De novo assembly and annotation of the Amblyomma hebraeum tick midgut transcriptome response to Ehrlichia ruminantium infection.

The South African bont tick Amblyomma hebraeum is a hematophagous vector for the heartwater disease pathogen Ehrlichia ruminantium in southern Africa. During feeding, the tick's enterocytes express proteins that perform vital functions in blood digestion, including proteins that may be involved in E. ruminantium acquisition, colonization or immunity. To delineate the molecular mechanism of midgut response to E. ruminantium infection, we performed comparative analyses of midgut transcriptomes of E. ruminantium infected engorged A. hebraeum nymphs, and infected adult male and female ticks with their corresponding matched uninfected controls, before and during feeding. A total of 102,036 unigenes were annotated in public databases and their expression levels analyzed for engorged nymphs as well as unfed and partly-fed adult ticks. There were 2,025 differentially expressed genes (DEGs) in midguts, of which 1,225 unigenes were up-regulated and 800 unigenes were down-regulated in the midguts of infected ticks. Annotation of DEGs revealed an increase in metabolic and cellular processes among E. ruminantium infected ticks. Notably, among the infected ticks, there was up-regulation in the expression of genes involved in tick immunity, histone proteins and oxidative stress responses. We also observed up-regulation of glycoproteins that E. ruminantium could potentially use as docking sites for host cell entry. Insights uncovered in this study offer a platform for further investigations into the molecular interaction between E. ruminantium and A. hebraeum.

Gene co-expression network identifies critical genes, pathways and regulatory motifs mediating the progression of rift valley fever in Bostaurus.

Rift Valley Fever (RVF) is a mosquito-borne viral disease caused by the Rift Valley Fever Virus. The disease is a zoonosis that largely affects domestic animals, including sheep, goats, and cattle, resulting in severe morbidity and mortality marked by massive storm abortions. To halt human and livestock deaths due to RVF, the development of efficacious vaccines and therapeutics is a compelling and urgent priority. We sought to identify potential key modules (gene clusters), hub genes, and regulatory motifs involved in the pathogenesis of RVF in Bos taurus that are amenable to inhibition. We analyzed 39 Bos taurus RNA-Seq samples using the weighted gene co-expression network analysis (WGCNA) R package and uncovered significantly enriched modules containing genes with potential pivotal roles in RVF progression. Moreover, regulatory motif analysis conducted using the Multiple Expectation Maximization for Motif Elicitation (MEME) suite identified motifs that probably modulate vital biological processes. Gene ontology terms associated with identified motifs were inferred using the GoMo human database. The gene co-expression network constructed in WGCNA using 5000 genes contained seven (7) modules, out of which four were significantly enriched for terms associated with response to viruses, response to interferon-alpha, innate immune response, and viral defense. Additionally, several biological pathways implicated in developmental processes, anatomical structure development, and multicellular organism development were identified. Regulatory motifs analysis identified short, repeated motifs whose function(s) may be amenable to disruption by novel therapeutics. Predicted functions of identified motifs include tissue development, embryonic organ development, and organ morphogenesis. We have identified several hub genes in enriched co-expressed gene modules and regulatory motifs potentially involved in the pathogenesis of RVF in B. taurus that are likely viable targets for disruption by novel therapeutics.

Draft genome sequences of two strains of Staphylococcus aureus isolated from mastitis-infected camel in Kajiado County, Kenya.

We report the draft genome sequences of two Staphylococcus aureus strains isolated from a mastitis-infected camel in Kajiado County, Kenya. The 2,739,512-bp and 3,025,943-bp draft genomes coding for 2,577 and 2,889 protein sequences, respectively, provide invaluable data for the computational design of a camel mastitis subunit vaccine.

Draft Genome Sequences of Enterococcus faecium, Enterococcus gallinarum, and Lactococcus lactis Strains Isolated from a Mastitis-Infected Camel in Isiolo County, Kenya.

We report the draft genome sequences and annotation of Enterococcus faecium, Enterococcus gallinarum, and Lactococcus lactis isolates that were recovered from a mastitis-infected camel in Isiolo County, Kenya. Collectively, these data provide an invaluable repository for data mining to support the development of a potential multicomponent mastitis subunit vaccine.

Draft Genome Sequence of Streptococcus agalactiae KALRO-LC1 Strain Isolated from a Mastitis-Infected Camel in Laikipia County, Kenya.

We report the draft genome sequence of Streptococcus agalactiae KALRO-LC1 strain obtained from a mastitis-infected camel in Laikipia County, Kenya. The 2,201,604-bp draft genome is assembled into 3 contigs with a GC content of 35.87% and is predicted to contain 1,192 protein-coding sequences.

Draft Genome Sequence of Enterococcus faecalis 1351, Isolated from a Mastitis-Affected Camel in Isiolo County, Kenya.

Enterococcus faecalis causes mastitis disease in livestock, leading to massive economic losses. Sequencing of isolates obtained from resource-poor regions will facilitate the design of novel sensitive diagnostics and efficacious vaccines. We announce the draft genome of E. faecalis strain 1351, which was obtained from a camel in Isiolo County, Kenya.

Potentially zoonotic gastrointestinal nematodes co-infecting free ranging non-human primates in Kenyan urban centres.

BACKGROUND: Natural infections with soil-transmitted nematodes occur in non-human primates (NHPs) and have the potential to cross primate-species boundaries and cause diseases of significant public health concern. Despite the presence of NHPs in most urban centres in Kenya, comprehensive studies on their gastrointestinal parasites are scant. OBJECTIVE: Conduct a cross-sectional survey to identify zoonotic nematodes in free-ranging NHPs found within four selected urban and peri-urban centres in Kenya. METHODS: A total of 86 NHPs: 41 African green monkeys [AGMs] (Chlorocebus aethiops), 30 olive baboons (Papio anubis), 5 blue monkeys (Cercopithecus mitis stuhlmanni) and 10 red-tailed monkeys (Cercopithecus ascanius) were sampled once in situ and released back to their habitat. Microscopy was used to identify nematodes egg and larvae stages in the samples. Subsequently, PCR coupled with high-resolution melting (PCR-HRM) analysis and sequencing were used to identify nodule worms. RESULTS: NHPs inhabiting densely populated urban environs in Kenya were found infected with a rich diversity of nematodes including three potentially zoonotic nematodes including Oesophagostomum stephanostomum, Oesophagostomum bifurcum and Trichostrongylus colubriformis and co-infections were common. CONCLUSION: Phylogenetic analysis showed that O. stephanostomum from red-tailed and blue monkeys have a close evolutionary relatedness to human isolates suggesting the zoonotic potential of this parasite. Moreover, we also report the first natural co-infection of O. bifurcum and O. stephanostomum in free-ranging AGMs.

Broad diversity of simian immunodeficiency virus infecting Chlorocebus species (African green monkey) and evidence of cross-species infection in Papio anubis (olive baboon) in Kenya.

BACKGROUND: Simian immunodeficiency virus (SIV) naturally infects African non-human primates (NHPs) and poses a threat of transmission to humans through hunting and consumption of monkeys as bushmeat. This study investigated the as of yet unknown molecular diversity of SIV in free-ranging Chlorocebus species (African green monkeys-AGMs) and Papio anubis (olive baboons) within Mombasa, Kisumu and Naivasha urban centres in Kenya. METHODS: We collected blood samples from 124 AGMs and 65 olive baboons in situ, and detected SIV by high-resolution melting analysis and sequencing of PCR products. RESULTS: Simian immunodeficiency virus prevalence was 32% in AGMs and 3% in baboons. High-resolution melting (HRM) analysis demonstrated distinct melt profiles illustrating virus diversity confirmed by phylogenetic analysis. CONCLUSIONS: There is persistent evolutionary diversification of SIVagm strains in its natural host, AGMs and cross-species infection to olive baboons is occurring. Further study is required to establish pathogenesis of the diverse SIVagm variants and baboon immunological responses.

Coevolution of hytrosaviruses and host immune responses.

BACKGROUND: Hytrosaviruses (SGHVs; Hytrosaviridae family) are double-stranded DNA (dsDNA) viruses that cause salivary gland hypertrophy (SGH) syndrome in flies. Two structurally and functionally distinct SGHVs are recognized; Glossina pallidipes SGHV (GpSGHV) and Musca domestica SGHV (MdSGHV), that infect the hematophagous tsetse fly and the filth-feeding housefly, respectively. Genome sizes and gene contents of GpSGHV (~ 190 kb; 160-174 genes) and MdSGHV (~ 124 kb; 108 genes) may reflect an evolution with the SGHV-hosts resulting in differences in pathobiology. Whereas GpSGHV can switch from asymptomatic to symptomatic infections in response to certain unknown cues, MdSGHV solely infects symptomatically. Overt SGH characterizes the symptomatic infections of SGHVs, but whereas MdSGHV induces both nuclear and cellular hypertrophy (enlarged non-replicative cells), GpSGHV induces cellular hyperplasia (enlarged replicative cells). Compared to GpSGHV's specificity to Glossina species, MdSGHV infects other sympatric muscids. The MdSGHV-induced total shutdown of oogenesis inhibits its vertical transmission, while the GpSGHV's asymptomatic and symptomatic infections promote vertical and horizontal transmission, respectively. This paper reviews the coevolution of the SGHVs and their hosts (housefly and tsetse fly) based on phylogenetic relatedness of immune gene orthologs/paralogs and compares this with other virus-insect models. RESULTS: Whereas MdSGHV is not vertically transmitted, GpSGHV is both vertically and horizontally transmitted, and the balance between the two transmission modes may significantly influence the pathogenesis of tsetse virus. The presence and absence of bacterial symbionts (Wigglesworthia and Sodalis) in tsetse and Wolbachia in the housefly, respectively, potentially contributes to the development of SGH symptoms. Unlike MdSGHV, GpSGHV contains not only host-derived proteins, but also appears to have evolutionarily recruited cellular genes from ancestral host(s) into its genome, which, although may be nonessential for viral replication, potentially contribute to the evasion of host's immune responses. Whereas MdSGHV has evolved strategies to counteract both the housefly's RNAi and apoptotic responses, the housefly has expanded its repertoire of immune effector, modulator and melanization genes compared to the tsetse fly. CONCLUSIONS: The ecologies and life-histories of the housefly and tsetse fly may significantly influence coevolution of MdSGHV and GpSGHV with their hosts. Although there are still many unanswered questions regarding the pathogenesis of SGHVs, and the extent to which microbiota influence expression of overt SGH symptoms, SGHVs are attractive 'explorers' to elucidate the immune responses of their hosts, and the transmission modes of other large DNA viruses.

Genome-Wide Identification and Evolutionary Analysis of Sarcocystis neurona Protein Kinases.

The apicomplexan parasite Sarcocystis neurona causes equine protozoal myeloencephalitis (EPM), a degenerative neurological disease of horses. Due to its host range expansion, S. neurona is an emerging threat that requires close monitoring. In apicomplexans, protein kinases (PKs) have been implicated in a myriad of critical functions, such as host cell invasion, cell cycle progression and host immune response evasion. Here, we used various bioinformatics methods to define the kinome of S. neurona and phylogenetic relatedness of its PKs to other apicomplexans. We identified 97 putative PKs clustering within the various eukaryotic kinase groups. Although containing the universally-conserved PKA (AGC group), S. neurona kinome was devoid of PKB and PKC. Moreover, the kinome contains the six-conserved apicomplexan CDPKs (CAMK group). Several OPK atypical kinases, including ROPKs 19A, 27, 30, 33, 35 and 37 were identified. Notably, S. neurona is devoid of the virulence-associated ROPKs 5, 6, 18 and 38, as well as the Alpha and RIO kinases. Two out of the three S. neurona CK1 enzymes had high sequence similarities to Toxoplasma gondii TgCK1-α and TgCK1-ß and the Plasmodium PfCK1. Further experimental studies on the S. neurona putative PKs identified in this study are required to validate the functional roles of the PKs and to understand their involvement in mechanisms that regulate various cellular processes and host-parasite interactions. Given the essentiality of apicomplexan PKs in the survival of apicomplexans, the current study offers a platform for future development of novel therapeutics for EPM, for instance via application of PK inhibitors to block parasite invasion and development in their host.

Correction: Genome-Wide Comparative Analysis of Chemosensory Gene Families in Five Tsetse Fly Species.

[This corrects the article DOI: 10.1371/journal.pntd.0004421.].

A proteomics approach reveals molecular manipulators of distinct cellular processes in the salivary glands of Glossina m. morsitans in response to Trypanosoma b. brucei infections.

BACKGROUND: Glossina m. morsitans is the primary vector of the Trypanosoma brucei group, one of the causative agents of African trypanosomoses. The parasites undergo metacyclogenesis, i.e. transformation into the mammalian-infective metacyclic trypomastigote (MT) parasites, in the salivary glands (SGs) of the tsetse vector. Since the MT-parasites are largely uncultivable in vitro, information on the molecular processes that facilitate metacyclogenesis is scanty. METHODS: To bridge this knowledge gap, we employed tandem mass spectrometry to investigate protein expression modulations in parasitized (T. b. brucei-infected) and unparasitized SGs of G. m. morsitans. We annotated the identified proteins into gene ontologies and mapped the up- and downregulated proteins within protein-protein interaction (PPI) networks. RESULTS: We identified 361 host proteins, of which 76.6 % (n = 276) and 22.3 % (n = 81) were up- and downregulated, respectively, in parasitized SGs compared to unparasitized SGs. Whilst 32 proteins were significantly upregulated (> 10-fold), only salivary secreted adenosine was significantly downregulated. Amongst the significantly upregulated proteins, there were proteins associated with blood feeding, immunity, cellular proliferation, homeostasis, cytoskeletal traffic and regulation of protein turnover. The significantly upregulated proteins formed major hubs in the PPI network including key regulators of the Ras/MAPK and Ca(2+)/cAMP signaling pathways, ubiquitin-proteasome system and mitochondrial respiratory chain. Moreover, we identified 158 trypanosome-specific proteins, notable of which were proteins in the families of the GPI-anchored surface glycoproteins, kinetoplastid calpains, peroxiredoxins, retrotransposon host spot multigene and molecular chaperones. Whilst immune-related trypanosome proteins were over-represented, membrane transporters and proteins involved in translation repression (e.g. ribosomal proteins) were under-represented, potentially reminiscent of the growth-arrested MT-parasites. CONCLUSIONS: Our data implicate the significantly upregulated proteins as manipulators of diverse cellular processes in response to T. b. brucei infection, potentially to prepare the MT-parasites for invasion and evasion of the mammalian host immune defences. We discuss potential strategies to exploit our findings in enhancement of trypanosome refractoriness or reduce the vector competence of the tsetse vector.

Comparative Analysis of Salivary Gland Proteomes of Two Glossina Species that Exhibit Differential Hytrosavirus Pathologies.

Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) is a dsDNA virus exclusively pathogenic to tsetse flies (Diptera; Glossinidae). The 190 kb GpSGHV genome contains 160 open reading frames and encodes more than 60 confirmed proteins. The asymptomatic GpSGHV infection in flies can convert to symptomatic infection that is characterized by overt salivary gland hypertrophy (SGH). Flies with SGH show reduced general fitness and reproductive dysfunction. Although the occurrence of SGH is an exception rather than the rule, G. pallidipes is thought to be the most susceptible to expression of overt SGH symptoms compared to other Glossina species that are largely asymptomatic. Although Glossina salivary glands (SGs) play an essential role in GpSGHV transmission, the functions of the salivary components during the virus infection are poorly understood. In this study, we used mass spectrometry to study SG proteomes of G. pallidipes and G. m. morsitans, two Glossina model species that exhibit differential GpSGHV pathologies (high and low incidence of SGH, respectively). A total of 540 host proteins were identified, of which 23 and 9 proteins were significantly up- and down-regulated, respectively, in G. pallidipes compared to G. m. morsitans. Whereas 58 GpSGHV proteins were detected in G. pallidipes F1 progenies, only 5 viral proteins were detected in G. m. morsitans. Unlike in G. pallidipes, qPCR assay did not show any significant increase in virus titers in G. m. morsitans F1 progenies, confirming that G. m. morsitans is less susceptible to GpSGHV infection and replication compared to G. pallidipes. Based on our results, we speculate that in the case of G. pallidipes, GpSGHV employs a repertoire of host intracellular signaling pathways for successful infection. In the case of G. m. morsitans, antiviral responses appeared to be dominant. These results are useful for designing additional tools to investigate the Glossina-GpSGHV interactions.

Resistance related metabolic pathways for drug target identification in Mycobacterium tuberculosis.

BACKGROUND: Increasing resistance to anti-tuberculosis drugs has driven the need for developing new drugs. Resources such as the tropical disease research (TDR) target database and AssessDrugTarget can help to prioritize putative drug targets. Hower, these resources do not necessarily map to metabolic pathways and the targets are not involved in dormancy. In this study, we specifically identify drug resistance pathways to allow known drug resistant mutations in one target to be offset by inhibiting another enzyme of the same metabolic pathway. One of the putative targets, Rv1712, was analysed by modelling its three dimensional structure and docking potential inhibitors. RESULTS: We mapped 18 TB drug resistance gene products to 15 metabolic pathways critical for mycobacterial growth and latent TB by screening publicly available microarray data. Nine putative targets, Rv1712, Rv2984, Rv2194, Rv1311, Rv1305, Rv2195, Rv1622c, Rv1456c and Rv2421c, were found to be essential, to lack a close human homolog, and to share >67 % sequence identity and >87 % query coverage with mycobacterial orthologs. A structural model was generated for Rv1712, subjected to molecular dynamic simulation, and identified 10 compounds with affinities better than that for the ligand cytidine-5'-monophosphate (C5P). Each compound formed more interactions with the protein than C5P. CONCLUSIONS: We focused on metabolic pathways associated with bacterial drug resistance and proteins unique to pathogenic bacteria to identify novel putative drug targets. The ten compounds identified in this study should be considered for experimental studies to validate their potential as inhibitors of Rv1712.

Genome-Wide Comparative Analysis of Chemosensory Gene Families in Five Tsetse Fly Species.

For decades, odour-baited traps have been used for control of tsetse flies (Diptera; Glossinidae), vectors of African trypanosomes. However, differential responses to known attractants have been reported in different Glossina species, hindering establishment of a universal vector control tool. Availability of full genome sequences of five Glossina species offers an opportunity to compare their chemosensory repertoire and enhance our understanding of their biology in relation to chemosensation. Here, we identified and annotated the major chemosensory gene families in Glossina. We identified a total of 118, 115, 124, and 123 chemosensory genes in Glossina austeni, G. brevipalpis, G. f. fuscipes, G. pallidipes, respectively, relative to 127 reported in G. m. morsitans. Our results show that tsetse fly genomes have fewer chemosensory genes when compared to other dipterans such as Musca domestica (n>393), Drosophila melanogaster (n = 246) and Anopheles gambiae (n>247). We also found that Glossina chemosensory genes are dispersed across distantly located scaffolds in their respective genomes, in contrast to other insects like D. melanogaster whose genes occur in clusters. Further, Glossina appears to be devoid of sugar receptors and to have expanded CO2 associated receptors, potentially reflecting Glossina's obligate hematophagy and the need to detect hosts that may be out of sight. We also identified, in all species, homologs of Ir84a; a Drosophila-specific ionotropic receptor that promotes male courtship suggesting that this is a conserved trait in tsetse flies. Notably, our selection analysis revealed that a total of four gene loci (Gr21a, GluRIIA, Gr28b, and Obp83a) were under positive selection, which confers fitness advantage to species. These findings provide a platform for studies to further define the language of communication of tsetse with their environment, and influence development of novel approaches for control.

A comparative analysis of trypanosomatid SNARE proteins.

The Kinetoplastida are flagellated protozoa evolutionary distant and divergent from yeast and humans. Kinetoplastida include trypanosomatids, and a number of important pathogens. Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. inflict significant morbidity and mortality on humans and livestock as the etiological agents of human African trypanosomiasis, Chagas' disease and leishmaniasis respectively. For all of these organisms, intracellular trafficking is vital for maintenance of the host-pathogen interface, modulation/evasion of host immune system responses and nutrient uptake. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are critical components of the intracellular trafficking machinery in eukaryotes, mediating membrane fusion and contributing to organelle specificity. We asked how the SNARE complement evolved across the trypanosomatids. An in silico search of the predicted proteomes of T. b. brucei and T. cruzi was used to identify candidate SNARE sequences. Phylogenetic analysis, including comparisons with yeast and human SNAREs, allowed assignment of trypanosomatid SNAREs to the Q or R subclass, as well as identification of several SNAREs orthologous with those of opisthokonts. Only limited variation in number and identity of SNAREs was found, with Leishmania major having 27 and T. brucei 26, suggesting a stable SNARE complement post-speciation. Expression analysis of T. brucei SNAREs revealed significant differential expression between mammalian and insect infective forms, especially within R and Qb-SNARE subclasses, suggesting possible roles in adaptation to different environments. For trypanosome SNAREs with clear orthologs in opisthokonts, the subcellular localization of TbVAMP7C is endosomal while both TbSyn5 and TbSyn16B are at the Golgi complex, which suggests conservation of localization and possibly also function. Despite highly distinct life styles, the complement of trypanosomatid SNAREs is quite stable between the three pathogenic lineages, suggesting establishment in the last common ancestor of trypanosomes and Leishmania. Developmental changes to SNARE mRNA levels between blood steam and procyclic life stages suggest that trypanosomes modulate SNARE functions via expression. Finally, the locations of some conserved SNAREs have been retained across the eukaryotic lineage.

Evolution and Structural Analyses of Glossina morsitans (Diptera; Glossinidae) Tetraspanins.

Tetraspanins are important conserved integral membrane proteins expressed in many organisms. Although there is limited knowledge about the full repertoire, evolution and structural characteristics of individual members in various organisms, data obtained so far show that tetraspanins play major roles in membrane biology, visual processing, memory, olfactory signal processing, and mechanosensory antennal inputs. Thus, these proteins are potential targets for control of insect pests. Here, we report that the genome of the tsetse fly, Glossina morsitans (Diptera: Glossinidae) encodes at least seventeen tetraspanins (GmTsps), all containing the signature features found in the tetraspanin superfamily members. Whereas six of the GmTsps have been previously reported, eleven could be classified as novel because their amino acid sequences do not map to characterized tetraspanins in the available protein data bases. We present a model of the GmTsps by using GmTsp42Ed, whose presence and expression has been recently detected by transcriptomics and proteomics analyses of G. morsitans. Phylogenetically, the identified GmTsps segregate into three major clusters. Structurally, the GmTsps are largely similar to vertebrate tetraspanins. In view of the exploitation of tetraspanins by organisms for survival, these proteins could be targeted using specific antibodies, recombinant large extracellular loop (LEL) domains, small-molecule mimetics and siRNAs as potential novel and efficacious putative targets to combat African trypanosomiasis by killing the tsetse fly vector.

Evolutionary genomics of Glossina morsitans immune-related CLIP domain serine proteases and serine protease inhibitors.

Several species of haematophagous tsetse flies (genus Glossina) are vectors for trypanosomes, the parasitic protozoans that cause Human African Trypanosomiasis (HAT). Although there was a reduced incidence of HAT in the mid 1960s, decreased disease surveillance has led to a resurgence of HAT in sub-Saharan Africa. Despite being efficient vectors for HAT transmission, the prevalence of G. morsitans infection by trypanosomes in the wild is surprisingly minimal. The precise mechanisms by which G. morsitans remain refractory to trypanosome infection are largely unknown although it has been demonstrated that G. morsitans mounts a strong immune response to invading pathogens. This study identifies G. morsitans immune-related CLIP domain serine proteases and their inhibitors, serine protease inhibitors (serpin) genes. It further establishes their evolutionary relationships with counterparts in Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Manduca sexta and Culex quinquefasciatus. Multiple sequence alignments show conservation of most secondary structure elements for both CLIPs and serpins. Amino acid composition of the serpin reactive site loop (RSL) indicates that the G. morsitans serpins act through an inhibitory mechanism to the target serine protease. Similar to D. melanogaster and unlike A. gambiae, the transcriptome data suggest that G. morsitans does not contain gene expansions in their CLIP-domain serine protease and serpin families. The presence of alternatively spliced variants in the G. morsitans serpins transcriptome data mirrors that of the D. melanogaster transcriptome.