Visualization of the Attachment Organelle and Cytadherence Proteins of Mycoplasma pneumoniae by Immunofluorescence Microscopy

Abstract

A method was developed for protein localization in Mycoplasma pneumoniae by immunofluorescence microscopy. The P1 adhesin protein was revealed to be located at least at one cell pole in all adhesive cells, as has been observed by immunoelectron microscopy. Cell images were classified according to P1 localization and assigned by DNA content. Cells with a single P1 focus at one cell pole had a lower DNA content than cells with two foci, at least one of which was positioned at a cell pole. Those with one focus at each cell pole had the highest DNA content, suggesting that the nascent attachment organelle is formed next to the old one and migrates to the opposite cell pole before cell division. Double staining revealed that the accessory proteins for cytadherence-HMW1, HMW3, P30, P90, P40, and P65-colocalized with the P1 adhesin in all cells. The localization of cytadherence proteins was also examined in cytadherence-deficient mutant cells with a branched morphology. In M5 mutant cells, which lack the P90 and P40 proteins, HMW1, HMW3, P1, and P30 were focused at the cell poles of short branches, and P65 showed no signal. In M7 mutant cells, which produce a truncated P30 protein, HMW1, HMW3, P1, P90, and P40 were focused, and P65 showed no signal. In M6 mutant cells, which express no HMW1 and a truncated P30 protein, the P1 adhesin was distributed throughout the entire cell body, and no signal was detected for the other proteins. These results suggest that the cytadherence proteins are sequentially assembled to the attachment organelle with HMW1 first, HMW3, P1, P30, P90, and P40 next, and P65 last.Mycoplasmas are parasitic bacteria with a small genome size and no peptidoglycan layer (36). Several mycoplasmas have terminal structures which enable them to adhere to the host cell surface for colonization and nutrient acquisition. The terminal structure of Mycoplasma pneumoniae, designated the attachment organelle, has been well described (19,20). It is a membrane protrusion supported by a cytoskeleton-like structure and characterized by a dense cluster of the adhesin protein known as P1 (35).Electron microscopic images have suggested that M. pneumoniae cells divide by binary fission and that the formation and migration of the attachment organelle are coordinated with the cell division process (6). However, the actual order of cell images relative to the cell cycle must be known, and information about the timing of DNA replication is required, in order to substantiate this model. In previous works we quantified and localized the chromosomal DNA through the observation of 4Ј,6Ј-diamidino-2-phenylindole (DAPI)-stained cells of Mycoplasma capricolum by fluorescence microscopy (40, 41). This technique may also be useful for examining the cell division process of M. pneumoniae, although it does not provide the required information about the position of the attachment organelle. Recently, immunofluorescence microscopy was used to study the subcellular localization of bacterial proteins (27). This technique, combined with DAPI...

Terminal organelle development in the cell wall-less bacterium Mycoplasma pneumoniae

“…Limits of resolution and the small size of the mycoplasma cell restrict the conclusions that might be drawn by light microscopy, but cell images by electron microscopy (17) as well as data correlating DNA content and the number and location of terminal organelles in fixed cells (18) are consistent with this model. Here we used fluorescent protein fusions with terminal organelle proteins P30, P41, and P65 and time-lapse digital imaging to observe directly the formation and maturation of the terminal organelle in individual cells during M. pneumoniae growth.…”

“…However, the exceptionally small size of M. pneumoniae cells, the limits of resolution, and therefore the inability to identify terminal organelles definitively, made a conclusive determination impossible. More recently Seto et al correlated the number and position of terminal organelles with genome content (18), but their technique required cell fixation, hence it was not possible to document the sequence of events that led to those images or to rule out the potential introduction of fixation artifacts. In the current study we used fluorescent protein fusions and digital image analysis to examine assembly and function of the terminal organelle over time in growing cultures, confirming that M. pneumoniae terminal organelle duplication and separation precede cell division.…”

“…The loss of certain M. pneumoniae terminal organelle proteins results in accelerated turnover of others and is thought to reflect sequential requirements in the assembly process (9). Cataloging the downstream consequences of loss of essential binding partners has yielded a model for terminal organelle assembly whereby some components are predicted to be incorporated early and others, including P30 and P65, are incorporated later (9,18,23). To begin to test that model, we examined fluorescence patterns in growing mycoplasma cultures producing either P41-YFP or P65-YFP and P30-cyan fluorescent protein (CFP) for their relative timing of appearance.…”

Attachment organelle ultrastructure correlates with phylogeny, not gliding motility properties, in Mycoplasma pneumoniae relatives

“…The special relationship between attachment organelle formation and DNA replication in M. pneumoniae might highlight a role for the electron-dense core in coordination between the two events. An increase in the amount of cellular DNA is accompanied by appearance of a second attachment organelle adjacent to the first one in M. pneumoniae, and the distance between the two organelles increases with continually increasing DNA levels (Seto et al, 2001). Furthermore, time-lapse microcinematography of dividing M. pneumoniae cells reveals that a new attachment organelle generally appears adjacent to the old one, and the new one anchors the cell in place while the old one glides away from it (Hasselbring et al, 2006a), resulting in the two structures being present at opposite poles.…”

“…Gliding appears to be important for spreading from the initial infection site (Jordan et al, 2007). Duplication of the M. pneumoniae attachment organelle at a site adjacent to the existing one accompanies initiation of DNA replication (Seto et al, 2001).…”