The Leishmania donovani Lipophosphoglycan Excludes the Vesicular Proton-ATPase from Phagosomes by Impairing the Recruitment of Synaptotagmin V

We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galβ1,4Manα1-PO4 units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification

Genetic Transformation of an Obligate Anaerobe, P. gingivalis for FMN-Green Fluorescent Protein Expression in Studying Host-Microbe Interaction

The recent introduction of “oxygen-independent” flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) is of major interest to both eukaryotic and prokaryotic microbial biologists. Accordingly, we demonstrate for the first time that an obligate anaerobe, the successful opportunistic pathogen of the oral cavity, Porphyromonas gingivalis, can be genetically engineered for expression of the non-toxic green FbFP. The resulting transformants are functional for studying dynamic bacterial processes in living host cells. The visualization of the transformed P. gingivalis (PgFbFP) revealed strong fluorescence that reached a maximum emission at 495 nm as determined by fluorescence microscopy and spectrofluorometry. Human primary gingival epithelial cells (GECs) were infected with PgFbFP and the bacterial invasion of host cells was analyzed by a quantitative fluorescence microscopy and antibiotic protection assays. The results showed similar levels of intracellular bacteria for both wild type and PgFbFP strains. In conjunction with organelle specific fluorescent dyes, utilization of the transformed strain provided direct and accurate determination of the live/metabolically active P. gingivalis' trafficking in the GECs over time. Furthermore, the GECs were co-infected with PgFbFP and the ATP-dependent Clp serine protease-deficient mutant (ClpP-) to study the differential fates of the two strains within the same host cells. Quantitative co-localization analyses displayed the intracellular PgFbFP significantly associated with the endoplasmic reticulum network, whereas the majority of ClpP- organisms trafficked into the lysosomes. Hence, we have developed a novel and reliable method to characterize live host cell-microbe interactions and demonstrated the adaptability of FMN-green fluorescent protein for studying persistent host infections induced by obligate anaerobic organisms

Data from: Assessing conservation risks to populations of an anadromous Arctic salmonid, the northern Dolly Varden (Salvelinus malma malma), via estimates of effective and census population sizes and approximate Bayesian computation

Census population size (Nc) is crucial to the development of resource management strategies, however, monitoring the effective population size (Ne) of managed populations has proliferated because of this parameter’s relationship to the short-term impacts of genetic stochasticity and long-term population viability. Thus, having a sound understanding of both Nc and Ne, including population connectivity, provides valuable insights into both the demographic and genetic risks to extinction. Here, we assessed microsatellite DNA variation in four (of five known) anadromous northern Dolly Varden (NDV, Salvelinus malma malma) populations from Canada’s western Arctic region, to estimate Ne using both temporal-based and single-sample estimators and to test for associations between Ne and Nc. We also employed approximate Bayesian computation (ABC) to evaluate several evolutionary scenarios that have potentially shaped contemporary population structure in this species, focusing particularly on population size and connectivity. We found evidence for moderate to large contemporary and historical Ne, suggesting that short- and long-term extinction risks are low for these populations. Estimates of contemporary and long-term Ne were variable within and among populations and overall estimates could not be reliably linked with Nc or available spawning habitat. The overall estimate of Ne/Nc, was 0.152 and ranged from 0.024 to 0.442 when including errors around the estimate of Ne and Nc. Finally, ABC analyses suggest that NDV had a common origin followed by divergence in isolation while maintaining large effective sizes, but also that these populations were bottlenecked in the past, likely the result of post-glacial colonization processes. These results corroborate indications of limited gene flow at present, indicating independent demographic and evolutionary trajectories that imply NDV is best managed on a per-river-population basis. Overall, the results of this study further our general understanding of Ne, Ne/Nc and demographic independence in NDV, and provide a comprehensive and quantitative assessment of the potential genetic and demographic risk status of Arctic anadromous salmonids, including baselines for future monitoring

Data from: Evolution and origin of sympatric shallow-water morphotypes of Lake Trout, Salvelinus namaycush, in Canada's Great Bear Lake

Range expansion in north-temperate fishes subsequent to the retreat of the Wisconsinan glaciers has resulted in the rapid colonization of previously unexploited, heterogeneous habitats and, in many situations, secondary contact among conspecific lineages that were once previously isolated. Such ecological opportunity coupled with reduced competition likely promoted morphological and genetic differentiation within and among post-glacial fish populations. Discrete morphological forms existing in sympatry, for example, have now been described in many species, yet few studies have directly assessed the association between morphological and genetic variation. Morphotypes of Lake Trout, Salvelinus namaycush, are found in several large-lake systems including Great Bear Lake (GBL), Northwest Territories, Canada, where several shallow-water forms are known. Here, we assess microsatellite and mitochondrial DNA variation among four morphotypes of Lake Trout from the five distinct arms of GBL, and also from locations outside of this system to evaluate several hypotheses concerning the evolution of morphological variation in this species. Our data indicate that morphotypes of Lake Trout from GBL are genetically differentiated from one another, yet the morphotypes are still genetically more similar to one another compared with populations from outside of this system. Furthermore, our data suggest that Lake Trout colonized GBL following dispersal from a single glacial refugium (the Mississippian) and support an intra-lake model of divergence. Overall, our study provides insights into the origins of morphological and genetic variation in post-glacial populations of fishes and provides benchmarks important for monitoring Lake Trout biodiversity in a region thought to be disproportionately susceptible to impacts from climate change

Cell biology - The extraordinary phagosome

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62698/1/418286a.pd

Data from: Life-history characteristics and landscape attributes as drivers of genetic variation, gene flow and fine-scale population structure in Northern Dolly Varden (Salvelinus malma malma) in Canada

The Northern Dolly Varden (Salvelinus malma malma) displays variable life-history types and occupies freshwater habitats with varying levels of connectivity. Here, we assayed microsatellite DNA variation in Northern Dolly Varden from the western Canadian Arctic to resolve landscape and life history variables driving variation in genetic diversity and population structure. Overall, genetic variation was highest in anadromous populations and lowest in those isolated above waterfalls with stream-resident forms intermediate between the two. Anadromous and isolated populations were genetically divergent from each other while no genetic differentiation was detectable between sympatric anadromous and stream-resident forms. Population structure was stable over 25 years, hierarchically organized and conformed to an isolation-by-distance pattern, but stream-isolated forms often deviated from these patterns. Gene flow occurred primarily among Yukon North Slope populations and between sympatric anadromous and resident forms. These results were sex-dependent to some extent, but were influenced more by reproductive status and life history. Our study provides novel insights into the life history, population demographic and habitat variables that shape the distribution of genetic variation and population structure in Arctic fluvial habitats while providing a spatial context for management and conservation

Genepop Data File

Genepop data file (3 digit format) used for analyses - sample codes are listed in Table 1 of the manuscript

Genepop File

This file contains all microsatellite data for all samples/individuals. The year the samples were collected is included as well as the life history type (either freshwater resident (res) or freshwater isolated (iso) forms - note that no code is used for anadromous individuals which constitutes the majority of the samples)