In Vitro Spatial and Temporal Analysis of Mycoplasma pneumoniae Colonization of Human Airway Epithelium

Abstract

Mycoplasma pneumoniae is an important cause of respiratory disease, especially in school-age children and young adults. We employed normal human bronchial epithelial (NHBE) cells in air-liquid interface culture to study the interaction of M. pneumoniae with differentiated airway epithelium. These airway cells, when grown in air-liquid interface culture, polarize, form tight junctions, produce mucus, and develop ciliary function. We examined both qualitatively and quantitatively the role of mycoplasma gliding motility in the colonization pattern of developing airway cells, comparing wild-type M. pneumoniae and mutants thereof with moderate to severe defects in gliding motility. Adherence assays with radiolabeled mycoplasmas demonstrated a dramatic reduction in binding for all strains with airway cell polarization, independent of acquisition of mucociliary function. Adherence levels dropped further once NHBE cells achieved terminal differentiation, with mucociliary activity strongly selecting for full gliding competence. Analysis over time by confocal microscopy demonstrated a distinct colonization pattern that appeared to originate primarily with ciliated cells, but lateral spread from the base of the cilia was slower than expected. The data support a model in which the mucociliary apparatus impairs colonization yet cilia provide a conduit for mycoplasma access to the host cell surface and suggest acquisition of a barrier function, perhaps associated with tethered mucin levels, with NHBE cell polarization.M ycoplasma pneumoniae is a human respiratory tract pathogen primarily associated with tracheobronchitis and pneumonia. Infections are typically not life threatening but can be life altering due to the long-term lung damage that can result, including asthma and chronic obstructive pulmonary disease (1). M. pneumoniae initiates colonization of the airway mucosal epithelium via its terminal organelle (2-4). This highly differentiated polar structure functions in adhesion to host cell receptors, gliding motility, and cell division (5-8). Adhesin proteins P1 and P30 localize to the terminal organelle surface, where they participate directly in adherence to host cells and gliding motility (5, 6, 9, 10).Colonization of the human airways requires circumvention of mucociliary defenses, which effectively obstruct, capture, and remove inhaled substances, limiting access to the epithelium (11-13). Previous M. pneumoniae colonization models employed submerged organ and tissue culture systems and have contributed to our current understanding of pathogen-host cell interactions, but they are limited in their ability to accurately reflect the environment of the airway mucosa (3, 4, 14-17). Mycoplasma-host interactions in vivo typically begin at mucosal barriers (11-13), which we define here as including ciliary motion, mucus production, and tight-junction formation (11, 18). Gliding motility is required for lung colonization in experimentally infected hamsters and mice (19,20), and we speculate that this requirement begins with...

Insights into the pathogenesis of Mycoplasma pneumoniae

“…In addition to pseudo-stratified columnar ciliated epithelia, M. pneumoniae can also adhere to red blood cells, HeLa cells, fibroblasts, macrophages and tracheal organ cultures in vitro , and can adhere to the surfaces of glass or plastics (7). M. pneumoniae is asymmetric under electron microscopy (8).…”

“…Studies have shown that M. pneumoniae can invade A549 lung cancer cells, evidenced by its detection in the cytoplasm and nucleus, and the invasive ability depends on the duration and temperature of infection (24). In cell culture in vitro , M. pneumoniae has been shown to invade non-phagocytes, survive for >6 months and synthesize DNA inside cells (7). When the clinically isolated RYC15989 strain was utilized to infect human Hep-G2 cells and rat N2A cells, intracellular Mycoplasma were observed under laser confocal microscopy, and the intracellular invasion damaging ability of M. pneumoniae was also confirmed (25).…”

Modelling persistentMycoplasma pneumoniaeinfection of human airway epithelium

“…We utilise primary normal human bronchial epithelium (NHBE) cells in air–liquid interface (ALI) culture to model M. pneumoniae colonisation of the airways (Jordan et al, ; Krunkosky et al, ; Prince, Krunkosky, & Krause, ). ALI‐grown NHBE cells present a polarised, pseudostratified, and fully differentiated mucociliary epithelium, allowing initiation of infection on the apical surface, which is exposed to air, in the same manner as occurs with aerosol transmission in the human airways.…”

“…ALI‐grown NHBE cells present a polarised, pseudostratified, and fully differentiated mucociliary epithelium, allowing initiation of infection on the apical surface, which is exposed to air, in the same manner as occurs with aerosol transmission in the human airways. Using this model, we previously demonstrated that mycoplasma gliding is essential to overcome mucociliary defences and characterized temporal and spatial features of M. pneumoniae infection over the first 4 hr, where mycoplasmas initially localise to the tips of cilia, then accumulate transiently at the base of the cilia, and gradually spread laterally, with an affinity for intercellular junctions (Jordan et al, ; Prince et al, ).…”

Structural Study of MPN387, an Essential Protein for Gliding Motility of a Human-Pathogenic Bacterium, Mycoplasma pneumoniae

“…Detailed analyses of gliding in M. pneumoniae have shown that the gliding cells move continuously and pivot around the protrusion (7). This motility, combined with the ability to adhere to epithelial cells, is involved in the infection process, enabling the bacteria to translocate from the tip of bronchial cilia to the host cell surface (10). Previous studies, including genome analyses, have shown that this motility is not related to other known mechanisms of bacterial movement and does not involve motor proteins known to be associated with eukaryotic cell motility (6,(11)(12)(13)(14)(15)(16).…”