Association between rat decompression sickness resistance, transthyretin single nucleotide polymorphism, and expression: A pilot study.

Decompression sickness (DCS) is a systemic syndrome that can occur after an environmental pressure reduction. Previously, we showed that the plasmatic tetrameric form of transthyretin (TTR) nearly disappeared in rats suffering DCS but not in asymptomatic ones. In this pilot study, we assessed whether the resistance to DCS could be associated with polymorphism of the gene of TTR. For this study, Sanger sequencing was performed on purified PCR products from the liver of 14-week-old male and female standard and DCS-resistant rats (n = 5 per group). Hepatic TTR mRNA expression was assessed by RT-qPCR in 18-19 week-old male and female standard and resistant rats (n = 6 per group). There is a synonymous single nucleotide polymorphism (SNP) on the third base of codon 46 (c.138 C > T). The thymine allele was present in 90% and 100% of males and females standard, respectively. However, this allele is present in only 30% of DCS-resistant males and females (p = 0.0002301). In the liver, there is a significant effect of the resistance to DCS (p = 0.043) and sex (p = 0.047) on TTR expression. Levels of TTR mRNA were lower in DCS-resistant animals. To conclude, DCS resistance might be associated with a SNP and a lower expression of TTR.

External pH modulation during the growth of Vibrio tapetis, the aetiological agent of brown ring disease.

AIMS: Brown ring disease (BRD) is an infection of the Manila clam Ruditapes philippinarum due to the pathogen Vibrio tapetis. During BRD, clams are facing immunodepression and shell biomineralization alteration. In this paper, we studied the role of pH on the growth of the pathogen and formulated hypothesis on the establishment of BRD by V. tapetis. METHODS AND RESULTS: In this study, we monitored the evolution of pH during the growth of V. tapetis in a range of pH and temperatures. We also measured the pH of Manila clam haemolymph and extrapallial fluids (EPFs) during infection by V. tapetis. We highlighted that V. tapetis modulates the external pH during its growth, to a value of 7·70. During the development of BRD, V. tapetis also influences EPFs and haemolymph pH in vitro in the first hours of exposure and in vivo after 3 days of infection. CONCLUSIONS: Our experiments have shown a close interaction between V. tapetis CECT4600, a pathogen of Manila clam that induces BRD, and the pH of different compartments of the animals during infection. These results indicate that the bacterium, through a direct mechanism or as a consequence of physiological changes encountered in the animal during infection, is able to interfere with the pH of Manila clam fluids. This pH modification might promote the infection process or at least create an imbalance within the animal that would favour its persistence. This last hypothesis should be tested in future experiment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first observation of pH modifications in the context of BRD and might orient future research on the fine mechanisms of pH modulation associated with BRD.

A proteomic study of resistance to Brown Ring disease in the Manila clam, Ruditapes philippinarum.

Marine mollusk aquaculture has more than doubled over the past twenty years, accounting for over 15% of total aquaculture production in 2016. Infectious disease is one of the main limiting factors to the development of mollusk aquaculture, and the difficulties inherent to combating pathogens through antibiotic therapies or disinfection have led to extensive research on host defense mechanisms and host-pathogen relationships. It has become increasingly clear that characterizing the functional profiles of response to a disease is an essential step in understanding resistance mechanisms and moving towards more effective disease control. The Manila clam, Ruditapes philippinarum, is a main cultured bivalve species of economic importance which is affected by Brown Ring disease (BRD), an infection induced by the bacterium Vibrio tapetis. In this study, juvenile Manila clams were subjected to a 28-day controlled challenge with Vibrio tapetis, and visual and molecular diagnoses were carried out to distinguish two extreme phenotypes within the experimental clams: uninfected ("RES", resistant) and infected ("DIS", diseased) post-challenge. Total protein extractions were carried out for resistant and diseased clams, and proteins were identified using LC-MS/MS. Protein sequences were matched against a reference transcriptome of the Manila clam, and protein intensities based on label-free quantification were compared to reveal 49 significantly accumulated proteins in resistant and diseased clams. Proteins with known roles in pathogen recognition, lysosome trafficking, and various aspects of the energy metabolism were more abundant in diseased clams, whereas those with roles in redox homeostasis and protein recycling were more abundant in resistant clams. Overall, the comparison of the proteomic profiles of resistant and diseased clams after a month-long controlled challenge to induce the onset of Brown Ring disease suggests that redox homeostasis and maintenance of protein structure by chaperone proteins may play important and interrelated roles in resistance to infection by Vibrio tapetis in the Manila clam.

Cyclin D1 mediates resistance to apoptosis through upregulation of molecular chaperones and consequent redistribution of cell death regulators.

Cyclin D1 is a key regulator of cell proliferation. It also controls other aspects of the cell fate, such as cellular senescence, apoptosis and tumourigenesis. We used B-lymphoid cell lines producing cyclin D1 to investigate the role of this protein in B-cell lymphomas and leukaemias. Constitutive low levels of cyclin D1 had no effect per se on cell proliferation, but conferred resistance to various apoptotic stimuli in B cells. Activation of the pro-apoptotic protein, Bax, was reduced and mitochondrial permeabilization and phosphatidylserine exposure following cytokine withdrawal were delayed in cyclin D1-producing cells. Proteomic analysis showed that the presence of cyclin D1 led to intracellular accumulation of various molecular chaperones. The chaperone, heat shock protein (Hsp)70, bound to both Bax and the mitochondrial apoptosis inducing factor following cytokine withdrawal, and impeded inhibitors of kappaB (IkappaB)-mediated inhibition of nuclear factor-kappaB anti-apoptotic signalling. Impairment of Hsp70 activity--using a pharmacological Hsp inhibitor or transfecting cells with an Hsp70-blocking antibody--restored the cellular response to mitochondrial apoptosis triggering. Thus, constitutive de-novo cyclin D1 production in B cells delays commitment to apoptosis by inducing Hsp70 chaperoning activity on pre- and post-mitochondrial pro-apoptotic factors.

Maltose utilization in Enterococcus faecalis.

AIMS: The aim of this research was to characterize the metabolic pathway for maltose utilization in Enterococcus faecalis. METHODS AND RESULTS: Screening a library of Enterococcus faecalis insertional mutants allowed the isolation of mutants affected in maltose utilization. Genetic analysis of the insertion loci revealed insertions in neighbour genes encoding an EII component of a phosphotransferase system (PTS) transporter (malT) and a maltose phosphorylase homologue (malP). The malP gene forms part of an operon which also includes genes encoding a phosphoglucomutase (malB), a mutarotase (aldose 1-epimerase) (malM) and a transcriptional regulator (malR). Analysis of (14)C-labelled carbohydrates uptake revealed that more than 97% of maltose enters the cells by the PTS transporter MalT. CONCLUSIONS: Both experimental data and genetic organization of the malPBMR operon strongly suggest that in Enterococcus faecalis, maltose enters using a PTS, leaving maltose-6-phosphate inside the cells which is hydrolysed by a maltose phosphate phosphorylase (MalP). SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a new pathway for maltose utilization in lactic acid bacteria.

Survival of Enterococcus faecalis in seawater microcosms is limited in the presence of bacterivorous zooflagellates.

The survival and persistence of growing and starved cells of Enterococcus faecalis in untreated and differentially filtered (20 microm, 5 microm, 3 microm, 1.2 microm, and 0.1 microm) seawater was analyzed in samples taken at different times over a 1-year period by plate counts and scanning electron microscopy. Whereas seawater filtered through a 0.1-microm mesh was not at all or only slightly bactericidal during incubation at 16 degrees C in the dark, culturability of E. faecalis in the other systems decreased as a function of increasing pore size of the filters. Recovery of culturable, glucose pre-starved cells was always higher than that of cells harvested from the exponential growth phase. Electron microscopic analysis showed that the disappearance of enterococci appeared related to the presence and multiplication of various zooflagellates.

The stress proteome of Enterococcus faecalis.

Enterococcus faecalis is a resident bacterium of the intestinal tract of humans and animals. This bacterium can be responsible for serious diseases and is one of the largest causes of hospital-based infections. This hardy organism resists many kinds of stresses and is used as a major indicator of the hygienic quality of food, milk, and drinking water. On the other side, enterococci seem to have beneficial role in the development of cheese aroma and are added in certain starter cultures. Since ten years, our laboratory has used the two-dimensional electrophoresis (2-DE) technique to study the response of E. faecalis to physical or chemical stresses as well as to glucose and total starvation. Twenty-seven protein spots on 2-D gels have been identified by N-terminal sequencing or Western blotting which make up the first proteome database of this species. The proteins were classified in four different groups according to their function and their regulation. The first group comprises well-characterized proteins with known protective functions towards stresses. The second group contains enzymes of catabolic pathways. Their implication in stress resistance seems not obvious. A third group are proteins induced in glucose-starved cells belonging to the CcpA regulon. Induction of these enzymes under starvation may serve to increase the scavenging capacity of the cells for nutrients or may be important to mobilize endogenous energetic reserves. Lastly, nine N-terminal amino acid sequences or open reading frames (ORF) showed no homologies with sequences in databases. A comprehensive description of stress proteins of E. faecalis and analysis of their patterns of expression under different environmental conditions would greatly increase our understanding of the molecular mechanisms underlying the extraordinary capacity of this bacterium to survive under hostile conditions.

Changes in protein synthesis and morphology during acid adaptation of Propionibacterium freudenreichii.

Survival of bacteria in changing environments depends on their ability to adapt to abiotic stresses. Microorganisms used in food technology face acid stress during fermentation processes. Similarly, probiotic bacteria have to survive acid stress imposed within the stomach in order to reach the intestine and play a beneficial role. Propionibacteria are used both as cheese starters and as probiotics in human alimentation. Adaptation to low pH thus constitutes a limit to their efficacy. Acid stress adaptation in the probiotic SI41 strain of Propionibacterium freudenreichii was therefore investigated. The acid tolerance response (ATR) was evidenced in a chemically defined medium. Transient exposure to pH 5 afforded protection toward acid challenge at pH 2. Protein neosynthesis was shown to be required for optimal ATR, since chloramphenicol reduced the acquired acid tolerance. Important changes in genetic expression were observed with two-dimensional electrophoresis during adaptation. Among the up-regulated polypeptides, a biotin carboxyl carrier protein and enzymes involved in DNA synthesis and repair were identified during the early stress response, while the universal chaperonins GroEL and GroES corresponded to a later response. The beneficial effect of ATR was evident at both the physiological and morphological levels. This study constitutes a first step toward understanding the very efficient ATR described in P. freudenreichii.

Uptake of choline from salmon flesh and its conversion to glycine betaine in response to salt stress in Shewanella putrefaciens.

When cultured in M63 minimal medium plus 0.6 M NaCl, the growth of Shewanella putrefaciens was strongly inhibited. The addition of an extract from smoked salmon to this medium restored the growth almost to the unstressed level. A comparison of the 13C NMR spectra of intracellular solutes extracted from S. putrefaciens cells cultured in both conditions revealed the accumulation of glycine betaine (GB) from the smoked salmon extract (SSE). Analysis of the osmoprotective properties of this extract for several strains of Escherichia coli (which differ from each other in their ability to accumulate GB (i) from the surrounding environment, and (ii) from its hydroxylated precursor choline), demonstrated the absence of GB in the SSE. From the overall results, we inferred that salt-stressed S. putrefaciens cells accumulated GB from choline present in the SSE. Furthermore, the use of [14C]-labeled betaines gave evidence that S. putrefaciens (i) oxidised choline to GB, (ii) accumulated GB as a non-metabolisable osmolyte (up to 1300 nmol (mg dw)(-1) when cultured in a medium containing 0.5 M NaCl and either 1 mM choline or 1 mM GB), and (iii) both choline and GB uptake activities were osmotically upregulated (both activities were increased more than 50-fold in media containing 0.4 to 0.6 M NaCl). In all, our results suggest that in salted smoked salmon, S. putrefaciens imports and oxidises choline, leading to the intracellular accumulation of GB.

Identification and characterization of gsp65, an organic hydroperoxide resistance (ohr) gene encoding a general stress protein in Enterococcus faecalis.

The Enterococcus faecalis general stress protein Gsp65 has been purified from two-dimensional gel electrophoresis. Determination of its N-terminal sequence and characterization of the corresponding gene revealed that the gsp65 product is a 133-amino-acid protein sharing homologies with organic hydroperoxide resistance (Ohr) proteins. Transcriptional analysis of gsp65 gave evidence for a monocistronic mRNA initiated 52 nucleotides upstream of the ATG start codon and for an induction in response to hydrogen peroxide, heat shock, acid pH, detergents, ethanol, sodium chloride, and tert-butylhydroperoxide (tBOOH). A gsp65 mutant showed increased sensitivity to the organic hydroperoxide tBOOH and to ethanol.

Starvation and osmotic stress induced multiresistances. Influence of extracellular compounds.

Growth restriction due to stasis and/or hyperosmolarity is a common situation encountered by microorganisms in nature. Therefore, they have developed defence systems allowing them to withstand these periods. Bacteria respond to these conditions by a metabolic reprogramming which leads to a cellular state of enhanced resistance. This communication reviews recent advances in knowledge of the molecular basis of this phenomenon in different bacteria.

Nanomolar levels of dimethylsulfoniopropionate, dimethylsulfonioacetate, and glycine betaine are sufficient to confer osmoprotection to Escherichia coli.

We combined the use of low inoculation titers (300 +/- 100 CFU/ml) and enumeration of culturable cells to measure the osmoprotective potentialities of dimethylsulfoniopropionate (DMSP), dimethylsulfonioacetate (DMSA), and glycine betaine (GB) for salt-stressed cultures of Escherichia coli. Dilute bacterial cultures were grown with osmoprotectant concentrations that encompassed the nanomolar levels of GB and DMSP found in nature and the millimolar levels of osmoprotectants used in standard laboratory osmoprotection bioassays. Nanomolar concentrations of DMSA, DMSP, and GB were sufficient to enhance the salinity tolerance of E. coli cells expressing only the ProU high-affinity general osmoporter. In contrast, nanomolar levels of osmoprotectants were ineffective with a mutant strain (GM50) that expressed only the low-affinity ProP osmoporter. Transport studies showed that DMSA and DMSP, like GB, were taken up via both ProU and ProP. Moreover, ProU displayed higher affinities for the three osmoprotectants than ProP displayed, and ProP, like ProU, displayed much higher affinities for GB and DMSA than for DMSP. Interestingly, ProP did not operate at substrate concentrations of 200 nM or less, whereas ProU operated at concentrations ranging from 1 nM to millimolar levels. Consequently, proU(+) strains of E. coli, but not the proP(+) strain GM50, could also scavenge nanomolar levels of GB, DMSA, and DMSP from oligotrophic seawater. The physiological and ecological implications of these observations are discussed.

Disaccharides as a new class of nonaccumulated osmoprotectants for Sinorhizobium meliloti.

Sucrose and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) are very unusual osmoprotectants for Sinorhizobium meliloti because these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed S. meliloti cells. Here, we show that, in fact, sucrose and ectoine belong to a new family of nonaccumulated sinorhizobial osmoprotectants which also comprises the following six disaccharides: trehalose, maltose, cellobiose, gentiobiose, turanose, and palatinose. Also, several of these disaccharides were very effective exogenous osmoprotectants for strains of Rhizobium leguminosarum biovars phaseoli and trifolii. Sucrose and trehalose are synthesized as endogenous osmolytes in various bacteria, but the other five disaccharides had never been implicated before in osmoregulation in any organism. All of the disaccharides that acted as powerful osmoprotectants in S. meliloti and R. leguminosarum also acted as very effective competitors of [14C]sucrose uptake in salt-stressed cultures of these bacteria. Conversely, disaccharides that were not osmoprotective for S. meliloti and R. leguminosarum did not inhibit sucrose uptake in these bacteria. Hence, disaccharide osmoprotectants apparently shared the same uptake routes in these bacteria. Natural-abundance 13C nuclear magnetic resonance spectroscopy and quantification of cytosolic solutes demonstrated that the novel disaccharide osmoprotectants were not accumulated to osmotically significant levels in salt-stressed S. meliloti cells; rather, these compounds, like sucrose and ectoine, were catabolized during early exponential growth, and contributed indirectly to enhance the cytosolic levels of two endogenously synthesized osmolytes, glutamate and the dipeptide N-acetylglutaminylglutamine amide. The ecological implication of the use of these disaccharides as osmoprotectants is discussed.

Toxicity and osmoprotective activities of analogues of glycine betaine obtained by solid phase organic synthesis towards Sinorhizobium meliloti.

Seven analogues of the bacterial osmoprotectant glycine betaine (GB, trimethylammonioacetate), in which the methyl groups of the Me3N+ moiety are replaced by various substituents, were obtained by SPOS using Wang resin. Their biological activities (osmoprotection vs toxicity), appeared closely related to their uptake efficiency and their catabolism in the betaine-demethylating model bacterium Sinorhizobium meliloti.

Sucrose is a nonaccumulated osmoprotectant in Sinorhizobium meliloti.

Intracellular accumulation of sucrose in response to lowered water activity seems to occur only in photosynthetic organisms. Here we demonstrate, for the first time, the potent ability of this common sugar, supplied exogenously, to reduce growth inhibition of Sinorhizobium meliloti cells in media of inhibitory osmolarity. Independently of the nature of the growth substrates and the osmotic agent, sucrose appears particularly efficient in promoting the recovery of cytoplasmic volume after plasmolysis. Surprisingly, sucrose is not accumulated by the bacteria at an osmotically efficient level. Instead, it strongly stimulates the accumulation of the main endogenous osmolytes glutamate and N-acetylglutaminylglutamine amide (NAGGN). Examining cell volume changes during the hyperosmotic treatment, we found a close correlation between the enhancement of the osmotically active solute pool and the increase in cell volume. Sucrose shares several features with ectoine, another nonaccumulated osmoprotectant for S. meliloti. Overall, osmoregulation in S. meliloti appears to be strongly divergent from that in most bacteria.

Differential Effects of Dimethylsulfoniopropionate, Dimethylsulfonioacetate, and Other S-Methylated Compounds on the Growth of Sinorhizobium meliloti at Low and High Osmolarities.

An extract from the marine alga Ulva lactuca was highly osmoprotective in salt-stressed cultures of Sinorhizobium meliloti 102F34. This beneficial activity was due to algal 3-dimethylsulfoniopropionate (DMSP), which was accumulated as a dominant compatible solute and strongly reduced the accumulation of endogenous osmolytes in stressed cells. Synthetic DMSP also acted as a powerful osmoprotectant and was accumulated as a nonmetabolizable cytosolic osmolyte (up to a concentration of 1,400 nmol/mg of protein) throughout the growth cycles of the stressed cultures. In contrast, 2-dimethylsulfonioacetate (DMSA), the sulfonium analog of the universal osmoprotectant glycine betaine (GB), was highly toxic to unstressed cells and was not osmoprotective in stressed cells of wild-type strains of S. meliloti. Nonetheless, the transport and accumulation of DMSA, like the transport and accumulation of DMSP and GB, were osmoregulated and increased fourfold in stressed cells of strain 102F34. Strikingly, DMSA was not toxic and became highly osmoprotective in mutants that are impaired in their ability to demethylate GB and DMSA. Furthermore, 2-methylthioacetate and thioglycolic acid (TGA), the demethylation products of DMSA, were excreted, apparently as a mechanism of cellular detoxification. Also, exogenous TGA and DMSA displayed similar inhibitory effects in strain 102F34. Thus, on the basis of these findings and other physiological and biochemical evidence, we infer that the toxicity of DMSA in wild-type strains of S. meliloti stems from its catabolism via the GB demethylation pathway. This is the first report describing the toxicity of DMSA in any organism and a metabolically stable osmoprotectant (DMSP) in S. meliloti.

Transient Accumulation of Glycine Betaine and Dynamics of Endogenous Osmolytes in Salt-Stressed Cultures of Sinorhizobium meliloti.

The fate of exogenously supplied glycine betaine and the dynamics of endogenous osmolytes were investigated throughout the growth cycle of salt-stressed cultures of strains of Sinorhizobium meliloti which differ in their ability to use glycine betaine as a growth substrate, but not as an osmoprotectant. We present (sup13)C nuclear magnetic resonance spectral and radiotracer evidence which demonstrates that glycine betaine is only transiently accumulated as a cytoplasmic osmolyte in young cultures of wild-type strains 102F34 and RCR2011. Specifically, these strains accumulate glycine betaine as a preferred osmolyte which virtually prevents the accumulation of endogenous osmolytes during the lag and early exponential phases of growth. Then, betaine levels in stressed cells decrease abruptly during the second half of the exponential phase. At this stage, the levels of glutamate and the dipeptide N-acetylglutaminylglutamine amide increase sharply so that the two endogenous solutes supplant glycine betaine in the ageing culture, in which it becomes a minor osmolyte because it is progressively catabolized. Ultimately, glycine betaine disappears when stressed cells reach the stationary phase. At this stage, wild-type strains of S. meliloti also accumulate the disaccharide trehalose as a third major endogenous osmolyte. By contrast, glycine betaine is always the dominant osmolyte and strongly suppresses the buildup of endogenous osmolytes at all stages of the growth cycle of a mutant strain, S. meliloti GMI766, which does not catabolize this exogenous osmoprotectant under any growth conditions.