Monday, 10 February 2014

Bacterial heat shock proteins? Inducing invertebrate immune responses?!

   Previous posts discussing infection management in aquaculture have referred to the paradigm that invertebrates do not have acquired immune systems like us vertebrates. Without antibodies, how does the invertebrate immune system rely completely on innate (non-specific) defences? This study details an interesting mechanism of how the innate immune system responds to pathogens.
   Litopenaeus vannamei is a shrimp widely devoured by Homo sapiens and is assumed to have an innate immune system, defending the arthropod using melanisation, hemolymph clotting, foreign particle trapping and good old phagocytosis. The hemolymph enzyme transglutaminase (TGase) is instrumental in all of these processes and inhibiting its production in shrimp results in bacteria rich hemolymph and very sick crustaceans. Hemocytes produce prophenoloxidase (proPOase), the precursor to another important immune enzyme, phenoloxidase (POase). Once activated, PO facilitates phenol oxidation to antimicrobial quinones, which can also polymerize into melanin to encapsulate pathogens. Basically, TGase and proPO are what makes the crustacean innate immune system tick.
   As you know, heat shock proteins (HSPs) are a highly multifunctional family of proteins used by all organisms. The family HSP70 is the most highly conserved and studied; recently it has been flagged as a notable regulator in the initial innate immune system response, improving POase activity in Artemia infected with Vibrio. But such studies are controversial; many have used a recombinant HSP70 extracted from E. coli called DnaK, which may have been contaminated with endotoxins able to activate the immune system in a similar way.
   This study aimed to determine whether or not HSP70 homologue DnaK induces immune response in L. vannamei, by measuring changes in the expression of proPOase and TGase genes in response to an injection of either recombinant HSP70 (DnaK) from E. coli or a contaminant-free control of chemically synthesised HSP70 (synDnaK). All shrimp used were specific pathogen free (SPF) and given a high and a low dose injection. Gene expression was measured by synthesising cDNA from isolated RNA, then amplifying it by quantitative PCR with TGase and proPOase primers. TGase gene expression increased very significantly in response to low and high doses of DnaK, low doses of synDnaK, but did not change for high dose synDnaK. Expression of a proPOase gene increased significantly for low and high doses of DnaK; a similar pattern was observed for synDnaK. DnaK contamination was measured also, showing endotoxin levels much lower than concentrations found to induce crustacean immune response in other studies.
Recombinant DnaK and synthetic DnaK induced similar patterns of immune response, so contaminants are not the only contributors to stimulation of proPOase and TGase up-regulation. A novel mechanism for TGase and proPOase increase in response to pathogens has been demonstrated. Bizarrely, lower injections of both treatments induced stronger responses; this inverted dose-response relationship points to an unknown mechanism of immune system inhibition by DnaK. In conclusion, this study proposes the hypothesis that bacterial derived HSP70 acts as an alarm signal, activating host TGase and proPOase genes. I wonder how this response compares between organisms with only innate immune system and those with both acquired and innate immune system; is it stronger in the former because it is the main immune response? When pathogens initially colonise a host, they are probably exposed to a sudden range of stresses and therefore increase HSP production. This could be why the innate immune system has evolved to respond to HSP, because high bacterial HSP levels coincide with early infection.

Hu, B., Phuoc, L. H., Sorgeloos, P., & Bossier, P. (2014). Bacterial HSP70 (DnaK) is an efficient immune stimulator in Litopenaeus vannameiAquaculture418, 87-93.


  1. Very interesting summary. Is interesting following on from my shrimp based paper which talks of the quasi-immune response (immune-like system) as a basis for allowing testing into vaccines.

    Clearly this paper backs up this possibility, detailing a possible method for inducing a response. Conclusion in which Bacterial produced HSP is very interesting, the immune-like system seeing it as a sign in which external pathogens may have colonised.

    When you say that contamination of DnaK was less contaminated than in other experiments that have produced an immune response, does this mean that it was still contaminated? I imagine that is where the synDnaK comes in to control for contamination?

    1. Immune system interactions with microbes is a great topic and one I will post about again. I bet invertebrate immune systems do have their own way of remembering pathogens.
      They say their DnaK endotoxin levels were less than the concentrations of pure endotoxins used in other studies to induce immune responses. But they were still contaminated, which is the reason for the uncontaminated control.