Ingestion of bacteria overproducing DnaK attenuates Vibrio infection of Artemia franciscana larvae

Abstract

Feeding of bacterially encapsulated heat shock proteins (Hsps) to invertebrates is a novel way to limit Vibrio infection. As an example, ingestion of Escherichia coli overproducing prokaryotic Hsps significantly improves survival of gnotobiotically cultured Artemia larvae upon challenge with pathogenic Vibrio campbellii. The relationship between Hsp accumulation and enhanced resistance to infection may involve DnaK, the prokaryotic equivalent to Hsp70, a major molecular chaperone in eukaryotic cells. In support of this proposal, heat-stressed bacterial strains LVS 2 (Bacillus sp.), LVS 3 (Aeromonas hydrophila), LVS 8 (Vibrio sp.), GR 8 (Cytophaga sp.), and GR 10 (Roseobacter sp.) were shown in this work to be more effective than nonheated bacteria in protecting gnotobiotic Artemia larvae against V. campbellii challenge. Immunoprobing of Western blots and quantification by enzymelinked immunosorbent assay revealed that the amount of DnaK in bacteria and their ability to enhance larval resistance to infection by V. campbellii are correlated. Although the function of DnaK is uncertain, it may improve tolerance to V. campbellii via immune stimulation, a possibility of significance from a fundamental perspective and also because it could be applied in aquaculture, a major method of food production.

Efficacy of heterologous and homologous heat shock protein 70s as protective agents to Artemia franciscana challenged with Vibrio campbellii

“…1B and D). Similarly, DnaK was also upregulated in arabinose-induced YS2 strain, yielding a polypeptide of approximately 83 kDa visible on Western blot probed with antibody to DnaK (result not shown, see Sung et al [13]). The increase in molecular mass of approximately 13 kDa in the induced A native and YS2 strains, as compared with the normal mass of Hsp70 or DnaK, was because of the amino-terminal incorporation of thioredoxin encoded by the TOPO Ò cloning vector.…”

“…Subsequently, L-arabinose at 0.5 mg ml À1 , which gave the best induction in the dose-response experiment, was tested for different time intervals (1, 2, 3 and 4 h) [18]. Maximum production of Artemia Hsp70 in A native cells was obtained at 0.5 mg ml À1 L-arabinose for 4 h, as obtained for YS2 cells overproducing DnaK [13]. The respective bacteria after induction were transferred to sterile tubes, centrifuged at 2200 Â g for 15 min at 28 C, suspended in filtered (0.2 mm) autoclaved sea water, and fed immediately to Artemia larvae.…”

“…1B, control). Control strains, such as, induced or non-induced YS1, do not produce a protein recognised by monoclonal antibody 8E2/2, raised in mouse to DnaK (result not shown, see Sung et al [13]). Already with 0.5 mg ml À1 arabinose and 1 h induction, maximal induction could be obtained.…”

“…Strains YS2 (which expresses the 70-kDa bacterial Hsp, DnaK, upon arabinose induction) and YS1 (which does not express DnaK upon arabinose induction) were described previously [13].…”

“…Recently, our laboratory has explored the hypothesis that Hsps control disease in aquaculture and found that induction of Hsp in Artemia (eukaryotic Hsp70) through a non-lethal heat shock is associated with protection against virulent Vibrios [12]. In another study [13], the same authors observed that ingestion of Escherichia coli overproducing DnaK (prokaryotic equivalent to Hsp70) significantly improved the survival of gnotobiotically cultured Artemia upon challenge with V. campbellii. These studies suggest that Hsp70 (either prokaryotic or eukaryotic) conferred protection to Vibriochallenged Artemia.…”