Molecular Analysis of <i>Pfs47</i>-Mediated <i>Plasmodium</i> Evasion of Mosquito Immunity

<div><p>Malaria is a life-threatening disease caused by <i>Plasmodium falciparum</i> parasites that is transmitted through the bites of infected anopheline mosquitoes. <i>P</i>. <i>falciparum</i> dispersal from Africa, as a result of human migration, required adaptation of the parasite to several different indigenous anopheline species. The mosquito immune system can greatly limit infection and <i>P</i>. <i>falciparum</i> evolved a strategy to evade these responses that is mediated by the <i>Pfs47</i> gene. <i>Pfs47</i> is a polymorphic gene with signatures of diversifying selection and a strong geographic genetic structure at a continental level. Here, we investigated the role of single four amino acid differences between the <i>Pfs47</i> gene from African (GB4 and NF54) and a New World (7G8) strains that differ drastically in their ability to evade the immune system of <i>A</i>. <i>gambiae</i> L35 refractory mosquitoes. Wild type NF54 and GB4 parasites can survive in this mosquito strain, while 7G8 parasites are eliminated. Our studies indicate that replacement in any of these four single amino acids in <i>Pfs47</i> from the NF54 strain by those present in 7G8, completely disrupts the ability of NF54 parasites to hide from the mosquito immune system. One of these amino acid replacements had the opposite effect on <i>A</i>. <i>albimanus</i> mosquitoes, and enhanced infection. We conclude that malaria transmission involves a complex interplay between the genetic background of the parasite and the mosquito and that <i>Pfs47</i> can be critical in this interaction as it mediates <i>Plasmodium</i> immune evasion through molecular interactions that need to be precise in some parasite/vector combinations.</p></div

Effect of LRIM1 silencing in <i>A</i>. <i>albimanus</i> mosquitoes on its infection with <i>P</i>. <i>falciparum Pfs47</i> NF54 V247A and I248L.

<p>Effect of silencing the leucine-rich repeat immune protein 1 (LRIM1) in <i>A</i>. <i>albimanus</i> on infection with NF54 <i>Pfs47</i> haplotypes V247A and I248L. Each dot represents the number of parasite on an individual mosquito and the median is indicated with a black line (n = number of midguts examined). The orange area of the pie charts indicates the prevalence of infection. All parasite phenotypes were confirmed in two independent experiments. Median number of oocysts were compared using the Mann–Whitney test. ****P < 0.0001.</p

Molecular Analysis of <i>Pfs47</i>-Mediated <i>Plasmodium</i> Evasion of Mosquito Immunity - Fig 2

<p><b>(A) Schematic representation and alignment of Pfs47 Central Domain Haplotypes sequences used in this study.</b> Variable positions between the NF54 haplotype and the 7G8 haplotype along with the point mutations constructs used in this study to complement the Pfs47KO line are marked as orange. The known infection phenotype of these strains in the <i>A</i>. <i>gambiae</i> R strain midgut is shown on the right. (B) Schematic representation of the integrase-mediated complementation of different <i>Pfs47</i> haplotypes on the Pfs47KO locus by the plasmid pPfs47attP. The drug selection cassettes hDHFR and BSD the recombination adaptor sites attP/attB are showed. Representation is not in scale and is for illustration purposes only. (C) Schematic representation of the amino acids substituted on every single <i>Pfs47</i> point mutation construct. Nucleophilic amino acids are shaded in cyan and hydrophobic amino acids are shaded in orange.</p

Infection phenotype of different <i>Pfs47</i> complement <i>P</i>. <i>falciparum</i> lines (T236I, S242L, V247A and I248L) in the <i>A</i>. <i>gambiae</i> R strain, <i>A</i>. <i>albimanus</i> and <i>A</i>. <i>stephensi</i> 7–9 d post-feeding representing two pooled independent experiments (Experiment 1 and Experiment 2, S1–S3 Tables).

<p>(A) Infection of <i>A</i>. <i>gambiae</i> R strain. The pie charts indicate the proportion of live (orange) and melanized (black) parasites. (B) Infection of <i>A</i>. <i>stephensi</i> Nijmegen, and (C) <i>A</i>. <i>albimanus</i> mosquitoes with <i>P</i>. <i>falciparum</i> NF54 Pfs47KO complemented derivatives expressing <i>Pfs47</i> haplotypes NF54 T236I, S242L, V247A and I248L. The orange area of the pie charts indicates the prevalence of infection in <i>A</i>. <i>albimanus</i>. Each dot represents the number of parasite on an individual mosquito and the median is indicated with a black line (n = number of midguts examined). Melanization prevalences in <i>A</i>. <i>gambiae</i> R strain were compared with the X² test relative to Pfs47 KO + NF54, infection prevalences in <i>A</i>. <i>albimanus</i> were compared with the X² test, ns = no significant difference,** p<0.01, **** p<0.0001. All parasite phenotypes were confirmed in two or three independent experiments.</p

Participation of the JNK Pathway in L3–5 Mosquitoes Refractory (R) responses to <i>Plasmodium berghei</i> Infection.

<p>(A) Basal mRNA expression of genes from the JNK pathway in the midgut and hemocytes of G3 susceptible (S) (gray) and R (blue) mosquitoes. (B) Expression of effector genes regulated by the JNK pathway in S (gray) and R (blue) mosquitoes. Basal mRNA levels of HPx2 and NOX5 in the midgut, and of TEP1 and FBN9 in hemocytes. Graphs represent the expression level in R females, relative to S females, that were adjusted to a value of “1”; for R females samples the bars represent the SEM of three biological replicates from independent experiments (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s010" target="_blank">Table S4</a>). P-values determined by paired Student's-T test after log2 transformation; *, p<0.05, **, p<0.01, ***, <i>p</i><0.001. (C) Effect of silencing JNK (right panel) in the number of melanized and live parasites on individual midguts of R mosquitoes. Red dots indicate the number of parasites on an individual midgut, live (y-axis) and melanized (x-axis). Green horizontal bars indicate median infection intensities. Inset pie graphs represent the percentage of total parasites for each group displaying a live (green) or melanized (black) phenotype; percentage displayed refers to melanized parasites. Graphs represent data from three biological replicates (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s016" target="_blank">Table S10</a>). (n = number of midguts analyzed).</p

Infection phenotype of different <i>P</i>. <i>falciparum</i> lines (NF54 WT, Pfs47 KO, Pfs47 KO + NF54) in the <i>A</i>. <i>gambiae</i> R strain midgut 7–9 d postfeeding.

<p>Live and melanized parasites on individual mosquito midguts in the <i>A</i>. <i>gambiae</i> R strain and confirmation of gametocyte viability in the susceptible <i>A</i>. <i>stephensi</i> Nijmegen using <i>P</i>. <i>falciparum</i> NF54 (A), Pfs47 KO (B) and Pfs47 KO + NF54 (C). The medians are indicated with black lines and proportion of live (orange) and melanized (black) parasites are indicated with pie charts. Each dot represents the number of parasite on an individual mosquito and the median is indicated with a black line (n = number of midguts examined). The differences in the proportion of melanized parasites relative to NF54 WT were analyzed using the X² test, **** p<0.0001, ns = not significant. All parasite phenotypes were confirmed in two independent experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168279#pone.0168279.s001" target="_blank">S1 Fig</a>).</p

The JNK Pathway and <i>Plasmodium berghei</i> infection in <i>Anopheles gambiae</i>.

<p>(A) Diagram representing the organization of the JNK signaling cascade based on functional studies from vertebrates and <i>Drosophila</i>. Five <i>An. gambiae</i> orthologs were functionally characterized, including two kinases, hemipterous <i>(hep)</i> and c-Jun N-terminal kinase <i>(jnk)</i>; a phosphatase, <i>puc</i>; and two transcription factors, Jun <i>(jun)</i> and Fos <i>(fos)</i> (B) Basal mRNA expression of putative genes from the JNK pathway in adult females. <i>Hemipterous (Hep)</i>, Jun N-terminal kinase <i>(JNK)</i>, <i>Jun</i> and <i>Fos</i> transcription factors and <i>puckered (puc)</i> mRNA levels in different organs of sugar-fed females. Mg, midgut; H, head; Th, thorax; Ab, abdomen; Hc, hemocyte; Ov, ovaries. Expression in different tissues relative to midgut levels, for which the mean was given a value of “1”. Error bars indicate SEM of two biological replicates. (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s017" target="_blank">Table S11</a> for gene ID numbers and primer sequences) (C) Midgut expression of members of the JNK pathway in response to <i>Plasmodium</i> infection in three independent experiments. Ratio of expression in infected/control blood-fed mosquitoes of <i>Hep</i>, <i>JNK</i>, <i>Puc</i>, <i>Jun</i> and <i>Fos</i> mRNA levels in midguts of mosquitoes from 3 independent experiments (green, red and blue bars). Error bars indicate SEM of two technical replicates. The expression analysis in each biological replicate is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s009" target="_blank">Table S3</a>. P-values determined by Student's-T test after log 2 transformation; **, <i>p</i><0.01, ***, <i>p</i><0.001. *, p<0.05; **, p<0.01, ***, p<0.001. (D) Effect of silencing JNK pathway members on <i>P. berghei</i> infection. (E) Effect of silencing the transcription factor <i>jun</i> alone or and co-silencing <i>jun</i> and the negative regulator <i>puc</i> on <i>Plasmodium</i> infection. For (D) and (E), the green dots represent oocyst counts from individual midguts and the horizontal red bar indicates the median infection level. Groups were compared using the KS, Mann-Whitney and Kruskal-Wallis tests with Dunn's post test (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s009" target="_blank">Table S3</a>). The P-values for the Mann-Whitney tests are shown. Graphs represent samples pooled from three biological replicates with comparable (not statistically different) medians in their <i>dsLacZ</i>-injected groups (n = number of midguts).</p

The JNK Pathway Is a Key Mediator of <i>Anopheles gambiae</i> Antiplasmodial Immunity

<div><p>The innate immune system of <i>Anopheles gambiae</i> mosquitoes limits <i>Plasmodium</i> infection through multiple molecular mechanisms. For example, midgut invasion by the parasite triggers an epithelial nitration response that promotes activation of the complement-like system. We found that suppression of the JNK pathway, by silencing either <i>Hep</i>, <i>JNK</i>, <i>Jun</i> or <i>Fos</i> expression, greatly enhanced <i>Plasmodium</i> infection; while overactivating this cascade, by silencing the suppressor <i>Puckered</i>, had the opposite effect. The JNK pathway limits infection via two coordinated responses. It induces the expression of two enzymes (HPx2 and NOX5) that potentiate midgut epithelial nitration in response to <i>Plasmodium</i> infection and regulates expression of two key hemocyte-derived immune effectors (TEP1 and FBN9). Furthermore, the <i>An. gambiae</i> L3–5 strain that has been genetically selected to be refractory (R) to <i>Plasmodium</i> infection exhibits constitutive overexpression of genes from the JNK pathway, as well as midgut and hemocyte effector genes. Silencing experiments confirmed that this cascade mediates, to a large extent, the drastic parasite elimination phenotype characteristic of this mosquito strain. In sum, these studies revealed the JNK pathway as a key regulator of the ability of <i>An. gambiae</i> mosquitoes to limit <i>Plasmodium</i> infection and identified several effector genes mediating these responses.</p></div

Previous reports of insect immune memory have identified evidence for specific, nonspecific, transgenerational, and long-term immune memory depending on the insect and pathogen model.

Previous reports of insect immune memory have identified evidence for specific, nonspecific, transgenerational, and long-term immune memory depending on the insect and pathogen model.</p

Immune priming in <i>An</i>. <i>gambiae</i> mosquitoes.

(A) Plasmodium infection induces the expression HPX7 and HPX8 that mediate PGE2 synthesis by the midgut following microbiota contact with epithelial cells during ookinete invasion. The release of PGE2 triggers the production of the HDF by DBLOX-positive fat body oenocytes that proliferate following a Tip60-dependent mechanism. At the hemolymph, HDF induces the proliferation of circulating granulocytes, which are attracted to the midgut during reinfection following the PGE2 signal. Granulocyte release microvesicles (HdMv) at the site of recruitment, which mediates complement-like activation and Plasmodium elimination. Thus, the intensity of the mosquito immune response to Plasmodium can be enhanced by a previous infection. (B) Upon PGE2-dependent priming, the production of HDF in response to ookinete midgut invasion is constitutively enhanced following the first challenge, and this induces a constitutive increase in the proportion of circulating granulocytes. After the initial challenge, hemocyte association with the mosquito midgut goes back to basal levels. However, reprogramming of hemocytes during this first exposure results in enhanced hemocyte recruitment and a stronger immune response to a subsequent infection [37–40,43]. DBLOX, double-peroxidase; FB, fat body; HDF, hemocyte differentiation factor; HdMv, hemocyte-derived microvesicle; PGE2, prostaglandin E2.</p