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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. Johnson1,2, Rodolphe Barrangou1,2, and Todd R. Klaenhammer1,2
1Graduate Program in Microbiology, North Carolina State University, Raleigh, NC, USA2Department 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
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10 kDa
NCFMNCK936
NCK777
NCK778
NCK776
NCK246
L. h
elve
ticu
s
L. h
elve
ticu
s
L. c
risp
atus
L. g
alli
nar
um
L. a
myl
ovor
us
NCFMNCK230
NCK246
NCK1088
NCK953
NCK1351
NCK1560
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elve
ticu
s
L. c
risp
atus
L. g
alli
nar
um
NCFMSLAPs
NCK948
NCK334
NCK702
NCK779
NCK125
NCK1561
L. jo
hnso
nii
c
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asse
ri
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uter
i
L. jo
hnso
nii
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sei
L. d
elbr
. bul
g.
Me
thod =
Ward
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L. crispatus CZ6
L. amylovorus ATCC 3620
L. casei ATCC 393
L. bulgaricus
L. helveticus CNRZ32
L. helveticus 481-C
L. helveticus ATCC 15009
L. crispatus ATCC 33820
L. crispatus chicken isolate
Den
dro
gram
Hierarch
ical Clu
stering
Background Information & Methodology
SLAP extraction and Identification
Heat Map Clustering of Identified Proteins
Conclusions & References
L. crispatus CZ6
L. amylovorus ATCC 3620
L. casei ATCC 393
L. bulgaricus
L. helveticus CNRZ32
L. helveticus 481-C
L. helveticus ATCC 15009
L. crispatus ATCC 33820
L. crispatus chicken isolate
L. acidophilus NCFM
0 10 20 30 40 50 60 70 80 90 100
Uncharacterized protein
Bacterial 3 Ig-like domain protein
Putative S-layer protein
Sugar transport protein
Ribosomal protein
Intracellular/moonlighting protein
Fibronectin-binding protein
Cell division-related protein
Peptide transport protein
Bacteriocin/quorum sens-ing protein
% Distribution
ABC transporter
ABC transporter
L. c
risp
atus
CZ6
L. c
risp
atus
AT
CC 3
38
20
L. c
risp
atus
ch
icke
n is
ol.
L. a
myl
ovor
us A
TCC
36
20
L. c
asei
AT
CC 3
93
L. d
elbr
. su
bsp
. bul
gari
cus
L. h
elve
ticu
s CN
RZ3
2
L. h
elve
ticu
s 4
81
-C
L. h
elve
ticu
s AT
CC 1
50
09
Uncharacterized protein
S-layer protein
Bacterial group 3 Ig-like protein
Uncharacterized protein
Cell separation protein
Uncharacterized protein
Uncharacterized protein
Uncharacterized protein
S-layer protein
Bacterial group 3 Ig-like protein
Bacterial group 3 Ig-like protein
Bacterial group 3 Ig-like protein
Bacterial group 3 Ig-like protein
S-layer protein
Uncharacterized protein
S-layer protein
Surface layer protein
S-layer protein
Uncharacterized protein
Uncharacterized protein
Uncharacterized protein
Uncharacterized protein
Uncharacterized protein
L. crispatus SLAPsC
L. c
risp
atus
CZ6
L. c
risp
atus
AT
CC 3
38
20
L. c
risp
atus
ch
icke
n is
ol.
L. a
myl
ovor
us A
TCC
36
20
L. c
asei
AT
CC 3
93
L. d
elbr
. su
bsp
. bul
gari
cus
L. h
elve
ticu
s CN
RZ3
2
L. h
elve
ticu
s 4
81
-C
L. h
elve
ticu
s AT
CC 1
50
09
SlpX
Putative uncharacterized protein
Cell separation protein
Putative uncharacterized protein
Putative uncharacterized protein
Putative uncharacterized protein
S-layer protein
S-layer protein
Putative uncharacterized protein
Cell separation 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
Uncharacterized protein
S-layer protein
S-layer protein
S-layer protein
Putative uncharacterized protein
L. amylovorus SLAPsBM
eth
od
= W
ard
02.8
81
45.7
62
78.6
44
111
.525
14
.40
741
2.1
380
9.8
412
07.6
16
05.3
20
03
L. c
ris
patu
sch
icke
n iso
late
L.
bu
lgari
cu
s
De
nd
rog
ram
Hie
rarc
hic
al C
lus
teri
ng
20000
1600
1400
800
400
15
12
8
6
4
L. c
risp
atus
CZ6
L. c
risp
atus
AT
CC 3
38
20
L. c
risp
atus
ch
icke
n is
ol.
L. a
myl
ovor
us A
TCC
36
20
L. c
asei
AT
CC 3
93
L. d
elbr
. su
bsp
. bul
gari
cus
L. h
elve
ticu
s CN
RZ3
2
L. h
elve
ticu
s 4
81
-C
L. h
elve
ticu
s AT
CC 1
50
09
Bacterial Ig-like domain 3 protein
Putative uncharacterized protein
Surface layer protein
Putative bacterial surface layer protein
Putative bacterial surface layer protein
Putative bacterial surface layer protein
Putative uncharacterized protein
Uncharacterized protein
Uncharacterized protein
Uncharacterized protein
Uncharacterized protein
Uncharacterized protein
Cell envelope-associated proteinase
Cell envelope-associated proteinase
Cell envelope-associated proteinaseCell separation protein
Cell separation protein
Cell separation proteinCell separation proteinPutative surface layer protein
L. helveticus SLAPsA
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).
Lactobacillus crispatus ST1
Lactobacillus amylovorus GRL1112
Lactobacillus acidophilus NCFM
Lactobacillus helveticus CNRZ32
Lactobacillus delbrueckii subsp. bulgaricus
Lactobacillus johnsonii NCC533
Lactobacillus gasseri ATCC33323
Lactobacillus casei ATCC334
0.01
Surface-layer
Surface-layer associated proteins
Peptidoglycan
Lipid Membrane
Lipoteichoicacid (LTA)
Wall teichoicacid (WTA)
Surface-layer proteins (Slps)
Surface-layer associated proteins (SLAPs)
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. 2013. Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM. Microbiology 159:2269-2282.
• 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.
