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The Aedes aegypti peritrophic matrix controls arbovirus vector competence through HPx1, a heme–induced peroxidase [1]

['Octavio A. C. Talyuli', 'Instituto De Bioquímica Médica Leopoldo De Meis', 'Universidade Federal Do Rio De Janeiro', 'Rio De Janeiro', 'Jose Henrique M. Oliveira', 'Departamento De Microbiologia', 'Imunologia E Parasitologia', 'Universidade Federal De Santa Catarina', 'Florianópolis', 'Vanessa Bottino-Rojas']

Date: 2023-05 

Aedes aegypti mosquitoes are the main vectors of arboviruses. The peritrophic matrix (PM) is an extracellular layer that surrounds the blood bolus. It acts as an immune barrier that prevents direct contact of bacteria with midgut epithelial cells during blood digestion. Here, we describe a heme-dependent peroxidase, hereafter referred to as heme peroxidase 1 (HPx1). HPx1 promotes PM assembly and antioxidant ability, modulating vector competence. Mechanistically, the heme presence in a blood meal induces HPx1 transcriptional activation mediated by the E75 transcription factor. HPx1 knockdown increases midgut reactive oxygen species (ROS) production by the DUOX NADPH oxidase. Elevated ROS levels reduce microbiota growth while enhancing epithelial mitosis, a response to tissue damage. However, simultaneous HPx1 and DUOX silencing was not able to rescue bacterial population growth, as explained by increased expression of antimicrobial peptides (AMPs), which occurred only after double knockdown. This result revealed hierarchical activation of ROS and AMPs to control microbiota. HPx1 knockdown produced a 100-fold decrease in Zika and dengue 2 midgut infection, demonstrating the essential role of the mosquito PM in the modulation of arbovirus vector competence. Our data show that the PM connects blood digestion to midgut immunological sensing of the microbiota and viral infections.

Arboviruses transmitted by Aedes aegypti are a major public health threat. The peritrophic matrix (PM) is an extracellular layer that surrounds the blood bolus. It acts as an immune barrier that limits microbiota interaction with midgut epithelial cells. However, its usually assumed that the viral infection precedes the PM formation, which does not impact vector competence. We identified a PM-associated peroxidase that controls PM integrity and intestinal immunology and does so because it is controlled by a transcription factor that integrates inputs from dietary heme and blood meal triggered ecdysone signaling. Therefore, PM-driven intestinal homeostasis is pivotal to dengue and Zika infection, unveiling a new physiological role of this barrier for arboviral establishment in the mosquito.

Funding: The study is provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to OACT, JHMO, VBR, GOS, PHA, GOPS, and PLO; and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to GOPS and PLO. Support also received from the Financiadora de Estudos e Projetos (FINEP) to PLO and from the Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) to GOPS and PLO and the National Institutes of Health – NIH (Z01AI000947) for ABFB, AMK and CBM. The funders had no role in study design, data collection, and analysis, decision to publish, or manuscript preparation.

Here, we show that HPx1, a heme peroxidase associated with the A. aegypti PM, has a dual role, acting in the PM assembly crucial for its barrier function and as an antioxidant hydrogen peroxide-detoxifying enzyme. This role of HPx1 in midgut physiology and immunity highlights that dietary heme is a signal that by triggering HPx1 expression and PM function, produces a homeostatic response that controls ROS and AMP immune effectors, microbiota expansion, and viral infection.

Kumar et al. (2010) showed that heme peroxidase 15 (HPx15), also referred to as immunomodulatory peroxidase (IMPer), is expressed in the A. gambiae midgut and uses the hydrogen peroxide generated by DUOX as a substrate to crosslink proteins of the mucous layer in the ectoperitrophic space, limiting diffusion of immune elicitors from the gut microbiota and thus preventing activation of midgut antimicrobial responses to commensal bacteria. IMPer silencing results in constant activation of epithelial immune responses against both bacteria and Plasmodium parasites [ 22 ]. A similar immune barrier role for the PM against parasite infection has also been shown in tsetse flies infected with T. brucei and sandflies infected with Leishmania [ 12 , 23 ]. Therefore, most of the studies on the PM of insect disease vectors have focused on its role as a barrier for parasites, but much less is known about the influence of PM on viral infections or its contribution to gut homeostasis and immune responses in A. aegypti.

There are several studies on the role of reactive oxygen species (ROS) and redox metabolism on the gut immune response to pathogens. In Drosophila melanogaster, ROS production by a dual oxidase enzyme (DUOX, an NADPH oxidase family member) is triggered by pathogenic bacteria. The self-inflicted oxidative damage arising from DUOX activation is prevented by hydrogen peroxide scavenging via an immune-regulated catalase (IRC) [ 13 , 14 ]. In Anopheles gambiae, Plasmodium ookinete midgut invasion triggers a complex epithelial response mediated by nitric oxide and hydrogen peroxide that is crucial to mount an effective mosquito antiplasmodial response [ 15 ]. Furthermore, an Anopheles gambiae strain genetically selected to be refractory to Plasmodium infection exhibits enhanced activation of JNK-mediated oxidative stress responses [ 16 , 17 ]. In Aedes aegypti, it has been proposed that the dengue NS1 viral protein decreases hydrogen peroxide levels, preventing an oxidative intestinal environment, which is an adverse condition for both dengue and Zika viral infection [ 18 , 19 ]. Catalase silencing in the Aedes aegypti gut reduces the dengue infection prevalence rate [ 20 ]. The ROS generation by DUOX plays a key role in modulating proliferation of the indigenous microbiota, growth of opportunistic pathogenic bacteria, and dengue virus infection [ 7 , 18 , 21 ].

The peritrophic matrix (PM) in mosquitoes is a semi-permeable chitinous acellular layer secreted by intestinal cells after blood feeding. The PM completely envelopes the blood bolus, and its structure avoids direct contact of the digestive bolus with the midgut epithelia [ 3 , 4 ]. The PM is the site of deposition of most of the heme produced from blood hemoglobin hydrolysis, thus limiting exposure of the midgut cells to harmful concentrations of heme, a pro-oxidant molecule [ 5 ]. Extensive gut microbiota proliferation occurs in most hematophagous insects after a blood meal. Therefore, the PM is a barrier that limits interaction of the tissue with the intestinal microbiota [ 6 – 8 ], playing a role analogous to the mammalian intestinal mucous layer [ 8 ]. The PM is mainly composed of chitin and proteins, and correct assembly of this structure is crucial to its barrier function. Additionally, the PM is a barrier for parasites such as Plasmodium, Trypanosoma brucei, and Leishmania major, which must attach to or traverse the PM to complete their development in an insect vector [ 9 – 12 ].

Mosquito-borne viruses are emerging as global threats to public health. Female mosquitos ingest infected blood from a host and transmit the virus to another host during the next blood-feeding. As the first insect tissue infected by the virus, the midgut is the initial barrier that the virus must overcome to establish itself in the mosquito [ 1 ]. Because blood digestion occurs in the midgut concomitantly with viral infection of epithelial cells, digestion-triggered physiological events have a major influence on the course of intestinal infection [ 2 ].

A) Dengue 2 titers in midguts 7 days postinfection. B) Mosquitoes were treated with anitbiotics in sugar solution 3 days prior of DENV2 infection. Titers in midguts at 7 days postinfection. C) Zika titers in midguts at 4 days postinfection. Viral titers were assessed by the plaque assay. Each dot represents an individual mosquito gut, and bars indicate the median. * p< 0.05; Mann–Whitney test for A and B. The prevalence statistical analysis was performed by the Chi-square test followed by Fisher’s exact test.

The midgut epithelium is the first tissue a virus must invade to establish a successful infection. By modulating diffusion of immune elicitors, HPx1 is crucial to gut homeostasis, allowing the proliferation of the gut microbiota without triggering an immune response, which led us to investigate HPx1 role in arbovirus infection. Dengue infection prevalence was reduced in HPx1-silenced females, while the infection intensity was not affected ( Fig 5A ). On the other hand, Zika (ZIKV) infection intensity was reduced upon HPx1 silencing without affecting the infection prevalence ( Fig 5B ). This result suggests that DENV and ZIKV infection are differentially regulated by the HPx1 activity. As microbiota-derived elicitors activate ROS generation by the gut epithelia, we tested whether HPx1 silencing in mosquitoes treated with antibiotics to reduce microbiota levels would still impact gut infection. Fig 5B shows that the antibiotic treatment followed by the HPx1 silencing rescued the infection phenotype, confirming that microbiota-derived factors trigger a viral restriction response in the gut. Addressing the PM role by silencing the enzyme chitin synthase, responsible for the synthesis of a canonical PM structural component, affects the DENV infection in a way almost identical to what was observed in HPx1-silenced insects (SUP 4).

A) Representative images of ROS levels measured by DHE oxidation in individual midguts at 18 h ABM. B) Quantitative analysis of the fluorescence intensity of oxidized DHE (dsLacZ, n = 38; dsHPx1, n = 43; dsDUOX, n = 21; dsHPx1+DUOX, n = 31). C) The intestinal microbiota load analyzed through eubacterial ribosomal 16S gene expression by qPCR at 24 h ABM (dsLacZ n = 6; dsHPx1 n = 6; dsHPx1+DUOX n = 4). D-F) Immune-related gene expression upon HPx1 silencing at 24 h ABM by qPCR (at least n = 5 for each condition). F) Schematic panel of intestinal immune activation showing that the PM integrity mediated by HPx1 activity isolates the gut microbiota, and once this integrity is lost, DUOX and antimicrobial peptides (AMPs) are activated. **p<0.005, ***p<0.001, ****p<0.0001, ns = not significant. Data are the mean +/- SEM. One-way ANOVA with Tukey’s post-test for A’, B, and C.

NADPH-oxidases are a family of ROS-producing enzymes related to the immune system. Members of the dual oxidase (DUOX) group have been shown to play an essential role against bacterial challenge in the insect intestinal environment [ 7 , 13 , 22 ]. Silencing HPx1 alone significantly increased ROS levels ( Fig 4A ). In contrast, ROS levels were similar to those of dsLacZ controls in females in which HPx1 and DUOX were co-silenced ( Fig 4A and 4B ), indicating that DUOX activity is the source of ROS when HPx1 is silenced. Unexpectedly, despite lowered ROS levels in double-silenced mosquitoes, bacterial levels remained reduced ( Fig 4C ). This antibacterial response appears to be mediated by activation of canonical immune signaling pathways, as evidenced by increased expression of antimicrobial peptides and PGRP-LB in HPx1/DUOX co-silenced females ( Fig 4D–4F ).

A) Midgut transverse slices at 18 h ABM supplemented with dextran. Green: dextran—FITC. Blue: DAPI nuclear staining. Insets highlight dextran localization. B) Representative images of ROS levels measured by DHE oxidation in individual midguts at 18 h ABM. B’) Quantitative analysis of the fluorescence intensity of oxidized DHE (dsLacZ, n = 35; dsHPx1, n = 39). C) Quantitative analysis of the fluorescence intensity of oxidized DHE from individual midguts at 18 h ABM (dsLacZ, n = 32; dsHPx1—Ab, n = 34; dsHPx1 + Ab, n = 27). D) Mitosis index in the mosquito midgut at 18 h ABM (phospho-histone H3) (dsLacZ n = 33; dsHPx1 n = 38). E) The intestinal microbiota load analyzed through eubacterial ribosomal 16S gene expression by qPCR at 24 h ABM (dsLacZ n = 6; dsHPx1 n = 7). F) Immune-related gene expression upon HPx1 silencing at 24 h ABM by qPCR (dsLacZ n = 8; dsHPx1 n = 7). *p<0.05, ****p<0.0001, ns = not significant. Data are the mean +/- SEM. The T test for B’, D, E and F, and one-way ANOVA with Tukey’s posttest for C.

The PM is a semipermeable matrix that controls the traffic of molecules between the intestinal lumen and the epithelia, and its correct assembly is essential to fulfilling its barrier function. The PM morphology (observed by the staining of chitin fibers present in the intestinal lumen) was not heavily compromised by the HPx1 silencing (SUP 3C). Fig 3A shows that, when fed fluorescent dextran particles, different from silenced insects, control mosquitoes retained the polymer on the gut luminal side, a proxy of the normal barrier function of the PM. In contrast, HPx1-silenced mosquitoes presented strong fluorescence in the epithelial layer, suggesting a role for HPx1 in the proper assembly of the PM, as its permeability barrier function was compromised by HPx1 silencing. It has been proposed that the A. gambiae IMPer mediates a protein crosslinking by dityrosine bound, which maintains the PM barrier structure. Silencing HPx1 also reduces the dityrosine residues in the gut epithelia in A. aegypti (SUP3A-B). As this alteration in permeability might expose the epithelium to bacterial elicitors from the proliferative microbiota, we evaluated ROS production, known as an antimicrobial defense. Fig 3B and 3B’ show that HPx1 silencing increased ROS levels in the midgut. The increase in ROS in HPx1-silenced mosquitoes was due to increased exposure of the gut epithelia to either the microbiota or microbiota-derived immune elicitors, once oral administration of the antibiotics prevented the increase in the ROS levels, as observed in HPx1-silenced mosquitos ( Fig 3C ). Exposure to damage signals and elevated ROS has been shown to activate intestinal stem cell mitosis (30). Indeed, HPx1 silencing increased phosphorylated H3-histone levels ( Fig 3D ) in midgut epithelial cells, indicative of mitotic activity and suggestive of epithelial remodeling in response to oxidative imbalance. This highlights the key role of HPx1 in tissue homeostasis. The native midgut bacterial load was significantly reduced after HPx1 silencing ( Fig 3E ). However, this was not due to the activation of canonical immune signaling pathways, as the expression of two antimicrobial peptides, Attacin and Cecropin G, was not significantly different from dsLacZ controls ( Fig 3F ). Interestingly, the bacterial sensor PGRP-LB decreased its expression as the total amount of microbiota was also reduced ( Fig 3F ). Overall, it highly suggests that ROS levels control the intestinal microbiota proliferation.

A) HPx1 expression in the midgut of sugar-fed or at 24 h after SBM feeding (without heme or supplemented with 50 μM of heme) (Sugar n = 5; -Hm n = 14; +Hm n = 14). B) Catalytic activity of PMs from mosquitoes fed different diets at 24 h postfeeding (Blood n = 3 pools of 10 PM each; -Hm n = 4 pools of 10 PM each; +Hm n = 4 pools of 10 PM each). C) HPx1 expression in midguts from control (dsLacZ) and dsE75-injected mosquitoes at 24 h ABM (dsLacZ n = 6; dsE75 n = 7). D) Schematic model of molecular signaling for HPx1 expression in the mosquito midgut. *p<0.05, **p<0.005, ns = not significant. Data are the mean +/- SEM. One-way ANOVA with Dunnett’s post-test for A and B and the T test for C.

A blood meal triggers large changes in the gene expression pattern of A. aegypti and, among several factors, the heme released upon hemoglobin proteolysis acts as a pleiotropic modulator of transcription [ 26 ]. Feeding the insects with SBM with or without heme revealed that heme significantly regulated HPx1 gene expression ( Fig 2A ). Accordingly, lower hydrogen peroxide decomposition activity was observed in the PM secreted by females fed SBM without heme compared to the blood-fed insects, a phenotype rescued by heme supplementation ( Fig 2B ). In silico analysis of the HPx1 promoter gene region revealed putative binding sites for E75, a hormone-responsive transcription factor that functions as a heme and redox sensor (SUP2A) [ 27 , 28 ]. E75 knockdown significantly reduced HPx1 gene expression in the midgut after blood feeding, suggesting a molecular mechanism for triggering HPx1 by heme ( Fig 2C ; E75 silencing efficiency: SUP2B). Proliferation of the gut microbiota in response to blood feeding is known to induce expression of several genes in the midgut. However, neither microbiota depletion by oral administration of antibiotics (aseptic) nor reintroduction of a bacterial species (Enterobacter cloacae) into antibiotic-treated mosquitoes affected HPx1 gene expression (SUP2C). Thus, we propose the molecular signaling model shown in Fig 2D .

We hypothesized that the observed PM hydrogen peroxide detoxifying activity should be attributed to another enzyme encoded by the mosquito genome. Peroxidases are a multigene family of enzymes and the genome of A. aegypti, as with most other organisms, has many peroxidases. Peroxidases are grouped in three large families: glutathione, heme, and thioredoxin peroxidases. As the PM is an extracellular structure, we initially searched for peroxidases with a predicted signal peptide. Interestingly, this search identified ten peroxidases, all of them belonging to the heme peroxidase family, which also includes the secreted peroxidases of Anopheles gambiae [ 22 ] and Drosophila melanogaster [ 14 ]. Phylogenetic analysis showed that A. aegypti heme peroxidase 1 (HPx1) is a close homolog of the peroxidase HPx15/IMPer that promotes the crosslink of extracellular proteins in the gut lumen of A. gambiae ( Fig 1C ). As the HPx15/IMPer from A. gambiae was shown to be secreted by the midgut epithelia, we used the presence of a secretion signal peptide as an additional feature to indicate HPx1 as the A. aegypti PM enzyme responsible for decomposing hydrogen peroxide. Fig 1D shows that the blood meal induced HPx1 gene expression in the gut. Moreover, western blotting showed that most of the HPx1 in the midgut was bound to the PM, with a minor fraction being associated with the epithelia ( Fig 1E ), and RNAi silencing of HPx1 expression significantly decreased hydrogen peroxide detoxification by the PM ( Fig 1E ; HPx1 silencing efficiency: SUP1F).

A) PMs were dissected and pooled at different times after a blood meal (ABM), and their catalase specific activity was measured (12 h n = 5, 24 h n = 6; 36 h n = 5 pools of 5 PM). B) Catalase activity of PMs dissected from control (dsLacZ-injected) and catalase (AAEL013407-RB)-knockdown insects at 24 h AMB (dsLacZ n = 9; dsCat n = 9). C) Phylogenetic tree of heme peroxidases from Aedes aegypti (AAEL), Anopheles gambiae, and Drosophila melanogaster. Maximum likelihood analysis was performed, and the numbers in each branch represent bootstraps. (D) HPx1 expression in midguts at 24 h ABM relative to the sugar-fed control (Sugar n = 11; Blood n = 11). (E) Western blot of the HPx1 protein in 20 μg of gut epithelia and PM extracts at 18 h ABM and catalase activity of PMs dissected from control and HPx1-knockdown insects (dsLacZ n = 8; dsHPx1 n = 13). The full western blot membrane is shown in S1E Fig . Data are the mean +/- SEM. **p> 0.005, ***p<0.001 for the T test for D and E.

After a blood meal, the antioxidant capacity of the Aedes aegypti midgut is increased by expressing enzymes and low molecular weight radical scavengers [ 24 ]. These protective mechanisms are complemented by the capacity of the PM to sequester most of the heme produced during blood digestion, which has been proposed to be a preventive antioxidant defense, as heme is a pro-oxidant molecule [ 5 , 25 ]. Fig 1A shows that A. aegypti PM exhibited hydrogen peroxide detoxifying activity up to 24 h after blood-meal (ABM) followed by a sharp decrease at 36 h, close to the end of blood digestion. The specific activity of the PM hydrogen peroxide scavenging activity was comparable to the activity found in the midgut epithelia 24 h after blood-feeding, which is attributed to a canonical intracellular catalase (SUP1A). However, silencing of the “canonical” intracellular catalase (AAEL013407-RB) did not alter the PM’s ability to detoxify hydrogen peroxide at 24 h after feeding ( Fig 1B ), contrasting with previous reports showing that silencing of this gene effectively decreased epithelial cellular catalase activity, therefore suggesting that the activity in the PM is not due to the midgut intracellular catalase (AAEL013407-RB) [ 20 ]. This hypothesis received support from the observation that the hydrogen peroxide decomposing activity of the PM and midgut epithelia showed distinct in vitro sensitivity to the classical catalase inhibitor amino triazole (SUP1B). Moreover, neither depletion of the native microbiota by antibiotic treatment (SUP1C) nor feeding the insect an artificial diet (cell-free meal) devoid of catalase (SUP1D) altered PM hydrogen peroxide detoxification, additionally excluding the hypothesis of an enzyme originating from the microbiota or host red blood cells.

Discussion

The Aedes aegypti PM is an acellular layer that surrounds the blood bolus throughout the digestion process and limits direct contact of the epithelium with the midgut content and the intestinal microbiota, which undergoes massive proliferation upon blood feeding. In female mosquitoes, PM formation occurs in response to ingestion of a blood meal, following a time course that is finely coordinated with the pace of blood digestion. However, the signaling pathways that trigger PM secretion in adult mosquito females have not been elucidated nor has the impact of this structure on viral infection. Here, we characterize an intestinal secreted peroxidase (HPx1) that functions in PM assembly, contributing to its barrier function and promoting microbiota growth by preventing an antimicrobial response. This PM function has a permissive role for viral replication in the mosquito gut, thus constituting a novel determinant of vector competence. Importantly, dietary heme triggers HPx1 gene expression using the heme-dependent transcription factor E75, allowing synchronization of PM maturation with blood digestion by sensing the free heme released as hemoglobin is digested.

Similar to all blood-feeding organisms, mosquitoes face an oxidative challenge due to large amounts of heme—a pro-oxidant molecule—released by hemoglobin degradation. Therefore, preventing oxidative damage through ROS detoxification is a hallmark of their physiology [24]. HPx1 mediates a novel mechanism to promote redox balance in the Aedes midgut through its hydrogen peroxide scavenging activity and by modulating PM barrier function. We also show that HPx1 allows proliferation of the gut microbiota without activating a DUOX-mediated oxidative burst by limiting exposure of gut epithelial cells to microbial immune elicitors. We have previously shown that Aedes aegypti catalase (AAEL013407-RB), the main intracellular hydrogen peroxide scavenger, is induced in the blood-fed midgut of females [20]. Here, we demonstrate that HPx1 contributes to the overall peroxide scavenging capacity of the gut in a way that is independent of epithelial intracellular catalase but at comparable activity levels (SUP 1A). Although peroxidases are less efficient in decomposing hydrogen peroxide than catalases, it is known that some heme peroxidases also have high catalase activity [29,30].

A. gambiae IMPer/HPx15 belongs to anopheline-specific expansion of the heme peroxidase family [31] nested in the same branch of the peroxidase family of A. aegypti HPx1 and immune-regulated catalase (IRC; CG8913) of D. melanogaster [14,32,33]. However, Drosophila IRC has a conserved heme peroxidase domain structure (Pfam PF03098) but lacks a catalase domain (Pfam PF00199), despite having a high hydrogen peroxide dismutation activity [14], which is a feature of catalases but uncommon among peroxidases. Interestingly, IMPer/HPx15 of Anopheles is also expressed in the female reproductive tract, induced by the ecdysone transferred along with the sperm during insemination, due to ecdysone-responsive elements present in the promoter region [34]. Ecdysone-responsive element sequences close to the Aedes HPx1 gene have been identified in silico [35]. Therefore, Aedes HPx1, IRC, and AgIMPer are secreted enzymes that modulate interactions of the midgut with commensal and pathogenic bacteria. Nevertheless, their role in the biology of the reproductive organs has not been well established. Although our data reveal that HPx1, IRC, and IMPer share sequence and functional homology, it is not possible at present to speculate which roles are ancestral and which were acquired secondarily during the evolution of dipterans.

A. gambiae IMPer has been proposed to crosslink external matrix proteins by forming dityrosine bridges, reducing the accessibility of microbial elicitors to the intestinal cells [22]. In Drosophila, however, a transglutaminase enzyme crosslinks PM proteins, also protecting the midgut epithelia from damage [36,37]. In this study, we show that HPx1 associates with the PM and modulates gut permeability in A. aegypti (Fig 4) and promotes protein crosslink by dityrosine bounds (SUP 3A-B). When the PM permeability was compromised by HPx1 silencing, we observed immediate responses from the epithelium that increased ROS levels, which was attributed mainly to DUOX activation by microbial elicitors. The role of PM in preventing elevated ROS production in the gut epithelium was also observed when the PM was compromised by the chitin inhibitor diflubenzuron administered in a blood meal for A. aegypti females [38]. A simple hypothesis to explain how elevated ROS might lower virus infection is that they directly attack the virus. However, arbovirus infection of midgut cells is thought to occur early during digestion, several hours before proliferation of microbiota occurs and before the PM is secreted [39]. Therefore, it is unlikely that extracellular ROS produced in response to bacterial elicitors in HPx1-silenced insects would directly attack virus particles that will be localized intracellularly when these molecules start to increase in the lumen. In general, cellular antiviral mechanisms are most plausibly responsible for hampering viral infection upon HPx1 silencing. Among these possible mechanisms, the elevated extracellular ROS levels derived from DUOX activation are indeed sensed by the gut as a danger signal, evoking a tissue-repairing response the hallmark of which is stem cell proliferation (Fig 4), a homeostatic response coupled to cell death in response to insult and damage. Interestingly, Taracena et al. proposed that different degrees of resistance to infection among mosquito strains are related to different capacities to promote a rapid increase in stem cell proliferation; hence, faster cell death, followed by cell renewal from stem cell activation, is a process that reduces viral infection [38]. One could hypothesize that this mechanism is responsible for the decrease in ZIKV and DENV infection promoted by HPx1 silencing.

In mammals, the mucus layer allows commensal bacteria from the gut microbiota to thrive without eliciting microbicidal immune responses by the intestinal mucosa. In other words, the mammalian mucus layer acts as a physical barrier that leads to “immunological ignorance” by preventing a state of constant immune activation and chronic inflammation of the intestine in response to immune elicitors from the normal microbiota [40–42]. In mosquitos, the PM is secreted when the microbiota peaks in number to approximately 100–1000 times the population found before a blood meal [7], representing a potential massive immune challenge to this tissue. Hixson et al. suggested that immune tolerance to the indigenous microbiota might be mediated by high expression of caudal and PGRP genes, leading to low expression of antimicrobial peptides in epithelial cells from the posterior gut [43]. Here, we highlight the role of PM-associated HPx1 in limiting exposure of the epithelium to immune elicitors from the expanded microbiota observed postfeeding. Before blood feeding, the PM is absent, and bacteria interact with the epithelium, leading to ROS generation and damage-induced repair [7,38]. When the first line of immediate response to immune elicitors (redox mediated) is further prevented by simultaneous DUOX silencing, a reaction is activated (antibacterial peptides expression) to limit microbial growth. These data suggest hierarchal immune activation in the A. aegypti midgut fundamentally orchestrated by the PM integrity. This is similar to what was reported for A. gambiae, whereby bacterial elicitors lead to DUOX-mediated activation of IMPer, which promotes cross-linking of extracellular proteins [22]. However, in this report, an epithelial cell mucous layer (and not the PM) was indicated as the site of action of the peroxidase. Regarding the mode of activation of this pathway, the findings shown herein are also fairly similar to those for Drosophila, with ROS produced by DUOX being primarily triggered by bacterial pathogens, and the IMD pathway induces antimicrobial peptide production upon activation failure [21,43–45].

Traditionally, immunology has focused on how hosts eliminate pathogens while fighting infections, but in the last decade, there has been a growing interest in how hosts endure infection by utilizing disease tolerance, including diminishing both the direct damage caused by the pathogen and the self-inflicted damage due to the host immune reaction directed at the elimination of the pathogen. In this study, we showed that PM-associated HPx1 is pivotal to maintaining gut immune homeostasis, acting as a tolerance mechanism that prevents responses to the microbiota and, consequently, to the viral infection. This conclusion was reinforced by the fact that antibiotic treatment prevented the effect of HPx1-silencing on virus infection. The chitin synthase silencing, used as an approach to disrupting PM formation by an independent mechanism, recapitulates the HPx1 silencing impact on dengue 2 infection and stretches the claim that PM integrity is essential to gut homeostasis and has a permissive role for viral infection.

Additionally, our data revealed that, although belonging to the same family, ZIKV and DENV infection outcomes were distinct when HPx1 was silenced, once only the infection intensity was affected in the case of ZIKV, in contrast to a prevalence effect when DENV was evaluated. It has been reported that ZIKV and DENV do not necessarily exploit the same transcriptional response in the mosquito gut, indicating they play different roles in modulating the immune and physiological responses [46]. In this way, the HPx1 can have distinct roles toward each different viruses.

Together, our results indicate that the A. aegypti PM supports midgut homeostasis during blood digestion. Heme derived from blood hemoglobin digestion regulates expression of HPx1, an enzyme that has a central role in the assembly of a fully functional PM, through the heme-sensitive E75 transcription factor. Thus, HPx1 maintains immunological ignorance of the midgut epithelia toward the microbiota, allowing a state of microbiota and viral tolerance and preventing tissue damage.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011149

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