Construction and characterization of rtxA and rtxC mutants of auxotrophic O139 Vibrio cholerae.
Vibrio cholerae is a Gram-negative bacterium that causes diarrheal disease. V. cholerae O1 and O139 serogroups are toxigenic and are known to cause epidemic cholera. These serogroups produce cholera toxin and other accessory toxins such as accessory cholera enterotoxin, zonula occludens toxin, and multifunctional, autoprocessing repeat in toxin (MARTX). In the present study, we incorporated mutated rtxA and rtxC genes that encode MARTX toxin into the existing aminolevulinic acid (ALA) auxotrophic vaccine candidate VCUSM2 of V. cholerae O139 serogroup. The rtxC mutant was named VCUSM9 and the rtxC/rtxA mutant was named VCUSM10. VCUSM9 and VCUSM10 were able to colonize intestinal cells well, compared with the parent vaccine strain, and produced no fluid accumulation in a rabbit ileal loop model. Cell rounding and western blotting assays indicated that mutation of the rtxC gene alone (VCUSM9 strain) did not abolish MARTX toxicity. However mutation of both the rtxA and rtxC genes (VCUSM10) completely abolished MARTX toxicity. Thus we have produced a new, less reactogenic, auxotrophic rtxC/rtxA mutated vaccine candidate against O139 V. cholerae.
Cheong
Tan Gim
TG
Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia Health Campus, Kubang Kerian 16150, Kelantan, Malaysia.
Chan
Melissa
M
Kurunathan
Sinniah
S
Ali
Syed Atif
SA
ZiNing
Tan
T
Zainuddin
Zainul Fadziruddin
ZF
Lalitha
Pattabhiraman
P
Ravichandran
Manickam
M
D000217:Acyltransferases / Q000235:genetics*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics* / Q000633:toxicity; D045744:Cell Line, Tumor; D022121:Cholera Vaccines; D017353:Gene Deletion; D005798:Genes, Bacterial; D006801:Humans; D007422:Intestines / Q000382:microbiology; D051379:Mice; D016254:Mutagenesis, Insertional; D011817:Rabbits; D012703:Serotyping; D041242:Vibrio cholerae O139 / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity; D037521:Virulence Factors / Q000235:genetics
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D022121:Cholera Vaccines; C434464:RtxA protein, Vibrio cholerae; D037521:Virulence Factors; D000217:Acyltransferases; C434465:RtxC protein, Vibrio cholerae