“Probiotic” activates AMPK in mosquitoes, inhibiting Zika and Dengue viruses: The Shock and Live Approach links Viruses, Oocytes, Sperm, & Progeria

“Probiotic” activates AMPK in mosquitoes, inhibiting Zika and Dengue viruses: The Shock and Live Approach links Viruses, Oocytes, Sperm, & Progeria

https://www.linkedin.com/pulse/probiotic-activates-ampk-mosquitoes-inhibiting-zika-dengue-finley?trk=pulse_spock-articles

  

Reports last week from the Australia-based non-profit organization Eliminate Dengue provided details of an ongoing initiative to decrease the spread of dengue and Zika viruses through the coordinated release of male and female mosquitoes (called Aedes aegypti) that were purposely infected with a bacterium that inhibits the mosquito’s ability to transmit the two viruses to humans [1].  Set to launch in Colombia and Brazil early next year, the control campaigns will be funded with $18 million from donors including the U.S. and British governments as well as the Bill & Melinda Gates Foundation.  Interestingly, the Google health spin-off Verily also recently announced a similar initiative to prevent the spread of dengue and Zika virus transmission by using the same bacterium to infect male mosquitoes only. Infected male mosquitoes are unable to reproduce with normal/uninfected female mosquitoes [1,2].    

Interestingly, in addition to significantly inhibiting the transmission of the two viruses by Aedes aegypti to humans, the bacterium, called Wolbachia, also occurs naturally in many insects and is transmitted successfully to offspring by females after an infected female mates with an infected male, or an infected female mates with a normal/uninfected male [3].  However, a normal/uninfected female is only able to produce viable offspring with a normal/uninfected male, leading to a significant reproductive advantage for Wolbachia-infected females and thus propagation of Wolbachia throughout mosquitoes in the wild. Additionally, only female mosquitoes bite and require a blood meal to promote egg production, further highlighting the potential of reducing dengue and Zika virus transmission to humans via Wolbachia-infected female mosquitoes.

Although the method through which Wolbachia is transmitted to mosquito offspring has been well studied, how Wolbachia infection leads to a decrease in dengue and Zika virus replication in, and transmission by, mosquitoes is heavily debated.  Interestingly, although Wolbachia does not naturally occur in Aedes aegypti, Wolbachia appears to impart beneficial immunomodulatory effects in these mosquitoes not unlike that of certain beneficial bacteria that normally colonize the human gut.  Such bacteria, also known as “probiotics”, are typified by the genera Lactobacillus and Bifidobacterium and have been shown to significantly enhance natural and acquired immunity, induce reactive oxygen species (ROS) generation and the upregulation of Nrf2-dependent cytoprotective genes, produce short chain fatty acids (SCFAs) that promote T cell differentiation, and produce antimicrobial peptides that exhibit bactericidal activity [4-7].  Interestingly, the induction of cellular stress via ROS generation has been shown to activate the master metabolic regulator AMPK and AMPK activation has been shown to be critical for T cell activation and the mounting of an effective immune response in vivo to viral and bacterial infections [8-10].  Indeed, acetate, propionate, and butyrate, three SCFAs abundantly produced by probiotic bacteria, each activate AMPK, indicating that the immunomodulatory effects of probiotic bacteria are dependant on cellular stress-induced activation of AMPK [11]. Furthermore, AMPK has recently been discovered in Aedes aegypti and activation of AMPK in Aedes aegypti by compounds that induce cellular stress leads to an enhanced immune response and increased longevity in Aedes aegypti (see below), implicating the provocative assertion that inhibition of dengue and Zika virus replication via Wolbachia infection occurs as result of cellular stress-induced activation of AMPK [12].

As noted above, Wolbachia, which are found in reproductive tissues of arthropods, often engage in a mutualistic relationship with its host. Indeed, Wolbachia has been shown to induce resistance to RNA viral infections in the model organism Drosophila melanogaster [13]. D. melanogaster (also known as the fruit fly) has been studied extensively in biological research and activation of AMPK has been shown to extend lifespan as well as slow aging in D. melanogaster through autophagic induction via upregulation of Atg1 (ULK1 in mammals), reduction of insulin-like peptide levels in the brain, and an increase in 4E-BP [14,15]. 

Interestingly, insulin-like peptides and hyperphosphorylation of 4E-BP (i.e. inactivation) is positively associated with target of rapamycin (TOR) activation and egg development in Aedes aegypti (Ae. Aegypti) [16]. Mammalian target of rapamycin (mTORC1/TORC1) is a protein kinase that plays a critical role in promoting mRNA translation and is the primary target of rapamycin, a macrolide drug that extends lifespan in several model organisms including normal genetically heterogeneous mice and D. melanogaster [17,18].  Rapamycin was shown to delay yolk deposition in control Ae. aegypti eggs and inhibit insulin-mediated 4E-BP (a negative regulator of translation) phosphorylation. Silencing of 4E-BP also led to a reduction in lifespan of adult female mosquitoes, mirroring results obtained via AMPK activation in D. melanogaster [15,16,19]. Strikingly, in addition to delaying egg maturation in Ae. aegypti, rapamycin has also been shown to dramatically increase the number of Wolbachia in D. melanogaster oocytes via TORC1 inhibition, whereas somatic hyperactivation of TORC1 decreases Wolbachia titer in oocytes [20]. Interestingly, ablation of insulin-producing cells in the D. melanogaster brain abolished the yeast-induced reduction of Wolbachia in oocytes, mirroring previous results showing that AMPK-induced lifespan extension in D. melanogaster is associated with a decrease in insulin-like peptides in the brain [15,20].    

Because rapamycin, similar to AMPK activation by diverse compounds, induces autophagy, inhibits mTOR/TORC1, promotes mitochondrial biogenesis, and extends  lifespan in several model organisms, it would expected that rapamycin would also induce activation of AMPK, likely via the induction of cellular stress (i.e. increase in the levels of ROS, intracellular calcium increase, increase in the AMP:ADP/ATP ratio, etc). Indeed, two recent publications clearly demonstrated that rapamycin potently induced the activation of AMPK in vivo in normal elderly mice, along with an upregulation of ULK1 (mammalian orthologue of Atg1 critical for the induction of autophagy) and PGC-1a (a transcription factor essential for promoting mitochondrial biogenesis) [21,22].  As rapamycin has been shown to increase the number of Wolbachia in D. melanogaster oocytes, delay egg maturation and inhibit insulin-mediated 4E-BP phosphorylation in Ae. aegypti, and increase lifespan in D. melanogaster, the recent finding that activation of AMPK in Ae. aegypti also increases lifespan and improves the immune response (see below) further bolsters the notion that activation of AMPK via the induction of cellular stress likely promotes lifespan extension, immune system enhancement, and Wolbachia  propagation in oocytes in both D. melanogaster and Ae. aegypti.  Moreover, because certain bacteria (e.g. Lactobacillus) that colonize the human gut induces beneficial immune responses by inducing cellular stress (e.g. increased ROS levels) and also produce compounds that activate AMPK, the inhibition of dengue and Zika virus replication in, and transmission by, Ae. aegypti is likely the result of a Wolbachia-induced cellular stress response, leading to the up regulation of anti-viral mechanisms that are likely modulated by AMPK.

Indeed, a recent study by Wong et al. demonstrated that ROS/oxidative stress is positively correlated with Wolbachia-mediated antiviral protection in D. melanogaster [23]. The authors of the study observed that H2O2 (hydrogen peroxide) was increased 1.25- to 2- fold in flies that harbored protective strains of Wolbachia (e.g. wMelCS, wRi, wAu) compared to Wolbachia-free controls. Interestingly, flies with a null mutation in Cu/Zn SOD (an antioxidant enzyme) exhibited elevated endogenous levels of oxidative stress that mimicked Wolbachia-induced oxidative stress.  In these Wolbachia-free Cu/Zn SOD mutant flies, a 70% survival rate was observed at 4 days post-infection after Drosophila C virus infection compared to a less than 10% survival rate for non-mutant flies, indicating that elevated levels of oxidative stress induced by protective Wolbachia strains confers a survival advantage by decreasing susceptibility to viral infection via activation of signaling pathways that potentiate anti-viral immune responses [23].

As noted above, ROS/oxidative stress has been shown to activate AMPK and AMPK activation in both D. melanogaster and Ae. Aegypti leads to an increase in lifespan [8,12,15].  Interestingly, Sykiotis et al. showed that oxidants/oxidative stress promote lifespan extension in D. melanogaster males by activating the Nrf2 (CncC) pathway, a master antioxidant transcription factor that induces the expression of several antioxidant genes including NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) [24].  Curiously, Sykiotis et al. also observed that the synthetic dithiolthione oltipraz, which has been shown to activate AMPK, inhibit HIV-1 replication, and reverse accelerating aging defects in Hutchinson-Gilford progeria syndrome (see below), also induced Nrf2 signaling in flies [24-27].  AMPK has also been shown to increase the transcriptional activity of Nrf2 and increase Nrf2 nuclear retention via phosphorylation, suggesting that AMPK may represent a central node in ROS/oxidative stress-induced immune and anti-viral responses [28,29].

Indeed, a recent study by Pan et al. showed that infection of Ae. Aegypti with Wolbachia led to a ROS/oxidative stress-induced upregulation of genes associated with immunity and reduction-oxidation, thus enhancing inhibition of dengue virus replication [30]. Several gene transcripts related to the immunity and redox/stress/mitochondrion group were upregulated in Wolbachia-infected Ae. aegypti females before blood feeding and after infection with dengue virus (DENV) serotype 2, including the antimicrobial peptides cecropin D (CECD) and defensin C (DEFC) as well as the antioxidants glutathione peroxidase, CuZnSOD, MnSOD, and glutathione peroxidase [30].  A significant increase in the levels of H2O2 and in the transcript abundance of both NADPH oxidase M (NOXM) and dual oxidase 2 (DUOX2), two enzymes that generate ROS, were also observed in Wolbachia-infected mosquitoes compared to control mosquitoes.  Interestingly, the expression of several Toll pathway (a pathway that mediates the production of antioxidants and antimicrobial peptides) marker genes as well as the antimicrobial peptides CECD and DEFC were increased by the addition of H2O2 in a sugar solution given to female control/uninfected mosquitoes [30].  This effect was also mirrored in Wolbachia-infected female mosquitoes, wherein silencing of NOXM and DUOX2 deactivated the Toll pathway and suppressed the expression of CECD and DEFC, indicating that Wolbachia-induced ROS is required for activation of the Toll pathway and induction of antimicrobial peptides.  Importantly, individual or double RNAi-induced knockdown of DEFC and CECD significantly increased viral titers of DENV serotype 2 in Wolbachia-infected mosquitoes compared to controls, providing further evidence that Wolbachia-induced ROS leads to a beneficial cellular response characterized by antimicrobial-mediated inhibition of viral replication [30].

Because Wolbachia-induced ROS/oxidative stress leads to beneficial anti-viral cellular responses in both D. melanogaster and Ae. aegypti and because AMPK activation, which increases Nrf2 transcriptional activity, leads to increased lifespan in D. melanogaster, it would be expected that cellular stress-induced AMPK activation in Ae. aegypti would also lead to an increase in lifespan as well as an enhanced immune response.  Indeed, Nunes et al. showed that autophagy, immune system activation, and the average lifespan of Ae. Aegypti (a vector for dengue as well as chikungunya and Zika virus) is increased by feeding mosquitoes several plant-based polyphenols [31].  Interestingly, administration of resveratrol (derived from grapes), quercetin (found in many fruits and vegetables), epigallocathechin-3-gallate (EGCG, found in green tea), and genistein (found in soybeans) each increased average lifespan for male and female mosquitoes compared to controls.  Insects fed resveratrol also displayed higher levels of AMPK phosphorylation/activation compared to controls and compound C, a pharmacological inhibitor of AMPK, blocked the suppression of triglyceride (TG) content induced by resveratrol [31]. Resveratrol treatment also led to immunomodulatory effects, with reduced bacterial populations for female mosquitoes compared to controls, an effect that was mimicked by AICAR (a prototypical AMPK activator) but inhibited by compound C.  Interestingly, resveratrol only slightly decreased bacterial populations in vitro, indicating that the effects of resveratrol are indirect and likely associated with activation of the immune response in mosquitoes. Autophagy was also stimulated in resveratrol- and AICAR-fed mosquitoes which was abolished by silencing of AMPK [31].  

Interestingly, each of the polyphenols used in that study (resveratrol, quercetin, epigallocathechin-3-gallate, and genistein) have each been shown to induce cellular stress (e.g. ROS generation), similar to Wolbachia-induced ROS generation, in a number of mammalian cells in addition to activating AMPK [32-35].  As AMPK is activated by a number of different compounds (polyphenols, bacterial metabolites, etc.) and methodologies (e.g. electrical stimulation) that induce cellular stress, it is likely that Wolbachia-induced inhibition of dengue and Zika virus replication in and transmission by Ae. Aegypti is also modulated by AMPK, a master metabolic regulator that increases lifespan and improves immune and antioxidant responses in another Wolbachia-infected insect, D. melanogaster [36,37].  Although Nunes et al. did not determine if the polyphenols tested inhibited dengue virus replication in or transmission by Ae. Aegypti, recent studies have shown that in mammalian cells quercetin and analogs of resveratrol exhibit significant inhibitory activities against dengue virus type 2 [38,39].  EGCG has also recently been shown to inhibit Zika virus entry in Vero E6 cells (albeit at higher concentrations), indicating that cellular stress-induced activation of AMPK may represent a common mechanism of action that spans species boundaries to prevent the replication and/or transmission of dengue and Zika viruses [40].  Additionally, salidroside (derived from the plant Rhodiola rosea) and curcumin (derived from the plant Curcuma longa) have both been shown to exhibit anti-dengue virus activity in vitro and induce AMPK activation in vivo, providing further evidence that many structurally diverse compounds likely share a common mechanism of stress-induced AMPK activation to effectuate antiviral responses [41-44]. 

Remarkably, cellular stress-induced activation of AMPK (i.e. “Shock”) appears to link the antiviral effects mediated by a Wolbachia-induced increase in ROS production in A. aegypti with the amelioration of accelerated cellular aging defects in the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS), the reactivation of latent HIV-1 in infected T cells (to facilitate immune system detection and viral destruction), oocyte meiotic resumption, and the acrosome reaction in sperm (i.e. “Live”).

Indeed, the proteasome inhibitor MG132 has recently been shown to inhibit progerin (the toxic protein that causes accelerated cellular aging defects) in fibroblasts derived from HGPS patients, inhibit dengue, West Nile, and Yellow fever viruses, and activate AMPK [45-47]. 1α,25-dihydroxyvitamin D3, the most potent metabolite of vitamin D, has also been shown to delay premature senescence and significantly improve accelerated aging in patient-derived HGPS cells, reduce dengue virus infection in human myelomonocyte and hepatic cell lines, and activate AMPK [48-50].  Interestingly, nuclear aging defects in HGPS patient-derived cells were also reversed via activation of the antioxidant transcription factor Nrf2 by oltipraz. Oltipraz has also been shown to activate AMPK, inhibit HIV-1 replication, and induced Nrf2 signaling in D. melanogaster [24-27]. 

Furthermore, just as Wolbachia-induced ROS/hydrogen peroxide (H2O2) generation (i.e. “Shock”) leads to a compensatory upregulation of antimicrobial peptides and an enhanced antiviral immune response in Ae. Aegypti (i.e. “Live”), the exposure of HIV-1 latently infected monocyte or lymphocyte cell lines to H2O2 has also been shown to reactivate HIV-1 [51]. Additionally, ROS induces activation of AMPK and AMPK plays a critical role in T cell activation (and thus reactivation of latent HIV-1 that resides in T cell cells) [8,52].

AMPK activation has also been shown to play a critical role in the initiation of oocyte meiotic resumption and maturation (in preparation for oocyte activation) and the free radical-generating agent menadione (i.e. “Shock”) has been shown to induce AMPK-dependent meiotic resumption in cumulus-enclosed and denuded mouse oocytes (i.e. “Live”) via promotion of oxidative stress [53].  Lastly, ROS also plays a critical role in the induction of the acrosome reaction in sperm, a process that facilitates oocyte penetration through release of hydrolytic enzymes and is indispensable, along with oocyte activation, for the creation of all human life outside of a clinical setting [54].  Strikingly, AMPK has recently been found for the first time to be localized across the entire acrosome in human sperm and both H2O2/ROS and vitamin D (i.e. “Shock) have been shown to induce the acrosome reaction in human sperm (i.e. “Live”), providing compelling evidence that cellular stress-induced AMPK activation is critical for the induction of the acrosome reaction in human sperm and the creation of human life [55-57].

The antiviral effects mediated by Wolbachia-induced ROS generation paints a clear yet provocative portrait that cellular-stress mediated activation of AMPK represents a common mechanism of action that spans species boundaries, beneficially modulating cellular processes that are involved in the immune response, fertilization, and aging itself.  Indeed, structurally distinct compounds including MG132 and vitamin D have been shown to exert potent antiviral effects against dengue virus and significantly improve accelerated aging defects in HGPS.  More tellingly, H2O2/ROS/oxidative stress has been shown to reactivate latent HIV-1, promote oocyte meiotic resumption, and induce the acrosome reaction in human sperm.  Because AMPK is critical for oocyte meiotic resumption (and hence oocyte activation) and AMPK has recently been found for the first time to be localized across the entire acrosome in human sperm, AMPK activation may indeed be critical for the creation of all human life, as originally proposed in my recent publication [58].  Perhaps beyond perplexing on first glance, Wolbachia, mosquitoes, and the creation of all human life are likely connected.

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