Antigen-specific B cells direct T follicular-like helper cells into lymphoid follicles to mediate Mycobacterium tuberculosis control.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global cause of death. Granuloma-associated lymphoid tissue (GrALT) correlates with protection during TB, but the mechanisms of protection are not understood. During TB, the transcription factor IRF4 in T cells but not B cells is required for the generation of the TH1 and TH17 subsets of helper T cells and follicular helper T (TFH)-like cellular responses. A population of IRF4+ T cells coexpress the transcription factor BCL6 during Mtb infection, and deletion of Bcl6 (Bcl6fl/fl) in CD4+ T cells (CD4cre) resulted in reduction of TFH-like cells, impaired localization within GrALT and increased Mtb burden. In contrast, the absence of germinal center B cells, MHC class II expression on B cells, antibody-producing plasma cells or interleukin-10-expressing B cells, did not increase Mtb susceptibility. Indeed, antigen-specific B cells enhance cytokine production and strategically localize TFH-like cells within GrALT via interactions between programmed cell death 1 (PD-1) and its ligand PD-L1 and mediate Mtb control in both mice and macaques.

Direct Proteomic Detection and Prioritization of 19 Onchocerciasis Biomarker Candidates in Humans.

Onchocerca volvulus, the causative agent of onchocerciasis, infects over 20 million people and can cause severe dermatitis and ocular conditions including blindness. Current treatments employed in mass drug administration programs do not kill adult female worms, and common diagnostic tests cannot reliably assess viability of adult worms. There is an urgent need for better diagnostic tests to facilitate monitoring the efficacy of new treatments and disease elimination efforts. Here, eight plasma samples collected from individuals infected with O. volvulus and seven from uninfected individuals were analyzed by MS/MS spectrometry to directly identify O. volvulus proteins present in infected but absent in uninfected control samples. This direct proteomic approach for biomarker discovery had not been previously employed for onchocerciasis. Among all detected proteins, 19 biomarker candidates were supported by two or more unique peptides, identified in the plasma of at least three O. volvulus-infected human samples and absent in all control samples. Comprehensive analysis and ranking of these candidates included detailed functional annotation and a review of RNA-seq gene expression profiles. Isotope-labeled standard peptides were run in parallel and validated MS/MS peptide identifications for 15 peptides from 11 of the 19 proteins, and two infected urine and one uninfected urine sample was used for additional validation. A major antigen/OVOC11613 was identified as the most promising candidate with eight unique peptides across five plasma samples and one urine sample. Additional strong candidates included OVOC1523/ATP synthase, OVOC247/laminin and OVOC11626/PLK5, and along with OVOC11613, and were also detected in urine samples from onchocerciasis patients. This study has identified a promising novel set of proteins that will be carried forward to develop assays that can be used for diagnosis of O. volvulus infections and for monitoring treatment efficacy.

Nuclear Factor κB Signaling Deficiency in CD11c-Expressing Phagocytes Mediates Early Inflammatory Responses and Enhances Mycobacterium tuberculosis Control.

Early innate immune responses play an important role in determining the protective outcome of Mycobacterium tuberculosis (Mtb) infection. Nuclear factor κB (NF-κB) signaling in immune cells regulates the expression of key downstream effector molecules that mount early antimycobacterial responses. Using conditional knockout mice, we studied the effect of abrogation of NF-κB signaling in different myeloid cell types and its impact on Mtb infection. Our results show that the absence of IKK2-mediated signaling in all myeloid cells resulted in increased susceptibility to Mtb infection. In contrast, the absence of IKK2-mediated signaling in CD11c+ myeloid cells induced early proinflammatory cytokine responses, enhanced the recruitment of myeloid cells, and mediated early resistance to Mtb. Abrogation of IKK2 in MRP8-expressing neutrophils did not affect disease pathology or Mtb control. Thus, we describe an early immunoregulatory role for NF-κB signaling in CD11c-expressing phagocytes and a later protective role for NF-κB in LysM-expressing cells during Mtb infection.

Intestinal cell diversity and treatment responses in a parasitic nematode at single cell resolution.

BACKGROUND: Parasitic nematodes, significant pathogens for humans, animals, and plants, depend on diverse organ systems for intra-host survival. Understanding the cellular diversity and molecular variations underlying these functions holds promise for developing novel therapeutics, with specific emphasis on the neuromuscular system's functional diversity. The nematode intestine, crucial for anthelmintic therapies, exhibits diverse cellular phenotypes, and unraveling this diversity at the single-cell level is essential for advancing knowledge in anthelmintic research across various organ systems. RESULTS: Here, using novel single-cell transcriptomics datasets, we delineate cellular diversity within the intestine of adult female Ascaris suum, a parasitic nematode species that infects animals and people. Gene transcripts expressed in individual nuclei of untreated intestinal cells resolved three phenotypic clusters, while lower stringency resolved additional subclusters and more potential diversity. Clusters 1 and 3 phenotypes displayed variable congruence with scRNA phenotypes of C. elegans intestinal cells, whereas the A. suum cluster 2 phenotype was markedly unique. Distinct functional pathway enrichment characterized each A. suum intestinal cell cluster. Cluster 2 was distinctly enriched for Clade III-associated genes, suggesting it evolved within clade III nematodes. Clusters also demonstrated differential transcriptional responsiveness to nematode intestinal toxic treatments, with Cluster 2 displaying the least responses to short-term intra-pseudocoelomic nematode intestinal toxin treatments. CONCLUSIONS: This investigation presents advances in knowledge related to biological differences among major cell populations of adult A. suum intestinal cells. For the first time, diverse nematode intestinal cell populations were characterized, and associated biological markers of these cells were identified to support tracking of constituent cells under experimental conditions. These advances will promote better understanding of this and other parasitic nematodes of global importance, and will help to guide future anthelmintic treatments.

Altered ratio of dendritic cell subsets in skin-draining lymph nodes promotes Th2-driven contact hypersensitivity.

Plasmacytoid dendritic cells (pDCs) specialize in the production of type I IFN (IFN-I). pDCs can be depleted in vivo by injecting diphtheria toxin (DT) in a mouse in which pDCs express a diphtheria toxin receptor (DTR) transgene driven by the human CLEC4C promoter. This promoter is enriched for binding sites for TCF4, a transcription factor that promotes pDC differentiation and expression of pDC markers, including CLEC4C. Here, we found that injection of DT in CLEC4C-DTR+ mice markedly augmented Th2-dependent skin inflammation in a model of contact hypersensitivity (CHS) induced by the hapten fluorescein isothiocyanate. Unexpectedly, this biased Th2 response was independent of reduced IFN-I accompanying pDC depletion. In fact, DT treatment altered the representation of conventional dendritic cells (cDCs) in the skin-draining lymph nodes during the sensitization phase of CHS; there were fewer Th1-priming CD326+ CD103+ cDC1 and more Th2-priming CD11b+ cDC2. Single-cell RNA-sequencing of CLEC4C-DTR+ cDCs revealed that CD326+ DCs, like pDCs, expressed DTR and were depleted together with pDCs by DT treatment. Since CD326+ DCs did not express Tcf4, DTR expression might be driven by yet-undefined transcription factors activating the CLEC4C promoter. These results demonstrate that altered DC representation in the skin-draining lymph nodes during sensitization to allergens can cause Th2-driven CHS.

A Praziquantel Treatment Study of Immune and Transcriptome Profiles in Schistosoma haematobium-Infected Gabonese Schoolchildren.

BACKGROUND: Although Schistosoma haematobium infection has been reported to be associated with alterations in immune function, in particular immune hyporesponsiveness, there have been only few studies that have used the approach of removing infection by drug treatment to establish this and to understand the underlying molecular mechanisms. METHODS: Schistosoma haematobium-infected schoolchildren were studied before and after praziquantel treatment and compared with uninfected controls. Cellular responses were characterized by cytokine production and flow cytometry, and in a subset of children RNA sequencing (RNA-Seq) transcriptome profiling was performed. RESULTS: Removal of S haematobium infection resulted in increased schistosome-specific cytokine responses that were negatively associated with CD4+CD25+FOXP3+ T-cells and accompanied by increased frequency of effector memory T-cells. Innate responses to Toll like receptor (TLR) ligation decreased with treatment and showed positive association with CD4+CD25+FOXP3+ T-cells. At the transcriptome level, schistosome infection was associated with enrichment in cell adhesion, whereas parasite removal was associated with a more quiescent profile. Further analysis indicated that alteration in cellular energy metabolism was associated with S haematobium infection and that the early growth response genes 2 and 3 (EGR 2 and EGR3), transcription factors that negatively regulate T-cell activation, may play a role in adaptive immune hyporesponsiveness. CONCLUSIONS: Using a longitudinal study design, we found contrasting effects of schistosome infection on innate and adaptive immune responses. Whereas the innate immune system appears more activated, the adaptive immunity is in a hyporesponsive state reflected in alterations in CD4+CD25+FOXP3+ T-cells, cellular metabolism, and transcription factors involved in anergy.

Genomes of Fasciola hepatica from the Americas Reveal Colonization with Neorickettsia Endobacteria Related to the Agents of Potomac Horse and Human Sennetsu Fevers.

Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis' gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in ruminants and humans.

Improving eukaryotic genome annotation using single molecule mRNA sequencing.

BACKGROUND: The advantages of Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology include long reads, low systematic bias, and high consensus read accuracy. Here we use these attributes to improve on the genome annotation of the parasitic hookworm Ancylostoma ceylanicum using PacBio RNA-Seq. RESULTS: We sequenced 192,888 circular consensus sequences (CCS) derived from cDNAs generated using the CloneTech SMARTer system. These SMARTer-SMRT libraries were normalized and size-selected providing a robust population of expressed structural genes for subsequent genome annotation. We demonstrate PacBio mRNA sequences based genome annotation improvement, compared to genome annotation using conventional sequencing-by-synthesis alone, by identifying 1609 (9.2%) new genes, extended the length of 3965 (26.7%) genes and increased the total genomic exon length by 1.9 Mb (12.4%). Non-coding sequence representation (primarily from UTRs based on dT reverse transcription priming) was particularly improved, increasing in total length by fifteen-fold, by increasing both the length and number of UTR exons. In addition, the UTR data provided by these CCS allowed for the identification of a novel SL2 splice leader sequence for A. ceylanicum and an increase in the number and proportion of functionally annotated genes. RNA-seq data also confirmed some of the newly annotated genes and gene features. CONCLUSION: Overall, PacBio data has supported a significant improvement in gene annotation in this genome, and is an appealing alternative or complementary technique for genome annotation to the other transcript sequencing technologies.

Functional and phylogenetic characterization of proteins detected in various nematode intestinal compartments.

The parasitic nematode intestine is responsible for nutrient digestion and absorption, and many other processes essential for reproduction and survival, making it a valuable target for anthelmintic drug treatment. However, nematodes display extreme biological diversity (including occupying distinct trophic habitats), resulting in limited knowledge of intestinal cell/protein functions of fundamental or adaptive significance. We developed a perfusion model for isolating intestinal proteins in Ascaris suum (a parasite of humans and swine), allowing for the identification of over 1000 intestinal A. suum proteins (using mass spectrometry), which were assigned to several different intestinal cell compartments (intestinal tissue, the integral and peripheral intestinal membranes, and the intestinal lumen). A multi-omics analysis approach identified a large diversity of biological functions across intestinal compartments, based on both functional enrichment analysis (identifying terms related to detoxification, proteolysis, and host-parasite interactions) and regulatory binding sequence analysis to identify putatively active compartment-specific transcription factors (identifying many related to intestinal sex differentiation or lifespan regulation). Orthologs of A. suum proteins in 15 other nematodes species, five host species, and two outgroups were identified and analyzed. Different cellular compartments demonstrated markedly different levels of protein conservation; e.g. integral intestinal membrane proteins were the most conserved among nematodes (up to 96% conservation), whereas intestinal lumen proteins were the most diverse (only 6% conservation across all nematodes, and 71% with no host orthologs). Finally, this integrated multi-omics analysis identified conserved nematode-specific intestinal proteins likely performing essential functions (including V-type ATPases and ABC transporters), which may serve as promising anthelmintic drug or vaccine targets in future research. Collectively, the findings provide valuable new insights on conserved and adaptive features of nematode intestinal cells, membranes and the intestinal lumen, and potential targets for parasite treatment and control.

An Integrated Multiomics Approach to Identify Candidate Antigens for Serodiagnosis of Human Onchocerciasis.

Improved diagnostic methods are needed to support ongoing efforts to eliminate onchocerciasis (river blindness). This study used an integrated approach to identify adult female Onchocerca volvulus antigens that can be explored for developing serodiagnostic tests. The first step was to develop a detailed multi-omics database of all O. volvulus proteins deduced from the genome, gene transcription data for different stages of the parasite including eight individual female worms (providing gene expression information for 94.8% of all protein coding genes), and the adult female worm proteome (detecting 2126 proteins). Next, female worm proteins were purified with IgG antibodies from onchocerciasis patients and identified using LC-MS with a high-resolution hybrid quadrupole-time-of-flight mass spectrometer. A total of 241 immunoreactive proteins were identified among those bound by IgG from infected individuals but not IgG from uninfected controls. These included most of the major diagnostic antigens described over the past 25 years plus many new candidates. Proteins of interest were prioritized for further study based on a lack of conservation with orthologs in the human host and other helminthes, their expression pattern across the life cycle, and their consistent expression among individual female worms. Based on these criteria, we selected 33 proteins that should be carried forward for testing as serodiagnostic antigens to supplement existing diagnostic tools. These candidates, together with the extensive pan-omics dataset generated in this study are available to the community (http://nematode.net) to facilitate basic and translational research on onchocerciasis.

Helminth.net: expansions to Nematode.net and an introduction to Trematode.net.

Helminth.net (http://www.helminth.net) is the new moniker for a collection of databases: Nematode.net and Trematode.net. Within this collection we provide services and resources for parasitic roundworms (nematodes) and flatworms (trematodes), collectively known as helminths. For over a decade we have provided resources for studying nematodes via our veteran site Nematode.net (http://nematode.net). In this article, (i) we provide an update on the expansions of Nematode.net that hosts omics data from 84 species and provides advanced search tools to the broad scientific community so that data can be mined in a useful and user-friendly manner and (ii) we introduce Trematode.net, a site dedicated to the dissemination of data from flukes, flatworm parasites of the class Trematoda, phylum Platyhelminthes. Trematode.net is an independent component of Helminth.net and currently hosts data from 16 species, with information ranging from genomic, functional genomic data, enzymatic pathway utilization to microbiome changes associated with helminth infections. The databases' interface, with a sophisticated query engine as a backbone, is intended to allow users to search for multi-factorial combinations of species' omics properties. This report describes updates to Nematode.net since its last description in NAR, 2012, and also introduces and presents its new sibling site, Trematode.net.

Functional variants of human papillomavirus type 16 demonstrate host genome integration and transcriptional alterations corresponding to their unique cancer epidemiology.

BACKGROUND: Human papillomaviruses (HPVs) are a worldwide burden as they are a widespread group of tumour viruses in humans. Having a tropism for mucosal tissues, high-risk HPVs are detected in nearly all cervical cancers. HPV16 is the most common high-risk type but not all women infected with high-risk HPV develop a malignant tumour. Likely relevant, HPV genomes are polymorphic and some HPV16 single nucleotide polymorphisms (SNPs) are under evolutionary constraint instigating variable oncogenicity and immunogenicity in the infected host. RESULTS: To investigate the tumourigenicity of two common HPV16 variants, we used our recently developed, three-dimensional organotypic model reminiscent of the natural HPV infectious cycle and conducted various "omics" and bioinformatics approaches. Based on epidemiological studies we chose to examine the HPV16 Asian-American (AA) and HPV16 European Prototype (EP) variants. They differ by three non-synonymous SNPs in the transforming and virus-encoded E6 oncogene where AAE6 is classified as a high- and EPE6 as a low-risk variant. Remarkably, the high-risk AAE6 variant genome integrated into the host DNA, while the low-risk EPE6 variant genome remained episomal as evidenced by highly sensitive Capt-HPV sequencing. RNA-seq experiments showed that the truncated form of AAE6, integrated in chromosome 5q32, produced a local gene over-expression and a large variety of viral-human fusion transcripts, including long distance spliced transcripts. In addition, differential enrichment of host cell pathways was observed between both HPV16 E6 variant-containing epithelia. Finally, in the high-risk variant, we detected a molecular signature of host chromosomal instability, a common property of cancer cells. CONCLUSIONS: We show how naturally occurring SNPs in the HPV16 E6 oncogene cause significant changes in the outcome of HPV infections and subsequent viral and host transcriptome alterations prone to drive carcinogenesis. Host genome instability is closely linked to viral integration into the host genome of HPV-infected cells, which is a key phenomenon for malignant cellular transformation and the reason for uncontrolled E6 oncogene expression. In particular, the finding of variant-specific integration potential represents a new paradigm in HPV variant biology.

Discovery of anthelmintic drug targets and drugs using chokepoints in nematode metabolic pathways.

Parasitic roundworm infections plague more than 2 billion people (1/3 of humanity) and cause drastic losses in crops and livestock. New anthelmintic drugs are urgently needed as new drug resistance and environmental concerns arise. A "chokepoint reaction" is defined as a reaction that either consumes a unique substrate or produces a unique product. A chokepoint analysis provides a systematic method of identifying novel potential drug targets. Chokepoint enzymes were identified in the genomes of 10 nematode species, and the intersection and union of all chokepoint enzymes were found. By studying and experimentally testing available compounds known to target proteins orthologous to nematode chokepoint proteins in public databases, this study uncovers features of chokepoints that make them successful drug targets. Chemogenomic screening was performed on drug-like compounds from public drug databases to find existing compounds that target homologs of nematode chokepoints. The compounds were prioritized based on chemical properties frequently found in successful drugs and were experimentally tested using Caenorhabditis elegans. Several drugs that are already known anthelmintic drugs and novel candidate targets were identified. Seven of the compounds were tested in Caenorhabditis elegans and three yielded a detrimental phenotype. One of these three drug-like compounds, Perhexiline, also yielded a deleterious effect in Haemonchus contortus and Onchocerca lienalis, two nematodes with divergent forms of parasitism. Perhexiline, known to affect the fatty acid oxidation pathway in mammals, caused a reduction in oxygen consumption rates in C. elegans and genome-wide gene expression profiles provided an additional confirmation of its mode of action. Computational modeling of Perhexiline and its target provided structural insights regarding its binding mode and specificity. Our lists of prioritized drug targets and drug-like compounds have potential to expedite the discovery of new anthelmintic drugs with broad-spectrum efficacy.

Temporal Dynamics of Growth and Photosynthesis Suppression in Response to Jasmonate Signaling.

Biotic stress constrains plant productivity in natural and agricultural ecosystems. Repression of photosynthetic genes is a conserved plant response to biotic attack, but how this transcriptional reprogramming is linked to changes in photosynthesis and the transition from growth- to defense-oriented metabolism is poorly understood. Here, we used a combination of noninvasive chlorophyll fluorescence imaging technology and RNA sequencing to determine the effect of the defense hormone jasmonate (JA) on the growth, photosynthetic efficiency, and gene expression of Arabidopsis (Arabidopsis thaliana) rosette leaves. High temporal resolution was achieved through treatment with coronatine (COR), a high-affinity agonist of the JA receptor. We show that leaf growth is rapidly arrested after COR treatment and that this effect is tightly correlated with changes in the expression of genes involved in growth, photosynthesis, and defense. Rapid COR-induced expression of defense genes occurred concomitantly with the repression of photosynthetic genes but was not associated with a reduced quantum efficiency of photosystem II. These findings support the view that photosynthetic capacity is maintained during the period in which stress-induced JA signaling redirects metabolism from growth to defense. Chlorophyll fluorescence images captured in a multiscale time series, however, revealed a transient COR-induced decrease in quantum efficiency of photosystem II at dawn of the day after treatment. Physiological studies suggest that this response results from delayed stomatal opening at the night-day transition. These collective results establish a high-resolution temporal view of how a major stress response pathway modulates plant growth and photosynthesis and highlight the utility of chlorophyll fluorescence imaging for revealing transient stress-induced perturbations in photosynthetic performance.

A novel antigen biomarker for detection of high-level of Loa loa microfilaremia.

BACKGROUND: Loiasis is a disease caused by the nematode Loa loa. Serious adverse events sometimes occur in people with heavy L. loa microfilaremia after ivermectin treatment. In regions of Central Africa where loiasis is endemic, this significantly impedes global elimination programs for lymphatic filariasis and onchocerciasis that use mass distribution of ivermectin. Improved diagnostic tests to identify individuals at increased risk of serious adverse events could facilitate efforts to eliminate lymphatic filariasis and onchocerciasis in this region. METHODS AND FINDINGS: We previously identified the L. loa protein Ll-Bhp-1 in loiasis patient sera. Here, we further characterize Ll-Bhp-1 and report development of an antigen capture ELISA to detect this antigen. This assay detected Ll-Bhp-1 in 74 of 116 (63.8%) loiasis patient sera. Ll-Bhp-1 levels were significantly correlated with L. loa microfilarial counts, and the sensitivity of the assay was highest for samples from people with high counts, (94% and 100% in people with ≥20,000 and ≥50,000 microfilaria per milliliter of blood, respectively). The antigen was not detected in 112 sera from people with other filarial infections, or in 34 control sera from the USA. CONCLUSIONS: This Ll-Bhp-1 antigen assay is specific for loiasis, and highly sensitive for identifying people with high L. loa microfilarial counts who are at increased risk for serious adverse events after ivermectin treatment. L. loa antigen detection has the potential to facilitate loiasis mapping efforts and programs to eliminate lymphatic filariasis and onchocerciasis in Central Africa.

Spatial proteomics of Onchocerca volvulus with pleomorphic neoplasms shows local and systemic dysregulation of protein expression.

Onchocerca volvulus is the agent of onchocerciasis (river blindness) and targeted by WHO for elimination though mass drug administration with ivermectin. A small percentage of adult worms develop pleomorphic neoplasms (PN) that are positively associated with the frequency of ivermectin treatment. Worms with PN have a lower life expectancy and a better understanding about the proteins expressed in PN, and how PN affect protein expression in different tissues could help to elucidate the mechanisms of macrofilaricidal activity of ivermectin. Within a clinical trial of drug combinations that included ivermectin, we detected 24 (5.6%) O. volvulus females with PN by histology of paraffin embedded nodules. To assess the protein inventory of the neoplasms and to identify proteins that may be associated with tumor development, we used laser capture microdissection and highly sensitive mass spectrometry analysis. Neoplasm tissue from three female worms was analyzed, and compared to normal tissues from the body wall, uterus and intestine from the same worms, and to tissues from three females without PN. The healthy females showed all intact embryogenesis. In PN worms, 151 proteins were detected in the body wall, 215 proteins in the intestine, 47 proteins in the uterus and 1,577 proteins in the neoplasms. Only the uterus of one PN female with some stretched intrauterine microfilariae had an elevated number of proteins (601) detectable, while in the uteri of the healthy females 1,710 proteins were detected. Even in tissues that were not directly affected by PN (intestine, body wall), fewer proteins were detected compared to the corresponding tissue of the healthy controls. Immunolocalization of the calcium binding protein OvDig-1 (OVOC8391) confirmed the detection in PN by mass spectrometry. In conclusion we identified proteins that are potentially linked to the development of PN, and systemic dysregulation of protein expression may contribute to worm mortality. Author summary: Onchocerca volvulus , the causative agent of onchocerciasis (river blindness), is targeted for elimination by WHO. The primary strategy involves mass administration of ivermectin. A small proportion of adult female worms develop pleomorphic neoplasms (PN). Here, we used laser capture microdissection and highly sensitive mass spectrometry analysis to determine the protein inventory of PN to identify proteins that may be associated with tumor development. Neoplasm tissue from female worms was analyzed, and compared to normal tissue from the body wall, uterus and intestine from the same worms, and to tissues from females without PN. When compared, PN and healthy control (HC) worms display a different set of proteins, the PN tissue being the one with the highest number of proteins (1,390). From these, 594 were not present in any HC worm tissue. Despite the large number of proteins identified in PN tissue, their low abundance suggests also in PN dysregulation of protein expression. Immunolocalization of a calcium binding protein detected in PN confirmed the mass spectrometry results. In conclusion, we have developed a system to analyze the proteome of O. volvulus from nodule sections and identified proteins that are potentially linked to the development of PN and may contribute to worm mortality.

Optimal timepoint sampling in high-throughput gene expression experiments.

MOTIVATION: Determining the best sampling rates (which maximize information yield and minimize cost) for time-series high-throughput gene expression experiments is a challenging optimization problem. Although existing approaches provide insight into the design of optimal sampling rates, our ability to utilize existing differential gene expression data to discover optimal timepoints is compelling. RESULTS: We present a new data-integrative model, Optimal Timepoint Selection (OTS), to address the sampling rate problem. Three experiments were run on two different datasets in order to test the performance of OTS, including iterative-online and a top-up sampling approaches. In all of the experiments, OTS outperformed the best existing timepoint selection approaches, suggesting that it can optimize the distribution of a limited number of timepoints, potentially leading to better biological insights about the resulting gene expression patterns. AVAILABILITY: OTS is available at www.msu.edu/∼jinchen/OTS.

Escherichia coli O157:H7 induces stronger plant immunity than Salmonella enterica Typhimurium SL1344.

Consumption of fresh produce contaminated with bacterial human pathogens has resulted in various, sometimes deadly, disease outbreaks. In this study, we assessed plant defense responses induced by the fully pathogenic bacteria Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium SL1344 in both Arabidopsis thaliana and lettuce (Lactuca sativa). Unlike SL1344, O157:H7 induced strong plant immunity at both pre-invasion and post-invasion steps of infection. For instance, O157:H7 triggered stomatal closure even under high relative humidity, an environmental condition that generally weakens plant defenses against bacteria in the field and laboratory conditions. SL1344 instead induced a transient stomatal immunity. We also observed that PR1 gene expression was significantly higher in Arabidopsis leaves infected with O157:H7 compared with SL1344. These results suggest that plants may recognize and respond to some human pathogens more effectively than others. Furthermore, stomatal immunity can diminish the penetration of human pathogens through the leaf epidermis, resulting in low bacterial titers in the plant apoplast and suggesting that additional control measures can be employed to prevent food contamination. The understanding of how plant responses can diminish bacterial contamination is paramount in preventing outbreaks and improving the safety of food supplies.

The proteome of extracellular vesicles of the lung fluke Paragonimus kellicotti produced in vitro and in the lung cyst.

Paragonimiasis is a zoonotic, food-borne trematode infection that affects 21 million people globally. Trematodes interact with their hosts via extracellular vesicles (EV) that carry protein and RNA cargo. We analyzed EV in excretory-secretory products (ESP) released by Paragonimus kellicotti adult worms cultured in vitro (EV ESP) and EV isolated from lung cyst fluid (EV CFP) recovered from infected gerbils. The majority of EV were approximately 30-50 nm in diameter. We identified 548 P. kellicotti-derived proteins in EV ESP by mass spectrometry and 8 proteins in EV CFP of which 7 were also present in EV ESP. No parasite-derived proteins were reliably detected in EV isolated from plasma samples. A cysteine protease (MK050848, CP-6) was the most abundant protein found in EV CFP in all technical and biological replicates. Immunolocalization of CP-6 showed strong labeling in the tegument of P. kellicotti and in the adjacent cyst and lung tissue that contained worm eggs. It is likely that CP-6 present in EV is involved in parasite-host interactions. These results provide new insights into interactions between Paragonimus and their mammalian hosts, and they provide potential clues for development of novel diagnostic tools and treatments.

Poly(ADP-ribose) polymerase 9 mediates early protection against Mycobacterium tuberculosis infection by regulating type I IFN production.

The ADP ribosyltransferases (PARPs 1-17) regulate diverse cellular processes, including DNA damage repair. PARPs are classified on the basis of their ability to catalyze poly-ADP-ribosylation (PARylation) or mono-ADP-ribosylation (MARylation). Although PARP9 mRNA expression is significantly increased in progressive tuberculosis (TB) in humans, its participation in host immunity to TB is unknown. Here, we show that PARP9 mRNA encoding the MARylating PARP9 enzyme was upregulated during TB in humans and mice and provide evidence of a critical modulatory role for PARP9 in DNA damage, cyclic GMP-AMP synthase (cGAS) expression, and type I IFN production during TB. Thus, Parp9-deficient mice were susceptible to Mycobacterium tuberculosis infection and exhibited increased TB disease, cGAS and 2'3'-cyclic GMP-AMP (cGAMP) expression, and type I IFN production, along with upregulation of complement and coagulation pathways. Enhanced M. tuberculosis susceptibility is type I IFN dependent, as blockade of IFN α receptor (IFNAR) signaling reversed the enhanced susceptibility of Parp9-/- mice. Thus, in sharp contrast to PARP9 enhancement of type I IFN production in viral infections, this member of the MAR family plays a protective role by limiting type I IFN responses during TB.