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. Proteomic analysis of secreted cell surface proteins in S-layer and non-S-layer forming species of the Lactobacillus acidophilus complex Abstract Brant R. Johnson 1,2, Rodolphe Barrangou 1,2, and Todd R. Klaenhammer 1,2 1 Graduate Program in Microbiology, North Carolina State University, Raleigh, NC, USA 2 Department of Food, Bioprocessing, and Nutrition Science, North Carolina State University, Raleigh, NC, USA 250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 20 kDa 15 kDa 10 kDa NCFM NCK 936 NCK 777 NCK 778 NCK 776 NCK 246 L. helveticus L. crispatus L. gallinarumL. amylovorus NCFM NCK 230 NCK 246 NCK 1088 NCK 953 NCK 1351 NCK 1560 L. helveticus L. crispatus L. gallinarum NCFM SLAPs NCK 948 NCK 334 NCK 702 NCK 779 NCK 125 NCK 1561 L. johnsonii cL. gasseriL.reuteriL. johnsoniiL.caseiL. delbr. bulg. Background Information & Methodology SLAP extraction and Identification Heat Map Clustering of Identified Proteins Conclusions & References ABC transporter L. crispatus CZ6 L. crispatus ATCC 33820L. crispatus chicken isol. L. amylovorus ATCC 3620 L. casei ATCC 393L. delbr. subsp. bulgaricusL. helveticus CNRZ32L. helveticus 481-C L. helveticus ATCC Uncharacterized protein S-layer protein Bacterial group 3 Ig-like protein Uncharacterized protein Cell separation protein Uncharacterized protein S-layer protein Bacterial group 3 Ig-like protein S-layer protein Uncharacterized protein S-layer protein Surface layer protein S-layer protein Uncharacterized protein L. crispatus SLAPs C L. crispatus CZ6 L. crispatus ATCC 33820L. crispatus chicken isol.L. amylovorus ATCC 3620 L. casei ATCC 393 L. delbr. subsp. bulgaricus L. helveticus CNRZ32 L. helveticus 481-C L. helveticus ATCC SlpX Putative uncharacterized protein Cell separation protein Putative uncharacterized protein S-layer protein Putative uncharacterized protein Cell separation protein Putative uncharacterized protein Uncharacterized protein Putative bacterial surface layer protein Uncharacterized protein Glycosyl hydrolase family 25 Oligopeptide ABC transporter substrate Lysin Uncharacterized protein Putative uncharacterized protein Fibronectin domain protein Uncharacterized protein S-layer protein Putative uncharacterized protein L. amylovorus SLAPs B L. crispatus CZ6 L. crispatus ATCC 33820L. crispatus chicken isol.L. amylovorus ATCC 3620L. casei ATCC 393 L. delbr. subsp. bulgaricus L. helveticus CNRZ32 L. helveticus 481-C L. helveticus ATCC Bacterial Ig-like domain 3 protein Putative uncharacterized protein Surface layer protein Putative bacterial surface layer protein Putative uncharacterized protein Uncharacterized protein Cell envelope-associated proteinase Cell separation protein Putative surface layer protein L. helveticus SLAPs A 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 S- layer forming strains (highlighted in pink box) phylogenetically cluster distinct from the non-S-layer forming lactobacilli (highlighted in blue box). Bacterial surface (S-) layers are crystalline arrays of self- assembling, 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. A B C 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 S- layer 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 LC-MS/MS and categorized based on their predicted function. Left: The distribution of identified proteins among the nine strains tested, as well as L. acidophilus NCFM for comparison. In the S-layer forming lactobacilli, there is a marked increase in uncharacterized proteins (dark blue), while in the non- S-layer strains (L. bulgaricus and L. casei ) there is an increase in intracellular proteins (light blue & yellow). 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. 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. Johnson, B., K. Selle, S. O’Flaherty, Y.J. Goh, and T.R. Klaenhammer Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM. Microbiology 159: 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. 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.