| Aeromonas hydrophila is a Gram-negative bacterium that is pathogenic in fish ,causing motile aeromonad septicaemia.lt has been speculated that A. hydrophila virulence could involve several extracellular enzymes including proteases,hemolysins,and enterotoxins,and it can enter (invade) culture cells, and survive as an intracellular parasite.In this work, the invasion pathway and the extracellular protease(ECPase) of a fish isolate in our country was studied.The host-pathogen interaction and sinal trasduction pathway were studied by screening sinal trasduction inhibitors using HEp-2 cells and a virulent strain,AhJ-1.Internalization of AhJ-1 involoved microfilaments and protein tyrosine kinase since cytochalasin D (an inhibitor of microfilament polymerization) and genistein (an inhibitor of protein tyrosine kinase) prevented internalization. Staurosporine(a protein kinase C inhibitor) and Sodium orthovanadate (a protein tyrosine phosphatase inhibitor) accelerated internalization of AhJ-1 into HEp-2 cells. GFP marked AhJ-lwas used to study the invasion pathway in vivo (Carassius auratus). The fish was challenged in a bath way with the following dealed: (1) fish with artificial wounds; (2) fish with a reduced epidernal mucus layer caused by remover of the mucus layer on two occasions by a swabbing procedure;(3) a control group of untreated fish. Twenty-four hours post the bath challenge,the number of the bacteria in kidney, muscle,blood and the liver was higher in the experiment groups. This show the wound and the skin may be the ways of Aeromonas hydrophila invasion.The optimal medium for ECPase productionwas as follows: 5 g- L-1 sucrose, 5 g - L-1 tryptose, 0.02 mol/L KC1, 0.06 mol/L K2HPO4 and pH 7.5. In this case, Ah J-l was culturedat 28 C for 65 hours on a shaker with a speed of 150 r '.min'1, the ECPase production could reach 20.8 U - mL-1.The pathogenicity of ECPase was study using Carrassius auratus ibelio injected intraperitioneally or intramuscularly, The injected fish out came the similar characters to natural diseased ones, and their typical symptoms were as follows: body surface congested, anus swollen and red with inflated abdomen, and a great quantity of ascites in abdominal cavity. ECPase could cause an alterative pathological changes in fish live and kidney .And another study was conducted to screen the protease-dificient mutant by Tn916 transposon.Unlike the parent strain, the growth rate and serum resistance of mutant strain MJ-1 was decreased significantly, and it did not produce lesions in Carrassius auratus ibebio after intramuscular injection with 5 X106CFU germs. The 50% lethal dose of MJ-1 for the fish was more than 108CFU.In order to investigate the relationship of the structure and function of extracellular protease ,The effects of protein modification reagents on the Aeromonas hydrophila extracellular protease ECPase54 activity have been studied. The protease was not affected by EDC,PMSF, 2,3-Diacetyl and Acetic anhydride modification , indicating that Carboxyl groups, Serine residues Guanidine groups and Amino groups were non-essential to the enzyme activity. The enzyme activity was significantly decreased after DEPC, NBS, N-AI, 2-ME modification and was slightly decreased after PCMB modification .These results indicated that the histidine residues , tryptophane residues, tyrosine residues and disulfides groups seemed to be essential to catalytic activity or located at the active site of ECPase54.The PCR products of a serine protease of AhJ-1 were cloned into pGEM-T vector and sequenced. The sequence was compared with the corresponding regions of the GenBank strains and analyzed by DNAstar software .The nucleotide sequence showed 87% homology to the sequence of Ahe2 gene .The nucleotide sequence was predicted to encode a 343-aa protein with the molecular weight of 35700 and with highly antigenic.Based on the published 16S rDNA gene sequence of Aeromonas spp and aerolysingene sequence of Aeromonas hydrophila, the synthetic oligonucleotide primers were used in a p...
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