Genetic dissection of Anopheles gambiae gut epithelial responses to Serratia marcescens.

Genetic variation in the mosquito Anopheles gambiae profoundly influences its ability to transmit malaria. Mosquito gut bacteria are shown to influence the outcome of infections with Plasmodium parasites and are also thought to exert a strong drive on genetic variation through natural selection; however, a link between antibacterial effects and genetic variation is yet to emerge. Here, we combined SNP genotyping and expression profiling with phenotypic analyses of candidate genes by RNAi-mediated silencing and 454 pyrosequencing to investigate this intricate biological system. We identified 138 An. gambiae genes to be genetically associated with the outcome of Serratia marcescens infection, including the peptidoglycan recognition receptor PGRPLC that triggers activation of the antibacterial IMD/REL2 pathway and the epidermal growth factor receptor EGFR. Silencing of three genes encoding type III fibronectin domain proteins (FN3Ds) increased the Serratia load and altered the gut microbiota composition in favor of Enterobacteriaceae. These data suggest that natural genetic variation in immune-related genes can shape the bacterial population structure of the mosquito gut with high specificity. Importantly, FN3D2 encodes a homolog of the hypervariable pattern recognition receptor Dscam, suggesting that pathogen-specific recognition may involve a broader family of immune factors. Additionally, we showed that silencing the gene encoding the gustatory receptor Gr9 that is also associated with the Serratia infection phenotype drastically increased Serratia levels. The Gr9 antibacterial activity appears to be related to mosquito feeding behavior and to mostly rely on changes of neuropeptide F expression, together suggesting a behavioral immune response following Serratia infection. Our findings reveal that the mosquito response to oral Serratia infection comprises both an epithelial and a behavioral immune component.

Peptide antagonism as a mechanism for NK cell activation.

Inhibition of natural killer (NK) cells is mediated by MHC class I receptors including the killer cell Ig-like receptor (KIR). We demonstrate that HLA-C binding peptides can function as altered peptide ligands for KIR and antagonize the inhibition mediated by KIR2DL2/KIR2DL3. Antagonistic peptides promote clustering of KIR at the interface of effector and target cells, but do not result in inhibition of NK cells. Our data show that, as for T cells, small changes in the peptide content of MHC class I can regulate NK cell activity.

Asaia accelerates larval development of Anopheles gambiae.

Arthropod borne diseases cause significant human morbidity and mortality and, therefore, efficient measures to control transmission of the disease agents would have great impact on human health. One strategy to achieve this goal is based on the manipulation of bacterial symbionts of vectors. Bacteria of the Gram-negative, acetic acid bacterium genus Asaia have been found to be stably associated with larvae and adults of the Southeast Asian malaria vector Anopheles stephensi, dominating the microbiota of the mosquito. We show here that after the infection of Anopheles gambiae larvae with Asaia the bacteria were stably associated with the mosquitoes, becoming part of the microflora of the midgut and remaining there for the duration of the life cycle. Moreover they were passed on to the next generation through vertical transmission. Additionally, we show that there is an increase in the developmental rate when additional bacteria are introduced into the organism which leads us to the conclusion that Asaia plays a yet undetermined crucial role during the larval stages. Our microarray analysis showed that the larval genes that are mostly affected are involved in cuticle formation, and include mainly members of the CPR gene family.

Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1.

The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I-III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.

Crystallographic insight into collagen recognition by discoidin domain receptor 2.

The discoidin domain receptors, DDR1 and DDR2, are widely expressed receptor tyrosine kinases that are activated by triple-helical collagen. They control important aspects of cell behavior and are dysregulated in several human diseases. The major DDR2-binding site in collagens I-III is a GVMGFO motif (O is hydroxyproline) that also binds the matricellular protein SPARC. We have determined the crystal structure of the discoidin domain of human DDR2 bound to a triple-helical collagen peptide. The GVMGFO motifs of two collagen chains are recognized by an amphiphilic pocket delimited by a functionally critical tryptophan residue and a buried salt bridge. Collagen binding results in structural changes of DDR2 surface loops that may be linked to the process of receptor activation. A comparison of the GVMGFO-binding sites of DDR2 and SPARC reveals a striking case of convergent evolution in collagen recognition.