Native microbiota shape insect vector competence for human pathogens

Summary

The resident microbiota of insect vectors can impede transmission of human pathogens. Recent studies have highlighted the capacity of endogenous bacteria to decrease viral and parasitic infections in mosquito and tsetse fly vectors by activating their immune responses or directly inhibiting pathogen development. These microbes may prove effective agents for manipulating the vector competence of malaria and other important human pathogens.

Introduction

Insects serve as the vectors of many pathogens of public health importance, such as those causing malaria, dengue, and trypanosomiasis. These vectors harbor a diversity of microbes from bacteria to fungi that are often found in close proximity to the pathogens that the vectors transmit. The recently demonstrated importance of the microbial communities within multicellular organisms has led to renewed interest in the tripartite interactions that occur between an arthropod vector, the bacteria it harbors, and the pathogens it transmits.

The microbiota associated with insects play important roles in vector physiology, such as nutrition and digestion (reviewed in Dillon and Dillon, 2004) and maturation of the innate immune system (Weiss et al., 2011). Recent analyses using culture-dependent and culture-independent approaches have been conducted to survey the microbial diversity of mosquitoes (references found in Dong et al., 2009; Cirimotich et al., 2011) and tsetse flies (Lindh and Lehane, 2011). These surveys have revealed a diverse community of bacteria encompassing several phylogenetic classes. Most abundant of these is the bacterial class Proteobacteria, family Enterobacteriaceae. Common isolates include members of the genera Enterobacter, Pantoea, Pseudomonas and Serratia. With the exception of vertically-transmitted bacteria of the genus Asaia in mosquitoes (Favia et al., 2007) and symbiotic bacteria in tsetse flies, the originating source of commensal bacteria in insect vectors has not yet been fully resolved. The intimate relationship that often exists between some of these microbes and their arthropod hosts supports investigations of their use in vector-borne disease control strategies.

The application of microbial symbionts to reduce vector competence is a novel approach to controlling the spread of arthropod-transmitted pathogens. Proof of concept for this approach comes from observations in mosquitoes and tsetse flies. For instance, a higher bacterial load in the gut is associated with a lower infection rate in Anopheles mosquitoes exposed to Plasmodium falciparum (Cirimotich et al., 2011; Dong et al., 2009; Pumpuni et al., 1993). Furthermore, it appears that not all bacteria have the potential to produce a resistant phenotype. Some bacterial isolates have a stronger effect than others in reducing parasite survival in the mosquito gut (Cirimotich et al., 2011; Gonzalez-Ceron et al., 2003; Pumpuni et al., 1993). For example, currently, there is no evidence that the symbiotic Asaia bacteria directly reduce pathogen infection in mosquitoes, but trypanosome infection of tsetse flies may be modulated by the mutualistic bacterial symbiont Wigglesworthia sp. (Pais et al., 2008).

Another approach, paratransgenesis, makes use of this information but goes a step further to effectively target the pathogen. Through the genetic modification of bacterial symbionts to produce anti-pathogen molecules in the proper tissues or compartments of the insect, vector competence for pathogens can be reduced. The pioneering paratransgenic strategy involved the disruption of Trypanosoma cruzi transmission in the triatomine bug Rhodnius prolixus via expression of antimicrobial peptides and single-chain antibodies by the genetically modified symbiotic bacterium Rhodococcus rhodnii (reviewed in Beard et al., 2002). The unique transmission of Rhodococcus through coprophagy makes this association optimal for paratransgenic pathogen control, but the concept serves as a foundation for exploratory studies in other systems.

There is a functional overlap in the antibacterial, anti-parasitic and anti-viral innate immune responses of disease vectors. When microbe-associated molecular patterns are detected by vector pattern recognition receptors, immune pathways are activated, culminating in an increased abundance of effector molecules that defend against infection with human pathogens such as P. falciparum and dengue virus (Dong et al., 2009; Xi et al., 2008). Recent studies have shown that antibacterial responses mounted against commensal midgut bacteria in the mosquito and symbiotic bacteria in the tsetse fly also protect the vector against infection by viruses and parasites, thereby interrupting the transmission cycle of these pathogens. This review summarizes the current advances in our understanding of vector-microbiota-pathogen tripartite interactions and discusses future perspectives on the application of microorganisms as novel control strategies.

The native insect microbiota influence parasite transmission

The Anopheles gambiae antibacterial immune response is at least partially responsible for inhibition of Plasmodium infection (Figure 1). Microarray analyses have shown a substantial overlap in innate immune gene up-regulation in A. gambiae mosquitoes that have been challenged with systemic bacterial infection or oral Plasmodium infection (Dong et al., 2006). Some of these genes have been found to protect mosquitoes against bacterial infection, whereas others protect against both bacterial and Plasmodium infection. However, no genes were found to protect specifically against parasite infection, suggesting that A. gambiae uses the antibacterial arm of the innate immune response to control Plasmodium infection (Dong et al., 2006). While functional overlap does exist within the mosquito innate immune pathways, the immune deficiency (IMD) pathway, sharing conserved features with the tumor necrosis factor signaling pathway involved in lipid metabolism, insulin resistance and other activities in humans, is primarily important for controlling human malaria parasite infection in the mosquito (Garver et al., 2009).

Figure 1.

The microbiota harbored in the digestive tract of arthropods impacts the ability of vector-borne pathogens to infect the vector. (1) Commensal and symbiotic bacteria associated with the arthropod gut stimulate an antibacterial immune response that also negatively impacts pathogen development. (2) A cross-linked protein layer that inhibits over-activation of the immune system may also protect pathogens in the gut (Kumar et al., 2010). (3) Bacterial populations expand rapidly in the gut following blood ingestion and may provide a physical barrier to pathogen interaction with the gut epithelium. (4) Molecules secreted by bacteria, such as reactive oxygen intermediates or secondary metabolites, may kill or interfere with parasites in the gut before infection of the vector occurs (Cirimotich et al., 2011).

Commensal bacteria within the mosquito midgut stimulate production of basal levels of effector molecules that control the proliferation of the bacterial populations as well as the Plasmodium parasite populations at the ookinete and oocyst stages of development (Dong et al., 2009). A. gambiae mosquitoes are more susceptible to Plasmodium infection when the midgut-commensal bacteria including Chryseobacterium, Enterobacter, and Serratia species are removed from the midgut via antibiotic treatment prior to an infectious blood feed, and the resistance phenotype can be recapitulated by the introduction of non-native or midgut-isolated bacteria into antibiotic-treated mosquitoes (Dong et al., 2009; Meister et al., 2009). In these analyses, the impact on parasite development is to a significant extent linked to the abundance of multiple immune effector molecules that are regulated by the basal activation of the IMD pathway through the transmembrane peptidoglycan recognition protein LC (Dong et al., 2009; Meister et al., 2009).

In support of a role for innate immunity in Plasmodium infection, P. falciparum susceptibility in field-captured mosquitoes can be linked to a single genomic locus comprised of numerous innate immune genes with potent anti-Plasmodium potential (Riehle et al., 2006). In these analyses, it was observed that a majority of mosquito pedigrees were completely refractory to parasite infection, suggesting that refractoriness is the dominant phenotype, with infection occurring as a result of immune repression or failure (Riehle et al., 2006). A. gambiae mosquitoes are protected from an over-activation of their immune responses following blood ingestion by a network of cross-linked proteins (Figure 1). In a series of reactions, immune peroxidase(ImPer) and dual oxidase (DUOX) enzymes coordinate to form dityrosine bonds between proteins in the midgut lumen (Kumar et al., 2010). This network resides between the midgut epithelium and peritrophic matrix and minimizes the interaction of the mosquito midgut with microbial immune elicitors that would over-stimulate innate immune responses (Kumar et al., 2010). However, mosquitoes depleted of ImPer or DUOX produced higher levels of immune factors and harbored lower parasite burdens, showing that the matrix also confers protection to the Plasmodium parasites within the midgut by providing a more hospitable environment for parasite development (Kumar et al., 2010).

Endosymbiotic bacteria in tsetse flies have been implicated in an inhibition of trypanosomes that involves the stimulation of antibacterial immune responses (Wang et al., 2009) (Figure 1). Wigglesworthia bacteria are vertically transmitted from mother to offspring, reside in a specialized organ within the fly midgut, and are essential for reproductive success in the fly. Homeostasis between symbiotic bacteria and the tsetse fly is maintained through the peptidoglycan recognition protein LB (PGRP-LB), which is up-regulated by the presence of Wigglesworthia (Wang et al., 2009). It is thought that tsetse PGRP-LB scavenges the immune-stimulating peptidoglycan released by Wigglesworthia, decreasing the antibacterial response directed toward the bacteria. However, PGRP-LB and effector molecules of the tsetse fly IMD pathway have direct anti-trypanosome activity, suggesting that a balance between a tolerance for symbiotic microorganisms and control of pathogenic microbes is required (Wang et al., 2009). These experiments have also shown that parasite control mediated by antibacterial immune responses is a conserved mechanism in mosquitoes and tsetse flies and suggest that a direct antiparasite response in these vectors may not be present.

Bacteria may also directly inhibit pathogen development, either by hindering necessary interactions between the pathogen and vector epithelium or through the production of anti-pathogen molecules (reviewed in Azambuja et al., 2005) (Figure 1). Recently, it was found that an Enterobacter bacterium isolated from wild Anopheles populations in Zambia decreases Plasmodium development in an in vitro culture system, showing that a mosquito-mounted response is not required for the observed inhibition (Cirimotich et al., 2011). The production of reactive oxygen molecules was established as the basis for the inhibition exhibited by the Enterobacter bacterium (Cirimotich et al., 2011). In this study, other Gram-negative bacteria inhibited Plasmodium development to different levels at the ookinete stage but all isolates nearly eliminated oocyst formation, suggesting that the microbial exposure of wild mosquito populations, and thus the composition of the midgut bacteria, can determine vector competence for malaria parasites (Cirimotich et al., 2011). These observations could help to partially explain the variability in transmission dynamics exhibited by mosquitoes of the same species in endemic regions, although this hypothesis requires much more experimental attention.

Native mosquito microbiota influence arbovirus infection

Relatively few studies have examined how the mosquito microbiota influences arbovirus infection and transmission. Similar to the bacteria-dependent infection intensities in other vector-pathogen systems, removal of gut bacteria through antibiotic treatment results in higher dengue virus type 2 (family Flaviviridae) loads in Aedes aegypti mosquitoes (Xi et al., 2008). Although the mechanism(s) responsible for this phenotype has (have) not been characterized, innate immune responses elicited by the midgut bacteria, especially the Toll pathway may be involved (Xi et al., 2008) (Figure 1). Mosquitoes in which the Toll pathway has been activated prior to dengue virus challenge are better able to defend against virus infection, implicating this pathway in mosquito antiviral defense (Xi et al., 2008).

An alternative mechanism could be that midgut bacterial populations interfere with proper binding of virus with the respective receptors on the mosquito midgut epithelium (Figure 1). This could be mediated through a direct interaction between the bacteria and virus in the blood bolus or bacterial secretion of a factor that inhibits virus binding and/or entry into the midgut epithelial cells. Incubation of La Crosse virus (family Bunyaviridae) with bacterial isolates from field collected A. albopictus mosquitoes, a regional vector of the virus, reduces virus infectivity in vitro (Joyce et al., 2011). The mechanism of inactivation has not been identified and no studies of this potential interaction within the mosquito vector have been published. However, bacteria in this study were removed through filtration prior to challenging the in vitro cell culture with virus, suggesting that bacterial production of a virus neutralizing or killing factor may also influence arbovirus infection (Joyce et al., 2011). The identification of antiviral molecules produced by bacteria could lead to paratransgenic strategies of arbovirus control.

Current and future applications of microbiota to decrease pathogen transmission

Recently, the introduction into mosquitoes of non-native intracellular bacterial symbionts of the genus Wolbachia has been shown to reduce infection by dengue and chikungunya viruses, filarial nematodes, and Plasmodium parasites (reviewed in Iturbe-Ormaetxe et al., 2011). These bacteria and entomopathogenic fungi also decrease the ability of mosquitoes to transmit pathogens by decreasing the longevity of infected individuals (reviewed in Kanzok and Jacobs-Lorena, 2006; Iturbe-Ormaetxe et al., 2011). Wolbachia-infected mosquitoes are currently being pursued in field trials for eventual use as interventions to disrupt vector-borne disease transmission (Enserink, 2010).

Genetically modified bacterial symbionts have been used to effectively decrease parasite transmission by triatomine bugs (reviewed in Beard et al., 2002). In a progressive study for the development of paratransgenic techniques to control mosquito-borne disease, a Pantoea agglomerans bacterium isolated from Anopheles mosquitoes was engineered to secrete known synthetic anti-Plasmodium molecules (Bisi and Lampe, 2011). While the potential inhibition of mosquito infection has not been published, this proof-of-principle study will further the efforts of developing paratransgenic strategies in mosquitoes and tsetse flies. However, new bacterial transformation techniques require development and the source of mosquito-commensal midgut bacteria will need to be identified before these strategies can be implemented. The eventual engineering of bacteria that stably associate with vector insects, such as Asaia, Wolbachia, Wigglesworthia or Sodalis species could provide novel tools for combating vector-borne diseases.

Currently, no interventions based on native mosquito bacteria are yet in the field stage of development. Along with paratransgenic strategies, novel interventions aimed at increasing the prevalence of inhibitory commensal bacteria in wild vector populations may eventually decrease the burden of vector-borne disease transmission. As new strategies to combat pathogen transmission are being developed, it will be important to examine the native microbiota and their impact on disease transmission as part of an integrated strategy for disease control.