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Host Glycan Remodeling - Sweet Cross Talk between the Host and the Microbe
Narine Arabyan1, D. Park2, H. Yu3, S. Foutouhi1, A. M. Weis1, B. C. Huang1, C. C. Williams2, P. Desai1†, J. Shah1‡,
R. Jeannotte1£, N. Kong1, X. Chen3, C. B. Lebrilla2,3 and B. C. Weimer1
1Department of Population Health and Reproduction, School of Veterinary Medicine, UC Davis, CA 95616, USA; 2Department of Chemistry, School of Medicine, UC Davis, CA 95616, USA;
3Department of Biochemistry and Molecular Medicine, School of Medicine, UC Davis, CA 95616, USA; †Present Address: Zoetis, 333 Portage Street, Kalamazoo, MI 49007, USA; ‡Present Address:
4626 Dover Hills Dr., Apt 302, Kalamazoo, MI 49009, USA. £Present Address: Department of Plant Sciences, College of Agricultural and Environmental Sciences, UC Davis, CA 95616, USA
ABSTRACT
The host gut epithelial membrane is shielded by complex glycans to protect the cell. Gastroenteritis
begins when invasive pathogens bind and degrade the glyans at the epithelial barrier to gain access
of host membrane. While glycan degradation is crucial for infection, this process is poorly
understood. We hypothesized that Salmonella deploys its glycosyl hydrolases (GHs) to target and
degrade host glycans leading to altered infection and glycan remodeling. Salmonella was grown in
defined medium containing 18 different synthetic glycans modeled after human glycans as the sole
carbon source. Significantly differentially expressed (q<0.05) GHs were identified and genetically
deleted to determine the specific effect on adhesion and invasion in vitro with differentiated colonic
epithelial cells (Caco-2). HPLC-Chip-QTOF MS was used to determine the glycan composition and
structure with specific set of enzymes used during infection. Growth analysis showed that
Salmonella is able to digest and metabolize synthetic glycans. GHs recognized terminal
monosaccharides and significantly (p<0.05) altered invasion in vitro. Sialic acid depletion reduced
adherence of Salmonella during infection. Salmonella used its two GHs nanH and malS for
internalization. Host glycans were altered during Salmonella association via the induction of N-
glycan biosynthesis pathways leading to host glycan remodeling by increasing fucosylation,
mannosylation, and hybrid glycan content, while decreasing sialylation. Gene expression analysis
indicated that the host cell responded by regulating more than 50 genes showing that remodeled
glycans are in response to Salmonella infection. Our study established the glycan structures on
colonic epithelial cells, determined that Salmonella required two GHs for internalization, the host cell
remodeled the glycan during infection, and host glycan landscape influences the host-microbe
interaction. Microbial GHs are understudied and unrecognized virulence factors that may be new
therapeutic targets.
INTRODUCTION
INTRODUCTION
EXPERIMENTAL DESIGN
RESULTS RESULTS
DISCUSSION
CONTACT INFORMATION AND FUNDING
Bart C. Weimer, Ph.D. (bcweimer@ucdavis.edu)
Narine Arabyan (narabyan@ucdavis.edu)
UC Davis (VM:PHR) VetMed3B - Room 4016
1089 Veterinary Medicine Dr. Davis, CA 95616
(530)752-6426
http://weimermicrolab.org
http://www.100kgenomes.org
Salmonella tops GI illnesses associated with food
Salmonella
USA (CDC/FDA/USDA)
•1.4-1.6 million annual cases
•1000 deaths
•$ 2.3 billion / year
WHO (Worldwide)
•1.5 billion cases
USA
Mechanisms of virulence
Type 3
Secretion
System
(T3SS)
T3SS
needle
Salmonella Salmonella
inside epithelial
cells within a
lysosome
Mucin layer and glycocalyx as barriers to bacterial infections
EPITHELIAL CELLS ?
Does Salmonella
degrade the
glycocalyx layer?
Does
Salmonella
bypass the
glycocalyx
layer?
Both routes results in a
lysosome with a
surface glycan on the
inside
How does Salmonella interact with the glycans in the gut to
cross the epithelium?
Salmonella cleaves sialic and
releases to other community
members
Community members metabolize the
released molecules into small molecules
and FAs
The glycan barrier is poorly understudied
as a mechanism of microbe association
We hypothesized that Salmonella deploys its glycosyl
hydrolases (GHs) to target and degrade host glycans leading to
altered infection and glycan remodeling
CmR
CmR
ACTCTTCTGTGCTACTCGTCACATAAACAGAAATAACGGTAATCATATGTGTGTAGGCTGGAGCTGCTTC GGACCATGGCTAATTCCCATACATCAAAAAATAGCCATTGCTGCTATATAACATATAGCAGCAGTCTCTA
CmR
1132 bp
Gene expression
Enzymatic release of N-linked glycans
Infection of Caco-2 cells
Glycans were analyzed using Agilent HPLC-Chip-QTOF MS
Glycan mass profiling and structure elucidation with tandem MS-MS
N-Glycans were identified by composition with a retrosynthetic library using accurate
mass according to mass tolerance, retention times and abundance information
Construct GH deletion strains
Cell membrane extraction
Verified
with WGS
Salmonella is able to digest and metabolize synthetic glycans
Salmonella LT2 growth rate comparison in CDM with substrates
CDM
CDM+3% Lactose
1-3GlcNAc(OH)
CDM+Gal 1-3GalNAc(OH)
CDM+Gal
1-4Glc(OH)
CDM+Gal
3
ProN
1-4Glc
CDM+Gal 1-4GlcNAc(OH)
CDM+Gal
1-4Glc(OH)
1-3Gal
1-4GlcNAc
CDM+Gal 1-3GlcNAc(OH)
2-3Gal
CDM+Neu5Ac 1-3GalNAc(OH)
2-3Gal
CDM+Neu5Ac
1-4Glc(OH)
2-3Gal
CDM+Neu5Ac
3
ProN
1-4Glc
2-3Gal
CDM+Neu5Ac
2-3Gal(OH)
CDM+Neu5Ac
1-4Glc(OH)
2-6Gal
CDM+Neu5Ac
3
ProN
1-4Glc
2-6Gal
CDM+Neu5Ac
2-6Gal(OH)
CDM+Neu5Ac
1-3(Neu5Aca2-6)Galb1-4Glc(OH)
1-4GlcNAc
2-6Gal
CDM+Neu5Ac
1-4Glc(OH)
1-3Gal
CDM+GlcNAc
1-4Glc(OH)
1-3Gal
1-3)GlcNAc
1-4(Fuc
CDM+Gal 1-4Glc(OH)
1-3Gal
1-4GlcNAc
2-3Gal
CDM+Neu5Ac
0.00
0.01
0.02
0.03
0.04
Growth rate
Salmonella LT2 Highest yield OD(595) comparison in CDM with substrates
CDM
CDM+3% Lactose
1-3GlcNAc(OH)
CDM+Gal 1-3GalNAc(OH)
CDM+Gal 1-4Glc(OH)
CDM+Gal
3
ProN
1-4Glc
CDM+Gal 1-4GlcNAc(OH)
CDM+Gal 1-4Glc(OH)
1-3Gal
1-4GlcNAc
CDM+Gal 1-3GlcNAc(OH)
2-3Gal
CDM+Neu5Ac 1-3GalNAc(OH)
2-3Gal
CDM+Neu5Ac 1-4Glc(OH)
2-3Gal
CDM+Neu5Ac
3
ProN
1-4Glc
2-3Gal
CDM+Neu5Ac 2-3Gal(OH)
CDM+Neu5Ac 1-4Glc(OH)
2-6Gal
CDM+Neu5Ac
3
ProN
1-4Glc
2-6Gal
CDM+Neu5Ac 2-6Gal(OH)
CDM+Neu5Ac
1-3(Neu5Aca2-6)Galb1-4Glc(OH)
1-4GlcNAc
2-6Gal
CDM+Neu5Ac 1-4Glc(OH)
1-3Gal
CDM+GlcNAc 1-4Glc(OH)
1-3Gal
1-3)GlcNAc
1-4(Fuc
CDM+Gal 1-4Glc(OH)
1-3Gal
1-4GlcNAc
2-3Gal
CDM+Neu5Ac
0.0
0.1
0.2
0.3
0.4
0.5
0.6
High yield OD
* * * * *
*
* P < 0.05
*
Lipid Raft
(Control)
treatment
Sialic acid depletion reduces adherence of Salmonella during infection
Salmonella WT
invA (STM2896)
melA (STM4298)
CHPNeu (STM1252)
nanH (STM0928)
malS (STM3664)
glgX (STM3537)
nanT (STM3338)
xylR (STM3662)
bax (STM3663)
-250
-225
-200
-175
-150
-125
-100
-75
-50
-25
0
25
50
75
100
125
150
Adhesion
Invasion
Salmonella WT (CFU)/Caco-2 cell
* * *
*
*
* * *
* *
*
* * *
NS
NS
* * *
* * *
* * *
Putative
substrates Sugar
metabolism
Amylose
Mannose
Hexose
Sialic
acid
Galactose
Caco-2 + Salmonella WT
-2 0 2
Salmonella WT
Depletion of cell surface sialic acid led to a significant reduction in
Salmonella association, further implicating sialic acid in the adherence
process via sialidases
Glycan degrading enzymes alter host membrane access during
invasion of Salmonella
Deletion of genes related to metabolism of
sugars released from glycans did not
change association compared to WT
nanH and malS were used for
internalization by targeting different glycan
structures
The invasion levels of ΔnanH and ΔmalS
were not significant compared to ΔinvA
nanH and malS may represent new
virulence genes in Salmonella infections
Host glycome is altered during infection with Salmonella WT
C
B A
F
E D
Fucosylated
Sialylated Sialylated
Fucosylated
Sialylated Sialylated Fucosylated Fucosylated
Fucosylated
Sialylated
Fucosylated
Fucosylated
Salmonella infection led to alteration in host N-glycome
0
20
40
60
80
100
Clatherin-mediated Endocytosis
Caveolar-mediated Endocytosis
Integrin Signaling
PI3K/AKT Signaling
Inositol Phosphate Metabolism
Fructose/ Mannose Metabolism
N-glycan Biosynthesis
Percentage
A B C
D E
Host cells modify their own glycan during microbial glycan degradation
Refinement of the glycans during Salmonella infection
Salmonella must overcome the protective glycoclyx layer that coats the epithelium in the gut
by 1st being able to attach to and 2nd by degrading the glycans
Salmonella establishes infection by using sialic acid as a potential receptor for adherence
process
Refinement of glycans during infection
Induction of host genes related to N-glycan biosynthesis supports the observation that new
glycans were produced de novo
Host response to glycan modification during infection suggests that a dynamic shift in the
glycan is caused by Salmonella degradation coupled with the host remodeling to produce
different structures
Salmonella and the epithelium cooperate to produce a new glycan surface during the infection
process
Salmonella infection results in the accumulation
of fucose and depletion of sialic acid
Rapid fucosylation of intestinal epithelial cells
may be a protective mechanism that utilizes the
host’s resources to maintain host–microbe
interactions during pathogen-induced stress
NIH
1RO1HD065122-01A1 (BCW)
U24-DK097154 (BCW)
RO1GM049077(CLB)
Agilent Technologies
Thought Leader Award (BCW)
Weimer Lab
100K Pathogen
Genome Project