Transposon mutagenesis reinforces the correlation between Mycoplasma pneumoniae cytoskeletal protein HMW2 and cytadherence

Abstract

A new genetic locus associated with Mycoplasma pneumoniae cytadherence was previously identified by transposon mutagenesis with Tn4001. This locus maps approximately 160 kbp from the genes encoding cytadherence-associated proteins HMW1 and HMW3, and yet insertions therein result in loss of these proteins and a hemadsorption-negative (HA ؊ ) phenotype, prompting the designation cytadherence-regulatory locus (crl). In the current study, passage of transformants in the absence of antibiotic selection resulted in loss of the transposon, a wild-type protein profile, and a HA ؉ phenotype, underscoring the correlation between crl and M. pneumoniae cytadherence. Nucleotide sequence analysis of crl revealed open reading frames (ORFs) orfp65, orfp216, orfp41, and orfp24, arranged in tandem and flanked by a promoter-like and a terminator-like sequence, suggesting a single transcriptional unit, the P65 operon. The 5 end of orfp65 mRNA was mapped by primer extension, and a likely promoter was identified just upstream. The product of each ORF was identified by using antisera prepared against fusion proteins. The previously characterized surface protein P65 is encoded by orfp65, while the 190,000 M r cytadherence-associated protein HMW2 is a product of orfp216. Proteins with sizes of 47,000 and 41,000 M r and unknown function were identified for orfp41 and orfp24, respectively. Structural analyses of HMW2 predict a periodicity highly characteristic of a coiled-coil conformation and five leucine zipper motifs, indicating that HMW2 probably forms dimers in vivo, which is consistent with a structural role in cytadherence. Each transposon insertion mapped to orfp216 but affected the levels of all products of the P65 operon. HMW2 is thought to form a disulfide-linked dimer, formerly designated HMW5, and examination of an hmw2 deletion mutant confirms that HMW5 is a product of the hmw2 gene.Mycoplasma pneumoniae attachment to host respiratory epithelium is a multifactorial process, the complexity of which is only beginning to be appreciated (20). This cell wall-less prokaryote is a leading cause of pneumonia in older children and young adults. Colonization requires binding to host cell receptors (cytadherence) by mycoplasma surface proteins localized primarily to a differentiated terminal structure termed the attachment organelle (reviewed in reference 34). The membrane proteins P1 (169 kDa [19]) and P30 (30 kDa [2]) are thought to be directly involved in receptor recognition (34), although the evidence for P30 is limited. Their functionality is dependent upon accessory proteins, including HMW1-HMW3 and A, B, and C (20, 34), which collectively maintain the proper distribution and/or disposition of the adhesins in the mycoplasma membrane (1, 20). In addition, proteins P65 (32) and P200 (33) share characteristic structural features with HMW1 and HMW3, suggesting a common function. Most are elements of a cytoskeleton-like network in M. pneumoniae, consistent with a possible scaffolding role (20). In particular, HMW3 is a major componen...

Protein Kinase/Phosphatase Function Correlates with Gliding Motility in Mycoplasma pneumoniae

“…While this raises the possibility that P1 is an intermediate in PrkC-dependent phosphorylation of HMW1, it should be noted that the absence of P1 in the mutant IV-22 did not affect HMW1 phosphorylation levels. Moreover, if P1 were an intermediate in HMW1 phosphorylation, then one might also expect P1 hyperphosphorylation in mutant H9, where HMW1 is present at greatly reduced levels (23,27), but this was not the case ( Fig. 6A and 7).…”

“…Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting (33, 34) using a monoclonal P30-specific antibody (at a 1:1,000 dilution) (35) and rabbit antisera to HMW1 (1:10,000) (36), HMW2/P28 (1:2,000) (23), HMW3 (1:10,000) (37), P1 (1:2,000) (38), P200 (MPN567) (1:2,000) (39), TopJ (MPN119) (1:2,000) (40), P65 (1: 3,000) (41), B (1:5,000) and C (1:1,000) (10), P41 (1:1,000), and P24 (MPN312) (1:250) (23).…”

Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence

“…The function of these MG218 derivatives, not identified in M. pneumoniae, is still unknown. In contrast, the 25 kDa C-terminal MG218 derivative, which could arise from the translation of MG218 starting at either methionine 1574 or 1615 (Supplementary Table S1), is probably the orthologue of M. pneumoniae P28 protein (Krause et al, 1997). The presence of this small derivative in both mycoplasma species suggests that it plays a significant biological function that still remains unknown.…”

“…Western blots were performed as described previously (Burgos et al, 2006), using MG218 monoclonal and polyclonal antibodies developed in this work, or rabbit antiserum to P41 (Krause et al, 1997), HMW1 (Stevens & Krause, 1991) and HMW3 proteins (Stevens & Krause, 1992). Monoclonal antibodies to MG218 protein were obtained by repeated Microcinematography.…”

Spreading Factors of Mycoplasma alligatoris , a Flesh-Eating Mycoplasma

“…Therefore, both spreading and nutrient acquisition capabilities are probably mediated by the M. alligatoris hyaluronidase. The only other currently confirmed mycoplasmal proteins as large as NagH, besides the nucleic acid polymerases and helicases, are the adhesin P1 (1,628 amino acids) and cytoskeletal protein HMW2 (1,819 amino acids) involved in cytadherence and pathogenicity of M. pneumoniae (24,45).…”