Interaction of Mycoplasma pneumoniae with human lung fibroblasts: role of receptor sites.

The biochemical nature of the neuraminidase-sensitive Mycoplasma pneumoniae receptor site on human lung fibroblast cells was studied. Purified, mixed sialoglycolipid (ganglioside) preparations from human and bovine tissues did not bind to M. pneumoniae organisms and block their subsequent attachment to fibroblasts. Fibroblasts incubated for 24 h in sialoglycolipid solutions to increase the ganglioside content of their membranes did not show increased pathogen attachment when later incubated with mycoplasmas. HeLa cells grown in the presence of sodium butyrate to increase GM3 ganglioside levels likewise did not have significantly increased uptake of M. pneumoniae organisms. Treatment of fibroblasts with enzymes indicated that the mycoplasma receptor site is trypsin and papain resistant but Pronase sensitive. Pronase digests of fibroblast membranes contained a product(s) which combined with M. pneumoniae cellls and cosedimented with them during centrifugation. Glycoproteins, purified from fibroblast membranes by a lithium diiodosalicylate solubilization technique, similarly bound to M. pneumoniae organisms. Collectively, these data suggest that the major component of the M. pneumoniae receptor site is a sialoglycoprotein with little or no lipid.     

Alterations in nucleotide content of human lung fibroblasts infected with Mycoplasma pneumoniae.

The nucleotide content of normal MRC-5 human lung fibroblasts and fibroblasts infected with Mycoplasma pneumoniae PI 1428 was determined. Nucleotides from control and infected fibroblasts were extracted with 5% trichloracetic acid. After neutralization of the extracts, the nucleotides in the extracts were separated by anion-exchange chromatography. Significant differences were found between the nucleotide content of the control and infected cells. Nucleotide triphosphate levels were twofold higher in the control fibroblasts than in the infected fibroblasts 4 h after the initiation of infection. At the same time, nucleotide diphosphate and monophosphate levels were higher in the infected fibroblasts than in the control fibroblasts. Determination of the energy charge ratio for each set of nucleotides (adenosine, guanosine, cytidine, and uridine) demonstrated a shift of nucleotide content in the infected fibroblasts. Immediately after infection, the energy charge for each set of nucleotides was higher for the control fibroblasts than it was for the infected fibroblasts. This pattern continued throughout the infection period with only minor exceptions. The work presented here indicates a loss of energy charge in fibroblasts infected with M. pneumoniae and may help to explain some of the metabolic changes and cell damage which accompany infection.     

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.     

Interaction of Pseudomonas aeruginosa with human lung fibroblasts: role of bacterial elastase.

Colonization of cell surfaces by Pseudomonas aeruginosa is mediated by bacterial adherence, which, in turn, is influenced by both host and microbial factors. Previous studies with this organism suggest that elastase contributes to tissue invasion and necrosis. We studied the effects of Pseudomonas elastase (PE) on the adherence of P. aeruginosa to human lung fibroblast monolayers. Treatment of fibroblasts with PE (1 microgram/ml or 0.06 U/ml) increased adherence of 35S-labeled P. aeruginosa to cells, but heat-inactivated PE did not affect bacterial adhesion. Immunocytochemistry of cultured cells showed that PE (0.06 to 0.63 U/ml) decreased fibronectin (Fn) on the cell surface and extracellular matrix of cultured human lung fibroblasts. Data obtained by cytofluorography indicated that elastase also decreased Fn receptors on fibroblasts. Additional evidence for Fn degradation was provided by SDS-PAGE analysis of soluble Fn and proteins from surface iodinated cell monolayers treated with PE. We conclude that the increased bacterial adherence to fibroblasts may be due, in part, to elastase-induced proteolysis of Fn and its receptors on cell surfaces. Degradation of Fn could thus influence the extent and course of Pseudomonas infection in the lungs.     

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.     

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.     

Interaction of Mycoplasma pneumoniae with alveolar macrophages: viability of adherent and ingested mycoplasmas.

Guinea pig peritoneal or alveolar macrophages were inoculated with Mycoplasma pneumoniae cells. Extracellular mycoplasms were killed by complement treatment, and the effect of macrophage action on the number of the remaining viable mycoplasmas was observed. The complement killing was to some extent inhibited by the presence of the macrophages, but the mechanism of this protection remains unknown. Opsonized mycoplasmas were ingested, and approximately 98% were killed within 4 h. The killing rate was somewhat lower than comparable data for bacteria, but lack of cell wall and high lipid content of the membrane apparently do not cause a significant delay in intracellular destruction.     

Isolation of Mycoplasma pneumoniae from the human urogenital tract.

Mycoplasma pneumoniae is a common etiologic agent of lower respiratory tract infections in humans. However, it has been reported previously that the organism has occasionally been isolated from sites other than the oropharynx and respiratory tract. We report the isolation of 24 strains of M. pneumoniae from urogenital specimens obtained from 22 female patients. Most isolates were of cervical origin from patients attending several local gynecological clinics over a 2-year period. Strains were also isolated from the urethra of one of three healthy male sexual partners of female patients positive for the organism. Single serum specimens obtained from three female patients and three different male sexual partners showed antibody levels suggestive of either recent respiratory infection or genital tract colonization with M. pneumoniae. Although there is no apparent definitive explanation for the localized outbreak of the organism at these unusual sites, the possible transfer through sexual and/or orogenital contact remains the most likely mode of transmission. The occurrence of an organism with obvious pathogenicity for human epithelial tissue in the urogenital tract suggests such transfer could play a role in genital tract infection.     

Interaction of Mycoplasma pneumoniae with erythrocyte glycolipids of I and i antigen types.

The role of sialoglycolipids (gangliosides) as receptors for the human pathogen Mycoplasma pneumoniae was investigated by using purified gangliosides of known carbohydrate structures as inhibitors of the binding of 51Cr-labeled erythrocytes to sheet cultures of M. pneumoniae. We found that sialoglycolipids with long carbohydrate backbones of the poly-N-acetyllactosamine type were more potent inhibitors of M. pneumoniae binding than those with short carbohydrate chains. This is in accord with earlier inhibition data for glycoproteins and oligosaccharides. Thus, the inhibitory activity of a fraction of bovine erythrocyte gangliosides containing long backbone structures of I antigen type was approximately 200 times greater than that of the short chain gangliosides GM3 and GT1b. The binding of M. pneumoniae to erythrocytes of I and i antigen types was found to be comparable, indicating that M. pneumoniae in its adhesive specificity may not distinguish between the branched carbohydrate backbones of I type and the linear structures of i type. Thus, the production of autoantibodies to the backbone structures of I type rather than i type after infection with this agent may simply reflect a greater abundance of branched carbohydrate receptors of I type on the surface of host cells with which the mycoplasma forms immunogenic complexes.     

Identification and characterization of human surfactant protein A binding protein of Mycoplasma pneumoniae

Mycoplasma pneumoniae infections represent a major primary cause of human respiratory diseases, exacerbate other respiratory disorders, and are associated with extrapulmonary pathologies. Cytadherence is a critical step in mycoplasma colonization, aided by a network of mycoplasma adhesins and cytadherence accessory proteins which mediate binding to host cell receptors. Furthermore, the respiratory mucosa is enriched with extracellular matrix components, including surfactant proteins, fibronectin, and mucin, which provide additional in vivo targets for mycoplasma parasitism. In this study we describe interactions between M. pneumoniae and human surfactant protein-A (hSP-A). Initially, we found that viable M. pneumoniae cells bound to immobilized hSP-A in a dose- and calcium (Ca(2+))-dependent manner. Mild trypsin treatment of intact mycoplasmas reduced binding markedly (80 to 90%) implicating a surface-associated mycoplasma protein(s). Using hSP-A-coupled Sepharose affinity chromatography and polyacrylamide gel electrophoresis, we identified a 65-kDa hSP-A binding protein of M. pneumoniae. The presence of Ca(2+) enhanced binding of the 65-kDa protein to hSP-A, which was reduced by the divalent cation-chelating agent, EDTA. The 65-kDa hSP-A binding protein of M. pneumoniae was identified by sequence analysis as a novel protein (MPN372) possessing a putative S1-like subunit of pertussis toxin at the amino terminus (amino acids 1 to 226), with the remaining amino acids (227 to 591) exhibiting no homology with other subunits of pertussis toxin, other known toxins, or any reported proteins. Recombinant MPN372 (MPN372) bound to hSP-A in a dose-dependent manner, which was markedly reduced by preincubation with mouse recombinant MPN372 antisera. Also, adherence of viable M. pneumoniae cells to hSP-A was inhibited by recombinant MPN372 antisera, demonstrating that MPN372, a previously designated hypothetical protein, is surface exposed and mediates mycoplasma attachment to hSP-A.     

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.     

Cold hemagglutinin cross-reactivity with Mycoplasma pneumoniae.

The data presented indicate that one of the primary actions of Pseudomonas aeruginosa exotoxin during experimental infection is the inactivation of elongation factor 2 (EF-2) in various mouse organs. Organs from mice infected with the toxigenic P. aeruginosa strain PA103 contained considerably less EF-2 activity than did organs from uninfected controls. Whereas EF-2 activity was reduced in all organs examined from PA103-infected animals, the largest decrease was observed in the liver, where the active EF-2 levels were reduced by 70 to 90%. In addition, consistent inhibition of protein synthesis in livers but not in other organs was observed in mice infected with the toxigenic PA103 strain. Treatment of mice with antitoxin before infection with strain PA103 prevented inactivation of EF-2. When mice were infected with lethal doses of the nontoxigenic P. aeruginosa WR5 strain, tissue EF-2 levels were not markedly reduced below those derived from uninfected control animals.     

Opsonin-reversible resistance of Mycoplasma pneumoniae to in vitro phagocytosis by alveolar macrophages.

Several species of mycoplasmas are responsible for respiratory disease in animals and man. As yet, little is known about the interaction of these pathogens with alveolar macrophages, one of the primary components of pulmonary resistance to infections. The present study was undertaken to develop an in vitro model to examine this organism-cell interaction, using a human pathogen, mycoplasma pneumoniae, and normal guinea pig alveolar macrophages. During a 24-h incubation of M. pneumoniae with a monolayer of macrophages, mycoplasmas were found to attach directly to the surface of the cells without inducing significant phagocytosis. Ultrastructurally, the organisms appeared bound to the cell membrane by their characteristic attachment organelles. Only after the addition of specific anti-mycoplasma serum were cells able to engulf attached and surrounding organisms. These data suggest that the interaction of M. pneumoniae and alveolar macrophages is a potentially important aspect of disease pathogenesis, and immune factors which might alter this interaction merit further examination.     

Detection of Mycoplasma pneumoniae adhesin (P1) in the nonhemadsorbing population of virulent Mycoplasma pneumoniae.

Mycoplasma pneumoniae organisms possessing a hemadsorbing-negative (HA-) phenotype comprise more than 50% of the population of virulent M. pneumoniae cultures. Monoclonal antibody to P1, the major adhesin of M. pneumoniae reacts with this HA- mycoplasma fraction based upon radioimmunoprecipitation and immunoblotting. Demonstration of P1 in the entire mycoplasma population suggests that topological organization of this adhesin in the membrane or the physiological state of the mycoplasmas may determine hemadsorbing capabilities.     

Mycoplasma pneumoniae protein involved in the antibody response in human infection

Antigen from purified Mycoplasma pneumoniae organisms treated with Tween-80-ether was used in a solid phase enzyme immunoassay and compared with the conventional lipid containing complement fixation antigen for measuring antibodies in sera from patients with aseptic or bacterial meningitis or with apparent M pneumoniae infection. In immunoblotting of the enzyme immunoassay antigen, enzyme immunoassay positive sera detected a polypeptide at Mr = 180.000-200.000, while enzyme immunoassay negative sera whether positive or negative in the complement fixation test did not. These results indicate that the enzyme immunoassay antigen containing the high molecular weight polypeptide can be used to measure M pneumoniae antibodies more specifically than the conventional lipid containing complement fixation antigen.     

A Mycoplasma genitalium protein resembling the Mycoplasma pneumoniae attachment protein.

In previous studies with hyperimmune rabbit sera and monoclonal antibodies against the P1 protein of Mycoplasma pneumoniae, we obtained evidence of a shared antigenic determinant with a single protein of Mycoplasma genitalium. Because of biologic and morphologic similarities between these two human Mycoplasma species, attempts were made to characterize this cross-reacting protein of M. genitalium (designated MgPa). The protein was surface exposed and had an estimated molecular size of 140 kilodaltons. Electron microscopy with monoclonal antibodies produced against either MgPa or P1 demonstrated that MgPa is located over the surface of the terminal structure of M. genitalium which is covered by a nap layer. These immunologic and morphologic findings suggest that the MgPa protein of M. genitalium could be the counterpart of the P1 protein of M. pneumoniae.     

In vitro characterization of natural and synthetic dermal matrices cultured with human dermal fibroblasts

The ideal dermal matrix should be able to provide the right biological and physical environment to ensure homogenous cell and extracellular matrix (ECM) distribution, as well as the right size and morphology of the neo-tissue required. Four natural and synthetic 3D matrices were evaluated in vitro as dermal matrices, namely (1) equine collagen foam, TissuFleece, (2) acellular dermal replacement, Alloderm, (3) knitted poly(lactic-co-glycolic acid) (10:90)-poly(-caprolactone) (PLGA-PCL) mesh, (4) chitosan scaffold. Human dermal fibroblasts were cultured on the specimens over 3 weeks. Cell morphology, distribution and viability were assessed by electron microscopy, histology and confocal laser microscopy. Metabolic activity and DNA synthesis were analysed via MTS metabolic assay and [(3)H]-thymidine uptake, while ECM protein expression was determined by immunohistochemistry. TissuFleece, Alloderm and PLGA-PCL mesh supported cell attachment, proliferation and neo-tissue formation. However, TissuFleece contracted to 10% of the original size while Alloderm supported cell proliferation predominantly on the surface of the material. PLGA-PCL mesh promoted more homogenous cell distribution and tissue formation. Chitosan scaffolds did not support cell attachment and proliferation. These results demonstrated that physical characteristics including porosity and mechanical stability to withstand cell contraction forces are important in determining the success of a dermal matrix material.     

Intoxication of cultured human lung fibroblasts with Clostridium difficile toxin.

The cytopathogenic effect of partially purified toxin from Clostridium difficile on cultured human lung fibroblasts was studied. Conditions for determination of 50% tissue culture dose were standardized. The cytopathogenic effect of the toxin was dependent on toxin concentration, exposure time, and density of the cells. Transfer of the cells to 0 degrees C did not inhibit binding of toxin to the fibroblast surface, but prevented the development of the cytopathogenic effect. Both binding of toxin and some intracellular step(s) were prevented by 2,4-dinitrophenol. These preventative effects were reversible. Before and concomitantly with the appearance of the cytopathogenic effect, the cellular uptake of uridine and of amino acids was markedly stimulated. Protein synthesis was depressed when 100% of the cells showed the cytopathogenic effect, but the synthesis of nucleic acids was inhibited only several hours later. The primary cellular target for the toxin is still unknown.