Allison F. Carey

Odorant reception in the malaria mosquito Anopheles gambiae.

The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa. It locates its human hosts primarily through olfaction, but little is known about the molecular basis of this process. Here we functionally characterize the Anopheles gambiae odorant receptor (AgOr) repertoire. We identify receptors that respond strongly to components of human odour and that may act in the process of human recognition. Some of these receptors are narrowly tuned, and some salient odorants elicit strong responses from only one or a few receptors, suggesting a central role for specific transmission channels in human host-seeking behaviour. This analysis of the Anopheles gambiae receptors permits a comparison with the corresponding Drosophila melanogaster odorant receptor repertoire. We find that odorants are differentially encoded by the two species in ways consistent with their ecological needs. Our analysis of the Anopheles gambiae repertoire identifies receptors that may be useful targets for controlling the transmission of malaria.

Molecular basis of odor coding in the malaria vector mosquito Anopheles gambiae

A systematic functional analysis across much of the conventional Anopheles gambiae odorant receptor (AgOR) repertoire was carried out in Xenopus oocytes using two-electrode, voltage-clamp electrophysiology. The resulting data indicate that each AgOR manifests a distinct odor-response profile and tuning breadth. The large diversity of tuning responses ranges from AgORs that are responsive to a single or small number of odorants (specialists) to more broadly tuned receptors (generalists). Several AgORs were identified that respond robustly to a range of human volatiles that may play a critical role in anopheline host selection. AgOR responses were analyzed further by constructing a multidimensional odor space representing the relationships between odorants and AgOR responses. Within this space, the distance between odorants is related to both chemical class and concentration and may correlate with olfactory discrimination. This study provides a comprehensive overview of olfactory coding mechanisms of An. gambiae that ultimately may aid in fostering the design and development of olfactory-based strategies for reducing the transmission of malaria and other mosquito-borne diseases.

Insect olfaction from model systems to disease control

Great progress has been made in the field of insect olfaction in recent years. Receptors, neurons, and circuits have been defined in considerable detail, and the mechanisms by which they detect, encode, and process sensory stimuli are being unraveled. We provide a guide to recent progress in the field, with special attention to advances made in the genetic model organism Drosophila. We highlight key questions that merit additional investigation. We then present our view of how recent advances may be applied to the control of disease-carrying insects such as mosquitoes, which transmit disease to hundreds of millions of people each year. We suggest how progress in defining the basic mechanisms of insect olfaction may lead to means of disrupting host-seeking and other olfactory behaviors, thereby reducing the transmission of deadly diseases.

Calcium dynamics of Plasmodium berghei sporozoite motility.

Calcium is a key signalling molecule in apicomplexan parasites and plays an important role in diverse processes including gliding motility. Gliding is essential for the malaria parasite to migrate from the skin to the liver as well as to invade host tissues and cells. Here we investigated the dynamics of intracellular Ca2+ in the motility of Plasmodium berghei sporozoites by live imaging and flow cytometry. We found that cytosolic levels of Ca2+ increase when sporozoites are activated in suspension, which is sufficient to induce the secretion of integrin‐like adhesins that are essential for gliding motility. By increasing intracellular Ca2+ levels artificially with an ionophore, these adhesins are secreted onto the sporozoite surface, however, the parasite is not capable of gliding. A second level of Ca2+ modulation was observed during attachment to and detachment from a solid substrate, leading to a further increase or a decrease in the cytoplasmic levels of Ca2+ respectively. We also observed oscillations in the intracellular Ca2+ level during gliding. Finally, an intracellular Ca2+ chelator, an inhibitor of phosphoinositide‐specific phospholipase C (PI‐PLC), and an inhibitor of the inositol triphosphate (IP3) receptor blocked the rise in intracellular Ca2+, adhesin secretion, and motility of activated sporozoites, indicating that intracellular stores supply Ca2+ during sporozoite gliding. Our study indicates that a rise in intracellular Ca2+ is necessary but not sufficient to activate gliding, that Ca2+ levels are modulated in several ways during motility, and that a PI‐PLC/IP3 pathway regulates Ca2+ release during the process of sporozoite locomotion.

A bug’s life in the granuloma

The granuloma is the defining feature of the host response to infection with Mycobacterium tuberculosis (Mtb). Despite knowing of its existence for centuries, much remains unclear regarding the host and bacterial factors that contribute to granuloma formation, heterogeneity of presentation, and the forces at play within. Mtb is highly adapted to life within the granuloma and employs many unique strategies to both create a niche within the host as well as survive the stresses imposed upon it. Adding to the complexity of the granuloma is the vast range of pathology observed, often within the same individual. Here, we explore some of the many ways in which Mtb crafts the immune response to its liking and builds a variety of granuloma features that contribute to its survival. We also consider the multitude of ways that Mtb is adapted to life in the granuloma and how variability in the deployment of these strategies may result in different fates for both the bacterium and the host. It is through better understanding of these complex interactions that we may begin to strategize novel approaches for tuberculosis treatments.

Scoring sporozoite motility.

Sporozoites, the stage of Plasmodium infectious to vertebrates when injected in the skin by a mosquito vector, are highly motile cells. Their unusual form of gliding motility is essential for infectivity, allowing the parasite to travel through both the mosquito and mammalian hosts, invading different cell types and escaping immune cell-mediated death. In this chapter, we describe techniques to study gliding motility of sporozoites in vitro and in vivo.

TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities

Once considered a phenotypically monomorphic bacterium, there is a growing body of work demonstrating heterogeneity among Mycobacterium tuberculosis (Mtb) strains in clinically relevant characteristics, including virulence and response to antibiotics. However, the genetic and molecular basis for most phenotypic differences among Mtb strains remains unknown. To investigate the basis of strain variation in Mtb, we performed genome-wide transposon mutagenesis coupled with next-generation sequencing (TnSeq) for a panel of Mtb clinical isolates and the reference strain H37Rv to compare genetic requirements for in vitro growth across these strains. We developed an analytic approach to identify quantitative differences in genetic requirements between these genetically diverse strains, which vary in genomic structure and gene content. Using this methodology, we found differences between strains in their requirements for genes involved in fundamental cellular processes, including redox homeostasis and central carbon metabolism. Among the genes with differential requirements were katG, which encodes the activator of the first-line antitubercular agent isoniazid, and glcB, which encodes malate synthase, the target of a novel small-molecule inhibitor. Differences among strains in their requirement for katG and glcB predicted differences in their response to these antimicrobial agents. Importantly, these strain-specific differences in antibiotic response could not be predicted by genetic variants identified through whole genome sequencing or by gene expression analysis. Our results provide novel insight into the basis of variation among Mtb strains and demonstrate that TnSeq is a scalable method to predict clinically important phenotypic differences among Mtb strains.

Concurrent infection with Mycobacterium tuberculosis confers robust protection against secondary infection in macaques

For many pathogens, including most targets of effective vaccines, infection elicits an immune response that confers significant protection against reinfection. There has been significant debate as to whether natural Mycobacterium tuberculosis (Mtb) infection confers protection against reinfection. Here we experimentally assessed the protection conferred by concurrent Mtb infection in macaques, a robust experimental model of human tuberculosis (TB), using a combination of serial imaging and Mtb challenge strains differentiated by DNA identifiers. Strikingly, ongoing Mtb infection provided complete protection against establishment of secondary infection in over half of the macaques and allowed near sterilizing bacterial control for those in which a secondary infection was established. By contrast, boosted BCG vaccination reduced granuloma inflammation but had no impact on early granuloma bacterial burden. These findings are evidence of highly effective concomitant mycobacterial immunity in the lung, which may inform TB vaccine design and development.