Heterogeneity of theYersiniaYopM protein

Microbial Pathogenesis 1998; 25: 343–348 Article No. mi980247

MICROBIAL PATHOGENESIS

SHORT COMMUNICATION

Heterogeneity of the Yersinia YopM protein Anne Boland, Sophie Havaux & Guy R. Cornelis∗ Microbial Pathogenesis Unit, Christian de Duve Institute of Cellular Pathology and Faculte´ de Me´decine, Universite´ Catholique de Louvain, UCL 74-49. B-1200 Brussels, Belgium (Received July 14, 1998; accepted in revised form September 14, 1998)

Virulent Yersinia species (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica) possess a 70kb virulence plasmid that encodes the Yop virulon. This virulence system allows extracellular bacteria adhering at the surface of eukaryotic cells to secrete and inject bacterial effector proteins, called Yops, into the cytosol of these cells in order to disarm them. These secreted Yop proteins are remarkably conserved among the different species. A Y. enterocolitica O:8 strain was found to secrete a protein antigenically related to YopM but significantly larger. Sequencing of the corresponding gene showed that the protein was a YopM variant with three repeats of one domain. Comparison of the yopM gene of various Yersinia strains by PCR amplification, as well as analysis of the secreted Yop proteins by SDS-PAGE and Western blotting revealed that, unlike the other Yops, the YopM protein shows some heterogeneity.  1998 Academic Press Key words: Yersinia enterocolitica, virulence, Yops, type III secretion.

Introduction Among the Yersinia genus, only three species are pathogenic for humans, namely Y. pestis, Y. pseudotuberculosis and Y. enterocolitica. Pathogenic Yersinia strains all harbour a plasmid of 70kb called pYV that encodes the Yop virulon. This system allows extracellular bacteria adhering to the surface of the eukaryotic cells to inject bacterial effector proteins, called Yops, into the cytosol of the eukaryotic cells in order to disable them [for review, see 1]. The Yop virulon basically ∗Author for correspondence: Dr. G. Cornelis, Microbial Pathogenesis Unit, Avenue Hippocrate 74, UCL box 74-49, B-1200 Brussels, Belgium. 0882–4010/98/120343+06 $30.00/0

consists of a type-III secretion machinery (Ysc) devoted to Yop secretion [2–10], a set of Yop effectors, namely YopE, YopH, YopO/YpkA, YopM, YopP and YopT [11–19, Sory et al., unpublished results] and at least three Yop proteins, YopB, YopD and LcrV required for translocation of the effectors across the eukaryotic cell membrane [11–21]. Sequence comparisons showed that YopE, YopH, YopB and YopD are more than 95% conserved between the different species [22–27]. In contrast, some heterogeneity between the species and strains has been reported for LcrV [28]. The yopM gene has been sequenced in Y. pestis [29] and in Y. enterocolitica W22703 [16]. In both cases it was found to encode a protein of 367 residues with a calculated molecular weight of 41.6kDa and an isolectric point of 4.09. Unlike the  1998 Academic Press

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Figure 1. (a) SDS-PAGE (Coomassie blue stained) showing the proteins secreted by strain E40 (lane 1), strain A127/90 (lane 2), strain MRS40(pABS2) (lane 3). The arrows point to the additional protein of strains A127/90 and MRS40(pABS2). The letters identify the different Yop proteins. (b) Western blot with anti-YopM antibodies. Lane 1: strain E40; lane 2: strain A127/90.

other Yops, YopM contains repeated motifs [29] as well as leucine-rich repeats [16, 30]. Although YopM was first thought to bind thrombin and to have an extracellular role [31, 32], recent results show that YopM is delivered into the cytosol of eukaryotic cells [16, 33]. The Y. enterocolitica strains are classified into many serotypes and biotypes [34], only a few of which are pathogenic [35]. In Europe, the strains most frequently encountered belong to serotypes O:3, O:9 and O:5,27. Serotypes O:4, O:8, O:13a, 13b, O:18, O:20 and O:21, which are more virulent, were initially encountered only in North America [35, 36]. In 1990, the first case outside of North America of a human infection by a Y. enterocolitica O:8 strain was reported in Japan [37].

(b)

YopM ref. 367 residues

A

YopM A127/90 515 residues

B

Figure 2. (a) Amino acid sequence of the YopM protein from strain A127/90. The underlined residues are conserved in the YopM proteins of Y. enterocolitica (strain W22703, serotype 0:9) and Y. pestis. The repeated domains (residues 220 to 279, 280 to 339, 340 to 399, 400 to 459) are aligned. (b) Schematic representations of the YopM proteins. Alignment of the YopM protein of Y. pestis and Y. enterocolitica (A) with the YopM protein of strain A127/90 (B). The boxes represent the conserved domains and the line the domain that is different. The hatched box shows the domain that is repeated in the YopM protein of 57kDa.

Results and discussion Analysis of the Yop proteins secreted by strain A127/90 We compared the SDS-PAGE profile of the Yops secreted by the O:8 strain isolated in Japan (called hereafter strain A127/90; gift from G. Wauters) with that of our reference strain Y. enterocolitica O:9 E40 [14]. Figure 1(a) (lanes 1 and 2) shows that there are three major differences: in the case of the A127/90 strain, the bands corresponding

to the 41–kDa YopM and to the LcrV proteins are missing, while an additional 55kDa band is present. Immunoblot analysis with specific antiYop antibodies revealed that LcrV is present but not well separated from YopB (data not shown) and that the 55kDa protein strongly reacts with an anti-YopM polyclonal serum [Fig. 1(b)]. These results suggested that no Yop protein is missing in strain A127/90, but that the YopM protein is different.

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Table 1. MiPA number Strain

Origin

Serotype

1087 1119 812 1015 1068 1074 955 1091 1089 1078 1083 421

Japan — USA (Ohio) — — — — Canada Canada — USA —

O:8 O:9 O:8 O:9 O:9 O:3 O:3 O:13a O:13b O:21 1 1b 3

Y. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y.

enterocolitica A127/90 enterocolitica E40 enterocolitica 8081 enterocolitica W22703 enterocolitica AT319 enterocolitica W804 enterocolitica C1432 enterocolitica A285 enterocolitica A11/86 pseudotuberculosis pseudotuberculosis pseudotuberculosis pIB1

Cloning and sequencing of the yopM gene of strain A127/90

Comparison of YopM in various Yersinia strains

In order to elucidate this major difference, we cloned and sequenced the yopM gene from strain A127/90. A 4kb EcoRI fragment containing the entire yopM gene was identified by Southern blotting, and cloned in pBluescript KS-, leading to plasmid pABS2. To ensure that this plasmid indeed contained the entire yopM gene, we introduced it by electroporation in strain MRS40, a bla derivative of strain E40 [38] and we analysed the proteins secreted by the recombinant strain. An additional 55kDa protein was secreted by strain MRS40(pABS2) [Fig. 1(a), lane 3], indicating that the pABS2 plasmid indeed contains the entire yopM gene. We then sequenced plasmid pABS2 and several deletion clones thereof in order to obtain the entire sequence of the A127/ 90 yopM gene on one strand. This gene turned out to be 1515 nucleotides long and to encode a protein of 505 residues with a calculated molecular weight of 56.9kDa, which fits with that of the observed protein. The isoelectric point of the predicted protein is 4.2. Alignment of the YopM amino acid sequence from strain A127/90 with those of Y. pestis and Y. enterocolitica revealed that the first 200 and the 100 last residues are highly conserved [Fig. 2(a), underlined sequence]. A more detailed analysis showed that the central part of the YopM A127/90 protein consists of the triple repetition of a 60-amino acid-domain [hatched in Fig. 2(b)], which is present at the Cterminal end of the reference YopM protein [see Fig. 2(b)]. The larger size of the YopM protein in strain A127/90 is thus due to the triplication of a portion of the gene.

To investigate if this variation in the YopM protein was a peculiarity of strain A127/90, or if it was also encountered in other strains, we amplified the yopM gene from several strains (listed in Table 1) by PCR and analysed the various PCR products on a 0.7% agarose gel. Figure 3a shows that, as expected, the yopM gene of the A127/90 strain is 1.5kb long while the yopM genes of our reference strains E40 and W22703 and of the Y. enterocolitica 8081 O:8 strain have a size around 1.1kb. Among the strains tested, the three Y. pseudotuberculosis strains and the Y. enterocolitica O:21 strain also showed some heterogeneity in the yopM gene. These results were confirmed by analysis of the secreted proteins by immunoblotting with a polyclonal anti-YopM serum [Fig. 3(b)]. Comparison of the Yop profiles in SDS-PAGE confirmed that the other Yops are well conserved [Fig. 3(c)]. Taken together, these observations demonstrate the YopM, contrary to the other Yop proteins, can display size polymorphism among the pathogenic Yersinia strains. This is in good agreement with the observation that monoclonal antibodies directed against the Y. enterocolitica O:9 YopM protein can react with proteins of different sizes in other Y. enterocolitica serotypes and in Y. pseudotuberculosis [39]. The presence of a modified YopM protein apparently does not alter the virulence of the strain, since the A127/90 strain was reported as highly virulent [37]. The presence of differences in the YopM protein is obviously not related to a particular serotype, and it has thus probably no epidemiological significance.

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(a) ATG

TAG

MIPA 375

MIPA 283

induction of the yop regulon and Yop proteins analysis were carried out as previously described [27].

SDS-PAGE and immunobloting 2 kb 1.6 kb

Secreted Yop proteins were separated on a 14% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% non fat dry milk in phosphate-buffered saline, the membrane was probed with rabbit polyclonal antiYopM antibodies (dilution 1/500). Immunoreactive bands were visualized by incubation for 1h with goat anti-rabbit antibodies conjugated to horseradish peroxidase (dilution 1/1000; Dako), followed by revelation with enhanced chemiluminescence reagents (Pierce).

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H M B V D N P E Q

Figure 3. Analysis of the yopM genes of strains Y. enterocolitica A127/90 (lane 1), Y. enterocolitica E40 (lane 2), Y. enterocolitica 8081 (lane 3), Y. enterocolitica W22703 (lane 4), Y. enterocolitica AT319 (lane 5), Y. enterocolitica W804 (lane 6), Y. enterocolitica C1423 (lane 7), Y. enterocolitica A285 (lane 8), Y. enterocolitica A11/86 (lane 9), Y. pseudotuberculosis (serotype 1) (lane 10), Y. pseudotuberculosis (serotype 1b) (lane 11), Y. pseudotuberculosis pIB1 (serotype 3) (lane 12). (a) PCR amplification of the yopM gene. (b) Western blot analysis of the secreted proteins with a polyclonal anti-YopM serum. (c) Analysis of the secreted proteins by SDS-PAGE (Coomassie blue stained). The arrowheads show the different YopM proteins. Note that there is little or no variation for YopO, YopH, YopB, YopD, YopN, YopP, YopE and YopQ. LcrV cannot be identified in all the lanes.

However, since the activity of YopM inside eukaryotic cells is not known for the moment, we could not test whether these modifications alter the function of the protein.

Molecular cloning and sequencing procedures To clone the yopM gene of strain A127/90, pYV plasmid DNA was subjected to Southern blot analysis using the entire yopM gene of Y. enterocolitica W22703 as a probe. This allowed us to identify a 4kb EcoRI fragment containing the entire yopM gene. This EcoRI fragment was cloned in pBluescript KS-, leading to plasmid pABS2. This plasmid was then submitted to gradual deletions by exonuclease III. Sequencing of plasmid pABS2 and several deletion clones allowed us to obtain the entire sequence of the yopM gene on one strand. Sequencing was done by the Sanger method, using the Taq cycle sequencing kit from Amersham and an automated sequencer (Licor).

PCR amplification The yopM gene of the various strains was amplified from pYV plasmid DNA using amplimers MIPA 375 (5′-TTCGAATTCGGCATTCAATATGTTCA-3′) and MIPA 283 (5′-CGACAAGCTTATGAACGCTCTTGC-3′), located upstream and downstream of the yopM gene respectively. The various PCR products were then analysed on a 0.7% agarose gel.

Materials and methods Bacterial strains and growth conditions

Acknowledgements

All the Yersinia strains used in this study are listed in Table 1. Bacterial growth conditions,

We thank M. Iriarte for helpful discussion. I. Lambermont for her help during the sequencing and A.

Yersinia and YopM

Boyd for critical reading of the manuscript. We also thank G. Wauters for the gift of the Yersinia strains. A.B was research assistant funded by the Belgian ‘Fonds National de la Recherche Scientifique’. The Yersinia project is supported by the Belgian ‘Fonds National de la Recherche Scientifique Me´dicale’ (Convention 3.4595.97), the ‘Direction ge´ne´rale de la Recherche Scientifique-Communaute´ Franc¸aise de Belgique’ (Action de Recherche Concerte´e 94/99172) and by the ‘Interuniversity Poles of Attraction Program – Belgian State, Prime Minister’s Office, Federal Office for Scientific, Technical and Cultural Affairs’ (PAI 4/03).

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