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bioRxiv2019

Development of a potent and protective germline-like antibody lineage against Zika virus in a convalescent human

Zika virus (ZIKV) specific neutralizing antibodies hold a great promise for antibody-based interventions and vaccine design against ZIKV infection. However, their development in infected patients remain unknown. Here, we report on the dynamic development of a potent and protective ZIKV-specific human antibody ZK2B10 initially isolated from a ZIKV convalescent individual using next-generation sequencing (NGS). The unbiased repertoire analysis showed dramatic changes in many families of heavy and light chain variable regions. However, lineage tracing of ZK2B10 revealed limited somatic hypermutation throughout the 12 months since the onset of symptom. In particular, NGS-derived germline-like somatic variants neutralized and protected mice from lethal challenge of ZIKV without detectable cross-reactivity with Dengue virus (DENV). Site-directed mutagenesis identified two residues within {lambda} chain, N31 and S91 that are essential to the functional maturation. The dynamic features unveiled here will assist us to better understand the pathogenesis of ZIKV infection and inform rational design of vaccines.\n\nAuthor summaryRecently emerged ZIKV is associated with severe neurological complications such as Guillain-Barre syndrome in adults and congenital microcephaly in newborns. No ZIKV-specific therapeutics or vaccines are currently available. We and others have identified a number of neutralizing antibodies capable of protecting experimental animals from ZIKV infection. However, the development of these potent antibodies during ZIKV natural infection remains unknown. Here, we report on the longitudinal analysis of one such antibody ZK2B10 using next-generation sequencing (NGS), bioinformatics and functional analysis. We found that the ZK2B10 germline-like antibodies possess strong neutralizing activity in vitro and impressive protectivity against lethal ZIKV infection in vivo. These findings suggest that the potent and protective antibody response against ZIKV can be generated within relative short term with high germline identity which provide great hope and promise for successful vaccine development against ZIKV.

microbiology↗

bioRxiv2019

In vitro and in vivo efficacy of the combination of colistin and endolysins against clinical strains of Multi-Drug Resistant (MDR) pathogens

The multidrug resistance (MDR) among pathogenic bacteria is jeopardizing the worth of antimicrobials, which had previously changed medical sciences. In this study, we used bioinformatic tools to identify the endolysins ElyA1 and ElyA2 (GH108-PG3 family) present in the genome of bacteriophages Ab1051{Phi} and Ab1052{Phi}, respectively. The muralytic activity of these endolysins over MDR clinical isolates (Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae) was tested using the turbidity reduction assay. The minimal inhibitory concentrations (MICs) of endolysin, colistin and their combination were determined using the microdilution checkerboard method. The antimicrobial activity of the combinations was confirmed by time kill curves and in vivo assays in larvae of Galleria mellonella. Our results showed that ElyA1 displayed activity against all 25 strains of A. baumannii and P. aeruginosa tested and against 13 out of 17 strains of K. pneumoniae. No activity was detected when assays were done with endolysin ElyA2. The combined antimicrobial activity of colistin and endolysin ElyA1 yielded a reduction in the colistin MIC for all strains studied, except K. pneumoniae. These results were confirmed in vivo in G. mellonella survival assays. In conclusion, the combination of colistin with new endolysins such as ElyA1 could increase the bactericidal activity and reduce the MIC of the antibiotic, thus also reducing the associated toxicity.\n\nIMPORTANCEThe development of multiresistance by pathogen bacteria increases the necessity of the development of new antimicrobial strategies. In this work, we combined the effect of the colistin with a new endolysin, ElyA1, from a bacteriophage present in the clinical strain of Acinetobacter baumannii Ab105. ElyA1 is a lysozyme-like family (GH108-GP3), whose antimicrobial activity was described for first time in this work. Also, another endolysin, ElyA2, with the same origin and family, was characterized but in this case no activity was detected. ElyA1 presented lytic activity over a broad spectrum of strains from A. baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. When colistin was combined with ElyA1 an increase of the antimicrobial activity was observed with a reduced concentration of colistin, and this observation was also confirmed in vivo in Galleria mellonella larvae. The combination of colistin with new endolysins as ElyA1 could increase the bactericidal activity and lowering the MIC of the antibiotic, thus also reducing the associated toxicity.

microbiology↗

bioRxiv2019

Next generation taxonomy: integrating traditional species description with the holobiont concept and genomic approaches - The in-depth characterization of a novel Euplotes species as a case study

In 1991 Margulis defined holobionts as the assemblage of \"two or more organisms, members of different species\" which remain associate \"throughout a significant portion of the life history\". In recent times, holobionts have been described among many and far-related groups of living beings, such as plants, algae, insects, corals, and even humans. These studies have arisen an increasing interest in different contexts but, to our knowledge, the holobiont concept has not been applied in taxonomy. Here we propose a new approach to modern taxonomy, aimed to integrate the holobiont concept and genomic and bioinformatic analyses with the classical/morphological tools traditionally used in taxonomy. The inclusion of symbiont morphology, and of mitochondrial and symbiont genomes will allow the discipline to move toward what could become the \"next generation taxonomy\". As an example of this new paradigm in the characterization of holobionts, we herein provide the taxonomic description of the ciliate protist Euplotes vanleeuwenhoeki sp. nov. (Euplotia, Ciliophora) and its bacterial endosymbiont \"Candidatus Pinguicoccus supinus\" gen. nov., sp. nov. (Opitutae, Verrucomicrobia). Interestingly, we found that this endosymbiont has an extremely reduced genome (~163 Kbp), which is suggestive of a high integration with the host and represents the first case of such an extreme reduction in Verrucomicrobia, and the first case in a protist host.

zoology↗

bioRxiv2019

Screening and functional analysis of differentially expressed genes in an animal model of EBV-associated lymphomas

Epstein-Barr virus (EBV) is an important human oncogenic virus. This paper is to explore how EBV induce malignant transformation of human lymphocytes and the related mechanism of lymphomagenesis. We have constructed hu-PBL/SCID chimeric mice and established a model of EBV-associated human-derived lymphomas. By using Agilent human whole genome microarray and a series of bioinformatic analyses, a total of 202 differentially expressed genes were screened from the EBV-induced lymphomas in hu-PBL/SCID mice, including 44 up-regulated and 158 down-regulated genes. Calculation of the rank score (RS) values of these genes in the HIPPIE protein interaction networks showed that topoisomerase II alpha (TOP2A), ubiquitin like with PHD and ring finger domains 1 (UHRF1), histone cluster 2 H2B family member E (HIST2H2BE), phosphoglycerate dehydrogenase (PHGDH), vinculin (VCL), insulin-like growth factor 1 receptor (IGF1R), Fos proto-oncogene (FOS), snail family transcriptional repressor 1 (SNAI1), PDZ binding kinase (PBK), and ring finger protein 144B (RNF144B) were the top 10 key node genes of EBV-induced lymphoma. In which, PBK, an up-regulated genes with the highest number of GO annotations, was verified by cellular function experiments and clinical lymphoma samples.\n\nAuthor summaryEB virus is closely associated with human lymphoma and nasopharyngeal carcinoma. Since the susceptible hosts of EBV limit to human and cottontop tammarins, there are no appropriate animal models so far to study the EBV-associated oncogenesis. In our previous experiments, the EBV-associated lymphomas were induced in hu-PBL/SCID chimera (a new humanized mouse model). However, the cellular and molecular mechanisms of malignant transformation of normal human cells and tumor formation induced by EBV remain unclear. In this study, we examined and compared the gene expression profiles of EBV-induced lymphomas and normal human lymphocytes of the same origin in SCID mice. By constructing the gene-function relationship network, we preliminarily found that TOP2A, UHRF1, HIST2H2BE, PHGDH, VCL, IGF1R, FOS, SNAI1, PBK, and RNF144B may be the key genes in EBV-induced lymphomas. These findings suggest that the induction of lymphoma by EBV is a complex process that involves multiple genes and pathways.

cancer biology↗

bioRxiv2019

A robust metaproteomics pipeline for a holistic taxonomic and functional characterization of microbial communities from marine particles

This study aimed to establish a robust, reproducible and reliable metaproteomic pipeline for an in-depth characterization of marine particle-associated (PA) bacteria. To this end, we compared six well-established protein extraction protocols together with different MS-sample preparation techniques using particles sampled during a North Sea spring algae bloom in 2009. In this optimized workflow, proteins are extracted using a combination of SDS-containing lysis buffer and cell disruption by bead-beating, separated by SDS-PAGE, in-gel digested and analysed by LC-MS/MS, before MASCOT search against a metagenome-based database and data processing/visualization with the in-house-developed bioinformatics tools Prophane and Paver.\n\nAs proof of principle, free-living (FL) and particulate communities sampled in April 2009 were analysed, resulting in an as yet unprecedented number of 9,354 and 5,034 identified protein groups for FL and PA bacteria, respectively. Our data revealed that FL and PA communities appeared similar in their taxonomic distribution, with notable exceptions: eukaryotic proteins and proteins assigned to Flavobacteriia, Cyanobacteria, and some proteobacterial genera were found more abundant on particles, whilst overall proteins belonging to Proteobacteria were more dominant in the FL fraction. In contrast, significant functional differences including proteins involved in polysaccharide degradation, sugar- and phosphorus uptake, adhesion, motility, and stress response were detected.\n\nOriginality-Significance StatementMarine particles consist of organic particulate matter (e.g. phyto- or zooplankton) and particle-associated (PA) microbial communities, which are often embedded in a sugary matrix. A significant fraction of the decaying algal biomass in marine ecosystems is expected to be mineralized by PA heterotrophic communities, which are thus greatly contributing to large-scale carbon fluxes. Whilst numerous studies have investigated the succession of planktonic marine bacteria along phytoplankton blooms, the community structure and functionality of PA bacterial communities remained largely unexplored and knowledge on specific contributions of these microorganisms to carbon cycling is still surprisingly limited. This has been mostly been due to technical problems, i.e. to the difficulty to retrieve genomic DNA and proteins from these polysaccharide-rich entities, their enormous complexity and the high abundance of eukaryotic microorganisms.\n\nOur study presents an innovative, robust, reproducible, and reliable metaproteomics pipeline for marine particles, which will help to address and fill the above-described knowledge gap. Employing the here established workflow enabled us to identify more than 5,000 PA proteins, which is, at least to our knowledge, the largest number of protein groups ever assigned to marine particles. Notably, the novel pipeline has been validated by a first, comparative metaproteome analysis of free-living and PA bacterial communities indicating a significant functional shift enabling surface-associated bacteria to adapt to particle-specific living conditions. In conclusion, our novel metaproteomics pipeline presents a solid and promising methodological groundwork for future culture-independent analyses of seasonal taxonomic and functional successions of PA microbial communities in aquatic habitats.

microbiology↗

bioRxiv2019

Mitochondrial genomes of Columbicola feather lice are highly fragmented, indicating repeated evolution of minicircle-type genomes in parasitic lice

Most animals have a conserved mitochondrial genome structure composed of a single chromosome. However, some organisms have their mitochondrial genes separated on several smaller circular or linear chromosomes. Highly fragmented circular chromosomes (\"minicircles\") are especially prevalent in parasitic lice (Insecta: Phthiraptera), with 16 species known to have between 9 and 20 mitochondrial minicircles per genome. All of these species belong to the same clade (mammalian lice), suggesting a single origin of drastic fragmentation. Nevertheless, other work indicates a lesser degree of fragmentation (2-3 chromosomes/genome) is present in some avian feather lice (Ischnocera: Philopteridae). In this study, we tested for minicircles in four species of the feather louse genus Columbicola (Philopteridae). Using whole genome shotgun sequence data, we applied three different bioinformatic approaches for assembling the Columbicola mitochondrial genome. We further confirmed these approaches by assembling the mitochondrial genome of Pediculus humanus from shotgun sequencing reads, a species known to have minicircles. All three methods indicated Columbicola spp. genomes are highly fragmented into 15-17 minicircles between 1,119 and 3,173 bp in length, with 1-4 genes per minicircle. Subsequent annotation of the minicircles indicated that tRNA arrangements of minicircles varied substantially between species. These mitochondrial minicircles for species of Columbicola represent the first feather lice (Philopteridae) for which minicircles have been found in a full mitochondrial genome assembly. Combined with recent phylogenetic studies of parasitic lice, our results provide strong evidence that highly fragmented mitochondrial genomes, which are otherwise rare across the Tree of Life, evolved multiple times within parasitic lice.

evolutionary biology↗

bioRxiv2019

SKU5-Similar3 gene could be alternatively spliced into two variants at an unreported splicing site in Arabidopsis

Alternative splicing largely enhanced the diversity of transcriptome and proteome in eukaryas. Along with technical development, more and more alternatively splicing was demonstrated. Here, we report an unexpected alternative splicing of SKU5-Similar 3 (SKS3) within a special splicing site in Arabidopsis. Based on bioinformatics database, SKS3 was predicted to be alternatively transcribed into two variants, SKS3.1 and SKS3.2, which encoded a GPI-anchored protein and a soluble secretory protein respectively. But, instead of SKS3.2, a novel variant, SKS3.3, which encoded a protein with transmembrane region at its C-terminus, was demonstrated based on our experimental data. Interestingly, it exhibited a different organ-specific expression pattern from SKS3.1, and its intron splicing site did not follow GT-AG rule or any reported rules.

plant biology↗

bioRxiv2019

Comprehensive characterisation of molecular host-pathogen interactions in influenza A virus-infected human macrophages

Macrophages in the lung detect and respond to influenza A virus (IAV), determining the nature of the immune response. Using terminal depth 5-RNA sequencing (CAGE) we quantify transcriptional activity of both host and pathogen over a 24-hour timecourse of IAV infection in primary human monocyte-derived macrophages (MDM). We use a systems approach to describe the transcriptional landscape of the host response to IAV contrasted with bacterial lipopolysaccharide treated MDMs, observing a failure of IAV-treated MDMs to induce feedback inhibitors of inflammation. Systematic comparison of host RNA sequences incorporated into viral mRNA (\"snatched\") against a complete survey of background RNA in the host cell enables an unbiased quantification of over-represented features of snatched host RNAs. We detect preferential snatching of RNAs associated with snRNA transcription and demonstrate that cap-snatching avoids transcripts encoding host ribosomal proteins, which are required by IAV for replication.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC=\"FIGDIR/small/670919v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (34K):\norg.highwire.dtl.DTLVardef@b05478org.highwire.dtl.DTLVardef@79a519org.highwire.dtl.DTLVardef@462739org.highwire.dtl.DTLVardef@1b91eb6_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Abstract(A) Overview of bioinformatics pipeline. (B) Host gene expression reveals that human macrophages exposed to IAV exhibit sustained production of key inflammatory mediators and failure to induce expression of feedback inhibitors of inflammation. (C) Unbiased comparison with total background RNA expression demonstrates that IAV cap-snatching has a strong preference for, and aversion to, different groups of host transcripts.

immunology↗

bioRxiv2019

Exploring novel probiotic mechanisms of Streptococcus A12 with functional genomics

Health-associated biofilms in the oral cavity are composed of a diverse group of microbial species that can foster an environment that is less favorable for the outgrowth of dental caries pathogens, like Streptococcus mutans. A novel oral bacterium, designated Streptococcus A12, was previously isolated from supragingival dental plaque of a caries-free individual, and was shown to interfere potently with the growth and virulence properties of S. mutans. Here, we apply functional genomics to begin to identify molecular mechanisms used by A12 to antagonize, and to resist the antagonistic factors of, S. mutans. Using bioinformatics, genes that could encode factors that enhance the ability of A12 to compete with S. mutans were identified. Selected genes, designated as potential competitive factors (pcf), were deleted. Certain mutant derivatives showed a reduced capacity to compete with S. mutans compared to the parental strain. The A12 pcfO mutant lost the ability to inhibit comX-inducing peptide (XIP) signaling by S. mutans, while mutants in the pcfFEG locus were impaired in sensing of, and were more sensitive to, the lantibiotic nisin. Loss of PcfV, annotated as a colicin V biosynthetic protein, resulted in diminished antagonism of S. mutans. Collectively, the data provide new insights into the complexities and variety of factors that affect biofilm ecology and virulence. Continued exploration of the genomic and physiologic factors that distinguish commensals from truly beneficial members of the oral microbiota will lead to a better understanding of the microbiome and new approaches to promote oral health.\n\nImportanceAdvances in defining the composition of health-associated biofilms have highlighted the important role for beneficial species in maintaining health. Comparatively little, however, has been done to address the genomic and physiological basis underlying the probiotic mechanisms of beneficial commensals. In this study, we explored the ability of a novel oral bacterial isolate, Streptococcus A12, to compete with the dental pathogen Streptococcus mutans, using various gene products with diverse functions. A12 displayed enhanced competitiveness by: i) disrupting intercellular communication pathways of S. mutans, ii) sensing and resisting antimicrobial peptides, and iii) producing factors involved in the production of a putative antimicrobial compound. Research on the probiotic mechanisms employed by Streptococcus A12 is providing essential insights into how beneficial bacteria may help maintain oral health, which will aid in the development of biomarkers and therapeutics that can improve the practice of clinical dentistry.

microbiology↗

bioRxiv2019

Inherited duplications of PPP2R3B promote naevi and melanoma via a novel C21orf91-driven proliferative phenotype

The majority of the heredity of melanoma remains unexplained, however inherited copy number changes have not yet been systematically studied. The genetic environment is highly relevant to treatment stratification, and new gene discovery is therefore desirable. Using an unbiased whole genome screening approach for copy number we identify here a novel melanoma predisposing factor, familial duplications of gene PPP2R3B, encoding a regulatory unit of critical phosphatase PP2A. Significant correlation between expression of PPP2R3B in tumour tissue and survival in a large melanoma cohort was confirmed, and associated with a non-immunological expression profile. Mechanistically, construction and extensive characterization of a stable, inducible cellular model for PPP2R3B overexpression revealed induction of pigment cell switching towards proliferation and away from migration. Importantly, this was independent of the known microphthalmia-associated transcription factor (MITF)-controlled pigment cell phenotype switch, and was instead driven by uncharacterised gene C21orf91. Bioinformatic studies point to C21orf91as a novel target of MITF, and therefore a potential hub in the control of phenotype switching in melanoma. This study identifies novel germline copy number variants in PPP2R3B predisposing to melanocytic neoplasia, and uncovers a new potential therapeutic target C21orf91 in the control of pigment cell proliferation.

genomics↗

bioRxiv2019

A predicted deleterious allele of the essential meiosis gene MND1, present in ~3% of East Asians, does not disrupt reproduction in mice.

Infertility is a major health problem affecting ~15% of couples worldwide. Except for cases involving readily-detectable chromosome aberrations, confident identification of a causative genetic defect is problematic. Despite the advent of genome sequencing for diagnostic purposes, the preponderance of segregating genetic variants complicates identification of culprit genetic alleles or mutations. Many algorithms have been developed to predict the effects of \"variants of unknown significance\" (VUS), typically SNPs (single nucleotide polymorphisms), but these predictions are not sufficiently accurate for clinical action. As part of a project to identify population variants that impact fertility, we have been generating CRISPR-Cas9 edited mouse models of suspect SNPs in genes that are known to be required for fertility in mice. Here, we present data on a non-synonymous (amino acid altering) SNP (rs140107488) in the meiosis gene Mnd1, which is predicted bioinformatically to be deleterious to protein function. We report that when modeled in mice, this allele (MND1K85M), which is present allele frequency of ~3% in East Asians, has no discernable effect upon fertility, fecundity, or gametogenesis, although it may cause sex skewing of progeny from homozygous males. In sum, this appears to be a benign allele that can be eliminated or de-prioritized in clinical genomic analyses of infertility patients.

genetics↗

bioRxiv2019

Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repression. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, these homologs have been characterized to bind a 10-base consensus sequence T/GACAnnTGTA. Here, we bioinformatically and empirically characterize these operator sites across species using S. pneumoniae CopY as a guide for binding. By examining the 21-base repeat operators for the pneumococcal cop operon and comparing binding of recombinant CopY to this, and the operator sites found in Enterococcus hirae, we show using biolayer interferometry that the T/GACAnnTGTA sequence is essential to binding, but it is not sufficient. We determine a more comprehensive S. pneumoniae CopY operator sequence to be RnYKACAAATGTARnY (where \"R\" is purine, \"Y\" is pyrimidine, and \"K\" is either G or T) binding with an affinity of 28 nM. We further propose that the cop operon operator consensus site of pneumococcal homologs be RnYKACAnnYGTARnY. This study illustrates the necessity to explore bacterial operator sites further to better understand bacterial gene regulation.

microbiology↗

bioRxiv2019

Stress-responsive Entamoeba topoisomerase II: a potential anti-amoebic target

Topoisomerases are ubiquitous enzymes, involved in all DNA processes across the biological world. These enzymes are also targets for various anticancer and antimicrobial agents. The causative organism of amoebiasis, Entamoeba histolytica (Eh), has seven unexplored genes annotated as putative topoisomerases. One of the seven topoisomerases in this parasite was found to be highly up-regulated during heat shock and oxidative stress. The bioinformatic analysis shows that it is a eukaryotic type IIA topoisomerase. Its ortholog was also highly up-regulated during the late hours of encystation in E. invadens (Ei), the encystation model of Eh. Immunoprecipitated endogenous EhTopoII showed topoisomerase II activity in vitro. Immunolocalization studies show that this enzyme colocalized with newly forming nuclei during encystation, which is a significant event in maturing cysts. Double-stranded RNA mediated down-regulation of the TopoII both in Eh and Ei reduced the viability of actively growing trophozoites and also reduced the encystation efficiency in Ei. Drugs, targeting eukaryotic topoisomerase II, e.g., etoposide, ICRF193, and amsacrine, show 3-5 times higher EC50 in Eh than that of mammalian cells. Sequence comparison with human TopoII showed that key amino acid residues involved in the interactions with etoposide and ICRF193 are different in Entamoeba TopoII. Interestingly, ciprofloxacin an inhibitor of prokaryotic DNA gyrase showed about six times less EC50 value in Eh than that of human cells. The parasites notable susceptibility to prokaryotic topoisomerase drugs in comparison to human cells opens up the scope to study this invaluable enzyme in the light of an antiamoebic target.

biochemistry↗

bioRxiv2019

Show me your secret(ed) weapons: a multifaceted approach reveals novel type III-secreted effectors of a plant pathogenic bacterium

Many Gram-negative plant and animal pathogenic bacteria employ a type III secretion system (T3SS) to secrete protein effectors into the cells of their hosts and promote disease. The plant pathogen Acidovorax citrulli requires a functional T3SS for pathogenicity. As with Xanthomonas and Ralstonia spp., an AraC-type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III-secreted effectors (T3Es) in A. citrulli. A previous study reported eleven T3E genes in this pathogen, based on the annotation of a sequenced strain. We hypothesized that this was an underestimation. Guided by this hypothesis, we aimed at uncovering the T3E arsenal of the A. citrulli model strain, M6. We carried out a thorough sequence analysis searching for similarity to known T3Es from other bacteria. This analysis revealed 51 A. citrulli genes whose products are similar to known T3Es. Further, we combined machine learning and transcriptomics to identify novel T3Es. The machine learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA-Seq revealed differential gene expression between wild-type M6 and a mutant defective in HrpX. Data combined from these approaches led to the identification of seven novel T3E candidates, that were further validated using a T3SS-dependent translocation assay. These T3E genes encode hypothetical proteins, do not show any similarity to known effectors from other bacteria, and seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study not only uncovered the arsenal of T3Es of an important pathogen, but it also places A. citrulli among the \"richest\" bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.\n\nAuthor summaryAcidovorax citrulli is a Gram-negative bacterium that causes bacterial fruit blotch (BFB) disease of cucurbits. This disease represents a serious threat to cucurbit crop production worldwide. Despite the agricultural importance of BFB, the knowledge about basic aspects of A. citrulli-plant interactions is rather limited. As many Gram-negative plant and animal pathogenic bacteria, A. citrulli employs a complex secretion system, named type III secretion system, to deliver protein virulence effectors into the host cells. In this work we aimed at uncovering the arsenal of type III-secreted effectors (T3Es) of this pathogen by combination of bioinformatics and experimental approaches. We found that this bacterium possesses at least 51 genes that are similar to T3E genes from other pathogenic bacteria. In addition, our study revealed seven novel T3Es that seem to occur only in A. citrulli strains and in other plant pathogenic Acidovorax species. We found that two of these T3Es localize to the plant cell nucleus while one partially interacts with the endoplasmic reticulum. Further characterization of the novel T3Es identified in this study may uncover new host targets of pathogen effectors and new mechanisms by which pathogenic bacteria manipulate their hosts.

microbiology↗

bioRxiv2019

Effector prediction and characterization in the oomycete pathogen Bremia lactucae reveal host-recognized WY domain proteins that lack the canonical RXLR motif

Pathogens infecting plants and animals use a diverse arsenal of effector proteins to suppress the host immune system and promote infection. Identification of effectors in pathogen genomes is foundational to understanding mechanisms of pathogenesis, for monitoring field pathogen populations, and for breeding disease resistance. We identified candidate effectors from the lettuce downy mildew pathogen, Bremia lactucae, using comparative genomics and bioinformatics to search for the WY domain. This conserved structural element is found in Phytophthora effectors and some other oomycete pathogens; it has been implicated in the immune-suppressing function of these effectors as well as their recognition by host resistance proteins. We predicted 54 WY domain containing proteins in isolate SF5 of B. lactucae that have substantial variation in both sequence and domain architecture. These candidate effectors exhibit several characteristics of pathogen effectors, including an N-terminal signal peptide, lineage specificity, and expression during infection. Unexpectedly, only a minority of B. lactucae WY effectors contain the canonical N-terminal RXLR motif, which is a conserved feature in the majority of cytoplasmic effectors reported in Phytophthora spp. Functional analysis effectors containing WY domains revealed eleven out of 21 that triggered necrosis, which is characteristic of the immune response on wild accessions and domesticated lettuce lines containing resistance genes. Only two of the eleven recognized effectors contained a canonical RXLR motif, suggesting that there has been an evolutionary divergence in sequence motifs between genera; this has major consequences for robust effector prediction in oomycete pathogens.\n\nAuthor SummaryThere is a microscopic battle that takes place at the molecular level during infection of plants and animals by pathogens. Some of the weapons that pathogens battle with are known as \"effectors,\" which are secreted proteins that enter host cells to alter physiology and suppress the immune system. Effectors can also be a liability for plant pathogens because plants have evolved ways to recognize these effectors, triggering a defense response leading to localized cell death, which prevents the spread of the pathogen. Here we used computer models to predict effectors from the genome of Bremia lactucae, the causal agent of lettuce downy mildew. Three effectors were demonstrated to suppress the basal immune system of lettuce. Eleven effectors were recognized by one or more resistant lines of lettuce. In addition to contributing to our understanding of the mechanisms of pathogenesis, this study of effectors is useful for breeding disease resistant lettuce, decreasing agricultural reliance on fungicides.

plant biology↗

bioRxiv2019

Estimation of Speciation Times Under the Multispecies Coalescent

MotivationThe multispecies coalescent model is now widely accepted as an effective model for incorporating variation in the evolutionary histories of individual genes into methods for phylogenetic inference from genome-scale data. However, because model-based analysis under the coalescent can be computationally expensive for large data sets, a variety of inferential frameworks and corresponding algorithms have been proposed for estimation of species-level phylogenies and associated parameters, including speciation times and effective population sizes. ResultsWe consider the problem of estimating the timing of speciation events along a phylogeny in a coalescent framework. We propose a maximum a posteriori estimator based on composite likelihood (MAPCL) for inferring these speciation times under a model of DNA sequence evolution for which exact site pattern probabilities can be computed under the assumption of a constant{theta} throughout the species tree. We demonstrate that the MAPCL estimates are statistically consistent and asymptotically normally distributed, and we show how this result can be used to estimate their asymptotic variance. We also provide a more computationally efficient estimator of the asymptotic variance based on the nonparametric bootstrap. We evaluate the performance of our method using simulation and by application to an empirical dataset for gibbons. Availability and implementationThe method has been implemented in the PAUP* program, freely available at https://paup.phylosolutions.com for Macintosh, Windows, and Linux operating systems. Contactpeng.650@osu.edu Supplementary informationSupplementary data are available at Bioinformatics online.

evolutionary biology↗

bioRxiv2019

Identification and characterization of cis-regulatory elements for photoreceptor type-specific transcription in zebrafish

Tissue-specific or cell type-specific transcription of protein-coding genes is controlled by both trans-regulatory elements (TREs) and cis-regulatory elements (CREs). However, it is challenging to identify TREs and CREs, which are unknown for most genes. Here, we describe a protocol for identifying two types of transcription-activating CREs--core promoters and enhancers--of zebrafish photoreceptor type-specific genes. This protocol is composed of three phases: bioinformatic prediction, experimental validation, and characterization of the CREs. To better illustrate the principles and logic of this protocol, we exemplify it with the discovery of the core promoter and enhancer of the mpp5b apical polarity gene (also known as ponli), whose red, green, and blue (RGB) cone-specific transcription requires its enhancer, a member of the rainbow enhancer family. While exemplified with an RGB cone-specific gene, this protocol is general and can be used to identify the core promoters and enhancers of other protein-coding genes.

developmental biology↗

bioRxiv2019

QTL analysis of macrophages from an AKR/JxDBA/2J intercross identified the Gpnmb gene as a modifier of lysosome function

Our prior studies found differences in the AKR/J and DBA/2J strains in regard to atherosclerosis and macrophage phenotypes including cholesterol ester loading, cholesterol efflux, and autolysosome formation. The goal of this study was to determine if there were differences in macrophage lysosome function, and if so to use quantitative trait locus (QTL) analysis to identify the causal gene. Lysosome function was measured by incubation with an exogenous double-labeled ovalbumin indicator sensitive to proteolysis. DBA/2J vs. AKR/J bone marrow macrophages had significantly decreased lysosome function. Macrophages were cultured from 120 mice derived from an AKR/JxDBA/2J F4 intercross. We measured lysosome function and performed a high density genome scan. QTL analysis yielded two genome wide significant loci on chromosomes 6 and 17, called macrophage lysosome function modifier (Mlfm) loci Mlfm1 and Mlfm2. After adjusting for Mlfm1, two additional loci were identified. Based on proximity to the Mlfm1 peak, macrophage mRNA expression differences with AKR/J >> DBA/2J, and a protein coding nonsense variant in DBA/2J, the Gpnmb gene, encoding a lysosomal membrane protein, was our top candidate. To test this candidate, Gpnmb expression was knocked down with siRNA in AKR/J macrophages; and, to express the wildtype Gpnmb in DBA/2J macrophages, we obtained a DBA/2 substrain, DBA/2J-Gpnmb+/SjJ, which was isolated from the parental strain prior to its acquiring the nonsense mutation, and subsequently back crossed to the modern DBA/2J background. Knockdown of Gpnmb in AKR/J macrophages decreased lysosome function, while restoration of the wildtype Gpnmb allele in DBA/2J macrophages increased lysosome function. However, this modifier of lysosome function was not responsible for the strain differences in macrophage cholesterol ester loading or cholesterol efflux. In conclusion, we identified the Gpnmb gene as the major modifier of lysosome function and we showed that the QTL in a dish strategy is efficient in identifying modifier genes.\n\nAuthor SummaryInbred strains of mice differ in both their genetic backgrounds as well as in many traits; and, classical mouse genetics allows the mapping of genes responsible for these traits. We identified many traits that differ between the inbred strains AKR/J and DBA/2J, including atherosclerosis susceptibility, macrophage cholesterol metabolism, and in the current study, macrophage protein degradation via an organelle called the lysosome. Using mouse genetic mapping and bioinformatics we identified a candidate gene, called Gpnmb, responsible for modifying lysosome function; and, the DBA/2J strain carries a mutation in this gene. Here we demonstrate that the Gpnmb gene is a modifier of lysosome function by either correcting this Gpnmb mutation in DBA/2J macrophages, or by knocking down Gpnmb expression in AKR/J macrophages. This study is noteworthy as the human GPNMB gene has been implicated in many diseases including cancer, kidney injury, obesity, non-alcoholic steatohepatitis, Parkinson disease, osteoarthritis, and lysosome storage disorders.

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