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Mycoplasma gallisepticum is a significant respiratory and reproductive pathogen of domestic poultry. While the complete genomic sequence of the virulent, low-passage M. gallisepticum strain R (R low ) has been reported, genomic determinants responsible for differences in virulence and host range remain to be completely identified. Here, we utilize genome sequencing and microarray-based comparative genomic data to identify these genomic determinants of virulence and to elucidate genomic variability among strains of M. gallisepticum. Analysis of the high-passage, attenuated derivative of R low , R high , indicated that relatively few total genomic changes (64 loci) occurred, yet they are potentially responsible for the observed attenuation of this strain. In addition to previously characterized mutations in cytadherence-related proteins, changes included those in coding sequences of genes involved in sugar metabolism. Analyses of the genome of the M. gallisepticum vaccine strain F revealed numerous differences relative to strain R, including a highly divergent complement of vlhA surface lipoprotein genes, and at least 16 genes absent or significantly fragmented relative to strain R. Notably, an R low isogenic mutant in one of these genes (MGA_1107) caused significantly fewer severe tracheal lesions in the natural host compared to virulent M. gallisepticum R low . Comparative genomic hybridizations indicated few genetic loci commonly affected in F and vaccine strains ts-11 and 6/85, which would correlate with proteins affecting strain R virulence. Together, these data provide novel insights into inter-and intrastrain M. gallisepticum genomic variability and the genetic basis of M. gallisepticum virulence.

, the primary etiologic agent of chronic respiratory disease (CRD) in poultry, leads to prolonged recruitment and activation of inflammatory cells in the respiratory mucosa. This is consistent with the current model of immune dysregulation that ostensibly allows the organism to evade clearance mechanisms and establish chronic infection. To date, studies using quantitative reverse transcription-PCR (qRT-PCR) and microarrays have shown a significant transient upregulation of cytokines and chemokines from tracheal epithelial cells (TECs) and tracheal tissue in response to virulent strain R that contributes to the infiltration of inflammatory cells into the tracheal mucosa. To expand upon these experiments, RNA was isolated from tracheas of 20 chickens infected with R and 20 mock-infected animals at days 1, 3, 5, and 7 postinoculation, and samples were analyzed for differential gene expression using Illumina RNA sequencing. A rapid host response was observed 24 h postinfection, with over 2,500 significantly differentially expressed genes on day 3, the peak of infection. Many of these genes have immune-related functions involved in signaling pathways, including Toll-like receptor (TLR), mitogen-activated protein kinase, Jak-STAT, and the nucleotide oligomerization domain-like receptor pathways. Of interest was the increased expression of numerous cell surface receptors, including TLR4 and TLR15, which may contribute to the production of cytokines. Metabolic pathways were also activated on days 1 and 3 postinfection, ostensibly due to epithelial cell distress that occurs upon infection. Early perturbations in tissue-wide gene expression, as observed here, may underpin a profound immune dysregulation, setting the stage for disease manifestations characteristic of infection.

Many lipoproteins are expressed on the surfaces of mycoplasmas, and some have been implicated as playing roles in pathogenesis. Family 2 lipoproteins of Mycoplasma pneumoniae have a conserved "mycoplasma lipoprotein X" central domain and a "mycoplasma lipoprotein 10" C-terminal domain and are differentially expressed in response to environmental conditions. Homologues of family 2 lipoproteins are Mycoplasma specific and include the lipoprotein of Mycoplasma gallisepticum, encoded by the MGA0674 gene. Comparative transcriptomic analysis of the M. gallisepticum live attenuated vaccine strain F and the virulent strain R low , reported in this study, indicated that MGA0674 is one of several differentially expressed genes. The MGA0674-encoded lipoprotein is a proteolytically processed, immunogenic, TX-114 detergent-phase protein which appears to have antigenic divergence between field strains R low and S6. We examined the virulence of an R low ⌬MGA0674 mutant (P1H9) in vivo and observed reduced recovery and attenuated virulence in the tracheas of experimentally infected chickens. The virulence of two additional R low ⌬MGA0674 mutants, 2162 and 2204, was assessed in a second in vivo virulence experiment. These mutants exhibited partial to complete attenuation in vivo, but recovery was observed more frequently. Since only Mycoplasma species harbor homologues of MGA0674, the gene product has been renamed "Mycoplasma-specific lipoprotein A" (MslA). Collectively, these data indicate that MslA is an immunogenic lipoprotein exhibiting reduced expression in an attenuated strain and plays a role in M. gallisepticum virulence.Mycoplasma gallisepticum is a respiratory and reproductive tract pathogen of poultry, and disease results in major economic losses on commercial farms. M. gallisepticum can be transmitted via inhalation of aerosolized respiratory secretions and can also be spread vertically to the offspring of infected hens. Attachment of the bacterium to host respiratory epithelium results in inflammation, metaplasia, and loss of cilia. M. gallisepticum infection also places infected chickens at increased risk of developing a more severe, and potentially fatal, polymicrobial disease known as chronic respiratory disease (CRD) (18). Several live attenuated vaccines (LAV) have been generated to manage M. gallisepticum disease on chicken farms, but little is known about the genetic basis for their attenuation. We recently undertook an investigation of the genetic means by which the LAV strain F became avirulent by sequencing its complete genome and comparing it with the genome of the virulent strain R low . Deletions and mutations in many genes were identified as significantly altering the coding sequence and may have an effect on virulence. We showed that a transposon knockout of the "hypothetical" gene MGA1107 in R low (gene deleted in strain F) resulted in attenuation of the organism in vivo, indicating that this gene plays a role in the pathogenesis of M. gallisepticum (33).Lipoproteins (LPs) reside on the surfaces of th...

Background: Allergic asthma is a major cause of worldwide morbidity and results from inadequate immune regulation in response to innocuous, environmental antigens. The need exists to understand the mechanisms that promote nonreactivity to human-relevant allergens such as house dust mite (HDM) in order to develop curative therapies for asthma. The aim of our study was to compare the effects of short-, intermediate- and long-term HDM administration in a murine asthma model and determine the ability of long-term HDM exposure to suppress allergic inflammation. Methods: C57BL/6 mice were intranasally instilled with HDM for short-term (2 weeks), intermediate-term (5 weeks) and long-term (11 weeks) periods to induce allergic airway disease (AAD). The severity of AAD was compared across all stages of the model via both immunological and pulmonary parameters. Results: Short- and intermediate-term HDM exposure stimulated the development of AAD that included eosinophilia in the bronchoalveolar lavage fluid (BALF), pronounced airway hyperreactivity (AHR) and evidence of lung inflammation. Long-term HDM exposure promoted the suppression of AAD, with a loss of BALF eosinophilia and AHR despite persistent mononuclear inflammation in the lungs. Suppression of AAD with long-term HDM exposure was associated with an increase in both Foxp3+ regulatory T cells and IL-10-positive alveolar macrophages at the site of inflammation. Conclusions: This model recapitulates the key features of human asthma and may facilitate investigation into the mechanisms that promote immunological tolerance against clinically relevant aeroallergens.

Mycoplasma gallisepticum, a significant respiratory and reproductive pathogen of domestic poultry, has since 1994 been recognized as an emergent pathogen of the American house finch (Carpodacus mexicanus). Epizootic spread and pathognomonic characteristics of house finchassociated Mycoplasma gallisepticum (HFMG) have been studied as a model of an emergent to endemic pathogen in a novel host. Here we present comparative analysis of eight HFMG genomes, including one from an index isolate and seven isolates separated spatially and temporally (1994)(1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008) across the epizootic, and notably having differences in virulence. HFMG represented a monophyletic clade relative to sequenced poultry isolates, with genomic changes indicating a novel M. gallisepticum lineage and including unique deletions of coding sequence. Though most of the HFMG genome was highly conserved among isolates, genetic distances correlated with temporal-spatial distance from the index. The most dramatic genomic differences among HFMG involved phase-variable and immunodominant VlhA lipoprotein genes, including those variable in presence and genomic location. Other genomic differences included tandem copy number variation of a 5 kbp repeat, changes in and adjacent to the clustered regularly interspaced short palindromic repeats, and small-scale changes affecting coding potential and association of genes with virulence. Divergence of monophyletic isolates from similar time/space in the epizootic indicated local diversification of distinct HFMG sublineages. Overall, these data identify candidate virulence genes and reveal the importance of phase-variable lipoproteins during the evolution of M. gallisepticum during its emergence and dissemination in a novel host in nature, likely mediating an important role at the interface between pathogen virulence and host immunity.

Hydrogen peroxide (H 2 O 2 ) is a by-product of glycerol metabolism in mycoplasmas and has been shown to cause cytotoxicity for cocultured eukaryotic cells. There appears to be selective pressure for mycoplasmas to retain the genes needed for glycerol metabolism. This has generated interest and speculation as to their function during infection. However, the actual effects of glycerol metabolism and H 2 O 2 production on virulence in vivo have never been assessed in any Mycoplasma species. To this end, we determined that the wild-type (WT) R low strain of the avian pathogen Mycoplasma gallisepticum is capable of producing H 2 O 2 when grown in glycerol and is cytotoxic to eukaryotic cells in culture. Transposon mutants with mutations in the genes present in the glycerol transport and utilization pathway, namely, glpO, glpK, and glpF, were identified. All mutants assessed were incapable of producing H 2 O 2 and were not cytotoxic when grown in glycerol. We also determined that vaccine strains ts-11 and 6/85 produce little to no H 2 O 2 when grown in glycerol, while the naturally attenuated F strain does produce H 2 O 2 . Chickens were infected with one of two glpO mutants, a glpK mutant, R low , or growth medium, and tracheal mucosal thickness and lesion scores were assessed. Interestingly, all glp mutants were reproducibly virulent in the respiratory tracts of the chickens. Thus, there appears to be no link between glycerol metabolism/H 2 O 2 production/cytotoxicity and virulence for this Mycoplasma species in its natural host. However, it is possible that glycerol metabolism is required by M. gallisepticum in a niche that we have yet to study.

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. Mycoplasma pneumoniae is a chronic human pathogen and the etiological agent of many cases of bronchitis and community-acquired pneumonia. Infection with this bacterium can also cause and/or exacerbate other diseases including asthma, myocarditis, sickle cell disease, and encephalitis. Outbreaks are common at such institutions as military bases, schools, and hospitals, where individuals are in close contact for long periods of time, reflecting the community-acquired nature of infections. Diagnosis is difficult as most medical laboratories do not screen for this pathogen, since quick and inexpensive tests are not readily available. Common first-line ␤-lactam antibiotics are ineffective treatments since mycoplasmas lack a cell wall; consequently, this pathogen is often overlooked during diagnosis of affected individuals, and common treatments do not target the source of the disease. This results in considerable economic and societal hardships due to lost and ineffective work/school time, making this pathogen a burden to public health (1, 42).Several virulence determinants of M. pneumoniae have been previously identified and characterized. This bacterium is thought primarily to exploit an extracellular niche and requires a complex tip structure to attach to its host's mucosal epithelium. The tip structure is also involved in gliding motility and cell division. Densely clustered on the surface of the tip structure are the major cytadhesins P1 and P30 and cytadherence-accessory proteins B/C, which are supported by a framework of interdependent cytoskeletal proteins, including HMW1, HMW2, HMW3, P200, P65, P41, and P24 (reviewed in reference 2). Cytadhesin protein P30 has been shown to be critical for both attachment (11, 32) and gliding motility, although its requirements for each are distinguishable (18). Thus, mutant II-...