Biology subjects

Dimopoulos, G.

Publications and source records attributed to Dimopoulos, G..

13 recordsLinked to original sources

bioRxiv2025

Rhodo-Box: a Synthetic Biology Toolbox to Facilitate Metabolic Engineering of Rhodobacter sphaeroides

Rhodobacter sphaeroides is a purple non-sulphur alphaproteobacterium with a highly versatile metabolism. This microorganism holds promise as a chassis for sustainable biomanufacturing of numerous chemicals. Yet, its potential is constrained by a lack of standardized, well-characterized genetic elements to tune gene expression such as transcriptional promoters and ribosome binding sites (RBSs). In this study, we present Rhodo-Box, a comprehensive toolkit for R. sphaeroides created by adapting and extending the Zymo-Parts modular cloning framework. Using Rhodo-Box we built and characterized: (a) three broad-host origins of replication (pBBR1, RK2 and RSF1010), (b) a set of 13 promoters, (c) four inducible expression systems (NahR-PsalTTC, LacI-PlacT7A1_O3O4, VanR-PvanCC, and XylS-Pm), (d) 11 RBSs, and (e) four transcriptional terminators. Furthermore, we present a semi-automated, user-friendly cloning approach which enables rapid construction of R. sphaeroides strains. The Rhodo-Box toolkit equips R. sphaeroides with a standardized, automation-compatible collection of parts and workflows essential for efficient design-build-test-learn cycles and advanced metabolic engineering. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/685836v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fcb172org.highwire.dtl.DTLVardef@1e5eb44org.highwire.dtl.DTLVardef@1b8df28org.highwire.dtl.DTLVardef@42eb2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Enhancing Tissue-Specific Antiviral Immunity to Disrupt Arbovirus Transmission by Mosquitoes

Arboviruses, including dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV), pose a significant global health and economic burden, with Aedes aegypti serving as their primary vector. Arbovirus infection in Ae. aegypti progresses sequentially through the midgut (MG), carcass (CA), and salivary glands (SG), with each tissue exhibiting distinct antiviral responses. Here, we investigate tissue-specific antiviral mechanisms, focusing on the small interfering RNA (siRNA) pathway in SGs. Our results reveal that SGs possess weaker antiviral defense and are more susceptible to arboviral infection compared to MGs and CAs. Notably, overexpression of Dicer2 (Dcr2), a key component of the siRNA pathway, in SGs leads to a significant decrease in arboviral replication. Conversely, Dcr2 overexpression in fat bodies, the primary tissue in CAs, only moderately suppresses DENV2 infection and has no notable effect on Mayaro virus (MAYV) infection. Remarkably, the simultaneous overexpression of Dcr2 in both MGs and SGs enhances antiviral activity, effectively blocking the transmission of multiple arboviruses. These findings reveal the tissue-specific dynamics of mosquito antiviral immunity and underscore the potential for targeting SG-specific immunity to disrupt arbovirus transmission, providing a promising approach for controlling mosquito-borne diseases.

microbiology↗

bioRxiv2024

Dietary L-3,4-dihydroxyphenylalanine (L-DOPA) augments cuticular melanization in Anopheles mosquitos while reducing their lifespan and malaria parasite burden

L-3,4-dihydroxyphenylalanine (L-DOPA), a naturally occurring tyrosine derivative, is prevalent in environments that include mosquito habitats, potentially serving as part of their diet. Given its role as a precursor for melanin synthesis we investigate the effect of dietary L-DOPA on mosquito physiology and immunity to Plasmodium falciparum and Cryptococcus neoformans infection. Dietary L-DOPA is incorporated into mosquito melanin via a non-canonical pathway and has a profound transcriptional effect associated with enhanced immunity, increased pigmentation, and reduced lifespan. Increased melanization results in an enhanced capacity to absorb electromagnetic radiation that affects mosquito temperatures. Bacteria in the mosquito microbiome act as sources of dopamine, a substrate for melanization. Our results illustrate how an environmentally abundant amino acid analogue can affect mosquito physiology and suggest its potential usefulness as an environmentally friendly vector control agent to reduce malaria transmission, warranting further research and field studies.

microbiology↗

bioRxiv2024

Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insects innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

microbiology↗

bioRxiv2023

Anopheles gambiae mosGILT regulates innate immune genes and zpg expression

Gene-edited mosquitoes lacking a gamma-interferon-inducible lysosomal thiol reductase-like protein, namely (mosGILTnull) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILTnull A. gambiae was therefore compared to wild type (WT) by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILTnull A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg, an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILTnull mosquitoes. These results provide the crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.

genomics↗

bioRxiv2022

CRISPR Mediated Transactivation in the Human Disease Vector Aedes aegypti

As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ( dead) Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2). This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may lead to the development of innovative vector control tools.

bioengineering↗

bioRxiv2022

Bacterial communities of Aedes aegypti mosquitoes differ between crop and midgut tissues

Microbiota studies of Aedes aegypti and other mosquitoes generally focus on the bacterial communities found in adult female midguts. However, other compartments of the digestive tract maintain communities of bacteria which remain almost entirely unstudied. For example, the Dipteran crop stores nectar and other sugars, but few studies have looked at the microbiome of crops in mosquitoes, and only a single previous study has investigated the crop in Ae. aegypti. In this study, we used both culture-dependent and culture-independent methods to compare the bacterial communities in midguts and crops of laboratory-reared Ae. aegypti. Both methods revealed a trend towards higher abundance, but also higher variability, of bacteria in the midgut than the crop. When present, bacteria from the genus Elizabethkingia (family Weeksellaceae) dominated midgut bacterial communities. In crops, we found a higher diversity of bacteria, and these communities were generally dominated by acetic acid bacteria (family Acetobacteriaceae) from the genera Tanticharoenia and Asaia. These three taxa drove significant community structure differences between the tissues. We used FAPROTAX to predict the metabolic functions of these communities and found that crop bacterial communities were significantly more likely to contain bacteria capable of methanol oxidation and methylotrophy. Both the presence of acetic acid bacteria (which commonly catabolize sugar to produce acetic acid) and the functional profile that includes methanol oxidation (which is correlated with bacteria found with natural sources like nectar) may relate to the presence of sugar in the crop. A better understanding of what bacteria are present in the digestive tract of mosquitoes and how these communities assemble will inform how the microbiota impacts mosquito physiology and the full spectrum of functions provided by the microbiota. It may also facilitate better methods of engineering the mosquito microbiome for vector control or prevention of disease transmission. Author summaryBacteria inside mosquitoes guts have been found to have an impact on mosquito life history traits (such as longevity and fecundity) as well as their susceptibility to infection by human pathogens. Engineering these communities may provide an effective and safe way to control mosquitoes and reduce the impact of the pathogens they spread. In this work, we assayed the bacteria found in midgut and crop tissues of a medically important mosquito, Aedes aegypti. Our results show that these tissues harbor communities of bacteria that differ in composition and function and vary in abundance. Experiments like ours are important to better understand where bacteria are found in an insects body and how these communities assemble. This knowledge may help future researchers more successfully engineer bacterial communities in mosquitoes.

microbiology↗

bioRxiv2021

Transcriptome Profiles of Anopheles gambiae Harboring Natural Low-Level Plasmodium Infection Reveal Adaptive Advantages for the Mosquito

Anopheline mosquitoes are the sole vectors for the Plasmodium pathogens responsible for malaria, which is among the oldest and most devastating of human diseases. The continuing global impact of malaria reflects the evolutionary success of a complex vector-pathogen relationship that accordingly has been the long-term focus of both debate and study. An open question in the biology of malaria transmission is the impact of naturally occurring low-level Plasmodium infections of the vector on the mosquitos health and longevity as well as critical behaviors such as host- preference/seeking. To begin to answer this, we have completed a comparative RNAseq-based transcriptome profile study examining the effect of biologically salient, salivary gland transmission- stage Plasmodium infection on the molecular physiology of Anopheles gambiae s.s. head, sensory appendage, and salivary glands. When compared with their uninfected counterparts, Plasmodium infected mosquitoes exhibit increased transcript abundance of genes associated with olfactory acuity as well as a range of synergistic processes that align with increased fitness based on both anti-aging and reproductive advantages. Taken together, these data argue against the long-held paradigm that malaria infection is pathogenic for anophelines and, instead, suggests there are biological and evolutionary advantages for the mosquito that drive the preservation of its high vectorial capacity.

molecular biology↗

bioRxiv2021

Analysis of melanotic Plasmodium spp. capsules in mosquitoes reveal eumelanin-pheomelanin composition and identify AgMesh as a modulator of parasite infection

Melanins are structurally complex pigments produced by organisms in all domains of life. In insects, melanins are essential for survival and have key roles in cuticle sclerotization, wound healing and innate immunity. In this study, we used a diverse set of molecular, biochemical, and imaging approaches to characterize mosquito melanin involved in innate immune defense (melanotic capsules). We observed that melanotic capsules enclosing Plasmodium berghei ookinetes were composed of an acid-resistant and highly hydrophobic material with granular appearance, which are characteristic properties of melanins. Spectroscopical analyses reveal chemical signatures of eumelanins and pheomelanin. Furthermore, we identified a set of 14 acid-resistant mosquito proteins embedded within the melanin matrix possibly related to an anti-Plasmodium response. Among these, AgMESH, a mucin-related protein highly conserved among insects that is associated with the midgut brush border microvilli proteome of Anopheles gambiae and A. albimanus. AgMESH gene silencing in mosquitos was associated with reduced Plasmodium parasite infection, compromised integrity of the peritrophic matrix, and inability to synthesize a dityrosine network. Our results provide a new approach to study aspects of insect melanogenesis that revealed proteins associated with melanotic capsule, one of which was strongly implicated in the stabilization of the peritrophic matrix and pathogenesis of Plasmodium spp. mosquito infection. Given the conservation of AgMESH among disease-transmitting insect vector species, future analysis of this protein could provide fertile ground for the identification of strategies that block transmission of vector borne diseases to humans. Significance StatementMalaria is a parasitic disease transmitted by mosquito bites. Here, we adapt methodologies to study fungal melanogenesis to explore the melanin-based immune response of Anopheles gambiae against malaria parasites. We reveal that melanotic capsules against Plasmodium are composed of pheomelanin and eumelanin. We demonstrate that melanin-encapsulated Plasmodium is associated to acid-resistant mosquito gut proteins and identify several putative factors of the melanin-mediated immunity. Disruption of AgMESH, a surface-associated protein conserved among other mosquito vectors, demonstrates its ability to impaired formation of the dityrosine network and peritrophic matrix compromising parasite development within the mosquito gut. Our study provides a new approach to investigate the melanin-based defense mechanism in insects and identified a potential host molecule for developing novel universal vector-control schemes.

microbiology↗

bioRxiv2020

Field-deployable molecular diagnostic platform for arbovirus and Wolbachia detection in Aedes aegypti

BackgroundSurveillance of mosquito infection status is critical for planning and deployment of proper mosquito control initiatives. Concurrently, Wolbachia is being widely used as a control method for arboviral transmission. Point-of-care (POC) detection assays are necessary for monitoring the infection prevalence and geographic range of viruses as well as Wolbachia in mosquito vector populations. We therefore assessed the novel qPCR bCUBE molecular diagnostic system as a tool for virus and Wolbachia detection. ResultsWe developed a reliable, specific, and sensitive diagnostic assay for detecting Zika virus and dengue virus serotype 2 using the real-time qPCR platform bCUBE. With bCUBE-based qRT-PCR, both Wolbachia bacterium and virus RNA could be reliably detected in individually infected Ae. aegypti mosquitoes and in pools of 5, 10, or 15 mosquitoes. ConclusionsThe portable qPCR bCUBE diagnostic platform is capable of detecting Zika and dengue virus as well as Wolbachia in mosquitoes and therefore has potential as a practical field-deployable diagnostic test for vector-borne disease surveillance programs.

microbiology↗

bioRxiv2019

Malaria parasite immune evasion and adaptation to its mosquito host is influenced by the acquisition of multiple blood meals

A minimum of two blood meals are required for a mosquito to acquire and transmit malaria, yet Anopheles mosquitoes frequently obtain additional blood meals during their adult lifespan. To determine the impact of subsequent blood-feeding on parasite development in Anopheles gambiae, we examined rodent and human Plasmodium parasite infection with or without an additional non-infected blood meal. We find that an additional blood meal significantly reduces P. berghei immature oocyst numbers, yet does not influence mature oocysts that have already begun sporogony. This is in contrast to experiments performed with the human parasite, P. falciparum, where an additional blood meal does not affect oocyst numbers. These observations are reproduced when mosquitoes were similarly challenged with an artificial protein meal, suggesting that parasite losses are due to the physical distension of the mosquito midgut. We provide evidence that feeding compromises the integrity of the midgut basal lamina, enabling the recognition and lysis of immature P. berghei oocysts by the mosquito complement system. Moreover, we demonstrate that additional feeding promotes P. falciparum oocyst growth, suggesting that human malaria parasites exploit host resources provided with blood-feeding to accelerate their growth. This contrasts experiments with P. berghei, where the size of surviving oocysts is independent of an additional blood meal. Together, these data demonstrate differences in the ability of Plasmodium species to evade immune detection and adapt to utilize host resources at the oocyst stage, representing an additional, yet unexplored component of vectorial capacity that has important implications for transmission of malaria.

microbiology↗

bioRxiv2018

Use of IFNγ/IL10 ratio for stratification of hydrocortisone therapy in patients with septic shock

BackgroundLarge clinical trials testing hydrocortisone therapy in septic shock have produced conflicting results. Subgroups may however benefit depending on their individual immune response.\n\nMethodsWe performed an exploratory analysis of the CORTICUS trial database employing machine learning to a panel of 137 variables collected from 83 patients (60 survivors, 23 non-survivors) including demographic and clinical measures, organ failure scores, leukocyte counts and circulating cytokine levels. The identified biomarker was validated against data collected from patients enrolled into a cohort of the Hellenic Sepsis Study Group (HSSG) (n=162) and two data sets of two other clinical trials. Ex vivo studies were performed on this biomarker to assess a possible mechanistic role.\n\nResultsA low serum IFN{gamma}/IL10 ratio predicted increased survival in the hydrocortisone group whereas a high ratio predicted better survival in the placebo group. Using this ratio for a decision rule, we found significant improvement in survival in the groups of patients being in compliance with the prediction rule (discovery set: OR=3.03 [95% Cl: 1.05-8.75], P=0.031, validation set: OR=2.01 [95% CI: 1.04-3.88], P=0.026). Applying the rule to two further, smaller datasets showed the same tendency. Mechanistic studies revealed that IFN{gamma}/IL10 was negatively associated with pathogen load in spiked human blood. An in silico analysis of published IFN{gamma} and IL10 values in bacteremic and non-bacteremic SIRS patients supported this association between the ratio and pathogen burden.\n\nConclusionIf confirmed prospectively, the IFN{gamma}/IL10 ratio could be used as a rapidly available theranostic for use of hydrocortisone therapy in septic shock.

immunology↗

bioRxiv2018

Regulation of midgut cell proliferation impacts Aedes aegypti susceptibility to dengue virus

Aedes aegypti is the vector of some of the most important vector-borne diseases like Dengue, Chikungunya, Zika and Yellow fever, affecting millions of people worldwide. The cellular processes that follow a blood meal in the mosquito midgut are directly associated with pathogen transmission. We studied the homeostatic response of the midgut against oxidative stress, as well as bacterial and dengue virus (DENV) infections, focusing on the proliferative ability of the intestinal stem cells (ISC). Inhibition of the peritrophic matrix (PM) formation led to an increase in ROS production by the epithelial cells in response to contact with the resident microbiota, suggesting that maintenance of low levels of ROS in the intestinal lumen is key to keep ISCs division in balance. We show that dengue virus infection induces midgut cell division in both DENV susceptible (Rockefeller) and refractory (Orlando) mosquito strains. However, the susceptible strain delays the activation of the regeneration process compared with the refractory strain. Impairment of the Delta/Notch signaling, by silencing the Notch ligand Delta using RNAi, significantly increased the susceptibility of the refractory strains to DENV infection of the midgut. We propose that this cell replenishment is essential to control viral infection in the mosquito. Our study demonstrates that the intestinal epithelium of the blood fed mosquito is able to respond and defend against different challenges, including virus infection. In addition, we provide unprecedented evidence that the activation of a cellular regenerative program in the midgut is important for the determination of the mosquito vectorial competence.

cell biology↗