Characterization of a Mycoplasma pneumoniae hmw3 mutant: implications for attachment organelle assembly

The proteins required for adherence of the pathogen Mycoplasma pneumoniae to host respiratory epithelial cells are localized to a polar structure, the attachment organelle. A number of these proteins have been characterized functionally by analysis of noncytadhering mutants, and many are components of the mycoplasma cytoskeleton. Mutations in some cytadherence-associated proteins have pleiotropic effects, including decreased stability of other proteins, loss of adherence and motility, and abnormal morphology. The function of protein HMW3, a component of the attachment organelle, has been difficult to discern due to lack of an appropriate mutant. In this paper, we report that loss of HMW3 resulted in decreased levels and more diffuse localization of cytoskeletal protein P65, subtle changes in morphology, inability to cluster the adhesin P1 consistently at the terminal organelle, reduced cytadherence, and, in some cells, an atypical electron-dense core in the attachment organelle. This phenotype suggests a role for HMW3 in the architecture and stability of the attachment organelle.     

Comparison of the efficacy of egg yolk extract and horse serum for growth of Mycoplasma pneumoniae

The usefulness of chicken egg yolk extract as a substitute for horse serum in culture media for Mycoplasma pneumoniae was investigated. As a growth-supporting factor in the growth medium for M. pneumoniae and some other mycoplasmal species, the primary isolation medium for M. pneumoniae, and the metabolism-inhibition test medium for diagnosis of M. pneumoniae infection, egg yolk extract may be an excellent substitute for horse serum. The particular superiority of egg yolk extract to horse serum is that egg yolk extract, unlike horse serum, did not show any inhibitory effect on the growth of M. pneumoniae.     

Cimulative hemagglutination by Mycoplasma pneumoniae and other agglutinins

John, T. Jacob (University of Colorado School of Medicine, Denver), Marlene Stahl, and Vincent A. Fulginiti. Cumulative hemagglutination by Mycoplasma pneumoniae and other agglutinins. J. Bacteriol. 92:1002-1004. 1966.-The phenomenon of cumulative hemagglutination, or hemagglutination by two agglutinins, each in subagglutinating concentration, was demonstrated by use of four different systems, namely, horse serum and Mycoplasma pneumoniae, horse serum and measles antigen, M. pneumoniae and measles antigen, and parainfluenza 2 virus and M. pneumoniae. Cumulative hemagglutination appears to be the mechanism by which a horse-serum diluent causes high hemagglutination titers of M. pneumoniae, since both contain hemagglutinins against vervet erythrocytes. It was also shown that antibodies against either one of the two antigens may cause inhibition of such hemagglutination.     

Detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in patients with community acquired pneumonia

A total of 292 patients with pneumonia confirmed by clinical, roentgenological and laboratory methods, admitted to the hospital from closed communities during the one year period, were examined. The sputa of patients with pneumonia in the acute stage were studied in the polymerase chain reaction (PCR). The predominence of M. pneumoniae and C. pneumoniae causualties in autumn and winter period was established. The monitoring of atypical infective agents with the use of the PCR techniques gives evidence for conclusion on their ever growing role in the etiological structure of community acquired pneumonia in groups of closely interacting young people.     

Analysis of Pulmonary Inflammation and Function in the Mouse and Baboon after Exposure to Mycoplasma pneumoniae CARDS Toxin

Mycoplasma pneumoniae produces an ADP-ribosylating and vacuolating toxin known as the CARDS (Community Acquired Respiratory Distress Syndrome) toxin that has been shown to be cytotoxic to mammalian cells in tissue and organ culture. In this study we tested the ability of recombinant CARDS (rCARDS) toxin to elicit changes within the pulmonary compartment in both mice and baboons. Animals responded to a respiratory exposure to rCARDS toxin in a dose and activity-dependent manner by increasing the expression of the pro-inflammatory cytokines IL-1α, 1β, 6, 12, 17, TNF-α and IFN-γ. There was also a dose-dependent increase in several growth factors and chemokines following toxin exposure including KC, IL-8, RANTES, and G-CSF. Increased expression of IFN-γ was observed only in the baboon; otherwise, mice and baboons responded to CARDS toxin in a very similar manner. Introduction of rCARDS toxin to the airways of mice or baboons resulted in a cellular inflammatory response characterized by a dose-dependent early vacuolization and cytotoxicity of the bronchiolar epithelium followed by a robust peribronchial and perivascular lymphocytic infiltration. In mice, rCARDS toxin caused airway hyper-reactivity two days after toxin exposure as well as prolonged airway obstruction. The changes in airway function, cytokine expression, and cellular inflammation correlate temporally and are consistent with what has been reported for M. pneumoniae infection. Altogether, these data suggest that the CARDS toxin interacts extensively with the pulmonary compartment and that the CARDS toxin is sufficient to cause prolonged inflammatory responses and airway dysfunction.     

Severe asthma exacerbation: role of acute Chlamydophila pneumoniae and Mycoplasma pneumoniae infection

Background

Chlamydophila pneumoniae and Mycoplasma pneumoniae are associated with acute exacerbation of bronchial asthma (AEBA). The aim of this study was to evaluate the correlation between these acute bacterial infections and the severity of AEBA.

Methods

We prospectively analysed consecutive patients admitted to the Emergency Department with acute asthma exacerbation. In every patient peak expiratory flow (PEF) measurement was performed on admission, and spirometry during follow-up. Serology for Chlamydophila and Mycoplasma pneumoniae was performed on admission and after 4–8 weeks.

Results

Fifty-eight patients completed the study. Acute atypical infections (AAI) was observed in 22/58 cases; we found single acute C. pneumoniae in 19 cases, single acute M. pneumoniae in 2 cases, and double acute infection in one case. Functional impairment on admission was greater in patients with AAI than in patients without AAI (PEF 205 ± 104 L/min vs 276 ± 117 p = 0.02) and persisted until visit 2 (FEV1% 76.30 ± 24.54 vs FEV1% 92.91 ± 13.89, p = 0.002). Moreover, the proportion of patients who presented with severe AEBA was significantly greater in the group with AAI than in the group without AAI (15/22 vs 12/36, p = 0.01; OR 4.29, 95% CI 1.38–13.32).

Conclusion

Our data suggest an association between acute atypical infection and a more severe AEBA.     

Mycoplasma pneumoniae respiratory disease symposium: summation and significance

The symposium on M. pneumoniae respiratory disease has examined the clinical expression of infection in adults and children, the pathophysiologic disturbances which occur, and the laboratory diagnosis by isolation and serology. That these infections are very common has been well documented; however, a variable incidence over periods of several years tends to minimize importance of the disease for many clinicians. While good laboratory diagnostic methods exist, they provide retrospective insight predominantly and are not useful for early diagnosis or therapeutic decision making. Development of rapid diagnostic methods which are sensitive and specific is an important goal for future research. Success would facilitate our understanding and control of M. pneumoniae disease.     

Internalization and intracellular survival of Mycoplasma pneumoniae by non-phagocytic cells

Current theory holds that mycoplasmas remain attached to the surface of epithelial cells although some mycoplasmas have evolved mechanisms for entering host cells that are not naturally phagocytic. The ability of Mycoplasma pneumoniae strain M129 to invade and survive within host cells was studied using a HeLa cell line and a human lung carcinoma cell line (A549). The invasion process into the eukaryotic cells was studied qualitatively by confocal laser scanning microscopy and quantitatively by the gentamicin resistance assay. Internalization was found with A549 cells but not with HeLa cells. Internalization was dependent on the duration of the infection and on temperature. The organism, detected in the cytoplasm and perinuclear regions, survived within the host cells for prolonged periods of time. The intracellular location of M. pneumoniae is obviously a privileged niche, well protected from the immune system and from the action of many antibiotics and may explain the pathogenic potential of this organism.     

Typing of isolates of Mycoplasma pneumoniae and Mycoplasma salivarium by growth inhibitors derived from mycoplasmal cells treated with chloroform

Isolates of Mycoplasma pneumoniae and M. salivarium could be subclassified at the strain level by inhibitors (ch-Mcin) derived from mycoplasmal cells treated with chloroform. Sixty-one isolates of M. pneumoniae obtained from oral cavities of patients were divided into three types by their ch-Mcin: a definite type which completely inhibits the growth of M. fermentans PG18, an indefinite type and a noinhibition type. Sixty-seven isolates of M. salivarium were also divided into similar types by their ch-Mcin which does or does not inhibit the growth of M. salivarium Hup127. In the case of isolates of M. pneumoniae and M. salivarium belonging to the indefinite type which gave ambiguous patterns in the ch-Mcin typing, it was demonstrated that they could be clearly typed by further testing after repeated cloning.     

Detection and discrimination of Mycoplasma pneumoniae and Mycoplasma genitalium by the in vitro DNA amplification.

By using the primers designed on the bases of the sequences of the 16S rRNA genes of Mycoplasma pneumoniae and Mycoplasma genitalium, respectively, specific and sensitive in vitro DNA amplification assay system for the detection and discrimination of these two mycoplasmas was established. The detection limit of the assay was 100 cells for M. pneumoniae and 1,000 cells for M. genitalium. Neither other human mycoplasmas nor oral bacteria existing in human saliva showed any cross-reactions with these primers.     

R-roscovitine Reduces Lung Inflammation Induced by Lipoteichoic Acid and Streptococcus pneumoniae

Bacterial pneumonia remains associated with high morbidity and mortality. The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Lipoteichoic acid (LTA) is an important proinflammatory component of the gram-positive bacterial cell wall. R-roscovitine, a purine analog, is a potent cyclin-dependent kinase (CDK)-1, −2, −5 and −7 inhibitor that has the ability to inhibit the cell cycle and to induce polymorphonuclear cell (PMN) apoptosis. We sought to investigate the effect of R-roscovitine on LTA-induced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LTA or viable S. pneumoniae in vivo. In vitro R-roscovitine enhanced apoptosis in PMNs and reduced tumor necrosis factor (TNF)-α and keratinocyte chemoattractant (KC) production in MH-S (alveolar macrophage) and MLE-12/MLE-15 (respiratory epithelial) cell lines. In vivo R-roscovitine treatment reduced PMN numbers in bronchoalveolar lavage fluid during LTA-induced lung inflammation; this effect was reversed by inhibiting apoptosis. Postponed treatment with R-roscovitine (24 and 72 h) diminished PMN numbers in lung tissue during gram-positive pneumonia; this step was associated with a transient increase in pulmonary bacterial loads. R-roscovitine inhibits proinflammatory responses induced by the gram-positive stimuli LTA and S. pneumoniae. R-roscovitine reduces PMN numbers in lungs upon LTA administration by enhancing apoptosis. The reduction in PMN numbers caused by R-roscovitine during S. pneumoniae pneumonia may hamper antibacterial defense.     

Manifestations and complications of Mycoplasma pneumoniae disease: a review

Over the past 20 years the annual number of reports on extrapulmonary symptoms during Mycoplasma (M.) pneumoniae disease has increased. Clinical and epidemiological data indicate that symptoms from the skin and mucous membranes, from the central nervous system, from the heart, and perhaps from other organs as well are not quite uncommon manifestations of M. pneumoniae disease. Reports on unusual courses of the disease have also accumulated, including cases of severe respiratory symptoms, sometimes seen in patients with underlying disease or with a concomitant viral infection. Serious extrapulmonary manifestations have been common in fatal cases of M. pneumoniae disease. Some observations and experimental data on these manifestations and on the possible pathogenic mechanisms are dealt with. The conclusion is that such mechanisms are still largely unknown.     

Growth Factors for Mycoplasma pneumoniae in Yeast and Tissue Extracts

1. Many tissue extracts have been shown to have growth factor activity towards M. pneumoniae notably bovine lung. Yeast extract is therefore not unique in this respect. The materials in lung digest and in yeast extract are very similar in properties and could be identical.
2. The growth factor is a low molecular weight water soluble material. Despite its stability to heat it decomposes on storage, presumably by oxidation.
3. Chromatographic procedures, on both Dowex 50W columns and on paper, indicated the presence of two active components.     

Functional Analysis of the Superfamily 1 DNA Helicases Encoded by Mycoplasma pneumoniae and Mycoplasma genitalium

The DNA recombination and repair machinery of Mycoplasma pneumoniae is composed of a limited set of approximately 11 proteins. Two of these proteins were predicted to be encoded by neighboring open reading frames (ORFs) MPN340 and MPN341. Both ORFs were found to have sequence similarity with genes that encode proteins belonging to the DNA helicase superfamily 1 (SF1). Interestingly, while a homolog of the MPN341 ORF is present in the genome of Mycoplasma genitalium (ORF MG244), MPN340 is an M. pneumoniae-specific ORF that is not found in other mycoplasmas. Moreover, the length of MPN340 (1590 base pairs [bp]) is considerably shorter than that of MPN341 (2148 bp). Examination of the MPN340-encoded amino acid sequence indicated that it may lack a so-called 2B subdomain, which is found in most SF1 DNA helicases. Also, the MPN340-encoded amino acid sequence was found to differ between subtype 1 strain M129 and subtype 2 strain FH at three amino acid positions. Both protein variants, which were termed PcrA^s _M129 and PcrA^s _FH, respectively, as well as the MPN341- and MG244-encoded proteins (PcrA_Mpn and PcrA_Mge, respectively), were purified, and tested for their ability to interact with DNA. While PcrA_Mpn and PcrA_Mge were found to bind preferentially to single-stranded DNA, both PcrA^s _M129 and PcrA^s _FH did not demonstrate significant DNA binding. However, all four proteins were found to have divalent cation- and ATP-dependent DNA helicase activity. The proteins displayed highest activity on partially double-stranded DNA substrates carrying 3′ single-stranded extensions.