The virus, the vector and the microbiome
If it has a digestive tract, it has microbiota
Despite their often-negative billing as disease-causing germs, gut-living microbes are now recognized as a virtual “organ”. In anything with a digestive tract–from the tiniest fruit fly to the largest whale–the gut microbiota plays a crucial role in modulating host defense against pathogens and is indispensable to the host health. Mosquitoes–the vector for malaria, dengue, yellow fever, and Zika–also host diverse communities of commensal bacteria in their gut. This raises an important question: How does the mosquitoes microbiome affect these human pathogens?
Insect microbiota are MOSTLY beneficial
The coexistence of the insect and its microbiota is mostly harmonious and generally beneficial to the insect. In mosquitoes, it seems that gut bacteria protect their host against invading pathogens by stimulating the insect immune response (1, 2). Multiple studies have found that removing the gut microbiota with antibiotics makes the mosquito more susceptible to infection by the dengue virus (DENV) (3-4) and other pathogens (5-6-7-8).
Does a mosquito gut bacterium give dengue a helping hand?
However, recent studies have shown that resident bacteria can also promote infection by incoming pathogens. In their recent work Wu et al. demonstrate that the presence of Serratia marcescens in the mosquito gut is sufficient to enhance arbovirus infection of the host mosquito.
Using a well-established laboratory-adapted mosquito strain (A. aegypti Rockefeller strain), Wu et al. detect a 10-fold decrease in DENV infectivity after killing the gut bacteria with antibiotics. This suggests that some bacteria of the Rockefeller strain might facilitate viral infection.
Serratia marcescens: A bad resident
To follow up on this initial discovery, the authors isolated 21 bacterial species from the mosquito gut and tested their role in DENV infection of the mosquito. Among these bacteria, the oral re-introduction of Serratia marcescens enhanced the infectivity of DENV in the antibiotic-treated mosquitos. This finding could be repeated for Zika and Sindbis viruses, suggesting that S. marcescens regulates the susceptibility of the Rockefeller strain to arboviruses. The role of S. marcescens was then validated in vivo using interferon receptor-deficient mice, an established model for DENV infection. Mice infected with DENV were subjected to daily biting by antibiotic-treated Rockefeller Aegypty reconstituted orally with or without S. marcescens. DENV load was higher in the mosquito fed with S. marcescens, suggesting that the presence of this commensal bacterium enhances mosquito susceptibility to arboviral infection.
What’s the mechanism?
To identify the mechanism behind this enhanced susceptibility, Wu et al. used the S. marcescens cell lysate or culture supernatant mixed with blood and DENV to feed antibiotic-treated mosquitos, and found that the culture supernatant resulted in a significant enhancement of the DENV infectivity. This result indicates that an extracellular effector secreted by S. marcescens is responsible for DENV infectivity.
Enhancin enhances infection
By mass spectrometry, the authors identified Enhancin (a secreted S. marcescens peptidase) as the culprit! Gut colonization of S. marcescens boosted DENV infection of antibiotic-treated mosquitoes, which could be blocked by a polyclonal antiserum against Enhancin. Consistent with these findings, feeding a mutant strain of S. marcescens that lacks the Enhancin-encoding gene failed to boost DENV infection.
Enhancin breaks down the mosquito gut barrier
Enhancins have proteolytic activity against insect intestinal mucins (glycoproteins that protect and lubricate the epithelial luminal surfaces) and disrupt the epithelium integrity, thereby enabling pathogens to easily penetrate the gut epithelia. Therefore, Wu et al. hypothesized that Enhancin digests the mucin layer on the gut epithelia, allowing DENV easy entry and spread to other tissues.
Although many bacterial species can secrete Enhancin-like proteins, when the authors fed Enhancin-like molecules (29–37% identity) to the mosquito, DENV infectivity was not enhanced. This result suggests that only Enhancin from S. marcescens (not the other bacterial Enhancins) damages the mosquito membrane-bound mucins.
Membrane-bound mucins block DENV in mosquitos
Six mucin genes were found to be highly expressed in the mosquito midgut. RNA-mediated silencing of two of these genes (the only two carrying a transmembrane domain) enhanced DENV infection, suggesting that only mucins with a transmembrane domain play a refractory role against arboviral infection.
Do these findings apply in real-world field strains?
The authors next tested for the presence of S. marcescens in five field-derived mosquitoes; two from regions where DENV is not prevalent (Hainan and Jiangsu strains) and three from regions with recent DENV epidemics (Guangzhou, Foshan, and Tainan strains). Indeed, Wu et al. found S. marcescens in the guts of those mosquitos from the DENV epidemic regions (accounting for up to 18% of the cultivable bacteria colonies), but not in the non-DENV regions (barely detectable). Introduction of S. marcescens into the two deficient strains resulted in enhanced DENV infection, suggesting that the presence of this bacterium in the mosquito gut is necessary and sufficient to enhance infection.
Host-gut microbiome-virus interactions
This is an intriguing and challenging study, which implies that one particular bacterium in the mosquito microbiome is required for high-level DENV infection. Could this also explain the low prevalence of DENV in certain areas of the world where, however, the same types of mosquito thrive? Is S. marcescent absent in the gut of these mosquitos?
Although the authors show that Enhancin is able to cut through mucins that, when present, restrict DENV infection, the study doesn’t prove that mucin cleavage is the only mode of action of Enhancin, as this would have required the challenging development of inactive mutants. Given the delicate balance between the commensal gut microbiota and the immune system of any organism, it cannot be excluded that the introduction of S. marcescens in the mosquito after antibiotic treatment may have additional consequences.
Neverthless, this is a interesting mechanistic study that highlights once again the importance and complexity of the microbiome for viral transmission and dissemination. Can this information be exploited to inform novel strategies of prevention of vector-borne disease transmission?
Article discussed in this blog:
References
1) Weiss & Aksoy, Microbiome influences on insect host vector competence, 2011, Cell
2) Guegan et al., The mosquito holobiont: fresh insight into mosquito-microbiota interactions, 2018, Microbiome
3) Xi et al., The Aedes aegypti toll pathway controls dengue virus infection, 2008, PLoS Pathog.
4) Moreira et al., A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium, 2009, Cell
5) Dong et al., Implication of the Mosquito Midgut Microbiota in the defense against Malaria Parasites, 2009, PLoS Pathog.
6) Gendrin et al., Antibiotics in Ingested Human Blood Affect the Mosquito Microbiota and Capacity to Transmit Malaria, 2015, Nature Com.
7) Cirimotich et al., Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae, 2011, Science
8) Cirimotich et al., Native Microbiota Shape Insect Vector Competence for Human Pathogens, 2011, Cell Host Microbe
