Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

Chlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells

ENERGETIC COSTS AND STRATEGIES OF POST‐JUVENAL MOLT IN AN EQUATORIAL BIRD, THE RUFOUS‐COLLARED SPARROW (ZONOTRICHIA CAPENSIS)

Abstract  ∙ Many tropical birds have slow‐paced life history strategies, exhibiting lower metabolic rates, reduced annual investment in reproduction, and longer lifespans relative to birds at higher latitudes. Life history strategies have been relatively well documented in adult individuals in the tropics, but we know comparatively little about the immature life history stage. Here we examine strategies of feather replacement (molt) and fattening in immature Rufous‐collared Sparrows (Zonotrichia capensis) in a high elevation equatorial population, following a parallel, previous study on an arctic congener, the White‐crowned Sparrow (Zonotrichia leucophrys gambelii). In captivity, Rufous‐ collared Sparrows incurred energetic costs of experimentally induced feather growth, similar to those previously described for Zonotrichia at higher latitudes. In contrast, free‐ranging immature Rufous‐collared Sparrows in natural molt had fat stores that declined over time, opposite to patterns evident in arctic Zonotrichia that fatten before migration. Equatorial birds in good condition molted more heavily (controlling for fat stores), suggesting that body condition limits the intensity of molt. Heavily molting equatorial sparrows also had lower amounts of fat (controlling for body condition), suggesting a trade‐off between allocation of resources to fat stores versus feather growth. Molt progressed slowly in Rufous‐collared Sparrows relative to previously described patterns in their arctic congener, which is concordant with a slower pace‐of‐life syndrome in tropical, as compared with high latitude, birds. Resumen ∙ Costos energéticos y estrategias de muda post‐juvenil en un ave ecuatorial, el Chingolo (Zonotrichia capensis) Muchas especies de aves tropicales presentan historias de vida lenta, exhibiendo bajas tasas metabólicas, esfuerzo reproductivo anual reducido, y mayor longevidad que las especies que habitan en latitudes más altas. La variación en historia de vida en especies tropicales ha sido bien documentada para individuos adultos, pero sabemos comparativa‐ mente poco de los individuos inmaduros. Aquí estudiamos las estrategias de muda y deposición de grasa en individuos inmaduros de Chingolo (Zonotrichia capensis) en una población ecuatorial de altura y comparamos los resultados con un estudio similar realizado en una población ártica del congénere Chingolo Coroniblanco (Zonotrichia leucophrys gambelii). En cautiverio, individuos juveniles de Z. capensis incurrieron costos energéticos debidos a la muda inducida, similar lo encontrado en Z. leucophrys gambelii. En contraste, Z. capensis inmaduros mudando en libertad presentaron depósitos de grasa que disminuyeron a lo largo del tiempo, lo opuesto a Z. l. gambelii, que deposita más grasa antes de migrar. Z. capensis inmaduros en buen estado nutricional (controlando por diferencias en deposición de grasa) mudaron de manera más intensa, lo que sugiere que el estado nutricional limita la muda. Individuos inmaduros de Z. capensis mudando de manera intensa presentaron menor cantidad de grasa depositada (controlando por diferencias en estado nutricional), lo que sugiere un balance entre la deposición de grasa y la muda. La muda fue más lenta en la población ecuatorial de Z. capensis comparado con la de Z. l. gambelii, lo que concuerda con lo esperado debido a la historia de vida más lenta en aves tropicales

VE-cadherin and claudin-5: it takes two to tango

Endothelial barrier function requires the adhesive activity of VE-cadherin and claudin-5, which are key components of adherens and tight endothelial junctions, respectively. Emerging evidence suggests that VE-cadherin controls claudin-5 expression by preventing the nuclear accumulation of FoxO1 and -catenin, which repress the claudin-5 promoter. This indicates that a crosstalk mechanism operates between these junctional structures

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle. IMPORTANCE: Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms

Timing of HIV Seroreversion Among HIV-Exposed, Breastfed Infants in Malawi: Type of HIV Rapid Test Matters

Introduction Rapid HIV serological tests are a cost-effective, point-of-care test among HIV exposed infants but cannot distinguish between maternal and infant antibodies. The lack of data on the timing of decay of maternal antibodies in young infants hinders the potential use of rapid tests in exposed infants. We aimed to determine the time to seroreversion for two commonly used rapid tests in a prospective cohort of HIV-exposed breastfeeding infants ages 3-18 months of life. Methods We collected data on the performance of two commonly used rapid tests (Determine and Unigold) in Malawi between 2008 and 2012 or at the University of North Carolina between 2014 and 2015. Time to seroreversion was estimated for both rapid tests using the Kaplan-Meier product limit estimator which allows for interval censored data. Results At 3 months of age, 3 % of infants had seroreverted according to Determine and 7 % had seroreverted according to Unigold. About one in four infants had achieved seroreversion by 4 months using Unigold, but only about one in twelve infants by 4 months when using Determine. More than 95 % of all infants had seroverted by 7 months according to Unigold and by 12 months according to the Determine assay. Discussion We show that the time of seroreversion depends greatly on the type of test used. Our results highlight the need for recommendations to specify the timing and type of test used in the context of infant HIV detection in resource-poor settings, and base the interpretation of test result on knowledge of time to seroreversion of the selected test

RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP)

Many biological processes are RNA-mediated, but higher-order structures for most RNAs are unknown, making it difficult to understand how RNA structure governs function. Here we describe SHAPE mutational profiling (SHAPE-MaP) that makes possible de novo and large-scale identification of RNA functional motifs. Sites of 2’-hydroxyl acylation by SHAPE are encoded as non-complementary nucleotides during cDNA synthesis, as measured by massively parallel sequencing. SHAPE-MaP-guided modeling identified greater than 90% of accepted base pairs in complex RNAs of known structure and was used to define a second-generation model for the HIV-1 RNA genome. The HIV-1 model contains all known structured motifs and previously unknown elements, including experimentally validated pseudoknots. SHAPE-MaP yields accurate and high-resolution secondary structure models, enables analysis of low abundance RNAs, disentangles sequence polymorphisms in single experiments, and will ultimately democratize RNA structure analysis

Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization

West Nile virions incorporate 180 envelope (E) proteins that orchestrate the process of virus entry and are the primary target of neutralizing antibodies. The E proteins of newly synthesized West Nile virus (WNV) are organized into trimeric spikes composed of pre-membrane (prM) and E protein heterodimers. During egress, immature virions undergo a protease-mediated cleavage of prM that results in a reorganization of E protein into the pseudo-icosahedral arrangement characteristic of mature virions. While cleavage of prM is a required step in the virus life cycle, complete maturation is not required for infectivity and infectious virions may be heterogeneous with respect to the extent of prM cleavage. In this study, we demonstrate that virion maturation impacts the sensitivity of WNV to antibody-mediated neutralization. Complete maturation results in a significant reduction in sensitivity to neutralization by antibodies specific for poorly accessible epitopes that comprise a major component of the human antibody response following WNV infection or vaccination. This reduction in neutralization sensitivity reflects a decrease in the accessibility of epitopes on virions to levels that fall below a threshold required for neutralization. Thus, in addition to a role in facilitating viral entry, changes in E protein arrangement associated with maturation modulate neutralization sensitivity and introduce an additional layer of complexity into humoral immunity against WNV

Genome Copy Number Regulates Inclusion Expansion, Septation, and Infectious Developmental Form Conversion in Chlamydia trachomatis

DNA replication is essential for the growth and development of Chlamydia trachomatis, however it is unclear how this process contributes to and is controlled by the pathogen's biphasic lifecycle. While inhibitors of transcription, translation, cell division, and glucose-6-phosphate transport all negatively affect chlamydial intracellular development, the effects of directly inhibiting DNA polymerase have never been examined. We isolated a temperature sensitive dnaE mutant (dnaEts ) that exhibits a ∼100-fold reduction in genome copy number at the non-permissive temperature (40°C), but replicates similarly to the parent at the permissive temperature of 37°C. We measured higher ratios of genomic DNA nearer the origin of replication than the terminus in dnaEts at 40°C, indicating that this replication deficiency is due to a defect in DNA polymerase processivity. dnaEts formed fewer and smaller pathogenic vacuoles (inclusions) at 40°C, and the bacteria appeared enlarged and exhibited defects in cell division. The bacteria also lacked both discernable peptidoglycan and polymerized MreB, the major cell division organizing protein in Chlamydia responsible for nascent peptidoglycan biosynthesis. We also found that absolute genome copy number, rather than active genome replication, was sufficient for infectious progeny production. Deficiencies in both genome replication and inclusion expansion reversed when dnaEts was shifted from 40°C to 37°C early in infection, and intragenic suppressor mutations in dnaE also restored dnaEts genome replication and inclusion expansion at 40°C. Overall, our results show that genome replication in C. trachomatis is required for inclusion expansion, septum formation, and the transition between the microbe's replicative and infectious forms.SIGNIFICANCE Chlamydiae transition between infectious, extracellular elementary bodies (EBs) and non-infectious, intracellular reticulate bodies (RBs). Some checkpoints that govern transitions in chlamydial development have been identified, but the extent to which genome replication plays a role in regulating the pathogen's infectious cycle has not been characterized. We show that genome replication is dispensable for EB to RB conversion, but is necessary for RB proliferation, division septum formation, and inclusion expansion. We use new methods to investigate developmental checkpoints and dependencies in Chlamydia that facilitate the ordering of events in the microbe's biphasic life cycle. Our findings suggest that Chlamydia utilizes feedback inhibition to regulate core metabolic processes during development, likely an adaptation to intracellular stress and a nutrient-limiting environment

Sinkhole Distribution and Density of Renault Quadrangle, Monroe County, Illinois

Relief shown by contours and spot heights"Geology based on field work by S.V. Panno, J.C. Angel, D.O. Nelson, C.P. Weibel, and J.A. Devera, 2000.""Digital cartography by J. Domier, D. Nelson, S. Geegan, and S. Radil, Illinois State Geological Survey."Includes text, 1 location map with index diagram, and 2 aerial photosIncludes bibliographical references (p. 5-6 of text