Draft Genome Sequences of Salmonella enterica Serovar Typhimurium LT2 with Deleted Chitinases That Are Emerging Virulence Factors

Abstract

Chitinases are glycosyl hydrolases that catalyze the hydrolysis of the β-1,4 linkages in complex carbohydrates and those that contain GlcNAc. These enzymes are considered emerging virulence factors during infection because the host glycan changes. This is the release of four single chitinase deletion mutants in Salmonella enterica serovar Typhimurium LT2.

Full text

Draft Genome Sequences of Salmonella
enterica Serovar Typhimurium LT2 with
Deleted Chitinases That Are Emerging
Virulence Factors
Narine Arabyan,
a,b
Bihua C. Huang,
a,b
Bart C. Weimer
a,b
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California,
Davis, California, USA
a
; 100K Pathogen Genome Project, University of California, Davis, California, USA
b
ABSTRACT Chitinases are glycosyl hydrolases that catalyze the hydrolysis of the
␤
-1,4 linkages in complex carbohydrates and those that contain GlcNAc. These en-
zymes are considered emerging virulence factors during infection because the host
glycan changes. This is the release of four single chitinase deletion mutants in Sal-
monella enterica serovar Typhimurium LT2.
Chitinases are glycosyl hydrolases (GHs) that belong to the GH18 and GH19 families
(1–6). GH enzymes play a significant role in virulence by altering the host glycan
structure during infection and gaining access to the host epithelial cells, which results
in the microbe binding to terminal monosaccharides to initiate glycan degradation on
the host epithelial cell (7, 8). Chitinases are emerging virulence factors because they
recognize host GlcNAc-containing glycans in mucin and other N-glycosylated proteins
in the host membrane, which enable host association as well as glycan digestion, to
gain access to the cell membrane to initiate invasion (9, 10). Glycans with GlcNAc
molecules with a
␤
-1,4-glycosidic bond (11) are found on intestinal epithelial cells (IECs)
and are hydrolyzed during association (1, 10). This provides Salmonella spp. a method
to degrade the glycan and digest the glycocalyx to establish intracellular infections.
Deletion of chitinase genes in Listeria monocytogenes led to a reduction in bacterial
counts in the liver and spleen of infected mice (12). An adherent-invasive Escherichia
coli (AIEC) LF82 deletion of the chiA gene significantly reduced the adhesion to IECs
compared to that of the wild type (13). Furthermore, AIEC LF82 interacted with an
N-glycosylated chitin-binding protein (CHI3L1) on the host cell to mediate close
interaction between the host membrane and bacterial cell, which is regulated in animal
models of colitis and in human inflammatory bowel diseases (IBDs) (14). Microarray
analysis showed that SL0018 (chiA) gene in the Salmonella SL1344 strain was strongly
induced during the infection of murine macrophage cells (15, 16). These data indicate
that chitinases relandscape the host glycan to promote the attachment of bacteria to
the host cells through the interaction with mucin or N-glycosylated glycans during
association. The genus Salmonella contains four chitinases that were derived from
bacteria and phages. Park et al. (8) also demonstrated that Salmonella initiates glycan
relandscaping during infection via host gene expression changes and microbe grazing
to degrade the glycan, making these enzymes important for infection.
The 100K Pathogen Genome Project (http://www.100kgenomes.org) is a large-scale
sequencing consortium that offers the use of new next-generation sequencing meth-
ods to provide cutting-edge methods for pathogen detection and control in the food
supply. This project is focused on sequencing genomes of bacteria from the environ-
ment, plants, animals, and humans worldwide, providing new insights into the genetic
diversity of pathogens and the microbiome. Four chitinase deletions (⌬STM0018,
Received 23 May 2017 Accepted 25 May
2017 Published 3 August 2017
Citation Arabyan N, Huang BC, Weimer BC.
2017. Draft genome sequences of Salmonella
enterica serovar Typhimurium LT2 with deleted
chitinases that are emerging virulence factors.
Genome Announc 5:e00659-17. https://doi
.org/10.1128/genomeA.00659-17.
Copyright © 2017 Arabyan et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Bart C. Weimer,
bcweimer@ucdavis.edu.
PROKARYOTES
crossm
Volume 5 Issue 31 e00659-17 genomea.asm.org 1

⌬STM0233,⌬STM0907, and ⌬STM1869A) were constructed in the Weimer laboratory
(University of California, Davis) (7) as described by Datsenko and Wanner (17). Cultures
were prepared for sequencing as described previously (18–25). Genome sequences
were de novo assembled using CLC Workbench version 6.5.1 with default parameters
(18).
Accession number(s). All sequences are publicly available and can be found at the
100K Pathogen Genome Project BioProject (NCBI PRJNA186441) in the Sequence Read
Archive (http://www.ncbi.nlm.nih.gov/sra). NCBI GenBank accession numbers for the
genome assemblies are listed in Table 1.
ACKNOWLEDGMENTS
B.C.W. is grateful for the funding contributed by the NIH (1R01HD065122-01A1;
NIH-U24-DK097154) and an Agilent Technologies Thought Leader Award.
REFERENCES
1. Frederiksen RF, Paspaliari DK, Larsen T, Storgaard BG, Larsen MH, Ingmer
H, Palcic MM, Leisner JJ. 2013. Bacterial chitinases and chitin-binding
proteins as virulence factors. Microbiology 159:833– 847. https://doi.org/
10.1099/mic.0.051839-0.
2. Davies G, Henrissat B. 1995. Structures and mechanisms of glycosyl
hydrolases. Structure 3:853– 859. https://doi.org/10.1016/S0969
-2126(01)00220-9.
3. Henrissat B. 1991. A classification of glycosyl hydrolases based on amino
acid sequence similarities. Biochem J 280:309 –316. https://doi.org/10
.1042/bj2800309.
4. Henrissat B, Bairoch A. 1993. New families in the classification of glycosyl
hydrolases based on amino acid sequence similarities. Biochem J 293:
781–788. https://doi.org/10.1042/bj2930781.
5. Henrissat B, Bairoch A. 1996. Updating the sequence-based classification
of glycosyl hydrolases. Biochem J 316:695– 696. https://doi.org/10.1042/
bj3160695.
6. Henrissat B, Davies G. 1997. Structural and sequence-based classification
of glycoside hydrolases. Curr Opin Struct Biol 7:637– 644. https://doi.org/
10.1016/S0959-440X(97)80072-3.
7. Arabyan N, Park D, Foutouhi S, Weis AM, Huang BC, Williams CC, Desai
P, Shah J, Jeannotte R, Kong N, Lebrilla CB, Weimer BC. 2016. Salmonella
degrades the host glycocalyx leading to altered infection and glycan
remodeling. Sci Rep 6:29525. https://doi.org/10.1038/srep29525.
8. Park D, Arabyan N, Williams CC, Song T, Mitra A, Weimer BC, Maverakis E,
Lebrilla CB. 2016. Salmonella typhimurium enzymatically landscapes the
host intestinal epithelial cell (IEC) surface glycome to increase invasion. Mol
Cell Proteomics 15:3653–3664. https://doi.org/10.1074/mcp.M116.063206.
9. Jacobs H, Mink SN, Duke K, Bose D, Cheng ZQ, Howlett S, Ferrier GR,
Light RB. 2005. Characterization of membrane N-glycan binding sites of
lysozyme for cardiac depression in sepsis. Intensive Care Med 31:
129 –137. https://doi.org/10.1007/s00134-004-2487-y.
10. Tran HT, Barnich N, Mizoguchi E. 2011. Potential role of chitinases and
chitin-binding proteins in host-microbial interactions during the devel-
opment of intestinal inflammation. Histol Histopathol 26:1453–1464.
https://doi.org/10.14670/HH-26.1453.
11. Stanley P, Schachter H, Taniguchi N. 2009. N-Glycans, chap. 8. In Varki A,
Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler
ME (ed.), Essentials of glycobiology, 2nd ed. Cold Spring Harbor Labo-
ratory Press, Cold Spring Harbor, NY.
12. Chaudhuri S, Bruno JC, Alonzo F III, Xayarath B, Cianciotto NP, Freitag NE.
2010. Contribution of chitinases to Listeria monocytogenes pathogenesis.
Appl Environ Microbiol 76:7302–7305. https://doi.org/10.1128/AEM
.01338-10.
13. Low D, Tran HT, Lee IA, Dreux N, Kamba A, Reinecker HC, Darfeuille-
Michaud A, Barnich N, Mizoguchi E. 2013. Chitin-binding domains of
Escherichia coli ChiA mediate interactions with intestinal epithelial cells
in mice with colitis. Gastroenterology 145:602– 612. https://doi.org/10
.1053/j.gastro.2013.05.017.
14. Mizoguchi E. 2006. Chitinase 3-like-1 exacerbates intestinal inflammation by
enhancing bacterial adhesion and invasion in colonic epithelial cells. Gas-
troenterology 130:398 – 411. https://doi.org/10.1053/j.gastro.2005.12.007.
15. Eriksson S, Lucchini S, Thompson A, Rhen M, Hinton JC. 2003. Unravel-
ling the biology of macrophage infection by gene expression profiling of
intracellular Salmonella enterica. Mol Microbiol 47:103–118. https://doi
.org/10.1046/j.1365-2958.2003.03313.x.
16. Larsen T, Petersen BO, Storgaard BG, Duus JØ, Palcic MM, Leisner JJ. 2011.
Characterization of a novel Salmonella Typhimurium chitinase which hydro-
lyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate.
Glycobiology 21:426 – 436. https://doi.org/10.1093/glycob/cwq174.
17. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal
genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci
U S A 97:6640 – 6645. https://doi.org/10.1073/pnas.120163297.
18. Arabyan N, Weis AM, Huang BC, Weimer BC. 2017. Implication of siali-
dases in Salmonella infection: genome release of sialidase knockout
strains from Salmonella enterica serovar Typhimurium LT2. Genome
Announc 5(19):e00341-17. https://doi.org/10.1128/genomeA.00341-17.
19. Jeannotte R, Lee E, Kong N, Ng W, Kelly L, Weimer BC. 2014. High-
throughput analysis of foodborne bacterial genomic DNA using Agilent
2200 TapeStation and genomic DNA ScreenTape system. Agilent Tech-
nologies Application Note. https://doi.org/10.13140/RG.2.1.3354.6961.
20. Kong N, Ng W, Lee V, Kelly L, Weimer BC. 2013. Production and analysis
of high molecular weight genomic DNA for NGS pipelines using Agilent
DNA extraction kit (p/n 200600). Agilent Technologies Application Note.
https://doi.org/10.13140/RG.2.1.2961.4807.
21. Weis AM, Huang BC, Storey DB, Kong N, Chen P, Arabyan N, Gilpin B, Mason
C, Townsend AK, Smith WA, Byrne BA, Taff CC, Weimer BC. 2017. Large-scale
release of campylobacter draft genomes: resources for food safety and
public health from the 100K pathogen genome project. Genome Announc
5(1):e00925-00916. https://doi.org/10.1128/genomeA.00925-16.
22. Kong N, Ng W, Foutouhi A, Huang BH, Kelly L, Weimer BC. 2014. Quality
control of high-throughput library construction pipeline for KAPA HTP
library using an Agilent 2200 TapeStation. Agilent Technologies Appli-
cation Note. https://doi.org/10.13140/RG.2.1.4927.5604.
TABLE 1 Salmonella enterica serovar Typhimurium LT2 chitinase deletion mutants
GenBank
accession no.
SRA
accession no.
Isolate
name
Gene
deleted Enzyme activity
No. of
contigs
Coverage
(ⴛ)
Total genome
size (bp)
No. of coding
sequences
MZNQ00000000 SRR5288763 BCW_8404 ⌬STM0018 Exochitinase 61 139 4,893,048 4,810
MXBA00000000 SRR5288762 BCW_8406 ⌬STM0233 Endochitinase 63 162 4,894,557 4,808
MXBB00000000 SRR5288761 BCW_8409 ⌬STM0907 Prophage chitinase 61 188 4,895,634 4,808
MZYL00000000 SRR5288732 BCW_8417 ⌬STM1869A Putative chitinase 63 177 4,895,461 4,811
Arabyan et al.
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23. Kong N, Thao K, Huang C, Appel M, Lappin S, Knapp L, Kelly L, Weimer
BC. 2014. Automated library construction using KAPA library prepa-
ration kits on the Agilent NGS workstation yields high-quality librar-
ies for whole-genome sequencing on the Illumina platform. Agilent
Technologies Application Note. https://doi.org/10.13140/RG.2.1.2306
.1203.
24. Lüdeke CH, Kong N, Weimer BC, Fischer M, Jones JL. 2015. Complete
genome sequences of a clinical isolate and an environmental isolate of
Vibrio parahaemolyticus. Genome Announc 3(2):e00216-00215. https://
doi.org/10.1128/genomeA.00216-15.
25. Weis AM, Clothier KA, Huang BC, Kong N, Weimer BC. 2016. Draft
genome sequences of Campylobacter jejuni strains that cause abortion in
livestock. Genome Announc 4(6):e01324-16. https://doi.org/10.1128/
genomeA.01324-16.
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