Delftia tsuruhatensis TC1 symbiont suppresses malaria transmission by anopheline mosquitoes.

Malaria control demands the development of a wide range of complementary strategies. We describe the properties of a naturally occurring, non-genetically modified symbiotic bacterium, Delftia tsuruhatensis TC1, which was isolated from mosquitoes incapable of sustaining the development of Plasmodium falciparum parasites. D. tsuruhatensis TC1 inhibits early stages of Plasmodium development and subsequent transmission by the Anopheles mosquito through secretion of a small-molecule inhibitor. We have identified this inhibitor to be the hydrophobic molecule harmane. We also found that, on mosquito contact, harmane penetrates the cuticle, inhibiting Plasmodium development. D. tsuruhatensis TC1 stably populates the mosquito gut, does not impose a fitness cost on the mosquito, and inhibits Plasmodium development for the mosquito's life. Contained field studies in Burkina Faso and modeling showed that D. tsuruhatensis TC1 has the potential to complement mosquito-targeted malaria transmission control.

γδ T cells suppress Plasmodium falciparum blood-stage infection by direct killing and phagocytosis.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum-infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria-γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs.

Th2-like T Follicular Helper Cells Promote Functional Antibody Production during Plasmodium falciparum Infection.

CD4+ T follicular helper cells (Tfh) are key drivers of antibody development. During Plasmodium falciparum malaria in children, the activation of Tfh is restricted to the Th1 subset and not associated with antibody levels. To identify Tfh subsets that are associated with antibody development in malaria, we assess Tfh and antibodies longitudinally in human volunteers with experimental P. falciparum infection. Tfh cells activate during infection, with distinct dynamics in different Tfh subsets. Th2-Tfh cells activate early, during peak infection, while Th1-Tfh cells activate 1 week after peak infection and treatment. Th2-Tfh cell activation is associated with the functional breadth and magnitude of parasite antibodies. In contrast, Th1-Tfh activation is not associated with antibody development but instead with plasma cells, which have previously been shown to play a detrimental role in the development of long-lived immunity. Thus, our study identifies the contrasting roles of Th2 and Th1-Tfh cells during experimental P. falciparum malaria.

Effect of naturally occurring Wolbachia in Anopheles gambiae s.l. mosquitoes from Mali on Plasmodium falciparum malaria transmission.

A naturally occurring Wolbachia strain (wAnga-Mali) was identified in mosquitoes of the Anopheles gambiae complex collected in the Malian villages of Dangassa and Kenieroba. Phylogenetic analysis of the nucleotide sequence of two 16S rRNA regions showed that wAnga-Mali clusters with Wolbachia strains from supergroup A and has the highest homology to a Wolbachia strain isolated from cat fleas (Ctenocephalides). wAnga-Mali is different from two Wolbachia strains previously reported in A. gambiae from Burkina Faso (wAnga_VK5_STP and wAnga_VK5_3.1a). Quantitative analysis of Wolbachia and Plasmodium sporozoite infection in field-collected mosquitoes indicates that the prevalence and intensity of Plasmodium falciparum sporozoite infection is significantly lower in Wolbachia-infected females. The presence of Wolbachia in females from a laboratory Anopheles coluzzii (A. gambiae, M form) colony experimentally infected with P. falciparum (NF54 strain) gametocyte cultures slightly enhanced oocyst infection. However, Wolbachia infection significantly reduced the prevalence and intensity of sporozoite infection, as observed in the field. This indicates that wAnga-Mali infection does not limit early stages of Plasmodium infection in the mosquito, but it has a strong deleterious effect on sporozoites and reduces malaria transmission.

Environmental Correlation Analysis for Genes Associated with Protection against Malaria.

Genome-wide searches for loci involved in human resistance to malaria are currently being conducted on a large scale in Africa using case-control studies. Here, we explore the utility of an alternative approach-"environmental correlation analysis, ECA," which tests for clines in allele frequencies across a gradient of an environmental selection pressure-to identify genes that have historically protected against death from malaria. We collected genotype data from 12,425 newborns on 57 candidate malaria resistance loci and 9,756 single nucleotide polymorphisms (SNPs) selected at random from across the genome, and examined their allele frequencies for geographic correlations with long-term malaria prevalence data based on 84,042 individuals living under different historical selection pressures from malaria in coastal Kenya. None of the 57 candidate SNPs showed significant (P < 0.05) correlations in allele frequency with local malaria transmission intensity after adjusting for population structure and multiple testing. In contrast, two of the random SNPs that had highly significant correlations (P < 0.01) were in genes previously linked to malaria resistance, namely, CDH13, encoding cadherin 13, and HS3ST3B1, encoding heparan sulfate 3-O-sulfotransferase 3B1. Both proteins play a role in glycoprotein-mediated cell-cell adhesion which has been widely implicated in cerebral malaria, the most life-threatening form of this disease. Other top genes, including CTNND2 which encodes δ-catenin, a molecular partner to cadherin, were significantly enriched in cadherin-mediated pathways affecting inflammation of the brain vascular endothelium. These results demonstrate the utility of ECA in the discovery of novel genes and pathways affecting infectious disease.

Interactions between a fungal entomopathogen and malaria parasites within a mosquito vector.

BACKGROUND: Mosquitoes are becoming increasingly resistant to the chemical insecticides currently available for malaria vector control, spurring interest in alternative management tools. One promising technology is the use of fungal entomopathogens. Fungi have been shown to impact the potential for mosquitoes to transmit malaria by reducing mosquito longevity and altering behaviour associated with flight and host location. Additionally, fungi could impact the development of malaria parasites within the mosquito via competition for resources or effects on the mosquito immune system. This study evaluated whether co-infection or superinfection with the fungal entomopathogen Beauveria bassiana affected malaria infection progress in Anopheles stephensi mosquitoes. METHODS: The study used two parasite species to examine possible effects of fungal infection at different parasite development stages. First, the rodent malaria model Plasmodium yoelii was used to explore interactions at the oocyst stage. Plasmodium yoelii produces high oocyst densities in infected mosquitoes and thus was expected to maximize host immunological and resource demands. Second, fungal interactions with mature sporozoites were evaluated by infecting mosquitoes with the human malaria species Plasmodium falciparum, which is highly efficient at invading mosquito salivary glands. RESULTS: With P. yoelii, there was no evidence that fungal co-infection (on the same day as the blood meal) or superinfection (during a subsequent gonotrophic cycle after parasite infection) affected the proportion of mosquitoes with oocysts, the number of oocysts per infected mosquito or the number of sporozoites per oocyst. Similarly, for P. falciparum, there was no evidence that fungal infection affected sporozoite prevalence. Furthermore, there was no impact of infection with either malaria species on fungal virulence as measured by mosquito survival time. CONCLUSIONS: These results suggest that the impact of fungus on malaria control potential is limited to the well-established effects on mosquito survival and transmission behaviour. Direct or indirect interactions between fungus and malaria parasites within mosquitoes appear to have little additional influence.

Chromobacterium Csp_P reduces malaria and dengue infection in vector mosquitoes and has entomopathogenic and in vitro anti-pathogen activities.

Plasmodium and dengue virus, the causative agents of the two most devastating vector-borne diseases, malaria and dengue, are transmitted by the two most important mosquito vectors, Anopheles gambiae and Aedes aegypti, respectively. Insect-bacteria associations have been shown to influence vector competence for human pathogens through multi-faceted actions that include the elicitation of the insect immune system, pathogen sequestration by microbes, and bacteria-produced anti-pathogenic factors. These influences make the mosquito microbiota highly interesting from a disease control perspective. Here we present a bacterium of the genus Chromobacterium (Csp_P), which was isolated from the midgut of field-caught Aedes aegypti. Csp_P can effectively colonize the mosquito midgut when introduced through an artificial nectar meal, and it also inhibits the growth of other members of the midgut microbiota. Csp_P colonization of the midgut tissue activates mosquito immune responses, and Csp_P exposure dramatically reduces the survival of both the larval and adult stages. Ingestion of Csp_P by the mosquito significantly reduces its susceptibility to Plasmodium falciparum and dengue virus infection, thereby compromising the mosquito's vector competence. This bacterium also exerts in vitro anti-Plasmodium and anti-dengue activities, which appear to be mediated through Csp_P -produced stable bioactive factors with transmission-blocking and therapeutic potential. The anti-pathogen and entomopathogenic properties of Csp_P render it a potential candidate for the development of malaria and dengue control strategies.

Midgut microbiota of the malaria mosquito vector Anopheles gambiae and interactions with Plasmodium falciparum infection.

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.

Mosquito ingestion of antibodies against mosquito midgut microbiota improves conversion of ookinetes to oocysts for Plasmodium falciparum, but not P. yoelii.

The mosquito midgut is a site of complex interactions between the mosquito, the malaria parasite and the resident bacterial flora. In laboratory experiments, we observed significant enhancement of Plasmodium falciparum oocyst production when Anopheles gambiae (Diptera: Culicidae) mosquitoes were membrane-fed on infected blood containing gametocytes from in vitro cultures mixed with sera from rabbits immunized with A. gambiae midguts. To identify specific mechanisms, we evaluated whether the immune sera was interfering with the usual limiting activity of gram-negative bacteria in An. gambiae midguts. Enhancement of P. falciparum infection rates occurred at some stage between the ookinete and oocyst stage and was associated with greater numbers of oocysts in mosquitoes fed on immune sera. The same immune sera did not affect the sporogonic development of P. yoelii, a rodent malaria parasite. Not only did antibodies in the immune sera recognize several types of midgut-derived gram-negative bacteria (Pseudomonas spp. and Cedecea spp.), but gentamicin provided in the sugar meal 3 days before an infectious P. falciparum blood meal mixed with immune sera eliminated the enhancing effect. These results suggest that gram-negative bacteria, which normally impair P. falciparum development between the ookinete and oocyst stage, were altered by specific anti-bacterial antibodies produced by immunizing rabbits with non-antibiotic-treated midgut lysates. Because of the differences in developmental kinetics between human and rodent malaria species, the anti-bacterial antibodies had no effect on P. yoelii because their ookinetes leave the midgut much earlier than P. falciparum and so are not influenced as strongly by resident midgut bacteria. While this study highlights the complex interactions occurring between the parasite, mosquito, and midgut microbiota, the ultimate goal is to determine the influence of midgut microbiota on Plasmodium development in anopheline midguts in malaria endemic settings.

Treatment and control of mycoplasma contamination in Plasmodium falciparum culture.

A comparative efficacy of four antibiotics, plasmocin (macrolid), Biomyc-1, -2, (tetracycline), and Biomyc-3, and Mycoplasma Removing Agent (quinolone derivatives) was determined for elimination of mycoplasma from Plasmodium falciparum culture. Presence of mycoplasma was detected using enzyme-PCR-based mycoplasma detection kit and survival of malaria parasite was determined in Giemsa's stained smear made from treated and untreated cultures. It was observed that a combination of Biomyc-1 and -2 killed malaria parasites within 24 h, whereas plasmocin and Biomyc-3 caused slow death of malaria parasite stretched over a period of 6 days. The only compound which did not kill malaria parasite and eradicated mycoplasma from P. falciparum culture was observed to be MRA.

Interference by malaria in the diagnosis of typhoid using Widal test alone.

A total of 270 febrile patients (130 males and 140 females) aged between 15 and 59 were screened using thick and thin blood film stains for malaria, bacteriologic culture and Widal test for enteric fevers. Sixty (22%) were positive for malaria while 38 (14%) were positive for enteric fevers out of which 16 (26.6%) concomitantly had malaria parasite. Cases without malaria parasite (MP) or enteric fever organism were 172 (63.7%) and classified as pyrexia of unknown origin (PUO). Forty-four were strictly malaria cases out of which 36 (82%) were due to Plasmodium falciparum, and all had antibody Widal titres > or = 160 to 0 antigen while 4 (9%) were due to Plasmodium malariae, 3 (6.8%) were due to P. ovale and 1 (2.3%) was due to P. vivax. Twenty (52.6%) of the 38 patients with enteric fever had typhoid, all had Widal titres > or = 160 to 0 antigen. In all, antibody reaction Widal titres to H antigen were < 20. There was no statistical significant difference [chi2 = 327.2, P > 0.05] between Widal titres of malaria and typhoid cases. Hence using Widal test alone, one cannot differentiate typhoid fever from malaria. In another 250 healthy adults, of equal sex distribution, used as controls 12 (4.8%) had malaria parasite and 4 (1.6%) had enteric fever organisms. While only 4 (1.6%) gave Widal titre of 80 to 0 antigen the rest had antibody titres of < 20 to O antigen. Malaria could interfere with serological diagnosis of typhoid and hence lead to over diagnosis of typhoid in Nigeria.

Produccion del factor de necrosis tumoral en la malaria aguda / Production of tumor necrosis factor in acute malaria

El factor de necrosis tumoral (FNT) es una citocina producida por los macrofagos activados qeu parece tener importacia tanto en la proteccion como en la patologia de la malaria. En este estudio se evaluo la liberacion espontanea del factor en el sobrenadante de cultivos in vitro de celulas mononucleares de pacientes malaricos. Un grupo de 40 individuos con Plasmodium falciparum o P. vivax y otro de 20 personas normales se estudiaron mediante un bioensayo con fibroblastos murinos de la linea L929 para la determinacion de los niveles de FNT. Casi 50% de los sobrenadantes de los sujetos infectados presentaron altos niveles del factor, con cantidades 5-10 veces mayores que las cifras normales. A pesar de que algunas de estas personas tenian parasitemias altas (5%-9.2%) ninguna hizo cuadros complicados de la enfermedad y no hubo correlacion entre ella y el factor. Tanto al grupo control como al grupo de los infectados se les determinaron niveles de quimica sanguinea y fibrinogeno, sin que se observara ninguna correlacion con los niveles de FNT

Viruslike particles containing knob-associated histidine-rich protein are secreted into the culture medium of Plasmodium falciparum in vitro cultures.

This report describes the isolation of a viruslike particle from in vitro cultures of the human malaria parasite P. falciparum. Electronmicroscopic observations suggest that the particles are liberated into the culture medium by budding from the erythrocyte membrane. The density of the free particles is 1.16, they contain nucleic acid and two distinct molecular species of the knob-associated Histidine-rich protein. Proteins of the particles are recognized by sera from malaria patients. The previously described knobs may correspond to viral coats inserted in the membrane.

[Isolation of virus-like particles of in vitro cultures of Plasmodium falciparum]. / Isolement de particules de type viral dans les cultures in vitro de Plasmodium falciparum.

We have isolated virus-like particles from in vitro cultures of the malarial agent Plasmodium falciparum. These particles have a buoyant density of 1.16 g/cm3, contain DNA, and appear to arise from budding structures on the surface of parasitized erythrocytes.

Identificacion de proteinas de cubierta y de membrana en merozoitos de Plasmodium falciparum / Identification of coat and membrane proteins in merozoite of Plasmodium falciparum

Se identificaron proteinas que se desprenden facilmente del merozoito y proteinas intrinsecas de la membrana por el fraccionamiento de parasitos marcados endogenamente y purificados. La marcacion continua durante todo el ciclo aseguro la identificacion de las proteinas independientemente de su tiempo de sintesis. Este metodo permitio detectar proteinas de membrana, independientemente de su suceptiobilidad a la digestion enzimatica o a la marcacion exogena. Se identificaron 4 proteinas de 100. 75. 50 y 45 KD que probablemente son constituyentes de la cubierta del merozoito. En la fraccion de membranas, solubles en detergente, se detectaron 6 proteinas principales de 225, 86,82,75,72 y 40 KD y 4 proteinas menores de 200, 69, 45, y 43 KD. Este trabajo es una cotribucion a la caracterizacion de la superficie del merozoito del plasmodium falciparum.