Raffaele Ronca

Humoral response to the Anopheles gambiae salivary protein gSG6: a serological indicator of exposure to Afrotropical malaria vectors.

Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens.

The Anopheles gambiae salivary protein gSG6: an anopheline-specific protein with a blood-feeding role.

The Anopheles gambiae salivary gland protein 6 (gSG6) is a small protein specifically found in the salivary glands of adult female mosquitoes. We report here the expression of a recombinant form of the protein and we show that in vivo gSG6 is expressed in distal-lateral lobes and is secreted with the saliva while the female mosquito probes for feeding. Injection of gSG6 dsRNA into adult A. gambiae females results in decreased gSG6 protein levels, increased probing time and reduced blood feeding ability. gSG6 orthologs have been found so far only in the salivary glands of Anopheles stephensi and Anopheles funestus, both members of the Cellia subgenus. We report here the gSG6 sequence from five additional anophelines, four species of the A. gambiae complex and Anopheles freeborni, a member of the subgenus Anopheles. We conclude that gSG6 plays some essential blood feeding role and was recruited in the anopheline subfamily most probably after the separation of the lineage which gave origin to Cellia and Anopheles subgenera.

MarR-Like Transcriptional Regulator Involved in Detoxification of Aromatic Compounds in Sulfolobus solfataricus

A DNA binding protein, BldR, was identified in the crenarchaeon Sulfolobus solfataricus as a protein 5- to 10-fold more abundant in cells grown in the presence of toxic aldehydes; it binds to regulatory sequences located upstream of an alcohol dehydrogenase gene (Sso2536). BldR is homologous to bacterial representatives of the MarR (multiple antibiotic resistance) family of transcriptional regulators that mediate response to multiple environmental stresses. Transcriptional analysis revealed that the bldR gene was transcribed in a bicistronic unit composed of the genes encoding the transcriptional regulator (Sso1352) and a putative multidrug transporter (Sso1351) upstream. By homology to bacterial counterparts, the bicistron was named the mar-like operon. The level of mar-like operon expression was found to be increased at least 10-fold in response to chemical stress by aromatic aldehydes. Under the same growth conditions, similar enhanced in vivo levels of Sso2536 gene transcript were also measured. The gene encoding BldR was expressed in E. coli, and the recombinant protein was purified to homogeneity. DNA binding assays demonstrated that the protein is indeed a transcription factor able to recognize site specifically both the Sso2536 and mar-like promoters at sites containing palindromic consensus sequences. Benzaldehyde, the substrate of ADH(Ss), stimulates DNA binding of BldR at both promoters. The role of BldR in the auto-activation as well as in the regulation of the Sso2536 gene, together with results of increased operon and gene expression under conditions of exposure to aromatic aldehydes, indicates a novel coordinate regulatory mechanism in cell defense against stress by aromatic compounds.

Wide cross-reactivity between Anopheles gambiae and Anopheles funestus SG6 salivary proteins supports exploitation of gSG6 as a marker of human exposure to major malaria vectors in tropical Africa

BackgroundThe Anopheles gambiae gSG6 is an anopheline-specific salivary protein which helps female mosquitoes to efficiently feed on blood. Besides its role in haematophagy, gSG6 is immunogenic and elicits in exposed individuals an IgG response, which may be used as indicator of exposure to the main African malaria vector A. gambiae. However, malaria transmission in tropical Africa is sustained by three main vectors (A. gambiae, Anopheles arabiensis and Anopheles funestus) and a general marker, reflecting exposure to at least these three species, would be especially valuable. The SG6 protein is highly conserved within the A. gambiae species complex whereas the A. funestus homologue, fSG6, is more divergent (80% identity with gSG6). The aim of this study was to evaluate cross-reactivity of human sera to gSG6 and fSG6.MethodsThe A. funestus SG6 protein was expressed/purified and the humoral response to gSG6, fSG6 and a combination of the two antigens was compared in a population from a malaria hyperendemic area of Burkina Faso where both vectors were present, although with a large A. gambiae prevalence (>75%). Sera collected at the beginning and at the end of the high transmission/rainy season, as well as during the following low transmission/dry season, were analysed.ResultsAccording to previous observations, both anti-SG6 IgG level and prevalence decreased during the low transmission/dry season and showed a typical age-dependent pattern. No significant difference in the response to the two antigens was found, although their combined use yielded in most cases higher IgG level.ConclusionsComparative analysis of gSG6 and fSG6 immunogenicity to humans suggests the occurrence of a wide cross-reactivity, even though the two proteins carry species-specific epitopes. This study supports the use of gSG6 as reliable indicator of exposure to the three main African malaria vectors, a marker which may be useful to monitor malaria transmission and evaluate vector control measures, especially in conditions of low malaria transmission and/or reduced vector density. The Anopheles stephensi SG6 protein also shares 80% identity with gSG6, suggesting the attractive possibility that the A. gambiae protein may also be useful to assess human exposure to several Asian malaria vectors.

Structural analysis of BldR from Sulfolobus solfataricus provides insights into the molecular basis of transcriptional activation in archaea by MarR family proteins.

The multiple antibiotic resistance regulator (MarR) family constitutes a significant class of transcriptional regulators whose members control a variety of important biological functions such as regulation of response to environmental stress, control of virulence factor production, resistance to antimicrobial agents, and regulation of aromatic catabolic pathways. Although the majority of MarR family members have been characterized as transcriptional repressors, a few examples of transcriptional activators have also been reported. BldR is a newly identified member of this family that has been demonstrated to act as a transcriptional activator in stress response to aromatic compounds in the crenarchaeon Sulfolobus solfataricus. In this work, we report findings on the BldR X-ray crystal structure and present a molecular modeling study on the complex that this protein forms with its cognate DNA sequence, thus providing the first detailed description of the DNA-binding mechanism of an archaeal activator belonging to the MarR family. Two residues responsible for the high binding specificity of this transcriptional regulator were also identified. Our studies demonstrated that, in Archaea, the capability of MarR family members to act as activators or repressors is not related to a particular DNA-binding mechanism but rather could be due to the position of the binding site on the target DNA. Moreover, since genes encoding MarR proteins often control transcription of operons that encode for multisubstrate efflux pumps, our results also provided important insights for the identification of new tools to overcome the microorganisms multidrug resistance.

A novel E. coli biosensor for detecting aromatic aldehydes based on a responsive inducible archaeal promoter fused to the green fluorescent protein

A whole-cell bacterial biosensor for measuring aqueous concentrations of aromatic aldehydes was developed. It is based on the E. coli BL21DE3(RIL) expressing the green fluorescent protein under the control of an alcohol dehydrogenase inducible promoter belonging to the archaeon Sulfolobus solfataricus (Sso2536adh promoter). Since it was previously reported that the BldR regulatory protein is the transcription factor required for aromatic aldehyde response in S. solfataricus, the gene encoding for the sensor protein BldR was co-expressed in the biosensor strain on a different compatible plasmid. Gel mobility shift assays showed that the purified recombinant protein can bind specifically to the Sso2536adh promoter. We demonstrated the ability of the archaeal promoter and the BldR transcription factor to operate in a bacterial context to drive active gene expression in a hybrid archaeal/eukaryal fusion. Furthermore, the E. coli BL21DE3(RIL) biosensor strain displayed a specific response and high sensitivity to the different aromatic aldehydes used, suggesting its potential low-cost application to environmentally relevant samples.

Antibody acquisition models: A new tool for serological surveillance of malaria transmission intensity

Serology has become an increasingly important tool for the surveillance of a wide range of infectious diseases. It has been particularly useful to monitor malaria transmission in elimination settings where existing metrics such as parasite prevalence and incidence of clinical cases are less sensitive. Seroconversion rates, based on antibody prevalence to Plasmodium falciparum asexual blood-stage antigens, provide estimates of transmission intensity that correlate with entomological inoculation rates but lack precision in settings where seroprevalence is still high. Here we present a new and widely applicable method, based on cross-sectional data on individual antibody levels. We evaluate its use as a sero-surveillance tool in a Tanzanian setting with declining malaria prevalence. We find that the newly developed mathematical models produce more precise estimates of transmission patterns, are robust in high transmission settings and when sample sizes are small, and provide a powerful tool for serological evaluation of malaria transmission intensity.

Differential antibody response to the Anopheles gambiae gSG6 and cE5 salivary proteins in individuals naturally exposed to bites of malaria vectors

BackgroundMosquito saliva plays crucial roles in blood feeding but also evokes in hosts an anti-saliva antibody response. The IgG response to the Anopheles gambiae salivary protein gSG6 was previously shown to be a reliable indicator of human exposure to Afrotropical malaria vectors. We analyzed here the humoral response to the salivary anti-thrombin cE5 in a group of individuals from a malaria hyperendemic area of Burkina Faso.MethodsELISA was used to measure the anti-cE5 IgG, IgG1 and IgG4 antibody levels in plasma samples collected in the village of Barkoumbilen (Burkina Faso) among individuals of the Rimaibé ethnic group. Anti-gSG6 IgG levels were also determined for comparison. Anopheles vector density in the study area was evaluated by indoor pyrethrum spray catches.ResultsThe cE5 protein was highly immunogenic and triggered in exposed individuals a relatively long-lasting antibody response, as shown by its unchanged persistence after a few months of absent or very low exposure (dry season). In addition cE5 did not induce immune tolerance, as previously suggested for the gSG6 antigen. Finally, IgG subclass analysis suggested that exposed individuals may mount a Th1-type immune response against the cE5 protein.ConclusionsThe anti-cE5 IgG response is shown here to be a sensitive indicator of human exposure to anopheline vectors and to represent an additional tool for malaria epidemiological studies. It may be especially useful in conditions of low vector density, to monitor transiently exposed individuals (i.e. travellers/workers/soldiers spending a few months in tropical Africa) and to evaluate the impact of insecticide treated nets on vector control. Moreover, the gSG6 and cE5 salivary proteins were shown to trigger in exposed individuals a strikingly different immune response with (i) gSG6 evoking a short-lived IgG response, characterized by high IgG4 levels and most likely induction of immune tolerance, and (ii) cE5 eliciting a longer-living IgG response, dominated by anti-cE5 IgG1 antibodies and not inducing tolerance mechanisms. We believe that these two antigens may represent useful reagents to further investigate the so far overlooked role of Anopheles saliva and salivary proteins in host early immune response to Plasmodium parasites.

The Anopheles gambiae cE5, a tight- and fast-binding thrombin inhibitor with post-transcriptionally regulated salivary-restricted expression.

Mosquito saliva carries a large number of factors with anti-hemostatic, anti-inflammatory and immuno-modulatory activities. The cE5 protein was initially identified during an Anopheles gambiae salivary gland transcriptome study and later shown to share sequence similarity with anophelin, a thrombin inhibitor from the saliva of the New World mosquito Anopheles albimanus. The cE5 gene was found to encode different mRNA isoforms coexisting in several tissues of both male and female mosquitoes, a highly unusual profile for a gene potentially encoding an anti-thrombin and involved in blood feeding. Expression of the cE5 protein and assessment of its activity and inhibitory properties showed that it is a highly specific and tight-binding thrombin inhibitor, which differs from the A. albimanus orthologue for the fast-binding kinetics. Despite the widespread occurrence of cE5 transcripts in different mosquito tissues the corresponding protein was only found in female salivary glands, where it undergoes post-translational modification. Therefore, tissue-specific restriction of the A. gambiae cE5 is not achieved by transcriptional control, as common for mosquito salivary genes involved in hematophagy, but by post-trascriptional gene regulatory mechanisms. Our observations provide a paradigm of post-transcriptional regulation as key determinant of tissue specificity for a protein from an important disease vector and point out that transcriptomic data should be interpreted with caution in the absence of concomitant proteomic support.

Polystyrene nanoparticles affect Xenopus laevis development

Exposing living organisms to nanoparticulates is potentially hazardous, in particular when it takes place during embryogenesis. In this investigation, we have studied the effects of 50-nm-uncoated polystyrene nanoparticles (PSNPs) as a model to investigate the suitability of their possible future employments. We have used the standardized Frog Embryo Teratogenesis Assay-Xenopus test during the early stages of larval development of Xenopus laevis, and we have employed either contact exposure or microinjections. We found that the embryos mortality rate is dose dependent and that the survived embryos showed high percentage of malformations. They display disorders in pigmentation distribution, malformations of the head, gut and tail, edema in the anterior ventral region, and a shorter body length compared with sibling untreated embryos. Moreover, these embryos grow more slowly than the untreated embryos. Expressions of the mesoderm markers, bra (T-box Brachyury gene), myod1 (myogenic differentiation1), and of neural crest marker sox9 (sex SRY (determining region Y-box 9) transcription factor sox9), are modified. Confocal microscopy showed that the nanoparticles are localized in the cytoplasm, in the nucleus, and in the periphery of the digestive gut cells. Our data suggest that PSNPs are toxic and show a potential teratogenic effect for Xenopus larvae. We hypothesize that these effects may be due either to the amount of NPs that penetrate into the cells and/or to the “corona” effect caused by the interaction of PSNPs with cytoplasm components. The three endpoints of our study, i.e., mortality, malformations, and growth inhibition, suggest that the tests we used may be a powerful and flexible bioassay in evaluating pollutants in aquatic embryos.