Antimicrobial activity of LAAO

One of the important features of LAAO is its antimicrobial activity. Skarnes first reported the antibacterial nature of LAAO in 1970 (Skarnes, 1970). Therein it was stated that LAAO from C. adamanteus is active against both gram positive and gram-negative bacteria. One of the interesting points in this article concerned D-amino acid oxidase (DAAO). DAAO is an enzyme similar to LAAO and produces the same products hydrogen peroxide and ammonia upon oxidative deamination of D-amino acids. However, it was shown to have no antibacterial activity. They used mainly D-serine as the substrate for DAAO, at all the used concentrations of D-D-serine, it was proved not to be antibacterial (Skarnes, 1970). These findings showed that there could be different mechanisms for antimicrobial activity by LAAO.

Later on Stiles et al in 1991 made quite extensive studies on the antibacterial effects of snake venoms (Stiles et al., 1991). They tried venoms from different snakes such as C. scutulatus, C. adamanteus, Psuedechis australis and Echis carinatus. All these venoms proved to be antibacterial. More specifically they could isolate the antibacterial components from the snake venom of Psuedechis australis (Australian king brown or mulga snake) and these antimicrobial components were found to be LAAO 1 and LAAO 2 (Stiles et al., 1991). They also concluded that the majority of antibacterial effects seen in the elapid venoms were due to the L-amino acid oxidase.

They used different aeromonas strains of bacteria, which are infectious agents in humans, reptiles and amphibians. Compared to a tetracycline drug, which is generally used for the infections caused by these aeromonas strains, the antibacterial effects of LAAO 1 and LAAO 2 are 70 and 17.5 times more effective respectively than tetracycline (Stiles et al., 1991). In the above reports, it has been shown that hydrogen peroxide generated by the enzyme played a key role in the antibacterial activities.

Addition of catalase, a prominent scavenger of hydrogen peroxide prevented the antibacterial activity (Skarnes, 1970; Stiles et al., 1991).

Further studies were made on the antibacterial activity of achacin; a glycoprotein isolated from the African giant snail and is a close homologue of LAAO (Ogawa et al., 1999). It was shown to be active against gram positive and gram-negative bacteria.

It appears to attack the plasma membrane of the bacteria and was shown to induce extensive filamentation in E. coli (Otsuka-Fuchino et al., 1993). The report by Ehara et al, showed that achacin inhibits the growth of S. aureus and E. coli. At minimum inhibitory concentrations, achacin could produce about 0.2 to 0.4 mM of hydrogen peroxide. But the minimum inhibitory concentrations of hydrogen peroxide required to kill the bacteria was 0.7 to 1 mM. These results indicate that hydrogen peroxide produced by achacin was not sufficient to kill the bacteria. However, they showed that achacin specifically binds to the growth phase specific bacteria and this binding is responsible for exerting maximum antibacterial activity even though it produces low amounts of hydrogen peroxide which is not sufficient to kill the bacteria (Ehara et al., 2002). So, hydrogen peroxide produced in the medium and the local concentrations of hydrogen peroxide generated by binding may have a cumulative effect and that helped achacin to have antimicrobial activity. These findings are similar to those of Suhr et al (1999), wherein it is shown that LAAO binds to the cells and generates a local concentration of hydrogen peroxide.

Another factor they looked at was, whether sialic acid (Neu5Ac) could inhibit the interaction of achacin to the bacteria. According to this study, concentrations up to 2 mg/ml could not block the interaction of this enzyme to the bacterial cells. At higher concentrations of Neu5Ac, it inhibited the enzyme activity of achacin (Ehara et al., 2002).

Other possible reason for the antibacterial activity of LAAO has been put forward by Mitsuru et al in 2003 (Mitsuru et al., 2003). According to them, aplysianin A, a homologue of LAAO induced antibacterial effect on Bacillus subtilis. This effect was mainly contributed to the hydrogen peroxide generated by this enzyme. Interestingly, aplysianin A in the presence of catalase could induce antibacterial activity. The reason for the latter effect was shown to be rapid depletion of L-arginine from the medium

(Mitsuru et al., 2003). This shows that also depletion of amino acids might play a role in the antibacterial activity of the LAAO.

A new LAAO from Crotalus durissus cascavella venom has been isolated recently (Toyama et al., 2006). It presented high sequence similarities with other snake venom LAAO’s such as Calloselasma rhodostoma and Crotalus adamanteus. This showed antibacterial properties against gram positive and gram-negative bacteria (Toyama et al., 2006). Antibacterial activity exhibited by the enzyme was mainly contributed for the hydrogen peroxide produced by this enzyme. In the presence of catalase, antibacterial activity was completely suppressed. Hydrogen peroxide produced by this enzyme induced bacterial membrane rupture and consequently promoted extravasation of plasmatic content to the out side of the cells. This was the first report that indicated that LAAO can cause the bacterial membrane rupture (Toyama et al., 2006).

From all the above literature reports it appears that LAAO’s exhibit antibacterial properties. The primary attribute to this effect was, for sure, production of hydrogen peroxide. In this context its worth mentioning the recent work by Zhang et al, where it is shown that LAAO causes antibacterial activity through binding to the cell surface as previously reported by Ehara et al, in 2002. One more interesting fact they put forward was the antibacterial action of DAAO. They could find out that DAAO could elicit antibacterial effect and at the same time it interacts with the cell surface (Zhang et al., 2004b). This is contradictory to the work by Skarnes (1970), wherein it was reported that DAAO did not elicit any antibacterial effect.

Apart from the antibacterial effects, LAAO’s have been reported to possess antiviral activity (Zhang et al., 2003). According to them, LAAO from Trimeresurus stejnegeri has the potential to inhibit the HIV-1 virus. It inhibited the replicative action and infection of virus. But, addition of catalase did not result in the complete inhibition of the antiviral activity. At the same time, exogenous hydrogen peroxide did not show any antiviral activity (Zhang et al., 2003). These results once again pinpoint the

specific action of LAAO that appears to be different from that of exogenous hydrogen peroxide.

Leishmania species are responsible for causing leishmaniasis or sleeping sickness in human beings. This disease affects around 12 million people annually worldwide. The snake venom of Bothrops moojeni was shown to inhibit the growth of Leishmania species. LAAO that was present in this venom was shown to be the causative agent.

Hydrogen peroxide produced by LAAO was the sole cause for this killing and can be completely rescued by the addition of catalase (Tempone et al., 2001).

In conclusion, LAAOs from different organisms exhibit different properties such as antibacterial activity, antiviral activity and they are also active against intracellular parasites such as Leishmania species. More detailed understanding on the mechanisms of these effects caused by LAAO will help to gives us more clues and if possible making it a wonderful drug in the field of therapeutic approaches.