Priming the prophenoloxidase system of Artemia franciscana by heat shock proteins protects against Vibrio campbellii challenge

Like other invertebrates, the brine shrimp Artemia franciscana relies solely on innate immunity, which by definition lacks adaptive characteristics, to combat against invading pathogens. One of the innate mechanisms is melanisation of bacteria mediated by the activation of the prophenoloxidase (proPO) system. The 70 kDa heat shock proteins (Hsp70) derived from either prokaryote (Escherichia coli) or eukaryote (Artemia), well conserved and immune-dominant molecules, protect Artemia against Vibrio campbellii. However, the molecular mechanisms by which these proteins protect Artemia against Vibrio campbellii infection are unknown. Here we demonstrated that feeding gnotobiotically grown Artemia with either Artemia Hsp70 or the E. coli Hsp70 equivalent DnaK, each overproduced in E. coli, followed by V. campbellii challenge enhanced the proPO system, at both mRNA and protein activity levels. Additionally, the Artemia fed with these proteins survived well in a Vibrio challenge assay. These results indicated that Hsp70s derived from either prokaryotic or eukaryotic sources generate protective immunity in the crustacean Artemia against V. campbellii infection by priming the proPO system. This is apparently the first in vivo report on priming activity of Hsp70 in an invertebrate.     

Poly-beta-hydroxybutyrate-accumulating bacteria protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii

A poly-beta-hydroxybutyrate (PHB)-accumulating enrichment culture was obtained using activated sludge from a polyphosphate-accumulating reactor as inoculum. PHB accumulated by the enrichment culture significantly enhanced the survival of Artemia nauplii, infected with the virulent pathogen Vibrio campbellii LMG 21363. A strain was isolated from the enrichment culture, based on its ability to accumulate PHB, and 16S rRNA gene sequencing of the isolate revealed 99% sequence similarity to Brachymonas denitrificans AS-P1. The isolate, named PHB2, showed good PHB-accumulating activity (up to 32% of the cell dry weight). PHB accumulated by isolate PHB2 was able to protect Artemia completely from the V. campbellii strain. Our data indicate that PHB-accumulating bacteria, such as B. denitrificans PHB2, could be used as an an effective and economically interesting alternative strategy to control infections in aquaculture.     

Non-lethal heat shock protects gnotobiotic Artemia franciscana larvae against virulent Vibrios

Brine shrimp Artemia were exposed under gnotobiotic conditions to a non-lethal heat shock (NLHS) from 28 to 32, 37 and 40 degrees C. Different recovery periods (2, 6, 12 and 24h) and different heat-exposure times (15, 30, 45 and 60 min) were tested. After these NLHS, Artemia was subsequently challenged with Vibrio. Challenge tests were performed in stressed and unstressed nauplii at concentrations of 10(7) cells ml(-1) of pathogenic bacteria, Vibrio campbellii and Vibrio proteolyticus. A NLHS with an optimal treatment of 37 degrees C for 30 min and a subsequent 6h recovery period resulted in a cross-protection against pathogenic Vibrio. A 100% increase in the larval survival (P < 0.05) was observed. We have also demonstrated by Western blot that a NLHS increases the expression of HSP-70 in heat-shocked (HS) treated animals. This report is the first to reveal a cross protection of a NLHS against deleterious bacterial challenges in living crustaceans. The putative role of heat shock proteins (HSPs) in this process is discussed.     

Exposure of gnotobiotic Artemia franciscana larvae to abiotic stress promotes heat shock protein 70 synthesis and enhances resistance to pathogenic Vibrio campbellii

Larvae of the brine shrimp Artemia franciscana serve as important feed in fish and shellfish larviculture; however, they are subject to bacterial diseases that devastate entire populations and consequently hinder their use in aquaculture. Exposure to abiotic stress was shown previously to shield Artemia larvae against infection by pathogenic Vibrio, with the results suggesting a mechanistic role for heat shock protein 70. In the current report, combined hypothermic/hyperthermic shock followed by recovery at ambient temperature induced Hsp70 synthesis in Artemia larvae. Thermotolerance was also increased as was protection against infection by Vibrio campbellii, the latter indicated by reduced mortality and lower bacterial load in challenge tests. Resistance to Vibrio improved in the face of declining body mass as demonstrated by measurement of ash-free dry weight. Hypothermic stress only and acute osmotic insult did not promote Hsp70 expression and thermotolerance in Artemia larvae nor was resistance to Vibrio challenge augmented. The data support a causal link between Hsp70 accumulation induced by abiotic stress and enhanced resistance to infection by V. campbellii, perhaps via stimulation of the Artemia immune system. This possibility is now under investigation, and the work may reveal fundamental properties of crustacean immunity. Additionally, the findings are important in aquaculture where development of procedures to prevent bacterial infection of feed stock such as Artemia larvae is a priority.     

The bacterial storage compound poly-beta-hydroxybutyrate protects Artemia franciscana from pathogenic Vibrio campbellii

Infections caused by antibiotic-resistant luminescent Vibrios can cause dramatic losses in aquaculture. In this study, the short-chain fatty acid beta-hydroxybutyrate and its polymer poly-beta-hydroxybutyrate were investigated as possible new biocontrol agents. beta-Hydroxybutyrate was shown to completely inhibit the growth of pathogenic Vibrio campbelli at 100 mM. Moreover, the addition of 100 mM of this fatty acid to the culture water of Artemia nauplii infected with the V. campbelli strain significantly increased the survival of the nauplii. As Artemia is a non-selective and particle filter feeder, we also investigated whether poly-beta-hydroxybutyrate particles could be used to protect Artemia from the pathogenic V. campbellii. The addition of 100 mg l(-1) poly-beta-hydroxybutyrate or more to the Artemia culture water offered a preventive and curative protection from the pathogen as a significantly enhanced survival was noticed. If added as a preventive treatment, a complete protection of infected nauplii (no significant mortality compared with uninfected nauplii) was observed at 1000 mg l(-1) poly-beta-hydroxybutyrate. Our data indicate that the use of poly-beta-hydroxybutyrate might constitute an ecologically and economically sustainable alternative strategy to fight infections in aquaculture.     

Quorum sensing-disrupting brominated furanones protect the gnotobiotic brine shrimp Artemia franciscana from pathogenic Vibrio harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates

Autoinducer 2 (AI-2) quorum sensing was shown before to regulate the virulence of Vibrio harveyi towards the brine shrimp Artemia franciscana. In this study, several different pathogenic V. harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates were shown to produce AI-2. Furthermore, disruption of AI-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic Artemia from the pathogenic isolates in in vivo challenge tests.     

The heat shock response of adult Artemia franciscana

1. We studied responses of adult brine shrimp, Artemia franciscana, to high temperature, including LT₅₀ determination, induced thermotolerance (ITT), the Hsp-70 family of stress proteins and protein synthesis before and after heat shock.

2. Adults were grown in laboratory cultures from encysted embryos (cysts) obtained from San Francisco Bay (SF) and much warmer culture ponds in Vietnam (V).

3. Adults from V cysts were more tolerant of high temperatures than those from SF cysts, but this difference essentially disappeared in the second generation of adults.

4. Levels of constitutive Hsc-70 were very low in adults of both groups, but were strongly upregulated by a sublethal heat shock (37°C, 30 min), with V adults showing the greater degree of upregulation. Heat shock also induced Hsp-67, to a greater extent in V compared to SF adults

5. Incorporation of ¹⁴C-leucine into protein did not result in the “classic” heat shock response, possibly due to increased permeability of heat-shocked animals to the tracer.

Quorum Sensing-Disrupting Brominated Furanones Protect the Gnotobiotic Brine Shrimp Artemia franciscana from Pathogenic Vibrio harveyi, Vibrio campbellii, and Vibrio parahaemolyticus Isolates

Autoinducer 2 (AI-2) quorum sensing was shown before to regulate the virulence of Vibrio harveyi towards the brine shrimp Artemia franciscana. In this study, several different pathogenic V. harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates were shown to produce AI-2. Furthermore, disruption of AI-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic Artemia from the pathogenic isolates in in vivo challenge tests.     

Aerobic heat shock activates trehalose synthesis in embryos of Artemia franciscana.

Encysted embryos (cysts) of the brine shrimp, Artemia franciscana, contain large amounts of trehalose which they use as a major substrate for energy metabolism and biosynthesis for development under aerobic conditions at 25 degrees C. When cysts are placed at 42 degrees C (heat shock) these pathways stop, and the cysts re-synthesize the trehalose that was utilized during the previous incubation at 25 degrees C. Glycogen and glycerol, produced from trehalose at 25 degrees C, appear to be substrates for trehalose synthesis during heat shock. Anoxia prevents trehalose synthesis in cysts undergoing heat shock. These results are consistent with the view that trehalose may play a protective role in cells exposed to heat shock, and other environmental insults, in addition to being a storage form of energy and organic carbon for development.     

Functional analysis of a small heat shock/alpha-crystallin protein from Artemia franciscana. Oligomerization and thermotolerance.

Oviparously developing embryos of the brine shrimp, Artemia franciscana, synthesize abundant quantities of a small heat shock/alpha-crystallin protein, termed p26. Wild-type p26 functions as a molecular chaperone in vitro and is thought to help encysted Artemia embryos survive severe physiological stress encountered during diapause and anoxia. Full-length and truncated p26 cDNA derivatives were generated by PCR amplification of p26-3-6-3, then cloned in either pET21(+) or pRSETC and expressed in Escherichia coli BL21(DE3). All constructs gave a polypeptide detectable on Western blots with either p26 specific antibody, or with antibody to the His(6) epitope tag encoded by pRSETC. Full-length p26 in cell-free extracts of E. coli was about equal in mass to that found in Artemia embryos, but p26 lacking N- and C-terminal residues remained either as monomers or small multimers. All p26 constructs conferred thermotolerance on transformed E. coli, although not all formed oligomers, and cells expressing N-terminal truncated derivatives of p26 were more heat resistant than bacteria expressing p26 with C-terminal deletions. The C-terminal extension of p26 is seemingly more important for thermotolerance than is the N-terminus, and p26 protects E. coli against heat shock when oligomer size and protein concentration are low. The findings have important implications for understanding the functional mechanisms of small heat shock/alpha-crystallin proteins.     

The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi

Aims: To study the potential biocontrol activity of bioflocs technology. Methods and Results: Glycerol‐grown bioflocs were investigated for their antimicrobial and antipathogenic properties against the opportunistic pathogen Vibrio harveyi. The bioflocs did not produce growth‐inhibitory substances. However, bioflocs and biofloc supernatants decreased quorum sensing‐regulated bioluminescence of V. harveyi. This suggested that the bioflocs had biocontrol activity against this pathogen because quorum sensing regulates virulence of vibrios towards different hosts. Interestingly, the addition of live bioflocs significantly increased the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged to V. harveyi. Conclusions: Bioflocs grown on glycerol as carbon source inhibit quorum sensing‐regulated bioluminescence in V. harveyi and protect brine shrimp larvae from vibriosis. Significance and Impact of the Study: The results presented in this study indicate that in addition to water quality control and in situ feed production, bioflocs technology could help in controlling bacterial infections within the aquaculture pond.     

Truncation attenuates molecular chaperoning and apoptosis inhibition by p26, a small heat shock protein from Artemia franciscana

The small heat shock proteins (sHSPs), which prevent irreversible protein denaturation and inhibit apoptosis, consist of an amino-terminus, the canonical α-crystallin domain, and a carboxy-terminal extension. It remains difficult, however, to define sHSP structure-function relationships and with this in mind p26, an sHSP from the crustacean Artemia franciscana, was truncated by deletion mutagenesis. Wild-type p26 cDNA and three truncated variants inserted into the eukaryotic expression vector pcDNA3.1/HisC were used to generate stably transfected 293H cells. p26 shielded transfected cells against death upon exposure to heat and oxidative stress. Truncation reduced chaperone activity, with cells synthesizing the p26 α-crystallin domain being the least resistant. Wild-type p26 inhibited apoptosis in transfected cells, with protection against oxidation-generated apoptosis being more effective than that against heat-induced apoptosis. Truncation reduced p26 apoptotic inhibitory activity, with the α-crystallin domain again being the least effective. The results show that a crustacean sHSP functions effectively in mammalian cells, demonstrating interchangeability of these proteins between distantly related organisms and indicating similarities in their mechanisms of action. Moreover, maximal activity was observed for full-length p26, indicating that structural elements required for chaperone activity and apoptosis inhibition reside throughout the protein.     

Oligomerization, chaperone activity, and nuclear localization of p26, a small heat shock protein from Artemia franciscana

Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the last characterized by profound metabolic dormancy and extreme stress resistance. Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development. To understand better the role of p26 in Artemia embryos, the structural and functional characteristics of full-length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined. p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization. When produced in either prokaryotic or eukaryotic cells the p26 alpha-crystallin domain consisting of amino acid residues 61-152 existed predominantly as monomers, and p26 variants lacking the amino-terminal domain but with intact carboxyl-terminal extensions were mainly monomers and dimers. The amino terminus was, therefore, required for efficient dimer formation. Assembly of higher order oligomers was enhanced by the carboxyl-terminal extension, although removing the 10 carboxyl-terminal residues had relatively little effect on oligomerization and chaperoning. Full-length and carboxyl-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells; however, variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered the nuclei. A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress. However, when examined in Artemia, the p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer dissociation.     

Molecular cloning and expression analysis of heat-shock protein 70 in orange-spotted grouper Epinephelus coioides following heat shock and Vibrio alginolyticus challenge

In this study, the complementary (c)DNA encoding heat-shock protein 70 (Hsp70) of orange-spotted grouper Epinephelus coioides (OsgHsp70) was cloned. OsgHsp70 was 2206 bp and encoded 652 amino acids with predicted molecular mass of 70·89 kDa and theoretical isoelectric point of 5·48. Three Hsp70 family signatures, bipartite nuclear localization signal sequence (NLS) and cytoplasmic characteristic motif (EEVD) were observed in the OsgHsp70, which shared high similarity in amino-acid sequences with the Hsp70 gene of other vertebrates. The results indicated that the OsgHsp70 is a member of the heat-shock protein 70 family. The Hsp70 messenger (m)RNA expressions were quantified by real-time PCR following heat shock, bacterial infection and immunization with formalin-killed Vibrio alginolyticus, a kind of bacterial pathogen that causes septicaemia. Hsp70 mRNA expression in gill, kidney, spleen, thymus gland, muscle and total-blood samples increased at first and then decreased gradually following heat shock. A similar time-dependent pattern was observed following V. alginolyticus pathogen challenge, in which Hsp70 mRNA expression peaked at 24 h after live bacterial infection and 3 days after dead bacterial vaccination. The results indicated that the Hsp70 gene was inducible and involved in the fish immune response.     

Genetic evidence for a protective role for heat shock factor 1 and heat shock protein 70 against colitis

Inflammatory bowel disease (IBD) involves infiltration of leukocytes into intestinal tissue, resulting in intestinal damage induced by reactive oxygen species (ROS). Pro-inflammatory cytokines and cell adhesion molecules (CAMs) play important roles in this infiltration of leukocytes. The roles of heat shock factor 1 (HSF1) and heat shock proteins (HSPs) in the development of IBD are unclear. In this study, we examined the roles of HSF1 and HSPs in an animal model of IBD, dextran sulfate sodium (DSS)-induced colitis. The colitis worsened or was ameliorated in HSF1-null mice or transgenic mice expressing HSP70 (or HSF1), respectively. Administration of DSS up-regulated the expression of HSP70 in colonic tissues in an HSF1-dependent manner. Expression of pro-inflammatory cytokines and CAMs and the level of cell death observed in colonic tissues were increased or decreased in DSS-treated HSF1-null mice or transgenic mice expressing HSP70, respectively, relative to control wild-type mice. Relative to macrophages from control wild-type mice, macrophages prepared from HSF1-null mice or transgenic mice expressing HSP70 displayed enhanced or reduced activity, respectively, for the generation of pro-inflammatory cytokines in response to lipopolysaccharide stimulation. Suppression of HSF1 or HSP70 expression in vitro stimulated lipopolysaccharide-induced up-regulation of CAMs or ROS-induced cell death, respectively. This study provides the first genetic evidence that HSF1 and HSP70 play a role in protecting against DSS-induced colitis. Furthermore, this protective role seems to involve various mechanisms, such as suppression of expression of pro-inflammatory cytokines and CAMs and ROS-induced cell death.     

Characterization of novel sequence motifs within N- and C-terminal extensions of p26, a small heat shock protein from Artemia franciscana

The small heat shock proteins function as molecular chaperones, an activity often requiring reversible oligomerization and which protects against irreversible protein denaturation. An abundantly produced small heat shock protein termed p26 is thought to contribute to the remarkable stress resistance exhibited by encysted embryos of the crustacean, Artemia franciscana. Three novel sequence motifs termed G, R and TS were individually deleted from p26 by site-directed mutagenesis. G encompasses residues G8-G29, a glycine-enriched region, and R includes residues R36-R45, an arginine-enhanced sequence, both in the amino terminus. TS, composed of residues T169-T186, resides in the carboxy-extension and is augmented in threonine and serine. Deletion of R had more influence than removal of G on p26 oligomerization and chaperoning, the latter determined by thermotolerance induction in Escherichia coli, protection of insulin and citrate synthase from dithiothreitol- and heat-induced aggregation, respectively, and preservation of citrate synthase activity upon heating. Oligomerization of the TS and R variants was similar, but the TS deletion was slightly more effective than R as a chaperone. The extent of p26 structural perturbation introduced by internal deletions, including modification of intrinsic fluorescence, 1-anilino-8-naphthalene-sulphonate binding and secondary structure, paralleled reductions in oligomerization and chaperoning. Three-dimensional modeling of p26 based on wheat Hsp16.9 crystal structure indicated many similarities between the two proteins, including peptide loops associated with secondary structure elements. Loop 1 of p26 was deleted in the G variant with minimal effect on oligomerization and chaperoning, whereas loop 3, containing beta-strand 6 was smaller than the corresponding loop in Hsp16.9, which may influence p26 function.     

Heat shock protein 70 or heat shock protein 27 overexpressed in human endothelial cells during posthypoxic reoxygenation can protect from delayed apoptosis

Overexpression of heat shock protein (Hsp) 70 and Hsp27 in vivo was proclaimed as a potential tool in therapy of ischemia-reperfusion injury. However, it was so far not known whether these Hsps can beneficially act when increased in cells just at the stage of postischemic reperfusion. This issue was examined in a model of ischemia-reperfusion stress when cultures of endothelial cells (EC) from human umbilical vein were infected with virus-based vectors expressing Hsp70 or Hsp27, or Hsp56, or green fluorescent protein (GFP) and exposed to 20 hours of hypoxia followed by reoxygenation. The infection was performed either 10 hours before hypoxia or immediately after hypoxia, or at different time points of reoxygenation. Only low cell death was detected during hypoxia, but later, up to 40% of the treated cells died via caspase-dependent apoptosis between 6 and 12 hours of reoxygenation. The percentage of apoptotic cells was 1.6- to 3-fold greater in Hsp56- and GFP-infected EC than in Hsp70- or Hsp27-infected EC. The last 2 groups exhibited a lesser extent of procaspase-9 and procaspase-3 activation within 6-9 hours of reoxygenation. The cytoprotective effects of overexpressed Hsp70 and Hsp27 were observed not only in the case of infection before hypoxia but also when EC were infected at the start of reoxygenation or 1-2 hours later. An increase in the Hsp70 and Hsp27 levels in infected EC correlated well with their resistance to apoptosis under reoxygenation. These findings suggest that overexpression of Hsp70 or Hsp27, if it occurs in the involved cells at the early stage of postischemic reperfusion, can still be cytoprotective.     

Influence of different yeast cell-wall mutants on performance and protection against pathogenic bacteria (Vibrio campbellii) in gnotobiotically-grown Artemia

A selection of isogenic yeast strains (with deletion for genes involved in cell-wall synthesis) was used to evaluate their nutritional and immunostimulatory characteristics for gnotobiotically-grown Artemia. In the first set of experiments the nutritional value of isogenic yeast strains (effected in mannoproteins, glucan, chitin and cell-wall bound protein synthesis) for gnotobiotically-grown Artemia was studied. Yeast cell-wall mutants were always better feed for Artemia than the isogenic wild type mainly because they supported a higher survival but not a stronger individual growth. The difference in Artemia performance between WT and mutants feeding was reduced when stationary-phase grown cells were used. These results suggest that any mutation affecting the yeast cell-wall make-up is sufficient to improve the digestibility in Artemia. The second set of experiments, investigates the use of a small amount of yeast cells in gnotobiotic Artemia to overcome pathogenicity of Vibrio campbellii (VC). Among all yeast cell strains used in this study, only mnn9 yeast (less cell-wall bound mannoproteins and more glucan and chitin) seems to completely protect Artemia against the pathogen. Incomplete protection against the pathogen was obtained by the gas1 and chs3 mutants, which are lacking the gene for a particular cell-wall protein and chitin synthesis, respectively, resulting in more glucan. The result with the chs3 mutant is of particular interest, as its nutritional value for Artemia is comparable to the wild type. Hence, only with the chs3 strain, in contrast to the gas1 or mnn9 strains, the temporary protection to VC is not concomitant with a better growth performance under non-challenged conditions, suggesting non-interference of general nutritional effects.