown significantly reduced chlamydial titers and inclusion size. Here, we sought to determine whether nectin-1 was required for chlamydial development in vivo by intravaginally infecting nectin-1-/- mice with Chlamydia muridarum and monitoring chlamydial shedding by chlamydial titer assay. We observed a significant reduction in chlamydial shedding in female nectin-1-/- mice compared to nectin-1+/+ control mice, an observation that was confirmed by PCR. Immunohistochemical staining in mouse cervical tissue confirmed that there are fewer chlamydial inclusions in Chlamydia-infected nectin-1-/- mice. Notably, anorectal chlamydial infections are becoming a substantial health burden, though little is known regarding the pathogenesis of these infections. We therefore established a novel male murine model of rectal chlamydial infection, which we used to determine whether nectin-1 is required for anorectal chlamydial infection in male mice. In contrast to the data from vaginal infection, no difference in rectal chlamydial shedding was observed when male nectin-1+/+ and nectin-1-/- mice were compared. Through the use of these two models, we have demonstrated that nectin-1 promotes chlamydial infection in the female genital tract but does not appear to contribute to rectal infection in male mice. These models could be used to further characterize tissue and sex related differences in chlamydial infection

Flavanone 3-Hydroxylase Expression in Citrus Paradisi and Petunia Hybrida Seedlings

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression

Yeast knockout library allows for efficient testing of genomic mutations for cell-free protein synthesis

Cell-free protein synthesis (CFPS) systems from crude lysates have benefitted from modifications to their enzyme composition. For example, functionally deleting enzymes in the source strain that are deleterious to CFPS can improve protein synthesis yields. However, making such modifications can take substantial time. As a proof-of-concept to accelerate prototyping capabilities, we assessed the feasibility of using the yeast knockout collection to identify negative effectors in a Saccharomyces cerevisiae CFPS platform. We analyzed extracts made from six deletion strains that targeted the single deletion of potentially negative effectors (e.g., nucleases). We found a statistically significant increase in luciferase yields upon loss of function of GCN3, PEP4, PPT1, NGL3, and XRN1 with a maximum increase of over 6-fold as compared to the wild type. Our work has implications for yeast CFPS and for rapidly prototyping strains to enable cell-free synthetic biology applications

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

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

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