Anti-CPB2 monoclonal antibody, clone BIUBGJ-5027

Removal of the pro-domain of a cysteine protease is essential for activation of the enzyme. We have engineered a cysteine protease (CPB2.8) of the protozoan parasite Leishmania mexicana by site-directed mutagenesis to remove the active site cysteine (to produce CPBC25G). When CPBC25G was expressed in a L, mexicana mutant lacking all CPB genes, the inactive pro-enzyme was processed to the mature protein and trafficked to the lysosome. These results show that auto-activation is not required for correct processing of CPB in vivo. When CPBC25G was expressed in a L. mexicana mutant lacking both CPA and CPB genes, the majority of the pro-enzyme remained unprocessed and accumulated in the flagellar pocket. These data reveal that CPA can directly or indirectly process CPBC25G and suggest that cysteine proteases are targeted to lysosomes via the flagellar pocket. Moreover, they show that another protease can process CPB in the absence of either CPA air CPB, albeit less efficiently. Abolition of the glycosylation site in the mature domain of CPB did not affect enzyme processing, targeting or in vitro activity towards gelatin. This indicates that glycosylation is not required for trafficking. Together these findings provide evidence that the major route of trafficking of Leishmania cysteine proteases to lysosomes is via the flagellar pocket and therefore differs significantly from cysteine protease trafficking in mammalian cells.

Clostridium perfringens type A isolates carrying an enterotoxin (cpe) gene are an important cause of human gastrointestinal diseases, including food poisoning, antibiotic-associated diarrhoea (AAD) and sporadic diarrhoea (SD). Using polymerase chain reaction (PCR), the current study determined that the cpb2 gene encoding the recently discovered beta2 toxin is present in < 15% of food poisoning isolates, which typically carry a chromosomal cpe gene. However, > 75% of AAD/SD isolates, which usually carry a plasmid cpe gene, tested cpb2(+) by PCR. Western blot analysis demonstrated that > 97% of those cpb2(+)/cpe(+) AAD/SD isolates can produce CPB2. Additional PCR analyses, sequencing studies and pulsed field gel electrophoresis experiments determined that AAD/SD isolates carry cpb2 and cpe on the same plasmid when IS1151 sequences are present downstream of cpe, but cpb2 and cpe are located on different plasmids in AAD/SD isolates where IS1470-like sequences are present downstream of cpe. Those analyses also demonstrated that two different CPB2 variants (named CPB2h1 or CPB2h2) can be produced by AAD/SD isolates, dependent on whether IS1470-like or IS1151 sequences are present downstream of their cpe gene. CPB2h1 is similar to 10-fold more cytotoxic for CaCo-2 cells than is CPB2h2. Collectively, these results suggest that CPB2 could be an accessory toxin in C. perfringens enterotoxin (CPE)-associated AAD/SD.