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Implication of Sialidases in Salmonella Infection: Genome Release of Sialidase Knockout Strains from Salmonella enterica Serovar Typhimurium LT2

May 2017
Genome Announcements 5(19):e00341-17

DOI:10.1128/genomeA.00341-17

License
CC BY 4.0

Authors:

Narine Arabyan

University of California, Davis

Allison M Weis

University of Utah

Carol Huang

University of California, Davis

Bart C Weimer

University of California, Davis

Sialidases, which are widely distributed in nature, cleave the α-ketosidic bond of terminal sialic acid residue. These emerging virulence factors degrade the host glycan. We report here the release of seven sialidase and one sialic acid transporter deletion in Salmonella enterica serovar Typhimurium strain LT2, which are important in cellular invasion during infection.

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Implication of Sialidases in Salmonella

Infection: Genome Release of Sialidase

Knockout Strains from Salmonella

enterica Serovar Typhimurium LT2

Narine Arabyan,

a,b

Allison M. Weis,

a,b

Bihua C. Huang,

a,b

Bart C. Weimer

a,b

Department of Population Health and Reproduction, School of Veterinary Medicine, UC Davis, Davis, California,

USA

; 100K Pathogen Genome Project, UC Davis, Davis, California, USA

ABSTRACT Sialidases, which are widely distributed in nature, cleave the

␣

-ketosidic

bond of terminal sialic acid residue. These emerging virulence factors degrade the

host glycan. We report here the release of seven sialidase and one sialic acid trans-

porter deletion in Salmonella enterica serovar Typhimurium strain LT2, which are im-

portant in cellular invasion during infection.

Sialidases are widely distributed among microbes and are one of the least charac-

terized and ill-deﬁned glycosyl hydrolases. Sialidases have been associated with

several diseases. Sialidases play a critical role in microbiology by mediating metabolism,

adherence, and infection, and they are important regulators of alternate complement

pathway activation, red blood cell destruction, cell growth, cell adhesion, and tumor

metastasis in mammalian systems (1–5). Recently, the importance of sialidases in

infection and commensalism has come to light, opening the potential to use newly

measured genomic diversity as a means to investigate infection mechanisms. Though

antibiotics are available for treatment of bacterial infections, inhibitors of all sialidases

and new drug targets may be medically useful where sialidase activity has been

correlated with severe infection pathology.

The presence of sialidases is highly correlated with the progress and severity of the

disease, and the most probable role of sialidases is for successful attachment and

colonization. Microbes use sialidases to reveal the cell surface that holds sialic acid-

containing cell membrane receptors during infection. Sialidases play an important role

in infection by altering the host glycan structure to gain access of the host epithelial

cells by binding to terminal sialic acid receptors to initiate glycan degradation (6). The

two sialidases (⌬nanH and ⌬STM1252) from Salmonella enterica serovar Typhimurium

LT2 have the same domains and function as sialidases, but they are structurally very

different, indicating domain shufﬂing and lack of structural conservation; therefore, this

difference led to different invasion phenotypes during the in vitro infection of differ-

entiated colonic epithelial cells (Caco-2) (6).

The 100K Pathogen Genome Project (http://www.100kgenomes.org) is a large-scale

sequencing consortium that offers the use of new next-generation sequencing methods to

provide cutting-edge methods for pathogen detection and control in the food supply. This

project is focused on producing genomes of pathogenic isolates from the environment,

plants, animals, and humans worldwide, providing new insights into the genetic diversity

of Salmonella spp. and other foodborne pathogens. These seven sialidase and one sialic

acid transporter mutant strain were constructed in the Weimer Laboratory (UC Davis, Davis,

CA, USA) (6) as described by Datsenko and Wanner (7). Cultures were grown on 1.5%

Luria–Bertani agar (Difco, Franklin Lakes, NJ, USA), with 10

␮

g/mL of chloramphenicol at

37°C, and then lysed (8). Genomic DNA was extracted (9), checked for quality (10), and

Received 21 March 2017 Accepted 27 March

2017 Published 11 May 2017

Citation Arabyan N, Weis AM, Huang BC,

Weimer BC. 2017. Implication of sialidases in

Salmonella infection: genome release of

sialidase knockout strains from Salmonella

enterica serovar Typhimurium LT2. Genome

Announc 5:e00341-17. https://doi.org/10.1128/

genomeA.00341-17.

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 19 e00341-17 genomea.asm.org 1

fragmented (11). The 350- to 500-bp libraries (12,13) were indexed (96 genomes/lane) and

sequenced (Illumina HiSeq 3000; 150-bp paired-end) (14–16) at the UC Davis DNA Tech-

nologies Core. Paired-end reads were de novo assembled using CLC Workbench version 6

with default parameters. Here, the 100K Pathogen Genome Project has assembled seven

genomes of single and double sialidases and one sialic acid transporter deletion strain of

S. Typhimurium LT2.

Accession number(s). All sequences are publicly available and can be found at the

100K Pathogen Genome Project (NCBI PRJNA186441) in the Sequence Read Archive

(http://www.ncbi.nlm.nih.gov/sra); genome assemblies can be found in NCBI GenBank

(Table 1).

ACKNOWLEDGMENTS

B.C.W. gratefully acknowledges funding for this project from Mars, Inc., NIH (1R01HD065122-

01A1 and U24-DK097154), and Agilent Technologies (Thought Leader Award).

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safety and public health from the 100K pathogen genome project.

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.00925-16.

TABLE 1 S. Typhimurium LT2 sialidase and sialic acid transporter deletion mutants

GenBank accession no. SRA accession no. Isolate name Gene deleted

No. of

contigs Coverage (ⴛ)

Total genome

size (bp)

No. of coding

sequences

MWQQ00000000 SRR5279339 BCW_7500 ⌬nanT 65 143 4,895,101 4,810

MWVC00000000 SRR5288771 BCW_7514 ⌬invA::⌬nanH 240 15 4,892,397 4,898

MWQR00000000 SRR5279338 BCW_7515 ⌬invA::⌬STM1252 74 130 4,892,686 4,819

MWQS00000000 SRR5279337 BCW_7516 ⌬melA::⌬nanH 72 142 4,870,638 4,785

MWQT00000000 SRR5279336 BCW_7517 ⌬melA::⌬STM1252 59 302 4,895,400 4,805

MWQU00000000 SRR5279335 BCW_7518 ⌬nanH::⌬STM1252 54 106 4,893,964 4,803

MWQV00000000 SRR3622954 BCW_8441 ⌬STM1252 57 165 4,894,714 4,806

MWQW00000000 SRR3622955 BCW_8442 ⌬nanH 60 139 4,894,435 4,815

Arabyan et al.

Volume 5 Issue 19 e00341-17 genomea.asm.org 2

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Jan 2014

The Agilent DNA Extraction Kit (p/n 200600) was compared to standard methods such as beadbeating and enzyme treatment for preparation of genomic DNA from the prokaryote Listeria monocytogenes. Using this extraction kit, with modifications, to lyse the bacteria and isolate high molecular weight DNA reproducibly yielded high quality DNA suitable for further applications such as polymerase chain reactions to produce amplicons, or for next-generation DNA sequencing. The quality of the high molecular weight DNA, and the comparison of extraction methods, was shown on the Agilent 2200 TapeStation with the Agilent Genomic DNA ScreenTape (p/n 5067-5365) and Agilent Genomic DNA Reagents (p/n 5067-5366). 2

Automated Library Construction Using KAPA Library Preparation Kits on the Agilent NGS Workstation Yields High-Quality Libraries for Whole-Genome Sequencing on the Illumina Platform

Technical Report

Full-text available

May 2014

A new method was developed to automate the KAPA HTP Library Preparation kit for microbial whole genome sequencing. This method uses the Agilent NGS Workstation, consisting of the NGS Bravo liquid handling platform with its accessories for heating, cooling, shaking, and magnetic bead manipulations in a 96-well format. User intervention in multistep protocols is minimized through the use of other components of the workstation such as the BenchCel 4R Microplate Handler and Labware MiniHub for labware storage and movement. This method has been validated for sequencing on the Illumina platform and consists of three protocols: the first is for end repair to post-ligation cleanup; the second is used for library amplification setup; and the third is for the post-amplification cleanup. The modular design provides the end-user with the flexibility to complete library construction over two days, and is suitable for the construction of high-quality libraries from bacteria of various GC content. This combined solution produced a workflow that is suitable for production-scale sequencing projects such as the 100K Pathogen Genome Project.

High-Throughput Analysis of Foodborne Bacterial Genomic DNA Using Agilent 2200 TapeStation and Genomic DNA ScreenTape System

Technical Report

Full-text available

Jan 2014

The initial step in Next Generation Sequencing is to construct a library from genomic DNA. To gain the optimum result, extracted DNA must be of high molecular weight with limited degradation. High-throughput sequencing projects, such as the 100K Pathogen Genome Project, require methods to rapidly assess the quantity and quality of genomic DNA extracts. In this study, assessment of the applicability of the Agilent 2200 TapeStation was done using genomic DNA from nine foodborne pathogens using several accepted high-throughput methods. The Agilent 2200 TapeStation System with Genomic DNA ScreenTape and Genomic DNA Reagents was easy to use with minimal manual intervention. An important advantage of the 2200 TapeStation over other high-throughput methods was that high molecular weight genomic DNA quality and quantity can be quantified apart from lower molecular weight size ranges, providing a distinct advantage in the library construction pipeline and over other methods available for this important step in the Next Generation Sequencing process.

Quality Control of High-Throughput Library Construction Pipeline for KAPA HTP Library Using an Agilent 2200 TapeStation

Technical Report

Full-text available

Jan 2014

Next Generation Sequencing requires the input of high molecular weight genomic DNA to construct quality libraries for whole genome bacterial sequencing. Large scale sequencing projects, such as the 100K Pathogen Genome Project, require methods to rapidly assess the quantity and quality of the input DNA using high-throughput methods that are fast and cost effective. In this study, the Agilent 2200 TapeStation and Agilent 2100 Bioanalyzer Systems were used to assess a few critical quality control steps for library construction. With minimal manual intervention , the Agilent 2200 TapeStation System determined the quality of genomic DNA, fragmented DNA, and final libraries constructed from multiple types of foodborne pathogens. The Agilent 2200 TapeStation System provided a single platform that effectively evaluated the necessary quality control steps, which provided a distinct advantage to decrease the time needed for library construction and a common instrument methodology for quality control.

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