Papel de las actividades superóxido dismutasa y catalasa en la ...

Journal of Fish Diseases 2006, 29, 355–364
PDíaz-Rosales et al. Superoxide dismutase and catalase in P. damselae ssp. piscicida
Multiplication of P. damselae ssp. piscicida inside
several fish cell lines has also been reported. Elkamel
& Thune (2003) observed that the bacteria multiply
in EPC, CCO, and FHM cells and López-
Dóriga, Barnes, dos Santos & Ellis (2000) using
EPC cells observed that both virulent and avirulent
isolates were able to adhere to and invade cells.
Results obtained from this study show that
P. damselae ssp. piscicida is able to survive inside
sole phagocytes at least for 5 h, the survival rates
being higher for the virulent isolate. Although the
bacterium was able to survive, the rates obtained
always indicated a certain degree of bacterial
inactivation inside phagocytes.
Survival of the non-virulent strain in contact with
sole phagocytes was significantly lower compared
with the virulent strain. The non-virulent strain also
showed lower catalase activity. These results suggest
that bacterial inactivation could be due to the
accumulation of hydrogen peroxide, the precursor
of hydroxyl radicals, after decomposition of superoxide
radicals by bacterial SOD. This accumulation
would not take place to such an extent in the virulent
strain, as levels of catalase are higher. The important
role of catalase in the protection against oxidative
damage in P. damselae ssp. piscicida has been pointed
out by Barnes et al. (1999a), who observed that the
addition of exogenous catalase to the medium
protected the bacteria from inactivation by photochemically
generated superoxide anions.
Both virulent and non-virulent strains assayed by
Barnes et al. (1999a) showed high susceptibility to
cell-free generated superoxide radicals. In contrast,
we have observed that a non-virulent strain, EPOY-
8803-II, is significantly more susceptible to killing
by sole phagocytes than a virulent strain (Lg h41/01 ).
Besides the lower catalase activity present in the
non-virulent strain, the lack of a capsule in cells of
EPOY-8803-II could contribute to the high inactivation
rates observed. Thus, the capsule could
protect bacterial cells from oxidative radicals or even
prevent activation of phagocytes (Miller & Britigan
1997; Arijo et al. 1998).
The important role of iron in microbial infections
has been pointed out by several authors
(Miller & Britigan 1997; Weinberg 2000). The
pathogen needs to obtain iron from the host, where
this metal is linked to high-affinity proteins and
iron availability is very low; also, a transition metal
catalyst such as iron plays an important role in the
generation of hydroxyl radicals in vivo. Indeed, at
physiological pH, generation of hydroxyl radical
from hydrogen peroxide and superoxide anions is of
little biological importance unless a metal such as
ferric iron is present (Haber–Weiss reaction) (Miller
& Britigan 1997). Photobacterium damselae ssp.
piscicida is more susceptible to killing by sole
phagocytes when bacterial cells have been cultured
under iron-depleted conditions. This could be due
to the lower levels of catalase detected in both the
virulent and avirulent cells, the lowest rates corresponding
to strain EPOY-8803-II. Thus, although
the presence of iron in environments where superoxide
and hydrogen peroxide are generated, such as
in phagocytes, may promote the generation of
highly toxic hydroxyl radicals, it is also true that
bacteria require iron for growth and replication and
synthesize SOD and catalase to deal with the
oxidizing anions. Thus, the ability to obtain iron
from the host seems to be crucial for P. damselae
ssp. piscicida. Indeed, it has been demonstrated that
immune-activated macrophages modify intracellular
distribution and dampen iron influx in order to
diminish iron availability for invaders (Weinberg
2000).
Photobacterium damselae ssp. piscicida posses a
high-affinity iron uptake system (Magariños et al.
1994; Naka, Hirono & Aoki 2005). However,
despite its ability to obtain iron from high-affinity
systems, several authors have reported that cells
grown under iron-limited conditions have a
reduced amount of capsular material covering the
cells (Do Vale, Ellis & Silva 2001). These cells with
reduced capsule would be more susceptible to
phagocytosis and oxidative stress. Our results show
that iron plays an important role in survival of
P. damselae ssp. piscicida in contact with sole
phagocytes; whether this is attributable to its
contribution to capsular material or SOD and
catalase synthesis by the bacterium needs to be
investigated.
In conclusion, we have shown that P. damselae
ssp. piscicida is able to survive in contact with sole
phagocytes, survival rates being higher for a virulent
strain. The increased levels of catalase activity
detected in the virulent strain indicate a possible
role for this enzyme in bacterial survival.
Acknowledgements
P. Díaz-Rosales thanks the Ministerio Español de
Educación y Ciencia for a F.P.U. scholarship. This
research has been supported in part by the Research
Project AGL-2002-01488 and PETRI 95-0657.01.
Ó 2006
Blackwell Publishing Ltd
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