Quantitative reduction of 2,3,4-triphenyl tetrazolium chloride by hamster trachea organ cultures: effects of Mycoplasma pneumoniae cells and membranes.

The ability of Mycoplasma pneumoniae cells and membranes to affect tetrazolium reduction by hamster trachea organ cultures was evaluated. Uninfected trachea explants reduced 2,3,5-triphenyl tetrazolium chloride (TTC) and nitro-blue tetrazolium when incubated at 37 C in the absence of air. Reduced tetrazolium salts (formazans) were extractable with acetone or ethylene glycol and could be quantitated spectrophotometrically. The optimal assay system involved the use of three or more tracheal rings incubated for 2 h in 0.12% TTC in Tyrode balanced salts supplemented with 1.2% sodium succinate. Formazan was extracted for 5 min with acetone, and the optical density (490 nm) was determined. Trachea explants with metabolic activity reduced or obliterated by freeze-thaw lysis, heat (56 C X 30 min), or cyanide (0.1 M NaCN X 30 min) had negligible ciliary activity and tetrazolium reduction activity (optical density at 490 nm [dry weight]). Tracheas exposed to mycoplasma cells or membranes also showed significantly decreased ciliary activity and tetrazolium reduction; e.g., only 5pc of the ciliary activity and reduction capacity remained after 5 days in culture when infected with M. pneumonia PI 1428 cells. The data indicate that the exposure of ciliated respiratory epithelium to mycoplasma cells or membranes results in diminished oxidative metabolism, and that the ability to reduce TTC to its formazan is correlated with relative ciliary activity.     

Effect of squamous metaplasia on infection of hamster trachea organ cultures with Mycoplasma pneumoniae.

An organ culture system for hamster trachea was developed for maintenance of the ciliated respiratory epithelium during periods of extended cultivation (i.e., greater than 20 days). Evaluation of five serum types showed that horse serum and fetal calf serum were best for the maintenance of epithelial ciliary activity and morphology. Rings that were opened on one side ("split rings") had the best maintenance of the ciliated epithelium as judged by the retention of ciliary activity and normal histological appearance after 3 to 4 weeks in culture. The in vitro induction of squamous metaplasia was achieved by cultivating explants in Waymouth MAB 87/3 (vitamin A-free) medium, without serum. This system allowed a direct comparison of the effects of Mycoplasma pneumoniae infection in two epithelial types, ciliated pseudostratified columnar and keratinizing squamous. Attachment of 14C-labeled mycoplasmas was more than twofold greater in the normal epithelium. Pretreatment of explants with neuraminidase decreased attachment for both squamous and pseudostratified epithelial surfaces to a similar basal level. Recovery of viable organisms from infected tissue of both epithelial types indicated that the organism titer remained essentially constant during the infection period, but was significantly higher for the pseudostratified ciliated epithelium. These results suggest that specific receptor sites for M. pneumoniae are markedly reduced by the induction of squamous metaplasia and, hence, appear to be specific for the normal respiratory surface containing goblet cells and pseudostratified, ciliated epithelial cells.     

Oxygen consumption by trachea organ cultures infected with Mycoplasma pneumoniae.

Hamster trachea organ cultres were shown to consume measurable amounts of oxygen when incubated in a closed micro-chamber. Several simple and complex media were evaluated, and the optimal system involved incubating 2 to 4 mg of tracheal tissue in Tyrodes balanced salt solution at 37 C for 60 min, with oxygen activity measured with a Clark electrode. Tracheas infected with Mycoplasma pneumoniae showed a significantly lower oxygen utilitzation than did uninfected controls. The effect was dose dependent when levels of 105 to 107 colony-forming units per ml were used to infect the tracheal rings. Virulent mycoplasmas caused significant decreases in relative ciliary activity and oxygen utilization, whereas attenuated mycoplasmas reduced ciliary activity and oxygen utilization levels to a lesser extent.     

Mycoplasma pneumoniae in hamster tracheal organ culture studied by scanning electron microscopy.

Hamster tracheal rings in organ culture were inoculated with a virulent strain of Mycoplasma pneumoniae and examined by scanning electron microscopy. A progressive increase in epithelial cell injury was detected from 48 to 96 h post-inoculation and was characterized by apparent loss of the apical portion of ciliated cells. M. pneumoniae attaching to the epithelial cell surfaces could be identified by comparison with the surface morphology of mycoplasmas grown on glass cover slips.     

Interaction of virulent Mycoplasma pneumoniae with hamster tracheal organ cultures.

Exposure of hamster tracheal organ cultures to virulent Mycoplasma pneumoniae leads to alterations in ribonucleic acid (RNA) and protein biosynthesis and metabolism of the respiratory epithelium. An examination of the turnover rates of RNA and protein in infected tracheal organ cultures indicates that the rates of degradation of both prelabeled host cell RNA and protein are similar to those of uninfected controls. Infected tracheal organ cultures shifted to a nonpermissive medium within 24 h after infection and further incubated in the nonpermissive medium for 72 or 96 h behaved as normal uninfected cultures in terms of metabolic precursor uptake. Under these conditions, mycoplasmas remained attached to the respiratory epithelium. Cell membranes prepared from virulent mycoplasmas by several procedures neither attached to nor altered the metabolic activity of tracheal cultures. These data indicate that the intimate contact between virulent mycoplasmas and the respiratory epithelium does not alone account for the subsequent interruption of host cell metabolism but must be accompanied by continued multiplication and biochemical function of attached mycoplasmas.     

Uptake of Streptococcus pneumoniae by respiratory epithelial cells.

Although Streptococcus pneumoniae is the leading cause of community-acquired pneumonia in humans, the mechanism whereby the organism penetrates lung tissue is poorly understood. In the present study we have examined the capacity of pneumococci to penetrate A549 cells, a human lung alveolar carcinoma (type II pneumocyte) cell line. Not all clinical S. pneumoniae isolates initially tested were capable of penetration of the cells, as judged by resistance to extracellular antibiotics. The presence of a polysaccharide capsule also significantly reduced the capacity to both adhere to and penetrate A549 cells. Electron micrographs showed the presence of pneumococci enclosed within vacuoles of intact A549 cells, but bacteria were also seen free in the cytoplasm of damaged cells. Ongoing bacterial DNA, RNA, or protein synthesis was not essential for uptake of pneumococci by A549 cells, and uptake was not diminished by pretreatment of the pneumococci with trypsin. However, inhibition of A549 microfilament assembly with cytochalasin D abolished the phenomenon.     

Interaction of Mycoplasma pneumoniae with HeLa cells.

The susceptibility of HeLa cells to Mycoplasma pneumoniae-induced injury was examined. Infections were initiated with relatively low mycoplasma doses, carried out in a culture medium incapable of supporting M. pneumoniae replication in the absence of host cells, and monitored for up to 10 days. Under these conditions, a time- and dose-dependent decline in the number of viable host cells compared with that of uninfected controls was observed. The effect of M. pneumoniae infection on host cell macromolecular synthesis was also evaluated. At high doses of infection, synthesis of both protein and RNA declined rapidly relative to that in control cells. At lower doses there was a biphasic response in protein synthesis, which was substantially lower than that in the uninfected control by day 1 postinfection, returned to control levels by day 4 postinfection, and was again less than that in control cells by day 7 postinfection. In contrast, no transient recovery was observed in RNA synthesis, which declined very gradually over 7 days in infected HeLa cells compared with that in uninfected control cells. The ability of HeLa cells to support the proliferation of M. pneumoniae under these experimental conditions was demonstrated by quantitation of mycoplasma CFU in the nonpermissive medium in the presence or absence of HeLa cells. A negligible increase in the number of M. pneumoniae was observed over 4 days when HeLa cells were absent, while CFU increased by almost 20-fold when M. pneumoniae was cultured in the presence of HeLa cells. The susceptibility and response in macromolecular synthesis in M. pneumoniae-infected HeLa cells differed from that recently described for a nontransformed culture of hamster trachea epithelial cells under the same experimental conditions (Y.-Y. Chen and D.C. Krause, Infect. Immun. 56: 570-576, 1988), underscoring the importance of the choice of host cell for in vitro modeling of M. pneumoniae pathogenesis.     

Regulation of proinflammatory cytokines in human lung epithelial cells infected with Mycoplasma pneumoniae

Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans. It has also been associated with chronic conditions, such as arthritis, and extrapulmonary complications, such as encephalitis. Although the interaction of mycoplasmas with respiratory epithelial cells is a critical early phase of pathogenesis, little is known about the cascade of events initiated by infection of respiratory epithelial cells by mycoplasmas. Previous studies have shown that M. pneumoniae can induce proinflammatory cytokines in several different study systems including cultured murine and human monocytes. In this study, we demonstrate that M. pneumoniae infection also induces proinflammatory cytokine expression in A549 human lung carcinoma cells. Infection of A549 cells resulted in increased levels of interleukin-8 (IL-8) and tumor necrosis factor alpha mRNA, and both proteins were secreted into culture medium. IL-1 beta mRNA also increased after infection and IL-1 beta protein was synthesized, but it remained intracellular. In contrast, levels of IL-6 and gamma interferon mRNA and protein remained unchanged or undetectable. Using protease digestion and antibody blocking methods, we found that M. pneumoniae cytoadherence is important for the induction of cytokines. On the other hand, while M. pneumoniae protein synthesis and DNA synthesis do not appear to be prerequisites for the induction of cytokine gene expression, A549 cellular de novo protein synthesis is responsible for the increased cytokine protein levels. These results suggest a novel role for lung epithelial cells in the pathogenesis of M. pneumoniae infection and provide a better understanding of M. pneumoniae pathology at the cellular level.     

Human ciliated epithelial cells from nasal polyps as an experimental model for Mycoplasma pneumoniae infection.

Human ciliated epithelial cells derived from nasal polyps and cultured in a monolayer were studied as an experimental model for Mycoplasma pneumoniae infection. Scanning electron microscopy revealed two types of cultured epithelial cells: one which was covered by microvilli only and another which had microvilli and actively beating cilia. M. pneumoniae adhered to both types of cells, and the adherence followed saturation kinetics as a function of time. Infection of the cells for 20 h resulted in 75% inhibition of their intracellular catalase activity and a 3.5-fold increase in their malonyldialdehyde levels compared with noninfected controls. This indicates the presence of cellular oxidative damage due to M. pneumoniae infection. It is suggested that human nasal ciliated epithelial cells may serve as a representative model for studying M. pneumoniae in relation to its natural host.     

Mycoplasma pneumoniae host-pathogen studies in an air-liquid culture of differentiated human airway epithelial cells

The interaction between Mycoplasma pneumonaie and the airway epithelium in vivo is complex and multifaceted. While multiple in vitro studies have been conducted studying this interaction with cell lines and animal cell and organ culture models, the interactions between M. pneumoniae and fully differentiated human airway epithelium in air-liquid interface culture remains unexplored. In the present study we investigated M. pneumoniae interactions with airway epithelium utilizing an air-liquid interface culture of differentiated normal human bronchial epithelial (NHBE) cells. Utilizing confocal microscopy we found that M. pneumoniae cells bound initially to ciliated epithelial cells, but colonization became more evenly distributed over the entire surface with time. M. pneumoniae infection resulted in stimulation of intercellular adhesion molecule 1 (ICAM-1) gene expression and soluble ICAM-1 production in this culture system.     

Ciliated respiratory epithelial monolayers: new model for Mycoplasma pneumoniae infection.

Hamster respiratory epithelial cells were cultured in a monolayer format, and 20% of the cells were ciliated. Mycoplasma pneumoniae attached to the epithelial cells in a neuraminidase-specific fashion and induced ciliostasis and cytonecrosis.     

Mycoplasma pneumoniae proteins that selectively bind to host cells.

Mycoplasma pneumoniae proteins that bind to hamster trachea epithelial cells were identified by incubating 125I-labeled, detergent-solubilized mycoplasmas with glutaraldehyde-fixed host cells. Analysis of the bound fraction by gel electrophoresis and autoradiography revealed that proteins P1, P2, and HMW3 (molecular weights, 165,000, 110,000, and 140,000, respectively), previously implicated in attachment, were among the predominant species. Unlabeled mycoplasma preparations competed with the binding of radiolabeled proteins, suggesting the involvement of a limited number of receptor sites on the host cells.     

Typing of Mycoplasma pneumoniae by PCR-mediated DNA fingerprinting.

PCR fingerprinting was used to characterize clinical isolates of Mycoplasma pneumoniae. Among 24 strains tested, two types were distinguished. Nineteen strains belonged to type 1, whereas only 5 strains belonged to type 2. The majority of strains isolated since 1991 in Belgium belong to type 1. No variations in fingerprinting patterns were observed within each group, confirming the highly conserved nature of the M. pneumoniae genome.     

Chlamydia pneumoniae entry into epithelial cells by clathrin-independent endocytosis

A gram-negative obligate intracellular bacterium, Chlamydia pneumoniae, is a common respiratory pathogen. Here, we examined the invasion and attachment of C. pneumoniae K6 into nonphagocytic HL epithelial cell line by manipulating host plasma membranes by using cholesterol-depleting methyl-beta-cyclodextrin (MβCD) and cholesterol-loading MβCD complexed cholesterol (chol-MβCD). The invasion was attenuated by MβCD-treatment while chol-MβCD augmented the attachment and invasion. In addition, the invasion was inhibited by cholesterol sequestering reagents, nystatin and filipin. Furthermore, exposure of host cells to sphingomyelinase inhibited the invasion. RNA interference was used to assay the role of clathrin and human scavenger receptor B, type I (SR-BI) in the entry of C. pneumoniae into A549 lung epithelial adenocarcinoma cells. In contrast to Chlamydia trachomatis L2, the entry of C. pneumoniae was found to be independent of clathrin. In addition, the entry was found to be SR-BI-independent, but interestingly, the chlamydial growth was attenuated in the SR-BI-silenced cells. These findings suggest that the attachment and invasion of C. pneumoniae into nonphagocytic epithelial cells is dependent on the formation of cholesterol- and sphingomyelin-rich plasma membrane microdomains, and the entry is a clathrin-independent process. In addition, our data indicate that SR-BI supports the growth of C. pneumoniae in epithelial cells.     

Exfoliation of respiratory epithelium in hamster tracheal organ cultures infected with Mycoplasma pneumoniae

Summary Hamster tracheal organ cultures were infected with M. pneumoniae and examined sequentially by transmission and scanning electron microscopy to correlate surface with intracellular alterations. Infection was established by culture and the demonstration of morphologically compatible organisms on the mucosal surface. Ciliated epithelial cells developed vacuolization of the apical and subnuclear cytoplasm and eventually fragmented along planes formed by coalescing vacuoles. Non-ciliated cells showed apical swelling and loss of microvilli during the course of infection. After degeneration and sloughing of both ciliated and non-ciliated cells, a flattened layer of intact basal cells covered the submucosa. It is likely that progressive vacuolization of epithelial cells leads to exfoliation of both cells and cell fragments in M. pneumoniae infection. Since organisms frequently are associated with these exfoliated cells, their potential presence in sputum and lavage specimens could prove to be of diagnostic importance.     

Serological diagnosis of Mycoplasma pneumoniae infection by enzyme immunoassay.

Antibodies against Mycoplasma pneumoniae antigen obtained by Tween-ether treatment from purified M. pneumoniae were measured by means of enzyme-linked immunosorbent assay (ELISA). Paired sera from 19 patients with pneumonia and from 13 patients with acute pancreatitis with a significant rise in complement fixing antibodies against M. pneumoniae were studied. Single sera from healthy 1-year-old children were used as controls. High levels of IgG and IgM class antibodies were seen in sera from patients with pneumonia while most patients with acute pancreatitis and all the children showed low levels of antibodies. The results indicate that ELISA using Tween-ether treated M. pneumoniae antigen could be used successfully in the specific laboratory diagnosis of M. pneumoniae infection.     

Antigenic determinants of the attachment protein of Mycoplasma pneumoniae shared by other pathogenic Mycoplasma species.

In previous studies with hyperimmune rabbit antisera, we found evidence of serologic cross-reactivity among Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum. Because of certain biologic and morphologic similarities of these species, attempts were made to determine if this cross-reactivity related to the attachment protein (P1) of M. pneumoniae. Monoclonal and monospecific antibodies against P1 were used to probe proteins of the other species by immunoblotting. One of the P1 monoclonal antibodies was reactive with a smaller protein of M. genitalium; rabbit antiserum raised by immunization with P1 excised from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel was found to react with a similar-sized protein of M. gallisepticum. These preliminary findings suggest antigenic sharing among the species examined; however, limitations of the methods used are discussed.