Biology subjects

Orfano, A.

Publications and source records attributed to Orfano, A..

3 recordsLinked to original sources

bioRxiv2025

Anopheles mosquitoes exposed to long-acting antimalarials via drug-spiked bloodmeal absorb drug but do not suffer fitness costs

The World Health Organizations recommendations regarding the use of antimalarials for the prevention of malaria in endemic areas have greatly expanded, allowing more flexibility in the demographic groups and regions where chemoprevention and mass treatment are acceptable. An overlooked aspect of expanding human population-level drug exposure is the downstream impact of ingested drug on the mosquito vector. Data suggest both infected and uninfected Anopheles mosquitoes re-feed often with [≥]4 blood meals during their lifespan. This provides repeated opportunities for mosquitoes to ingest drug via bloodmeals taken from people with antimalarials in the bloodstream and raises questions as to whether exposure may impact the mosquito itself, and/or parasites developing within the infected mosquito. We investigated the impact of exposure to physiologic levels of commonly used long-acting human antimalarials in the Anopheles mosquito via drug-spiked blood feeds. We did not observe any significant differences in mosquito feeding, behavior, fertility, or viability after ingestion of amodiaquine, desethylamodiaquine, piperaquine, and sulfadoxine-pyrimethamine in either lab-reared An. gambiae or field-derived An. coluzzii mosquitoes. Interrogating drug distribution within mosquitoes utilizing LC-MS/MS, desethylamodiaquine, the longer-acting active metabolite of amodiaquine, was fed at 2 concentrations (1/2X and 2X Cmax) with drug subsequently detected in a dose-dependent manner in pooled whole mosquitoes, midguts and hemolymph. This was significant for whole mosquitoes harvested at 24hrs and 120hrs, and midguts harvested at 24hrs. Between 24 to 120 hrs, drug decreased in midguts but increased in hemolymph. Our results show biochemical evidence of antimalarial absorption into Anopheles hemolymph following bloodmeal ingestion. These studies lay the foundation for future work to assess the impact of vector-stage antimalarial drug exposure on parasite progression throughout development in the mosquito, which could in turn have important implications for transmission dynamics and drug resistance spread. Author summaryDrug resistance to first-line antimalarials has emerged in multiple African countries. A better understanding of antimalarial drug resistance emergence and spread is critical in preventing further morbidity and mortality. Millions regularly receive antimalarials for prophylaxis and mass treatment that are purposefully long-acting. Anopheles mosquitoes re-feed frequently, and these malaria vectors (both infected and uninfected) routinely feed on people whose blood contains these long-acting antimalarials. Parasites take approximately 10 days to develop within the mosquito. There is published precedent that several antimalarials can act upon vector-stage parasites, yet any potential impact of antimalarials on mosquitoes and/or parasites developing within has been largely overlooked. We questioned whether antimalarials ingested in mosquito bloodmeals could influence parasite development and drug resistance selection. As initial investigations, we exposed uninfected Anopheles mosquitoes to commonly used long-acting antimalarials via drug-spiked bloodmeals, mimicking predicted physiologic drug exposure. Investigated drugs did not impact mosquito viability. However, mass spectrometry confirmed drug absorption in whole mosquitoes, as well as within midguts and circulatory fluid, several days after feeding, demonstrating that mosquitoes can ingest key drugs without suffering fitness costs, and these drugs can persist in mosquitoes. This highlights the potential for antimalarials to impact parasite development and drug resistance selection within the mosquito.

microbiology↗

bioRxiv2025

PGRP-LA regulates peritrophic matrix synthesis and influences trypanosome infection outcomes in tsetse flies

Peptidoglycan Recognition Proteins (PGRPs) are conserved pattern-recognition receptors that detect microbe-associated molecular patterns (MAMPs) and activate host immune responses. Compared to other dipterans, the tsetse fly (Glossina morsitans morsitans) genome encodes only five PGRPs-PGRP-LA, -LB, -LC, -SA, and -SB - far fewer than most dipterans, likely reflecting its sterile blood diet and streamlined microbiota. Here, we identify PGRP-LA as a critical regulator of peritrophic matrix (PM) integrity in the cardia (proventriculus), the tissue responsible for PM production. The PM is a chitinous sleeve-like barrier that separates the midgut epithelium from the ingested bloodmeal, supporting digestive homeostasis and infection resistance. We show that pgrp-la is prominently expressed in the cardia, transiently induced after a bloodmeal in newly eclosed flies, and reinduced following subsequent feedings, likely in response to blood-constituents or mechanical stretch. This induction is sustained during microbial exposure and prolonged in trypanosome-infected flies. RNAi-mediated reduction of pgrp-la significantly increased the prevalence of midgut trypanosome infections, indicating a protective role during early infection. PGRP-LA did not mediate infection resistance via canonical IMD pathway signaling, as its silencing did not affect antimicrobial peptide expression. Instead, PGRP-LA modulated the expression of PM-associated genes and gut barrier integrity. Silencing pgrp-la reduced PM structure, increased midgut weights and enhanced fly survival following oral challenge with entomopathogen Serratia marcescens, likely due to earlier epithelial immune responses through a compromised PM. Similar phenotypes were observed when flies were fed anti-PGRP-LA antibodies, supporting a structural role for PGRP-LA. In addition, soluble variant surface glycoproteins (sVSGs) from trypanosomes and knockdown of microRNA-275 (miR-275), also decreased pgrp-la expression, suggesting that PGRP-LA is part of a broader regulatory network, including the miR-275/Wingless signaling. Collectively, our results identify PGRP-LA as novel regulator of PM biogenesis and vector competence in tsetse, expanding the functional repertoire of PGRPs in insect gut barrier maintenance beyond canonical immune signaling pathways. Author SummaryInsect vectors such as tsetse flies can be infected with pathogens that cause devastating disease in mammals. To protect themselves insect vectors rely on pattern recognition receptors (PRRs) that detect pathogens and activate the production of antimicrobial peptides (AMPs). Physical barriers in the gut also play an important role in limiting infections. One such barrier is the peritrophic matrix (PM), a sleeve-like structure that lines the insect gut and separates the blood meal and its contents from the underlying cells. For trypanosome parasites, which cause sleeping sickness in humans, the PM is the first barrier they must traverse to colonize the tsetses gut. In this study, we identified a PRR, PGRP-LA, that, unlike related proteins in other insects that activate AMPs, regulates the integrity of the tsetses protective PM barrier. When PGRP-LA was disrupted, the gut barrier weakened, and flies became more susceptible to trypanosome infection. Our work highlights a previously unrecognized role for PGRP-LA in maintaining gut barrier integrity and suggest that targeting this pathway could be a strategy to help reduce parasite transmission.

immunology↗

bioRxiv2024

Spiroplasma endosymbiont reduction of host lipid synthesis and Stomoxyn-like peptide contribute to trypanosome resistance in the tsetse fly Glossina fuscipes

Tsetse flies (Glossina spp.) vector African trypanosomes that cause devastating diseases in humans and domestic animals. Within the Glossina genus, species in the Palpalis subgroup exhibit greater resistance to trypanosome infections compared to those in the Morsitans subgroup. Varying microbiota composition and species-specific genetic traits can significantly influence the efficiency of parasite transmission. Notably, infections with the endosymbiotic bacterium Spiroplasma have been documented in several Palpalis subgroup species, including Glossina fuscipes fuscipes (Gff). While Spiroplasma infections in Gff are known to hinder trypanosome transmission, the underlying mechanisms remain unknown. To investigate Spiroplasma-mediated factors affecting Gff vector competence, we conducted high-throughput RNA sequencing of the midgut tissue along with functional assays. Our findings reveal elevated oxidative stress in the midgut environment in the presence of Spiroplasma, evidenced by increased expression of nitric oxide synthase, which catalyzes the production of trypanocidal nitric oxide. Additionally, we observed impaired lipid biosynthesis leading to a reduction of this important class of nutrients essential for parasite and host physiologies. In contrast, trypanosome infections in Gffs midgut significantly upregulated various immunity-related genes, including a small peptide, Stomoxyn-like, homologous to Stomoxyns first discovered in the stable fly Stomoxys calcitrans. We observed that the Stomoxyn-like locus is exclusive to the genomes of Palpalis subgroup tsetse species. GffStomoxyn is constitutively expressed in the cardia (proventriculus) and synthetic GffStomoxyn exhibits potent activity against Escherichia coli and bloodstream form of Trypanosoma brucei parasites, while showing no effect against insect stage procyclic forms or tsetses commensal endosymbiont Sodalis in vitro. Reducing GffStomoxyn levels significantly increased trypanosome infection prevalence, indicating its potential trypanocidal role in vivo. Collectively, our results suggest that the enhanced resistance to trypanosomes observed in Spiroplasma-infected Gff may be due to the reduced lipid availability necessary for parasite metabolic maintenance. Furthermore, GffStomoxyn could play a crucial role in the initial immune response(s) against mammalian parasites early in the infection process in the midgut and prevent gut colonization. We discuss the molecular characteristics of GffStomoxyn, its spatial and temporal expression regulation and its microbicidal activity against Trypanosome parasites. Our findings reinforce the nutritional influences of microbiota on host physiology and host-pathogen dynamics. Author SummaryThe tsetse fly, Glossina fuscipes fuscipes (Gff) is of high public health relevance. Gff exhibits strong innate resistance to trypanosomes, especially when infected with the endosymbiotic bacterium Spiroplasma. This study investigated how the bacterium Spiroplasma inside Gff enables them to be resistant to trypanosome infection. Our results indicate alterations in host lipid metabolism with reduction in levels of triglycerides, suggesting a potential metabolic barrier that limits the viability to parasite. In addition, we discovered a small peptide, stomoxyn, exclusively in Gff and related Palpalis tsetse species. We have shown that Gff synthetic Stomoxyn has antibacterial and antitrypanosomal properties and lowering Stomoxyn levels in Gff correlates with increased parasite prevalence. We suggest that strategies to increase Spiroplasma prevalence or enhance stomoxyn expression through paratransgenic approaches could be promising avenues for reducing trypanosomiasis transmission.

molecular biology↗