Steven J. Geary

P159 is a proteolytically processed, surface adhesin of Mycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryote cells

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine causing significant economic losses to swine production worldwide. Heparin is known to inhibit adherence of M. hyopneumoniae to porcine respiratory epithelial cilia. M. hyopneumoniae cells bind heparin but the identity of the heparin‐binding proteins is limited. Proteomic analysis of M. hyopneumoniae lysates identified 27 kDa (P27), 110 kDa (P110) and 52 kDa (P52) proteins representing different regions of a 159 kDa (P159) protein derived from mhp494. These cleavage fragments were surface located and present at all growth stages. Following purification of four recombinant proteins spanning P159 (F1P159, F2P159, F3P159 and F4P159), only F3P159 and F4P159 bound heparin in a dose‐dependent manner (Kd values 142.37 ± 22.01 nM; 75.37 ± 7.34 nM respectively). Scanning electron microscopic studies showed M. hyopneumoniae bound intimately to porcine kidney epithelial‐like cells (PK15 cells) but these processes were inhibited by excess heparin and F4P159. Similarly, latex beads coated with F2P159 and F4P159 adhered to and entered PK15 cells, but heparin, F2P159 and F4P159 was inhibitory. These findings indicate that P159 is a post‐translationally cleaved, glycosaminoglycan‐binding adhesin of M. hyopneumoniae.

Molecular and biochemical analysis of a 105 kDa Mycoplasma gallisepticum cytadhesin (GapA).

The identification of a gene (gapA) from Mycoplasma gallisepticum with homology to the P1 cytadherence gene of Mycoplasma pneumoniae is reported. The gapA gene is a 2895 bp ORF encoding a protein with a molecular mass of 105 kDa. Nucleotide sequence analysis of the gapA gene revealed 45% homology to the M. pneumoniae P1 gene, 46% homology to the Mycoplasma genitalium MgPa gene and 47% homology to the Mycoplasma pirum P1-like protein gene. It has a 64 mol % A+T content compared to 46, 60 and 72 mol % respectively for the P1, MgPa and the P1-like protein genes. As with the P1 and MgPa genes, gapA is a central gene in a multi-gene operon, but unlike the P1 and MgPa genes, there is only a single copy of gapA in the genome. GapA is a trypsin-sensitive surface-exposed protein. Chicken tracheal-ring inhibition-of-attachment assays, using anti-GapA Fab fragments, resulted in 64% inhibition of attachment. These results indicated that GapA plays a role in cytadherence of M. gallisepticum to host cells.

GapA and CrmA Coexpression Is Essential for Mycoplasma gallisepticum Cytadherence and Virulence

ABSTRACT It was previously demonstrated that avirulent Mycoplasma gallisepticum strain Rhigh (passage 164) is lacking three proteins that are expressed in its virulent progenitor, strain Rlow (passage 15). These proteins were identified as the cytadhesin molecule GapA, the putative cytadhesin-related molecule CrmA, and a component of a high-affinity transporter system, HatA. Complementation of Rhigh with wild-type gapA restored expression in the transformant (GT5) but did not restore the cytadherence phenotype and maintained avirulence in chickens. These results suggested that CrmA might play an essential role in the M. gallisepticum cytadherence process. CrmA is encoded by the second gene in the gapA operon and shares significant sequence homology to the ORF6 gene of Mycoplasma pneumoniae, which has been shown to play an accessory role in the cytadherence process. Complementation of Rhigh with wild-type crmA resulted in the transformant (SDCA) that lacked the cytadherence and virulence phenotype comparable to that found in Rhigh and GT5. In contrast, complementation of Rhigh with the entire wild-type gapA operon resulted in the transformant (GCA1) that restored cytadherence to the level found in wild-type Rlow. In vivo pathogenesis trials revealed that GCA1 had regained virulence, causing airsacculitis in chickens. These results demonstrate that both GapA and CrmA are required for M. gallisepticum cytadherence and pathogenesis.

Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase (lpd), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants

ABSTRACT To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.

Rapid Preclinical Detection of Sheeppox Virus by a Real-Time PCR Assay

ABSTRACT Sheeppox virus (SPPV) is a member of the Capripoxvirus (CaPV) genus of the Poxviridae family. Members of this genus, which also include goatpox and lumpy skin disease viruses, cause economically significant disease in sheep, goats, and cattle. A rapid diagnostic assay for CaPV would be useful for disease surveillance as well as for detection of CaPV in clinical samples and for outbreak management. Here we describe a fluorogenic probe hydrolysis (TaqMan) PCR assay designed for rapid detection of CaPV and tested on sheep experimentally infected with a virulent strain of SPPV. This assay can detect SPPV in buffy coats, nasal swabs, oral swabs, scabs, and skin lesions as well as in lung and lymph nodes collected at necropsy. This single-tube diagnostic assay can be performed in 2 h or less and can detect viral DNA in preclinical, clinical, and postmortem samples.

Analysis of Cytadherence-Deficient, GapA-Negative Mycoplasma gallisepticum Strain R

ABSTRACT Comparison of the phenotypic expression of Mycoplasma gallisepticum strain R low (passage 15) to that of strain R high (passage 164) revealed that three proteins, i.e., the cytadhesin molecule GapA, a 116-kDa protein (p116), and a 45-kDa protein (p45), are missing in strain R high. Sequence analysis confirmed that the insertion of an adenine 105 bp downstream of the gapAtranslational start codon resulted in premature termination of translation in R high. A second adenine insertion had also occurred at position 907. Restoration of expression of wild-type gapAin R high (clone designated GT5) allowed us to evaluate the extent to which the diminished cytadherence capacity could be attributed to GapA alone. The results indicated that GT5 attached to the same limited extent as the parental R high, from which it was derived. The cytadherence capability of the parental R high was not restored solely by gapA complementation alone, indicating that either p116 or p45 or both may play a role in the overall cytadherence process. The gene encoding p116 was found to be immediately downstream ofgapA in the same operon and was designatedcrmA. This gene exhibited striking homology to genes encoding molecules with cytadhesin-related functions in bothMycoplasma pneumoniae and Mycoplasma genitalium. Transcriptional analysis revealed thatcrmA is not transcribed in R high. We are currently constructing a shuttle vector containing both the wild-typegapA and crmA for transformation into R high to assess the role of CrmA in the cytadherence process.

Identification of Fibronectin-Binding Proteins in Mycoplasma gallisepticum Strain R

ABSTRACT We have determined that virulent Mycoplasma gallisepticum strain Rlow is capable of binding the extracellular matrix protein fibronectin. Fibronectin was found to be present in M. gallisepticum Rlow protein extracts by Western blotting and peptide sequencing. Mycoplasma gallisepticum Rhigh, the attenuated, high-passage derivative of Rlow, is deficient in this ability. MGA_1199, the M. gallisepticum homologue of the cytadherence-associated protein P65 from Mycoplasma pneumoniae, and MGA_0928, the M. gallisepticum homologue of the M. pneumoniae cytoskeletal protein HMW3, were identified as fibronectin-binding proteins. Peptides from the regions of MGA_1199 and MGA_0928 exhibiting the highest degree of homology with known fibronectin-binding proteins were shown to bind the gelatin/heparin-binding domain of fibronectin. MGA_1199 and MGA_0928 were shown to be absent and aberrant, respectively, in Rhigh, explaining its lack of fibronectin-binding capability. Consistent with its M. pneumoniae counterpart, MGA_1199 (renamed PlpA) was demonstrated to be surface exposed, despite a lack of classical membrane-spanning domains. Due to its demonstrated topology and the strength of interaction between its binding peptide and fibronectin, we propose that PlpA functions as a fibronectin-binding protein in vivo and may possess atypical transmembrane domains.

Identification of Lipoprotein MslA as a Neoteric Virulence Factor of Mycoplasma gallisepticum

ABSTRACT 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 Rlow, 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 Rlow and S6. We examined the virulence of an Rlow ΔMGA0674 mutant (P1H9) in vivo and observed reduced recovery and attenuated virulence in the tracheas of experimentally infected chickens. The virulence of two additional Rlow ΔMGA0674 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.

Correlates of Immune Protection in Chickens Vaccinated with Mycoplasma gallisepticum Strain GT5 following Challenge with Pathogenic M. gallisepticum Strain Rlow

ABSTRACT Colonization of the avian respiratory tract with Mycoplasma gallisepticum results in a profound inflammatory response in the trachea, air sacs, conjunctiva, and lungs. A live attenuated M. gallisepticum vaccine strain, GT5, was previously shown to be protective in chickens upon challenge; however, the mechanisms by which this vaccine and others confer protection remain largely unknown. The current study evaluated several potential correlates of GT5 vaccine-mediated immune protection following challenge with the pathogenic M. gallisepticum strain Rlow. GT5-vaccinated chickens developed mild tracheal lesions, consisting of few and scattered, discrete, lymphofollicular aggregates in the lamina propria. In addition, low numbers of aggregated B, CD4+, and CD8+ cells were observed to infiltrate the trachea, in stark contrast to the large numbers infiltrating the tracheas of sham-vaccinated chickens challenged with Rlow. Lymphofollicular aggregates were rarely observed prior to day 12 postchallenge in sham-vaccinated chickens. Instead, they contained an increasingly more cellular inflammatory response characterized by expansion of the lamina propria by lymphoplasmacytic and histiocytic infiltrates. This was due in part to expansion of interfollicular zones by large numbers of infiltrating CD4+ and CD8+ cells and a sizeable population of immunoglobulin A (IgA)- and IgG-secreting plasma cells. GT5-vaccinated chickens also had higher serum IgG concentrations, and significantly higher numbers of M. gallisepticum-specific IgG- and IgA-secreting plasma/B cells within the trachea, than did sham-vaccinated chickens. These responses were observed as early as day 4 postchallenge, indicating the importance of antibody-mediated clearance of mycoplasma in GT5-vaccinated chickens.

Comparative Genomic Analyses of Attenuated Strains of Mycoplasma gallisepticum

ABSTRACT 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 (Rlow) 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 Rlow, Rhigh, 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 Rlow isogenic mutant in one of these genes (MGA_1107) caused significantly fewer severe tracheal lesions in the natural host compared to virulent M. gallisepticum Rlow. 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.