Comparative genome analysis of Mycoplasma pneumoniae.

BACKGROUND: Mycoplasma pneumoniae is a common pathogen that causes upper and lower respiratory tract infections in people of all ages, responsible for up to 40% of community-acquired pneumonias. It also causes a wide array of extrapulmonary infections and autoimmune phenomena. Phylogenetic studies of the organism have been generally restricted to specific genes or regions of the genome, because whole genome sequencing has been completed for only 4 strains. To better understand the physiology and pathogenicity of this important human pathogen, we performed comparative genomic analysis of 15 strains of M. pneumoniae that were isolated between the 1940s to 2009 from respiratory specimens and cerebrospinal fluid originating from the USA, China and England. RESULTS: Illumina MiSeq whole genome sequencing was performed on the 15 strains and all genome sequences were completed. Results from the comparative genomic analysis indicate that although about 1500 SNP and indel variants exist between type1 and type 2 strains, there is an overall high degree of sequence similarity among the strains (>99% identical to each other). Within the two subtypes, conservation of most genes, including the CARDS toxin gene and arginine deiminase genes, was observed. The major variation occurs in the P1 and ORF6 genes associated with the adhesin complex. Multiple hsdS genes (encodes S subunit of type I restriction enzyme) with variable tandem repeat copy numbers were found in all 15 genomes. CONCLUSIONS: These data indicate that despite conclusions drawn from 16S rRNA sequences suggesting rapid evolution, the M. pneumoniae genome is extraordinarily stable over time and geographic distance across the globe with a striking lack of evidence of horizontal gene transfer.

Catalase Enhances Growth and Biofilm Production of Mycoplasma pneumoniae.

Mycoplasma pneumoniae causes chronic respiratory disease in humans. Factors thought to be important for colonization include the ability of the mycoplasma to form a biofilm on epithelial surfaces and the production of hydrogen peroxide to damage host tissue. Almost all of the mycoplasmas, including M. pneumoniae, lack superoxide dismutase and catalase and a balance should exist between peroxide production and growth. We show here that the addition of catalase to cultures enhanced the formation of biofilms and altered the structure. The incorporation of catalase in agar increased the number of colony-forming units detected and hence could improve the clinical diagnosis of mycoplasmal diseases.

Type 1 and type 2 strains of Mycoplasma pneumoniae form different biofilms.

Several mycoplasma species have been shown to form biofilms that confer resistance to antimicrobials and which may affect the host immune system, thus making treatment and eradication of the pathogens difficult. The present study shows that the biofilms formed by two strains of the human pathogen Mycoplasma pneumoniae differ quantitatively and qualitatively. Compared with strain UAB PO1, strain M129 grows well but forms biofilms that are less robust, with towers that are less smooth at the margins. A polysaccharide containing N-acetylglucosamine is secreted by M129 into the culture medium but found in tight association with the cells of UAB PO1. The polysaccharide may have a role in biofilm formation, contributing to differences in virulence, chronicity and treatment outcome between strains of M. pneumoniae. The UAB PO1 genome was found to be that of a type 2 strain of M. pneumoniae, whereas M129 is type 1. Examination of other M. pneumoniae isolates suggests that the robustness of the biofilm correlates with the strain type.

EPS-I polysaccharide protects Mycoplasma pulmonis from phagocytosis.

Few mycoplasmal polysaccharides have been described and little is known about their role in pathogenesis. The infection of mice with Mycoplasma pulmonis has been utilized in many in vivo and in vitro studies to gain a better understanding of host-pathogen interactions during chronic respiratory infection. Although alveolar macrophages have a primary role in host defence, M. pulmonis is killed inefficiently in vitro. One antiphagocytic factor produced by the mycoplasma is the family of phase- and size-variable Vsa lipoproteins. However, bacteria generally employ multiple strategies for combating host defences, with capsular polysaccharide often having a key role. We show here that mutants lacking the EPS-I polysaccharide of M. pulmonis exhibit increased susceptibility to binding and subsequent killing by alveolar macrophages. These results give further insight into how mycoplasmas are able to avoid the host immune system and sustain a chronic infection.

Mycoplasma polysaccharide protects against complement.

Although they lack a cell wall, mycoplasmas do possess a glycocalyx. The interactions between the glycocalyx, mycoplasmal surface proteins and host complement were explored using the murine pathogen Mycoplasma pulmonis as a model. It was previously shown that the length of the tandem repeat region of the surface lipoprotein Vsa is associated with susceptibility to complement-mediated killing. Cells producing a long Vsa containing about 40 repeats are resistant to complement, whereas strains that produce a short Vsa of five or fewer repeats are susceptible. We show here that the length of the Vsa protein modulates the affinity of the M. pulmonis EPS-I polysaccharide for the mycoplasma cell surface, with more EPS-I being associated with mycoplasmas producing a short Vsa protein. An examination of mutants that lack EPS-I revealed that planktonic mycoplasmas were highly susceptible to complement killing even when the Vsa protein was long, demonstrating that both EPS-I and Vsa length contribute to resistance. In contrast, the mycoplasmas were resistant to complement even in the absence of EPS-I when the cells were encased in a biofilm.

The Vsa shield of Mycoplasma pulmonis is antiphagocytic.

The infection of mice with Mycoplasma pulmonis is a model for studying chronic mycoplasmal respiratory disease. Many in vivo and in vitro studies have used the organism to gain a better understanding of host-pathogen interactions in chronic respiratory infection. The organism's Vsa proteins contain an extensive tandem repeat region. The length of the tandem repeat unit varies from as few as 11 amino acids to as many as 19. The number of tandem repeats can be as high as 60. The number of repeats varies at a high frequency due to slipped-strand mispairing events that occur during DNA replication. When the number of repeats is high, e.g., 40, the mycoplasma is resistant to lysis by complement but does not form a robust biofilm. When the number of repeats is low, e.g., 5, the mycoplasma is killed by complement when the cells are dispersed but has the capacity to form a biofilm that resists complement. Here, we examine the role of the Vsa proteins in the avoidance of phagocytosis and find that cells producing a protein with many tandem repeats are relatively resistant to killing by macrophages. These results may be pertinent to understanding the functions of similar proteins that have extensive repeat regions in other microbes.

Fewer essential genes in mycoplasmas than previous studies suggest.

Here, we describe mutants of Mycoplasma pulmonis that were obtained using a minitransposon, Tn4001TF1, which actively transposes but is then unable to undergo subsequent excision events. Using Tn4001TF1, we disrupted 39 genes previously thought to be essential for growth. Thus, the number of genes required for growth has been overestimated. This study also revealed evidence of gene duplications in M. pulmonis and identified chromosome segregation proteins that are dispensable in mycoplasmas but essential in Bacillus subtilis.

Identification of exopolysaccharide-deficient mutants of Mycoplasma pulmonis.

The presence of capsular exopolysaccharide (EPS) in Mollicutes has been inferred from electron micrographs for over 50 years without conclusive data to support the production of complex carbohydrates by the organism. Mycoplasma pulmonis binds the lectin Griffonia simplicifolia I (GS-I), which is specific for terminal beta-linked galactose residues. Mutants that failed to produce the EPS bound by GS-I were isolated from a transposon library. All of the mutants had the transposon located in open reading frame MYPU_7410 or MYPU_7420. These overlapping genes are predicted to code for a heterodimeric pair of ABC transporter permeases and may code for part of a new pathway for synthesis of EPS. Analysis by lectin-affinity chromatography in conjunction with gas chromatography demonstrated that the wild-type mycoplasma produced an EPS (EPS-I) composed of equimolar amounts of glucose and galactose that was lacking in the mutants. Phenotypic analysis revealed that the mutants had an increased propensity to form a biofilm on glass surfaces, colonized mouse lung and trachea efficiently, but had a decreased association with the A549 lung cell line. Confounding the interpretation of these results is the observation that the mutants missing EPS-I had an eightfold overproduction of an apparent second EPS (EPS-II) containing N-acetylglucosamine.

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.

Biofilms protect Mycoplasma pulmonis cells from lytic effects of complement and gramicidin.

The length of the tandem repeat region of the Vsa protein of Mycoplasma pulmonis has previously been shown to modulate the susceptibility of mycoplasmas to killing by complement: cells that produce a short form of the Vsa protein are highly sensitive, and cells producing the long Vsa protein are resistant. In contrast to their differing susceptibilities to complement, the mycoplasmas were highly sensitive to gramicidin irrespective of the length of the Vsa protein produced. We show here that when encased within a biofilm, cells of M. pulmonis producing a short form of the Vsa protein were more resistant to complement and gramicidin than mycoplasmas that were dispersed. The resistance appeared to be localized to those mycoplasmas within tower structures of the biofilms. Biofilm formation may be a mechanism that protects mycoplasmas from host immunity.

A stochastic mechanism for biofilm formation by Mycoplasma pulmonis.

Bacterial biofilms are communities of bacteria that are enclosed in an extracellular matrix. Within a biofilm the bacteria are protected from antimicrobials, environmental stresses, and immune responses from the host. Biofilms are often believed to have a highly developed organization that is derived from differential regulation of the genes that direct the synthesis of the extracellular matrix and the attachment to surfaces. The mycoplasmas have the smallest of the prokaryotic genomes and apparently lack complex gene-regulatory systems. We examined biofilm formation by Mycoplasma pulmonis and found it to be dependent on the length of the tandem repeat region of the variable surface antigen (Vsa) protein. Mycoplasmas that produced a short Vsa protein with few tandem repeats formed biofilms that attached to polystyrene and glass. Mycoplasmas that produced a long Vsa protein with many tandem repeats formed microcolonies that floated freely in the medium. The biofilms and the microcolonies contained an extracellular matrix which contained Vsa protein, lipid, DNA, and saccharide. As variation in the number of Vsa tandem repeats occurs by slipped-strand mispairing, the ability of the mycoplasmas to form a biofilm switches stochastically.

Resistance of Mycoplasma pulmonis to complement lysis is dependent on the number of Vsa tandem repeats: shield hypothesis.

The Vsa proteins are associated with the virulence of the murine respiratory pathogen Mycoplasma pulmonis. The antigens consist of a conserved N-terminal region that is combined with one of several different variable C-terminal regions comprised of tandem repeats. M. pulmonis strains that produce VsaA with about 40 tandem repeats do not adhere to polystyrene or erythrocytes and are highly resistant to complement killing. Strains that produce VsaA with three tandem repeats adhere strongly to polystyrene and erythrocytes and are highly susceptible to complement killing. We report here that the resistance to complement lysis was not due to a lack of activation of the complement cascade. Isolation and analysis of M. pulmonis strains that produced Vsa proteins other than VsaA (VsaG and VsaI) with either long or short repeat regions indicated that adherence to polystyrene and resistance to complement were dependent on the length of the repeat region but not on the Vsa type. Furthermore, M. pulmonis Vsa variants were susceptible to the polypeptide pore-forming molecule gramicidin D, independent of the Vsa type and length. Collectively, the data indicate the Vsa proteins nonspecifically mediate M. pulmonis surface interactions and function to sterically hinder access of complement to the mycoplasma cell membrane while permitting access of smaller molecules.

Bacteriophage MAV1 is not associated with virulence of Mycoplasma arthritidis.

Previous studies demonstrated that Mycoplasma arthritidis strain 158 acquired a high degree of virulence upon lysogenization with bacteriophage MAV1. In the present study, the association between MAV1 and virulence was reexamined by creating new lysogens of 158 and of a relatively avirulent mutant, strain 158-1. In the absence of lysogenization, 158 was more virulent than expected. The virulence of 158 and 158-1 did not increase upon lysogenization. A major antigenic difference between 158 and 158-1 was identified that is unrelated to MAV1 and could account for the difference in virulence.

Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression.

Although the fibroproliferative response to lung injury occurs with a high frequency in patients with clinical acute lung injury, the mechanisms that initiate this response are largely unknown. This study was undertaken first to identify fibroblast mitogenic factors in pulmonary edema fluid, and second to examine the human lung fibroblast's gene expression profile in response to pulmonary edema fluid. The edema fluid obtained from patients with early lung injury has an eightfold higher concentration of IL-1beta and a twofold greater IL-1beta-dependent mitogenic effect than does fluid obtained from control patients with hydrostatic pulmonary edema. Furthermore, fibroblasts responded to acute lung injury patient-derived edema fluid through production of soluble mediators that possess an autocrine mitogenic effect. Gene array analysis reveals that acute lung injury edema fluid induces several inflammation-modulating and proliferation-related genes in fibroblasts, whose inductions are similarly dependent on bioactive IL-1beta. Most notably, the 20-fold induction of IL-6 mRNA and protein was completely blocked by IL-1 receptor antagonist. The combined addition of IL-1beta and IL-6 was mitogenic, and the proliferative response to conditioned medium from IL-1beta-exposed cells was blocked by antagonistically acting Abs to IL-6 or to gp130. These novel findings indicate that soluble IL-1beta bioactivity and autocrine IL-1beta-dependent IL-6 up-regulation are critical initiators of fibroblast activation and proliferation and that they likely play a role in the fibroproliferative response seen in human acute lung injury.

The Vsa proteins modulate susceptibility of Mycoplasma pulmonis to complement killing, hemadsorption, and adherence to polystyrene.

The variable surface antigens (Vsa) of the murine respiratory pathogen Mycoplasma pulmonis are associated with the virulence of the microorganism in the lung. In strain UAB CT, the antigens consist of an N-terminal region that is combined with one of seven different C-terminal variable regions comprised of tandem repeats. M. pulmonis producing a VsaA protein with about 40 tandem repeats (R40) does not adhere to red blood cells or polystyrene. Strains that produce VsaH contain a short C-terminal region that lacks tandem repeats and adhere to red blood cells and plastic. We isolated and analyzed M. pulmonis strain CT variants (CT182 and derivatives) that produced a VsaA protein with only three tandem repeats (R3). These variants adhered to plastic and red blood cells similarly to the VsaH-producing strain. When the R3-producing CT182 strain or the VsaH-producing strains were incubated with normal guinea pig serum, they were efficiently killed. Killing was abolished when the serum was heat inactivated. In contrast, the M. pulmonis strains that produced VsaA R40 were highly resistant to complement killing. CT182R3 variants that survived the complement killing reactions all produced the R40 form of VsaA and were resistant to complement killing. VsaA R40 is the first mycoplasmal protein shown to be associated with resistance to complement. As both VsaH and VsaA can mediate adherence to plastic, cytadherence, and susceptibility to complement, we propose that Vsa modulates these phenotypes by nonspecific interactions.

Gene transfer in Mycoplasma pulmonis.

Experiments were undertaken to examine gene transfer in Mycoplasma pulmonis. Parent strains containing transposon-based tetracycline and chloramphenicol resistance markers were combined to allow transfer of markers. Two mating protocols were developed. The first consisted of coincubating the strains in broth culture for extended periods of time. The second protocol consisted of a brief incubation of the combined strains in a 50% solution of polyethylene glycol. Using either protocol, progeny that had acquired antibiotic resistance markers from both parents were obtained. Analysis of the progeny indicated that only the transposon and not flanking genomic DNA was transferred to the recipient cell. Gene transfer was DNase resistant and probably the result of conjugation or cell fusion.