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Infection and Immunity, July 2003, p. 3812-3820, Vol. 71, No. 7
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.7.3812-3820.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cytadherence-Deficient Mutants of Mycoplasma gallisepticum Generated by Transposon Mutagenesis

Sigalit Mudahi-Orenstein,1 Sharon Levisohn,2 Steven J. Geary,3,4 and David Yogev1*

Department of Membrane and Ultrastructure Research, Hadassah Medical School, The Hebrew University, Jerusalem 91120,1 Mycoplasma Unit, Kimron Veterinary Institute, Beit Dagan 50250, Israel,2 Department of Pathobiology,3 Center of Excellence for Vaccine Research, The University of Connecticut, Storrs, Connecticut 06269-30894

Received 26 November 2002/ Returned for modification 28 January 2003/ Accepted 26 March 2003

Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gmr HA-negative (HA-) colonies displaying a stable HA- phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA- mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA- mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.

* Corresponding author. Mailing address: Department of Membrane and Ultrastructure Research, Hadassah Medical School, The Hebrew University, Jerusalem 91120, Israel. Phone: 972-2-6758176. Fax: 972-2-6784010. E-mail: yogev{at}cc.huji.ac.il.

Editor: V. J. DiRita

Infection and Immunity, July 2003, p. 3812-3820, Vol. 71, No. 7
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.7.3812-3820.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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