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Cholesterol-dependent cytolysin (CDC) is a bacterial toxin that binds to eukaryotic cholesterol-containing membranes, forms oligomeric complexes, and is inserted into the bilayer to create large aqueous pores. Recently, we reported a species-specific duplication of the hemolysin gene in group III Clostridium botulinum. The duplicated genes (bly1 and bly2) encoded two separate CDC proteins (botulinolysins; BLY1 and BLY2). Here, we aimed to investigate whether BLY1 and BLY2 exert differential cytotoxicity. We isolated two bly genes from C. botulinum and evaluated the cytotoxicity of two recombinant BLY proteins (rBLY1 and rBLY2) in HeLa, IEC-6, and NRK cells. rBLYs were cytotoxic to equine erythrocytes. rBLY1 showed higher hemolytic activity than rBLY2. rBLY2 showed no or very weak cytotoxicity to the HeLa, IEC-6, and NRK cells, whereas rBLY1 showed high cytotoxicity to these cells. The comparison of the amino acid sequence of BLYs with those of other CDCs revealed that the already-known amino acid residues involved in cholesterol-containing membrane recognition, oligomerization, and insertion into membranes are well conserved in both BLYs. However, several amino acid substitutions were observed in the conserved regions, particularly in L2 and L3 regions involved in cell binding. These findings suggest that gene duplication in group III C. botulinum evolved distinct functional specializations, and differential cytotoxicity of BLY1 and BLY2 could be due to the amino acid substitution in the conserved regions. However, the structural and functional comparisons of the two BLYs are essential to gain insights into the function of the CDCs.

Clostridium botulinum produces seven botulinum neurotoxin (BoNT) serotypes. In nature, BoNT exists as a part of the progenitor toxin complex (PTC) through associations with neurotoxin associated proteins (NAPs), including nontoxic nonhemagglutinin and hemagglutinin (HA) complex, consists of HA-70, HA-17 and HA-33. Because PTC displays higher oral toxicity than pure BoNTs, NAPs play a critical role in food poisoning. In a previous study, we demonstrated that the NAP complex in mature large-sized PTC (L-PTC) from serotypes C and D concomitantly induced cell death and cytoplasmic vacuolation in the rat intestinal epithelial cell line IEC-6. Here, we found that the serotype D NAP complex induces only cytoplasmic vacuolation in the normal rat kidney cell line NRK-52E without reducing cell viability. NAP complexes from serotype A and B L-PTCs did not affect cell viability or cytoplasmic vacuolation in IEC-6 and NRK-52E cells. Furthermore, we assessed the effect of immature L-PTCs with fewer HA-33/HA-17 trimers (two HA-33 and one HA-17) than mature L-PTCs on cell viability and cytoplasmic vacuolation in IEC-6 and NRK-52E cells. As a result, mature L-PTCs with the maximum number of HA-33/HA-17 trimers displayed the greatest potency. Consequently, the reduction in cell viability and vacuolation induction are related to the number of HA-33/HA-17 trimers in PTC. The discovery of an epithelial cell model where botulinum PTC specifically induces vacuolization may help clarify the unknown cytotoxicity of PTC, which plays an important role in the trans-epithelial transport of the toxin.

A lactic acid bacterium, Lactiplantibacillus plantarum P1-2 (LpP1-2), can reduce environmental fatty acid hydroperoxides. The administration of LpP1-2 to oxygen-sensitive short-lived nematode mutants and iron-overloaded rats reduced the oxidative stress-related index. Since young piglets have a weak defense system against oxidative stress and are vulnerable to environmental stress, antimicrobial agents have been administered in the rearing. Based on these results, we investigated the effect of LpP1-2 administration to prepartum sows and infant piglets until weaning without antimicrobial agents on the growth of young piglets.The group including both sows and piglets that were administrated with lactic acid bacteria containing LpP1-2 (LABLp) until the end of lactation showed the growth-promoting effect of piglet from lactation to early regular rearing, and even in late regular rearing. Blood biochemical markers were in the normal ranges in both LABLp-administrated and non-administrated groups, but various disease-related markers tended to decrease in the administrated group.To investigate the effects of LABLp administration on postpartum piglets, the piglets born from prenatally LABLp-administrated sows were divided into two groups and then administrated with or without LABLp. The piglets in the LABLp-administrated group tended to grow very slightly higher than those in the non-administrated group from lactation to early regular rearing. After that, the growth in both groups was almost the same. These results suggest that LABLp administration to prepartum sows is essential for the growth-promoting effect.The postnatal LABLp-administrated piglets showed a lower serum lipid peroxidation index than the non-administrated piglets, and had higher numbers of lactic acid bacteria and bifidobacteria in feces at the end of LABLp treatment. In fattening performances, the LABLp-administrated group showed a significant improvement in meat quality.We also discuss the growth and physiological effects by LpP1-2 administration with iron on piglets because iron administration is another important issue in piglet farming.