Lactobacillus casei Low-Temperature, Dairy-Associated Proteome

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Lactobacillus casei Low-Temperature, Dairy-Associated Proteome Promotes Persistence in the Mammalian Digestive Tract Bokyung Lee, Sybille Tachon, Richard A. Eigenheer, Brett S Phinney, and Maria L Marco J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00387 • Publication Date (Web): 07 Jul 2015 Downloaded from http://pubs.acs.org on July 12, 2015

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Journal of Proteome Research

Lactobacillus casei Low-Temperature, Dairy-Associated Proteome Promotes Persistence in the Mammalian Digestive Tract Bokyung Lee,† Sybille Tachon,† Richard A Eigenheer,‡ Brett S Phinney,‡ and Maria L Marco*† †

Department of Food Science & Technology, University of California, Davis

‡

Proteomics Core Facility, Genome Center, University of California, Davis

*Corresponding Author: Maria L. Marco One Shields Avenue University of California Davis, CA 95616 Phone: 530-752-1516 Fax: 530-752-4759 Email: [email protected]

Running Title: Milk-carrier effects on Lactobacillus casei in vivo

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ABSTRACT We found that incubation of probiotic Lactobacillus casei BL23 in milk at 4 °C prior to ingestion increased its survival in the mammalian digestive tract. To investigate the specific molecular adaptations of L. casei to milk, we used tandem mass spectrometry to compare proteins produced by L. casei BL23 at 4 °C in milk to those in exponential and stationary phase cells in laboratory culture medium at either 37 °C or 4 °C. These comparisons revealed a core of expressed L. casei proteins as well as proteins produced in either a growth-phase or temperature-specific manner. In total, 205 L. casei proteins were uniquely expressed or detected in higher abundance specifically as a result of incubation in milk and included an over-representation of proteins for cell surface modification, fatty acid metabolism, amino acid transport and metabolism, and inorganic ion transport. Genes for DltD (D-alanine transfer protein), FabH (3-oxoacyl-ACP synthase), RecA (recombinase A), and Sod (superoxide dismutase) were targeted for inactivation. The competitive fitness of the mutants was altered in the mouse intestine compared to wild-type cells. These results show that the food matrix can have a profound influence on dietary (probiotic) bacteria and their functional significance in the mammalian gut.

KEYWORDS: proteomics, probiotics, Lactobacillus casei, dairy, milk, low temperature, microbiota, IBD, intestinal fitness, mass spectrometry


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INTRODUCTION Probiotic bacteria are typically targeted for delivery to the digestive tract where they are intended to maintain or improve host health.1 Health benefits conferred by probiotic strains are generally understood to be mediated either through modulation of the intestinal microbiota, alteration of the immune system, or direct interactions with intestinal epithelial cells.2,3 The specific probiotic cell components responsible for these effects are only partially known and thus far encompass a very limited number of cell-surface and secreted compounds including lipoteichoic acids and certain exopolysaccharides, proteins, and small metabolites.3,4 Dairy products are common carriers of probiotics in commercial products and clinical trials.5 Presently, the importance of consuming probiotics in dairy products as opposed to other food/beverage/supplement formats is not well understood. Numerous reports using simulated gastric conditions in vitro have indicated that milk provides sufficient buffering capacity for probiotic survival under acidic conditions in the stomach.6-9 Specific components of milk such as calcium, lactoferrin, and casein-derived peptides were suggested to support the growth of Grampositive probiotic bacteria while inhibiting some Gram-negative pathogens.10 Moreover, probiotics might benefit from access to milk derived carbohydrates such as lactose or oligosaccharides during transit through the intestine.11-15 To this regard, carbohydrate and amino acid metabolism were among several functional categories induced in probiotic Lactobacillus and Bifidobacterium during growth in milk.11,16-18 These bacteria can also undergo dynamic changes in gene expression over-time in milk at optimal temperatures as well as express genes with putative probiotic function.16 However, the relationships between probiotic bacteria adaptations expressed in milk and the intestinal persistence and efficacy of those strains have yet to be examined.



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Strains of Lactobacillus casei are among the most commonly applied probiotics in commercial dairy products.19,20 In clinical studies, L. casei was shown to reduce the duration of respiratory infections and diarrhea21-24 and reduce inflammation in rheumatoid arthritis.25 L. casei is a member of lactic acid bacteria, a diverse group of Gram-positive bacteria in the Firmicutes phylum that produces lactic acid as the primary end-product of fermentative growth. L. casei versatile species and multi-locus sequence typing, genome sequencing, and other genetic approaches have revealed a broad genetic diversity as well as niche-specific adaptations of L. casei to dairy and other environments.26-33 For example, strains of L. casei are able to persist and remain metabolically active in the human gut for at least several days after consumption.34,35 Herein, we investigate L. casei BL23, a strain that was previously found to confer probiotic effects in pre-clinical animal models of inflammatory bowel disease (IBD)36-38 as well as provided protection against Listeria monocytogenes in gnotobiotic mice.39 L. casei BL23 is also a model organism for elucidating carbohydrate metabolism, stress response pathways, and hostmicrobe interactions of the L. casei species.40-43 Proteome and transcriptome level analyses of L. casei BL23 and other strains in vitro have shown that these bacteria express different genes and proteins depending on their environmental context.44-47 What remains to be understood is whether there are specific adaptations expressed by L. casei in dairy products that are relevant for probiotic function in the intestine. In this study, we hypothesized that L. casei BL23 adapts to the dairy delivery matrix under low-temperature storage conditions (4 ºC) and that those adaptations contribute to the survival of this bacterial strain in the mammalian digestive tract. We first monitored the survival of this strain in the murine digestive tract after incubation in milk compared to other incubation conditions and delivery matrices. Shot-gun proteomics was then used to identify the proteins


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expressed by L. casei BL23 in milk and determine which proteins were uniquely expressed or more abundant in milk compared to cells incubated in standard laboratory culture medium (MRS) at different temperatures and phases of growth. Several milk-inducible genes of strain BL23 were then targeted for knockout mutagenesis to quantify their roles in L. casei survival in milk and the murine digestive tract.



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EXPERIMENTAL SECTION Bacterial Strains and Culture Conditions Bacterial strains and plasmids used in this study are listed in Table 1. L. casei strains were routinely grown under static conditions at 37 °C in a modified deMan, Rogosa and Sharpe (MRS) medium48 containing 2 % lactose or in Ultra High Temperature (UHT)-processed 2 % reduced fat milk (Gossner Foods, Inc., Logan, Utah). Escherichia coli DH5α was grown under aeration at 200 rpm at 37 °C in Luria Bertani Lennox broth (LB, Fisher Scientific, Pittsburgh, PA). For effective and selective recovery of L. casei BL23 in mouse intestinal contents, all experiments employed a spontaneous, rifampicin-resistant mutant of L. casei BL23. Rifampicin-resistant mutants have been used for examination of other Lactobacillus strains as well as other bacterial species in complex microbial environments such as the digestive tract.49-51 The rifampicinresistant mutant, BL23-R, was selected from single colony isolates of L. casei BL23 cells grown on MRS agar containing 2 % glucose and 50 µg/ml of rifampicin. This mutant was used for all experiments in this study, including the construction of the gene knockout mutants (see below) and referred to as the wild-type strain throughout this work. When appropriate, the following antibiotics were added to the media at the following concentrations: erythromycin (Erm), 5 µg/ml; rifampicin, 50 µg/ml; and ampicillin (Amp), 50 µg/ml.

Proteome Analysis For proteome profiling, L. casei BL23-R (here referred to as wild-type) was grown for 10 h in MRS at 37 °C until exponential-phase (OD600=1) (L0.5, µ = 0.4 h-1) and 24 h until stationary phase (OD600=9) (L1). Cells were also collected from L1 cultures for inoculation and incubation in MRS (L5) or UHT milk (M5) for 5 d at 4 °C (Supplemental Figure S1A). L. casei cell


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numbers did not increase in milk or MRS at 4 °C during the 5 d incubation, however, the pH of those cultures declined between 0.5 to 0.3 pH units in both media types during that time. Cultures were prepared in triplicate for each of the culture conditions tested. Cell-associated proteins were extracted from 500 ml of each culture (approximately 2.5 x 1011 cells) as described previously52 with minor modifications. Briefly, 165 ml 1 M trisodium citrate and 65 ml buffered saline solution (0.145 M NaCl, 0.016 M sodium β-glycerophosphate, and 0.1% Tween 80, pH 7.0) were added to avoid the precipitation of caseins.52 L. casei was then harvested by centrifugation at 8000 x g for 10 min at 4 °C and cell pellets were washed twice with ice-cold buffer (5 mM sodium phosphate, 1 mM EDTA, and 2 mM β-mercaptoethanol, pH 7.0) 52 and suspended in 50 mM ammonium bicarbonate. The cell suspensions were then lysed by mechanical agitation in ammonium bicarbonate in a MP Fastprep-24 instrument (MP Biomedicals, Santa Ana, CA) with 0.5 g of 0.1 mm zirconia/silica beads (Bio Spec Products Inc., Bartlesville, OK) at maximum speed for 40 sec with cooling on ice for 1 min between two runs. The soluble lysate was collected by centrifugation (2 min at 13,000 x g) and stored at – 20 °C. Protein abundance was quantified by the Bradford assay (Bio-rad Protein assay kit II, Bio-Rad, Hercules, CA) and visualized on a 12 % sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel (Supplemental Figure S1B). An amount of 55 µg protein from each culture was separated on a 12 % SDS-PAGE gel and each lane distributed in three gel slices for in-gel trypsin digestion as described previously.53 Digested peptides were then analyzed by high-performance liquid chromatography (LC) coupled with tandem mass spectrophotometry (MS/MS) analysis on a Thermo Scientific (Thermo Scientific, Waltham, MA) linear ion trap (LTQ) with Michrom Paradigm LC (Michrom Biosciences, Auburn, CA) and CTC Pal autosampler (CTC Analytics, Zwingen, Swizerland).



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The peptides were directly loaded onto an Agilent ZORBAX 300SB C18 reverse phase trap cartridge, which, after loading, was switched in-line with the Michrom Magic C18 AQ 200 µm x 150 mm nano-LC column and through a Michrom Advance Plug and Play nano-spray source connected to the mass spectrometer. Peptides were eluted using a gradient of 0.1% formic acid (A) and 100% acetonitrile (B) with a flow rate of 2ul/min. A 60 minute gradient was ran with 5% to 35% B over 50% to 80% B over three minutes, 80% B for one minute, 80% to 5% B over one minute, and finally held at 5% B for five minutes. MS and MS/MS spectra were acquired using a top 10 method, where the top 10 ions in the MS scan were subjected to automated, low- energy collision induced dissociation. Precursor isolation window was set to 2 Da, relative collision energy was set to 35% and dynamic exclusion was enabled with a repeat count of two and a repeat duration of 30 seconds.

Data Analysis Tandem mass spectra were extracted by the Xcalibur software (version 2.2, Thermo Scientific, San Jose, CA). Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using X!Tandem (The GPM, thegpm.org; version TORNADO (2010.01.01.4)). X!Tandem was set up to search a 2011 NCBI NR Lactobacillus casei BL23 database (12756 entries including an equal number of reverse sequences) assuming digestion with trypsin. X!Tandem was searched with a fragment ion mass tolerance of 0.40 Da and a parent ion tolerance of 1.8 Da. The iodoacetamide derivative of cysteine was specified in X!Tandem as a fixed modification. Deamidation of asparagine and glutamine, oxidation of methionine and tryptophan, sulphone of methionine, tryptophan oxidation to formylkynurenin of tryptophan, and acetylation of the n-terminus were specified in X!Tandem as variable 8 ACS Paragon Plus Environment

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modifications. Scaffold (version Scaffold_3.5.1), Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 80.0% probability by the Peptide Prophet algorithm.54 Protein identifications were accepted if they could be established at greater than 80.0%, and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.55 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. The peptide and protein target-decoy false discovery rates (FDR) were calculated as (0.04% peptide, 1.1% protein) according to previously described methods.56 To perform downstream analyses, a protein was regarded to be present in a growth condition when it was detected in a minimum of two out of the three replicate cultures for assigned spectral count. To allow multiple comparisons between different sample conditions, the normalized spectral abundance factor (NSAF) was applied to all identified proteins.57 Proteins present in levels at least 2-fold greater or lower amounts in the L0.5, L5, or M5 cultures compared to L1 or showed significant differences (p

Lactobacillus casei Low-Temperature, Dairy-Associated Proteome

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