The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis

Mycoplasma pulmonis is a wall-less eubacterium belonging to the Mollicutes (trivial name, mycoplasmas) and responsible for murine respiratory diseases. The genome of strain UAB CTIP is composed of a single circular 963 879 bp chromosome with a G + C content of 26.6 mol%, i.e. the lowest reported among bacteria, Ureaplasma urealyticum apart. This genome contains 782 putative coding sequences (CDSs) covering 91.4% of its length and a function could be assigned to 486 CDSs whilst 92 matched the gene sequences of hypothetical proteins, leaving 204 CDSs without significant database match. The genome contains a single set of rRNA genes and only 29 tRNAs genes. The replication origin oriC was localized by sequence analysis and by using the G + C skew method. Sequence polymorphisms within stretches of repeated nucleotides generate phase-variable protein antigens whilst a recombinase gene is likely to catalyse the site-specific DNA inversions in major M.pulmonis surface antigens. Furthermore, a hemolysin, secreted nucleases and a glyco-protease are predicted virulence factors. Surprisingly, several of the genes previously reported to be essential for a self-replicating minimal cell are missing in the M.pulmonis genome although this one is larger than the other mycoplasma genomes fully sequenced until now.     

A nomenclature for restriction enzymes,DNA methyltransferases,homing endonucleases and their genes

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.     

Epigenetic regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in cancer

Epigenetics refers to heritable changes in gene expression that are independent of alterations in DNA sequence. It is now accepted that disruption of epigenetic mechanisms plays a key role in the pathogenesis of cancer: culminating in altered gene function and malignant cellular transformation. DNA methylation and histone modifications are the most widely studied changes but non-coding RNAs such as miRNAs are also considered part of the epigenetic machinery. The insulin-like growth factor (IGF) axis is composed of two ligands, IGF-I and –II, their receptors and six high affinity IGF binding proteins (IGFBPs). The IGF axis plays a key role in cancer development and progression. As IGFBP genes have consistently been identified among the most common to be aberrantly altered in tumours, this review will focus on epigenetic regulation of IGFBP-3 in cancer for which the majority of evidence has been obtained.     

Nucleotide sequence of Mycoplasma mycoides subspecies Mycoides plasmid pKMK1.

To facilitate the development of mycoplasmal cloning vectors, we have determined the nucleotide sequence of pKMK1, a cryptic plasmid isolated from Mycoplasma mycoides subsp. mycoides. It is 1875 bp in length and contains two open reading frames (ORFs) that share homology with ORFs from members of a large family of gram-positive bacterial plasmids which replicate via a single-stranded DNA intermediate. Putative origins of replication and candidate cloning sites have been identified.     

Housing Conditions Modulate the Severity of Mycoplasma pulmonis Infection in Mice Deficient in Class A Scavenger Receptor

Mycoplasmosis is a frequent causative microbial agent of community-acquired pneumonia and has been linked to exacerbation of chronic obstructive pulmonary disease. The macrophage class A scavenger receptor (SRA) facilitates the clearance of noxious particles, oxidants, and infectious organisms by alveolar macrophages. We examined wildtype and SRA^−/− mice, housed in either individually ventilated or static filter-top cages that were cycled with fresh bedding every 14 d, as a model of gene–environment interaction on the outcome of pulmonary Mycoplasma pulmonis infection. Intracage NH₃ gas measurements were recorded daily prior to infection. Mice were intranasally infected with 1 × 10⁷ cfu M. pulmonis UAB CT and evaluated at 3, 7, and 14 d after inoculation. Wildtype mice cleared 99.5% of pulmonary M. pulmonis by 3 d after infection but remained chronically infected through the study. SRA^−/− mice were chronically infected with 40-fold higher mycoplasma numbers than were wildtype mice. M. pulmonis caused a chronic mixed inflammatory response that was accompanied with high levels of IL1β, KC, MCP1, and TNFα in SRA^−/− mice, whereas pulmonary inflammation in WT mice was represented by a monocytosis with elevation of IL1β. Housing had a prominent influence on the severity and persistence of mycoplasmosis in SRA^−/− mice. SRA^-/- mice housed in static cages had an improved recovery and significant changes in surfactant proteins SPA and SPD compared with baseline levels. These results indicate that SRA is required to prevent chronic mycoplasma infection of the lung. Furthermore, environmental conditions may exacerbate chronic inflammation in M. pulmonis-infected SRA^−/− mice.Abbreviations: BAL, bronchoalveolar lavage; COPD, chronic obstructive pulmonary disease; KC, keratinocyte-derived chemokine (CXCL1); MCP1, monocyte chemotactic protein 1; SPA, surfactant protein A (SFTPA1); SPB, surfactant protein B (SFTPB); SPD, surfactant protein D (SFTPD); SRA, class A scavenger receptor (MSR1); WT, wildtypeThere are numerous options for the housing and husbandry of rodents in the laboratory setting. Various available choices in caging, bedding material, and cage-change frequency have the potential to effect physiologic values and thus experimental outcomes.^20,108 In many facilities, current practices involve performing cage changes every 1 to 2 wk, with some facilities exploring the possibility of extending these practices to every 4 wk.⁹⁷ Cage-change frequency practices are established at various institutions after consideration of several variables that affect animal health, welfare, and cost. Ideally, an appropriate sanitation program provides clean and dry bedding, adequate air quality, and clean cage surfaces and accessories.⁴⁴ When establishing performance standards for a sanitation program that are different from those which are recommended in the Guide for the Care and Use of Animals in Research,⁴⁴ microenvironmental conditions, including intracage humidity, temperature, animal behavior and appearance, microbiologic loads, and levels of pollutants such as CO₂ and NH₃, should be evaluated and verified. Although there are currently no established NH₃ exposure limits for laboratory animals, the human occupational exposure limit of 25 ppm as an 8-h time-weighted average, established by the National Institute for Occupational Safety and Health, is often referenced as a guideline for animals.⁹⁵ Multiple factors, such as animal cage density, sex, age, bedding type, reusable compared with disposable caging, static caging compared with IVC, and cage-change frequency, influence intracage and ambient NH₃ levels.^82,83,97 Only limited information is available that addresses the effect of natural intracage NH₃ levels on respiratory function in experimental rodents and whether exposure to high NH₃ levels under current standard practices affects the results of respiratory disease research.Ammonia is an alkaline, corrosive, and irritant gas that is very water soluble. It reacts with the moisture of the mucous membranes of the eyes, mouth, and respiratory tract to form ammonium hydroxide in an exothermic reaction, resulting in thermal and chemical burns.⁶⁸ Clinical symptoms in humans exposed to high levels of NH₃ include eye irritation, headaches, and multiple acute and chronic respiratory symptoms, such as irritation of the nose, pharynx, and sinuses, and in severe cases, development of bronchitis and hyper-reactive airway disease.⁷⁹ Animals are similarly susceptible to NH₃-induced pulmonary disease.^23,31,48Mice exposed to naturally increasing levels of intracage NH₃ can develop lesions in the rostral nasal cavity, with decreasing severity of the lesions moving caudally into the nasopharynx, and no lesions in the lung.⁹⁷ However, dust is another common environmental pollutant that is often present in animal settings. Dust particles readily absorb NH₃, which then serve as a source of NH₃ deposition into the lower respiratory tract. Dust particulate can range from large (300 µm), minimally respirable particles to very fine (< 50 µm) particulate matter, which can settle deep within the alveoli.^10,102 The mucociliary system of the respiratory tract is the first line of defense against inspired noxious stimuli and pathogens. Exposure of the ciliated respiratory epithelium to the damaging effects of NH₃ are known to cause decreased mucociliary beating.⁵⁶ Disruption of the respiratory mucociliary escalator initiated by NH₃ exposure can then promote establishment of chronic infections and inflammation of the airway mucosa.^11,87 Therefore, NH₃ potentially can cause pathophysiologic changes of the lung in the absence of histopathologic lesions.Our primary goal was to analyze the effect of 2 housing modalities, which result in different intracage NH₃ concentrations, on mice that were challenged with a respiratory pathogen. Mycoplasma pulmonis was chosen as a model because it is a well-established model in rodents which causes chronic mycoplasmosis and reproduces the features of M. pneumoniae in humans.^22,41 M. pneumoniae infection is a frequent and contagious etiology of community-acquired pneumonia causing tracheobronchitis, sneezing, cough, and inflammation of the respiratory tract.^8,12,47,63 Moreover, atypical and difficult-to-detect respiratory pathogens such as Chlamydophila pneumoniae and Mycoplasma pneumoniae that can establish chronic asymptomatic infections may contribute to both the development and exacerbation of COPD^{26,45,57,58,62,63,66,72,96,103} and asthma.^8,51,65 Infection with M. pulmonis in rodents causes rhinitis, otitis media, tracheitis, and pneumonia, which can be exacerbated by housing conditions and genetic background.^14,32,85 The mechanism of pathogenicity of mycoplasmas continues to be an area of interest in the research.The innate host factors protecting against pulmonary mycoplasmosis include the secreted surfactant protein opsonins SPA and SPD, surfactant phospholipids, and the molecular pattern-recognition receptor TLR2.^15,16,54,74 Therefore, compared with their wildtype (WT) counterparts, SPA-deficient mice infected with either M. pulmonis or M. pneumoniae develop more severe inflammation and have decreased capacity to clear these infections from the lungs.⁴³ In addition, TLR2-deficient mice exhibit decreased clearance and increased inflammation in response to mycoplasma infection.^60,104Second, we wanted to study the effects of SRA deficiency in mycoplasmosis. The class A scavenger receptor (SRA) modulates inflammatory responses and mediates the clearance of airborne oxidants, particulates, and respiratory pathogens.^{3,17,18,49,88,101} Inhibition of SRA expression in alveolar macrophages in an elastase–LPS model of COPD was associated with decreased clearance of Haemophilus influenzae.³³ Lack of SRA similarly impaired alveolar macrophage-mediated clearance of Streptococcus pneumoniae,⁵ environmental particles,⁶ and ozone-oxidized lipids¹⁸ by alveolar macrophages. Absence of SRA also enhanced hyperoxia-induced lung injury⁴⁹ and exacerbated inflammation in response to Staphylococcus aureus infection.⁸⁸ SRA appears to have antiinflammatory properties with the capacity to modify macrophage phenotype and suppress polarization toward the M1 alternative macrophage activation state.¹³ The SRA gene (MSR1) is polymorphic in both mice and humans.^19,29,105 Genetic association studies in humans, however, showed that subjects with truncations or point mutations in MSR1 have significantly increased risk for the development of pulmonary diseases such as COPD^33,38,71,94 and asthma.⁵ Our understanding of the immune factors that contribute to mycoplasmosis is far from complete.In the present study, by investigating the role of SRA in mycoplasmosis jointly with the effects of housing, we demonstrated that genetic and environmental factors both serve as critical players in disease progression. We show that SRA-deficient mice are susceptible to chronic colonization with M. pulmonis and development of chronic mycoplasma-induced bronchopneumonia characterized by persistent multicellular inflammation. Furthermore, we show that housing conditions influence the effect of SRA deficiency on the severity of mycoplasmosis. Taken together, these results indicate that lack of SRA function impairs host protection against both infectious and environmental insults.     

The usage of marker traits in plants for evaluation of resistance of the winter wheat to pest insects

A new method of selection of the winter wheat varieties has been tested for resistance to the pest insects' complex by the traits of plants that are the markers of plant resistance. It makes it possible to use this method from year to year independently of the pests' density.     

Mycoplasma biofilms ex vivo and in vivo

Biofilms are communities of microorganisms that are encased in polymeric matrixes and grow attached to biotic or abiotic surfaces. Despite their enhanced ability to resist antimicrobials and components of the immune system in vitro , few studies have addressed the interactions of biofilms with the host at the organ level. Although mycoplasmas have been shown to form biofilms on glass and plastic surfaces, it has not been determined whether they form biofilms on the tracheal epithelium. We developed a tracheal organ-mounting system that allowed the entire surface of the tracheal lumen to be scanned using fluorescence microscopy. We observed the biofilms formed by the murine respiratory pathogen Mycoplasma pulmonis on the epithelium of trachea in tracheal organ culture and in experimentally infected mice and found similar structure and biological characteristics as biofilms formed in vitro . This tracheal organ-mounting system can be used to study interactions between biofilms formed by respiratory pathogens and the host epithelium and to identify the factors that contribute to biofilm formation in vivo .