Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579

To monitor the ability of the food-borne opportunistic pathogen Bacillus cereus to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42 degrees C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50 degrees C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42 degrees C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of B. subtilis. These observations indicate that heat adaptation of B. cereus involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic B. cereus, which may result in increased survival in foods.     

Adaptive larval thermotolerance and induced cross-tolerance to propoxur insecticide in mosquitoes Anopheles stephensi and Aedes aegypti

Abstract. Fourth-instar larvae of mosquitoes Anopheles stephensi and Aedes aegypti normally died within 90 min at 43C. Pre-exposure to high but sublethal temperatures conferred adaptive thermotolerance, dependent on the temperature and the duration of pre-exposure. Adaptive cross-tolerance to propoxur (a carbamate insecticide) was also induced in larvae by pre-exposing them to sublethal temperatures. Pre-exposure to sublethal concentrations of propoxur was found to confer cross-thermotolerance to a lower extent. These results suggest that the shock proteins (e.g. heat shock proteins) induced by unrelated stress factors play an important role in the development of adaptive cross-protection (stress response) to other stress conditions.     

Defense against lethal treatments and de novo protein synthesis induced by NaCl in Enterococcus faecalis ATCC 19433

Enterococcus faecalis was strongly resistant to high osmotic pressure in complex medium; however, when it was subjected to a moderate osmotic stress [6.5% (w/v) NaCl or 52% (w/v) sucrose] for 2 h, it showed cross-protection against ethanol (22%), detergents stresses [bile salts (0.3%) and SDS (0.017%)], hydrogen peroxide challenge (45 mM), and to a minor extent against lethal temperature (62° C). In response to salt stress [6.5% (w/v) NaCl], E. faecalis induced a large number of stress proteins. In addition, NaCl strongly induced the synthesis of many proteins more than tenfold. Although the acquired thermotolerance was inhibited markedly by chloramphenicol, the other NaCl-induced cross-tolerances seemed not to be correlated with de novo protein synthesis. The relationship between the stress protein synthesis and the induction of different types of cross-protection is discussed. Received: 7 September 1995 / Accepted: 29 January 1996     

Chemical Induction of Stress Proteins Does Not Induce Splicing Thermotolerance under Conditions Producing Survival Thermotolerance

Cell survival during a severe heat stress can be enhanced when heat shock proteins are induced prior to the severe heat treatment. Induction can be accomplished either by heat or chemical treatments. The increase in survival at these severe elevated temperatures after pretreatment has been referred to as thermotolerance, which we now refer to as survival thermotolerance. It has also been shown previously that mild heat treatment allows splicing in cells subjected to a severe heat treatment, now referred to as splicing thermotolerance. The experiments shown here demonstrate that even though chemical induction of the heat shock proteins leads to survival thermotolerance, this same treatment does not induce splicing thermotolerance. These are the first results that demonstrate at least two distinct aspects of thermotolerance.     

In vivo stress preconditioning

The heat shock or stress protein response is a highly conserved defense mechanism. Activation of the stress protein response by mild hyperthermia or by pharmacological agents allows cells to withstand a subsequent metabolic insult that would otherwise be lethal, a phenomenon referred as "thermotolerance" or "preconditioning." Heat shock response is characterized by increased expression of stress proteins that provide cellular protection, e.g., via increased chaperoning activity in all organisms, from bacteria to animals and humans. Indeed, there is experimental evidence that overexpression of specific heat shock proteins or heat shock factors produce protective effects similar to those observed after stress preconditioning. The purpose of this review is first to discuss the methods used to induce in vivo thermotolerance with mild hyperthermia or pharmacological agents. Then, as an example of the organ protection provided by in vivo stress preconditioning, the second part of this paper will examine how the induction of thermotolerance modulates the lung inflammatory response associated with acute lung injury, thus providing broad organ and tissue protection against oxidative stress associated this syndrome.     

Identification of Proteins Involved in the Heat Stress Response of Bacillus cereus ATCC 14579

To monitor the ability of the food-borne opportunistic pathogen Bacillus cereus to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42°C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50°C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42°C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of B. subtilis. These observations indicate that heat adaptation of B. cereus involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic B. cereus, which may result in increased survival in foods.     

Slow Growth Induces Heat-Shock Resistance in Normal and Respiratory-deficient Yeast

Yeast cells respond to a variety of environmental stresses, including heat shock and growth limitation. There is considerable overlap in these responses both from the point of view of gene expression patterns and cross-protection for survival. We performed experiments in which cells growing at different steady-state growth rates in chemostats were subjected to a short heat pulse. Gene expression patterns allowed us to partition genes whose expression responds to heat shock into subsets of genes that also respond to slow growth rate and those that do not. We found also that the degree of induction and repression of genes that respond to stress is generally weaker in respiratory deficient mutants, suggesting a role for increased respiratory activity in the apparent stress response to slow growth. Consistent with our gene expression results in wild-type cells, we found that cells growing more slowly are cross-protected for heat shock, i.e., better able to survive a lethal heat challenge. Surprisingly, however, we found no difference in cross-protection between respiratory-deficient and wild-type cells, suggesting induction of heat resistance at low growth rates is independent of respiratory activity, even though many of the changes in gene expression are not.     

Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in<Emphasis Type="Italic"> Propionibacterium freudenreichii</Emphasis>

Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, respectively. Traditionally used as Swiss-type cheese starters, propionibacteria also constitute promising human probiotics. Stress tolerance and cross-protection in Propionibacterium freudenreichii were thus examined after exposure to heat, acid, or bile salts stresses. Adapted cells demonstrated acquired homologous tolerance. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, bile salts pretreatment sensitized cells to acid challenge and vice versa. Surprisingly, heat and acid responses did not present significant cross-protection in P. freudenreichii. During adaptations, important changes in cellular protein synthesis were observed using two-dimensional electrophoresis. While global protein synthesis decreased, several proteins were overexpressed during stress adaptations. Thirty-four proteins were induced by acid pretreatment, 34 by bile salts pretreatment, and 26 by heat pretreatment. Six proteins are common to all stresses and represent general stress-response components. Among these polypeptides, general stress chaperones, and proteins involved in energetic metabolism, oxidative stress response, or SOS response were identified. These results bring new insight into the tolerance of P. freudenreichii to heat, acid, and bile salts, and should be taken into consideration in the development of probiotic preparations.     

Anticancer drugs as inducers of thermotolerance in yeast

Yeast cell viability was evaluated microscopically following exposure to heat shock for 30 min at 53°C. The cells were previously grown in the presence of potential stressors (anticancer drugs;e.g., 5-fluorouracil, methotrexate, cisplatin, bleomycin, mitomycin-C and camptothecin-11). The induction of thermotolerance was documented by significantly increased viability after heat shock. This effect, which was reversed by cycloheximide, was comparable to that observed following exposure to a mild heat stress. These data demonstrate that pretreatment with sub-toxic concentrations of some of the clinically used antineoplastic agents conferres thermotolerance to yeast, possibly through the synthesis of protein components.     

Atypical adaptive and cross-protective responses against peroxide killing in a bacterial plant pathogen, Agrobacterium tumefaciens

Physiological adaptive and cross-protection responses to oxidants were investigated in Agrobacterium tumefaciens. Exposure of A. tumefaciens to sublethal concentrations of H2O2 induced adaptive protection to lethal concentrations of H2O2. Similar treatments with organic peroxide and menadione did not produce adaptive protection to subsequent exposure to lethal concentrations of these oxidants. Pretreatment of A. tumefaciens with an inducing concentration of menadione conferred cross-protection against H2O2, but not to tert-butyl hydroperoxide (tBOOH), killing. The menadione induced cross-protection to H2O2 was due to the compound's ability to highly induce the peroxide scavenging enzyme, catalase. The levels of catalase directly correlated with the bacterium's ability to survive H2O2 treatment. Some aspects of the oxidative stress response of A. tumefaciens differ from other bacteria, and these differences may be important in plant/microbe interactions.     

The effects of glucose on protein synthesis and thermosensitivity in Chinese hamster ovary cells

Glucose deprivation induces the major glucose regulated proteins (GRPs) in Chinese hamster ovary cells. When these cells are then returned to a glucose containing environment, GRP synthesis is repressed while concurrently other proteins, identified as heat shock proteins, are induced. The induction of the GRPs is found to mark precisely the onset of a decline in the cell's ability to survive a thermal stress while the expression of heat shock proteins, when glucose is restored, is paralleled by significant increases in survival protection or thermotolerance.     

Acquisition of thermotolerance in bay scallops, Argopecten irradians irradians, via differential induction of heat shock proteins

Many cells and organisms are rendered transiently resistant to lethal heat shock by short exposure to sublethal temperatures. This induced thermotolerance is thought to be related to increased amounts of heat shock proteins (HSPs) which, as molecular chaperones, protect cells from stress-induced damage. As part of a study on bivalve stress and thermotolerance, work was undertaken to examine the effects of sublethal heat shock on stress tolerance of juveniles of the northern bay scallop, Argopecten irradians irradians, in association with changes in the levels of cytoplasmic HSP70 and 40. Juvenile bay scallops heat-shocked at a sublethal temperature of 32 °C survived an otherwise lethal heat treatment at 35 °C for at least 7 days. As determined by ELISA, acquisition of induced thermotolerance closely paralleled HSP70 accumulation, whereas HSP40 accrual appeared less closely associated with thermotolerance. Quantification of scallop HSPs following lethal heat treatment, with or without conditioning, suggested a causal role for HSP70 in stress tolerance, with HSP40 contributing to a lesser, but significant extent. Overall, this study demonstrated that sublethal heat shock promotes survival of A. irradians irradians juveniles upon thermal stress and the results support the hypothesis that HSPs have a role in this induced thermotolerance. Exploitation of the induced thermotolerance response shows promise as a means to improve survival of bay scallops in commercial culture.     

The Effect of Sodium Azide on Basic and Induced Thermotolerance in Saccharomyces cerevisiae

The action mechanism of the mitochondrial inhibitor sodium azide on thermotolerance in Saccharomyces cerevisiae was studied. At ambient growth temperature, pretreatment with sodium azide was shown to improve the thermotolerance of parent cells and the hsp104 mutant. Treating with the inhibitor during a mild heat shock suppressed the development of induced thermotolerance due to the inhibition of heat shock protein (Hsp104) synthesis. Treating with the inhibitor immediately before lethal heat shock produced a variety of effects on thermotolerance depending on whether the yeast metabolism was oxidative or fermentative. The conclusions are: (1) the protective effect of sodium azide on the thermotolerance of S. cerevisiae cells grown on glucose-containing medium is not related to Hsp104 functioning, and (2) the mechanisms of basic and induced thermotolerance differ considerably.     

Interleukin-6 induces thermotolerance in cultured Caco-2 cells independent of the heat shock response

In recent studies, induction of the heat shock response increased IL-6 production in gut mucosa in vivo and in cultured Caco-2 cells in vitro. The heat shock response is associated with increased survival of cells exposed to otherwise lethal hyperthermia, so called thermotolerance, but the role of IL-6 in the induction of thermotolerance is not known. We tested the hypothesis that treatment of cultured Caco-2 cells with IL-6 results in the development of thermotolerance. Cells were treated with human recombinant IL-6 for 1h followed by 3 h recovery in cytokine-free medium whereafter cells were exposed to heat stress (48 degrees C for 2 h). In untreated cells, the heat stress resulted in an approximately 80% cell death. In cells treated with IL-6, cell viability after heat stress was significantly improved and was doubled at an IL-6 concentration of 20 ng/ml. Treatment of the cells with other cytokines (IL-4, IL-10, IL-1beta, or TNFalpha) did not induce thermotolerance, suggesting that the effect of IL-6 may be specific for this cytokine. The induction of thermotolerance by IL-6 was blocked by an IL-6 receptor antibody, suggesting that the development of thermotolerance was receptor-mediated. Treatment of cells with IL-6 did not induce an heat shock response as suggested by unaltered heat shock protein 70 and 90 levels and unaffected heat shock factor DNA binding activity. In addition, the IL-6-induced thermotolerance was not inhibited by quercetin. The present study provides the first evidence of IL-6-induced thermotolerance and suggests that this effect of IL-6 is independent of the heat shock response.     

Heat shock-induced SRSF10 dephosphorylation displays thermotolerance mediated by Hsp27

Gene regulation in response to environmental stress is critical for the survival of all organisms. From Saccharomyces cerevisiae to humans, it has been observed that splicing of mRNA precursors is repressed upon heat shock. However, a mild heat pretreatment often prevents splicing inhibition in response to a subsequent and more severe heat shock, a phenomenon called splicing thermotolerance. We have shown previously that the splicing regulator SRSF10 (formerly SRp38) is specifically dephosphorylated by the phosphatase PP1 in response to heat shock and that dephosphorylated SRSF10 is responsible for splicing repression caused by heat shock. Here we report that a mild heat shock protects SRSF10 from dephosphorylation during a second and more severe heat shock. Furthermore, this "thermotolerance" of SRSF10 phosphorylation, like that of splicing, requires de novo protein synthesis, specifically the synthesis of heat shock proteins. Indeed, overexpression of one of these proteins, Hsp27, inhibits SRSF10 dephosphorylation in response to heat shock and does so by interaction with SRSF10. Our data thus provide evidence that splicing thermotolerance is acquired through maintenance of SRSF10 phosphorylation and that this is mediated at least in part by Hsp27.