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Chlamydia trachomatis (CT) is a sexually transmitted infection that can lead to adverse reproductive health outcomes. CT prevalence estimates are primarily derived from screening using nucleic acid amplification tests (NAATs). However, screening guidelines in the United States only include particular subpopulations, and NAATs only detect current infections. In contrast, seroassays identify past CT infections, which is important for understanding the public health impacts of CT, including pelvic inflammatory disease and tubal factor infertility. Older seroassays have been plagued by low sensitivity and specificity and have not been validated using a consistent reference measure, making it challenging to compare studies, define the epidemiology of CT, and determine the effectiveness of control programs. Newer seroassays have better performance characteristics. This narrative review summarizes the "state of the science" for CT seroassays that have been applied in epidemiologic studies and provides practical considerations for interpreting the literature and employing seroassays in future research. [ABSTRACT FROM AUTHOR]

We tested whether cystic fibrosis (CF) airway epithelia have larger innate immune responses than non-CF or cystic fibrosis transmembrane conductance regulator (CFTR)-corrected cells, perhaps resulting from ER stress due to retention of [DELTA]F508CFTR in the endoplasmic reticulum (ER) and activation of cytosolic [Ca.sup.2+] ([Ca.sub.i] and nuclear factor (NF)-[kappa]B signaling. Adenovirus infections of a human CF ([DELTA]F508/[DELTA]F508) nasal cell line (CF15) provided isogenic comparisons of wild-type (wt) CFTR and [DELTA]F508CFTR. In the absence of bacteria, there were no or only small differences among CF15, CF15-1acZ ([beta]-galactosidase-expressing), CF15-wtCFTR (wtCFTR-corrected), and CF15-[DELTA]F508CFTR (to test ER retention of [DELTA]F508CFTR) cells in NF-[kappa]B activity, interleukin (IL)-8 secretion, [Ca.sub.i] responses, and ER stress. Non-CF and CF primary cultures of human bronchial epithelial cells (HBE) secreted IL-8 equivalently. Upon infection with Pseudomonas aeruginosa (PA) or flagellin (key activator for airway epithelia), CF15, CF15lacZ, CF15-wtCFTR, and CF15[DELTA]F508CFTR cells exhibited equal PA binding, NF-[kappa]B activity, and IL-8 secretion; cells also responded similarly to flagellin when both CFTR (forskolin) and [Ca.sub.i] signaling (ATP) were activated. CF and non-CF HBE responded similarly to flagellin + ATP. Thapsigargin (Tg, releases ER [Ca.sup.2+]) increased flagellin-stimulated NF-[kappa]B and ER stress similarly in all cells. We conclude that ER stress, [Ca.sub.i], and NF-[kappa]B signaling and IL-8 secretion were unaffected by wt- or [DELTA]F508CFTR in control and during exposure to PA, flagellin, flagellin + ATP, or flagellin + ATP + forskolin. Tg, but not wt- or [DELTA]F508CFTR, triggered ER stress. Previous measurements showing hyperinflammatory responses in CF airway epithelia may have resulted from cell-specific, rather than CFTR- or [DELTA]F508CFTR-specific effects. nuclear factor-[kappa]B; interleukin-8; adenovirus; inflammation; endoplasmic reticulum stress; ire 1 [alpha]

Background: The lack of engineered C. trachomatis (CT) genetic mutant libraries continues to hamper our understanding of the underlying mechanisms of CT pathogenesis as well as the evaluation of live attenuated strains for vaccine potential. Here, we report the development of a targeted mutagenesis system for CT. This technological platform utilizes the lambda red homologous recombination system and enables the rapid construction of CT mutants with specific gene deletions. Methods: We designed a vector that encodes the lambda red genetic elements (gam, exo, and beta) under regulation of an inducible promoter, a selection cassette containing chloramphenicol acetyl transferase and GFP, homologous flanking sequences (~2 kb) for targeting the insert to chromosomal regions of interest, and flippase recognition target (FRT) sites for downstream marker removal. For the initial development of this system, the CT L2/434 strain was chosen as a target strain, and mutants were selected and propagated in McCoy cells under multiple rounds of chloramphenicol selection. Results: As a proof of principle, we used the system to delete the CT virulence factor IncA. We obtained incA deletion mutants within 2 rounds of CT growth, and the resulting clones (CTΔincA) were verified by PCR-based genotyping, immunofluorescence microscopy using a specific antibody to IncA, and functionally by phenotypic assessment of cells infected with CTΔincA. We next used the technology to target 4 additional genomic loci hypothesized to play important roles in CT pathogenesis and virulence: incDEFGA (5 genes), cdsZ, dub1dub2 (2 genes), and pmpE. Mutant CT strains were obtained and genetically confirmed for each of these targets. The ease of generating mutant clones for these 4 target loci highlights the versatility and efficiency of the lambda red system for CT, and we predict the system can be easily applied to other Chlamydia species, for example C. muridarum which is widely used for the mouse model of upper genital tract infection and immunological studies. Conclusion: The potential applications of this technology are vast and include the ability to study the virulence factors of Chlamydia, develop new drug targets, and explore vaccine candidates. [ABSTRACT FROM AUTHOR]

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial [Na.sup.+] channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ([R.sub.T]) are -5.9 mV and 829 [Omega] [multiplied by] [cm.sup.2], respectively. The apical membrane potential ([V.sub.A]) is -40.7 mV, and the mean ratio of apical to basolateral membrane resistance ([R.sub.A]/[R.sub.B]) is 2.8. Apical amiloride hyperpolarized [V.sub.A] by 19.7 mV and tripled [R.sub.A]/[R.sub.B]. A cAMP-elevating cocktail depolarized VA by 17.6 mV, decreased [R.sub.A]/[R.sub.B] by 60%, increased short-circuit current by 6 [micro]A/[cm.sup.2], decreased [R.sub.T] by 155 [Omega] [multiplied by] [cm.sup.2], and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited [Na.sup.+] currents [linear current-voltage (I-V) relation] and forskolin-stimulated [Cl.sup.-] currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2-4 [micro]l [multiplied by] [cm.sup.-2] [multiplied by] [h.sup.-1]). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by [Na.sup.+] transport via apical membrane ENaC, and fluid secretion is mediated, in part, by [Cl.sup.-] transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [[Na.sup.+]] and [[Cl.sup.-]]. amiloride; diphenylamine-2-carboxylate; milk secretion; patch clamp; microelectrodes; electrophysiology; cystic fibrosis; tight junctions; leaky and tight epithelia; epithelial sodium channel; cystic fibrosis transmembrane conductance regulator

Chlamydia secrete into host cells a diverse array of effector proteins, but progress in characterizing the spatiotemporal localization of these proteins has been hindered by a paucity of genetic approaches in Chlamydia and also by the challenge of studying these proteins within the live cellular environment. We adapted a split-green fluorescent protein (GFP) system for use in Chlamydia to label chlamydial effector proteins and track their localization in host cells under native environment. The efficacy of this system was demonstrated by detecting several known Chlamydia proteins including IncA, CT005 and CT694. We further used this approach to detect two chlamydial deubiquitinases (CT867 and CT868) within live cells during the infection. CT868 localized only to the inclusion membrane at early and late developmental stages. CT867 localized to the chlamydial inclusion membrane at an early developmental stage and was concomitantly localized to the host plasma membrane at a late stage during the infection. These data suggest that chlamydial deubiquitinase play important roles for chlamydial pathogenesis by targeting proteins at both the plasma membrane and the chlamydial inclusion membrane. The split-GFP technology was demonstrated to be a robust and efficient approach to identify the secretion and cellular localization of important chlamydial virulence factors. [ABSTRACT FROM AUTHOR]

Chlamydia are gram-negative obligate intracellular bacteria that replicate within a discrete cellular vacuole, called an inclusion. Although it is known that Chlamydia require essential nutrients from host cells to support their intracellular growth, the molecular mechanisms for acquiring these macromolecules remain uncharacterized. In the present study, it was found that the expression of mammalian cell glucose transporter proteins 1 (GLUT1) and glucose transporter proteins 3 (GLUT3) were up-regulated during chlamydial infection. Up-regulation was dependent on bacterial protein synthesis and Chlamydia-induced MAPK kinase activation. GLUT1, but not GLUT3, was observed in close proximity to the inclusion membrane throughout the chlamydial developmental cycle. The proximity of GLUT1 to the inclusion was dependent on a brefeldin A-sensitive pathway. Knockdown of GLUT1 and GLUT3 with specific siRNA significantly impaired chlamydial development and infectivity. It was discovered that the GLUT1 protein was stabilized during infection by inhibition of host-dependent ubiquitination of GLUT1, and this effect was associated with the chlamydial deubiquitinase effector protein CT868. This report demonstrates that Chlamydia exploits host-derived transporter proteins altering their expression, turnover and localization. Consequently, host cell transporter proteins are manipulated during infection as a transport system to fulfill the carbon source requirements for Chlamydia. [ABSTRACT FROM AUTHOR]

The precise strategies that intracellular pathogens use to exit host cells have a direct impact on their ability to disseminate within a host, transmit to new hosts, and engage or avoid immune responses. The obligate intracellular bacterium Chlamydia trachomatis exits the host cell by two distinct exit strategies, lysis and extrusion. The defining characteristics of extrusions, and advantages gained by Chlamydia within this unique double-membrane structure, are not well understood. Here, we define extrusions as being largely devoid of host organelles, comprised mostly of Chlamydia elementary bodies, and containing phosphatidylserine on the outer surface of the extrusion membrane. Extrusions also served as transient, intracellular-like niches for enhanced Chlamydia survival outside the host cell. In addition to enhanced extracellular survival, we report the key discovery that chlamydial extrusions are phagocytosed by primary bone marrow-derived macrophages, after which they provide a protective microenvironment for Chlamydia. Extrusion-derived Chlamydia staved off macrophage-based killing and culminated in the release of infectious elementary bodies from the macrophage. Based on these findings, we propose a model in which C. trachomatis extrusions serve as 'trojan horses' for bacteria, by exploiting macrophages as vehicles for dissemination, immune evasion, and potentially transmission. [ABSTRACT FROM AUTHOR]