Genetic variation among Mycoplasma agalactiae isolates detected by the variant surface lipoprotein gene (vspA) of Mycoplasma bovis

FEMS Microbiology Letters 156 (1997) 123^128

Genetic variation among Mycoplasma agalactiae isolates detected by the variant surface lipoprotein gene (vspA) of Mycoplasma bovis

Ravenna Flitman-Tene a b , Sharon Levisohn b , Ricardo Rosenbusch c , Eitan Rapoport d , David Yogev a * ;

;

a

Department of Membrane and Ultrastructure Research, The Hebrew University - Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel

b c d

Mycoplasma Unit, Kimron Veterinary Institute, Beit Dagan 50250, Israel

Veterinary Medical Research Institute, Iowa State University, Ames 50011, IA, USA

Small Ruminant Pathology, Israel Veterinary Services, P.O. Box 3, 18100 Afula, Israel

Received 30 May 1997 ; revised 30 August 1997; accepted 5 September 1997

Abstract

Multiple restriction fragments, homologous to the previously described Mycoplasma bovis vspA gene, were identified in the chromosome of Mycoplasma agalactiae. The vspA, a representative variable surface lipoprotein gene of the vsp gene family, and four synthetic oligonucleotides, representing sequences complementary to selected regions of the vsp genes, were used as probes against digested chromosomal DNAs of several M. agalactiae clinical isolates. The resulting Southern blot analysis demonstrated a marked DNA polymorphism of multiple vspA-related fragments among the isolates. An oligonucleotide representing a conserved 5P-region common to all known vsp genes, was found to hybridize to multiple M. agalactiae genomic fragments while the other three oligonucleotides, representing distinct repetitive structures within the coding region of three known vsp genes (vspA, vspE, and vspF), failed to react. These results argue for the possible existence of a gene family in M. agalactiae analogous to the vsp system of M. bovis but comprised of diverse genes. Keywords : Mycoplasma agalactiae

;

Mycoplasma bovis

; Variable surface lipoprotein; Gene family; Genetic variation

1. Introduction

Mycoplasma agalactiae is the major etiological agent of contagious agalactia, a disease of small ruminants of considerable economic importance, prevalent mainly in the Mediterranean countries [1]. This

* Corresponding author. Tel.: +972 (2) 6758-176; Fax: +972 (2) 6784-010; E-mail: [email protected]

mycoplasma species is phylogenetically closely related to Mycoplasma bovis [2], an important bovine pathogen causing mastitis, arthritis and respiratory diseases [3]. The close relationship between the two species is manifested by the high degree of homology in the 16S rRNA genes of both species [2], and by the presence of shared antigens detected with polyclonal or monoclonal antibodies [4,5]. On the other hand, DNA hybridization experiments have shown

0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 1 3 - 8

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only 40% homology between M. agalactiae and M. bovis genomes, a value which clearly indicates that these organisms are two distinct species [4]. Recently, a new system generating surface antigenic variation was discovered in M. bovis. A set of variable membrane surface lipoproteins designated Vsps, encoded by a cluster of related but divergent vsp genes, constitutes the vsp gene family. The Vsp proteins undergo spontaneous noncoordinate highfrequency phase variation between ON and OFF expression states as well as changes in size, giving rise to numerous possibilities of surface diversi¢cation [6,7]. The spontaneous high rate of Vsp phenotypic switching was recently shown to involve reversible DNA rearrangements occurring at a high frequency during oscillating phase transition of individual Vsps [7]. The antigenic relatedness between M. agalactiae and M. bovis, led us to explore the possibility that a genetic system analogous to the vsp gene family found in M. bovis [7] may also be present in M. agalactiae. Genes encoding surface antigens, in particular those which mediate high-frequency phenotypic switching, have not yet been explored in this organism. In this study, the vspA gene, a representative variable surface lipoprotein gene of the M. bovis vsp gene family, and several vsp-speci¢c synthetic oligonucleotides, representing sequences complementary to selected regions of known vsp genes [7], were used as probes to identify the homologous M. agalactiae genomic sequences. We provide evidence that (i) the chromosome of M. agalactiae possesses multiple vsp-related restriction fragments, (ii) these fragments exhibited marked DNA polymorphism among M. agalactiae clinical isolates, and (iii) the conserved 5P-region shared by all known vsp genes, is present in these multiple genomic fragments, while distinctive vsp repetitive sequences within the vsp structural genes of M. bovis were not detected in M. agalactiae. 2. Materials and methods

2.1. Mycoplasma strains

Type strain M. agalactiae PG2 was originally obtained from Dr. D.G.¡. Edward, Wellcome Research

Laboratories, Beckenham, Kent, England. M. bovis PG45 type strain was obtained from the collection of the Institute for Microbiology and Infectious Diseases of Animals, School of Veterinary Medicine, Hannover, Germany. Other mycoplasma species were obtained from the Jerusalem laboratory collection and included M. gallisepticum, M. imitans, M. capricolum, M. genitalium, M. synoviae, M. pirum, and M. penetrans. M. agalactiae ¢eld strains were isolated from typical cases of contagious agalactia in di¡erent areas of northern Israel during the period 1991^1995. Seven strains were isolated from milk of mastitic animals (#15445, #5512, #13605, #5854, and #80346/1 from sheep; #55003/1 and #5292 from goats) whereas strain #80346/2 was isolated from the eye of a lamb with conjunctivitis and strain #5026 from a joint of an arthritic sheep. Isolation was carried out by standard methods using mycoplasma medium based on Brain Heart Infusion broth (Difco) and 20% horse serum. Identi¢cation was based on the biochemical properties, with de¢nitive identi¢cation and di¡erentiation from M. bovis and other ruminant mycoplasmas by immuno£uorescence of the mycoplasma colonies using £uorescein conjugated antisera speci¢c for M. agalactiae and M. bovis (Dr. J.G. Tully, Frederick, MD, USA). Speci¢c antisera for M. agalactiae, used in the indirect immuno£uorescence test, were obtained from Dr. G. Jones (Edinburgh, UK) and for M. bovis from Dr. K. Sachse (Jena, Germany). 2.2. DNA manipulation, plasmids and oligonucleotides

Genomic DNA was digested by restriction enzymes, electrophoresed and hybridized as previously described [7]. The probes employed included: the plasmid pMC5 carrying one of highly conserved ribosomal ribonucleic acid (rRNA) operons from Mycoplasma capricolum [8], the plasmid pKA63 carrying the vspA gene on a 1.5 kb HindIII genomic fragment of M. bovis PG45 clonal isolate [7]. Oligonucleotide vsp-pro used for Southern blot hybridization was: 5PGGAGAGGATAAATTTATGA-3P. The nucleotide sequences of oligonucleotides RA 4, RF 2, RE 1 and the hybridization conditions were described elsewhere [9].

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EcoRI-digested

3. Results

125

chromosomal DNA of

M. agalactiae

PG2 (type strain) and of several clinical isolates of

M. agalactiae

As a ¢rst step it was necessary to genetically distinguish between was

achieved

by

M. bovis using

and

the

M. agalactiae.

recombinant

as well as of several other mycoplasma

species. Interestingly, a marked DNA polymorphism

This

of multiple

plasmid

vspA-related

restriction fragments of dif-

M.

pMC5 as a probe against genomic DNAs of the

ferent intensity and size was observed among the

two species digested by several restriction enzymes.

agalactiae

Comparison of the rRNA hybridization pro¢les of

bridization pattern is shown in Fig. 2. No homology

M. bovis

was detected with the other

and

M. agalactiae

(Fig. 1A lanes 1^3 and

isolates. An example of the

HindIII

mycoplasma species

lanes 4^6, respectively) indicated that the two species

(data not shown). On the other hand, all

can be clearly distinguished by this method. For ex-

tiae

ample, the

M. bovis HindIII

hy-

M. agalac-

isolates tested, including the PG2 strain, exhib-

ited a uniform rRNA hybridization pattern when

hybridization pattern

(Fig. 1A, lane 1) showed four distinct fragments

their

which are di¡erent in size from those obtained with

against the recombinant plasmid pMC5 (Fig. 1B).

the

M. agalactiae HindIII-restricted

as detected by the

M. agalactiae genomic fragments hoM. bovis vsp gene family, the rehybridization

Fig. 1. rRNA ¢ngerprints of

agalactiae

against

M. agalactiae

and

HindIII-

vspA

M. bovis.

or

with the restriction endonucleases

HindIII

hybridized

M. agalactiae

strains,

gene, suggests that the ob-

gene.

To further characterize the nature of the observed

Chromosomal DNA (4

PG2 (panel A, lanes 4^6) type strains, or from several

vspA

were

speci¢c genomic region with a high homology to the

combinant plasmid pKA63 was used as a probe in blot

DNAs

served DNA polymorphism may be restricted to a

mologous to the Southern

genomic

The heterogeneity among the

genomic DNA

(Fig. 1A, lane 4). To identify

restricted

M. agalactiae

Wg)

from

M. bovis

PG45 (panel A, lanes 1^3), from

M.

clinical isolates (panel B), were digested to completion

(panel A, lanes 1 and 4 and panel B, lanes 1^10), EcoRI (panel A, lanes 2 and 5), and EcoRV 32 P-labeled recombinant plasmid pMC5 carrying one of

(panel A, lanes 3 and 6), and subjected to Southern blot hybridization with the the rRNA operons of

M. capricolum. M. agalactiae

isolates included :

M. agalactiae

PG2 (lane 1), 5292 (lane 2), 80346/1 (lane 3), 80346/2

(lane 4), 13605 (lane 5), 15445 (lane 6), 5854 (lane 7), 5512 (lane 8), 5026 (lane 9), and 55003/1 (lane 10). Molecular size markers are indicated.

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R. Flitman-Tene et al. / FEMS Microbiology Letters 156 (1997) 123^128

126

vsp-5P-region-associated genomic fragM. agalactiae clinical isolates. Chromosomal DNA Wg) from M. agalactiae ¢eld isolates (depicted in Fig. 1, panel lanes 1^10) and also from M. bovis PG45 (lane 11, served as

Fig. 3. Identi¢cation of the Fig. 2. Restriction length fragment polymorphism of the sociated genomic fragments among Chromosomal DNA (4

Wg)

vspA-as-

M. agalactiae clinical isolates. M. agalactiae isolates

from several

(depicted in Fig. 1, panel B, lanes 1^10), was digested to completion with the restriction endonucleases Southern

blot hybridization 63 plasmid pKA carrying the

M. bovis

with

vspA

the

HindIII 32

and subjected to

P-labeled

recombinant

gene from a clonal isolate of

PG45 type strain. Molecular size markers are indicated.

vsp-associated homology between M. agalactiae and M. bovis, four synthetic oligonucleotides, representing selected regions of the vsp genes, were used as

ments among (4 B,

a positive control), was digested to completion with the restriction endonuclease

HindIII

and subjected to Southern blot hybrid-

ization with the synthetic oligonucleotide

vsp-pro.

Molecular size

markers are indicated.

tional oligonucleotides, designated

RE 1 ,

RA 4, RF 2,

and

representing distinct sequences complementary

probes in Southern blot hybridization. One oligonucleotide,

designated

vsp-pro

represents

complementary to the highly conserved stream region, shared by all

vsp

sequences

vsp-5P

up-

genes identi¢ed so

far ([7] ; Lysynansky, I. and Yogev, D., unpublished results). The hybridization patterns obtained using the

vsp-pro

HindIII-digested M. agalactiae strains depicted

oligonucleotide against

genomic DNAs of the

in Fig. 2, are shown in Fig. 3. Complex hybridization patterns of several bands of di¡erent intensity and exhibiting DNA polymorphism among the strains were observed. All these fragments were also detected by the

vspA

gene probe (Fig. 2).

As a positive control, the nomic DNA of

M. bovis

tides. Three oligonucleotides

HindIII-digested

ge-

PG45 was used (Fig. 3,

lane 11) to demonstrate the reaction of this probe with the multiple genomic fragments making up the

vsp

gene family of

M. bovis

[7]. When the three addi-

vsp-speci¢c oligonucleoRE 1 representing disknown vsp genes (vspA, vspF,

Fig. 4. Southern blot hybridization of

RA 4 , R F 2

tinct repetitive sequences of three and

vspE),

were used in Southern blot hybridization (panels A,

B, and C respectively) to probe 4 somal DNA of

agalactiae

M. bovis

Wg

of

HindIII-digested

chromo-

PG45 (lane 1 in each panel) or of

M.

PG2 (lane 2 in each panel). Molecular size markers

are indicated.

FEMSLE 7841 10-11-97

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R. Flitman-Tene et al. / FEMS Microbiology Letters 156 (1997) 123^128

127

to the unique repetitive coding sequences of the

homologous sequences downstream to the conserved

vspA, vspE

vsp-5P-region,

and

vspF

genes respectively [7], were

or perhaps the presence of distinct re-

used as probes against digested genomic DNAs of

petitive units within the corresponding genes of

M. agalactiae

agalactiae.

strains (depicted in Fig. 2), no homol-

ogy was detected (data not shown). An example demonstrating the positive reaction of and

RE 1

RA 4, RF 2,

oligonocleotides (Fig. 4, panels A, B and

C respectively) with digested genomic DNA of

bovis

M.

PG45 (Fig. 4, lane 1 in each panel) or the

negative

reaction

of

M. agalactiae

PG2

(Fig. 4,

M.

It is still possible, however, that other

vsp genes share homology with M. agalactiae genomic fragments. The

regions of the

the

identi¢ed

or-

ganizational and regional sequence similarity within

vsp

the 5P-region among the

genes in

M. bovis

[7],

o¡ers compelling evidence for gene duplication and divergence. It is, therefore, speculated that during evolution the two phylogenetically closely related

lane 2 in each panel) is shown.

mycoplasma species acquired the same genetic system for surface diversi¢cation and evasion of the host response, but underwent di¡erent host-speci¢c

4. Discussion

pressure. Evidence obtained in this study indicate that the

M. agalactiae

Surface antigens having tandem repetitive domains

chromosome possesses multiple ge-

are present in several pathogenic bacteria [11,12].

vspA gene of M. bovis was

Many of these are known or proposed as virulence

shown to consist of several related but also divergent

cluding evading the host immune response. Repeat

vsp genes [7], the vspA-associated fragments observed in M. agalactiae may represent an analogous multi-

structures in these molecules are thought to be li-

gene

phenotypic

tionary pressure in the bovine or in the small rumi-

switching in that organism. Several notable features

nant host is, therefore, likely to have a major e¡ect

nomic fragments homologous to the

M. bovis.

Since the

family

vsp

gene family in

mediating

high-frequency

of that putative system in ison to

vsp

gene family of

M. agalactiae in comparM. bovis, were revealed in

gand-binding domains [11,13]. The distinctive evolu-

on such repetitive antigens. A third potentially critical characteristic of the putative

this study. First, the

factors involved with pathogen^host interaction in-

M. agalactiae vspA-associated

fragments

contain sequences homologous to highly conserved 5P upstream regions shared by all known

vsp

genes

[7]. This was shown by Southern blot hybridization

vsp-pro

M. agalactiae

M. agalactiae emerged M. agalactiae patterns of multiple vspA-

system in

clinical isolates. Complex related

restriction

among the various

fragments

di¡ered

M. agalactiae

markedly

isolates were iden-

clin-

ti¢ed (Figs. 2 and 3). Our observation is not consis-

oligonucleotide probe.

tent with the ¢ndings reported by Tola et al. [15],

of genomic DNA of the selected ical isolates with the

vspA-related

from Southern blot analysis of selected

M. agalactiae

Conservation of the 5P regulatory region or of the N-

according to which

terminus

of

the PG2 type strain, are genetically homogenous.

genes comprising systems generating surface diversity

Solsona et al. [14] also reported on the homogeneity

portion

encoding

the

signal

peptide

in mycoplasmas, was documented for the

Mycoplasma hyorhinis genes of M. gallisepticum vsp genes of M. bovis [7]. of

vlp

genes

[10] and recently for the

heterogeneity of the

vsp-related genomic fragments M. agalactiae strains shown in this study also observed among several M. bovis strains

among

vspRF 2 and

comprising a signi¢cant portion of the struc-

vsp

strains, but with the excep-

tion of the PG2 type strain. The marked genetic

Second, in contrast to the conservation of the

tural region of these

M. agalactiae

[9] and for the pMGA

5P-region, distinct repetitive sequences (RA 4,

RE 1),

of several

isolates, including

was

(Kotser, S., Lysnyansky, I., Rosengarten, R. and Yogev, D., unpublished results). Recently, we have

genes ([7] ; Lysynansky, I.

shown that these complex hybridization patterns rep-

and Yogev, D., unpublished results), failed to react

resent di¡erent expression states of the correspond-

in Southern blot hybridization with restricted ge-

ing

nomic DNAs of the

M. agalactiae

clinical isolates

tested. This indicates the absence of these repetitive

vsp

genes, and that the observed phenotypic

switching of the Vsp lipoproteins in

M. bovis

is a

result of chromosomal rearrangements occurring at

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128

a high frequency during oscillating phase transition

vsp-analogous

of individual Vsps [7]. The putative system in

M. agalactiae

may, therefore, regulate in

a similar fashion. This is the ¢rst evidence, which indicates that the genome of

M. agalactiae

is sub-

jected to extensive variation which may occur at a high frequency and may also be linked with phenotypic switching. Characterization of the

M. agalactiae vsp-related

genes is underway to elucidate their structural features and the molecular basis of the genomic rearrangements in comparison to the

M. bovis.

vsp

gene system of

[5] Rasberry, U. and Rosenbusch, R.F. (1995) Membrane-associated and cytosolic species-speci¢c antigens of

vis

Mycoplasma bo-

recognized by monoclonal antibodies. Hybridoma 14, 481^

485. [6] Behrens, A., Heller, M., Kirchho¡, H., Yogev, D. and Rosengarten, R. (1994) A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of

vis.

Mycoplasma bo-

Infect. Immun. 62, 5075^5084.

[7] Lysnyansky, I., Rosengarten, R. and Yogev, D. (1996) Phenotypic switching of variable surface lipoproteins in

plasma bovis

Myco-

involves high-frequency chromosomal rearrange-

ments. J. Bacteriol. 178, 5395^5401. [8] Amikam, D., Razin, S. and Glaser, G. (1982) Ribosomal RNA genes in mycoplasma. Nucleic Acid Res. 10, 4215^4222. [9] Yogev, D., Rosengarten, R., Watson-McKown, R. and Wise, K.S. (1991) Molecular basis of

Mycoplasma

surface antigenic

variation : a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5P regulatory sequen-

Acknowledgments

ces. EMBO J. 10, 4069^4079. [10] Markham, P.F., Glew, M.D., Whithear, K.G. and Walker,

This work was supported by a grant (IS-2540-95R) from the United States-Israel Binational Agricultural Research

and

Development

Fund

(BARD).

skilled assistance of N. Hatib in isolation of

lactiae

I.D. (1993) Molecular cloning of a member of the gene family

Mycoplasma gallisep-

that encodes pMGA, a hemagglutinin of

ticum.

Infect. Immun. 61, 903^909.

The

[11] Dramsi, S., Dehoux, P. and Cossart, P. (1993) Common fea-

M. aga-

tures of Gram-positive bacterial proteins involved in cell rec-

from clinical samples is greatly appreciated.

ognition. Mol. Microbiol. 9, 1119^1122. [12] Kehoe, M.A. (1994) Cell-wall-associated proteins in Grampositive bacteria. In : Bacterial Cell Wall (Ghuysenm, J.-M. and Hakenbeck, R., Eds.), pp. 217^261. Elsevier Biomedical

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Lett.