Membrane vesicles released by Lacticaseibacillus casei BL23 inhibit the biofilm formation of Salmonella Enteritidis.

Biofilms represent a major concern in the food industry and healthcare. The use of probiotic bacteria and their derivatives as an alternative to conventional treatments to fight biofilm development is a promising option that has provided convincing results in the last decades. Recently, membrane vesicles (MVs) produced by probiotics have generated considerable interest due to the diversity of roles they have been associated with. However, the antimicrobial activity of probiotic MVs remains to be studied. In this work, we showed that membrane vesicles produced by Lacticaseibacillus casei BL23 (LC-MVs) exhibited strong antibiofilm activity against Salmonella enterica serovar Enteritidis (S. Enteritidis) without affecting bacterial growth. Furthermore, we found that LC-MVs affected the early stages of S. Enteritidis biofilm development and prevented attachment of bacteria to polystyrene surfaces. Importantly, LC-MVs did not impact the biomass of already established biofilms. We also demonstrated that the antibiofilm activity depended on the proteins associated with the LC-MV fraction. Finally, two peptidoglycan hydrolases (PGHs) were found to be associated with the antibiofilm activity of LC-MVs. Overall, this work allowed to identify the antibiofilm properties of LC-MVs and paved the way for the use of probiotic MVs against the development of negative biofilms.

Spontaneous Prophage Induction Contributes to the Production of Membrane Vesicles by the Gram-Positive Bacterium Lacticaseibacillus casei BL23.

The formation of membrane vesicles (MVs) by Gram-positive bacteria has gained increasing attention over the last decade. Recently, models of vesicle formation have been proposed and involve the digestion of the cell wall by prophage-encoded or stress-induced peptidoglycan (PG) hydrolases and the inhibition of PG synthesis by ß-lactam antibiotics. The impact of these mechanisms on vesicle formation is largely dependent on the strain and growth conditions. To date, no information on the production of vesicles by the lactobacilli family has been reported. Here, we aimed to characterize the MVs released by the Gram-positive bacteria Lacticaseibacillus casei BL23 and also investigated the mechanisms involved in vesicle formation. Using electron microscopy, we established that the size of the majority of L. casei BL23 vesicles ranged from 50 to 100 nm. Furthermore, we showed that the vesicles were released consistently throughout the growth of the bacteria in standard culture conditions. The protein composition of the vesicles released in the supernatant was identified and a significant number of prophage proteins was detected. Moreover, using a mutant strain harboring a defective PLE2 prophage, we were able to show that the spontaneous and mitomycin-triggered induction of the prophage PLE2 contribute to the production of MVs by L. casei BL23. Finally, we also demonstrated the influence of prophages on the membrane integrity of bacteria. Overall, our results suggest a key role of the prophage PLE2 in the production of MVs by L. casei BL23 in the absence or presence of genotoxic stress. IMPORTANCE The last few decades have demonstrated that membrane vesicles (MVs) produced by microorganisms can have a wide variety of functions. This diversity places MVs at the crossroads of major research topics in current microbiology such as antibiotic resistance, horizontal gene transfer, cell communication, biofilm development, bacteriophage resistance, and pathogenesis. In particular, vesicles produced by probiotic strains have been shown to play a significant role in their beneficial effects. Thus, the study of vesicle biogenesis is a key element for promoting and improving their release. Overall, our results suggest a key role of spontaneous and mitomycin-triggered prophage induction in MV production by the Gram-positive bacteria Lacticaseibacillus casei BL23. This phenomenon is of great interest as prophage-induced MVs could potentially influence bacterial behavior, stress resistance, and vesicle functions.

Lactobacillus stress protein GroEL prevents colonic inflammation.

BACKGROUND: We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. METHODS: We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli. RESULTS: We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1ß, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4-/- mice, and the activation of a non-canonical TLR4 pathway. CONCLUSIONS: L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics.

Intestinal release of biofilm-like microcolonies encased in calcium-pectinate beads increases probiotic properties of Lacticaseibacillus paracasei.

In this study, we show that calcium pectinate beads (CPB) allow the formation of 20 µm spherical microcolonies of the probiotic bacteria Lacticaseibacillus paracasei (formerly designated as Lactobacillus paracasei) ATCC334 with a high cell density, reaching more than 10 log (CFU/g). The bacteria within these microcolonies are well structured and adhere to a three-dimensional network made of calcium-pectinate through the synthesis of extracellular polymeric substances (EPS) and thus display a biofilm-like phenotype, an attractive property for their use as probiotics. During bacterial development in the CPB, a coalescence phenomenon arises between neighboring microcolonies accompanied by their peripheral spatialization within the bead. Moreover, the cells of L. paracasei ATCC334 encased in these pectinate beads exhibit increased resistance to acidic stress (pH 1.5), osmotic stress (4.5 M NaCl), the freeze-drying process and combined stresses, simulating the harsh conditions encountered in the gastrointestinal (GI) tract. In vivo, the oral administration of CPB-formulated L. paracasei ATCC334 in mice demonstrated that biofilm-like microcolonies are successfully released from the CPB matrix in the colonic environment. In addition, these CPB-formulated probiotic bacteria display the ability to reduce the severity of a DSS-induced colitis mouse model, with a decrease in colonic mucosal injuries, less inflammation, and reduced weight loss compared to DSS control mice. To conclude, this work paves the way for a new form of probiotic administration in the form of biofilm-like microcolonies with enhanced functionalities.

Resveratrol Favors Adhesion and Biofilm Formation of Lacticaseibacillus paracasei subsp. paracasei Strain ATCC334.

Bacterial strains of the Lactobacillaceae family are widely used as probiotics for their multifaceted potential beneficial properties. However, no official recommendations for their clinical use exist since, in many cases, oral administrations of these bacteria displayed limited beneficial effects in human. Additional research is thus needed to improve the efficiency of existing strains with strong potential. In this context, we assess in vitro the effects of nine polyphenols to stimulate biofilm formation by lactobacilli, a feature enhancing their functionalities. Among these polyphenols, we identify trans-Resveratrol (referred to hereafter as Resveratrol) as a potent inducer of biofilm formation by Lacticaseibacillus paracasei (formerly designated as Lactobacillus paracasei) ATCC334 strain. This effect is strain-dependent and relies on the enhancement of L. paracasei adhesion to abiotic and biotic surfaces, including intestinal epithelial cells. Mechanistically, Resveratrol modify physico-chemical properties of the bacterial surface and thereby enhances L. paracasei aggregation, subsequently facilitating adhesion and biofilm development. Together, our in vitro data demonstrate that Resveratrol might be used to modulate the behavior of Lactobacilli with probiotic properties. Combination of probiotics and polyphenols could be considered to enhance the probiotic functionalities in further in vivo studies.

Chemical Transfers Occurring Through Oenococcus oeni Biofilm in Different Enological Conditions.

Chardonnay wine malolactic fermentations were carried out to evaluate the chemical transfers occurring at the wood/wine interface in the presence of two different bacterial lifestyles. To do this, Oenococcus oeni was inoculated into must and wine in its planktonic and biofilm lifestyles, whether adhering or not to oak chips, leading to three distinct enological conditions: (i) post-alcoholic fermentation inoculation in wine in the absence of oak chips, (ii) post-alcoholic fermentation inoculation in wine in the presence of oak chips, and (iii) co-inoculation of both Saccharomyces cerevisiae and O. oeni directly in Chardonnay musts in the presence of oak chips. Classical microbiological and physico-chemical parameters analyzed during the fermentation processes confirmed that alcoholic fermentation was completed identically regardless of the enological conditions, and that once O. oeni had acquired a biofilm lifestyle in the presence or absence of oak, malolactic fermentation occurred faster and with better reproducibility compared to planktonic lifestyles. Analyses of volatile components (higher alcohols and wood aromas) and non-volatile components (Chardonnay grape polyphenols) carried out in the resulting wines revealed chemical differences, particularly when bacterial biofilms were present at the wood interface. This study revealed the non-specific trapping activity of biofilm networks in the presence of wood and grape compounds regardless of the enological conditions. Changes of concentrations in higher alcohols reflected the fermentation bioactivity of bacterial biofilms on wood surfaces. These chemical transfers were statistically validated by an untargeted approach using Excitation Emission Matrices of Fluorescence combined with multivariate analysis to discriminate innovative enological practices during winemaking and to provide winemakers with an optical tool for validating the biological and chemical differentiations occurring in wine that result from their decisions.

The Phenotypic Analysis of Lactobacillus plantarum shsp Mutants Reveals a Potential Role for hsp1 in Cryotolerance.

Small heat shock proteins (sHSPs) are ubiquitous, low molecular weight (MW) proteins that share a conserved alpha-crystallin domain. sHSPs oligomers exhibit chaperon-like activities by interacting with unfolded substrates, thereby preventing their aggregation and precipitation. Unlike most lactobacilli, which have single shsp genes, three different sHSP-encoding genes, i.e., hsp1, hsp2, and hsp3, were previously identified in the probiotic Lactobacillus plantarum WCFS1. Early studies, including the characterization of the knock out (KO) mutant for hsp2, indicated a different organization and transcriptional regulation of these genes and suggested that the three L. plantarum sHSPs might accomplish different tasks in stress response. To unravel the role of sHSPs, KO mutants of hsp1 and hsp3 were generated using a Cre-lox based system. Mutation of either genes resulted in impaired growth capacity under normal conditions, heat-stress and stresses typically found during host interactions and food technological process. However, survival to heat shock and the level of thermal stabilization of cytoplasmic proteins were similar between mutants and parental strain. Transcriptional analysis revealed that in the mutant genetic backgrounds there is an upregulated basal expression of the un-mutated mate hsps and other stress-related genes, which may compensate for the loss of HSP function, hence possibly accounting for the lack of a remarkable susceptibility to heat challenge. HSP3 seemed relevant for the induction of thermotolerance, while HSP1 was required for improved cryotolerance. Cell surface properties and plasma membrane fluidity were investigated to ascertain the possible membrane association of sHSP. Intriguingly, the loss of hsp1 was associated to a lower level of maximal membrane fluidity upon heat stress. A role for HSP1 in controlling and improving membrane fluidity is suggested which may pertains its cryoprotective function.

Resveratrol-Induced Xenophagy Promotes Intracellular Bacteria Clearance in Intestinal Epithelial Cells and Macrophages.

Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and the modulating inflammatory response. Several infectious and immune disorders are associated with autophagy defects, suggesting that stimulation of autophagy in these diseases should be beneficial. Here, we show that resveratrol is able to boost xenophagy, a selective form of autophagy that target invasive bacteria. We demonstrated that resveratrol promotes in vitro autophagy-dependent clearance of intracellular bacteria in intestinal epithelial cells and macrophages. These results were validated in vivo using infection in a transgenic GFP-LC3 zebrafish model. We also compared the ability of resveratrol derivatives, designed to improve the bioavailability of the parent molecule, to stimulate autophagy and to induce intracellular bacteria clearance. Together, our data demonstrate the ability of resveratrol to stimulate xenophagy, and thereby enhance the clearance of two invasive bacteria involved life-threatening diseases, Salmonella Typhimurium and Crohn's disease-associated Adherent-Invasive Escherichia coli. These findings encourage the further development of pro-autophagic nutrients to strengthen intestinal homeostasis in basal and infectious states.

Production of the small heat shock protein Lo18 from Oenococcus oeni in Lactococcus lactis improves its stress tolerance.

Lactococcus lactis is a lactic acid bacterium widely used in cheese and fermented milk production. During fermentation, L. lactis is subjected to acid stress that impairs its growth. The small heat shock protein (sHsp) Lo18 from the acidophilic species Oenococcus oeni was expressed in L. lactis. This sHsp is known to play an important role in protein protection and membrane stabilization in O. oeni. The role of this sHsp could be studied in L. lactis, since no gene encoding for sHsp has been detected in this species. L. lactis subsp. cremoris strain MG1363 was transformed with the pDLhsp18 plasmid, which is derived from pDL278 and contains the hsp18 gene (encoding Lo18) and its own promoter sequence. The production of Lo18 during stress conditions was checked by immunoblotting and the cellular distribution of Lo18 in L. lactis cells after heat shock was determined. Our results clearly indicated a role for Lo18 in cytoplasmic protein protection and membrane stabilization during stress. The production of sHsp in L. lactis improved tolerance to heat and acid conditions in this species. Finally, the improvement of the L. lactis survival in milk medium thanks to Lo18 was highlighted, suggesting an interesting role of this sHsp. These findings suggest that the expression of a sHsp by a L. lactis strain results in greater resistance to stress, and, can consequently enhance the performances of industrial strains.

Effect of Biofilm Formation by Oenococcus oeni on Malolactic Fermentation and the Release of Aromatic Compounds in Wine.

The winemaking process involves the alcoholic fermentation of must, often followed by malolactic fermentation (MLF). The latter, mainly carried out by the lactic acid bacterium Oenococcus oeni, is used to improve wine quality when acidity reduction is required. Moreover, it prevents microbial spoilage and improves the wine's organoleptic profile. Prior observations showed that O. oeni is able to resist several months in harsh wine conditions when adhered on oak barrels. Since biofilm is a prevailing microbial lifestyle in natural environments, the capacity of O. oeni to form biofilms was investigated on winemaking material such as stainless steel and oak chips. Scanning Electron Microscopy and Confocal Laser Scanning Microscopy showed that O. oeni was able to adhere to these surfaces and form spatially organized microcolonies embedded in extracellular substances. To assess the competitive advantage of this mode of life in wine, the properties of biofilm and planktonic cells were compared after inoculation in a fermented must (pH 3.5 or 3.2 and 12% ethanol) The results indicated that the biofilm culture of O. oeni conferred (i) increased tolerance to wine stress, and (ii) functional performance with effective malolactic activities. Relative gene expression focusing on stress genes and genes involved in EPS synthesis was investigated in a mature biofilm and emphasized the role of the matrix in increased biofilm resistance. As oak is commonly used in wine aging, we focused on the O. oeni biofilm on this material and its contribution to the development of wine color and the release of aromatic compounds. Analytical chromatography was used to target the main oak aging compounds such as vanillin, gaiacol, eugenol, whisky-lactones, and furfural. The results reveal that O. oeni biofilm developed on oak can modulate the wood-wine transfer of volatile aromatic compounds during MLF and aging by decreasing furfural, gaiacol, and eugenol in particular. This work showed that O. oeni forms biofilms consisting of stress-tolerant cells capable of efficient MLF under winemaking conditions. Therefore surface-associated behaviors should be considered in the development of improved strategies for the control of MLF in wine.

Biofilms of Lactobacillus plantarum and Lactobacillus fermentum: Effect on stress responses, antagonistic effects on pathogen growth and immunomodulatory properties.

Few studies have extensively investigated probiotic functions associated with biofilms. Here, we show that strains of Lactobacillus plantarum and Lactobacillus fermentum are able to grow as biofilm on abiotic surfaces, but the biomass density differs between strains. We performed microtiter plate biofilm assays under growth conditions mimicking to the gastrointestinal environment. Osmolarity and low concentrations of bile significantly enhanced Lactobacillus spatial organization. Two L. plantarum strains were able to form biofilms under high concentrations of bile and mucus. We used the agar well-diffusion method to show that supernatants from all Lactobacillus except the NA4 isolate produced food pathogen inhibitory molecules in biofilm. Moreover, TNF-α production by LPS-activated human monocytoid cells was suppressed by supernatants from Lactobacillus cultivated as biofilms but not by planktonic culture supernatants. However, only L. fermentum NA4 showed anti-inflammatory effects in zebrafish embryos fed with probiotic bacteria, as assessed by cytokine transcript level (TNF-α, IL-1ß and IL-10). We conclude that the biofilm mode of life is associated with beneficial probiotic properties of lactobacilli, in a strain dependent manner. Those results suggest that characterization of isolate phenotype in the biofilm state could be additional valuable information for the selection of probiotic strains.

Protective role of glutathione addition against wine-related stress in Oenococcus oeni.

Oenococcus oeni is the main species responsible for the malolactic fermentation (MLF) of wine due to its ability to survive in this environment. Some wine-related stress factors, such as ethanol and low pH, may alter the cell redox balance of O. oeni. For the first time, the ability to uptake glutathione (GSH), an almost universal tripeptide with antioxidant properties, has been associated to the improvement of stress response in O. oeni. Despite the inability of O. oeni to synthesize GSH, this bacterium can capture it from the media. The ability of 30 O. oeni strains to uptake GSH was assessed in this study. Although all of the strains tested were able to import GSH, substantial variability among them was detected. To assess the physiological function of GSH, three strains with different GSH-import capacities were selected. Significant changes in membrane fatty acids composition were observed due to GSH addition. The most relevant was the increase of cyclopropane fatty acids in cell membrane, in both the exponential and the stationary phases. Cells grown with GSH showed an improved survival against ethanol shock (14% v/v). GSH addition also increased biomass production during the adaptation to wine stress conditions (pH4, pH3.4 and 6% ethanol). The results suggest that GSH enrichment could improve the resistance to stress to O. oeni, which could be useful for the adaptation of MLF starter cultures.

Cellular and Molecular Connections between Autophagy and Inflammation.

Autophagy is an intracellular catabolic pathway essential for the recycling of proteins and larger substrates such as aggregates, apoptotic corpses, or long-lived and superfluous organelles whose accumulation could be toxic for cells. Because of its unique feature to engulf part of cytoplasm in double-membrane cup-shaped structures, which further fuses with lysosomes, autophagy is also involved in the elimination of host cell invaders and takes an active part of the innate and adaptive immune response. Its pivotal role in maintenance of the inflammatory balance makes dysfunctions of the autophagy process having important pathological consequences. Indeed, defects in autophagy are associated with a wide range of human diseases including metabolic disorders (diabetes and obesity), inflammatory bowel disease (IBD), and cancer. In this review, we will focus on interrelations that exist between inflammation and autophagy. We will discuss in particular how mediators of inflammation can regulate autophagy activity and, conversely, how autophagy shapes the inflammatory response. Impact of genetic polymorphisms in autophagy-related gene on inflammatory bowel disease will be also discussed.

Proton-transfer-reaction mass spectrometry for the study of the production of volatile compounds by bakery yeast starters.

The aromatic impact of bakery yeast starters is currently receiving considerable attention. The flavor characteristics of the dough and the finished products are usually evaluated by gas chromatography and sensory analysis. The limit of both techniques resides in their low-throughput character. In the present work, proton-transfer-reaction mass spectrometry (PTR-MS), coupled to a time-of-flight mass analyzer, was employed, for the first time, to measure the volatile fractions of dough and bread, and to monitor Saccharomyces cerevisiae volatile production in a fermented food matrix. Leavening was performed on small-scale (1 g) dough samples inoculated with different commercial yeast strains. The leavened doughs were then baked, and volatile profiles were determined during leavening and after baking. The experimental setup included a multifunctional autosampler, which permitted the follow-up of the leavening process on a small scale with a typical throughput of 500 distinct data points in 16 h. The system allowed to pinpoint differences between starter yeast strains in terms of volatile emission kinetics, with repercussions on the final product (i.e. the corresponding micro-loaves). This work demonstrates the applicability of PTR-MS for the study of volatile organic compound production during bread-making, for the automated and online real-time monitoring of the leavening process, and for the characterization and selection of bakery yeast starters in view of their production of volatile compounds.

The biofilm mode of life boosts the anti-inflammatory properties of Lactobacillus.

The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro-inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic-fed animals, suggesting that these bacteria have anti-inflammatory properties. We treated human macrophage-differentiated monocytic THP-1 cells with supernatants of L. casei ATCC334 grown in either biofilm or planktonic cultures. TNF-α production was suppressed and the NF-κB pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti-inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF-α were inversely correlated. We confirmed that biofilm development in other Lactobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of Lactobacillus sp. and that secreted GroEL is involved in this beneficial effect.

Adaptation of the wine bacterium Oenococcus oeni to ethanol stress: role of the small heat shock protein Lo18 in membrane integrity.

Malolactic fermentation in wine is often carried out by Oenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in O. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from O. oeni grown under optimal growth conditions (here, control liposomes), one was prepared from O. oeni grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from Bacillus subtilis or Lactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from O. oeni suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of O. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.

Biotechnical applications of small heat shock proteins from bacteria.

The stress responses of most bacteria are thought to involve the upregulation of small heat shock proteins. We describe here some of the most pertinent aspects of small heat shock proteins, to highlight their potential for use in various applications. Bacterial species have between one and 13 genes encoding small heat shock proteins, the precise number depending on the species considered. Major efforts have recently been made to characterize the protein protection and membrane stabilization mechanisms involving small heat shock proteins in bacteria. These proteins seem to be involved in the acquisition of cellular heat tolerance. They could therefore potentially be used to maintain cell viability under unfavorable conditions, such as heat shock or chemical treatments. This review highlights the potential roles of applications of small heat shock proteins in stabilizing overproduced heterologous proteins in Escherichia coli, purified bacterial small heat shock proteins in protein biochip technology, proteomic analysis and food technology and the potential impact of these proteins on some diseases. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

The oligomer plasticity of the small heat-shock protein Lo18 from Oenococcus oeni influences its role in both membrane stabilization and protein protection.

The ability of the small Hsp (heat-shock protein) Lo18 from Oenococcus oeni to modulate the membrane fluidity of liposomes or to reduce the thermal aggregation of proteins was studied as a function of the pH in the range 5-9. We have determined by size-exclusion chromatography and analytical ultracentrifugation that Lo18 assembles essentially as a 16-mer at acidic pH. Its quaternary structure evolves to a mixture of lower molecular mass oligomers probably in dynamic equilibrium when the pH increases. The best Lo18 activities are observed at pH 7 when the particle distribution contains a major proportion of dodecamers. At basic pH, particles corresponding to a dimer prevail and are thought to be the building blocks leading to oligomerization of Lo18. At acidic pH, the dimers are organized in a double-ring of stacked octamers to form the 16-mer as shown by the low-resolution structure determined by electron microscopy. Experiments performed with a modified protein (A123S) shown to preferentially form dimers confirm these results. The α-crystallin domain of Methanococcus jannaschii Hsp16.5, taken as a model of the Lo18 counterpart, fits with the electron microscopy envelope of Lo18.

Co-expression of the small heat shock protein, Lo18, with ß-glucosidase in Escherichia coli improves solubilization and reveals various associations with overproduced heterologous protein, GroEL/ES.

We developed a new system to improve the overproduction of soluble proteins in E. coli based on a plasmid encoding the small heat-shock protein, Lo18, derived from the lactic acid bacterium Oenococcus oeni. The efficiency of this system was compared with that of another system based on production of the E. coli universal chaperone GroEL/ES. A compatible plasmid encoding ß-glucosidase was constructed for the overproduction and aggregation of this enzyme. Co-expression with Lo18 resulted in an increase in soluble ß-glucosidase levels similar to that obtained in the GroEL/ES co-expression system. Lo18 was found preferentially in the insoluble fraction, associated with aggregated enzyme. By contrast, GroEL/ES was more abundant in the soluble fraction.

Quantification of HSP27 and HSP70 molecular chaperone activities.

Stress-inducible heat-shock proteins (HSPs, like HSP70 and HSP27) are molecular chaperones that -protect cells from stress damage by keeping cellular proteins in a folding competent state and preventing them from irreversible aggregation. HSP27 and HSP70 chaperone activities are useful indicators to test chemical products and physical stress impact on protein denaturation, to select HSP inhibitors, or to -determine the implication of the chaperone function in other HSP activities, such as apoptosis. We have developed two simple and fast chaperone activity tests for HSP27 and HSP70 that we initially set up to test the effect of potential HSP inhibitors obtained after screening of chemical and small molecule libraries. These chaperone quantification tests are based on the capacity of HSP to counteract chemical or thermal protein aggregation.