Proteomic Analysis Of A New Probiotic Bacterium Lactobacillus Casei Zhang

Lactobacillus casei Zhang, isolated from traditional home-made koumiss in Inner Mongolia of China, was considered as a new probiotic bacterium by probiotic selection tests, especially L. casei Zhang was able to anti-acid and bile salts.In this study, a proteomic research was carried out to identify and characterize proteins expressed by L. casei Zhang under acid or bile salts stress after optimizing the two-dimensional gel electrophoresis (2-DE) conditions.Cytosolic proteins of the strain cultivated in MRS broth in the exponential phase and stationary phase were resolved by two-dimensional gel electrophoresis (2-DE) using pI 4-7 linear gradients. The number of protein spots quantified from the gels was 487±21 (exponential phase) and 494±13 (stationary phase) respectively, among which a total of 110 proteins were identified by MALDI-TOF/MS and/or MALDI-TOF/TOF according to significant growth phase-related differences or high expression intensity proteins. Accompanied with analysis of the COG, CAI, GRAVY value of each protein identified to the whole set of genes encoding proteins of L. casei Zhang, the study provided a very first insight into the profile of protein expression as a reference map of L. casei.Forty-seven spots of L. casei Zhang were also found in the study showing statistically significant differences between exponential phase and stationary phase. Thirty-three of them increased at least 2.5-fold in the stationary phase in comparison with the exponential phase, and including nineteen protein spots (e.g., Hsp20, Dnak, GroEL, LuxS, PK, GalU) whose intensity up-shifted above 3.0-fold. The transcriptional profiles were conducted to confirm several important differential expression proteins by using real-time qRT-PCR. The analysis suggests that the differentially expressed proteins were mainly categorized as stress response proteins and key components of central and intermediary metabolism, implicating these proteins might play potential important role for the adaptation to the surroundings, especially the accumulation of lactic acid in the course of growth, and the physiological processes in bacteria cell. The furter proteomic analysis of L. casei XM2-1 in exponential phase and stationary phase found that sixteen spots were in the study showing statistically significant differences, including 12 proteins up-shifted and 2 proteins down-regulated. From one point of view, the similarity of 2-DE maps between L. casei XM2-1 and L. casei Zhang proved they are belonging to the species of L. casei. From another point, there was particularly differential expression proteins depended on the individual strain.Proteome of L. casei Zhang was studied to achieve a better understanding of the ability to anti-bile salts stress. The protein patterns obtained under growing with 1.5 % or bile salts free condition were compared by 2-DE. Twenty-six proteins were identified to display clearly differential expression, and function annotation demonstrated that they mainly belonged to stress response proteins, carbohydrates metabolism, maintaining membrane stability, amino acid metabolism and anti-oxidative stress response, translation, ribosomal protein, nucleotide metabolism, fat acid metabolism and unknown function. The findings indicated that the response of L. casei Zhang to bile salts stress involves a complex physiological mechanism. From the results of further proteomic study, which the L. casei Zhang instantaneous exposing in 1.0 % oxgall for 30min under changeless of pH condition, the conserved hypothetical protein LCAZH₀885, cysteine synthase, acetyltransferase, aminopeptidase C, and dioxygenase, which deduced by long time bile salts stress, were also found changing expression. Moreover, transcriptional expression of some genes was validated by using the real-time qRT-PCR. On these results, the analysis presumes that proteins involved in the stress response, maintaining cell membrane stability, and anti-oxidative stress response may be playing an important role for bile salts tolerance in L. casei strains.To identify proteins that are differentially expressed by two strains of L. casei Zhang and L. casei XM2-1 growing under conditions of low pH, whole cellular proteins extracted from bacteria grown in batch culture at pH 7.0 or 5.5 were analyzed by 2-DE electrophoresis. Fifteen and forteen proteins, including stess proteins, esterase, and translation factor, etc., altered expression at low pH respectively in L. casei Zhang and L. casei XM2-1. Tht futher analysis shows that there were similar deduced proteins by the acid stress in both L. casei strains, and also had the special expression proteins depended on each strain.