Sex-specific and blood meal-induced proteins of Anopheles gambiae midguts: analysis by two-dimensional gel electrophoresis

BACKGROUND: Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite. METHODS: We carried out two-dimensional gel electrophoresis of An. gambiae midgut proteins and compared protein profiles for midguts from males, sugar-fed females and females fed on human blood. RESULTS: Very few differences were detected between male and female mosquitoes for the approximately 375 silver-stained proteins. Male midguts contained ten proteins not detected in sugar-fed or blood-fed females, which are therefore probably involved in male-specific functions; conversely, female midguts contained twenty-three proteins absent from male midguts. Eight of these proteins were specific to sugar-fed females, and another ten, to blood-fed females. CONCLUSION: Mass spectrometry analysis of the proteins found only in blood-fed female midguts, together with data from the recent sequencing of the An. gambiae genome, should make it possible to determine the role of these proteins in blood digestion or parasite receptivity

Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible

Plasminogen controls inflammation and pathogenesis of influenza virus infections via fibrinolysis

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza

Plasminogen contributes to influenza pathogenesis.

<p>Survival and weight loss of PLG-KO (triangles) and WT (squares) mice infected with (A) IAV A/PR/8/34 (50,000 PFU; n = 11–21 or 500 PFU; n = 23–24), (B) A/Netherlands/602/09 (30,000 PFU; n = 7) or (C) A/chicken/Ivory-Coast/1787/2006 (10 EID50; n = 12). The proportion of survival was determined based on euthanasia criteria. Animals that lost 20% of their body weight were considered to have reached humane endpoints and were sacrificed according to the study protocol. It is of note that upon WT mice infection with A/chicken/Ivory-Coast/1787/2006, all infected mice lost weight but died before reaching −20% of their body weight, in contrast to PLG-KO mice, which explains the difference in mortality but not in weight loss. Weight loss data represent weight average ± s.e.m of the above indicated number of mice. n = mice per group.</p

The deleterious role of plasminogen is independent on virus replication.

<p>(A) Virus replication of IAV A/PR/8/34 and A/Netherlands/602/09 after inoculation of A549 cells in presence or absence (triangle) of plasminogen (square) or trypsin (circle). Data represent mean ± s.e.m of three independent experiments. (B) Western blot analysis of A/PR/8/34 and A/Netherlands/602/09 HA cleavage after infection of A549 cells in presence or absence of plasminogen (PLG) or trypsin (Try). Membranes were probed with anti-HA and anti-tubulin antibodies. kDa (apparent molecular weight). NI stands for uninfected. (C) Infectious A/PR/8/34 (n = 3–5) and A/Netherlands/602/09 (n = 3) lung virus titers at the indicated time points post-inoculation of WT (black bars) or PLG-KO mice (white bars). Data represent mean ± s.e.m of 3–5 individual mice per group. n = mice per group and per time-point.</p

Effect of Ancrod treatment on inflammation and IAV pathogenesis.

<p>(A) Survival and weight loss of mice treated with Ancrod (open symbols, n = 11) or not (closed symbols, n = 11) after infection with IAV A/PR/8/34 (squares) or uninfected mice (diamonds, n = 5). Weight loss data represent weight average ± s.e.m of the above indicated number of mice. (B) Cytokines levels in the BAL were measured by ELISA after A/PR/8/34 infection of WT and PLG-KO (KO) mice treated with Ancrod (white bars) or not (black bars). Data represent mean ± s.e.m. of n = 4 mice per group. (C) Survival rate (left panels) and weight loss (right panels) of WT (squares) and PLG-KO (triangles) mice treated with Ancrod (open symbols) or not (closed symbols) after intranasal inoculation with IAV A/PR/8/34 (n = 8–10 mice per group). Weight loss data represent weight average ± s.e.m of the above indicated number of mice.</p

Fibrinolysis is induced following severe influenza infections.

<p>(A) Levels of Plasminogen, Active Plasmin, FDP, D-dimer and Fibrinogen, were determined by ELISA in the BAL of A/PR/8/34 infected or uninfected (−) C57BL/6 mice after the indicated days post-inoculation. Markers were also evaluated in the BAL of WT or PLG-KO mice infected with A/Netherlands/602/09 virus. Data represent mean ± s.e.m of n = 3–6 mice per group. (B) Western blot analysis for the detection of fibrinogen and FDP in the lungs of IAV-infected mice on the indicated days post inoculation (representative of n = 3). kDa: apparent molecular weight. n = mice per group. (C) Presence of fibrinogen was assessed in the blood of mice treated or not with Ancrod by ELISA (left panel) or Western blot analysis (right panel). The results represent the mean values ± s.e.m from 3 individual animals per group for the ELISA. The western blot analysis is representative for results of 3 mice per group.</p

Schematic overview of the proposed model for Plasminogen-mediated influenza virus pathogenesis.

<p>During IAV infection, plasminogen is converted into plasmin. On the one hand, plasmin cleaves and activates the viral hemagglutinin, promoting IAV replication for some influenza strains. On the other hand, plasmin promotes inflammation via fibrinolysis and increases permeability.</p

Plasminogen-deficiency prevents severe inflammation and virus dissemination.

<p>(A) Cytokine levels in BAL were assessed by 23-multiplex Luminex kit (uninfected, white bars; infected, black bars) on the indicated days post inoculation of WT (top panel) and PLG-KO mice (bottom panel) with IAV A/PR/8/34. The levels of IL-2, IL-3, IL-4, IL-5, IL-9, IL-12(p70), IL-13, IL-17 and eotaxin were below the detection limit (not shown). Data represent mean ± s.e.m. of 2 individual mice per group from one experiment and is representative of 2 individual experiments (total n = 3–6 mice per group). (B) A/PR/8/34 virus titers in the indicated organs of WT (closed symbols) and PLG-KO mice (open symbols) was assessed 2 and 5 days post-inoculation.</p

Effect of 6-aminohexanoic acid and/or Ancrod treatment on the course of IAV infection.

<p>Survival and weight loss of IAV inoculated C57BL/6 mice treated with 6-AHA (circle) or not (squares). (A) Mice were inoculated with IAV A/PR8/34 (5,000 PFU, n = 28 or 500 PFU; n = 11) in presence (open symbols) or absence (closed symbols) of Ancrod. 6-AHA treatment was initiated on the day of inoculation. (B) Infectious A/PR/8/34 lung virus titers in 6-AHA treated or untreated mice. Data represent mean ± s.e.m of 3 individual mice per group. (C) Mice were inoculated with IAV A/PR/8/34 (n = 10), A/Netherlands/602/09 (n = 16) or A/chicken/Ivory-Coast/1787/2006 (n = 10) as indicated. 6-AHA treatment was initiated two days post-inoculation. n = per group. Weight loss data represent weight average ± s.e.m of the above indicated number of mice.</p