<i>Mycoplasma pneumoniae</i> J‐domain protein required for terminal organelle function

Cloward, Jason M.; Krause, Duncan C.

doi:10.1111/j.1365-2958.2009.06602.x

Cited by 27 publications

(48 citation statements)

References 66 publications

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“…Mycoplasmas were examined by immunofluorescence microscopy as described previously (6), except that mycoplasma cells were grown on poly-L-lysine-coated coverslips in 24-well dishes. Mouse monoclonal anti-P30 antibody was used at 1:50, and rabbit polyclonal anti-P65 antibody was used at 1:500.…”

Section: Methodsmentioning

confidence: 99%

Domain Analysis of Protein P30 in Mycoplasma pneumoniae Cytadherence and Gliding Motility

2011

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The cell wall-less prokaryote Mycoplasma pneumoniae causes bronchitis and atypical pneumonia in humans. Mycoplasma attachment and gliding motility are required for colonization of the respiratory epithelium and are mediated largely by a differentiated terminal organelle. P30 is a membrane protein at the distal end of the terminal organelle and is required for cytadherence and gliding motility, but little is known about the functional role of its specific domains. In the current study, domain deletion and substitution derivatives of P30 were engineered and introduced into a P30 null mutant by transposon delivery to assess their ability to rescue P30 function. Domain deletions involving the extracellular region of P30 severely impacted protein stability and adherence and gliding function, as well as the capacity to stabilize terminal organelle protein P65. Amino acid substitutions in the transmembrane domain revealed specific residues uniquely required for P30 stability and function, perhaps to establish correct topography in the membrane for effective alignment with binding partners. Deletions within the predicted cytoplasmic domain did not affect P30 localization or its capacity to stabilize P65 but markedly impaired gliding motility and cytadherence. The larger of two cytoplasmic domain deletions also appeared to remove the P30 signal peptide processing site, suggesting a larger leader peptide than expected. We propose that the P30 cytoplasmic domain may be required to link P30 to the terminal organelle core, to enable the P30 extracellular domain to achieve a functional conformation, or perhaps both.The cell wall-less prokaryote Mycoplasma pneumoniae is a human pathogen causing community-acquired respiratory disease in persons of all ages but especially older children and young adults (32). Although it is perhaps best known as the etiologic agent of atypical, or "walking," pneumonia, accounting for up to 20% of all cases of community-acquired pneumonia (41), infections most commonly manifest as tracheobronchitis. Chronic lung disease and extrapulmonary sequelae due to immunopathology or direct invasion can ensue, including a strong correlation with onset and exacerbation of asthma (3,29,42). Colonization of the epithelial mucosa of the conducting airways is mediated largely by the terminal organelle, a polar, differentiated, membrane-bound extension of the mycoplasma cell (7, 30). The adhesins P30 and P1 and several adherence accessory proteins localize to this structure (23), which is defined by a complex electron-dense core that is a prominent component of the mycoplasma cytoskeleton (2, 17, 18). In addition to its role in adherence, the terminal organelle is the motor for gliding motility (14), and its duplication precedes cell division (15).P30 is a membrane protein oriented with the C terminus (domain III) on the mycoplasma cell surface, based on accessibility to antibodies and protease (10, 25) (Fig. 1A), and the N terminus likely in the cell interior (domain I). Its predicted transmembrane (TM) domain a...

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Section: Methodsmentioning

confidence: 99%

Domain Analysis of Protein P30 in Mycoplasma pneumoniae Cytadherence and Gliding Motility

2011

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“…Like several other M. pneumoniae cytadherence-associated proteins, P65 has a large, low-complexity, acidic pro-rich (APR) domain (3,11,27,38), which is followed by a central coiled-coil domain and a C-terminal domain of mixed secondary structures (Fig. 1B).…”

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confidence: 99%

P65 Truncation Impacts P30 Dynamics during Mycoplasma pneumoniae Gliding

2012

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The cell wall-less prokaryote Mycoplasma pneumoniae is a major cause of community-acquired bronchitis and pneumonia in humans. Colonization is mediated largely by a differentiated terminal organelle, which is also the leading end in gliding motility. Cytadherence-associated proteins P30 and P65 appear to traffic concurrently to the distal end of developing terminal organelles. Here, truncation of P65 due to transposon insertion in the corresponding gene resulted in lower gliding velocity, reduced cytadherence, and decreased steady-state levels of several terminal organelle proteins, including P30. Utilizing fluorescent protein fusions, we followed terminal organelle development over time. New P30 foci appeared at nascent terminal organelles in P65 mutants, as in the wild type. However, with forward cell motility, P30 in the P65 mutants appeared to drag toward the trailing cell pole, where it was released, yielding a fluorescent trail to which truncated P65 colocalized. In contrast, P30 was only rarely observed at the trailing end of gliding wild-type cells. Complementation with the recombinant wild-type P65 allele by transposon delivery restored P65 levels and stabilized P30 localization to the terminal organelle. Mycoplasma pneumoniae is an important human respiratory tract pathogen causing community-acquired bronchitis and primary atypical or "walking" pneumonia, accounting for up to 30% of all cases of pneumonia requiring hospitalization. Chronic or permanent lung damage and extrapulmonary sequelae are not uncommon, and a growing body of evidence supports a contributing role for M. pneumoniae in the onset and exacerbation of asthma (1,46). Transmission by aerosol is facilitated by a characteristically persistent cough, leading to colonization of the mucosal epithelium of the conducting airways.M. pneumoniae cells have no cell wall but possess a polar terminal organelle that functions in adherence to host cells (cytadherence), gliding motility, and cell division (6,8,21,39). This membrane-bound extension of the mycoplasma cell is defined by a complex, electron-dense core that is part of a Triton X-100 (TX)-insoluble mycoplasma cytoskeleton (3,6,17,35) and is comprised of multiple substructures (25), including a terminal button with an arched row of discrete proteins that align with proteins on the inner and outer leaflets of the mycoplasma membrane at the distal end of the terminal organelle (Fig. 1A). These proteins are thought to correspond to cytadherence complexes that include major adhesins P1 and P30 and accessory proteins P65, B, and C, which localize to this region of the terminal organelle (4,5,14,16,26,36,42,43) with sufficient proximity to allow their chemical crosslinking (31, 32).P30 is a transmembrane protein found almost exclusively at the distal end of the terminal organelle on wild-type cells, where it has essential but distinct roles in gliding motility and cytadherence (12,20,40). The extracellular C-terminal domain of P30 is dominated by repeating pro-rich motifs and is required for P30 fun...

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“…In the absence of TopJ, all other known AO proteins are stable except for P24, and cores are normal in appearance, but the core frequently fails to protrude from the cell body either fully or at all (46,47). Mutations of amino acids in TopJ that are required for cochaperone function in its homolog DnaJ generally recapitulate phenotypes associated with TopJ loss (48), suggesting that chaperone activity lies at the heart of this aspect of AO positioning.…”

Section: The Ao Core and Its Coiled-coil And Proline-rich Componentsmentioning

confidence: 88%

“…TopJ (MPN119), the only AO protein with clear homology to proteins from other types of organisms, consists of a J domain, which is associated with protein folding (45), an APR domain, and several EAGR boxes (46). In the absence of TopJ, all other known AO proteins are stable except for P24, and cores are normal in appearance, but the core frequently fails to protrude from the cell body either fully or at all (46,47).…”

Section: The Ao Core and Its Coiled-coil And Proline-rich Componentsmentioning

confidence: 99%

Mycoplasma pneumoniae, an Underutilized Model for Bacterial Cell Biology

Balish

2014

J Bacteriol

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In recent decades, bacterial cell biology has seen great advances, and numerous model systems have been developed to study a wide variety of cellular processes, including cell division, motility, assembly of macromolecular structures, and biogenesis of cell polarity. Considerable attention has been given to these model organisms, which include Escherichia coli, Bacillus subtilis, Caulobacter crescentus, and Myxococcus xanthus. Studies of these processes in the pathogenic bacterium Mycoplasma pneumoniae and its close relatives have also been carried out on a smaller scale, but this work is often overlooked, in part due to this organism's reputation as minimalistic and simple. In this minireview, I discuss recent work on the role of the M. pneumoniae attachment organelle (AO), a structure required for adherence to host cells, in these processes. The AO is constructed from proteins that generally lack homology to those found in other organisms, and this construction occurs in coordination with cell cycle events. The proteins of the M. pneumoniae AO share compositional features with proteins with related roles in model organisms. Once constructed, the AO becomes activated for its role in a form of gliding motility whose underlying mechanism appears to be distinct from that of other gliding bacteria, including Mycoplasma mobile. Together with the FtsZ cytoskeletal protein, motility participates in the cell division process. My intention is to bring this deceptively complex organism into alignment with the better-known model systems. Mycoplasmas have a long-standing reputation for being something they are not: exceptionally simple. The common term "mycoplasma" encompasses the bacteria of the genera within the phylum Tenericutes and the class Mollicutes (1). These organisms are most distinctively characterized by the absence of any cell wall material, with the cell envelope consisting of a single lipid bilayer and its associated proteins and carbohydrates. Having undergone reductive evolution from Firmicutes ancestors (2), mycoplasma genomes are less complex than those of other bacteria that are capable of being grown axenically in a laboratory setting, which has led to their service as model organisms for studying genomes in a minimal setting (3) and as platforms for synthetic biology (4).This reduced genomic complexity has led to a perception that mycoplasmas are aberrantly uncomplicated organisms whose value to researchers, beyond dealing with the diseases that they cause, is only as simplified models for cell structure and genome analysis. But since the small genome size results from loss of major anabolic pathways and reduction in the complexity of gene expression regulation, mycoplasmas can still serve as models for other kinds of complex cellular processes. These include genesis of cell polarity, assembly of macromolecular structures, cell division, adherence, and motility.The human pathogen Mycoplasma pneumoniae (5) is particularly instructive. M. pneumoniae cells feature an attachment organelle (AO) or termina...

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Mycoplasma pneumoniae J‐domain protein required for terminal organelle function

Cited by 27 publications

References 66 publications

Domain Analysis of Protein P30 in Mycoplasma pneumoniae Cytadherence and Gliding Motility

Domain Analysis of Protein P30 in Mycoplasma pneumoniae Cytadherence and Gliding Motility

P65 Truncation Impacts P30 Dynamics during Mycoplasma pneumoniae Gliding

Mycoplasma pneumoniae, an Underutilized Model for Bacterial Cell Biology

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