Pinctada fucata martensii (P.f. Martensii) is a bivalve mollusk, and as it lacks a specific immune system its defense against pathogens relies entirely on cellular immunity and humoral immune factors. Various diseases have occurred more frequently in recent years, resulting in increased mortality of P.f. Martensii. Improving the disease resistance of P.f. Martensii is therefore an urgent concern that needs to be addressed. In this study, multi-step high performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify proteins with antibacterial activity from the serum of P.f. Martensii.
Oysters were challenged with a mixture of heat-killed Escherichia coli and Micrococcus luteus injected into the adductor muscle with a needle, and then returned to sea water. Hemolymph was collected and pretreated using the methods described by Mitta. First, plasma samples were fully eluted using a SunfireTM prep C18 column at a flow rate of 1 mL/min. Next, active fractions were loaded onto an analytical Vydac C18 RP HPLC column, eluted with acetonitrile of different gradients. The fractions with peaks were then manually collected and tested for antibacterial activity by freeze drying.
Hemolymph was collected from P.f. Martensii. Cells and debris were removed by centrifugation and the supernatant was loaded on to a Sunfire™ prep C18 column and eluted with a gradient of 5–60% acetonitrile, yielding the fraction having antibacterial activity. The fractions were subjected to analytical reversed phase HPLC using a Vydac C18 RP HPLC column. One of the purified proteins that eluted at approximately 29 min (Fig. 1, labelled C3) had bacteriostatic activity. The antibacterial activity of PmAMP-1 was determined using the microplate reader method. The growth of M. luteus (Fig. 2A) and E. coli (Fig. 2B) was significantly inhibited following the addition of PmAMP-1 (p < 0.05).