Biology subjects

Acosta-Serrano, A.

Publications and source records attributed to Acosta-Serrano, A..

12 recordsLinked to original sources

bioRxiv2025

The beta-triketone, nitisinone, kills insecticide-resistant mosquitoes through cuticular uptake

The efficacy of numerous vector control initiatives is compromised by growing insecticide resistance among disease-transmitting arthropods of agricultural, veterinary, and public health significance. Previous investigations on hematophagous (blood-feeding) arthropod vectors, including mosquitoes, have indicated that ingesting blood containing inhibitors of the second enzyme in the tyrosine metabolism pathway, 4-hydroxyphenylpyruvate dioxygenase (HPPD), results in high insect mortality. Building upon this foundation, we evaluated the insecticidal efficacy of the HPPD inhibitor, nitisinone, against susceptible and pyrethroid-resistant strains of three mosquito species: Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus. These mosquitoes are vectors of historical diseases such as malaria, emerged diseases such as Dengue and Zika and emerging viral diseases such as the Oropouche and Usutu viruses. We demonstrate, by employing standard screening assays designed to assess the cuticular uptake of mosquitocidal agents, that nitisinone has mosquitocidal activity when blood-fed mosquitoes contact a nitisinone-coated surface. Notably, there is no discernible disparity in susceptibility to nitisinone between an insecticide-susceptible strain of Anopheles gambiae and two strains carrying multiple insecticide-resistance mechanisms. We conclude that the mosquitocidal mode of action of nitisinone differs from any of the current 37 classes of insecticides as none have a mode of action that specifically interferes with blood digestion. By highlighting the efficacy of nitisinone as a contact-based insecticide, our findings support the potential expansion of vector control strategies where nitisinone is incorporated into classic interventions like treated bednets and indoor residual spraying.

zoology↗

bioRxiv2024

Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates of Trypanosoma brucei infection

BackgroundTsetse flies (Glossina sp.) are vectors of Trypanosoma brucei subspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitive T. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naive tsetse with T. brucei DNA via faeces when co-housed. Methodology/Principle FindingsInsectary-reared teneral G. morsitans morsitans were fed an infectious T. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naive tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive for T. brucei DNA using TBR-qPCR. However, naive tsetse also tested positive. Even at a ratio of 1 infected to 11 naive flies, 91% of naive tsetse gave positive TBR-qPCR results. Furthermore, the quantity of T. brucei DNA detected in naive tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]). Conclusions/SignificanceOur results show that infected tsetse can contaminate naive flies with T. brucei DNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives. Author SummaryTsetse flies (Glossina sp.) are vectors of Trypanosoma brucei parasites that cause human African trypanosomiasis, also known as sleeping sickness. As part of disease surveillance, tsetse can be captured in traps and checked for parasite presence. The molecular screening of disease vectors (such as mosquitoes, ticks and blackflies) for the presence of pathogen DNA has gained popularity in recent years. However, DNA contamination may occur at capture when live vectors are retained for a limited period in a trap cage. To explore this, we conducted experiments, initially with laboratory-reared tsetse and then field-caught tsetse from Tanzania. Our results show that infected tsetse can contaminate uninfected tsetse with T. brucei DNA when retained together in a trap cage, and that the level of contamination can be extensive. Infected tsetse consistently shed T. brucei DNA in their faeces, which in turn contaminates other tsetse. This can produce false-positive results, leading to inaccurate reporting of infection prevalence. These findings impact not only trypanosomiasis surveillance, but may also have ramifications for the xenomonitoring of other vector-borne neglected diseases. Future work should explore whether pathogen DNA contamination routes exist in other vector species and, if so, the methods to mitigate DNA contamination in entomological traps.

molecular biology↗

bioRxiv2022

Tsetse flies choose birthing sites guided by environmental but not pheromonal cues

Tsetse flies significantly impact public health and economic development in sub-Saharan African countries by transmitting the fatal disease African trypanosomiasis. Unusually, instead of laying eggs, tsetse birth a single larva that immediately burrows into the soil to pupate. Where the female chooses to larviposit is therefore crucial for offspring survival. Previous studies showed that a putative larval pheromone, n-pentadecane, attracts gravid female Glossina morsitans morsitans to appropriate larviposition sites. However, this attraction could not be reproduced in field experiments. Here, we resolve this disparity by designing naturalistic laboratory experiments that closely mimic the characteristics found in the wild. We show that gravid tsetse were neither attracted to the putative pheromone nor, interestingly, to pupae placed in the soil. In contrast, females appear to choose larviposition sites based on environmental cues. We conclude that it is the substrate, rather than larval pheromones, which drives larviposition site selection under naturalistic conditions.

animal behavior and cognition↗

bioRxiv2021

Oxidative phosphorylation is required for powering motility and development of the sleeping sickness parasite Trypanosoma brucei within the tsetse fly vector

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 parasites 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.

molecular biology↗

bioRxiv2021

Protein glycosylation is essential for SARS-CoV-2 infection

SARS-CoV-2 extensively N-glycosylates its spike proteins, which are necessary for host cell invasion and the target of both vaccines and immunotherapies. These sugars are predicted to help mediate spike binding to the host receptor by stabilizing its open conformation and evading host immunity. Here, we investigated both the essentiality of the host N-glycosylation pathway and SARS-CoV-2 N-glycans for infection. Inhibition of host N-glycosylation using RNAi or FDA-approved drugs reduced virus infectivity, including that of several variants. Under these conditions, cells produced less virions and some completely lost their infectivity. Furthermore, partial deglycosylation of intact virions showed that surface-exposed N-glycans are critical for cell invasion. Altogether, spike N-glycosylation is a targetable pathway with clinical potential for treatment or prevention of COVID-19.

microbiology↗

bioRxiv2020

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naive tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type glycans. In overlay assays, these sugars were differentially recognized by the C-type lectins mannose receptor and DC-SIGN. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

biochemistry↗

bioRxiv2020

Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis

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 insect pollinator species. Here we present a promising, environment-friendly alternative that targets insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded. RNAi silencing of either the first two enzymes in the tyrosine degradation pathway (TAT and 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, it did not affect bumblebee survival. A mathematical model shows that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current elimination programmes.

biochemistry↗

bioRxiv2020

Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis

During Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the hosts immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC-MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with Man5GlcNAc2 being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans.

biochemistry↗

bioRxiv2019

Gut-associated bacteria invade the midgut epithelium of Aedes aegypti and stimulate innate immunity and suppress Zika virus infection in cells.

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.

microbiology↗

bioRxiv2019

The Glossina Genome Cluster: Comparative Genomic Analysis of the Vectors of African Trypanosomes

BackgroundTsetse flies (Glossina sp.) are the sole vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood specific diet by both sexes and obligate bacterial symbiosis. This work describes comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans (G.m. morsitans), G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes) and Fusca (G. brevipalpis) which represent different habitats, host preferences and vectorial capacity. ResultsGenomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex linked scaffolds show increased rates of female specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse specific genes are enriched in protease, odorant binding and helicase activities. Lactation associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other characterized insects. Vision associated Rhodopsin genes show conservation of motion detection/tracking functions and significant variance in the Rhodopsin detecting colors in the blue wavelength ranges. ConclusionsExpanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

genomics↗

bioRxiv2019

Trypanosoma brucei colonises the tsetse gut via an immature peritrophic matrix in the proventriculus

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 brucei must 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.

microbiology↗

bioRxiv2018

The crystal structure and localization of Trypanosoma brucei invariant surface glycoproteins suggest a more permissive VSG coat in the tsetse-transmitted metacyclic stage

Trypanosoma brucei spp. 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 from T. 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 [A] 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.

microbiology↗