Vaccination of BALB/c mice with an avirulent Mycoplasma pneumoniae P30 mutant results in disease exacerbation upon challenge with a virulent strain.

Mycoplasma pneumoniae is a significant human respiratory pathogen that causes high morbidity worldwide. No vaccine to prevent M. pneumoniae infection currently exists, since the mechanisms of pathogenesis are poorly understood. To this end, we constructed a P30 cytadhesin mutant (P-130) with a drastically reduced capacity for binding to erythrocytes and an inability to glide on glass substrates. This mutant was determined to be avirulent and cannot survive in the lungs of BALB/c mice. We also ascertained that the previously identified P30 gliding motility mutant II-3R is avirulent and also cannot be recovered from the lungs of mice after infection. Mutant P130 was then assessed for its efficacy as a live attenuated vaccine candidate in mice after challenge with wild-type M. pneumoniae. After vaccination with the P-130 P30 mutant, mice showed evidence of exacerbated disease upon subsequent challenge with the wild-type strain PI1428, which appears to be driven by a Th17 response and corresponding eosinophilia. Our results are in accordance with other reports of vaccine-induced disease exacerbation in rodents and emphasize the need to better understand the basic mechanisms of M. pneumoniae pathogenesis.

Magnetic polarity of pillow basalts from reykjanes ridge.

A first attempt to measure directly the magnetic polarity of submarine basalts dredged from the Reykjanes Ridge indicates that the first two magnetic anomalies over the ridge resulted from a reversal of the earth's magnetic field. Volcanic criteria were used to determine the orientation the samples had before they were dredged from the sea floor.

Mycoplasma pneumoniae induces airway epithelial cell expression of MUC5AC in asthma.

As excess mucin expression can contribute to the exacerbation of asthma, the present authors hypothesised that Mycoplasma pneumoniae significantly induces MUC5AC (the major airway mucin) expression in airway epithelial cells isolated directly from asthmatic subjects. A total of 11 subjects with asthma and six normal controls underwent bronchoscopy with airway brushing. Epithelial cells were cultured at an air-liquid interface and incubated with and without M. pneumoniae for 48 h, and in the presence and absence of nuclear factor (NF)-kappaB and a toll-like receptor (TLR)2 inhibitor. Quantitative PCR was performed for MUC5AC and TLR2 mRNA. MUC5AC protein and total protein were determined by ELISA. M. pneumoniae exposure significantly increased MUC5AC mRNA and protein expression after 48 h in epithelial cells isolated from asthmatic, but not from normal control subjects, at all concentrations as compared to unexposed cells. TLR2 mRNA expression was significantly increased in asthmatic epithelial cells at 4 h compared with unexposed cells. NF-kappaB and TLR2 inhibition reduced MUC5AC expression to the level of the unexposed control in both groups. Mycoplasma pneumoniae exposure significantly increased MUC5AC mRNA and protein expression preferentially in airway epithelial cells isolated from asthmatic subjects. The toll-like receptor 2 pathway may be involved in this process.

Mycoplasma pneumoniae cytadherence: organization and assembly of the attachment organelle.

Mycoplasma pneumoniae has no cell wall but possesses a complex terminal structure that is required for polar localization of adhesins and is thought to participate in cell division. Several protein components of this structure have been identified by analysis of non-cytadhering mutants. Genetic manipulation of mycoplasmas now allows elucidation of the assembly and regulation of the terminal organelle.

Juxtaposition of the genes encoding Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW3.

The loss and reacquisition of high-Mr (HMW) proteins, HMW1, 2, 3, 4 and 5, by Mycoplasma pneumoniae correlates with cytadherence phase variation. We are cloning and characterizing the genes encoding HMW1-5 to understand the mechanism regulating their coordinate expression. HMW1 was purified by polyacrylamide-gel electrophoresis. Amino acid (aa) sequence data were obtained from enzymatically generated peptide fragments from HMW1. A degenerate 17-mer probe synthesized based upon the aa sequence of one peptide clearly identified a single 4.75-kb BamHI fragment of M. pneumoniae DNA under stringent hybridization conditions. This fragment was cloned into pUC19 to generate pKV16. Restriction mapping of the 4.75-kb BamHI fragment in pKV16 revealed a possible overlap with the 9.4-kb EcoRI fragment containing the gene encoding protein HMW3. Southern blotting and reciprocal hybridization studies confirmed this overlap, establishing the juxtaposition of the genes encoding HMW1 and HMW3. Finally, physical mapping analysis by probing restriction fragments of M. pneumoniae DNA resolved by pulsed-field gel electrophoresis with the cloned genes encoding HMW1 and HMW3 revealed definitively that the hmw locus maps to a 106.8-kb ApaI fragment, rather than a 117.5-kb ApaI fragment, as had been reported previously for hmw3 [Krause and Mawn, J. Bacteriol. 172 (1990) 4790-4797].

Cloning and analysis of the gene encoding the cytadherence phase-variable protein HMW3 from Mycoplasma pneumoniae.

We have cloned the gene encoding the Mycoplasma pneumoniae cytadherence-accessory protein HMW3 into Escherichia coli to study its phase-variable expression. A truncated HMW3 protein (HMW3'; 113 kDa), identified using HMW3-specific, affinity-purified antibodies, was expressed under the control of the lacZ promoter in lambda gt11. The protein did not react with beta-galactosidase (beta Gal)-specific antibodies, however, indicating that HMW3' was not a beta Gal fusion protein. The direction of transcription was determined by examining gene expression from inserts in opposite orientations with respect to the lacZpo in pUC18 and pUC19, to generate pKV5 and pKV6. Amino acid sequence data were obtained from an enzymatically generated HMW3 peptide fragment and used to create a degenerate 17-mer probe. The degenerate 17-mer hybridized to the mycoplasma DNA insert in pKV6; both the 17-mer and the pKV6 insert hybridized to a 9.4-kb EcoRI fragment from wild-type (wt) M. pneumoniae chromosomal DNA. This EcoRI fragment was cloned from wt M. pneumoniae and an HMW3-deficient variant in both orientations into pUC18. The HMW3'-encoding region was localized to the center of the 9.4-kb EcoRI fragment, and no differences were observed in restriction patterns between the wt and variant. Although the 9.4-kb EcoRI fragment included the DNA segment encoding HMW3', neither this protein, nor derivatives thereof, were detected in IPTG-induced E. coli containing the EcoRI fragment from either wt or variant M. pneumoniae, in either orientation in pUC18.

Sequence analysis and characterization of the hmw gene cluster of Mycoplasma pneumoniae.

Mycoplasma pneumoniae (Mp) cytadherence requires the proper anchoring of cytadhesin proteins in the mycoplasmal membrane at an attachment organelle through their interaction with a cytoskeleton-like network of accessory proteins that includes HMW1 and HMW3. Approximately 8.25 kb of Mp DNA was sequenced, beginning at the 3' end of the hmw3 gene and continuing through hmw1. Comparison of the resulting deduced amino acid (aa) sequence with N terminus and internal peptide aa sequences from purified HMW1 permitted definitive identification of hmw1. HMW1 was characterized with respect to structure, hydrophobicity, possible phosphoacceptor sites and expression of the Mp recombinant protein in Escherichia coli. In addition, HMW1 membrane topography was examined for antibody accessibility on the mycoplasmal surface. hmw3 and hmw1 flank four open reading frames (ORFs) spanning approximately 4.3 kb and in the same orientation as the hmw genes. The sequences of their deduced products were evaluated for likely structural features and comparison with protein data banks. Finally, the Mp rpsD analog was identified immediately downstream from hmw1.

Nucleotide sequence of the Rickettsia prowazekii ATP/ADP translocase-encoding gene.

The Rickettsia prowazekii ATP/ADP translocase (Tlc) gene (tlc), previously cloned in Escherichia coli was localized to a 1.6-kb chromosomal fragment. Nucleotide sequence analysis of this fragment revealed an open reading frame of 1494 bp that could encode a hydrophobic protein of 497 amino acids (aa) with an Mr of 56,668. Analysis of the deduced aa sequence revealed that it contained twelve potential membrane-spanning regions. Comparisons between the deduced aa sequence of the R. prowazekii ATP/ADP Tlc and the sequences of mitochondrial (mt) Tlc revealed no detectable homologies between the rickettsial and mt sequences. The major protein synthesized in E. coli minicells containing the rickettsial gene exhibited and Mr of approx. 34,000.

Structure, function, and assembly of the terminal organelle of Mycoplasma pneumoniae.

Mycoplasmas are cell wall-less bacteria at the low extreme in genome size in the known prokaryote world, and the minimal nature of their genomes is clearly reflected in their metabolic and regulatory austerity. Despite this apparent simplicity, certain species such as Mycoplasma pneumoniae possess a complex terminal organelle that functions in cytadherence, gliding motility, and cell division. The attachment organelle is a membrane-bound extension of the cell and is characterized by an electron-dense core that is part of the mycoplasma cytoskeleton, defined here for working purposes as the protein fraction that remains after extraction with the detergent Triton X-100. This review focuses on the architecture and assembly of the terminal organelle of M. pneumoniae. Characterizing the downstream consequences of defects involving attachment organelle components has made it possible to begin to elucidate the probable sequence of certain events in the biogenesis of this structure.

Energy utilization in the induced release of gamma-aminobutyric acid from synaptosomes.

Newly accumulated gamma-aminobutyric acid (GABA) was released from synaptosomes by treatment with 30 mM K+ or the Ca2+ ionophore A23187. Release was Ca2+-dependent and energy-dependent. The induced release of GABA was inhibited by S-13, an uncoupler of oxidative phosphorylation, by azide, a blocker of mitochondrial respiration, and by oligomycin, efrapeptin, tributyltin and dicyclohexylcarbodiimide (DCCD), which are inhibitors of Ca2+/Mg2+-ATPases, including mitochondrial ATPase. Efrapeptin blocked GABA release induced by K+ but not A23187-induced release. Azide and oligomycin appeared to inhibit GABA release as a consequence of their effects on mitochondrial ATP synthesis. However, the inhibition of GABA release by the other compounds could not be totally accounted for by their effects on synaptosomal ATP stores. It is proposed that these compounds, in addition to affecting ATP synthesis, directly affect biochemical reactions involved in GABA release. Thus, these and similar inhibitors seem to be useful probes of the transmitter release process.

A spectrophotometric analysis of dentinal leakage in the resected root.

The purpose of this study was to compare dye leakage in the dentin of resected and nonresected roots. Fifty-four single rooted extracted teeth were used. The two groups were subdivided into young, middle age, and old age samples. Each sample was biomechanically prepared in a standard fashion and obturated using lateral condensation with sealer and gutta-percha. All root surfaces were sealed with nail polish, leaving the apical portion exposed. The apical portion of the roots were placed in 2% methylene blue dye for 72 h, rinsed, and placed in 15 ml of 35% HNO3 for 72 h. The supernatant was analyzed at 640 nm using a visible light spectrophotometer. The amount of leakage was extrapolated from a standard linear regression curve constructed from the stock 2% methylene blue dye solution. The percentage of concentration leakage in all samples ranged from less than 0.0600 to 0.1658. It was concluded that there is a greater amount of leakage in resected versus nonresected extracted teeth. The data also suggest that older teeth exhibit less leakage than younger teeth.

Cloning and expression in Escherichia coli of Mycoplasma gallisepticum antigens recognized by sera from infected chickens.

A clone bank of Mycoplasma gallisepticum (MG) strain A5969 DNA was prepared in the expression vector phage lambda gt11. Approximately 75% of the resulting phages were recombinants, based upon the insertional inactivation of the lacZ gene of the vector. Clones were screened immunologically with serum prepared from specific-pathogen-free white leghorn chickens that had been infected with aerosolized MG. Approximately 250 clones, or less than 1% of the recombinant phage, reacted positively to various degrees with the test serum and failed to react with serum from uninfected specific-pathogen-free control chickens. A single clone was chosen at random for comparison with a vector control by western immunoblot, revealing a polypeptide of 140,000 molecular weight in the clone profile but not the control profile that reacted with immune serum. Clones expressing MG antigens recognized during infection may provide an improved means for antigen preparation for serologic diagnosis of mycoplasmosis.

Mycoplasma pneumoniae cytadherence: unravelling the tie that binds.

Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults. Mycoplasma adherence to the respiratory epithelium (cytadherence) is required for colonization and pathogenesis. Although considered to be among the smallest and simplest known prokaryotes, this cell-wall-less bacterium possesses a highly differentiated terminal structure that is thought to be functional in mycoplasma cell division, gliding motility, and cytadherence. Mutant analysis has identified mycoplasma proteins associated with cytadherence, and revealed novel regulatory features. Ultrastructural and biochemical studies have established the subcellular location and interaction of key components, several of which are phosphorylated by ATP-dependent kinase(s) in a manner that is responsive to changing nutritional conditions. This review summarizes recent progress in defining the composition, organization and regulation of the attachment organelle. What emerges is a picture of M. pneumoniae cytadherence as a multifactorial process that extends well beyond adhesin-receptor recognition.

Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeletonlike Triton shell.

The location of the cytadherence-accessory high-molecular weight proteins 1 and 4 (HMW1/4) within Mycoplasma pneumoniae cells has been studied by both biochemical and electron microscopic techniques. Peptide mapping studies demonstrated that HMW1/4 share almost identical peptide profiles, suggesting that the two proteins are structurally related. Examination of thin sections of M. pneumoniae with antibodies to HMW1/4 and colloidal gold particles revealed distinct labeling of the filamentous extensions of the mycoplasma cells. Labeling was absent on thin sections of a cytadherence-deficient variant lacking HMW1/4. HMW1/4 partitioned in the detergent-insoluble fraction following Triton X-100 extraction, and analysis by sucrose density gradient centrifugation suggested that HMW1/4 are part of a high-molecular-weight, multiprotein complex. These results were confirmed by immunogold labeling of Triton X-100-extracted M. pneumoniae cells incubated with antibodies to HMW1/4: gold particles bound in specific clusters to detergent-insoluble filaments. Finally, immunogold labeling of whole cells revealed that HMW1/4 are exposed on the cell surface, although to a lesser degree than on the cell interior. These findings indicate that HMW1/4 are membrane proteins associated with the cytoskeletonlike triton shell of M. pneumoniae and localized primarily in the filamentous extensions of the mycoplasma cells.

Identification of a possible cytadherence regulatory locus in Mycoplasma pneumoniae.

Transposon mutagenesis was used to analyze Mycoplasma pneumoniae cytadherence. Mycoplasmas were electroporated with Tn4001, and transformants were identified by antibiotic selection using gentamicin. The resulting colonies were screened for hemadsorption (HA) as an indicator for cytadherence. Six HA- colonies from independent transformations were isolated, filter cloned, and characterized in more detail. Southern hybridization analysis revealed that all six transposon insertions mapped to the same 252-kbp ApaI fragment and 19.5-kbp XhoI fragment. More detailed analysis localized the insertion to two adjacent EcoRI fragments. This site is distinct from the locus containing the genes for the high-molecular weight cytadherence-accessory proteins HMW1 and HMW3, and yet these proteins were absent from the protein profiles of all six transformants. To determine if transposon insertion was responsible for the HA- phenotype, reversion frequencies of the transformants were assessed after passage in the presence of antibiotic selection. In contrast to a spontaneously arising HMW-deficient variant, which reverted to an HA+ phenotype readily, no HA+ revertants were identified for any of the six transformants. These observations suggest that a potential regulatory locus that may be important in the expression of the HMW cytadherence-accessory proteins has been identified.

Inhibition of mycoplasma pneumoniae hemadsorption and adherence to respiratory epithelium by antibodies to a membrane protein.

Antiserum and purified immunoglobulin directed against Mycoplasma pneumoniae membrane protein P1 were examined for their influence on mycoplasma viability, metabolism, and cytadsorption. Anti-P1 immunoglobulin inhibited adherence of M. pneumoniae to hamster tracheal rings in vitro by up to 80% and inhibited hemadsorption by greater than 90%. Cytadsorption was also inhibited by anti-P1 Fab fragments. Anti-P1 antibodies had no effect on M. pneumoniae viability or metabolism. The data indicate that anti-P1 antibody obstructs the interaction of M. pneumoniae adhesin P1 with its host receptors.

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.

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.