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Proteomic analysis of secreted cell surface proteins in S-layer and non-S-layer forming species of the Lactobacillus acidophilus complex 1,2 Johnson , Brant R. 1,2 Barrangou , Rodolphe and Todd R. 1,2 Klaenhammer Abstract The Lactobacillus acidophilus complex is a clade of homologous Gram-positive, lactic acid bacteria including L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. Although these bacteria are closely related, they have varied ecological lifestyles ranging from dairy and food fermentations, to allochthonous probiotics, and autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria, and their interaction with the intestinal mucosa. Notably, the L. acidophilus complex bacteria can be split based on their ability to produce S-layers, which are semi-porous, crystalline arrays of self-assembling, proteinaceous subunits found as the outermost layer of the bacterial cell wall. Based on previous data regarding the identification of S-layer associated proteins (SLAPs) in L. acidophilus, we employed a proteomic analysis of secreted surface proteins of the S-layer forming and non-S-layer forming bacteria of the L. acidophilus complex. Using a modified LiCl extraction protocol coupled with LC-MS/MS, we have proteomically identified the various extracellular proteins and SLAPs of the L. acidophilus complex, including annotated cell surface proteins, as well as conserved hypothetical proteins of unknown function. Analyses of these data highlight the proteomic complexity and differences of the cell surface of probiotic lactobacilli and reveal the potential for SLAPs to mediate intimate interactions with the intestinal mucosa. This opens new avenues for the selection of effective probiotics and the engineering of immunomodulatory bacteria. Background Information & Methodology Heat Map Clustering of Identified Proteins B C A 0.01 6 0 4 Surface-layer Surface-layer associated proteins Bacterial surface (S-) layers are crystalline arrays of selfassembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. Non-covalently bound cell surface proteins, such as Slp and S-layer associated proteins (SLAPs) can be extracted from cells using denaturing salts such as LiCl. Right: A schematic of the localization of Slp, SLAPs, and cell surface proteins in the cell wall of Lactobacillus species. Peptidoglycan Wall teichoic acid (WTA) Surface-layer proteins (Slps) 8 15 800 Surface-layer associated proteins (SLAPs) SlpX Putative uncharacterized protein Putative uncharacterized protein Surface layer protein Cell separation protein Putative bacterial surface layer protein Putative uncharacterized protein Putative bacterial surface layer protein Putative uncharacterized protein Uncharacterized protein Putative bacterial surface layer protein Putative uncharacterized protein Putative uncharacterized protein Cell separation protein Uncharacterized protein S-layer protein S-layer protein Uncharacterized protein Uncharacterized protein Putative uncharacterized protein Uncharacterized protein Uncharacterized protein Cell separation protein Uncharacterized protein Uncharacterized protein Cell separation protein Uncharacterized protein Putative uncharacterized protein Cell envelope-associated proteinase Uncharacterized protein Cell envelope-associated proteinase Putative bacterial surface layer protein Bacterial group 3 Ig-like protein Uncharacterized protein Bacterial group 3 Ig-like protein Bacterial group 3 Ig-like protein Lysin S-layer protein Uncharacterized protein Uncharacterized protein Surface layer protein S-layer protein Uncharacterized protein S-layer protein S-layer protein ABC transporter S-layer protein ABC transporter S-layer protein Uncharacterized protein Uncharacterized protein L. helveticus ATCC 15009 L. helveticus 481-C L. helveticus CNRZ32 L. delbr. subsp. bulgaricus L. casei ATCC 393 L. amylovorus ATCC 3620 L. crispatus chicken isol. Putative uncharacterized protein L. crispatus ATCC 33820 Uncharacterized protein Uncharacterized protein Uncharacterized protein With regard to the S-layer forming Lactobacillus species, there were three main groups of proteins: SLAPs specific to L. helveticus, SLAPs specific to L. amylovorus, and SLAPs specific to L. crispatus. In order to compare the SLAPs identified in these three groups, we focused on each corresponding area on the heat map to view the identified proteins (A, B, and C). Surprisingly, though each group had distinctive homologies, the same types of proteins were seen in each group. In fact, these proteins, including multiple putative uncharacterized proteins, cell surface proteases, and group 3 bacterial Ig-like domain proteins, were the same types of proteins identified as SLAPs in L. acidophilus NCFM. Notably, these putative SLAPs were absent in the non-S-layer producing strains tested, L. delbrueckii subsp. bulgaricus and L. casei. Therefore the exoproteomes of the S-layer species of the L. acidophilus complex are functionally conserved, yet proteomically distinct from each other. L. crispatus CZ6 37 kDa Bacterial group 3 Ig-like protein Uncharacterized protein L. crispatus CZ6 50 kDa S-layer protein Fibronectin domain protein 250 kDa 150 kDa 100 kDa 75 kDa Bacterial group 3 Ig-like protein Putative uncharacterized protein L. helveticus ATCC 15009 L. helveticus 481-C L. helveticus CNRZ32 NCK 1561 L. delbr. subsp. bulgaricus NCK 125 L. casei ATCC 393 NCK 779 L. amylovorus ATCC 3620 NCK 702 L. crispatus chicken isol. NCK 334 L. crispatus ATCC 33820 NCK 948 L. crispatus CZ6 L. delbr. bulg. NCFM SLAPs L.casei L. gallinarum NCK 1560 L. johnsonii NCK 1351 L.reuteri NCK 953 L. gasseri NCK 1088 L. johnsonii c NCFM L. crispatus NCK 776 L. helveticus L. helveticus NCK 778 L. amylovorus NCK 777 L. gallinarum L. crispatus L. helveticus NCFM NCK 936 NCK 246 S-layer protein Oligopeptide ABC transporter substrate Cell separation protein Cell separation protein Putative surface layer protein SLAP extraction and Identification Uncharacterized protein Glycosyl hydrolase family 25 Cell separation protein NCK 230 1600 Bacterial Ig-like domain 3 protein Cell envelope-associated proteinase Cell separation protein NCK 246 2000 1400 Above: The 2,929 identified proteins from the 7 S-layer (highlighted in pink) and 2 non-S-layer (highlighted in blue) were clustered based on the similarity of the identified proteins, and visualized using a red-blue heat map. The colors in the heat map represent the spectral counts of the identified proteins (semi-quantitative measure of protein abundance), with red being the most abundant, grey being somewhat present, and blue being slightly or not present. Observing the heat map, the proteins identified in the two non-S-layer strains, L. casei and L. delbrueckii subsp. bulgaricus, are unambiguously dissimilar to the other seven S-layer strains. Furthermore, almost all of the proteins identified in the non-S-layer strains were intracellular proteins, likely a result of the cell lysis occurring at stationary phase when the strains were subjected to LiCl treatment. C L. crispatus SLAPs B L. amylovorus SLAPs A L. helveticus SLAPs Lipid Membrane Lipoteichoic acid (LTA) 400 12 25 kDa 20 kDa 15 kDa 10 kDa Cell surface proteins, including Slp and SLAPs, were extracted from cells using treatment with LiCl, as described previously (Johnson et al., 2013). Above: When subjecting the samples to electrophoresis, the SLAP extractions from these sixteen strains revealed a diverse array of banding profiles in each of the Slayer producing strains. Notably, compared to S-layer strains, there were very few proteins extracted from the non-S-layer forming strains using LiCl. These data indicate that the exoproteomes of S-layer forming lactobacilli are more diverse than those without S-layers. We proteomically identified nine of the sixteen strains. Three L. crispatus strains, three L. helveticus strains, one L. amylovorus, one L. casei, and one L. delbrueckii subsp. bulgaricus were characterized by proteomic analyses. Proteins were identified using L. acidophilus NCFM LC-MS/MS and categorized Uncharacterized protein based on their predicted L. crispatus chicken isolate Bacterial 3 Ig-like domain function. Left: The protein L. crispatus ATCC 33820 distribution of identified Putative S-layer protein proteins among the nine L. helveticus ATCC 15009 Sugar transport protein strains tested, as well as L. Ribosomal protein L. helveticus 481-C acidophilus NCFM for Intracellular/moonlighting comparison. In the S-layer L. helveticus CNRZ32 protein forming lactobacilli, there is a Fibronectin-binding L. bulgaricus marked increase in protein Cell division-related uncharacterized proteins L. casei ATCC 393 protein Peptide transport protein (dark blue), while in the nonL. amylovorus ATCC 3620 S-layer strains (L. bulgaricus Bacteriocin/quorum and L. casei ) there is an sensing protein L. crispatus CZ6 increase in intracellular 0 20 40 60 80 100 proteins (light blue & yellow). % Distribution 1Graduate L. helveticus ATCC 15009 Lactobacillus casei ATCC334 L. helveticus 481-C Lactobacillus gasseri ATCC33323 L. helveticus CNRZ32 Lactobacillus johnsonii NCC533 L. delbr. subsp. bulgaricus Lactobacillus delbrueckii subsp. bulgaricus L. casei ATCC 393 Lactobacillus helveticus CNRZ32 L. amylovorus ATCC 3620 Lactobacillus acidophilus NCFM L. crispatus chicken isol. Lactobacillus amylovorus GRL1112 L. crispatus ATCC 33820 Lactobacillus crispatus ST1 The Lactobacillus acidophilus complex is comprised of L. acidophilus, L. crispatus, L. amylovorus, L. helveticus, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. Left: A phylogenetic tree of the 16S rRNA genes of the L. acidophilus complex. The tree was made using the neighbor-joining method, rooted with L. casei. Notably, the Slayer forming strains (highlighted in pink box) phylogenetically cluster distinct from the non-S-layer forming lactobacilli (highlighted in blue box). Conclusions & References • • • • • The exoproteomes of the L. acidophilus complex are distinctively diverse. S-layers appear to be important scaffolds for non-covalently bound extracellular cell surface proteins, including S-layer associated proteins (SLAPs). SLAPs in S-layer forming Lactobacillus species are functionally conserved but proteomically distinct. There is potential for SLAPs and cell surface proteins to mediate intimate interactions with the intestinal mucosa. Further functional characterization of these exoproteomes opens new avenues for the selection of effective probiotics, and the engineering of immunomodulatory bacteria. Johnson, B., K. Selle, S. O’Flaherty, Y.J. Goh, and T.R. Klaenhammer. 2013. Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM. Microbiology 159:2269-2282. This study was funded by the North Carolina Agricultural Foundation and DuPont Nutrition and Health. The authors wish to thank Dr. Sarah O’Flaherty and Dr. Yong Jun Goh, and Rosemary Sanozky-Dawes for helpful discussion and review. Program in Microbiology, North Carolina State University, Raleigh, NC, USA 2Department of Food, Bioprocessing, and Nutrition Science, North Carolina State University, Raleigh, NC, USA