Isolation and Characterization of Conidial Separation Mutants of \u3cem\u3eNeurospora crassa\u3c/em\u3e

Following ultraviolet irradiation, nine conidial separation mutants of N. crassa, wild type strain 74A, were isolated from 1481 colonies. Four of these mutants and one mutant previously isolated in this laboratory were analyzed genetically. All five mutants showed 1:1 segregation of the conidial separation trait in progeny of crosses to wild type 74a. Three of the mutants studied also carried the trait for osmotic sensitivity. This trait showed 1:1 segregation in two of the mutants, but not in the third. One of the stable mutants investigated in this study was osmotic remedial for conidial separation. Results of complementation tests indicate that these mutants not only represent separate loci for conidial separation, but are also new loci for conidial separation. A reversion experiment was carried out using an auxotrophic strain of the mutant SS-931, which had been previously isolated in this laboratory. No revertants were recovered from 42.07 x 106 viable conidia, indicating that the reversion rate for this mutation is less than 1 / 42.07 x 106. The mutants under investigation were also studied with light and scanning electron microscopy, and an investigation into the sensitivity of conidia of the various strains to exposure to elevated temperatures was conducted. The exposure of conidia to elevated temperatures demonstrated that the most sensitive of the strains investigated were the known osmotic sensitive mutants, and an osmotic sensitive conidial separation mutant isolated in this study

Modeling the transcriptome of genital tract epithelial cells and macrophages in healthy mucosa versus mucosa inflamed by Chlamydia muridarum infection

Chlamydia trachomatis urogenital serovars are intracellular bacteria that parasitize human reproductive tract epithelium. As the principal cell type supporting bacterial replication, epithelial cells are central to Chlamydia immunobiology initially as sentries and innate defenders, and subsequently as collaborators in adaptive immunity-mediated bacterial clearance. In asymptomatic individuals who do not seek medical care a decisive struggle between C. trachomatis and host defenses occurs at the epithelial interface. For this study, we modeled the immunobiology of epithelial cells and macrophages lining healthy genital mucosa and inflamed/infected mucosa during the transition from innate to adaptive immunity. Upper reproductive tract epithelial cell line responses were compared to bone marrow-derived macrophages utilizing gene expression microarray technology. Those comparisons showed minor differences in the intrinsic innate defenses of macrophages and epithelial cells. Major lineage-specific differences in immunobiology relate to epithelial collaboration with adaptive immunity including an epithelial requirement for inflammatory cytokines to express MHC class II molecules, and a paucity and imbalance between costimulatory and coinhibitory ligands on epithelial cells that potentially limits sterilizing immunity (replication termination) to Chlamydia-specific T cells activated with limited or unconventional second signals

Perforin is detrimental to controlling [corrected] C. muridarum replication in vitro, but not in vivo.

CD4 T cells are critical for clearing experimental Chlamydia muridarum genital tract infections. Two independent in vitro CD4 T cell mechanisms have been identified for terminating Chlamydia replication in epithelial cells. One mechanism, requiring IFN-γ and T cell-epithelial cell contact, terminates infection by triggering epithelial production of nitric oxide to chlamydiacidal levels; the second is dependent on T cell degranulation. We recently demonstrated that there are two independent in vivo clearance mechanisms singly sufficient for clearing genital tract infections within six weeks; one dependent on iNOS, the other on Plac8. Redundant genital tract clearance mechanisms bring into question negative results in single-gene knockout mice. Two groups have shown that perforin-knockout mice were not compromised in their ability to clear C. muridarum genital tract infections. Because cell lysis would be detrimental to epithelial nitric oxide production we hypothesized that perforin was not critical for iNOS-dependent clearance, but posited that perforin could play a role in Plac8-dependent clearance. We tested whether the Plac8-dependent clearance was perforin-dependent by pharmacologically inhibiting iNOS in perforin-knockout mice. In vitro we found that perforin was detrimental to iNOS-dependent CD4 T cell termination of Chlamydia replication in epithelial cells. In vivo, unexpectedly, clearance in perforin knockout mice was delayed to the end of week 7 regardless of iNOS status. The discordant in vitro/in vivo results suggest that the perforin's contribution to bacterial clearance in vivo is not though enhancing CD4 T cell termination of Chlamydia replication in epithelial cells, but likely via a mechanism independent of T cell-epithelial cell interactions

Chlamydia muridarum-Specific CD4 T-Cell Clones Recognize Infected Reproductive Tract Epithelial Cells in an Interferon-Dependent Fashion▿

During natural infections Chlamydia trachomatis urogenital serovars replicate predominantly in the epithelial cells lining the reproductive tract. This tissue tropism poses a unique challenge to host cellar immunity and future vaccine development. In the experimental mouse model, CD4 T cells are necessary and sufficient to clear Chlamydia muridarum genital tract infections. This implies that resolution of genital tract infection depends on CD4 T-cell interactions with infected epithelial cells. However, no laboratory has shown that Chlamydia-specific CD4 T cells can recognize Chlamydia antigens presented by major histocompatibility complex class II (MHC-I) molecules on epithelial cells. In this report we show that MHC-II-restricted Chlamydia-specific CD4 T-cell clones recognize infected upper reproductive tract epithelial cells as early as 12 h postinfection. The timing of recognition and degree of T-cell activation are dependent on the interferon (IFN) milieu. Beta IFN (IFN-β) and IFN-γ have different effects on T-cell activation, with IFN-β blunting IFN-γ-induced upregulation of epithelial cell surface MHC-II and T-cell activation. Individual CD4 T-cell clones differed in their degrees of dependence on IFN-γ-regulated MHC-II for controlling Chlamydia replication in epithelial cells in vitro. We discuss our data as they relate to published studies with IFN knockout mice, proposing a straightforward interpretation of the existing literature based on CD4 T-cell interactions with the infected reproductive tract epithelium

Cytokine profile of immune splenocytes from each experimental group infigure 1 (20 independent splenocyte populations) activated with UV-inactivated <i>C. muridarum.</i>

<p>3×10⁶ splenocytes were activated with 7.5×10⁶ UV-inactivated <i>C. muridarum</i> (multiplicity of 2.5 IFU per splenocyte) or mock-activated with equivalent volume of SPG buffer (con = control) in triplicate wells. Supernatants were collected at 72 h and analyzed by ELISA. Data representing each mouse strain/treatment condition were aggregated and compared to the aggregated data for the untreated C57BL/6J mice. Comparisons were made for each cytokine. There were no statistically significant differences in cytokine polarization between wild type and perforin knockout mice with or without MLA treatment.</p

Perforin-deficient CD4 T cells are superior to wild type CD4 T cells for terminating <i>C.</i> muridarum replication in epithelial cells.

<p>Top panels show IFU recovered per well, with means for the experimental wells shown in parentheses below their respective bars. The bottom panels show the T cell-mediated fold-reduction in IFU (calculated as IFU recovered from the “no T cell” control wells/IFU recovered in the experimental wells). A) Untreated C57epi.1 monolayers. B) C57epi.1 monolayers treated with 10 ηg recombinant murine IFN-γ for 10 h prior to infection. C) C57epi.1 monolayers treated with 10 ηg recobinant murine IFN-γ and 1 mM MLA for 10 h prior to infection. All monolayers were infected with <i>C. muridarum</i> at 3 IFU per cell. Inocula were removed 4 h later and infected epithelial cells were co-cultured without (no T cells) and with 2.5×10⁵ T cells in the absence and presence of MLA as indictated. 32 h post infection supernatants were collected and the wells harvested; <i>C. muridarum</i> was quantified on McCoy monolayers. In a single experiment, each experimental condition (no treatment, IFN-γ pretreatment, IFN-γ-MLA pretreatment) was done as triplicates for each of the 10 T cell lines and the “no T cell” control. Comparisons were made between aggregated data for the 5 wild type T cell lines (C57) and the 5 perforin knockout T cell lines (PrfKo) for each experimental condition. * = <i>p value</i> <0.05; *** = <i>p value</i> <0.0005; <i>NS</i> = not statistically significant.</p

Perforin-deficient CD4 T cells have a lesser cytopathic effect on infected C57 epithelial monolayers.

<p>A) Uninfected C57epi.1 monolayer at 32 h post mock infection (overgrown confluent monolayer). B) C57epi.1 monolayer 32 h post infection with <i>C. muridarum</i> at 3 IFU per cell. C) Infected C57epi.1 monolayer co-cultured with wild type polyclonal CD4 T cells at an effector to target ration of 1.25∶1 at 32 h post infection. D) Infected C57epi.1 monolayer co-cultured with perforin-deficient polyclonal CD4 T cells at an effector to target ration of 1.25∶1 at 32 h post infection. Representative DIC images at 200x magnification.</p