Identification of a possible cytadherence regulatory locus in Mycoplasma pneumoniae

C T Hedreyda, D C Krause
1995 Infection and Immunity  
Transposon mutagenesis was used to analyze Mycoplasma pneumoniae cytadherence. Mycoplasmas were electroporated with Tn4001, and transformants were identified by antibiotic selection using gentamicin. The resulting colonies were screened for hemadsorption (HA) as an indicator for cytadherence. Six HA ؊ colonies from independent transformations were isolated, filter cloned, and characterized in more detail. Southern hybridization analysis revealed that all six transposon insertions mapped to the
more » ... ame 252-kbp ApaI fragment and 19.5-kbp XhoI fragment. More detailed analysis localized the insertions to two adjacent EcoRI fragments. This site is distinct from the locus containing the genes for the high-molecular weight cytadherence-accessory proteins HMW1 and HMW3, and yet these proteins were absent from the protein profiles of all six transformants. To determine if transposon insertion was responsible for the HA ؊ phenotype, reversion frequencies of the transformants were assessed after passage in the presence of antibiotic selection. In contrast to a spontaneously arising HMW-deficient variant, which reverted to an HA ؉ phenotype readily, no HA ؉ revertants were identified for any of the six transformants. These observations suggest that a potential regulatory locus that may be important in the expression of the HMW cytadherence-accessory proteins has been identified. Attachment of the cell wall-less prokaryote Mycoplasma pneumoniae to the mucosal epithelium (cytadherence) is a crucial step in the successful colonization of the human respiratory tract, leading to atypical pneumonia and tracheobronchitis (8, 12). Cytadherence is generally mediated by a differentiated terminal structure (1, 4, 11, 18), seen as a membranebound extension of the mycoplasma cell, at what is the leading end as mycoplasmas move by gliding motility (6). The cytadhesin proteins P1 (1, 19) and P30 (2, 3, 7) are clustered at this attachment organelle (1, 4, 11, 18), apparently through their interaction with a complex network of cytadherence-accessory proteins (22, 23) . These include the high-molecular-weight proteins HMW1 to HMW5, which are components of a cytoskeleton-like scaffolding network in the mycoplasma cell (37, 38) . HMW3 appears to be a structural element of the attachment organelle, where it may anchor the adhesin proteins at the tip structure (38). HMW1 and HMW4, on the other hand, are associated exclusively with the filamentous extensions of the mycoplasma cell (37). While HMW1 and HMW4 are phosphorylated by an ATP-dependent Ser/Thr kinase (9, 26), their function remains unclear. The expression of HMW1 to HMW5 by M. pneumoniae is coordinately regulated (22, 23) . Spontaneously arising variants lacking HMW1 to HMW5 are avirulent, cytadhere very poorly, and fail to cluster the adhesin P1 at the attachment organelle, which has an altered shape from that seen in wild-type mycoplasmas (14, 15, 22, 23) . This is a reversible phenomenon, with reacquisition of HMW1 to HMW5 corresponding to a return to a virulent, cytadherence-positive phenotype. The genes for HMW1, HMW3, and P30 have been cloned and sequenced (7, 10, 21, 33, 34) and are closely linked on the mycoplasma chromosome. However, the mechanism regulating the phase-variable expression of HMW1 to HMW5 remains unclear. Recently we described the electroporation of M. pneumoniae
doi:10.1128/iai.63.9.3479-3483.1995 fatcat:atqzc753njbkpmx4jv7b5aylsu