Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
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Journal of Fish Diseases 2006, 29, 355–364<br />
PDíaz-Rosales et al. Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e in P. damse<strong>la</strong>e ssp. piscicida<br />
in both strains pulsed with hydrog<strong>en</strong> peroxi<strong>de</strong><br />
compared with stationary phase cultures.<br />
Discussion<br />
Enzymes such as SOD and cata<strong><strong>la</strong>s</strong>e, which neutralize<br />
ROS produced during aerobic metabolism or<br />
during respiratory burst in fish phagocytes are<br />
important virul<strong>en</strong>ce factors in many pathog<strong>en</strong>s<br />
(Barnes et al. 1996, 1999b; Yesilkaya, Kadioglu,<br />
Gingles, Alexan<strong>de</strong>r, Mitchell & Andrew 2000;<br />
Vattanaviboon & Mongkolsuk 2001; Uzzau et al.<br />
2002; Banin, Vassi<strong>la</strong>kos, Orr, Martínez & Ros<strong>en</strong>berg<br />
2003). In this study, all the strains of<br />
P. damse<strong>la</strong>e ssp. piscicida assayed showed a single<br />
band of SOD activity with i<strong>de</strong>ntical mobility on<br />
acry<strong>la</strong>mi<strong>de</strong> gels. A unique band simi<strong>la</strong>r in all the<br />
strains was also observed on cata<strong><strong>la</strong>s</strong>e activity gels.<br />
Simi<strong>la</strong>rly, Barnes, Balebona, Horne & Ellis<br />
(1999a), in a study that inclu<strong>de</strong>d a collection of<br />
P. damse<strong>la</strong>e ssp. piscicida strains iso<strong>la</strong>ted from<br />
gilthead seabream, Sparus aurata (L.), reported<br />
only one SOD located in the perip<strong><strong>la</strong>s</strong>mic space and<br />
one cytop<strong><strong>la</strong>s</strong>mic cata<strong><strong>la</strong>s</strong>e.<br />
Several studies have reported that microorganisms<br />
contain differ<strong>en</strong>t SOD and cata<strong><strong>la</strong>s</strong>e isozymes<br />
inducible un<strong>de</strong>r certain growth conditions (Storz,<br />
Tartaglia, Farr & Ames 1990; Privalle & Fridovich<br />
1992; Barnes et al. 1996; Yesilkaya et al. 2000;<br />
Geslin, L<strong>la</strong>nos, Prieur & Jeanthon 2001; Vattanaviboon<br />
& Mongkolsuk 2001). However, culture<br />
conditions assayed in this work have not induced<br />
new SOD or cata<strong><strong>la</strong>s</strong>e isozymes in P. damse<strong>la</strong>e ssp.<br />
piscicida. Mn-SOD activity has be<strong>en</strong> reported to be<br />
modu<strong>la</strong>ted by oxidative stress and iron-limiting<br />
conditions (Privalle & Fridovich 1992; Barnes<br />
et al. 1999b) but in the case of P. damse<strong>la</strong>e ssp.<br />
piscicida neither production of intracellu<strong>la</strong>r superoxi<strong>de</strong><br />
by methyl violog<strong>en</strong> nor culture un<strong>de</strong>r ironrestricted<br />
conditions induced the production of a<br />
differ<strong>en</strong>t type of SOD. Although further studies are<br />
necessary, this <strong>la</strong>ck of induction of a new SOD<br />
could be due to the pres<strong>en</strong>ce of only one sod g<strong>en</strong>e,<br />
i.e. sod B <strong>en</strong>coding Fe-SOD (Lynch & Kuramitsu<br />
2000).<br />
In contrast, differ<strong>en</strong>ces in the int<strong>en</strong>sity of the<br />
bands were observed in extracts obtained un<strong>de</strong>r<br />
differ<strong>en</strong>t culture conditions for both SOD and<br />
cata<strong><strong>la</strong>s</strong>e activities. As the amount of protein loa<strong>de</strong>d<br />
in the electrophoretic <strong>la</strong>nes was simi<strong>la</strong>r in all cases,<br />
the differ<strong>en</strong>t int<strong>en</strong>sities suggest variations in the<br />
levels of activity in the extracts <strong>de</strong>p<strong>en</strong>ding on the<br />
culture condition. These results are in agreem<strong>en</strong>t<br />
with those obtained by Barnes, Balebona, Horne &<br />
Ellis (1999a), who also <strong>de</strong>tected differ<strong>en</strong>ces in<br />
cultures carried out un<strong>de</strong>r iron replete and <strong>de</strong>pleted<br />
conditions and high- and low-aerated broths.<br />
The quantification of both SOD and cata<strong><strong>la</strong>s</strong>e<br />
activities carried out in this study corroborated that<br />
differ<strong>en</strong>t band int<strong>en</strong>sities correspon<strong>de</strong>d to variations<br />
in the levels of activity. The lowest levels of SOD<br />
activity were <strong>de</strong>tected wh<strong>en</strong> bacteria were grown<br />
un<strong>de</strong>r iron-restricted conditions. The ferric nature<br />
of P. damse<strong>la</strong>e ssp. piscicida SOD <strong>de</strong>scribed by<br />
Barnes et al. (1999a) could exp<strong>la</strong>in this lower<br />
activity in the pres<strong>en</strong>ce of an iron che<strong>la</strong>nt.<br />
Iron also influ<strong>en</strong>ced the levels of cata<strong><strong>la</strong>s</strong>e activity<br />
in P. damse<strong>la</strong>e ssp. piscicida. The role of iron as<br />
cofactor in this <strong>en</strong>zyme has be<strong>en</strong> <strong>de</strong>monstrated with<br />
inhibition studies. Thus, cata<strong><strong>la</strong>s</strong>e activity could not<br />
be <strong>de</strong>tected in the gels following exposure to<br />
sodium azi<strong>de</strong> and it was slightly reduced after<br />
treatm<strong>en</strong>t with potassium cyani<strong>de</strong>. These results<br />
suggest that the <strong>en</strong>zyme is an iron cofactored<br />
cata<strong><strong>la</strong>s</strong>e, as Mn-containing cata<strong><strong>la</strong>s</strong>es retain activity<br />
after treatm<strong>en</strong>t with azi<strong>de</strong> and cyani<strong>de</strong> and are<br />
inhibited by mercuric chlori<strong>de</strong> (Kono & Fridovich<br />
1983; Allgood & Perry 1986; Barnes et al. 1999b).<br />
This ferric nature of the cata<strong><strong>la</strong>s</strong>e may exp<strong>la</strong>in the<br />
lower cata<strong><strong>la</strong>s</strong>e activity observed in cultures with<br />
ad<strong>de</strong>d iron che<strong>la</strong>nt and lower survival with H 2 O 2<br />
observed by Díaz-Rosales, Chabrillón, Moriñigo &<br />
Balebona (2003).<br />
Lower survival of P. damse<strong>la</strong>e ssp. piscicida in sole<br />
phagocytes has be<strong>en</strong> observed for strain EPOY-<br />
8803-II compared with the virul<strong>en</strong>t strain. Contradictory<br />
results have be<strong>en</strong> reported on the ability of<br />
P. damse<strong>la</strong>e ssp. piscicida to survive insi<strong>de</strong> macrophages<br />
from several fish species. In a study using<br />
macrophages from sea bass, gilthead sea bream and<br />
rainbow trout, Skarmeta et al. (1995) conclu<strong>de</strong>d<br />
that head kidney macrophages from these fish<br />
species were able to kill the pathog<strong>en</strong>. However,<br />
Noya et al. (1995b) reported that whilst bacteria<br />
within granulocytes and macrophages from <strong>la</strong>rge<br />
gilthead sea bream were morphologically altered,<br />
bacteria insi<strong>de</strong> small fish remained unaffected. In<br />
addition, data on the ability of P. damse<strong>la</strong>e to<br />
survive insi<strong>de</strong> fish macrophages have be<strong>en</strong> reported<br />
by several authors who observed that bacteria can<br />
multiply insi<strong>de</strong> fish macrophages (Kubota et al.<br />
1970; Hawke, P<strong>la</strong>kas, Minton, McPherson, Zin<strong>de</strong>r<br />
& Guarino 1987; Noya et al. 1995a; Elkamel,<br />
Hawke, H<strong>en</strong>k & Thune 2003).<br />
Ó 2006<br />
B<strong>la</strong>ckwell Publishing Ltd<br />
361