Bacterial α(2)-macroglobulins: colonization factors acquired by horizontal gene transfer from the metazoan genome?

BACKGROUND: Invasive bacteria are known to have captured and adapted eukaryotic host genes. They also readily acquire colonizing genes from other bacteria by horizontal gene transfer. Closely related species such as Helicobacter pylori and Helicobacter hepaticus, which exploit different host tissues, share almost none of their colonization genes. The protease inhibitor α(2)-macroglobulin provides a major metazoan defense against invasive bacteria, trapping attacking proteases required by parasites for successful invasion. RESULTS: Database searches with metazoan α(2)-macroglobulin sequences revealed homologous sequences in bacterial proteomes. The bacterial α(2)-macroglobulin phylogenetic distribution is patchy and violates the vertical descent model. Bacterial α(2)-macroglobulin genes are found in diverse clades, including purple bacteria (proteobacteria), fusobacteria, spirochetes, bacteroidetes, deinococcids, cyanobacteria, planctomycetes and thermotogae. Most bacterial species with bacterial α(2)-macroglobulin genes exploit higher eukaryotes (multicellular plants and animals) as hosts. Both pathogenically invasive and saprophytically colonizing species possess bacterial α(2)-macroglobulins, indicating that bacterial α(2)-macroglobulin is a colonization rather than a virulence factor. CONCLUSIONS: Metazoan α(2)-macroglobulins inhibit proteases of pathogens. The bacterial homologs may function in reverse to block host antimicrobial defenses. α(2)-macroglobulin was probably acquired one or more times from metazoan hosts and has then spread widely through other colonizing bacterial species by more than 10 independent horizontal gene transfers. yfhM-like bacterial α(2)-macroglobulin genes are often found tightly linked with pbpC, encoding an atypical peptidoglycan transglycosylase, PBP1C, that does not function in vegetative peptidoglycan synthesis. We suggest that YfhM and PBP1C are coupled together as a periplasmic defense and repair system. Bacterial α(2)-macroglobulins might provide useful targets for enhancing vaccine efficacy in combating infections

Microbiota-Dependent Immune Responses to Intestinal Parasites

The digestive tract plays a central role in nutrient acquisition and harbors a vast and intricate community of bacteria, fungi, viruses and parasites, collectively known as the microbiota. In recent years, there has been increasing recognition of the complex and highly contextual involvement of this microbiota in the induction and education of host innate and adaptive immune responses under homeostasis, during infection and inflammation. The gut passage and colonization by unicellular and multicellular parasite species present an immense challenge to the host immune system and to the microbial communities that provide vital support for its proper functioning. In mammals, parasitic nematodes induce distinct shifts in the intestinal microbial composition. Vice versa, the commensal microbiota has been shown to serve as a molecular adjuvant and immunomodulator during intestinal parasite infections. Moreover, similar interactions occur within insect vectors of deadly human pathogens. The gut microbiota has emerged as a crucial factor affecting vector competence in Anopheles mosquitoes, where it modulates outcomes of infections with malaria parasites. In this review, we discuss currently known involvements of the host microbiota in the instruction, support or suppression of host immune responses to gastrointestinal nematodes and protozoan parasites in mice, as well as in the malaria mosquito vector. A deeper understanding of the mechanisms underlying microbiota-dependent modulation of host and vector immunity against parasites in mammals and mosquitoes is key to a better understanding of the host-parasite relationships and the identification of more efficient approaches for intervention and treatment of parasite infections of both clinical and veterinary importance

High-throughput Sorting of Mosquito Larvae for Laboratory Studies and for Future Vector Control Interventions

Background: Mosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever. Genetic control strategies require the release of large number of male mosquitoes into field populations, whether they are based on the use of sterile males (sterile insect technique, SIT) or on introducing genetic traits conferring refractoriness to disease transmission (population replacement). However, the current absence of high-throughput techniques for sorting different mosquito populations impairs the application of these control measures. Methods: A method was developed to generate large mosquito populations of the desired sex and genotype. This method combines flow cytometry and the use of Anopheles gambiae transgenic lines that differentially express fluorescent markers in males and females. Results: Fluorescence-assisted sorting allowed single-step isolation of homozygous transgenic mosquitoes from a mixed population. This method was also used to select wild-type males only with high efficiency and accuracy, a highly desirable tool for genetic control strategies where the release of transgenic individuals may be problematic. Importantly, sorted males showed normal mating ability compared to their unsorted brothers. Conclusions: The developed method will greatly facilitate both laboratory studies of mosquito vectorial capacity requiring high-throughput approaches and future field interventions in the fight against infectious disease vectors

Complement-Like Protein TEP1 Is a Determinant of Vectorial Capacity in the Malaria Vector Anopheles gambiae

AbstractAnopheles mosquitoes are major vectors of human malaria in Africa. Large variation exists in the ability of mosquitoes to serve as vectors and to transmit malaria parasites, but the molecular mechanisms that determine vectorial capacity remain poorly understood. We report that the hemocyte-specific complement-like protein TEP1 from the mosquito Anopheles gambiae binds to and mediates killing of midgut stages of the rodent malaria parasite Plasmodium berghei. The dsRNA knockdown of TEP1 in adults completely abolishes melanotic refractoriness in a genetically selected refractory strain. Moreover, in susceptible mosquitoes this knockdown increases the number of developing parasites. Our results suggest that the TEP1-dependent parasite killing is followed by a TEP1-independent clearance of dead parasites by lysis and/or melanization. Further elucidation of the molecular mechanisms of TEP1-mediated parasite killing will be of great importance for our understanding of the principles of vectorial capacity in insects

In Vivo Identification of Novel Regulators and Conserved Pathways of Phagocytosis in A. gambiae

SummaryAnopheles gambiae uses effective immune responses, including phagocytosis, to fight microbial infection. We have developed a semiquantitative phagocytosis test and used it in conjunction with dsRNA gene silencing to test the in vivo roles of 71 candidate genes in phagocytosis of Escherichia coli and Staphylococcus aureus. Here, we show that inactivation of 26 genes changes the phagocytic activity by more than 45% and that two pathways similar to those that mediate apoptotic cell removal in Caenorhabditis elegans are used in A. gambiae for phagocytosis of microorganisms. Simultaneous inactivation of the identified regulators of phagocytosis and conserved components defining each signaling pathway permitted provisional assignment of the novel regulators to one or the other pathway. Pathway inactivation enhances at least three times the ability of E. coli and S. aureus to proliferate in the mosquito. Interestingly, mosquito survival is not compromised even if both pathways are perturbed simultaneously

Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases

Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control

Fz2 and Cdc42 Mediate Melanization and Actin Polymerization but Are Dispensable for Plasmodium Killing in the Mosquito Midgut

The midgut epithelium of the mosquito malaria vector Anopheles is a hostile environment for Plasmodium, with most parasites succumbing to host defenses. This study addresses morphological and ultrastructural features associated with Plasmodium berghei ookinete invasion in Anopheles gambiae midguts to define the sites and possible mechanisms of parasite killing. We show by transmission electron microscopy and immunofluorescence that the majority of ookinetes are killed in the extracellular space. Dead or dying ookinetes are surrounded by a polymerized actin zone formed within the basal cytoplasm of adjacent host epithelial cells. In refractory strain mosquitoes, we found that formation of this zone is strongly linked to prophenoloxidase activation leading to melanization. Furthermore, we identify two factors controlling both phenomena: the transmembrane receptor frizzled-2 and the guanosine triphosphate–binding protein cell division cycle 42. However, the disruption of actin polymerization and melanization by double-stranded RNA inhibition did not affect ookinete survival. Our results separate the mechanisms of parasite killing from subsequent reactions manifested by actin polymerization and prophenoloxidase activation in the A. gambiae–P. berghei model. These latter processes are reminiscent of wound healing in other organisms, and we propose that they represent a form of wound-healing response directed towards a moribund ookinete, which is perceived as damaged tissue

Unbiased classification of mosquito blood cells by single-cell genomics and high-content imaging.

Mosquito blood cells are immune cells that help control infection by vector-borne pathogens. Despite their importance, little is known about mosquito blood cell biology beyond morphological and functional criteria used for their classification. Here, we combined the power of single-cell RNA sequencing, high-content imaging flow cytometry, and single-molecule RNA hybridization to analyze a subset of blood cells of the malaria mosquito Anopheles gambiae By demonstrating that blood cells express nearly half of the mosquito transcriptome, our dataset represents an unprecedented view into their transcriptional program. Analyses of differentially expressed genes identified transcriptional signatures of two cell types and provide insights into the current classification of these cells. We further demonstrate the active transfer of a cellular marker between blood cells that may confound their identification. We propose that cell-to-cell exchange may contribute to cellular diversity and functional plasticity seen across biological systems

Salivary gland-specific P. berghei reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes

Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines