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Glycan digestion during Salmonella enterica spp. enterica sv Typhimurium infection

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Abstract

Salmonella enterica spp. enterica sv Typhimurium infection accounts for 1.3 billion foodborne disease cases worldwide. Gastroenteritis is initiated at the epithelial barrier where Salmonella binds receptors to initiate infection. As a protective mechanism, the host membrane is covered with complex glycans including mucin (glycoproteins) and the glycocalyx (complex oligosaccharides). To invade the host cell, Salmonella must digest the complex oligosaccharides where additional glycolipids in the membrane are encountered that may also be used for infection. In this study it was hypothesized that of the 76 hydrolases in the Salmonella genome only a few of these enzymes are needed to degrade the glycan during the invasion process. Significantly differentially expressed (q<0.05) glycan degrading enzymes were identified and subsequently genetically deleted to determine the specific effect on adhesion and invasion in vitro with differentiated colonic epithelial cells (Caco-2). MALDI-FTICR-MS and Chip/TOF-MS were used to determine the glycan composition, structure, and specific set of enzymes used during infection. Salmonella infection resulted in digestion of the glycocalyx layer via induced hydrolases that recognized the galactose, N-Acetylneuraminic acid (sialic acid), and fucose as terminal sugars. The glycocalyx of Caco-2 cells was composed of 70 distinct glycan structures rich in mannose, galactosides, N-Acetylglucosamine, fucose, and sialic acid. The most abundant glycans were fucosylated, composed of galactose, and contained terminal sialic acid residues. In conclusion, Salmonella digested majority of the host glycan within 60 minutes of infection. Subsequent to glycan degradation, Salmonella gained access to glycolipids in the membrane that were used as a receptor for invasion. This study found that only few hydrolases were required for glycan digestion and invasion while others decreased, but did not inhibit invasion.
RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
Salmonella enterica
spp. enterica sv Typhimurium infection accounts for 1.3
billion foodborne disease cases worldwide. Gastroenteritis is initiated at the
epithelial barrier where
Salmonella
binds receptors to initiate infection. As a
protective mechanism, the host membrane is covered with complex glycans
including mucin (glycoproteins) and the glycocalyx (complex oligosaccharides).
To invade the host cell,
Salmonella
must digest the complex oligosaccharides
where additional glycolipids in the membrane are encountered that may also be
used for infection. In this study it was hypothesized that of the 76 hydrolases in
the
Salmonella
genome only a few of these enzymes are needed to degrade the
glycan during the invasion process. Significantly differentially expressed
(q<0.05) glycan degrading enzymes were identified and subsequently
genetically deleted to determine the specific effect on adhesion and invasion
in vitro
with differentiated colonic epithelial cells (Caco-2). MALDI-FTICR-MS
and Chip/TOF-MS were used to determine the glycan composition, structure,
and specific set of enzymes used during infection.
Salmonella
infection resulted
in digestion of the glycocalyx layer via induced hydrolases that recognized the
galactose, N-Acetylneuraminic acid (sialic acid), and fucose as terminal sugars.
The glycocalyx of Caco-2 cells was composed of 70 distinct glycan structures
rich in mannose, galactosides, N-Acetylglucosamine, fucose, and sialic acid.
The most abundant glycans were fucosylated, composed of galactose, and
contained terminal sialic acid residues. In conclusion,
Salmonella
digested
majority of the host glycan within 60 minutes of infection. Subsequent to glycan
degradation,
Salmonella
gained access to glycolipids in the membrane that
were used as a receptor for invasion. This study found that only few hydrolases
were required for glycan digestion and invasion while others decreased, but did
not inhibit invasion.
ABSTRACT
Salmonella enterica spp. enterica sv Typhimurium is one of the most important zoonotic foodborne pathogens that
causes gastroenteritis in humans [1]. In 2005 The World Health Organization (WHO) along with United States
Department of Agriculture (USDA) and Centers for Disease Control and Prevention (CDC) estimated that Salmonella
infections account for 1.4 to 1.6 million cases per year of foodborne illnesses in USA. The infection rate approaches
1.3 billion cases world-wide, representing approximately 14% of the world population affected yearly [2]. Further,
foodborne Salmonella outbreaks are not declining in spite of controls and reduction efforts in the food chain. This
problem is increasing due to emerging antibiotic resistance and hypervirulent strains [3]. These factors indicate that the
genetic evolution of Salmonella is actively progressing and leading to persistence and emergence of virulence factors
that are not well described.
Gastrointestinal tract (GIT) is protected with complex oligosaccharides composed of multiple layers glycoproteins
(mucin) and oligosaccharides (glycocalyx) and glycolipids [4]. These complex oligosaccharide layer is the first site
relative to the host membrane that the microbiota interact with host defense system. In the case of pathogens these
layers provide a barrier for microorganisms that exclude them from gaining access to the epithelial membrane [5].
Salmonella and other enteric invasive pathogens have evolved mechanisms to degrade this barrier and reach the
underlying epithelium where they can bind, invade, and cause disease. However, to date there are no studies that
describe how Salmonella degrades the host oligosaccharide barrier to gain access to the host membrane during
infection.
INTRODUCTION
Salmonella
colonizes the epithelial cells in the gut by first digesting the host’s
protective mucus and glycocalyx layer
Expression of specific oligosaccharide digesting enzymes in
Salmonella
regulate
digestion of the glycocalyx
Our understanding of the interaction between the pathogens that cause enteric
infections in the gut would be dramatically enhanced
This study can lead to novel stragegies to develop drug targets
The glycan profiles can be used as biomarkers to detect infection
RESULTS
DISCUSSION
REFERENCES
I am grateful to Dr. Weimer for his guidance and helpful suggestions and discussions. I wish to thank Dr.
Carlito Lebrilla, Dayoung Park, and Cynthia Williams for their help in glycan analysis. I also acknowledge
and appreciate all the help and support received from everyone in the Weimer Lab.
This work was supported by National Institute of Health.
CONTACT INFORMATION
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.wix.com/thelab
1Department of Population Health and Reproduction, School of Veterinary Medicine, University of California Davis, Davis, California, United States of America
2Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, California, United States of America
Narine Arabyan1, Dayoung Park 2, Prerak Desai1, Cynthia Williams2, Richard Jeanotte1,
Jigna Shah1, Nguyet Kong1, Mai Lee Yang1, Carlito Lebrilla2, & Bart C. Weimer1
Glycan digestion during Salmonella enterica spp. enterica sv Typhimurium infection
ACKNOWLEDGMENTS
1. Thiennimitr, P., S.E. Winter, and A.J. Baumler, Salmonella, the host and its microbiota. Curr Opin Microbiol, 2012. 15(1): p. 108-14.
2. Coburn, B., G.A. Grassl, and B.B. Finlay, Salmonella, the host and disease: a brief review. Immunol Cell Biol, 2007. 85(2): p. 112-8.
3. Heithoff, D.M., et al., Intraspecies variation in the emergence of hyperinfectious bacterial strains in nature. PLoS Pathog, 2012. 8(4): p.
e1002647.
4. Moran, A.P., A. Gupta, and L. Joshi, Sweet-talk: role of host glycosylation in bacterial pathogenesis of the gastrointestinal tract. Gut,
2011. 60(10): p. 1412-25.
5. McGuckin, M.A., et al., Mucin dynamics and enteric pathogens. Nat Rev Microbiol, 2011. 9(4): p. 265-78.
Figure 1. Gene expression
of Salmonella LT2 during
infection of Caco-2 cells.
Inducible carbohydrate-digestion enzymes alter access during invasion of Salmonella LT2.
Figure 2. Salmonella LT2 knockout strains
characterized for the alteration in adhesion and
invasion (A/I) using differentiated colonic epithelial
cells, Caco-2.
Figure 3. Glycan profiles during the course of infection.
EXPERIMENTAL APPROACH
Gene Expression
Construct Isogenic Mutants
Characterize Mutants (Gentamicin Assay)
Glycan Profiles
(MALDI-FTICR-MS AND ChiP/TOF-MS)
RESULTS
Monitored how individual glycans are affected over time
From 0 min to 45 min the overall glycan compositions appear similar
At 1 hour changes in the glycans and the abundances are observed
By 3 hour cells appear to recover and the glycan profile starts to resemble the uninfected
Caco-2 cells
Figure 4. Changes in relative abundance in specific glycans over time.
The infection time has a significant effect on individual glycans rather than on the global
glycosylation pattern
All the high mannose signals ranging from Man 5 to Man 9 showed the same trend of peaking at
the 1 hour time point
Figure 5. Glycan compositions found in Caco-2 before and after 1 hour of infection
with Salmonella LT2.
The non-decorated glycans (orange) and high mannose glycans (red) increased in signal at 1 hr
of infection
The large amount of terminal mannose residues on Caco-2 cells may have functional
significance in epithelial cells during bacterial infection
Figure 6. Changes in abundance of individual glycans after infection.
RESULTS
The first glycan (monofucosylated, bisialylated complex glycan) with m/z 2571.920 is the most
abundant glycan in the uninfected cell
The last glycan (bisecting N-acetylglucosamine at the mannosyl core) with m/z 2280.825 is the
most abundant glycan in the infected cell
Figure 7. Changes in relative abundance in specific glycans over time.
These enzymes appears to be specific to which sugar they digest
A monofucosylated glycan, Hex6HexNAc6Fuc1, showed a decrease in signal relative to the
uninfected cells
A monosialylated glycan, Hex5HexNAc5Fuc1Neu5Ac1, showed an increase in signal
A monosialylated-monofucosylated glycan, Hex4HexNAc5Fuc1Neu5Ac1, also showed an increase
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Glycan digestion during Salmonella enterica spp. enterica sv Typhimurium infection ACKNOWLEDGMENTS 1 Salmonella, the host and its microbiota
  • P Thiennimitr
  • S E Winter
  • A J Baumler
Glycan digestion during Salmonella enterica spp. enterica sv Typhimurium infection ACKNOWLEDGMENTS 1. Thiennimitr, P., S.E. Winter, and A.J. Baumler, Salmonella, the host and its microbiota. Curr Opin Microbiol, 2012. 15(1): p. 108-14.
and Cynthia Williams for their help in glycan analysis. I also acknowledge and appreciate all the help and support received from everyone in the Weimer Lab. This work was supported by National Institute of Health
  • Carlito Lebrilla
  • Dayoung Park
Carlito Lebrilla, Dayoung Park, and Cynthia Williams for their help in glycan analysis. I also acknowledge and appreciate all the help and support received from everyone in the Weimer Lab. This work was supported by National Institute of Health. CONTACT INFORMATION