Using bacteria to express and display anti-Plasmodium molecules in the mosquito midgut

Bacteria capable of colonizing mosquito midguts are attractive vehicles for delivering anti-malaria molecules. We genetically engineered Escherichia coli to display two anti-Plasmodium effector molecules, SM1 and phospholipase-A(2), on their outer membrane. Both molecules significantly inhibited Plasmodium berghei development when engineered bacteria were fed to mosquitoes 24h prior to an infective bloodmeal (SM1=41%, PLA2=23%). Furthermore, prevalence and numbers of engineered bacteria increased dramatically following a bloodmeal. However, E. coli survived poorly in mosquitoes. Therefore, Enterobacter agglomerans was isolated from mosquitoes and selected for midgut survival by multiple passages through mosquitoes. After four passages, E. agglomerans survivorship increased from 2 days to 2 weeks. Since E. agglomerans is non-pathogenic and widespread, it is an excellent candidate for paratransgenic control strategies.     

Inhibition of Plasmodium berghei Development in Mosquitoes by Effector Proteins Secreted from Asaia sp. Bacteria Using a Novel Native Secretion Signal

Novel interventions are needed to prevent the transmission of the Plasmodium parasites that cause malaria. One possible method is to supply mosquitoes with antiplasmodial effector proteins from bacteria by paratransgenesis. Mosquitoes have a diverse complement of midgut microbiota including the Gram-negative bacteria Asaia bogorensis. This study presents the first use of Asaia sp. bacteria for paratransgenesis against P. berghei. We identified putative secreted proteins from A. bogorensis by a genetic screen using alkaline phosphatase gene fusions. Two were secreted efficiently: a siderophore receptor protein and a YVTN beta-propeller repeat protein. The siderophore receptor gene was fused with antiplasmodial effector genes including the scorpine antimicrobial peptide and an anti-Pbs21 scFv-Shiva1 immunotoxin. Asaia SF2.1 secreting these fusion proteins were fed to mosquitoes and challenged with Plasmodium berghei-infected blood. With each of these effector constructs, significant inhibition of parasite development was observed. These results provide a novel and promising intervention against malaria transmission.     

Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes

Malaria kills millions of people every year, and new control measures are urgently needed. The recent demonstration that (effector) genes can be introduced into the mosquito germ line to diminish their ability to transmit the malaria parasite offers new hope toward the fight of the disease (Ito, J., Ghosh, A., Moreira, L. A., Wimmer, E. A. & Jacobs-Lorena, M. (2002) Nature, 417, 452-455). Because of the high selection pressure that an effector gene imposes on the parasite population, development of resistant strains is likely to occur. In search of additional antiparasitic effector genes, we have generated transgenic Anopheles stephensi mosquitoes that express the bee venom phospholipase A2 (PLA2) gene from the gut-specific and blood-inducible Anopheles gambiae carboxypeptidase (AgCP) promoter. Northern blot analysis indicated that the PLA2 mRNA is specifically expressed in the guts of transgenic mosquitoes with peak expression at approximately 4 h after blood ingestion. Western blot and immunofluorescence analyses detected PLA2 protein in the midgut epithelia of transgenic mosquitoes from 8 to 24 h after a blood meal. Importantly, transgene expression reduced Plasmodium berghei oocyst formation by 87% on average and greatly impaired transmission of the parasite to naive mice. The results indicate that PLA2 may be used as an additional effector gene to block the development of the malaria parasite in mosquitoes.     

Fitness of anopheline mosquitoes expressing transgenes that inhibit Plasmodium development

One potential strategy for the control of malaria and other vector-borne diseases is the introduction into wild vector populations of genetic constructs that reduce vectorial capacity. An important caveat of this approach is that the genetic construct should have minimal fitness cost to the transformed vector. Previously, we produced transgenic Anopheles stephensi expressing either of two effector genes, a tetramer of the SM1 dodecapeptide or the phospholipase A2 gene (PLA2) from honeybee venom. Mosquitoes carrying either of these transgenes were impaired for Plasmodium berghei transmission. We have investigated the role of two effector genes for malaria parasite blockage in terms of the fitness imposed to the mosquito vector that expresses either molecule. By measuring mosquito survival, fecundity, fertility, and by running population cage experiments, we found that mosquitoes transformed with the SM1 construct showed no significant reduction in these fitness parameters relative to nontransgenic controls. The PLA2 transgenics, however, had reduced fitness that seemed to be independent of the insertion site of the transgene. We conclude that the fitness load imposed by refractory gene(s)-expressing mosquitoes depends on the effect of the transgenic protein produced in that mosquito. These results have important implications for implementation of malaria control via genetic modification of mosquitoes.     

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.     

The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner.

We present here data showing that the Avr proteins HrmA and AvrPto are secreted in culture via the native Hrp pathways from Pseudomonas syringae pathovars that produce these proteins. Moreover, their secretion is strongly affected by the temperature and pH of the culture medium. Both HrmA and AvrPto were secreted at their highest amounts when the temperature was between 18 and 22 degrees C and when the culture medium was pH 6.0. In contrast, temperature did not affect the secretion of HrpZ. pH did affect HrpZ secretion, but not as strongly as it affected the secretion of HrmA. This finding suggests that there are at least two classes of proteins that travel the P. syringae pathway: putative secretion system accessory proteins, such as HrpZ, which are readily secreted in culture; and effector proteins, such as HrmA and AvrPto, which apparently are delivered inside plant cells and are detected in lower amounts in culture supernatants under the appropriate conditions. Because HrmA was shown to be a Hrp-secreted protein, we have changed the name of hrmA to hopPsyA to reflect that it encodes a Hrp outer protein from P. syringae pv. syringae. The functional P. syringae Hrp cluster encoded by cosmid pHIR11 conferred upon P. fluorescens but not Escherichia coli the ability to secrete HopPsyA in culture. The use of these optimized conditions should facilitate the identification of additional proteins traveling the Hrp pathway and the signals that regulate this protein traffic.     

Genetic engineering of malaria parasite resistance in vector mosquitoes

Malaria is the most significant vector‐borne disease and mostly affects people living in the lesser developed countries of tropical and sub‐tropical regions. Climate changes, rapid global transportation, immigration and invasion of exotic mosquito vectors bring the threat of introduction of the disease to developed nations. Sustainability of malaria control requires the discovery of therapeutic and prophylactic drugs, development of effective vaccines and control of vector mosquitoes. Drug development and vaccine research have been pursued aggressively over the past 20 years, and progress in novel approaches to vector control is now evident. Our long‐term objective is the production and utilization of strains of vector mosquitoes that are genetically refractory to the transmission of malaria parasites. These insects will be used to test the hypothesis that an increase in the frequency of a gene or allele that confers decreased vector competence to a population of mosquitoes will result in a reduction in the incidence and prevalence of malaria. Completed studies make it possible to develop strains of Anopheles mosquitoes expressing specific effector molecules that interfere completely with the transmission of the most lethal human malaria parasite, Plasmodium falciparum. Data are reviewed here that support the use of single‐chain monoclonal antibodies (scFv) that disable parasites in the midgut and hemolymph of transgenic mosquitoes.     

PSOP1, putative secreted ookinete protein 1, is localized to the micronemes of Plasmodium yoelii and P. berghei ookinetes

Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Activated gametocytes in the mosquito midgut egress from erythrocytes followed by fertilization and zygote formation. Zygotes differentiate into motile invasive ookinetes, which penetrate the midgut epithelium before forming oocysts beneath the basal lamina. Ookinete development and traversal across the mosquito midgut wall are major bottlenecks in the parasite life cycle. In ookinetes, surface proteins and proteins stored in apical organelles have been shown to be involved in parasite-host interactions. A group of ookinete proteins that are predicted to have such functions are named PSOPs (putative secreted ookinete protein). PSOP1 is possibly involved in migration through the midgut wall, and here its subcellular localization was examined in ookinetes by immunoelectron microscopy. PSOP1 localizes to the micronemes of Plasmodium yoelii and Plasmodium berghei ookinetes, indicating that it is stored and possibly apically secreted during ookinete penetration through the mosquito midgut wall.     

An age-structured model to evaluate the potential of novel malaria-control interventions: a case study of fungal biopesticide sprays

It has recently been proposed that mosquito vectors of human diseases, particularly malaria, may be controlled by spraying with fungal biopesticides that increase the rate of adult mortality. Though fungal pathogens do not cause instantaneous mortality, they can kill mosquitoes before they are old enough to transmit disease. A model is developed (i) to explore the potential for fungal entomopathogens to reduce significantly infectious mosquito populations, (ii) to assess the relative value of the many different fungal strains that might be used, and (iii) to help guide the tactical design of vector-control programmes. The model follows the dynamics of different classes of adult mosquitoes with the risk of mortality due to the fungus being assumed to be a function of time since infection (modelled using the Weibull distribution). It is shown that substantial reductions in mosquito numbers are feasible for realistic assumptions about mosquito, fungus and malaria biology and moderate to low daily fungal infection probability. The choice of optimal fungal strain and spraying regime is shown to depend on local mosquito and malaria biology. Fungal pathogens may also influence the ability of mosquitoes to transmit malaria and such effects are shown to further reduce vectorial capacity.     

Secretory delivery of recombinant proteins in attenuated Salmonella strains: potential and limitations of Type I protein transporters

Live attenuated Salmonella strains have been extensively explored as oral delivery systems for recombinant vaccine antigens and effector proteins with immunoadjuvant and immunomodulatory potential. The feasibility of this approach was demonstrated in human vaccination trials for various antigens. However, immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen. This is, at least partly, due to the lack of secretory expression systems, enabling large-scale extracellular delivery of vaccine and effector proteins by these strains. Because of their low complexity and the terminal location of the secretion signal in the secreted protein, Type I (ATP-binding cassette) secretion systems appear to be particularly suited for development of such recombinant extracellular expression systems. So far, the Escherichia coli hemolysin system is the only Type I secretion system, which has been adapted to recombinant protein secretion in Salmonella. However, this system has a number of disadvantages, including low secretion capacity, complex genetic regulation, and structural restriction to the secreted protein, which eventually hinder high-level in vivo delivery of recombinant vaccines and effector proteins. Thus, the development of more efficient recombinant protein secretion systems, based on Type I exporters can help to improve efficacies of live recombinant Salmonella vaccines. Type I secretion systems, mediating secretion of bacterial surface layer proteins, such as RsaA in Caulobacter crescentus, are discussed as promising candidates for improved secretory delivery systems.     

Plasmodium ookinete-secreted proteins secreted through a common micronemal pathway are targets of blocking malaria transmission

The mosquito midgut ookinete stage of the malaria parasite, Plasmodium, possesses microneme secretory organelles that mediate locomotion and midgut wall egress to establish sporogonic stages and subsequent transmission. The purpose of this study was 2-fold: 1) to determine whether there exists a single micronemal population with respect to soluble and membrane-associated secreted proteins; and 2) to evaluate the ookinete micronemal proteins chitinase (PgCHT1), circumsporozoite and TRAP-related protein (CTRP), and von Willebrand factor A domain-related protein (WARP) as immunological targets eliciting sera-blocking malaria parasite infectivity to mosquitoes. Indirect immunofluorescence localization studies in Plasmodium gallinaceum using specific antisera showed that all three proteins are distributed intracellularly with a similar granular cytoplasmic appearance and with focal concentration of PgCHT1 and PgCTRP, but not PgWARP, at the ookinete apical end. Immunogold double-labeling electron microscopy, using antisera against the membrane-associated protein CTRP and the soluble WARP, showed that these two proteins co-localized to the same micronemal population. Within the microneme CTRP was associated peripherally at the microneme membrane, whereas PgCHT1 and WARP were diffuse within the micronemal lumen. Sera produced against Plasmodium falciparum WARP significantly reduced the infectivity of P. gallinaceum to Aedes aegypti and P. falciparum to Anopheles mosquitoes. Antisera against PgCTRP and PgCHT1 also significantly reduced the infectivity of P. gallinaceum for A. aegypti. These results support the concept that ookinete micronemal proteins may constitute a general class of malaria transmission-blocking vaccine candidates.     

SOAP,a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development

An essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite-producing oocysts on the mosquito midgut wall. For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion. After invasion ookinete-to-oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components. Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP). The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus. It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine-rich domains. Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds. Moreover, SOAP interacts strongly with mosquito laminin in yeast-two-hybrid assays. Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts. These results identify SOAP as a key molecule for ookinete-to-oocyst differentiation in mosquitoes.     

Calcium signalling in malaria parasites

Ca²⁺ is a ubiquitous intracellular messenger in malaria parasites with important functions in asexual blood stages responsible for malaria symptoms, the preceding liver‐stage infection and transmission through the mosquito. Intracellular messengers amplify signals by binding to effector molecules that trigger physiological changes. The characterisation of some Ca²⁺ effector proteins has begun to provide insights into the vast range of biological processes controlled by Ca²⁺ signalling in malaria parasites, including host cell egress and invasion, protein secretion, motility and cell cycle regulation. Despite the importance of Ca²⁺ signalling during the life cycle of malaria parasites, little is known about Ca²⁺ homeostasis. Recent findings highlighted that upstream of stage‐specific Ca²⁺ effectors is a conserved interplay between second messengers to control critical intracellular Ca²⁺ signals throughout the life cycle. The identification of the molecular mechanisms integrating stage‐transcending mechanisms of Ca²⁺ homeostasis in a network of stage‐specific regulator and effector pathways now represents a major challenge for a meaningful understanding of Ca²⁺ signalling in malaria parasites.     

Delayed action insecticides and their role in mosquito and malaria control

There is considerable interest in the management of insecticide resistance in mosquitoes. One possible approach to slowing down the evolution of resistance is to use late-life-acting (LLA) insecticides that selectively kill only the old mosquitoes that transmit malaria, thereby reducing selection pressure favoring resistance. In this paper we consider an age-structured compartmental model for malaria with two mosquito strains that differ in resistance to insecticide, using an SEI approach to model malaria in the mosquitoes and thereby incorporating the parasite developmental times for the two strains. The human population is modeled using an SEI approach. We consider both conventional insecticides that target all adult mosquitoes, and LLA insecticides that target only old mosquitoes. According to linearised theory the potency of the insecticide affects mainly the speed of evolution of resistance. Mutations that confer resistance can also affect other parameters such as mean adult life span and parasite developmental time. For both conventional and LLA insecticides the stability of the malaria-free equilibrium, with only the resistant mosquito strain present, depends mainly on these other parameters. This suggests that the main long term role of an insecticide could be to induce genetic changes that have a desirable effect on a vital parameter such as adult life span. However, when this equilibrium is unstable, numerical simulations suggest that a potent LLA insecticide can slow down the spread of malaria in humans but that the timing of its action is very important.     

Engineering Plasmodium-refractory phenotypes in mosquitoes

A remarkable number of effector mechanisms have been developed for interfering with malaria parasite development in mosquitoes. These effector mechanisms affect different aspects of parasite biology and therefore could be targeted synergistically to reduce the probability of emergence of parasite resistance to any one mechanism. The use of these mechanisms will depend on how efficiently and safely they can be introduced into existing mosquito populations.     

Characterization of the NleF effector protein from attaching and effacing bacterial pathogens

Enterohemorrhagic Escherichia coli (EHEC) is a water- and food-borne pathogen that causes hemorrhagic colitis. EHEC uses a type III secretion system (T3SS) to translocate effector proteins that subvert host cell function. T3SS-substrates encoded outside of the locus of enterocyte effacement are important to E. coli pathogenesis. We discovered an EHEC secreted protein, NleF, encoded by z6020 in O-island 71 of E. coli EDL933 that we hypothesized to be a T3SS substrate. Experiments are presented that probe the function of NleF and its role in virulence. Immunoblotting of secreted and translocated proteins suggest that NleF is secreted by the T3SS and is translocated into host cells in vitro where it localizes to the host cytoplasm. Infection of HeLa cells with E. coli possessing or lacking nleF and transient expression of NleF-GFP via transfection did not reveal a significant role for NleF in several assays of bacterial adherence, host cytoskeletal remodeling, or host protein secretion. However, competitive coinfection of mice with Citrobacter rodentium strains possessing or lacking nleF suggested a contribution of NleF to bacterial colonization. Challenge of gnotobiotic piglets also revealed a role for NleF in colonization of the piglet colon and rectoanal junction.     

Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC. In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified. NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium, and was absent from non-pathogenic strains of E. coli and non-LEE-containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.     

Can Wolbachia be used to control malaria?

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites transmitted by the infectious bite of Anopheles mosquitoes. Vector control of malaria has predominantly focused on targeting the adult mosquito through insecticides and bed nets. However, current vector control methods are often not sustainable for long periods so alternative methods are needed. A novel biocontrol approach for mosquito-borne diseases has recently been proposed, it uses maternally inherited endosymbiotic Wolbachia bacteria transinfected into mosquitoes in order to interfere with pathogen transmission. Transinfected Wolbachia strains in Aedes aegypti mosquitoes, the primary vector of dengue fever, directly inhibit pathogen replication, including Plasmodium gallinaceum, and also affect mosquito reproduction to allow Wolbachia to spread through mosquito populations. In addition, transient Wolbachia infections in Anopheles gambiae significantly reduce Plasmodium levels. Here we review the prospects of using a Wolbachia-based approach to reduce human malaria transmission through transinfection of Anopheles mosquitoes.     

Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae

Endosymbiotic Wolbachia bacteria are potent modulators of pathogen infection and transmission in multiple naturally and artificially infected insect species, including important vectors of human pathogens. Anopheles mosquitoes are naturally uninfected with Wolbachia, and stable artificial infections have not yet succeeded in this genus. Recent techniques have enabled establishment of somatic Wolbachia infections in Anopheles. Here, we characterize somatic infections of two diverse Wolbachia strains (wMelPop and wAlbB) in Anopheles gambiae, the major vector of human malaria. After infection, wMelPop disseminates widely in the mosquito, infecting the fat body, head, sensory organs and other tissues but is notably absent from the midgut and ovaries. Wolbachia initially induces the mosquito immune system, coincident with initial clearing of the infection, but then suppresses expression of immune genes, coincident with Wolbachia replication in the mosquito. Both wMelPop and wAlbB significantly inhibit Plasmodium falciparum oocyst levels in the mosquito midgut. Although not virulent in non-bloodfed mosquitoes, wMelPop exhibits a novel phenotype and is extremely virulent for approximately 12-24 hours post-bloodmeal, after which surviving mosquitoes exhibit similar mortality trajectories to control mosquitoes. The data suggest that if stable transinfections act in a similar manner to somatic infections, Wolbachia could potentially be used as part of a strategy to control the Anopheles mosquitoes that transmit malaria.