Biology subjects

Povelones, M.

Publications and source records attributed to Povelones, M..

6 recordsLinked to original sources

bioRxiv2023

Establishment and comparative genomics of a high-quality collection of mosquito-associated bacterial isolates -- MosAIC (Mosquito-Associated Isolate Collection)

Mosquitoes transmit medically important human pathogens, including viruses like dengue virus and parasites such as Plasmodium spp., the causative agent of malaria. Mosquito microbiomes are critically important for the ability of mosquitoes to transmit disease-causing agents. However, while large collections of bacterial isolates and genomic data exist for vertebrate microbiomes, the vast majority of work in mosquitoes to date is based on 16S rRNA gene amplicon data that provides limited taxonomic resolution and no functional information. To address this gap and facilitate future studies using experimental microbiome manipulations, we generated a bacterial Mosquito-Associated Isolate Collection (MosAIC) consisting of 392 bacterial isolates with extensive metadata and high-quality draft genome assemblies that are publicly available for use by the scientific community. MosAIC encompasses 142 species spanning 29 bacterial families, with members of the Enterobacteriaceae comprising 40% of the collection. Phylogenomic analysis of three genera, Enterobacter, Serratia, and Elizabethkingia, reveal lineages of mosquito-associated bacteria isolated from different mosquito species in multiple laboratories. Investigation into species pangenomes further reveals clusters of genes specific to these lineages, which are of interest for future work to identify functions underlying mosquito host association. Altogether, we describe the generation of a physical collection of mosquito-associated bacterial isolates, their genomic data, and analyses of selected groups in context of genome data from closely related isolates, providing a unique, highly valuable resource to investigate factors for bacterial colonisation and adaptation within mosquito hosts. Future efforts will expand the collection to include broader geographic and host species representation, especially from individuals collected from field populations, as well as other mosquito-associated microbes, including fungi, archaea, and protozoa.

microbiology↗

bioRxiv2022

Adhesion of Crithidia fasciculata promotes a rapid change in developmental fate driven by cAMP signaling

Kinetoplastids are single-celled parasites responsible for human and animal disease. For the vast majority of kinetoplastids, colonization of an insect host is required for transmission. Stable attachment to insect tissues via the single flagellum coincides with differentiation and morphological changes. Although this process is essential for the generation of infectious forms, the molecular mechanism driving differentiation following adherence is not well understood. To study this process, we elaborate upon an in vitro model in which flagellated swimming cells of the kinetoplastid Crithidia fasciculata rapidly differentiate following adhesion to artificial substrates. Manipulation of culture parameters revealed that growth phase and time had a strong influence on the proportion of adherent cells. Live imaging of cells transitioning from swimming to an attached cell fate show parasites undergoing a defined sequence of events including an initial adhesion near the base of the flagellum, immediately followed by flagellar shortening, cell rounding, and the formation of a hemidesmosome-like structure between the tip of the shortened flagellum and the substrate. We have also assayed the role of the cyclic AMP (cAMP) signaling pathway in differentiation of C. fasciculata. Pharmacological inhibition of cAMP phosphodiesterases eliminated the ability of swimming cells to attach without affecting their growth rate. Further, treatment with inhibitor did not affect the growth rate of established attached cells, indicating its effect is limited to a critical window of time during the early stages of adhesion. Finally, in swimming parasites we have shown that a receptor adenylate cyclase localizes to the distal portion of the flagellum. In attached cells it is absent from the shortened flagellum and instead localizes to the cell body. Similarly, a putative phosphodiesterase, found along the length of the flagellum, also relocalizes to the cell body in attached parasites. These data suggest that in C. fasciculata cAMP signaling is required for adherence, that cAMP flux in the flagellum of swimming cells is spatially restricted, and that signaling domains may be reorganized during differentiation and attachment. These studies contribute to our understanding of the flagellum as a multi-functional organelle integrating processes related to motility, signaling, attachment, and differentiation and further develop C. fasciculata as a model kinetoplastid. Author SummaryParasites from the order Kinetoplastida are transmitted by insects and cause diseases such as Leishmaniasis, Chagas disease, and Human African Trypanosomiasis. A key aspect of the life cycle of these parasites is their ability to adhere to surfaces within the insect and subsequently differentiate into forms that are transmissable to their next host. Here, we explore the molecular mechanisms underpinning both adherence and differentiation using the mosquito parasite Crithidia fasciculata. This organism is closely related to pathogenic species but grows to high densities in culture and adheres robustly in vitro. We identified factors affecting the rate of adherence and defined morphological stages of this process including rapid shortening of the cells single flagellum. Using electron microscopy, we confirmed that adhesion to artificial substrates results in the formation of a filament-rich adhesive plaque at the tip of the flagellum that resembles the attachment to insect tissue. We also demonstrate the key role played by the cyclic AMP signaling pathway during adherence and differentiation. Specifically, pharmacological inhibition of enzymes that degrade cyclic AMP completely blocks adherence, and tagging of several proteins in this pathway show that their localization along the flagellum changes following differentiation to the attached form. These data provide insights into processes critical for all kinetoplastid life cycles.

microbiology↗

bioRxiv2022

Aedes aegypti Malpighian tubules are immunologically activated following systemic Toll activation

BackgroundCanine heartworm is a widespread and potentially fatal mosquito-borne disease caused by infections with the parasitic nematode, Dirofilaria immitis. We have previously shown that systemic activation of the Toll immune pathway via silencing of the negative regulator Cactus in Aedes aegypti blocks parasite development in the Malpighian tubules, the mosquito renal organ. However, it was not established whether the Malpighian tubules were directly responding to Toll activation or were alternatively responding to upregulated proteins or other changes to the hemolymph driven by other tissues. Distinguishing these possibilities is crucial for developing more precise strategies to block D. immitis while potentially avoiding the fitness cost to the mosquito associated with Cactus silencing. MethodsThis study defines the transcriptional response Malpighian tubules and changes to the hemolymph proteome of Ae. aegypti after systemic Toll activation via intra-thoracic injection of dsCactus. ResultsWe found Malpighian tubules significantly increase expression of Toll pathway target genes that significantly overlapped expression changes occurring in whole mosquitoes. Additionally, we identified a significant overlap between the transcriptional response of the Malpighian tubules and proteins upregulated in the hemolymph. ConclusionsOur data show that Malpighian tubules are capable of RNAi-mediated gene silencing and directly respond to dsCactus treatment by upregulating canonical Toll pathway targets. Though not definitive, the strong correspondence between the Malpighian tubule transcriptional and the hemolymph proteomic responses provides evidence that the tubules may contribute to mosquito humoral immunity.

immunology↗

bioRxiv2020

The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae

The arthropod melanization immune response is activated by extracellular protease cascades predominantly comprised of CLIP-domain serine proteases (CLIP-SPs) and serine protease homologs (CLIP-SPHs). In the malaria vector, Anopheles gambiae, the CLIP-SPHs SPCLIP1, CLIPA8, and CLIPA28 form the core of a hierarchical cascade downstream of mosquito complement that is required for melanization. However, our understanding of the regulatory relationship of the CLIP-SPH cascade with the catalytic CLIP-SPs driving melanization is incomplete. Here, we report on the development of a novel screen to identify melanization pathway components based on the quantitation of infection-induced excreta, eliminating the need for microdissections or hemolymph enzymatic assays. Using this screen, we identified CLIPC9 and subsequent functional analyses established that this protease is essential for the melanization of both Escherichia coli and the rodent malaria parasite Plasmodium berghei. Mechanistically, septic infection with E. coli promotes CLIPC9 cleavage and both full-length and cleaved CLIPC9 localize to this bacterium in a CLIPA8-dependent manner. The steady state level of CLIPC9 in the hemolymph is regulated by thioester-containing protein 1 (TEP1), suggesting it functions downstream of mosquito complement. In support, CLIPC9 cleavage is inhibited following SPCLIP1, CLIPA8, and CLIPA28 knockdown positioning it downstream of the CLIP-SPH cascade. Moreover, like CLIPA8 and CLIPA28, CLIPC9 processing is negatively regulated by serine protease inhibitor 2 (SRPN2). This report demonstrates how our novel excretion-based approach can be utilized to dissect the complex protease networks regulating mosquito melanization. Collectively, our findings establish that CLIPC9 is required for An. gambiae melanization and shed light on how the CLIP-SPH cascade regulates this potent immune response. Author SummaryMosquito vector competence for Plasmodium, antifungal defense, and lifespan are all influenced by the melanization response. Despite its importance, our understanding of the proteins comprising the An. gambiae melanization cascade is incomplete. To streamline the discovery of melanization pathway components in this disease vector, we developed a screening method that is able to identify proteins with far fewer mosquitoes than other approaches. This technique facilitated our discovery that the serine protease CLIPC9 is required for the melanization of bacteria and malaria parasites. CLIPC9 activation and localization to bacteria is regulated by members of the CLIPA subfamily, highlighting that these serine protease homologs broadly regulate the melanization immune response. Traditionally viewed as co-factors for the prophenoloxidase activating CLIPBs, this work demonstrates that CLIPAs can also control the activation of the catalytic proteases driving melanization. This work identifies a new player in the melanization hierarchy and provides the field with an efficient approach for dissecting the complex protease cascades controlling this important immune response.

microbiology↗

bioRxiv2020

A novel assay to measure the emergence of third-stage filarial nematodes in individual mosquitoes

BackgroundMosquitoes transmit filarial nematodes to both human and animal hosts, resulting in worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector. MethodsWe developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 {degrees}C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi infected Ae. aegypti. ResultsApproximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi produce emerging third-stage larvae. Once D. immitis third-stage larvae emerge at 13 days post infection, the proportion of mosquitoes producing them, and the number produced per mosquito remain stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12-14 days post infection. Increased uptake of D. immitis microfilariae increases the fitness cost to the mosquito but does not increase the number of emerging third-stage larvae. ConclusionsWe provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious L3 for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2019

C-type lectins CTL4 and CTLMA2: conserved heterodimeric structure and glycan specificity in Anopheles mosquitoes

AbstractThe C-type lectins CTL4 and CTLMA2 cooperatively influence Plasmodium infection in the malaria vector Anopheles. Here we report the purification and biochemical characterization of CTL4 and CTLMA2 from An. gambiae and An. albimanus. CTL4 and CTLMA2 are known to form a disulfide-bridged heterodimer via an N-terminal tri-cysteine CXCPC motif. We demonstrate in vitro that CTL4 and CTLMA2 intermolecular disulfide formation is promiscuous within this motif. Furthermore, CTL4 and CTLMA2 exhibit charge complementarity that promotes the formation of higher oligomeric states at physiological pH. Both lectins bind specific sugars, with an apparent preference for glycosaminoglycan motifs comprising {beta}1-3/{beta}1-4 linkages between glucose (Glc), galactose (Gal) and their respective hexosamines. Small-angle x-ray scattering data supports a compact heterodimer between the CTL domains. Recombinant CTL4/CTLMA2 is functional in vivo, reversing the enhancement of phenoloxidase activity in dsCTL4-treated mosquitoes. We propose these molecular features underline a common function for CTL4/CTLMA2 in mosquitoes, with species and strain-specific variation in degrees of activity in response to Plasmodium infection. Author SummaryMosquitoes of the genus Anopheles are vectors for the single-celled parasite Plasmodium, the causative agent of malaria. Mosquitoes, like all insects, utilize the process of melanization for both wound healing and defense against pathogens. CTL4 and CTLMA2 are two proteins found in Anopheles mosquitoes that act as inhibitors of melanization, so understanding their molecular function is important to understanding the immune response of Anopheles mosquitoes to Plasmodium infection. We have purified CTL4 and CTLMA2 from two species of Anopheles and studied their molecular properties with a variety of biochemical and biophysical techniques. We also verified that our purified protein is functional by injecting it into mosquitoes. We learned that CTL4 and CTLMA2 are joined together by a disulfide bond between any one of three cysteine residues near the N-terminus of each protein. The CTL4/CTLMA2 complex is compact, but can associate into larger structures in solution, probably because of a loop in each protein that carries an opposite charge. The proteins cooperatively bind calcium and sugars, specifically glycosaminoglycan sugars, which are typically present in the connective tissues of insects. This information will aid in further investigations of the function of CTL4 and CTLMA2.

biochemistry↗