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The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles1. Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space. Although unproven, it is generally accepted that trypanosomes reach the ectoperitrophic space by penetrating the peritrophic matrix in the anterior midgut2,3,4. Here, we revisited this event using fluorescence- and electron-microscopy methodologies. We show that trypanosomes penetrate the ectoperitrophic space in which the newly made peritrophic matrix is synthesized by the proventriculus. Our model describes how these proventriculus-colonizing parasites can either migrate to the ectoperitrophic space or become trapped within peritrophic matrix layers to form cyst-like bodies that are passively pushed along the gut as the matrix gets remodelled. Furthermore, early proventricular colonization seems to be promoted by factors in trypanosome-infected blood that cause higher salivary gland infections and potentially increase parasite transmission.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) extensively

The single-celled parasite Trypanosoma brucei causes sleeping sickness in humans and nagana in livestock and is transmitted by hematophagous tsetse flies. Lifecycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonises the glucose-poor insect midgut, its ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation. This process involves respiratory chain complexes and the F1FO-ATP synthase, and it requires protein subunits of these complexes that are encoded in the parasite’s mitochondrial DNA (kinetoplast or kDNA). Here we show that a progressive loss of kDNA-encoded functions correlates with an increasingly impaired ability of T. brucei to initiate and complete its development in the tsetse. First, parasites with a mutated F1FO-ATP synthase with a reduced capacity for oxidative phosphorylation can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonise the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonising or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1FO-ATP synthase complex that is completely unable to produce ATP by oxidative phosphorylation can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, mutant parasites lacking kDNA entirely can initiate differentiation but die within 24 h. Together, these three scenarios show that efficient ATP production via oxidative phosphorylation is not essential for initial colonisation of the tsetse vector, but it is required to power trypanosome migration within the fly.

SARS-CoV-2 extensively N-glycosylates its surface spike (S) proteins. This post-translational modification is essential to modulate protein conformation and host cell invasion. Each S monomer can be modified with up to 22 N-glycans. To meet the high demand of protein glycosylation during virus replication, SARS-CoV-2 upregulates the expression of host N-glycosylation genes. Although a substantial amount of detail is known about the structure of S protein N-glycans, the role of N-glycosylation in SARS-CoV-2 infection remains largely undetermined. Here, we investigated the essentiality of the host N-glycosylation pathway and viral N-glycans for SARS-CoV-2 infection. When either monkey or human cells were preincubated with glycosylation inhibitors, including FDA-approved iminosugars, virus infection was significantly reduced. This infection phenotype was confirmed after RNAi knockdown of several glycosylation genes. In addition, enzymatic deglycosylation of whole viral particles confirmed that accessible oligosaccharides on the SARS-CoV-2 surface are essential for host cell infection. Altogether, we show evidence that the normal functioning of the host N- glycosylation machinery is essential not only for SARS-CoV-2 to infect, but also to produce new functional virions. These findings open the door for developing new approaches targeting N-glycosylation against COVID-19.

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes., This study shows that tsetse flies, vectors of African trypanosomiasis, are highly susceptible to killing by nitisinone, a tyrosine catabolism inhibitor currently used to treat human metabolic diseases; this environment-friendly drug could facilitate elimination of African trypanosomiasis and other diseases transmitted by blood feeding insects.

As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal‐like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal‐like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air‐liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS‐CoV‐2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs., We have developed a protocol to generate airway epithelial basal‐like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways that are capable of supporting SARS‐CoV‐2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways.

The scientific community has responded to the coronavirus disease 2019 (COVID-19) pandemic by rapidly undertaking research to find effective strategies to reduce the burden of this disease. Encouragingly, researchers from a diverse array of fields are collectively working towards this goal. Research with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undertaken in high-containment laboratories; however, it is often desirable to work with samples at lower-containment levels. To facilitate the transfer of infectious samples from high-containment laboratories, we have tested methods commonly used to inactivate virus and prepare the sample for additional experiments. Incubation at 80°C, a range of detergents, Trizol reagents, and UV energies were successful at inactivating a high titer of SARS-CoV-2. Methanol and paraformaldehyde incubation of infected cells also inactivated the virus. These protocols can provide a framework for in-house inactivation of SARS-CoV-2 in other laboratories, ensuring the safe use of samples in lower-containment levels., SARS-CoV-2 research requires the safe use of samples in low-containment laboratories. Heat, UV, and chemical reagents inactivated high titers of virus in solution and cells. These methods will facilitate use of once-infectious samples in downstream procedures.

Microbiota within mosquitoes influence nutrition, immunity, fecundity, and the capacity to transmit pathogens. Despite their importance, we have a limited understanding of host-microbiota interactions, especially at the cellular level. It is evident bacterial symbionts that are localized within the midgut also infect other organs within the mosquito; however, the route these symbionts take to colonize other tissues is unknown. Here, utilizing the gentamicin protection assay, we showed that the bacterial symbionts Cedecea and Serratia have the capacity to invade and reside intracellularly within mosquito cells. Symbiotic bacteria were found within a vacuole and bacterial replication was observed in mosquito cell by transmission electron microscopy, indicating bacteria were adapted to the intracellular milieu. Using gene silencing, we determined that bacteria exploited host factors, including actin and integrin receptors, to actively invade mosquito cells. As microbiota can affect pathogens within mosquitoes, we examined the influence of intracellular symbionts on Zika virus (ZIKV) infection. Mosquito cells harbouring intracellular bacteria had significantly less ZIKV compared to uninfected cells or cells exposed to non-invasive bacteria. Intracellular bacteria were observed to substantially upregulate the Toll and IMD innate immune pathways, providing a possible mechanism mediating these anti-viral effects. Examining mono-axenically infected mosquitoes using transmission electron and fluorescent microscopy revealed that bacteria occupied an intracellular niche in vivo. Our results provided evidence that bacteria that associate with the midgut of mosquitoes have intracellular lifestyles which likely have implications for mosquito biology and pathogen infection. This study expands our understanding of host-microbiota interactions in mosquitoes, which is important as symbiont microbes are being exploited for vector control strategies.

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.

Background In the Kingdom of Saudi Arabia (KSA), Leishmania major and L. tropica are the main causative agents of Old World cutaneous leishmaniasis (CL). The national CL treatment regimen consists of topical 1% clotrimazole/2% fusidic acid cream followed by 1–2 courses of intralesional sodium stibogluconate (SSG); however, treatment efficacy is highly variable and the reasons for this are not well understood. In this study, we present a complete epidemiological map of CL and determined the efficacy of the standard CL treatment regime in several endemic regions of KSA. Results Overall, three quarters of patients in all CL-endemic areas studied responded satisfactorily to the current treatment regime, with the remaining requiring only an extra course of SSG. The majority of unresponsive cases were infected with L. tropica. Furthermore, the development of secondary infections (SI) around or within the CL lesion significantly favoured the treatment response of L. major patients but had no effect on L. tropica cases. Conclusions The response of CL patients to a national treatment protocol appears to depend on several factors, including Leishmania parasite species, geographical location and occurrences of SI. Our findings suggest there is a need to implement alternative CL treatment protocols based on these parameters. Electronic supplementary material The online version of this article (10.1186/s13071-019-3453-4) contains supplementary material, which is available to authorized users.

The peritrophic matrix (PM) of haematophagus insects is a chitinous structure that surrounds the bloodmeal, forming a protective barrier against oral pathogens and abrasive particles. To establish an infection in the tsetse midgut,Trypanosoma bruceimust colonise the ectoperitrophic space (ES), located between the PM and gut epithelium. Although unproven, it is generally accepted that trypanosomes reach the ES by directly penetrating the PM in the anterior midgut. Here we revisited this event by employing novel fluorescence and electron microscopy methodologies and found that instead, trypanosomes reach the ES via the newly secreted PM in the tsetse proventriculus. Within this model, parasites colonising the proventriculus can either migrate to the ES or become trapped within PM layers forming cysts that move along the entire gut as the PM gets remodelled. Early proventricular colonisation appears to be promoted by unidentified factors in trypanosome-infected blood, resulting in higher salivary gland infections and potentially increasing parasite transmission.

Trypanosoma bruceispp. develop into mammalian-infectious metacyclic trypomastigotes inside the tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, nothing is known about expression of invariant surface antigens by the metacyclic stage. Proteomic analysis of saliva fromT. brucei-infected flies revealed a novel family of hypothetical GPI-anchored surface proteins herein named Metacyclic Invariant Surface Proteins (MISP). MISP are encoded by five homolog genes and share ~80% protein identity. The crystal structure of MISP N-terminus at 1.82 Å resolution revealed a triple helical bundle that shares key features with other trypanosome surface proteins. However, molecular modelling combined with live fluorescent microscopy suggest that MISP N-termini are extended above the metacyclic VSG coat, exposing immunogenic epitopes. Collectively, we suggest that the metacyclic cell surface architecture appears more permissive than bloodstream forms in terms of expression of invariant GPI-anchored glycoproteins, which could be exploited for the development of novel vaccines against African trypanosomiases.

African trypanosomes are vector-borne hemoparasites of humans and animals. In the mammal, parasites evade the immune response through antigenic variation. Periodic switching of the variant surface glycoprotein (VSG) coat covering their cell surface allows sequential expansion of serologically distinct parasite clones. Trypanosome genomes contain many hundreds of VSG genes, subject to rapid changes in nucleotide sequence, copy number, and chromosomal position. Thus, analyzing, or even quantifying, VSG diversity over space and time presents an enormous challenge to conventional techniques. Indeed, previous population genomic studies have overlooked this vital aspect of pathogen biology for lack of analytical tools. Here we present a method for analyzing population-scale VSG diversity in Trypanosoma congolense from deep sequencing data. Previously, we suggested that T. congolense VSGs segregate into defined “phylotypes” that do not recombine. In our data set comprising 41 T. congolense genome sequences from across Africa, these phylotypes are universal and exhaustive. Screening sequence contigs with diagnostic protein motifs accurately quantifies relative phylotype frequencies, providing a metric of VSG diversity, called the “variant antigen profile.” We applied our metric to VSG expression in the tsetse fly, showing that certain, rare VSG phylotypes may be preferentially expressed in infective, metacyclic-stage parasites. Hence, variant antigen profiling accurately and rapidly determines the T. congolense VSG gene and transcript repertoire from sequence data, without need for manual curation or highly contiguous sequences. It offers a tractable approach to measuring VSG diversity across strains and during infections, which is imperative to understanding the host–parasite interaction at population and individual scales.

Trypanosome parasites are hiding in human skin, a discovery that may undermine efforts to eliminate sleeping sickness by 2020.