Addressing the Antibiotic Resistance Problem with Probiotics: Reducing the Risk of Its Double-Edged Sword Effect

Antibiotic resistance is a global public health problem that requires our attention. Indiscriminate antibiotic use is a major contributor in the introduction of selective pressures in our natural environments that have significantly contributed in the rapid emergence of antibiotic-resistant microbial strains. The use of probiotics in lieu of antibiotic therapy to address certain health conditions in both animals and humans may alleviate these antibiotic-mediated selective pressures. Probiotic use is defined as the actual application of live beneficial microbes to obtain a desired outcome by preventing diseased state or improving general health. Multiple studies have confirmed the beneficial effects of probiotic use in the health of both livestock and humans. As such, probiotics consumption is gaining popularity worldwide. However, concerns have been raised in the use of some probiotics strains that carry antibiotic resistance genes themselves, as they have the potential to pass the antibiotic resistance genes to pathogenic bacteria through horizontal gene transfer. Therefore, with the current public health concern on antibiotic resistance globally, in this review, we underscore the need to screen probiotic strains that are used in both livestock and human applications to assure their safety and mitigate their potential in significantly contributing to the spread of antibiotic resistance genes in our natural environments

Chlamydia trachomatis Immune Evasion via Downregulation of MHC Class I Surface Expression Involves Direct and Indirect Mechanisms

Genital C. trachomatis infections typically last for many months in women. This has been attributed to several strategies by which C. trachomatis evades immune detection, including well-described methods by which C. trachomatis decreases the cell surface expression of the antigen presenting molecules major histocompatibility complex (MHC) class I, MHC class II, and CD1d in infected genital epithelial cells. We have harnessed new methods that allow for separate evaluation of infected and uninfected cells within a mixed population of chlamydia-infected endocervical epithelial cells to demonstrate that MHC class I downregulation in the presence of C. trachomatis is mediated by direct and indirect (soluble) factors. Such indirect mechanisms may aid in priming surrounding cells for more rapid immune evasion upon pathogen entry and help promote unfettered spread of C. trachomatis genital infections

The major CD8 T cell effector memory subset in the normal and <it>Chlamydia trachomatis</it>-infected human endocervix is low in perforin

<p>Abstract</p> <p>Background</p> <p>The local tissue microenvironment plays an important role in the induction, homing, maintenance and development of effector functions of T cells. Thus, site-specific differences in phenotypes of mucosal and systemic T cell populations have been observed. <it>Chlamydia trachomatis</it> most commonly infects the endocervix in women, yet little is known about <it>Chlamydia</it>-specific effector T cell immunity at this unique mucosal site. Our previous flow-cytometry-based study of cervical-cytobrush retrieved cells indicated that CD8 T cells are significantly increased in the <it>C. trachomatis</it>-infected human endocervix. The cytolytic function of CD8 T cells is important in the protective immunity against many intracellular pathogens, and requires the cytolytic granule perforin to facilitate the entry of other molecules that mediate the lysis of target cells. Determination of perforin expression of the CD8 T cell population in the endocervix would therefore provide insights on the granule-mediated cytolytic potential of these cells at this site.</p> <p>Results</p> <p>Our histological data revealed that <it>C. trachomatis</it>-infected tissues have significantly higher numbers of CD3 and CD8 T cells compared to non-infected tissues (p<0.01), and that the majority of CD8⁺ cells do not express perforin <it>in situ</it>. A subsequent flow cytometric analysis of paired blood and endocervix-derived cells (n=16) revealed that while all the CD8 T cell subsets: naïve, effector memory (T_EM), central memory (T_CM) and terminally differentiated effector memory (T_EMRA) can be found in the blood, the endocervix is populated mainly by the T_EM CD8 T cell subset. Our data also showed that perforin expression in the T_EM population is significantly lower in the endocervix than in the blood of <it>C. trachomatis</it> positive women (n=15; p<0.0001), as well as in <it>C. trachomatis</it>-negative individuals (n=6; p<0.05). Interestingly, our <it>in vitro</it> co-culture study suggests that the exposure of HeLa 229 cervical epithelial cells to IFN gamma could potentially induce a decrease in perforin content in CD8 T_EM cells in the same microenvironment.</p> <p>Conclusions</p> <p>The low perforin content of CD8 T_EM cells in the endocervix, the local site of <it>C. trachomatis</it> infection in women, may reflect the unique immunological environment that balances immune protection against sexually transmitted infections and immune- tolerance to support conception.</p

The High-Risk Human Papillomavirus E6 Oncogene Exacerbates the Negative Effect of Tryptophan Starvation on the Development of <i>Chlamydia trachomatis</i>

<div><p><i>Chlamydia trachomatis</i> is an obligate intracellular pathogen that requires specific essential nutrients from the host cell, one of which is the amino acid tryptophan. In this context interferon gamma (IFNγ) is the major host protective cytokine against chlamydial infections because it induces the expression of the host enzyme, indoleamine 2,3-dioxygenase 1, that degrades tryptophan, thereby restricting bacterial replication. The mechanism by which IFNγ acts has been dissected <i>in vitro</i> using epithelial cell-lines such as HeLa, HEp-2, or the primary-like endocervical cell-line A2EN. All these cell-lines express the high-risk human papillomavirus oncogenes E6 & E7. While screening cell-lines to identify those suitable for <i>C</i>. <i>trachomatis</i> co-infections with other genital pathogens, we unexpectedly found that tryptophan starvation did not completely block chlamydial development in cell-lines that were HR-HPV negative, such as C33A and 293. Therefore, we tested the hypothesis that HR-HPV oncogenes modulate the effect of tryptophan starvation on chlamydial development by comparing chlamydial development in HeLa and C33A cell-lines that were both derived from cervical carcinomas. Our results indicate that during tryptophan depletion, unlike HeLa, C33A cells generate sufficient intracellular tryptophan via proteasomal activity to permit <i>C</i>. <i>trachomatis</i> replication. By generating stable derivatives of C33A that expressed HPV16 E6, E7 or E6 & E7, we found that E6 expression alone was sufficient to convert C33A cells to behave like HeLa during tryptophan starvation. The reduced tryptophan levels in HeLa cells have a biological consequence; akin to the previously described effect of IFNγ, tryptophan starvation protects <i>C</i>. <i>trachomatis</i> from clearance by doxycycline in HeLa but not C33A cells. Curiously, when compared to the known <i>Homo sapiens</i> proteome, the representation of tryptophan in the HR-HPV E6 & E6AP degradome is substantially lower, possibly providing a mechanism that underlies the lowered intracellular free tryptophan levels in E6-expressing cells during starvation.</p></div

Characteristics of the cell lines used in this study.

<p>Characteristics of the cell lines used in this study.</p

Intracellular free tryptophan concentrations in HeLa and C33A cells grown in Complete Media or after 20 Hours in Trp-Free Media.

<p>Intracellular free tryptophan concentrations in HeLa and C33A cells grown in Complete Media or after 20 Hours in Trp-Free Media.</p

Expression of the HPV16 E6 oncogene in C33A cells accentuates the effect of tryptophan starvation on <i>C</i>. <i>trachomatis</i> development.

<p>Stable C33A-derivative cell-lines were constructed by transducing C33A cells with a control retroviral vector (pLXSN), or pLXSN derivatives expressing the HPV16 E6, E6 & E7, or E7 oncogenes. Stable cell-lines were selected using G418 as described in the material and method section. A) Cell-lines were grown in tryptophan-free media for 12 hours after which immunoblots were used to query the phosphorylation status of eIF2α. Total eIF2α was used as a loading control. B) IFU/mL recovered at 42 h.p.i. after infected cells were grown in complete media or tryptophan free-media, as evaluated by infection of HeLa cells. As anticipated, growth in tryptophan-free media reduced IFU/mL recovered from all four cell-lines. IFU/mL recovery from C33A/E6 and C33A/E6+E7 cell-lines during tryptophan starvation were significantly lower than the IFU/mL recovered from the control C33A/pLXSN cell-line. The data represent three independent experiments (** indicates P < 0.01 by the Wilcoxon rank sum test).</p