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The cell-wall-less prokaryote Mycoplasma pneumoniae, long considered among the smallest and simplest cells capable of self-replication, has a distinct cellular polarity characterized by the presence of a differentiated terminal organelle which functions in adherence to human respiratory epithelium, gliding motility, and cell division. Characterization of hemadsorption (HA)-negative mutants has resulted in identification of several terminal organelle proteins, including P30, the loss of which results in developmental defects and decreased adherence to host cells, but their impact on M. pneumoniae gliding has not been investigated. Here we examined the contribution of P30 to gliding motility on the basis of satellite growth and cell gliding velocity and frequency. M. pneumoniae HA mutant II-3 lacking P30 was nonmotile, but HA mutant II-7 producing a truncated P30 was motile, albeit at a velocity 50-fold less than that of the wild type. HA-positive revertant II-3R producing an altered P30 was unexpectedly not fully wild type with respect to gliding. Complementation of mutant II-3 with recombinant wild-type and mutant alleles confirmed the correlation between gliding defect and loss or alteration in P30. Surprisingly, fusion of yellow fluorescent protein to the C terminus of P30 had little impact on cell gliding velocity and significantly enhanced HA. Finally, while quantitative examination of HA revealed clear distinctions among these mutant strains, gliding defects did not correlate strictly with the HA phenotype, and all strains attached to glass at wild-type levels. Taken together, these findings suggest a role for P30 in gliding motility that is distinct from its requirement in adherence.Mycoplasmas are cell-wall-less prokaryotes with minimal genomes and limited biosynthetic capabilities, dictating a strict dependence on host species for survival in nature (43). Mycoplasma pneumoniae is a human pathogen primarily colonizing the respiratory tract. While the most common clinical manifestations of infection are tracheobronchitis and atypical or "walking" pneumonia (7,12,14,30), recent studies indicate a strong correlation with asthma (5, 24, 38), and extrapulmonary complications are not uncommon (53). Adherence of M. pneumoniae cells to host respiratory epithelium (cytadherence) is required for colonization and pathogenesis (20) and is mediated largely by a differentiated terminal organelle (9, 39). This well-defined apical structure is a membrane-bound extension of the mycoplasma cell distinguished ultrastructurally by an electron-dense core (4), which is a major constituent of the M. pneumoniae cytoskeleton (17, 35).M. pneumoniae cells exhibit gliding motility, with the terminal organelle always the leading end (6), but details regarding the biological significance and the mechanism of gliding are largely unknown. Although the M. pneumoniae genome has been sequenced and twice annotated (11, 19), close inspection reveals no homology to proteins known to be involved in bacterial motility of any type in walled bact...

Mycoplasmas are cell wall-less bacteria considered among the smallest and simplest prokaryotes known, and yet several species including Mycoplasma pneumoniae have a remarkably complex cellular organization highlighted by the presence of a differentiated terminal organelle, a membrane-bound cell extension distinguished by an electron-dense core. Adhesin proteins localize specifically to the terminal organelle, which is also the leading end in gliding motility. Duplication of the terminal organelle is thought to precede cell division, but neither the mechanism of its duplication nor its role in this process is understood. Here we used fluorescent protein fusions and time-lapse digital imaging to study terminal organelle formation in detail in growing cultures of M. pneumoniae. Individual cells ceased gliding as a new terminal organelle formed adjacent to an existing structure, which then migrated away from the transiently stationary nascent structure. Multiple terminal organelles often formed before cytokinesis was observed. The separation of terminal organelles was impaired in a nonmotile mutant, indicating a requirement for gliding in normal cell division. Examination of cells expressing two different fluorescent protein fusions concurrently established their relative order of appearance, and changes in the fluorescence pattern over time suggested that nascent terminal organelles originated de novo rather than from an existing structure. In summary, spatial and temporal analysis of terminal organelle formation has yielded insights into the nature of M. pneumoniae cell division and the role of gliding motility in that process.cell division ͉ gliding motility ͉ adherence ͉ fluorescent protein fusion M ycoplasma pneumoniae causes chronic infections of the human respiratory tract, including bronchitis and primary atypical or ''walking'' pneumonia, accounting for up to 30% of all community-acquired pneumonia, particularly among older children and young adults. M. pneumoniae infections can result in chronic or permanent lung damage, and a growing body of evidence supports a correlation with the onset, exacerbation, and recurrence of asthma. Furthermore, extrapulmonary sequelae are not uncommon, reflecting both invasive and immunopathological components to M. pneumoniae disease (1).In addition to its significant impact on public health, M. pneumoniae is intriguing from a biological perspective. Mycoplasmas have no cell wall and are among the smallest known cells, with M. pneumoniae having a cell volume only Ϸ5% of that of Escherichia coli. Likewise, at 816 kb the M. pneumoniae genome is among the smallest known for a cell capable of a free-living existence, lacking genes for cell wall production, de novo synthesis of nucleotides and amino acids, and twocomponent or other common bacterial transcriptional regulators (2, 3). Nevertheless, a remarkable level of structural complexity underlies what are otherwise considered minimal cells (4). Thus, experimental evidence indicated the presence of cytoskeletal structure and fu...

BackgroundToll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3.Methodology/Principal FindingsUsing a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands.Conclusions/SignificanceLL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA.

The surface protein P65 is a constituent of the Mycoplasma pneumoniae cytoskeleton and is present at reduced levels in mutants lacking the cytadherence accessory protein HMW2. Pulse-chase studies demonstrated that P65 is subject to accelerated turnover in the absence of HMW2. P65 was also less abundant in noncytadhering mutants lacking HMW1 or P30 but was present at wild-type levels in mutants lacking proteins A, B, C, and P1. P65 exhibited a polar localization like that in wild-type M. pneumoniae in all mutants having normal levels of HMW1 and HMW2. Partial or complete loss of these proteins, however, correlated with severe reduction in the P65 level and the inability to localize P65 properly.Mycoplasma pneumoniae is a major cause of bronchitis and pneumonia in humans. Adherence of this cell wall-less bacterium to host respiratory epithelium (cytadherence) is pivotal to successful colonization and ensuing pathogenesis (6) and is mediated largely by a differentiated terminal structure, the attachment organelle, which is also believed to function in gliding motility and cell division (reviewed in reference 13). Protein P1 is a major adhesin (12) and localizes primarily to the attachment organelle in wild-type M. pneumoniae cells. Loss of HMW2, whether by frameshift mutation or transposon insertion, results in the inability to cytadhere and reduced levels of the cytadherence-associated proteins HMW1, HMW3, and P65 (8,11,15). These proteins are components of a Triton X-100 (TX)-insoluble network that comprises the mycoplasma cytoskeleton, or Triton shell, and are collectively required for the development of a fully functional attachment organelle, including the proper localization of the adhesin P1 to this structure (2,9,13,14,24).Proteins HMW1 and HMW3 are largely dissimilar but have in common a central acidic and proline-rich (APR) domain which is defined by its amino acid composition but not its sequence (1,7,20). The genes for HMW1 and HMW3 are coexpressed as part of a large transcriptional unit, the hmw operon (7, 30). Both proteins exhibit a distinctive subcellular distribution (25-27); HMW3 is a major component of the attachment organelle, while HMW1 localizes to the filamentous extensions of the mycoplasma cell, including the attachment organelle. The loss of HMW1 and HMW3 in hmw2 mutants occurs posttranslationally, probably a consequence of accelerated turnover by housekeeping protease activity (21). Newly synthesized HMW1 in the cytoplasmic pool associates with the mycoplasma cytoskeleton and is translocated to the mycoplasma surface as a peripheral membrane protein. Translocation and stabilization of HMW1 are much less efficient in the absence of HMW2, resulting in its removal (1).Like HMW1 and HMW3, protein P65 is a component of the M. pneumoniae Triton shell (23) and is found at significantly reduced levels in hmw2 mutants (15). Furthermore, like HMW1, P65 is peripherally associated with the mycoplasma membrane (22; M. F. Balish and D. C. Krause, unpublished data). P65 contains an APR domain (23), whic...