Here's a list of papers from which we got some ideas / inspiration and useful information

Asaia related

Bacteria of the Genus Asaia: A Potential Paratransgenic Weapon Against Malaria Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector isolation of Asaia from mosquitoes and expression of GFP Paternal transmission of symbiotic bacteria in malaria vectors Asaia transmission and stable Asaia mCherry strain Cloning of Escherichia coli lacZ and lacY Genes and Their Expression in Gluconobacter oxydans and Acetobacter liquefaciens Asaia plasmid

Blocking Plasmodium falciparum transmission

Bacteria expressing single-chain immunotoxin inhibit malaria parasite development in mosquitoes Immunotoxin against malaria parasite, composed of Shiva-1 and mouse single-chain antibody fragment against Pbs21. A single-chain antibody fragment specific for the Plasmodium berghei ookinete protein Pbs21 confers transmission blockade in the mosquito midgut 13.1 scFv single-chain antibody fragment against ookinete protein Pbs21 In vitro cytocidal effect of novel lytic peptides on Plasmodium falciparum and Trypanosoma cruzi Shiva-1 Ajay K. Saxena, Yimin Wu, and David N. Garboczi, Plasmodium P25 and P28 Surface Proteins: Potential Transmission-Blocking Vaccines, Eukaryot. Cell, Aug 2007; 6: 1260 - 1265 Link. Using antibodies to bind both ookinete surface proteins P25 and P28 could prevent the epithelial invasion.

Candidate effectors molecules to block malaria propagation:

A lytic peptide called SB-37 similar (in term of length and amino acids property) to Shiva-1 was used to kill P. falciparum in vitro [1]. SB-37 is a slightly modified version of a Cecropin, the Cecropin B [1], the sequence is in the paper [1]. Cecropins are peptides derived from the insect immune defence (a giant moth). Cecropin A was expressed in a simbiont of the bug Rhodnius prolixus in order to kill T. cruzi, a parasite that cause the Chagas disease [2]. More recently, It was also expressed in the mosquito’s gut (transgenic mosquitoes) to prevent the propagation of the malaria [3]. (Sequence: http://www.anaspec.com/products/product.asp?id=30699) Because of its hight homology the cecropins A and B, cecropin D could also be used (sequence: [4]). There exists 6 cecropins : cecropin A, … , F.

The Glossina attacin is an antimicrobial peptide effective on both gram-negative and protozoa. It was expressed in a symbiont of the tsetse fly in order to kill the pasasite T.brucei; We should determine if it acts on Asaia, attacins kill E.Coli but do not act on many other gram-positive and gram-negative bacteria (http://www.copewithcytokines.org/cope.cgi?key=Attacins). Sequence: [5].

Other effectors molecules against Plasmodium are cited by Jacobs-Lorena and al. [6]: - The salivary gland/midgut peptide (SM1), it is a dodecapeptide (sequence: [7]), It was found by testing a number of radom peptides, and it binds to both the gut and salivary gland bocking the Plasmodium at these two stages.

- An other attractive approach is to block chitinase present in the mousquito’s gut. This enzyme plays an important role in modelling of the peritrofic matrix (PM, that surround the bood meal) which is an important barrier for the Plasmodium. It has been shown that inhibiting chitinase make the PM thicker and efficiently blocks the plasmodium development. Chitinase is activated by cleaving an N-terminal propeptide, and it has been demonstrated, by feeding the mosquito with the peptide, that this same propeptide, called prochitinase peptide (13 amino-acids) blocks efficiently the chinase activity and the plasmodium developement in the mosquito’s gut (sequence: [8]).

- The Phospholypase A2 (PLA2) inhibits oocyst formation in the mousquito’s gut by feeding or expressing it in transgenic mousquitos (See [9] and [10]). Interestingly this enzyme comes from the snake or bee venom and its anti-malaria activity does not depend on its hydrolytic activity.

In summary we have those potential effector molecules:

• SB-37
• Ceropins (mainly A, B and D)
• Glossina attacin
• SM-1
• Prochitinase peptide
• P25, P28 proteins
• Immunotoxin

• [1] Jaynes et al. 1988
• [2] Durvasula et al. 1997
• [3] Kim et al. 2004
• [4] Hultmark et al. 1982
• [5] Wang et al., 2008
• [6] Jacobs-Lorena et al., 2005
• [7] Ghosh et al., 2001)
• [8] Bhatnagar et al., 2003
• [9] Zieler et al., 2001
• [10] Moreira et al., 2002

Ideas

1. Engineering BioBrick vectors from BioBrick parts This might help us to make an Asaia plasmid?