Infectivity of Trypanosoma brucei Cultivated at 28 C with Tsetse Fly Salivary Glands*

SYNOPSIS. When transformed procyclic noninfective trypanosomes of several unrelated stocks of Trypanosoma brucei were cultivated in T-30 Falcon flasks at 28 C in a liquid medium containing head-salivary gland explants of Glossina morsitans morsitans some of the organisms developed into forms infective for mice. Infective trypanosomes were detected 7 to 14 days after the cultures were prepared and they persisted for varying periods of up to 88 days when the cultures were terminated. A few of the salivary glands became invaded with parasites about the time infective organisms appeared in the cultures. Using T. brucei TREU 929, it was shown that trypanosomes grown with between 27 and 50 explants were capable of producing infections consistently for prolonged periods. On the other hand, trypanosomes cultivated with 25 or fewer explants rarely infected mice. Infectivity titrations on trypanosome suspensions from cultures of stocks TREU 1275 and TREU 929 revealed that the maximum number of infective organisms was present 26 to 50 days after initiation of the cultures. Control cultures of trypanosomes grown in medium alone were generally not infective but 2 of the 6 stocks gave rise to a few sporadic infections. A few epimastigote-like and metacyclic-like trypanosomes were seen in stained preparations of infective inocula.     

Identification of a Tsetse Fly Salivary Protein with Dual Inhibitory Action on Human Platelet Aggregation

Background

Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated.

Methodology/Principal Findings

Here we report on the functional characterisation of a 5′nucleotidase-related (5′Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5′Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5′nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5′Nuc/SUMO-fusion protein yielded an active apyrase enzyme with K_m and V_max values of 43±4 µM and 684±49 nmol Pi/min×mg for ATPase and 49±11 µM and 177±37 nmol Pi/min×mg for the ADPase activity. In addition, recombinant 5′Nuc was found to bind to GPIIb/IIIa with an apparent K_D of 92±25 nM. Consistent with these features, 5′Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5′Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process.

Conclusions/Significance

These data show that this 5′nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva.     

New Insight into Benign Tumours of Major Salivary Glands by Proteomic Approach

Major salivary gland tumours are uncommon neoplasms of the head and neck. The increase of precise pre-operative diagnosis is crucial for their correct management and the identification of molecular markers would surely improve the required accuracy. In this study we performed a comparative proteomic analysis of fine needle aspiration fluids of the most frequent benign neoplasms of major salivary glands, namely pleomorphic adenoma and Warthin''s tumour, in order to draw their proteomic profiles and to point out their significant features. Thirty-five patients submitted to parotidectomy were included in the study, 22 were identified to have pleomorphic adenoma and 14 Warthin''s tumour. Fine needle aspiration samples were processed using a two-dimensional electrophoresis/mass spectrometry-based approach. A total of 26 differentially expressed proteins were identified. Ingenuity software was used to search the biological processes to which these proteins belong and to construct potential networks. Intriguingly, all Warthin''s tumour up-regulated proteins such as Ig gamma-1 chain C region, Ig kappa chain C region and Ig alpha-1 chain C region and S100A9 were correlated to immunological and inflammatory diseases, while pleomorphic adenomas such as annexin A1, annexin A4, macrophage-capping protein, apolipoprotein E and alpha crystalline B chain were associated with cell death, apoptosis and tumorigenesis, showing different features of two benign tumours. Overall, our results shed new light on the potential usefulness of a proteomic approach to study parotid tumours and in particular up regulated proteins are able to discriminate two types of benign parotid lesions.     

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.     

Insights into the Evolution of Nuclear Dualism in the Ciliates Revealed by Phylogenetic Analysis of rRNA Sequences

The small subunit rRNA gene sequences of the karyorelictean ciliates, Loxodes striatus and Protocruzia sp., and the heterotrichian ciliates, Climacostomum virens and Eufolliculina uhligi , were used to test the evolution of nuclear dualism in the Phylum Ciliophora. Phylogenies derived using a least squares distance method, neighbour joining, and maximum parsimony demonstrate that the karyorelictean ciliates sensu Small and Lynn, 1985 do not form a monophyletic group. However, Loxodes and the heterotrich ciliates form the first branch in the ciliate lineage, and Protocruzia branches, in distance methods, basal to the spirotrich lineage. It is proposed that Protocruzia be removed from the Class Karyorelictea, and placed in closer taxonomic association with the spirotrich lineage. The distribution of nuclear division types along the phylogenetic tree is consistent with the notion that macronuclei incapable of division represent a derived rather than a primitive or "karyorelictid" character trait.     

Insights into the Biology of Hearing and Deafness Revealed by Single-Cell RNA Sequencing

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Structural Insights into the Interactions between Platelet Receptors and Fibrillar Collagen

Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin α₂β₁, glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of platelet collagen receptors onto the intact collagen fiber in three dimensions. This minireview will discuss these recent findings and their implications for platelet activation by collagen.     

Analysis of Multiple Tsetse Fly Populations in Uganda Reveals Limited Diversity and Species-Specific Gut Microbiota

The invertebrate microbiome contributes to multiple aspects of host physiology, including nutrient supplementation and immune maturation processes. We identified and compared gut microbial abundance and diversity in natural tsetse flies from Uganda using five genetically distinct populations of Glossina fuscipes fuscipes and multiple tsetse species (Glossina morsitans morsitans, G. f. fuscipes, and Glossina pallidipes) that occur in sympatry in one location. We used multiple approaches, including deep sequencing of the V4 hypervariable region of the 16S rRNA gene, 16S rRNA gene clone libraries, and bacterium-specific quantitative PCR (qPCR), to investigate the levels and patterns of gut microbial diversity from a total of 151 individuals. Our results show extremely limited diversity in field flies of different tsetse species. The obligate endosymbiont Wigglesworthia dominated all samples (>99%), but we also observed wide prevalence of low-density Sodalis (tsetse''s commensal endosymbiont) infections (<0.05%). There were also several individuals (22%) with high Sodalis density, which also carried coinfections with Serratia. Albeit in low density, we noted differences in microbiota composition among the genetically distinct G. f. fuscipes flies and between different sympatric species. Interestingly, Wigglesworthia density varied in different species (10⁴ to 10⁶ normalized genomes), with G. f. fuscipes having the highest levels. We describe the factors that may be responsible for the reduced diversity of tsetse''s gut microbiota compared to those of other insects. Additionally, we discuss the implications of Wigglesworthia and Sodalis density variations as they relate to trypanosome transmission dynamics and vector competence variations associated with different tsetse species.     

Analysis of Whitefly Transcriptional Responses to Beauveria bassiana Infection Reveals New Insights into Insect-Fungus Interactions

Background

The fungal pathogen, Beauveria bassiana, is an efficient biocontrol agent against a variety of agricultural pests. A thorough understanding of the basic principles of insect-fungus interactions may enable the genetic modification of Beauveria bassiana to enhance its virulence. However, the molecular mechanism of insect response to Beauveria bassiana infection is poorly understood, let alone the identification of fungal virulent factors involved in pathogenesis.

Methodology/Principal Findings

Here, next generation sequencing technology was applied to examine the expression of whitefly (Bemisia tabaci) genes in response to the infection of Beauveria bassiana. Results showed that, compared to control, 654 and 1,681genes were differentially expressed at 48 hours and 72 hours post-infected whiteflies, respectively. Functional and enrichment analyses indicated that the DNA damage stimulus response and drug metabolism were important anti-fungi strategies of the whitefly. Mitogen-activated protein kinase (MAPK) pathway was also likely involved in the whitefly defense responses. Furthermore, the notable suppression of general metabolism and ion transport genes observed in 72 hours post-infected B. tabaci might be manipulated by fungal secreted effectors. By mapping the sequencing tags to B. bassiana genome, we also identified a number of differentially expressed fungal genes between the early and late infection stages. These genes are generally associated with fungal cell wall synthesis and energy metabolism. The expression of fungal cell wall protein genes might play an important role in fungal pathogenesis and the dramatically up-regulated enzymes of carbon metabolism indicate the increasing usage of energy during the fungal infection.

Conclusions/Significance

To our knowledge, this is the first report on the molecular mechanism of fungus-whitefly interactions. Our results provide a road map for future investigations on insect-pathogen interactions and genetically modifying the fungus to enhance its efficiency in whitefly control.     

Insights into the biology of Escherichia coli through structural proteomics

Escherichia coli has historically been an important organism for understanding a multitude of biological processes, and represents a model system as we attempt to simulate the workings of living cells. Many E. coli strains are also important human and animal pathogens for which new therapeutic strategies are required. For both reasons, a more complete and comprehensive understanding of the protein structure complement of E. coli is needed at the genome level. Here, we provide examples of insights into the mechanism and function of bacterial proteins that we have gained through the Bacterial Structural Genomics Initiative (BSGI), focused on medium-throughput structure determination of proteins from E. coli. We describe the structural characterization of several enzymes from the histidine biosynthetic pathway, the structures of three pseudouridine synthases, enzymes that synthesize one of the most abundant modified bases in RNA, as well as the combined use of protein structure and focused functional analysis to decipher functions for hypothetical proteins. Together, these results illustrate the power of structural genomics to contribute to a deeper biological understanding of bacterial processes.