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

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RESULTS AND DISCUSSION resist 100 mM of peroxide, a lethal concentration without peroxide pretreatment. In the case of P. damselae subsp. piscicida, it is interesting to note that a significant increase in survival rates of the non-virulent strain was observed when cultures were pulsed with hydrogen peroxide compared to cells cultured until stationary phase. In contrast, this increase has not been observed for the virulent strain, which always showed higher survival regardless of the growth phase, or the pulse with hydrogen peroxide. On the other hand, culture under iron-restricted conditions results in a significant decrease in survival of both virulent and avirulent strains. The fact that this bacterial species contains a ferric catalase (article 1.2., Article section), whose activity is reduced under iron limiting conditions, may explain this result. Superoxide dismutase and catalase are metalloenzymes that can possess different metals in their active centre. Microorganisms capable to produce different superoxide dismutase and catalase isoenzymes, depending on culture conditions, have been described. These culture conditions include oxygen levels, low iron levels or growth until stationary phase (Storz et al., 1990; Privalle and Fridovich, 1992; Crockford et al., 1995; Schnell and Steinman, 1995; Barnes et al., 1996; Polack et al., 1996; St. John and Steinman, 1996; Lynch and Kuramitsu, 2000; Yesilkaya et al., 2000; Geslin et al., 2001; Vattanaviboon and Mongkolsuk, 2001). Results obtained in the present work show that P. damselae subsp. piscicida does not synthetize a new form of superoxide dismutase or catalase when cultured under assayed conditions. Thus, all strains show only one band of superoxide dismutase activity, with similar electrophoretic mobility to ferric superoxide dismutase described by Barnes et al. (1999a). Even oxidative stress, by hydrogen peroxide or paraquat (an oxygenic radical generator) addition, did not induce the synthesis of a different isoenzyme, unlike CuZnSOD and MnSOD described in Escherichia coli (Hassan and Fridovich, 1977; Privalle and Fridovich, 1992; Benov and Fridovich, 1994; Geslin et al., 2001), MnSOD in A. salmonicida (Barnes et al., 1996; Barnes et al., 1999b), Pseudomonas aeruginosa (Polack et al., 1996) and Streptococcus pneumoniae (Yesilkaya et al., 2000). Neither restrictive iron conditions induce MnSOD synthesis (Privalle and Fridovich, 1992; Barnes et al., 1999b). Although further studies would be 98
RESULTS AND DISCUSSION necessary, this lack of a new superoxide dismutase induction could be due to the presence of only one sod gene, sodB, encoding ferric superoxide dismutase (Lynch and Kuramitsu, 2000). In the same way all strains, under all assayed culture conditions, show only one band of catalase activity, with similar electrophoretic mobility to the band described by Barnes et al. (1999a). Treatment of catalase gels with inhibitors indicates that this bacterium contains an iron-cofactored enzyme, because catalases with manganese retain its activity after treatment with azida and cyanide, but they are inhibited with mercuric chloride (Kono and Fridovich, 1983; Allgood and Perry, 1986; Barnes et al., 1999b). Despite none assayed culture condition induced synthesis of more than one superoxide dismutase or catalase isoenzyme, differences in the intensity of the bands and activity levels, after spectrophotometrically quantification, are observed. These results are in agreement with those obtained by Barnes et al. (1999a), who also detected differences in cultures carried out under iron replete and depleted conditions and highand low- aerated broths. The quantification of both superoxide dismutase and catalase activities carried out in this study corroborated that different band intensities corresponded to variations in the levels of activity. The lowest levels of superoxide dismutase activity and catalase are detected in bacteria grown under iron-restricted conditions, attributable to the ferric nature of P. damselae subsp. piscicida superoxide dismutase and catalase. Under iron-limiting conditions the virulent strain shows higher activity levels of superoxide dismutase and catalase, than the non virulent strain. This result indicates the relevant role of iron uptake mechanisms for superoxide dismutase and catalase activity. Thus, a microorganism capable to obtain iron from the host would be able to express higher levels of antioxidant enzymes and could decompose superoxide and hydrogen peroxide radicals generated by the host. The importance of superoxide dismutase as antioxidant enzyme and its contribution to bacterial virulence have been demonstrated in several bacterial species such as Listeria monocytogenes (Welch et al., 1979), Shigella flexneri (Franzon et al., 1990) or A. salmonicida (Barnes et al., 1999a), however its role in P. damselae subsp. piscicida is not very clear and in the 99
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FACULTAD DE CIENCIAS DEPARTAMENTO D
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FACULTAD DE CIENCIAS DEPARTAMENTO D
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Los ensayos que constituyen esta Te
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- Díaz-Rosales, P, Arijo, S, Chabr
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Sabe esperar, aguarda que la marea
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ÍNDICE / INDEX Papel de las activi
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ÍNDICE / INDEX 2. Photobacterium d
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RESUMEN Photobacterium damselae sub
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INTRODUCCIÓN 1. LA ACUICULTURA. EL
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INTRODUCCIÓN saxatilis) (Hawke et
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INTRODUCCIÓN 2.2. SINTOMATOLOGÍA
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INTRODUCCIÓN las epidemias surgida
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INTRODUCCIÓN et al., 1997). En P.
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INTRODUCCIÓN 3.1. ESTALLIDO RESPIR
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INTRODUCCIÓN radicales generados e
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INTRODUCCIÓN Tabla 2 Ejemplos de m
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INTRODUCCIÓN de parasitismo en el
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INTRODUCCIÓN razón de la búsqued
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INTRODUCCIÓN araquidónico, están
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INTRODUCCIÓN simplificar la admini
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INTRODUCCIÓN sino también cuando
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INTRODUCCIÓN La necesidad de mejor
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OBJETIVOS El trabajo planteado en e
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MATERIA L Y MÉTODOS La metodologí
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RESULTADO S Y DISCUSIÓN El primer
Page 69 and 70: RESULTADO S Y DISCUSIÓN 1996; Lync
Page 71 and 72: RESULTADO S Y DISCUSIÓN Una vez de
Page 73 and 74: RESULTADO S Y DISCUSIÓN inmunoesti
Page 75 and 76: RESULTADO S Y DISCUSIÓN cruentum s
Page 77 and 78: RESULTADO S Y DISCUSIÓN permiten c
Page 79: RESULTADO S Y DISCUSIÓN harían fa
Page 83 and 84: CONCLUSIONES Tras los estudios real
Page 85: FACULTAD DE CIENCIAS DEPARTAMENTO D
Page 89: ABSTRACT Photobacterium damselae su
Page 93 and 94: INTRODUCTION 1. AQUACULTURE. THE CU
Page 95 and 96: INTRODUCTION With regard to Solea s
Page 97 and 98: INTRODUCTION 3.1. RESPIRATORY BURST
Page 99 and 100: INTRODUCTION Some catalases have al
Page 101 and 102: INTRODUCTION pathogens, study of th
Page 103 and 104: INTRODUCTION increase their bacteri
Page 105 and 106: INTRODUCTION 4.3.1. Porphyridium cr
Page 107 and 108: INTRODUCTION Different mechanisms h
Page 109: A I M S
Page 113: M A T E R I A L S A N D M E T H O D
Page 117: R E S U L T S A N D D I S C U S S I
Page 122 and 123: RESULTS AND DISCUSSION present work
Page 124 and 125: RESULTS AND DISCUSSION reason, once
Page 126 and 127: RESULTS AND DISCUSSION great number
Page 128 and 129: RESULTS AND DISCUSSION dominant spe
Page 131 and 132: CONCLUSIONS Studies carried out on
Page 133: R R E F E R E N C I A S E F E R E N
Page 136 and 137: REFERENCIAS / REFEREN CES Aoki, T,
Page 138 and 139: REFERENCIAS / REFEREN CES Bell, MV,
Page 140 and 141: REFERENCIAS / REFEREN CES Dalmo, RA
Page 142 and 143: REFERENCIAS / REFEREN CES Hamaguchi
Page 144 and 145: REFERENCIAS / REFEREN CES Kono, Y &
Page 146 and 147: REFERENCIAS / REFEREN CES Magariño
Page 148 and 149: REFERENCIAS / REFEREN CES Ortuño,
Page 150 and 151: REFERENCIAS / REFEREN CES Salinas,
Page 152 and 153: REFERENCIAS / REFEREN CES Thompson,
Page 155: S E C C I Ó N D E A R T Í C U L O
Page 159: A RTÍCULO 1.1. A RTICLE 1.1.
Page 162 and 163: Journal of Fish Diseases 2003, 26,
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Journal of Fish Diseases 2006, 29,
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Page 175 and 176:
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A RTÍCULO 2.1. A RTICLE 2.1.
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Abstract The stimulatory effect of
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alga Porphyridium cruentum. For thi
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Feeding assays were carried out wit
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(10 8 bacterias ml -1 ) and used to
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are in agreement with the data repo
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unvaccinated fish. However, the ser
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immune response of gilthead seabrea
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[27] Duncan, PL and Klesius, PH. Ef
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(Macrogard) and alginic acid (Ergos
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[66] Salinas, I, Díaz-Rosales, P,
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Figure 1a 3 2.5 Stimulation index 2
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Figure 1c Stimulation index 3 2.5 2
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Figure 2b Stimulation index 3 2.5 2
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Effect of the extracellular polysac
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1. Introduction Solea senegalensis
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On the other hand, β-1,3-glucan fr
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eaction was tested by adding supero
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Intraperitoneal inoculation of the
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defense mechanisms and protective i
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phagocytes from sole (Solea senegal
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Figure 1a Stimulation index 3 2.5 2
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Figure 2 Stimulation index 1.4 1.2
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Effect of dietary administration of
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1. Introduction Senegalese sole cul
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2.2. Fish and experimental design S
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2.5. Challenge with Photobacterium
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the densitometric curves of the pro
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most efficient probiotics for aquac
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acterial diversity only the dominan
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[9] Verschuere, L, Rombaut, G, Sorg
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[28] Burr, G, Gathin, D and Ricke,
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WB, editors. Techniques in Fish Imm
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and host-specific communities of ac
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Table 1 Primer Sequence (5’-3’)
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Figure 1b Stimulation index 2.5 2 1
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Table 2 Primer Control Pdp11 Pdp13
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Figure 3 Pearson correlation [0.0%-
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que perdieras parte de tu tiempo en
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