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A Peroxidase/Dual Oxidase System Modulates Midgut Epithelial Immunity in Anopheles gambiae

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Science  26 Mar 2010:
Vol. 327, Issue 5973, pp. 1644-1648
DOI: 10.1126/science.1184008

Mosquito Double Act

Peroxidase/dual oxidase (duox) systems act in concert to catalyze the nonspecific formation of dityrosine bonds, which cross-link a variety of proteins. Knowing that these reactions are involved in fine-tuning insect immune responses, Kumar et al. (p. 1644, published online 11 March) investigated how the peroxidase/duox system in malaria-vector mosquitoes protects the gut flora by modulating midgut antibacterial responses. Generating immune reactions resulted in a loss of mosquito egg viability, but modulating host responses allowed malaria parasites to persist among the surviving commensal flora. The peroxidase/duox system appears to promote dityrosine bond formation between proteins across the surface of midgut epithelial cells to form a layer that inhibits immune recognition and mediator release. Interference with the formation of this layer might provide a target for mosquito and malaria control.

Abstract

Extracellular matrices in diverse biological systems are cross-linked by dityrosine covalent bonds catalyzed by the peroxidase/oxidase system. We show that a peroxidase, secreted by the Anopheles gambiae midgut, and dual oxidase form a dityrosine network that decreases gut permeability to immune elicitors. This network protects the microbiota by preventing activation of epithelial immunity. It also provides a suitable environment for malaria parasites to develop within the midgut lumen without inducing nitric oxide synthase expression. Disruption of this barrier results in strong and effective pathogen-specific immune responses.

Insects, like most metazoa, harbor large numbers of commensal bacteria within their guts. Midgut epithelial cells need to protect the host from pathogenic organisms but must do so without mounting immune responses against the normal microbiota. This is especially challenging in blood-feeding insects because commensal bacteria proliferate extensively during blood digestion (1). In Drosophila, dual oxidase (Duox) has been shown to mediate a microbicidal response that prevents overproliferation of dietary bacteria and yeast (2, 3). Duox is a transmembrane protein that generates hydrogen peroxide, a substrate required by peroxidases (4).

In many hematophagous insects, including the mosquito Anopheles gambiae, the midgut secretes a peritrophic matrix (PM) in response to blood feeding. The PM is an acellular, semipermeable layer of chitin polymers that surrounds the blood meal and prevents blood cells and gut bacteria from coming into direct contact with midgut epithelial cells (5, 6). Mucins are secreted into the ectoperitrophic space (7) between the PM and the midgut epithelium. In this study, we characterize a heme peroxidase—immunomodulatory peroxidase (IMPer) [AGAP013327-PA or HPX15 (8)]—that is secreted by A. gambiae midgut epithelial cells in response to blood feeding. We found that IMPer, together with Duox, catalyzes protein cross-linking in the mucin layer, reduces permeability to immune elicitors, and prevents immune responses against bacteria and Plasmodium parasites. We uncovered an unexpected, previously unrecognized function of the peroxidase/Duox system that protects the gut microbiota.

Blood feeding induces IMPer mRNA, protein, and enzymatic activity in the midgut of A. gambiae females that peak around 12 hours after feeding and are effectively silenced by systemic injection of IMPer double-stranded (ds) RNA (Fig. 1, A to E) (9). IMPer enzymatic activity is localized in the periphery of the blood bolus (Fig. 1D) and is greatly reduced when IMPer is silenced (fig. S1). Antibodies to IMPer recognize two bands (56 and 57 kD) in midgut homogenates, close to the expected size for IMPer (56.5 kD) (Fig. 1E).

Fig. 1

Expression and localization of IMPer and effect of IMPer silencing on bacterial growth and immune activation. (A) IMPer mRNA expression and (B) effect of IMPer silencing on IMPer mRNA levels at different times after blood feeding (mean ± SEM). (C) Effect of IMPer silencing on midgut peroxidase activity detected with the use of 3,3′-diaminobenzidine (DAB) staining at different times after feeding. (D) Localization of inducible peroxidase activity detected with DAB staining (brown) in midgut sections 12 hours after feeding. N, nucleus of epithelial cells. Arrowheads indicate peroxidase activity. (E) Western blot of IMPer protein in midgut homogenates collected at different times after feeding in control (dsLacZ-injected) or IMPer-silenced mosquitoes. (F) Bacterial 16S rRNA levels in midguts collected at different times after blood feeding (mean ± SEM). (G) Effect of IMPer silencing on bacterial 16S rRNA of individual midguts 24 hours after feeding (mean ± SEM). Horizontal lines indicate the medians. (H) IMPer, cecropin, PGRP-S3, PGRP-LB, HPX8, and NOS mRNA levels in control (dsLacZ-injected) and IMPer-silenced midguts 24 hours after feeding (mean ± SEM). (I) Same as in (H), but in antibiotic-fed mosquitoes. Asterisks indicate significant differences relative to the dsLacZ control.

Midgut bacteria proliferation peaks around 30 hours after feeding (Fig. 1F). Unexpectedly, IMPer silencing reduces the median level of bacterial 16S ribosomal RNA (rRNA) in individual midguts collected 24 hours after feeding by 8.8-fold (P < 0.01) (Fig. 1G) and reduces bacterial genome copies ninefold (P < 0.01) (fig. S2). This indicates that IMPer does not mediate a microbicidal response but, on the contrary, it is required for bacterial survival.

We explored the possibility that IMPer modulates midgut antibacterial responses. Gene expression microarray analysis identified several putative midgut immune genes that are induced by a protein meal containing a large dose of heat-killed bacteria, which interact directly with epithelial cells before the PM barrier is formed (fig. S3 and table S1). Quantitative real-time fluorescence polymerase chain reaction confirmed the induction of 10 markers (fig. S4). Four of them—the antibacterial peptide cecropin, peptidoglycan recognition protein–S3 (PGRP-S3), PGRP-LB (a negative regulator of the Imd pathway) (10), and heme-peroxidase 8 (HPX8)—are induced when IMPer is silenced, whereas expression of nitric oxide synthase (NOS) is not affected (Fig. 1H). These markers are no longer induced when midgut bacteria are eliminated by pretreating mosquitoes with oral antibiotics (Fig. 1I).

In addition to its direct participation in antimicrobial activity, the Duox/peroxidase system also catalyzes cross-linking of extracellular matrices in very diverse biological systems by forming covalent bonds between tyrosine residues (dityrosine bonds) (11, 12). Our initial findings suggest that IMPer could be required to form a barrier that limits the rate of diffusion of immune elicitors and prevents immune activation. Given the negative effect of IMPer silencing on bacterial growth, we next asked whether IMPer also affects Plasmodium berghei (rodent malaria) survival. IMPer silencing reduces the median number of P. berghei oocysts present 7 days post infection (dpi) by 9.2-fold (Fig. 2A). This effect is already observed early in the invasion process (30 hours after feeding), when the number of intact ookinetes is greatly reduced (fig. S5). In IMPer-silenced females, ookinetes invade the midgut but are killed and appear fragmented (Fig. 2B). The drastic reduction of Plasmodium infection in IMPer-silenced mosquitoes is not due to activation of antibacterial responses, as it is also observed in females pretreated with oral antibiotics (Fig. 2C). As expected, antibacterial markers are not induced when antibiotic-treated females are infected with Plasmodium, indicating that IMPer silencing activates these genes only when bacterial elicitors are present in the midgut lumen. Instead, there is a dramatic induction of NOS, an enzyme that generates nitric oxide (a potent antiplasmodial effector molecule) (Fig. 2D). These findings indicate that IMPer is required for Plasmodium parasites to develop in the midgut without activating the immune pathway(s) that regulate NOS expression. We have previously shown that overactivation of the signal transducers and activators of transcription pathway induces high levels of NOS at 4 dpi that greatly reduce oocyst survival (13), but the abnormal induction of NOS in IMPer-silenced mosquitoes is observed earlier.

Fig. 2

Effect of IMPer silencing on Plasmodium infection and midgut immune activation. Effect of IMPer silencing on P. berghei infection (A) 7 dpi, (B) 30 hours post infection (hpi), and (C) 2 dpi in antibiotic-fed mosquitoes. Arrowheads in (B) indicate fragmented parasites. (D) IMPer, cecropin, PGRP-S3, PGRP-LB, HPX8, and NOS mRNA levels in control (dsLacZ-injected) and IMPer-silenced midguts of antibiotic-treated mosquitoes at 24 hpi (mean ± SEM). (E) Effect of IMPer silencing on P. falciparum infection at 8 dpi. (F) Effect of silencing IMPer or NOS (or co-silencing IMPer and NOS) on IMPer and NOS mRNA expression 12 hpi in antibiotic-fed mosquitoes infected with P. falciparum (mean ± SEM) and (G) P. falciparum infection at 7 dpi. Asterisks indicate significant differences relative to the dsLacZ control. Each circle represents the number of parasites in an individual midgut, and the horizontal lines indicate the medians.

Plasmodium falciparum infection is also greatly reduced in IMPer-silenced A. gambiae (Fig. 2, E and G) and A. stephensi (fig. S6) at 7 dpi. Furthermore, IMPer silencing in antibiotic-treated A. gambiae mosquitoes infected with P. falciparum also triggers a strong induction of midgut NOS expression 12 hours after feeding (Fig. 2F) and reduces infection (P < 0.0001) (Fig. 2G). At this time, immature ookinetes are still developing in the blood bolus within the PM matrix. When IMPer is not present, epithelial cells can detect Plasmodium immune elicitors, possibly glycosylphosphatidylinositols (GPIs), which activate NOS expression. Previous studies have shown that oral administration of Plasmodium GPIs activates midgut NOS expression through the Akt/protein kinase B pathway (14). To determine whether high levels of NOS mediate parasite killing, we silenced both IMPer and NOS. NOS mRNA levels are 30-fold less in the double-silenced mosquitoes than when only IMPer is silenced (Fig. 2F). Lower NOS levels prevent the deleterious effect of IMPer silencing on Plasmodium infection (Fig. 2G), indicating that the antiplasmodial effect of IMPer silencing is mediated by NOS, probably by increasing the rate of nitration when parasites invade gut epithelial cells (15).

Duox generates hydrogen peroxide, a substrate required for IMPer to be active, on the luminal surface of epithelial cells. We investigated whether Duox is also required to prevent activation of antiplasmodial responses. dsDuox silencing reduces midgut Duox mRNA levels by 77% (Fig. 3A) and drastically reduces Plasmodium infection in the presence (Fig. 3B) or absence (Fig. 3C) of bacteria. This phenotype is very similar to that observed when IMPer is silenced (Fig. 2A). In antibiotic-treated females infected with Plasmodium, Duox silencing does not induce expression of the antibacterial markers (Fig. 3E); however, NOS expression is highly induced. Together, our data support the hypothesis that IMPer and Duox are both required to prevent activation of midgut responses to Plasmodium immune elicitors.

Fig. 3

Effect of Duox silencing on Plasmodium infection and midgut immune activation. (A) Midgut Duox silencing at 4 days after injection of dsDoux. (B) Effect of Duox silencing on P. berghei infection at 7 dpi. (C) Same as in (B), but in antibiotic-fed females. (D) Effect of Duox silencing on cecropin, PGRP-S3, PGRP-LB, heme-peroxidase 8, and NOS expression in midguts of antibiotic-treated mosquitoes collected at 24 hpi (mean ± SEM).

We propose a model (fig. S7) in which the IMPer/Duox system mediates protein cross-linking by forming dityrosine bonds. This network of covalently linked proteins decreases the rate of diffusion of immune elicitors, decreasing their interaction with pathogen recognition receptors on the surface of midgut cells. In agreement with this model, silencing Duox (Fig. 4A) or IMPer (Fig. 4B) or reducing immune elicitors with oral antibiotics (fig. S8) significantly increases the rate of absorption of a fluorescent dextran administered in the blood meal, indicating that the dextran has increased access to the gut surface. Decreasing immune elicitors in IMPer-silenced mosquitoes further enhances gut permeability (Fig. 4C). The dityrosine network is dynamic and probably transient, as IMPer is expressed 6 to 18 hours after feeding, a time when bacteria proliferate but blood digestion is not fully active.

Fig. 4

Gut permeability and dityrosine network. Effect of (A) Duox or (B) IMPer silencing without or (C) with oral antibiotics (Ant) on midgut permeability to fluorescent dextran (4 kD). Each circle represents fluorescence in the hemolymph of an individual mosquito 18 to 20 hours after feeding. Horizontal lines indicate the medians. RFU, relative fluorescence units. (D to G) Immunofluorescence staining of midguts 14 hours after feeding. Dityrosine bonds (red) and muscle actin (green) in mosquitoes injected with (D) dsLacZ, (E) dsIMPer, or (F) dsDuox. (G) Enlargement of image in (D). DiTyr, dityrosine staining; Lu, lumen; Ba, basal; Mu, muscle; and Nu, nuclei.

We used monoclonal antibodies to detect dityrosine bonds on the midgut surface. A network of dityrosine-linked proteins is observed on the luminal surface of epithelial cells of blood-fed control mosquitoes injected with dsLacZ (Fig. 4, D and G) but is absent when either IMPer (Fig. 4E) or Duox (Fig. 4F) is silenced, in agreement with the proposed model.

In conclusion, A. gambiae, midgut epithelial cells have the ability to activate pathogen-specific responses to bacteria and Plasmodium and to modulate the permeability of the mucus layer to soluble molecules present in the blood bolus. The dityrosine network formed by the IMPer/Duox system allows bacteria to proliferate without activating epithelial immunity but also makes mosquitoes more susceptible to Plasmodium infection, as parasites can develop within the midgut lumen without being detected. In Drosophila, silencing of a secreted peroxidase results in high mortality when flies are fed live or dead bacteria, but not when they are fed sterile food. Furthermore, antioxidants can rescue this mortality, indicating that high levels of reactive oxygen species (ROS) are mediating death (16). Our studies suggest that this enzyme may also be involved in the formation of a dityrosine network in Drosophila and that disruption of this barrier could result in chronic immune activation and ROS generation. In mosquitoes, IMPer or Duox silencing does not affect survival (fig. S9), probably because mosquitoes are batch feeders, and gut bacteria and blood-meal remnants are expelled 2 to 3 days after feeding.

The Duox system appears to use a dynamic two-prong strategy to protect epithelial cells from potential pathogens. Bacterial elicitors are known to activate Duox activity quickly through the phospholipase Cβ signaling pathway (3, 17). This would generate hydrogen peroxide and activate IMPer, forming the dityrosine network and decreasing the permeability of the mucus layer to immune elicitors. Expression of microbicidal effector genes would be induced when this initial response cannot prevent contact of immune elicitors with pathogen recognition receptors on the surface of epithelial cells; for example, when pathogenic bacteria or Plasmodium parasites breach the PM and the mucus barriers. These two complementary mechanisms would allow an effective immune response while minimizing the deleterious effects that chronic activation of potent effector molecules could have on commensal bacteria and on the host.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1184008/DC1

Materials and Methods

Figs. S1 to S9

Tables S1 and S2

References and Notes

  • * These authors contributed equally to this work.

  • Present address: Biological Sciences Group, Faculty Division III, Birla Institute of Technology and Science–Pilani, Pilani 333031, Rajasthan, India.

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. This work was supported by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH. We thank A. Laughinghouse, K. Lee, T. Lehman, and R. Gwadz for insectary support; J. Kabat for assistance with confocal imaging; J. Ribeiro and J. Valenzuela for their comments and insight; and B. Marshall for editorial assistance. The complete data set for this microarray experiment has been submitted to the National Center for Biotechnology Information Gene Expression Omnibus database with accession number GSE20204.
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