UC Riverside

Genome Sequence and the Identification of Mosquitocidal Toxin operons in Clostridium bifermentans subsp. malaysia

by

Swati Chawla

Doctor of Philosophy Graduate Program in Environmental Toxicology

University of California, Riverside, March 2015

Dr. Sarjeet S. Gill, Chairperson

The management and control of mosquito vectors of human disease currently rely primarily on chemical insecticides. However, larvicidal treatments can be effective, and if based on biological insecticides, they can also ameliorate the risk posed to human health by chemical insecticides. The aerobic bacteria Bacillus thuringiensis and Lysinibacillus sphaericus have been used for vector control for a number of decades. But development of mosquito resistance to individual toxins has been seen in laboratory and in field. More recently, the anaerobic bacterium Clostridium bifermentans subsp. malaysia (Cbm) has been reported to have high mosquitocidal activity, and a number of proteins were identified as potentially mosquitocidal. However, the cloned proteins showed no mosquitocidal activity.

I determined genome sequence of the only known anaerobic mosquitocidal bacterium, Cbm by the next generation sequencing approach. I showed that the genome consists of a 3.7-Mb chromosome, seven small plasmids and a 109-kb megaplasmid that contains the two toxin operons responsible for mosquitocidal activity. Also, additional virulence-related factors could be identified for further work.

I co-authored and published that four toxins encoded by the Cry operon, Cry16A, Cry17A, Cbm17.1, and Cbm17.2, are all required for toxicity, and these toxins collectively show remarkable selectivity for Aedes rather than Anopheles mosquitoes, even though Cbm is more toxic to Anopheles. I also tried to improve the mosquitocidal activity of Cbm Cry operon by protein engineering of surface loop residues in domain II of Cry17Aa and wanted to test on 4 different species of important human disease vectors, Aedes aegypti, Anopheles gambiae, Anopheles stephensi and Culex quinquefasciatus.

I also identified the toxins that could target Anopheles are different from those expressed by the Cry operon and investigated the possibility that the genes encoded in second operon on pClosMP, the Clostridial Mosquitocidal Protein (Cmp) operon, could be toxic to Anopheles. The Cmp operon encodes seven genes, including a 150-kDa mosquitocidal toxin (CMP1) and HA gene. I show the three genes, CMP1, CNTNH, and CHA that play an important role in botulinum neurotoxicity are not active individually against mosquitoes. We need to express the native full length 16kb Cmp operon and CMP1-CNTNH-HA constructs to answer this question.

In summary, based on data in the literature and my work, I demonstrate that Cbm harbors its mosquitocidal activity on a large 109kb plasmid that encodes for two toxin operons. The Cry operon toxins work as a complex and are active against Aedes larvae. The Cmp operon toxins activity against Aedes and Anopheles larvae remains a vast field of study to be further explored.