Extracellular proteases and possible disease related virulence mechanisms of two marine bacteria implicated in an opportunistic bacterial infection of Nephrops norvegicus

Abstract

The extracellular products (ECP) secreted by two strains of gram-negative bacteria isolated from Nephrops norvegicus exhibiting signs of an opportunistic bacterial infection were investigated with the objective of understanding their role in the spoilage of host muscle tissue and identifying disease related virulence mechanisms. ECP from Vibrio sp . demonstrated no proteolytic activity. ECP from Pseudoalteromonas sp. (isolate N10) degraded several substrates, including azocasein and host muscle tissue. Proteolytic activity increased with temperature. Substrate-impregnated sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the effect of the isolates’ ECP on the molecular weight of proteins derived from abdominal muscle tissue revealed that the ECP of Pseudoalteromonas sp. selectively degraded the myosin heavy chain, troponin-T, troponin-I, paramyosin and several unidentified muscle proteins approximately 110 kDa in size. Topomyosin was also reduced in quantity. Degradation of SDS–PAGE gels impregnated with host muscle proteins, by the ECP of Pseudoalteromonas sp. revealed 3 zones of proteolysis, with estimated molecular weights between 100 and 30 kDa, indicating multiple proteases in the ECP. Through the API ZYM system, both isolates demonstrated strong leucine arylamidase activity, with the Vibrio sp. showing strong acid phosphatase activity. These enzymes have been identified as disease related virulence mechanisms in other bacterial pathogens. There is likely a complex pathway to the final condition, involving virulence factors of other species and the stresses involved in capture and transport.