INFECTION AND IMMUNITY, June 1990, p. 2017-2020 0019-9567/90/062017-04$02.00/0 Copyright C) 1990, American Society for Microbiology

Vol. 58, No. 6

Restriction Fragment Length Polymorphism in the Cytadhesin P1 Gene of Human Clinical Isolates of Mycoplasma pneumoniae SHATHA F. DALLO, JOHN R. HORTON, CHUNG J. SU, AND JOEL B. BASEMAN* Department of Microbiology, The University of Texas Health Science Center at San Antonio,

7703 Floyd Curl Drive, San Antonio, Texas 78284-7758 Received 16 November 1989/Accepted 14 February 1990

Genomic DNA obtained from Mycoplasma pneumoniae clinical isolates spanning a 30-year period was analyzed for the presence of polymorphism in their P1 cytadhesin genes. All clinical isolates expressed a 170-kilodalton P1 protein that reacted with anti-Pl monoclonal antibodies. However, Southern blot analysis of specific M. pneumoniae isolates with subclones of the P1 structural gene revealed the presence of restriction fragment length polymorphism, permitting the classification of their P1 genes into two distinct categories.

Mycoplasma pneumoniae is a common cause of acute respiratory infections in humans and provides an excellent model for the study of host-pathogen interactions. Virulent M. pneumoniae possesses a protein adhesin, designated P1, that mediates surface parasitism of respiratory epithelium (1, 4). The 170-kilodalton P1 protein clusters at the tip organelle of M. pneumoniae, permitting the recognition of host receptors, with subsequent colonization (1, 5, 6). Mutants that lack P1 or are unable to mobilize and concentrate the adhesin are avirulent (1, 7-9). Recently, we described the multiple-copy nature of regions of the P1 structural gene in wild-type M. pneumoniae M129-B16 (17). The possibility exists that P1-related sequences may recombine with the structural gene (18) and alter properties of the adhesin such as its affinity and specificity for host target receptors and its antigenicity (10). Therefore, we examined the P1 genes of different clinical isolates by Southern blot, analysis. Using genomic DNA from M. pneumoniae M129-B16, FH, and 25 clinical isolates derived from the 1960s, 1970s, and 1980s, we detected restriction length fragment polymorphism in the P1 structural genes of specific clinical isolates, permitting their categorization into two groups. Three clinical isolates of M. pneumoniae from the 1960s originated from the state of Washington and were provided by George Kenny (University of Washington, Seattle) (20); 16 isolates from the 1970s were derived from a military vaccine trial in South Carolina (21), and 6 strains from the 1980s were from France and were isolated by C. Bebear and H. Renaudin (both groups were provided by Joseph Tully, National Institute of Allergy and Infectious Diseases). Strain FH was purchased from the American Type Culture Collection (ATCC 15531). All strains, including the wild-type strain M129-B16 (ATCC 29342), were grown separately in 70 ml of SP-4 medium (19) in 32-oz. (ca. 950-ml) glass prescription bottles at 37°C for 72 h. Glass-adherent mycoplasmas were washed four times with phosphate-buffered saline (pH 7.2) and collected by centrifugation (9,500 x g for 20 min). Total protein profiles of the clinical isolates were determined by solubilizing mycoplasma pellets, separating proteins on 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels, and either staining gels with Coomassie brilliant blue or electrophoretically transferring proteins to nitrocellulose paper prior to immunoblotting (12). Examination of the stained polypeptides revealed no detectable differences in the one*

dimensional protein patterns of these isolates, including the 170-kilodalton region that represents cytadhesin P1 of virulent M. pneumoniae (Fig. 1). Furthermore, the P1 proteins in these clinical isolates were immunologically reactive with anti-Pl monoclonal antibodies generated against strain M129-B16, reinforcing the critical role of P1 in virulence. Nonetheless, we decided to monitor representative clinical isolates for possible differences in their P1 structural genes. Each clinical isolate was harvested, suspended in 2.7 ml of phosphate-buffered saline, lysed by the addition of 0.3 ml of 10% SDS, and incubated with 50 pug of RNase (Boehringer A B16 200-.

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FIG. 2. Southern blot analysis of M. pneumaniae genomic DNA from wild-type strain B16 (I) and a representative clinical isolate of M. pneumoniae (II) with subclones B through M, which encode the P1 structural gene (17; see Fig. 3). DNA was digested with different restriction enzymes (left to right: EcoRI, HindIII, Sacl, and SmaI), separated by electrophoresis, and transferred to nitrocellulose filters as described in Materials and Methods. Letters (B to M) over each group of profiles coincide with the restriction map shown in Fig. 3, and horizontal bars with values on the left indicate the sizes (in kilobases) of the Hindll-digested phage DNAs used as molecular weight markers. Subclones F, G, L, and M produce single-copy patterns of hybridization, while subclones B, C, D, E, H, I, J, and K hybridize to numerous bands, demonstrating the P1-related multicopy gene domains (17).

Mannheim Biochemicals) per ml for 30 min at 37°C. DNA preparations were extracted three times with an equal volume of phenol equilibrated with 1 M Tris (pH 8.0), once with phenol-chloroform (1:1), and once with chloroform-isoamyl alcohol (24:1). Sodium acetate (3 N; 0.1 volume) was added to the DNA preparation, and the DNA was precipitated with ethanol. Individual DNA preparations were digested to completion with different restriction enzymes (EcoRI, HindIII, Sacl, and SmaI) before electrophoretic separation on 0.7% agarose gels. DNA was transferred to nitrocellulose filter paper (15) with 20x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), rinsed once with 6x SSC, and then baked at 80°C for 2 h under vacuum. Filters were prehybridized overnight at 37°C in 20 ml of prehybridization solution

containing 6x SSC, 0.5% SDS, 5x Denhardt solution (bovine serum albumin, polyvinylpyrrolidone, and Ficoll [Pharmacia Fine Chemicals] each at 1 mg/ml), and 0.1 mg of denatured herring sperm DNA per ml. Hybridizations were carried out at 68°C in 10 ml of prehybridization solution plus 32P-labeled P1 gene subclone probes (17). After hybridization, filters were rinsed twice in 2x SSC-0.5% SDS at room temperature, washed at 68°C in 0.1 x SSC-0.5% SDS for 2 h with several buffer changes, dried at room temperature, and exposed to X-ray film. Twelve nonoverlapping subclones that encode the entire P1 gene of M. pneumoniae M129-B16 (17) were used as probes to hybridize to the genomic DNA of the clinical isolates. Panels labeled I in Fig. 2 represent M. pneumoniae M129-B16 genomic DNA cut with EcoRI, HindIII, SacI, and

VOL. 58, 1990

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FIG. 3. Restriction enzyme map of the P1 structural gene of wild-type strain B16 and map of a representative clinical isolate. H*, New restriction site of restriction enzyme HindIll in the P1 structural gene of the clinical isolate. 1, P1 structural gene of M. pneumoniae. Sites for restriction enzymes that cut more than once are numbered starting from the 5' end. Numbers in parentheses are site numbers.

SmaI (from left to right), and panels labeled II represent specific DNA from a representative clinical isolate cut with the same restriction enzymes. Each panel was hybridized separately with P1 subclones B through M (17; Fig. 3). Southern blot analysis of the clinical isolate revealed single and multiple P1-related DNA fragments (Fig. 2, panels labeled II), as previously reported for M. pneumoniae M129B16 (17). However, the restriction patterns of the P1 structural gene differed between strain M129-B16 and this representative isolate. For example, the P1 gene of wild-type M. pneumoniae M129-B16 hybridized to a major 4.4-kilobase (kb) HindIII fragment (subclones B through J) and a 2.1-kb band (subclones K through M). In the clinical isolate, HindIII digestion resulted in the disappearance of the 4.4-kb band when genomic DNA was hybridized with subclones B and C, and a new 1.3-kb band appeared. When subclones D through J were used for hybridization to genomic DNA of the clinical isolate, the 4.4-kb band disappeared and a new band of 'about 3.1 kb appeared. Subclones K through M gave a 4.1-kb band in the clinical isolate rather than the 2.1-kb band observed in M129-B16. These data suggested that the clinical isolate possessed restriction fragment length polymorphism in the subclones that spanned the P1 gene, when compared with wild-type strain M129-B16. To determine the extent to which this polymorphism existed, we performed similar Southern hybridizations on the remaining M. pneumoniae isolates. We detected patterns identical to those represented by category II (Fig. 2) in 22 of 25 clinical isolates plus strain FH. The three clinical isolates from the state of Washington displayed hybridization patterns identical to that of the wild-type strain M129-B16. From these results a map of the P1 structural gene was constructed for this distinct category (II) of clinical isolates (Fig. 3), indicating a new HindIII site inside the P1 structural gene. Also, a new HindIII site that was detected outside the

P1 structural gene gave rise to the 4.1-kb band rather the 2.1-kb band at the carboxy end of M129-B16. Other differences in the restriction enzyme patterns could be observed, but they were associated with the P1-related sequences. Since regions of the P1 gene appear as multiple copies, recombination between P1 and the related sequences homologous to P1 could result in the appearance of new restriction sites, thus providing the basis for variations in P1 genes among M. pneumoniae isolates. Genes for outer membrane proteins of microbial pathogens such as Neisseria gonorrheae have been reported to exist as multigene families (2, 3, 13, 16). It is known that gene conversion among different members of these gene families alters the structural and functional properties of these proteins (14, 16). DNA rearrangements can also cause antigenic variations in the surface proteins of Borrelia hermsii, an arthropod-borne pathogen that causes relapsing fever in humans (11). Our current and future studies will focus on the sequencing of P1 structural genes from representative clinical isolates. We intend to determine the role of restriction fragment length polymorphisms and of possible sequence alterations in the functionality of P1 cytadhesin and subsequent tissue tropism and virulence. This research was supported in part by Public Health Service grant Al 18540 from the National Institute of Allergy and Infectious Diseases. We thank Rose Garza for secretarial assistance.

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