Identification of a possible cytadherence regulatory locus in Mycoplasma pneumoniae

Abstract

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 same 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 phenoty...

Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium

“…These sizes are in agreement with the physical map of the M. pneumoniae genome digested with EcoRI [26]. The 9.2-klbp EcoRl fragment from pKV8 [ 161, carrying the ~30 and hmw3 genes upstream of the hmwl gene [17], hybridized to the same 9.2-kbp EcoRI fragment in wild-type and transformant 8R genomic DNAs digested with EcoRI (data not shown).…”

“…In M. pneumoniae transformant 8R, Tn4001 was localized in 14.3-kbp BamHI and 4%kbp EcoFU fragments (Fig. 4A, lanes 3 and 4; fragment sizes reflect a subtraction of the 4.7-kbp Tn4001 sequence from the calculated molecular weights [17,23] genomes of these Mycoplasma species did not carry Tn4001 sequences ( Fig. 4A and 4B, lanes 1 and 2).…”

“…Mycoplasma pneumoniae B 10 and Mycoplasma genitalium G37 strains were supplied by J. Tully (National Institute of Allergy and Infectious Diseases, Bethesda, MD) and grown in SP-4 medium [5,7] in tissue culture flasks. Plasmid pKV8 carrying the M. pneumoniae 9.4-kbp EcoRI DNA fragment, which encodes the P30 adhesin and HMW3 adhesin-related accessory protein [ 161, and plasmid pKV16 carrying the M. pneumoniae 5-kbp BumHI DNA fragment, which encodes the adhesin-related accessory protein HMWl [17,18], were provided by D. Krause (University of Georgia, Athens, GA). Plasmid pISMlOO1 carrying transposon Tn4001 [19] was a gift from C. Minion (Iowa State University, Ames, IA).…”

“…in 7). The DNA fragment sizes reflect a subtraction of 4.7-kbp (molecular weight of Tn4001) from the total calculated molecular weight [17,23]. Most gen-R isolates contained only one copy of Tn4001 although several clones possessed more than one copy.…”

Evaluation of Three Real-Time PCR Assays for Detection of Mycoplasma pneumoniae in an Outbreak Investigation

“…CS11301; Invitrogen) following the manufacturer's instructions. The CFU value per milliliter of each dilution was determined using a hemolytic plaque formation procedure as previously described (6). Each dilution was then tested with each assay in six replicates; values are reported in Table 1 as total CFU detected within each real-time PCR.…”