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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