Proteomic analysis of Lactobacillus pentosus for the identification of potential markers involved in acid resistance and their influence on other probiotic features (original) (raw)
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International Journal of Food Microbiology, 2016
Probiotic bacterial cultures require resistance mechanisms to avoid stress-related responses under challenging environmental conditions; however, understanding these traits is required to discern their utility in fermentative food preparations, versus clinical and agricultural risk. Here, we compared the proteomic responses of Lb. pentosus MP-10, a potentially probiotic lactic acid bacteria isolated from brines of naturally fermented Aloreña green table olives, exposed to sub-lethal concentrations of antibiotics (amoxicillin, chloramphenicol and tetracycline) and biocides (benzalkonium chloride and triclosan). Several genes became differentially expressed depending on antimicrobial exposure, such as the up-regulation of protein synthesis, and the downregulation of carbohydrate metabolism and energy production. The antimicrobials appeared to have altered Lb. pentosus MP-10 physiology to achieve a gain of cellular energy for survival. For example, biocide-adapted Lb. pentosus MP-10 exhibited a down-regulated phosphocarrier protein HPr and an unexpressed oxidoreductase. However, protein synthesis was over-expressed in antibiotic-and biocide-adapted cells (ribosomal proteins and glutamyl-tRNA synthetase), possibly to compensate for damaged proteins targeted by antimicrobials. Furthermore, stress proteins, such as NADH peroxidase (Npx) and a small heat shock protein, were only over-expressed in antibiotic-adapted Lb. pentosus MP-10. Results showed that adaptation to sub-lethal concentrations of antimicrobials could be a good way to achieve desirable robustness of the probiotic Lb. pentosus MP-10 to various environmental and gastrointestinal conditions (e.g., acid and bile stresses).
Applied and Environmental Microbiology, 2005
Two-component regulatory systems are one primary mechanism for environmental sensing and signal transduction. Annotation of the complete genome sequence of the probiotic bacterium Lactobacillus acidophilus NCFM revealed nine two-component regulatory systems. In this study, the histidine protein kinase of a two-component regulatory system (LBA1524HPK-LBA1525RR), similar to the acid-related system lisRK from Listeria monocytogenes (P. D. Cotter et al., J. Bacteriol. 181:6840-6843, 1999), was insertionally inactivated. A whole-genome microarray containing 97.4% of the annotated genes of L. acidophilus was used to compare genome-wide patterns of transcription at various pHs between the control and the histidine protein kinase mutant. The expression pattern of approximately 80 genes was affected by the LBA1524HPK mutation. Putative LBA1525RR target loci included two oligopeptide-transport systems present in the L. acidophilus genome, other components of the proteolytic system, and a LuxS homolog, suspected of participating in synthesis of the AI-2 signaling compound. The mutant exhibited lower tolerance to acid and ethanol in logarithmic-phase cells and poor acidification rates in milk. Supplementation of milk with Casamino Acids essentially restored the acid-producing ability of the mutant, providing additional evidence for a role of this two component system in regulating proteolytic activity in L. acidophilus.
Phenomic and proteomic characterization of Lactobacillus casei in response to acid stress
New Biotechnology, 2009
Although members of the Lactobacillus casei group are known to survive under acidic conditions, the underlying mechanisms of growth at acidic condition and the impact of low pH on the relative level of protein expression at the cell surface remain poorly studied. Material and Methods: After confirming the taxonomy of L. casei strain GCRL 12 which was originally isolated from cheese and confirmed by 16S rRNA sequence analysis, the impact of acidic pH on growth rate was determined. Results: Late log-phase cells cultured at pH 4.0 showed obvious changes in Gram staining properties while transmission electron microscopy analysis revealed evidence of structural distortions of the cell surface relative to the controls cultured at pH 6.5. When comparing cytosolic or whole cell preparations on SDS-PAGE, few changes in protein profiles were observed under the two growth conditions. However, analysis of surface protein extracted by 5M LiCl demonstrated changes in the proportions of proteins present in the molecular weight range of 10 to 80 kDa, with some proteins more dominant at pH 6.5 and other at pH 4. Conclusion: These data suggest that surface proteins of this strain are associated with growth and survival at low pH. The function of these proteins is subject to further investigation.
Comprehensive Reviews in Food Science & Food Safety, 2020
Probiotics are defined as live microorganisms that improve the health of the host when administered in adequate quantities. Nonetheless, probiotics encounter extreme environmental conditions during food processing or along the gastrointestinal tract. This review discusses different environmental stresses that affect probiotics during food preparation, storage, and along the alimentary canal, including high temperature , low temperature, low and alkaline pH, oxidative stress, high hydrostatic pressure, osmotic pressure, and starvation. The understanding of how probiotics deal with environmental stress and thrive provides useful information to guide the selection of the strains with enhanced performance in specific situations, in food processing or during gastrointestinal transit. In most cases, multiple biological functions are affected upon exposure of the cell to environmental stress. Sensing of sublethal environmental stress can allow for adaptation processes to occur, which can include alterations in the expression of specific proteins.
Proteomic studies on lactic acid bacteria: A review
Biochemistry and biophysics reports, 2018
Probiotics are amongst the most common microbes in the gastro-intestinal tract of humans and other animals. Prominent among probiotics are and . They offer wide-ranging health promoting benefits to the host which include reduction in pathological alterations, stimulation of mucosal immunity and interaction with mediators of inflammation among others. Proteomics plays a vital role in understanding biological functions of a cell. Proteomics is also slowly and steadily adding to the existing knowledge on role of probiotics. In this paper, the proteomics of probiotics, with special reference to lactic acid bacteria is reviewed with a view to understand i) proteome map, ii) mechanism of adaptation to harsh gut environment such as low pH and bile acid, iii) role of cell surface proteins in adhering to intestinal epithelial cells, and iv) as a tool to answer basic cell functions. We have also reviewed various analytical methods used to carry out proteome analysis, in which 2D-MS and LC-MS/...
PROTEOMICS, 2011
Lactobacillus acidophilus NCFM is a probiotic bacterium adapted to survive in the gastrointestinal tract and with potential health benefits to the host. Lactitol is a synthetic sugar alcohol used as a sugar replacement in low calorie foods and selectively stimulating growth of L. acidophilus NCFM. In the present study the whole-cell extract proteome of L. acidophilus NCFM grown on glucose until late exponential phase was resolved by 2-DE (pH 3-7). A total of 275 unique proteins assigned to various physiological processes were identified from 650 spots. Differential 2-DE (DIGE) (pH 4-7) of L. acidophilus NCFM grown on glucose and lactitol, revealed 68 spots with modified relative intensity. Thirty-two unique proteins were identified in 41 of these spots changing 1.6-12.7-fold in relative abundance by adaptation of L. acidophilus NCFM to growth on lactitol. These proteins included b-galactosidase small subunit, galactokinase, galactose-1-phosphate uridylyltransferase and UDP-glucose-4epimerase, which all are potentially involved in lactitol metabolism. This first comprehensive proteome analysis of L. acidophilus NCFM provides insights into protein abundance changes elicited by the prebiotic lactitol.
The acid-stress response in Lactobacillus sanfranciscensis CB1
2001
Lactobacillus sanfranciscensis CB1, an important sourdough lactic acid bacterium, can withstand low pH after initial exposure to sublethal acidic conditions. The sensitivity to low pH varied according to the type of acid used. Treatment of Lb. sanfranciscensis CB1 with chloramphenicol during acid adaptation almost completely eliminated the protective effect, suggesting that induction of protein synthesis was required for the acid-tolerance response. Two constitutively acid-tolerant mutants, CB1-5R and CB1-7R, were isolated using natural selection techniques after sequential exposure to lactic acid (pH 32). Two-dimensional gel electrophoresis analysis of protein expression by non-adapted, acid-adapted and acid-tolerant mutant cells of Lb. sanfranciscensis showed changes in the levels of 63 proteins. While some of the modifications were common to the acid-adapted and acid-tolerant mutant cells, several differences, especially regarding the induced proteins, were determined. The two mutants showed a very similar level of protein expression. Antibodies were used to identify heat-shock proteins DnaJ, DnaK,
Anaerobe, 2012
The viability of the probiotic strain L. acidophilus DSM 20079, after its passage through the simulated gastric and pancreatic juices, was evaluated as function of its pre-growth in a medium containing the known prebiotics pectin or inulin, and was compared to glucose used as control. The presence of pectin or inulin did not affect the growth (12.11 log10 colony forming units/mL and 12.08 log10 colony forming units/mL for pectin and inulin respectively versus 12.22 log10 colony forming units/mL obtained for glucose). Pectin and inulin, in contrast to glucose, induced cell stress resistance against gastrointestinal juices (∆ log10 1 and 2 colony forming units/mL respectively, versus ∆ log10 4.5 for glucose). The data were confirmed by the analysis of the protein pattern following stress treatments which, in the case of microbial cells grown with glucose, revealed a relevant protein degradation after the double passage through simulated gastric and intestinal juices. An impressive metabolic change, as function of the growth conditions, was demonstrated by analyzing the proteomic profile with a μ-2DE system, used herein for the first time as evaluation tool of prebiotic-probiotic interactions. The analysis M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 2 revealed a different pH protein distribution that was mostly acidic in the presence of pectin and neutral-alkaline in the presence of inulin. Both prebiotics stimulated the production of butyrate, a relevant healthy bio-molecule not detectable in the presence of glucose, that was measured by HPLC analysis to be 14.5 fold higher after growth in the presence of inulin, as compared to pectin. Three specific proteins were detected at pH 6 after growth in the presence of pectin or inulin. They could be correlated to the stress resistance and/or to the production of butyrate, the common phenotypic characteristics induced in the bacterial strain by the two prebiotics.
Proteome phenotyping of acid stress-resistant mutants ofLactococcus lactis MG1363
PROTEOMICS, 2007
To cope with medium acidity, Lactococcus lactis has evolved a number of inducible mechanisms commonly referred as acid stress response. To better understand the molecular basis of this response, several mutants constitutively tolerant to acidity were previously obtained by insertional random mutagenesis of L. lactis MG1363. Mutants in which the GMP synthase gene (i.e. guaA), the (p)ppGpp synthase gene (i.e. relA*) or the high affinity phosphate transport system (i.e. pstS) are inactivated are further characterized in this study. 2-DE was performed and showed that 42, 26, and 35 protein spots are positively deregulated in the guaA, relA*, and pstS mutants, respectively, as compared to the wild-type strain. Most of these proteins were identified by MS. Proteomes comparison of the mutants guaA, relA*, and pstS as well as the acid adaptation proteome of the wild-type strain revealed (i) the presence of numerous overlaps and (ii) that only five proteins were overexpressed in the four conditions, suggesting that these proteins play a crucial role in the constitutive acid stress tolerance of the mutants and in the acid tolerance response of the wild-type strain.
Research in Microbiology, 2010
F 1 F 0-ATPase has been identified as an operon directly involved in the tolerance of probiotic bacteria towards a hostile acidic environment encountered in the stomach. Expression of atpD (a key part of the F 1 F 0-ATPase operon) gene of the two putative probiotic Lactobacillus plantarum isolates (Lp9 and Lp91) under different in vitro pH conditions which closely mimic the physiological environment prevalent in the human gut was investigated by quantitative real-time PCR (RT-qPCR). A battery of housekeeping genes, i.e. gapB, dnaG, gyrA, ldhD, rpoD and 16S rRNA, were evaluated using geNorm 3.4 Excel-based application for normalizing atpD gene expression in Lp9 and Lp91. The most stably expressed genes were found to be gapB, gyrA and ldhD. Although both putative probiotic L. plantarum isolates investigated in this study were able to survive acid stress under in vitro conditions, amongst the two, Lp91 exhibited relatively greater acid tolerance, as revealed by 4.7-fold upregulation of the atpD gene as well as higher log counts at pH 2.5 after 90 min These results clearly demonstrate that expression of the 'atp' operon was chiefly instrumental in in vitro survival and tolerance of test cultures at acidic conditions encountered in the stomach.