Sequencing of Virulence Genes Shows Limited Genetic Variability in Yersinia pseudotuberculosis

Abstract

Yersinia pseudotuberculosis is a foodborne pathogen often detected and identified using polymerase chain reaction (PCR) with primers targeted to virulence genes. Sequence variability of the virulence genes in strains representing different serotypes is unknown. Sequence variability could hinder the recognition of this pathogen by PCR and affect the host–pathogen interactions. Sequencing of inv, virF, and yadA of 18 Y. pseudotuberculosis strains showed limited variability of inv and virF, whereas the sequences of yadA varied considerably.

Introduction

Y ersinia pseudotuberculosis belongs to the family of Enterobacteriaceae and is an important enteropathogen that has recently caused several outbreaks due to fresh produce in Finland (Nuorti et al., 2004; Jalava et al., 2006; Rimhanen-Finne et al., 2009; Kangas et al., 2008). Outbreaks have also been documented in Russia and Japan (Skurnik et al., 2009). The most typical symptoms of Y. pseudotuberculosis infections are fever and abdominal pain resulting from mesenteric adenitis and enteritis (Carniel et al., 2006). Symptoms can resemble those of appendicitis, leading to unnecessary appendectomies (Fredriksson-Ahomaa et al., 2010). Systemic manifestations, including joint pain, arthritis, and erythema nodosum, are also possible, and in East Asia, rash and desquamation are common (Carniel et al., 2006). Septicemia can occur in persons with underlying diseases (Carniel et al., 2006).

Y. pseudotuberculosis is currently divided into 21 serotypes, with O:1a and O:1b being the most common in Europe, Australasia, and North America, and O:4b and O:5b being prevalent in the East Asia (Carniel et al., 2006). Serotype O:15 strains are common in human patients in South Korea (Fukushima et al., 2001; Laukkanen-Ninios et al., 2011; De Castro et al., 2009). Serotypes O:6 to O:14 have been isolated mainly from animals and environmental sources (Carniel et al., 2006; Laukkanen-Ninios et al., 2011). The presence of the highly conserved 70-kb virulence plasmid (pYV) is required for pathogenicity and is found in all human and animal pathogenic Yersinia strains (Carniel et al., 2006). pYV has not been found in serotypes O:8, O:9, O:11, O:12, and O:13 isolated from animals and environmental sources (Carniel et al., 2006; Iwata et al., 2008).

To initiate Y. pseudotuberculosis infection, the chromosomally encoded outer membrane protein invasin (Inv) is used for passage of the bacterium through the intestinal epithelium in the terminal ileum (Leo and Skurnik, 2011). In addition to the role in traversing through the epithelium, Inv induces inflammatory response in intestinal epithelial cells, which contributes to the spread of Yersinia in the host (Grassl et al., 2003). After passage through the epithelium, the Yersinia adhesin YadA, encoded by pYV, mediates binding to collagen, fibronectin, and laminin, and also to intestinal mucus, epithelial cells, and macrophages (Leo and Skurnik, 2011). YadA protects Y. pseudotuberculosis from complement and from antimicrobial peptides generated by granulocytes. It also shields Yersinia cells from phagocytosis (Leo and Skurnik, 2011). In addition, YadA functions as a hemagglutinin and autoagglutinin, and it enhances biofilm formation (Leo and Skurnik, 2011; Heise and Dersch, 2006). VirF is a pYV-encoded transcriptional activator needed for the expression of several virulence genes, including those encoding the Yops (Yersinia outer protein) and yadA in Yersinia (Cornelis et al., 1998).

The isolation of pathogenic Yersinia, especially Y. pseudotuberculosis, is demanding due to slow growth and poor competition (Skurnik et al., 2009). Furthermore, identification of Y. pseudotuberculosis based on biochemical reactions is challenging because of its indistinguishable phenotype from the closely related Yersinia similis and Yersinia pekkanenii (Sprague et al., 2008; Niskanen et al., 2009; Murros-Kontiainen et al., 2011). To overcome these problems, detection and identification of Y. pseudotuberculosis is often based on polymerase chain reaction (PCR) with primers targeted to the virulence genes inv, virF, and yadA (Skurnik et al., 2009; Fredriksson-Ahomaa et al., 2010). However, while the genetic diversity within the species Y. pseudotuberculosis is generally narrow (Laukkanen-Ninios et al., 2011), there is little information on the sequence variability of virulence genes in the different serotypes of Y. pseudotuberculosis. Variation in virulence gene sequences would challenge the design of potent PCR primers and hinder detection, distorting the epidemiological understanding of this pathogen. The objective of this study was to reveal the sequence similarity of inv, virF, and yadA of 18 Y. pseudotuberculosis and two Y. similis strains originating from 12 different countries.

Materials and Methods

Bacterial strains and growth conditions

The 18 Y. pseudotuberculosis strains and two Y. similis strains (Laukkanen-Ninios et al., 2011) (Table 1) were grown on Luria-Bertani (LB) agar plates (BD, Franklin Lakes, NJ) or in LB broth (BD) at 30°C. The genomic DNA was extracted from overnight cultures by using Pitcher's method (Pitcher et al., 1989). Plasmid DNA was extracted using the Qiagen Plasmid Midi Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions.

Table 1.

Yersinia pseudotuberculosis and Yersinia similis Strains Used in the Study

Strain		BT	Serotype	pYV	ST	Origin	Country	Year of isolation	Source
Y. pseudotuberculosis
1	H346-36/89	2	3	+	19	Human	Germany	NK	MS
2	WS899K	1	1	+	NK	Capybara	Germany	2002	DFHEH
3	YLI16.9	1	2	+	NK	Song thrush	Sweden	2001	DFHEH
4	LE116.1K	2	2	+	NK	Cat	Finland	1998	DFHEH
5	A162.1K3	1	2a	+	NK	Lettuce	Finland	2000	DFHEH
6	553	1	4a	+	32	Soil	Russia	1982	MS
7	P30	1	1a	+	42	Buffalo	Brazil	NK	MS
8	S107	1	5b	+	87	Rabbit	China	NK	MS
9	476	2	R	+	19	Pig	Italy	NK	MS
10	43K.3	1	4	+	NK	Pig	England	2003	DFHEH
11	H5N6.1.1K2D	2	3	+	NK	Pig	Belgium	2006	DFHEH
12	T3.2.2K	1	1	+	NK	Pig	England	2004	DFHEH
13	T1.4.1K	2	5	+	NK	Pig	England	2004	DFHEH
14	14994/83	1	1	+	42	Human	Finland	NK	MS
15	T6.2.2C	1	1	+	NK	Pig	England	2004	DFHEH
16	2B1	1	1	+	NK	Roasted fish	Nigeria	2004	DFHEH
17	S₅3B1-3/IIIC	1	2	+	NK	Shrew	Finland	2005	DFHEH
18	283	1	1b	+	14	Human	Canada	NK	MS
Y. similis
19	R626R	2	9	-	83	Mole	Japan	1987	MS
20	R220	2	6	-	76	Field mouse	Japan	1987	MS

BT, biotype; pYV, virulence plasmid; ST, sequence type based on multilocus sequence analysis (Laukkanen-Ninios et al., 2011); NK, not known; MS, Mikael Skurnik; DFHEH, Department of Food Hygiene and Environmental Health, University of Helsinki, Finland.

PCR amplification and sequencing

Primers for amplification and sequencing were designed for the strain IP32953 (Chain et al., 2004) (GenBank accession number BX936398) by using the Primer3 software (Rozen and Skaletsky, 2000) and are listed in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/fpd). In PCR, Titanium Taq DNA polymerase (Clontech Laboratories Inc., Mountain View, CA,) was used according to the manufacturer's instructions. The total reaction volume of 50 μL contained 200 μM of dNTPs, 0.3 μM of both primers, and 100 ng of DNA template. For products under 1000 bp, the PCR run consisted of predenaturation at 95°C for 1 min, and 29 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min, followed by final extension at 72°C for 2 min. For products between 1000 and 1500 bp, the extension time was 90 s, and for products between 1500 and 2000 bp, the extension time was 2 min. PCR products were run in agarose gel, and the band of a correct size was purified by using the QIAquick Gel Extraction Kit (Qiagen GmbH) in accordance with the manufacturer's instructions. Sequencing was done at the Institute of Biotechnology, University of Helsinki, Finland.

Sequence analysis

Base-calling and quality assignment of the raw sequences were done using Phred (Ewing and Green, 1998). Gene sequences for each strain were constructed from multiple fragments with the gap4 program in the Staden package (Staden, 1996). Phylogenetic trees were constructed using MEGA (Tamura et al., 2007). Gene sequences were aligned with CLUSTAL and trimmed at both ends to have equal lengths. All nucleotide sequences were confirmed to contain an ATG start codon before converting them into amino acid sequences. Neighbor-joining trees with 1000 bootstrap replicates were constructed with the Kimura 2-parameter model for nucleotide sequences. Pairwise sequence similarities shown in Tables 2 –4 were calculated without evolution models.

Table 2.

Similarity (%) of Nucleotide (Upper Triangle) and Amino Acid (Lower Triangle) Sequences of inv Between 18 Yersinia pseudotuberculosis and Two Yersinia similis Strains

Strain	NT	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19 ^a	20 ^a
AA		99.2	100	99.8	99.7	99.8	99.6	100	99.3	99.2	99.8	99.2	100	99.2	100	100	99.3	99.8	99.4	93.4	96.7
1	99.4		99.2	99.2	98.9	99.2	99.3	99.2	99.2	100	99	100	99.2	100	99.2	99.2	99.9	99.2	99	92.7	97.1
2	100	99.4		99.8	99.7	99.8	99.6	100	99.3	99.2	99.8	99.2	100	99.2	100	100	99.3	99.8	99.4	93.4	96.7
3	99.8	99.2	99.8		99.5	100	99.6	99.8	99.2	99.2	99.7	99.2	99.8	99.1	99.8	99.8	99.2	100	99.4	93.2	96.6
4	99.4	98.8	99.4	99.2		99.5	99.3	99.7	99	98.9	99.5	98.9	99.7	98.9	99.7	99.7	99	99.5	99.1	93.1	96.4
5	99.8	99.2	99.8	100	99.2		99.6	99.8	99.2	99.2	99.7	99.2	99.8	99.1	99.8	99.8	99.2	100	99.4	93.2	96.6
6	99.8	99.2	99.8	99.6	99.2	99.6		99.6	99.4	99.3	99.4	99.3	99.6	99.3	99.6	99.6	99.3	99.6	99.5	93.2	96.5
7	100	99.4	100	99.8	99.4	99.8	99.8		99.2	99.2	99.8	99.2	100	99.1	99.9	99.9	99.3	99.8	99.4	93.4	96.6
8	99.2	99.4	99.2	99	98.6	99	99.2	99.2		99.2	99.1	99.2	99.2	99.2	99.2	99.2	99.2	99.2	98.9	92.8	96.6
9	99.4	100	99.4	99.2	98.8	99.2	99.2	99.4	99.4		99	100	99.2	100	99.2	99.2	99.9	99.2	99	92.7	97.1
10	100	99.4	100	99.8	99.4	99.8	99.8	100	99.2	99.4		99	99.8	99	99.8	99.8	99.1	99.7	99.3	93.4	96.7
11	99.4	100	99.4	99.2	98.8	99.2	99.2	99.4	99.4	100	99.4		99.2	100	99.2	99.2	99.9	99.2	99	92.7	97.1
12	100	99.4	100	99.8	99.4	99.8	99.8	100	99.2	99.4	100	99.4		99.1	99.9	99.9	99.3	99.8	99.4	93.4	96.6
13	99.4	100	99.4	99.2	98.8	99.2	99.2	99.4	99.4	100	99.4	100	99.4		99.1	99.1	99.9	99.1	99	92.7	97.1
14	99.9	99.3	99.9	99.7	99.3	99.7	99.7	99.9	99.1	99.3	99.9	99.3	99.9	99.3		99.9	99.3	99.8	99.4	93.4	96.6
15	100	99.4	100	99.8	99.4	99.8	99.8	100	99.2	99.4	100	99.4	100	99.4	99.9		99.3	99.8	99.4	93.4	96.6
16	99.4	100	99.4	99.2	98.8	99.2	99.2	99.4	99.4	100	99.4	100	99.4	100	99.3	99.4		99.2	99.1	92.7	97.2
17	99.8	99.2	99.8	100	99.2	100	99.6	99.8	99	99.2	99.8	99.2	99.8	99.2	99.7	99.8	99.2		99.4	93.2	96.6
18	99.3	98.7	99.3	99.1	98.7	99.1	99.1	99.3	98.5	98.7	99.3	98.7	99.3	98.7	99.2	99.3	98.7	99.1		93.1	96.3
19^a	92.4	91.7	92.4	92.2	91.7	92.2	92.2	92.4	91.5	91.7	92.4	91.7	92.4	91.7	92.3	92.4	91.7	92.2	92.1		95.5
20^a	95.7	96.3	95.7	95.5	95	95.5	95.5	95.7	95.7	96.3	95.7	96.3	95.7	96.3	95.6	95.7	96.3	95.5	94.9	95.5

Y. similis.

Column NT and row AA represent similarity with the reference strain IP32953.

NT, nucleotide; AA, amino acid.

Table 3.

Similarity (%) of Nucleotide (Upper Triangle) and Amino Acid (Lower Triangle) Sequences of virF Between 18 Yersinia pseudotuberculosis Strains

Strain	NT	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
AA		99.9	100	100	100	100	100	100	100	99.9	100	99.9	100	99.9	100	99.9	99.9	100	99.9
1	100		99.9	99.9	99.9	99.9	99.9	99.9	99.9	100	99.9	100	99.9	100	99.9	99.8	99.8	99.9	99.8
2	100	100		100	100	100	100	100	100	99.9	100	99.9	100	99.9	100	99.9	99.9	100	99.9
3	100	100	100		100	100	100	100	100	99.9	100	99.9	100	99.9	100	99.9	99.9	100	99.9
4	100	100	100	100		100	100	100	100	99.9	100	99.9	100	99.9	100	99.9	99.9	100	99.9
5	100	100	100	100	100		100	100	100	99.9	100	99.9	100	99.9	100	99.9	99.9	100	99.9
6	100	100	100	100	100	100		100	100	99.9	100	99.9	100	99.9	100	99.9	99.9	100	99.9
7	100	100	100	100	100	100	100		100	99.9	100	99.9	100	99.9	100	99.9	99.9	100	99.9
8	100	100	100	100	100	100	100	100		99.9	100	99.9	100	99.9	100	99.9	99.9	100	99.9
9	100	100	100	100	100	100	100	100	100		99.9	100	99.9	100	99.9	99.8	99.8	99.9	99.8
10	100	100	100	100	100	100	100	100	100	100		99.9	100	99.9	100	99.9	99.9	100	99.9
11	100	100	100	100	100	100	100	100	100	100	100		99.9	100	99.9	99.8	99.8	99.9	99.8
12	100	100	100	100	100	100	100	100	100	100	100	100		99.9	100	99.9	99.9	100	99.9
13	100	100	100	100	100	100	100	100	100	100	100	100	100		99.9	99.8	99.8	99.9	99.8
14	100	100	100	100	100	100	100	100	100	100	100	100	100	100		99.9	99.9	100	99.9
15	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100		99.8	99.9	99.8
16	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100		99.9	100
17	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100		99.9
18	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100

Column NT and row AA represent similarity with the reference strain IP32953.

NT, nucleotide; AA, amino acid.

Table 4.

Similarity (%) of Nucleotide (Upper Triangle) and Amino Acid (Lower Triangle) Sequences of yadA Between 18 Yersinia pseudotuberculosis Strains

Strain	NT	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
AA		99.1	100	98.8	100	98.8	99.6	99.9	99.9	98.8	99.8	99.1	100	99.1	99.9	99.8	99.1	98.8	99.1
1	97.7		99.1	98	99.1	98	99	99	99.1	99.6	99.2	99.9	99.1	99.9	99	99.1	98.2	98	98.2
2	100	97.7		98.8	100	98.8	99.6	99.9	99.9	98.8	99.8	99.1	100	99.1	99.9	99.8	99.1	98.8	99.1
3	96.5	95.1	96.5		98.8	100	98.9	98.8	98.9	97.8	98.8	98.1	98.8	98.1	98.8	98.7	98.9	100	98.9
4	100	97.7	100	96.5		98.8	99.6	99.9	99.9	98.8	99.8	99.1	100	99.1	99.9	99.8	99.1	98.8	99.1
5	96.5	95.1	96.5	100	96.5		98.9	98.8	98.9	97.8	98.8	98.1	98.8	98.1	98.8	98.7	98.9	100	98.9
6	99.1	97.2	99.1	96.9	99.1	96.9		99.5	99.7	98.7	99.8	99.1	99.6	99.1	99.5	99.7	99.1	98.9	99.1
7	99.8	97.4	99.8	96.5	99.8	96.5	98.8		99.8	98.7	99.8	99.1	99.9	99.1	100	99.7	99.1	98.8	99.1
8	99.8	97.9	99.8	96.7	99.8	96.7	99.3	99.5		98.9	99.9	99.2	99.9	99.2	99.8	99.8	99.1	98.9	99.1
9	97.7	99.5	97.7	95.1	97.7	95.1	97.2	97.4	97.9		99	99.7	98.8	99.7	98.7	98.9	98	97.8	98
10	99.8	97.9	99.8	96.7	99.8	96.7	99.3	99.5	100	97.9		99.3	99.8	99.3	99.8	99.9	99	98.8	99
11	97.9	99.8	97.9	95.3	97.9	95.3	97.4	97.7	98.1	99.8	98.1		99.1	100	99.1	99.2	98.3	98.1	98.3
12	100	97.7	100	96.5	100	96.5	99.1	99.8	99.8	97.7	99.8	97.9		99.1	99.9	99.8	99.1	98.8	99.1
13	97.9	99.8	97.9	95.3	97.9	95.3	97.4	97.7	98.1	99.8	98.1	100	97.9		99.1	99.2	98.3	98.1	98.3
14	99.8	97.4	99.8	96.5	99.8	96.5	98.8	100	99.5	97.4	99.5	97.7	99.8	97.7		99.7	99.1	98.8	99.1
15	99.8	97.9	99.8	96.7	99.8	96.7	99.3	99.5	100	97.9	100	98.1	99.8	98.1	99.5		98.9	98.7	98.9
16	98.1	95.8	98.1	97.2	98.1	97.2	97.9	98.1	97.9	95.8	97.9	96	98.1	96	98.1	97.9		98.9	100
17	96.7	94.8	96.7	99.8	96.7	99.8	97.2	96.7	96.9	94.8	96.9	95.1	96.7	95.1	96.7	96.9	97.4		98.9
18	98.1	95.8	98.1	97.2	98.1	97.2	97.9	98.1	97.9	95.8	97.9	96	98.1	96	98.1	97.9	100	97.4

Column NT and row AA represent similarity with the reference strain IP32953.

NT, nucleotide; AA, amino acid.

Results and Discussion

We sequenced the virulence genes in 18 Y. pseudotuberculosis strains originating from four continents and 11 countries, representing five different serotypes, and in two Y. similis strains (the sequence database accession numbers are HE805213–HE805268). Y. similis is a close relative to Y. pseudotuberculosis, but it is considered to be nonpathogenic due to lack of pYV (Fukushima et al., 2001; Laukkanen-Ninios et al., 2011; Sprague et al., 2008). The smallest sequence variation was observed in virF and the greatest one in yadA (Tables 2 –4).

The similarity of the nucleotide and amino acid sequences of inv encoding the invasin, a virulence factor needed for the passage of Y. pseudotuberculosis through the intestinal epithelium and spread in the host (Leo and Skurnik, 2011; Grassl et al., 2003), is presented in Table 2. At the amino acid level, the inv genes of the Y. pseudotuberculosis strains differed from each other at the most by 1.5% (Table 2). Nucleotide sequences of inv in the Y. pseudotuberculosis strains differed from each other at the most by 1.1% (Table 2). The token clustering of the Y. pseudotuberculosis strains based on inv sequences (Fig. 1) requires further support by a greater number of strains. Previously, three allelic variants were found when a 600-bp fragment of the 2958-bp inv was sequenced for 84 Y. pseudotuberculosis strains, 83 of which originated from the Russian Federation (Adgamov et al., 2010). The 84 strains belonged to serotypes 1 and 3 and originated from humans, wild rodents, and the environment (Adgamov et al., 2010). Allele distribution depended on the source of isolation of a strain, but the serotype and geographical origin were insignificant for allele distribution in the previous study (Adgamov et al., 2010).

FIG. 1.

Phylogenetic tree with bootstrap values on branches of inv of 18 Yersinia pseudotuberculosis and three Yersinia similis strains based on nucleotide sequences (EMBL accession numbers HE805213–HE805232). IP32953, sequence of inv of the reference strain IP32953.

Based on inv sequences, the Y. similis strains formed an outgroup (Fig. 1). The similarity of the nucleotide and amino acid sequences of the inv genes of Y. similis strains is presented in Table 2. The inv sequences of Y. similis strains R626R and R220, and that of another Y. similis strain N916Ysi (retrieved from whole genome sequence, accession number ERS008562) were compared with that of Y. pseudotuberculosis strain 283 (Supplementary Fig. S1). The inv sequences of the three Y. similis strains were not identical. When compared with inv of the strain 283, the Y. similis inv sequences contained a 1-bp deletion at position 40, causing a frame shift mutation. However, another start codon is located immediately downstream, at position 52–54. This is preceded by a perfect ribosomal binding site AGGAG (position 39–44). Furthermore, as the signal peptide of the predicted Y. pseudotuberculosis invasin is 48 amino acids long, a protein starting from the codon 52–54 would still contain a fully functional signal peptide of 31 amino acids. Translation of the Y. similis invasin protein likely initiates from this start codon.

When the published inv-specific PCR detection primers (Kaneko et al., 1995; Kageyama et al., 2002; Nakajima et al., 1992; Thoerner et al., 2003) were compared to the DNA sequences of the Y. pseudotuberculosis and Y. similis inv genes studied here, 0 to 1 and 0 to 9 mismatches per primer pair were found, respectively. Thus, the commonly used primers are expected to perform well in the detection of Y. pseudotuberculosis. However, also some Y. similis strains are likely to be detected with these primers, which may cause problems in diagnostics.

Only small differences were found in virF of the Y. pseudotuberculosis strains investigated at the nucleotide level, and none at the amino acid level (Table 3). Comparison of the virF-specific primer sequences (Kaneko et al., 1995; Kageyama et al., 2002; Thoerner et al., 2003; Harnett et al., 1996; Lambertz and Danielsson-Tham, 2005; Wren and Tabaqchali, 1990) to the virF sequences of our 18 Y. pseudotuberculosis strains revealed only 0–2 mismatches per primer pair. VirF is a member of the AraC family of transcriptional regulators and a key activator of virulence genes (Cornelis et al., 1998). Thus, the high conservation of virF in pathogenic Y. pseudotuberculosis is not surprising. The pYV plasmid encoding virF is absent in Y. similis (Fukushima et al., 2001; Laukkanen-Ninios et al., 2011; Sprague et al., 2008).

The similarity of the nucleotide and amino acid sequences of the pYV-encoded adhesin gene yadA of Y. pseudotuberculosis strains investigated is illustrated in Table 4. Y. similis does not carry the pYV (Fukushima et al., 2001; Laukkanen-Ninios et al., 2011; Sprague et al., 2008). The maximum difference between the Y. pseudotuberculosis strains at amino acid level was 5.2%, and at nucleotide level 2.2% (Table 4). The constructed phylogenetic tree (Fig. 2) did not support the presence of subgroups or clusters. However, the observed sequence differences can affect annealing of PCR primers, and thus, the ability to detect pathogenic Y. pseudotuberculosis by PCR. Hence, detection primers should be designed to the conserved areas of yadA (Supplementary Fig. S2). The commonly used yadA detection primers (Thoerner et al., 2003, Fukushima et al., 2003) have 2–3 predicted mismatches per primer pair with the 18 Y. pseudotuberculosis strains studied.

FIG. 2.

Phylogenetic tree with bootstrap values on branches of yadA of 18 Yersinia pseudotuberculosis strains based on nucleotide sequences (EMBL accession numbers HE805233–HE805250). IP32953, sequence of yadA of the reference strain IP32953.

Apart from challenging diagnostics, a high variation at the amino acid level of YadA could have an impact on the dissemination of this bacterium in host tissues (Heise and Dersch, 2006) or on the immune responses of the host. In Clostridium botulinum, a difference of 7% at the amino acid level in neurotoxin gene sequences has been shown to affect the antigenic properties of botulinum neurotoxin, but no data are available on the effects of smaller differences on the binding of antibodies (Lou et al., 2010).

Detailed comparison between the yadA sequences (Supplementary Fig. S2) revealed that most of the nucleotide substitutions resulted in amino acid substitutions; only for three out of the 40 affected codons were the nucleotide substitutions synonymous. The substitutions were heavily concentrated in the N-terminal proline-rich brim-like domain at the top of YadA head (Supplementary Fig. S3), and a few substitutions affected the head domain β-rolls, while the C-terminal half of the protein comprising the stalk and the membrane-anchoring barrel was virtually unaffected. In the left-handed parallel β-rolls, the 100% conserved glycine residues (highlighted in Supplementary Fig. S3) allow very tight turns (Nummelin et al., 2004). In each β-roll, the four amino acids preceding the glycine face inside the head, and the four residues following the glycine face outside. In a highly consistent manner, substitutions occurred only in residues following the glycine residue, indicating that these substitutions were tolerated and those introducing a charged residue could potentially change the surface properties of the YadA head. The changes affecting the proline-rich brim included, in addition to substitutions, deletions of 3 and 9 bp and an insertion of 6 bp; all of these affected the brim and could also potentially affect the functional properties of YadA.

Based on the distribution of the amino acid substitutions in the YadA sequences, the strains were divided into nine groups (Supplementary Fig. S2). Groups 2 and 3 differed only at one non-synonymous nucleotide residue from group 1, while group 4 differed at three positions. Group 6, in addition to nucleotide substitutions, had a 9-bp deletion, groups 7 and 8 had 3- and 9-bp deletions, and group 9 had a 6-bp insertion. It is possible that at some point the genes have been subjected to recombination since several identical substitutions are present in group 5, 6, and 9 sequences (Supplementary Fig. S2).

Finally, we made pairwise comparisons of the concatenated housekeeping genes (total length 2627 bp) for the nine strains for which the sequence type (ST) data was available (Supplementary Table S2) (Laukkanen-Ninios et al., 2011). Identity percentage between the concatenated sequences of Y. pseudotuberculosis strains ranged from 99.543% (12 mismatches) to 99.924% (two mismatches). It was 99.467% between the Y. similis strains (STs 76 and 83) and ranged from 95.166% to 95.965% between the Y. pseudotuberculosis and Y. similis strains. For Y. pseudotuberculosis, the corresponding identity percentage between inv and yadA followed roughly the ST percentage pattern (Tables 2 and 4 and Supplementary Table S2), but were throughout 0.057–0.795 smaller for inv and −0.043 to 1.595 smaller for yadA (Supplementary Table S2). Thus, the virulence genes represent relatively narrow diversity.

Conclusion

The virulence genes inv and virF are very similar among Y. pseudotuberculosis strains representing different serotypes and originating from diverse sources. In contrast, variation observed in yadA is reasonably high, which may affect the sensitivity or specificity of PCR-based detection, as well as the spread and immune responses induced by Y. pseudotuberculosis in the host.

Footnotes

Acknowledgments

This work was supported by the Finnish Center of Excellence in Microbial Food Safety Research, Academy of Finland (grants 118602 and 141140), the Doctoral Program of the Faculty of Veterinary Medicine of the University of Helsinki, the Finnish Veterinary Foundation, the Walter Ehrström Foundation, and the Medical Fund of the University of Helsinki.

Disclosure Statement

No competing financial interests exist.

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