Application of comprehensive proteomics to map metabolic pathways of Lactobacillus casei under carbohydrate starvation and growth under low pH

Lactic acid bacteria, and more particularly lactobacilli, have been used for the production of fermented foods for centuries. They are amongst the most common microorganisms in the gastrointestinal tract of human and other animals; several lactobacilli have been recognised as probiotics due to their wide range of health promoting effects. One such probiotic species Lactobacillus casei, first described in cheese, is widely distributed in fermented food products and present within the human intestinal tract and oral cavity. Lb. casei strains have been recognized as having probiotic traits and have therefore been applied in numerous commercial food-based and drink-based health supplement products. Survival of Lactobacillus species in harsh environments is one of the most important criteria for industrial and food-based applications. Environmental stresses affect the physiological properties, however, little is known about the molecular mechanisms underpinning survival of Lb. casei under stress conditions like those introduced during food fermentation, including changing composition of the food matrix, oxygen stress and acidity of the carrier food. To this end this thesis aims to provide information on the growth and physiological response of Lb. casei under nutritional stress (carbohydrate starvation) as well as growth under low pH conditions. An initial study employed an integrated global proteomic response of the non-starter lactic acid bacteria Lb. casei strain GCRL163 under carbohydrate depletion to understand aspects of its survival following cessation of fermentation. This study built on prior published work from our group on lactose starvation in Lb. casei which, based on partial proteomic analysis and single-point analysis of end-products of metabolism, indicated that some enzymes involved in the glycolytic pathway were upregulated during lactose starvation and that Tween 80 and other media constituents were potentially being utilized. The proteome of Lb. casei GCRL163 was analyzed quantitatively after growth in modified MRS (with and without Tween 80) with different levels of lactose (0% lactose, starvation; 0.2% lactose, growth limiting; 1% lactose, non-growth-limited control) using gel-free proteomics. Results revealed that carbohydrate starvation led to suppression of lactose and galactose catabolic pathways, which is consistent with the known mechanisms of induction of genes relating to lactose/galactose metabolism in the presence of the cognate substrate, as well as pathways for nucleotide and protein synthesis. Enzymes of the glycolysis/gluconeogenesis pathway, amino acid synthesis, and pyruvate and citrate metabolism became more abundant as well as other carbohydrate catabolic pathways, suggesting increased optimization of intermediary metabolism and scavenging alternative carbon and energy sources. This was consistent with temporal end-product analysis. Tween 80 did not affect growth yield; however, proteins related to fatty acid biosynthesis were repressed in the presence of Tween 80. These data suggested that Lb. casei adeptly switches to a scavenging mode, using both citrate and potentially Tween 80, and efficiently adjusts energetic requirements when carbohydrate starved and thus can sustain survival for weeks to months. The presence of caproic and octanoic acids in culture fluids suggested that Tween 80 was being degraded during lactose starvation. However, the mMRS basal medium contained both citrate and acetate as possible carbon sources, so it was not clear whether Tween 80 was serving as a carbon source or whether it aided growth in the absence of lactose by eliminating biotin requirement, as indicated in the early literature (Williams et al., 1947). To investigate whether Tween 80 could be degraded as a carbon source, Lb. casei GCRL163 was cultured in mMRS with combinations of citrate and acetate, with and without Tween 80. Gel-free proteomic analysis was used to provide insight into the metabolic changes which occurred when media either contained or lacked citrate and Tween 80. In the absence of carbohydrate, acetate and citrate as fermentable substrates, glycolysis and protein synthesis were strongly repressed, however moderate growth of GCRL163 was supported indicating that this strain has the ability to metabolise Tween 80. Growth could not be explained by release of oleic acid from Tween 80 following autoclaving and subsequent incubation of the medium at 30 oC, as <6% of the Tween 80 was released as oleic acid and this did not change in controls over the growth period. The presence of Tween 80 strongly affected metabolic pathways related to sorbitol/sorbose, glycerol metabolism and fatty acid biosynthesis, up-regulating these pathways. The metabolism of Tween 80 appears to have a substantial effect on providing protection against acid shock and, based on the proteome, does so by strongly promoting fatty acid biosynthesis, protein hydrolysis and amino acid accumulation, and also stimulates compatible solute uptake and cell wall proteins. This response appears to be caused by Tween 80 causing cell membrane disturbances and generalised stress responses. A further study was conducted to examine the growth of Lb. casei at low pH to simulate the acidic environments in food products which may affect the survival and thus the potential efficacy of probiotic bacteria. Acid stress adaptation responses of Lb. casei strains, including strain MJA12 which was isolated from the fermented milk product YakultTM (which claims to contain the Shirota probiotic strain) and a genetically distinct Cheddar cheese isolate GCRL163, were investigated using gel-free, label-free proteomic analysis. The strains were grown under anaerobic conditions in MRS broth adjusted to and maintained at pH 4.5 or pH 6.5 in fermenters, with biomass collected during mid-exponential and early stationary growth phase. Approximately 35% of the proteome of each strain was identified to a >95% confidence level. Independent of growth phase, physiological adaptations to grow at low pH as predicted by the proteomic responses were different between the strains, however both exhibited increased abundance in olipopeptide/dipepetide importers, peptidases/proteinases, branched chain aminotransferase, and the fatty acid biosynthetic pathway. These responses suggested exogenous amino acid accumulation, cell wall modulation and increased fatty acid unsaturation are the main means by which Lb. casei combats sudden acid stress, indicating that similar mechanisms are adopted during acid adaptation. Protein abundance data also revealed that acid adaptation varied between the strains in a growth phase-dependent manner, with strain MJA12 exhibiting most known acid adaptation-type responses when in the stationary growth phase, unlike strain GCRL163 which seems more directly responsive to acidic pH conditions earlier in growth. In addition, acid stress resulted in strain MJA12 (but not strain GCRL163) possessing increased levels of malolactic fermentation associated enzymes. The strain-dependent differences in the proteomes suggest adaptation of Lb. casei strains varies and thus strains could potentially react, behave and have different levels of persistence in either food or host systems. Furthermore, the effect of growth under acidic conditions on the adhesion ability of Lb. casei cheese isolate GCRL163 and fermented milk isolate MJA12 was examined using HT-29 cells as an in vitro model for intestinal epithelium cells. The strains were grown under anaerobic conditions in MRS broth adjusted and maintained at pH 4.5 or pH 6.5 in fermenters, with biomass collected during early stationary growth phase. Lb. casei showed increased numbers of bacterial cells attaching to the cell line after adaptation to grow at pH 4.5 compared with cultures grown at pH 6.5. Gel-free proteomic analysis was used to understand the nature of these changes. Treatment with 5 M LiCl, with the goal of enriching surface-associated proteins, demonstrated that proteins enriched in these fractions consisted mainly of transmembrane proteins, membrane-associated proteins, extracellular secreted proteins and weakly enriched peptidoglycan related proteins, including various small cytosolic proteins. Other proteins present in LiCl extracts included glycolytic proteins (glyceraldehydes-3-phosphate dehydrogenase, enolase and lactate dehydrogenase) and several proteins of unknown functionality, notably a highly enriched hydrolase. Exposing HT-29 cells to 10 ˜í¬¿g of dialysed LiCl extract decreased subsequent binding of both Lb. casei strains, implicating the involvement of proteins in these extracts in binding. Collectively, the results in this thesis increase the understanding of the physiological response of Lb. casei when grown under conditions that may be encountered in fermented foods and which pose specific stress conditions, namely carbohydrate limitation and acid stress. The study presents the functional analysis of proteins which also provide new insight into the metabolic pathways engaged by Lb. casei in dealing with food relevant stresses.