Gateway Entry Vector Library of Wolbachia pipientis Candidate Effectors from Strain wMel.

Wolbachia pipientis is an intracellular symbiont that modifies host biology using a type IV secretion system to inject bacterial effectors into the host cytoplasm. We utilized a bioinformatics approach to predict Wolbachia effectors and cloned the candidates into an entry vector, which can be utilized for subsequent analyses.

Large-Scale Identification of Wolbachia pipientis Effectors.

Wolbachia pipientis is an intracellular symbiont of arthropods well known for the reproductive manipulations induced in the host and, more recently, for the ability of Wolbachia to block virus replication in insect vectors. Since Wolbachia cannot yet be genetically manipulated, and due to the constraints imposed when working with an intracellular symbiont, little is known about mechanisms used by Wolbachia for host interaction. Here we employed a bioinformatics pipeline and identified 163 candidate effectors, potentially secreted by Wolbachia into the host cell. A total of 84 of these candidates were then subjected to a screen of growth defects induced in yeast upon heterologous expression which identified 14 top candidates likely secreted by Wolbachia. These predicted secreted effectors may function in concert as we find that their native expression is correlated and is highly upregulated at specific time points during Drosophila development. In addition, the evolutionary histories of some of these predicted effectors are also correlated, suggesting they may function together, or in the same pathway, during host infection. Similarly, most of these predicted effectors are limited to one or two Wolbachia strains-perhaps reflecting shared evolutionary history and strain specific functions in host manipulation. Identification of these Wolbachia candidate effectors is the first step in dissecting the mechanisms of symbiont-host interaction in this important system.

Identification and Characterization of a Candidate Wolbachia pipientis Type IV Effector That Interacts with the Actin Cytoskeleton.

UNLABELLED: Many bacteria live as intracellular symbionts, causing persistent infections within insects. One extraordinarily common infection is that of Wolbachia pipientis, which infects 40% of insect species and induces reproductive effects. The bacteria are passed from generation to generation both vertically (through the oocyte) and horizontally (by environmental transmission). Maintenance of the infection within Drosophila melanogaster is sensitive to the regulation of actin, as Wolbachia inefficiently colonizes strains hemizygous for the profilin or villin genes. Therefore, we hypothesized that Wolbachia must depend on the host actin cytoskeleton. In this study, we identify and characterize a Wolbachia protein (WD0830) that is predicted to be secreted by the bacterial parasite. Expression of WD0830 in a model eukaryote (the yeast Saccharomyces cerevisiae) induces a growth defect associated with the appearance of aberrant, filamentous structures which colocalize with rhodamine-phalloidin-stained actin. Purified WD0830 bundles actin in vitro and cosediments with actin filaments, suggesting a direct interaction of the two proteins. We characterized the expression of WD0830 throughout Drosophila development and found it to be upregulated in third-instar larvae, peaking in early pupation, during the critical formation of adult tissues, including the reproductive system. In transgenic flies, heterologously expressed WD0830 localizes to the developing oocyte. Additionally, overexpression of WD0830 results in increased Wolbachia titers in whole flies, in stage 9 and 10 oocytes, and in embryos, compared to controls, suggesting that the protein may facilitate Wolbachia's replication or transmission. Therefore, this candidate secreted effector may play a role in Wolbachia's infection of and persistence within host niches. IMPORTANCE: The obligate intracellular Wolbachia pipientis is a ubiquitous alphaproteobacterial symbiont of arthropods and nematodes and is related to the rickettsial pathogens Ehrlichia spp. and Anaplasma spp. Studies of Wolbachia cell biology suggest that this bacterium relies on host actin for efficient proliferation and transmission between generations. Here, we identified and characterized a Wolbachia protein that localizes to and manipulates the eukaryotic actin cytoskeleton, is expressed by Wolbachia during host development, and alters Wolbachia titers and localization in transgenic fruit flies. We hypothesize that WD0830 may be utilized by the bacterium to facilitate replication in or invasion of different niches during host development.

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine.

UNLABELLED: Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4At and the TraJ/VirD4At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4At domains bound this substrate in vitro We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCE: Bacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.

Wolbachia utilize host actin for efficient maternal transmission in Drosophila melanogaster.

Wolbachia pipientis is a ubiquitous, maternally transmitted bacterium that infects the germline of insect hosts. Estimates are that Wolbachia infect nearly 40% of insect species on the planet, making it the most prevalent infection on Earth. The bacterium, infamous for the reproductive phenotypes it induces in arthropod hosts, has risen to recent prominence due to its use in vector control. Wolbachia infection prevents the colonization of vectors by RNA viruses, including Drosophila C virus and important human pathogens such as Dengue and Chikungunya. Here we present data indicating that Wolbachia utilize the host actin cytoskeleton during oogenesis for persistence within and transmission between Drosophila melanogaster generations. We show that phenotypically wild type flies heterozygous for cytoskeletal mutations in Drosophila profilin (chic(221/+) and chic(1320/+)) or villin (qua(6-396/+)) either clear a Wolbachia infection, or result in significantly reduced infection levels. This reduction of Wolbachia is supported by PCR evidence, Western blot results and cytological examination. This phenotype is unlikely to be the result of maternal loading defects, defects in oocyte polarization, or germline stem cell proliferation, as the flies are phenotypically wild type in egg size, shape, and number. Importantly, however, heterozygous mutant flies exhibit decreased total G-actin in the ovary, compared to control flies and chic(221) heterozygous mutants exhibit decreased expression of profilin. Additionally, RNAi knockdown of profilin during development decreases Wolbachia titers. We analyze evidence in support of alternative theories to explain this Wolbachia phenotype and conclude that our results support the hypothesis that Wolbachia utilize the actin skeleton for efficient transmission and maintenance within Drosophila.

Passage of Wolbachia pipientis through mutant drosophila melanogaster induces phenotypic and genomic changes.

Wolbachia pipientis is a nearly ubiquitous, maternally transmitted bacterium that infects the germ line of insect hosts. Estimates are that Wolbachia infects 40 to 60% of insect species on the planet, making it one of the most prevalent infections on Earth. However, we know surprisingly little about the molecular mechanisms used by Wolbachia to infect its hosts. We passaged Wolbachia through normally restrictive Drosophila melanogaster hosts, bottlenecking Wolbachia through stochastic segregation while simultaneously selecting for mutants that could recolonize these previously restrictive hosts. Here, we show that Wolbachia alters its behavior when passaged through heterozygous mutant flies. After only three generations, Wolbachia was able to colonize the previously restrictive hosts at control titers. Additionally, the Wolbachia organisms passaged through heterozygous mutant D. melanogaster alter their pattern of tissue-specific Wsp protein production, suggesting a behavioral response to the host genotype. Using whole-genome resequencing, we identified the mutations accumulated by these lineages of Wolbachia and confirmed the existence and persistence of the mutations through clone library Sanger sequencing. Our results suggest that Wolbachia can quickly adapt to new host contexts, with genomic mutants arising after only two generations.

Molecular identification of Pilobolus species from Yellowstone National Park.

Pilobolus, a widely distributed coprophilous fungus, grows on herbivore dung. Species of Pilobolus traditionally are described with imprecise morphological characteristics potentially leading to misidentification. In this study we used PCR analysis of taxonomically informative sequences to provide more consistent species identification from isolates obtained in Yellowstone National Park. We collected Pilobolus park-wide from six taxa of herbivores over 9 y. Multiple transfers of single sporangium isolates provided pure cultures from which DNA was extracted. Sequence analysis of internal transcribed spacer (ITS) regions of DNA that code for rRNA genes were used to distinguish among similar species. We describe several species of Pilobolus associated with herbivores in various habitats, including two species not previously reported, P. heterosporus and P. sphaerosporus. Our results show that phylogenetic species identification of Pilobolus based on sequence analysis of pure culture isolates provides a more reliable means of identifying species than traditional methods.

Phylogeny of Pilobolaceae.

The three genera traditionally classified as Pilobolaceae have been identified on the basis of morphological characteristics. In the absence of distinctive morphological differences phylogenetic techniques have proven to be superior for developing phylogenies. Molecular techniques have been used primarily for studies of higher fungi; there are few investigations of the Zygomycota using genetic sequences for classification. DNA sequences coding for three regions of rRNA were used to investigate phylogenetic relationships of the three genera traditionally considered within the Pilobolaceae. Evidence indicates that Pilaira should be removed from Pilobolaceae and the family redescribed. Sporangiospore size is the morphological characteristic that most closely correlates with rDNA clades of phylogenetic trees. This study demonstrates that traditional morphological characteristics alone are not adequate to differentiate species of Pilobolus.

Mago Nashi, Tsunagi/Y14, and Ranshi form a complex that influences oocyte differentiation in Drosophila melanogaster.

During Drosophila melanogaster oogenesis, a germline stem cell divides forming a cyst of 16 interconnected cells. One cell enters the oogenic pathway, and the remaining 15 differentiate as nurse cells. Although directed transport and localization of oocyte differentiation factors within the single cell are indispensible for selection, maintenance, and differentiation of the oocyte, the mechanisms regulating these events are poorly understood. Mago Nashi and Tsunagi/Y14, core components of the exon junction complex (a multiprotein complex assembled on spliced RNAs), are essential for restricting oocyte fate to a single cell and for localization of oskar mRNA. Here we provide evidence that Mago Nashi and Tsunagi/Y14 form an oogenic complex with Ranshi, a protein with a zinc finger-associated domain and zinc finger domains. Genetic analyses of ranshi reveal that (1) 16-cell cysts are formed, (2) two cells retain synaptonemal complexes, (3) all cells have endoreplicated DNA (as observed in nurse cells), and (4) oocyte-specific cytoplasmic markers accumulate and persist within a single cell but are not localized within the posterior pole of the presumptive oocyte. Our results indicate that Ranshi interacts with the exon junction complex to localize components essential for oocyte differentiation within the posterior pole of the presumptive oocyte.

Yeast genomic expression patterns in response to low-shear modeled microgravity.

UNLABELLED: The low-shear microgravity environment, modeled by rotating suspension culture bioreactors called high aspect ratio vessels (HARVs), allows investigation in ground-based studies of the effects of microgravity on eukaryotic cells and provides insights into the impact of space flight on cellular physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) causes significant phenotypic changes of a select group of Saccharomyces cerevisiae genes associated with the establishment of cell polarity, bipolar budding, and cell separation. However, the mechanisms cells utilize to sense and respond to microgravity and the fundamental gene expression changes that occur are largely unknown. In this study, we examined the global transcriptional response of yeast cells grown under LSMMG conditions using DNA microarray analysis in order to determine if exposure to LSMMG results in changes in gene expression. RESULTS: LSMMG differentially changed the expression of a significant number of genes (1372) when yeast cells were cultured for either five generations or twenty-five generations in HARVs, as compared to cells grown under identical conditions in normal gravity. We identified genes in cell wall integrity signaling pathways containing MAP kinase cascades that may provide clues to novel physiological responses of eukaryotic cells to the external stress of a low-shear modeled microgravity environment. A comparison of the microgravity response to other environmental stress response (ESR) genes showed that 26% of the genes that respond significantly to LSMMG are involved in a general environmental stress response, while 74% of the genes may represent a unique transcriptional response to microgravity. In addition, we found changes in genes involved in budding, cell polarity establishment, and cell separation that validate our hypothesis that phenotypic changes observed in cells grown in microgravity are reflected in genome-wide changes. This study documents a considerable response to yeast cell growth in low-shear modeled microgravity that is evident, at least in part, by changes in gene expression. Notably, we identified genes that are involved in cell signaling pathways that allow cells to detect environmental changes, to respond within the cell, and to change accordingly, as well as genes of unknown function that may have a unique transcriptional response to microgravity. We also uncovered significant changes in the expression of many genes involved in cell polarization and bud formation that correlate well with the phenotypic effects observed in yeast cells when grown under similar conditions. These results are noteworthy as they have implications for human space flight.

The role of FLO11 in Saccharomyces cerevisiae biofilm development in a laboratory based flow-cell system.

A role of the FLO11 in Saccharomyces cerevisiae biofilm development in a flow cell system was examined. We carried out an ectopic FLO11 expression in the wild type (wt) BY4741 strain that has low levels of endogenous FLO11 transcript. In contrast to the nonadhesive wt, the FLO11 overexpression strain (BY4741 FLO11(+)) readily adhered to both liquid-hydrophobic and liquid-hydrophilic solid interfaces and was able to grow as a biofilm monolayer in a flow system. Cellular features associated with FLO11 were examined and found to be consistent with the previous studies conducted in different strains of S. cerevisiae. When grown in suspended liquid culture, BY4741 FLO11(+) formed larger cellular aggregates (clumps), consisting of from five to 60 cells, and displayed an increased cell surface hydrophobicity, without changes in the cell size or growth rate, compared to wt. However, the invasive growth associated with FLO11 expression was not observed in BY4741 FLO11(+). The significance of these findings is discussed in the context of clinically and industrially relevant biofilms.

Legionella species diversity in an acidic biofilm community in Yellowstone National Park.

Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30 degrees C) and higher (35 to 38 degrees C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.

PCR detection and analysis of the free-living amoeba Naegleria in hot springs in Yellowstone and Grand Teton National Parks.

Free-living thermotolerant amoebae pose a significant health risk to people who soak and swim in habitats suitable for their growth, such as hot springs. In this survey of 23 different hot springs in Yellowstone and Grand Teton National Parks, we used PCR with primer sets specific for Naegleria to detect three sequence types that represent species not previously described, as well as a fourth sequence type identified as the pathogen Naegleria fowleri.

Microbially mediated sulphide production in a thermal, acidic algal mat community in Yellowstone National Park.

Our objective in this study was to characterize prokaryotic sulphide production within the oxygenic, predominantly eukaryotic algal mat in an acidic stream, Nymph Creek, in Yellowstone National Park (YNP). We used microsensors to examine fluctuations in H2S and O2 concentrations over time through the vertical aspect of the approximately 3 mm mat in a 46-48 degrees C region of the creek. We also used analyses of PCR-amplified 16S rRNA gene sequences obtained from denaturing gradient gels, and PCR-amplified sequences of a functional gene associated with microbial sulphate respiration (dsrA) to characterize the bacterial community in the same region of the mat. During midday, photosynthesis rates were high within the first 500 micro m interval of the mat and high oxygen concentrations (600% air saturation) penetrated deeply (>1800 micro m) into the mat. During early evening and night, oxygen concentrations within the first 1100 micro m of the mat decreased over time from 60% air saturation (a.s) to 12% a.s. A precipitous decline in oxygen concentration occurred at a depth of 1100 micro m in all night measurements and anoxic conditions were present below 1200 micro m. Within this anoxic region, sulphide concentrations increased from nearly 0 micro M at 1200 micro m depth to 100 micro M at 2400 micro m depth. Enrichment cultures inoculated with Nymph Creek mat organisms also produced H2S. Sequence analyses of 16S rRNA and dsrA genes indicated the presence of at least five bacterial genera including species involved in dissimilative sulphate or sulphur reduction.

Detection of Naegleria sp. in a thermal, acidic stream in Yellowstone National Park.

An initial survey of sequences of PCR-amplified portions of the 18S rRNA genes from a community DNA clone library, prepared from an algal mat in a thermal, acidic stream in Yellowstone National Park, WY, USA, revealed among other sequences, several that matched Vahlkampfia. This finding prompted further investigation using primers specific for Naegleria. Sequences from a subsequent DNA clone library, prepared from the 5.8S rRNA gene and the adjacent internal transcribed spacer (ITS) regions of the rRNA, closely matched Naegleria and formed an independent lineage within a clade containing Naegleria sturti and Naegleria niuginiensis. The sequences may represent a new Naegleria species.