Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase (lpd), in Mycoplasma gallisepticum through In Vivo Scre
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感染与免疫杂志 2006年第2期
Center of Excellence for Vaccine Research Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, Connecticut 06269
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.
INTRODUCTION
Mycoplasma gallisepticum is the primary etiologic agent of the chronic respiratory disease complex in chickens and infectious sinusitis in turkeys. Primary inflammatory responses in the respiratory tract associated with infection are sinusitis, tracheitis, bronchitis, and airsacculitis. Colonization of the respiratory tract leads to ciliostasis and deciliation of the tracheal epithelium, allowing secondary infection by other bacterial and viral pathogens, such as Newcastle disease virus, infectious bronchitis virus, and Escherichia coli (24). Common signs of M. gallisepticum infection include nasal discharge, tracheal rales, weight loss, and decreased egg production. Mycoplasma gallisepticum is highly contagious in commercial chicken and turkey flocks, spreading horizontally in populations through aerosol, dust, and feathers and vertically transmitted through eggs (8, 10, 24, 40). Its economic impact on the poultry industry is significant, leading to millions of dollars of loss each year. Efforts to improve disease prevention and control programs require increased research to identify novel virulence-associated determinants. These determinants may provide the basis for new and promising vaccines or targets for other antimicrobial therapies (41).
In 1995, a global virulence determinant identification technique called signature-tagged mutagenesis was developed in the laboratory of David Holden. Using a pool of uniquely tagged transposons for random mutagenesis, Hensel et al. (17) were able to screen a large mutant population for organisms demonstrating attenuated virulence through negative selection in vivo. This approach allows a comprehensive screening of bacterial genomes for the identification of genes encoding novel virulence determinants. We developed a modification of this technique, termed signature sequence mutagenesis (SSM), and utilized it for the discovery of novel virulence-associated genes in M. gallisepticum. The genome of virulent M. gallisepticum strain R has recently been sequenced and annotated (33) and is the cornerstone of this SSM technique. Signature sequence mutagenesis avoids one of the primary problems of signature-tagged mutagenesis, i.e., the issue of cross-hybridizing transposon tags (19). The Southern hybridization step is replaced with primer sets consisting of a common forward primer annealing at the end of the transposon and a mutant-specific reverse primer annealing in the genomic sequence flanking the transposon. SSM allows the screening of multiple mutants in one animal challenge experiment, thus minimizing the number of animals used. Here, we describe the application of SSM to identify virulence-associated determinants in M. gallisepticum and specifically characterize the dihydrolipoamide dehydrogenase gene (lpd) as a virulence-associated gene.
MATERIALS AND METHODS
Organisms and culture conditions. M. gallisepticum strain R low (Rlow) (passage 3 after in vivo reisolation) was cultured at 37°C in Hayflick's medium (10% horse serum and 5% yeast extract) (8) or Hayflick's agar with 1% Noble agar (Difco, Franklin Lakes, NJ). Gentamicin sulfate (150 μg/ml in broth and 100 μg/ml in agar) was added to the media for the propagation of the Tn4001mod mutants. E. coli DH5 carrying pISM2062 (25) was propagated in Luria broth and on plates (10 g tryptone, 5 g yeast extract, and 5 g of NaCl per liter) with ampicillin (100 μg/ml).
Nucleic acid extraction. The genomic DNA of each Tn4001mod mutant was extracted from 30 ml of mid-logarithmic-phase culture using the Easy-DNA extraction kit (Invitrogen, Carlsbad, Calif.). DNA concentrations were determined using spectrophotometric-absorbance measurements at 260 nm; purities were verified by absorbance ratios at 260 nm and 280 nm. Plasmid DNA was extracted using the QIAGEN Plasmid Mini kit protocol following the manufacturer's protocol (QIAGEN Inc., Santa Clarita, Calif.).
M. gallisepticum Rlow transformation. M. gallisepticum Rlow was transformed by electroporation (Bio-Rad Gene Pulser, Hercules, Calif.) with the suicide plasmid pISM2062 (a gift of F. Chris Minion, Iowa State University). This plasmid carries the gram-positive transposon Tn4001mod, which contains the gentamicin resistance gene (5). M. gallisepticum cultures were grown to mid-logarithmic phase at 37°C in 5 ml of Hayflick's broth. The cells were harvested by centrifugation at 12,000 x g for 10 min at room temperature and then resuspended in 1 ml of electroporation buffer (8 mM HEPES, 272 mM sucrose, pH 7.4). After an additional centrifugation at 12,000 x g for 10 min, 60 μl electroporation buffer and 10 μg pISM2062 plasmid DNA were added, and the mixture was incubated on ice for 15 min. The cell-DNA suspension was transferred to a chilled 0.2-cm-gap electroporation cuvette and immediately pulsed (2.5 kV; 25 μF; 100 ). One milliliter of cold Hayflick's broth was added, and the mixture was incubated at room temperature for 10 min; the cells were then incubated at 37°C for 2 to 3 h (6, 12, 14). Fifty microliters of the transformation mixture was diluted 1:10, and then 200 μl of that dilution was spread onto Hayflick's agar plates with gentamicin (100 μg/ml). The plates were incubated at 37°C for 7 to 10 days. Single visible colonies were picked and then placed in 500 μl Hayflick's broth with gentamicin and grown to mid-logarithmic phase. These M. gallisepticum transformants were titered and then stored at –80°C as stock cultures.
Transposon insertion site identification. The Tn4001mod M. gallisepticum mutant genomic DNA was extracted and sequenced using the outward primer SG857, annealing 27 bp upstream from the end of the transposon. The primer was designed to utilize the unique BamHI-SmaI cut site located in one of the two insertion sequences (IS256) of the transposon.
Linear sequencing with BigDye Terminator Mix (PE-Applied Biosystems) was used to identify the site of transposon insertion for each Tn4001mod mutant. Each sequencing reaction mixture contained 16 μl BigDye Terminator Mix, 30 pmol of primer, and 2 to 3 μg genomic DNA in a total volume of 32 μl. The following sequencing conditions were used: initial denaturation at 95°C for 5 min, followed by 45 cycles at 95°C for 30 s, 55°C for 20 s, and 60°C for 4 min (15). Excess dye terminators were removed with AutoSeq G-50 spin columns (Amersham Biosciences, Piscataway, NJ), and the samples were vacuum dried. Sequencing was performed by the University of Connecticut Biotechnology Center. Using Artemis v. 5 (Sanger Centre, United Kingdom) and Vector NTI 8 (Invitrogen, Carlsbad, Calif.), the genomic-sequencing results from the mutants were compared to the sequenced and annotated genome of M. gallisepticum strain R (33).
Determining transposon insertion stability. Sterile 1.5-ml microcentrifuge tubes containing 0.5 ml Hayflick's broth were inoculated with 30 μl of Tn4001mod mutant stock and incubated at 37°C until growth was evident. These mutant cultures were passed 30 times in nonselective broth, and on every 10th passage, 30 μl of the culture was transferred and grown in 30 ml of Hayflick's broth with 150 μg/ml gentamicin for genomic-DNA extraction and transposon insertion site sequencing as described above. The resultant sequence was compared to the known insertion site sequence, and any transposon movement was documented. To determine the rate of transposon loss, 30 μl of each mutant was used to inoculate 0.5 ml of nonselective broth, and every day for 14 days, the cultures were passed once. After the 14th passage, the culture was serially diluted and duplicate plated on nonselective Hayflick's plates and those containing 100 μg/ml gentamicin. After incubation at 37°C, the CFU were quantified.
Growth curve analysis of the Tn4001mod mutants. Sterile 250-ml Erlenmeyer flasks containing 69 ml of Hayflick's broth were inoculated simultaneously with 1 ml of equal numbers of pretitered (based on previous optical-density values at 620 nm [OD620]) M. gallisepticum Rlow and each of the individual mutant stock cultures. The cells were incubated at 37°C in a 250-rpm orbital shaking incubator and were harvested at 2, 4, 6, 8, 10, 12, 14, 16, 28, 32, 40, 49, and 54 hours postinoculation. The growth was measured spectrophotometrically by assessing the OD620. All values were graphed semilogarithmically as OD620 values versus time. All growth curve analyses were performed in duplicate.
Design of the unique mutant identifier. The identification of the transposon insertion sites within the M. gallisepticum genome permitted the design of unique reverse primers for each Tn4001mod mutant. These reverse primers were used with the forward primer SG857 and each respective genomic DNA template to produce an amplicon of known size. Each amplicon ranged in size from 300 to 650 bp and represented a unique mutant identifier that distinguished one clone from another.
Animals. Female 4-week-old White Leghorn specific-pathogen-free chickens (SPAFAS, North Franklin, CT) were used in the challenge experiments. The birds were tagged upon arrival and placed in HEPA-filtered isolators (Controlled Isolator Systems, Pittsburgh, PA). After a 1-week period of acclimation (in accordance with approved Institutional Animal Care and Use Committees protocol), all challenge experiments were initiated.
In vivo virulence assay. Three chickens were used in the mutant pool assay. The chickens were challenged with a mixed population of 27 Rlow Tn4001mod mutants (input population) at 1.5 x 106 CFU of each unique transformant. One milliliter of pretitered stock aliquots of each mutant was treated as follows: 100 μl of each mutant culture was transferred to a common 15-ml plug-sealed tube, and the resultant mixed culture was pelleted by centrifugation at 12,000 x g and 4°C for 10 min. The remaining 900 μl of culture in each aliquot was centrifuged as stated above, decanted, and stored at –80°C as a template to produce an input population PCR profile for comparison to the output population PCR profile generated after mutant recovery. The pellet of the mixed culture was then thoroughly resuspended in 100 μl of Hayflick's medium, transferred to a 1.5-ml microcentrifuge tube, and placed on ice in preparation for the challenge. The above procedure was repeated for each chicken in the study. The chickens were infected by intratracheal inoculation using a P200 pipetter carrying 100-μl suspensions of the organism.
Two weeks postchallenge, the chickens were humanely killed by cervical dislocation and immediately necropsied, and samples of the tracheas and air sacs were removed for isolation of M. gallisepticum Tn4001mod mutants. Tissue samples and tracheal swabs were used to inoculate 30 ml of Hayflick's medium containing 150 μg/ml gentamicin, vortexed, and incubated at 37°C for 8 h. The cultures were then passed through 0.45-μm filters and transferred to new plug-sealed tubes, and incubation was continued. Cultures that were acidic (yellow) after being filtered were adjusted to pH 7.4 by the addition of 10 N NaOH, and incubation was continued at 37°C until growth was evident. The genomic DNA of each culture was extracted, and PCR was performed to determine the presence or absence of individual mutants in the output population by using primer sets containing a conserved forward primer (SG857) and a reverse primer specific for each transposon mutant (Table 2). Amplicons were visualized in an ethidium bromide-stained 0.8% agarose gel. The PCR profile of this output population was compared to the input population profile, and those mutants not present in the output population were considered potential virulence mutants.
Assessment of potential virulence mutants. Sixteen chickens were used in this assay. The chickens were divided into three groups, with two consisting of six chickens each (groups 1 and 2) and the third group consisting of four chickens (group 3). Group 1 chickens were challenged with 1.5 x 107 CFU of Rlow. The chickens in group 2 received 1.5 x 107 CFU of the individual mutant, and group 3 was given 1x phosphate-buffered saline (PBS) as a negative control. All groups were challenged on day 0 and day 2.
Two weeks postchallenge, the chickens were humanely killed by cervical dislocation and immediately necropsied, and samples of tracheas and air sacs were taken for M. gallisepticum recovery, if present. Tissue samples from groups 1 and 3 were placed in Hayflick's medium, while those from group 2 were grown in gentamicin selective medium. Using sterile instruments, samples of left and right thoracic and abdominal air sacs were removed and either placed in specific culture media (as detailed above) or fixed in 10% neutral buffered formalin for histopathological evaluation. The tracheas were divided into cranial, middle, and caudal thirds; cross-sections 3 to 5 mm in width were fixed in 10% neutral buffered formalin for histopathologic evaluation; and the remaining segments were placed in specific culture media. Formalin-fixed tissue samples were routinely processed, embedded in paraffin blocks, sectioned at 4 μm, and stained with hematoxylin and eosin according to standard histological protocols (39). Histopathological evaluations were performed in a blinded fashion and were based on criteria established by Nunoya et al. (31) and implemented in previous histopathologic studies of M. gallisepticum lesions in the trachea (34) and air sac (32). Lesion scores (LS) were assigned separately to trachea and air sacs.
The LS table adapted to this study to grade tracheal lesions was defined as follows: 0, no significant findings; 0.5, minimal multifocal lymphocytic or lymphofollicular infiltrates amounting to one to three discrete foci; 1, mild mucosal thickening resulting from either mild diffuse lymphocytic infiltrates or multifocal lymphocytic or lymphofollicular infiltrates amounting to four or more discrete foci without edema or heterophils; 2, moderate mucosal thickening resulting from multifocal to diffuse lymphocytic and histiocytic infiltrates with or without lymphofollicular infiltrates, intraepithelial and lamina proprial infiltrates of heterophils, and luminal exudates; and 3, severe mucosal thickening resulting from diffuse infiltrates of lymphocytes, histiocytes, and heterophils with flattening and attenuation of epithelium and luminal exudates. Lesions that had features intermediate in intensity between two scoring values were assigned midrange scores, e.g., 1.5 or 2.5.
The LS table adapted for scoring air sacs was defined as follows: 0, no significant findings; 0.5, minimal multifocal lymphocytic infiltrates with one to three discrete, small, widely separated foci; 1, mild stromal thickening due to either multifocal lymphocytic or lymphofollicular infiltrates amounting to four or more discrete foci or mild diffuse lymphocytic infiltrates without edema or heterophils; 1.5, lesions comparable to 1 with mild heterophilic infiltrates; 2, moderate stromal thickening resulting from multifocal to diffuse lymphocytic, histiocytic, and heterophilic infiltrates that included lymphofollicular aggregates and intraepithelial heterophils; 2.5, lesions comparable to 2 plus significant luminal heterophilic exudates; 3, severe stromal thickening resulting from multifocal to diffuse lymphocytic, histiocytic, and heterophilic infiltrates that might include epithelial attenuation or hyperplasia and luminal heterophilic or granulomatous exudates. In addition to qualitative scoring as described above, the tracheal mucosal width was measured using an ocular micrometer. The widths of the mucosa at four equidistant points along the circumference of each tracheal section were measured using the base of the cilia or the adluminal surface of deciliated or flattened epithelial cells as one boundary and the beginning of the collagenous stroma of the lamina propria-tunica submucosa as the opposite boundary. An average mucosal width was generated for each trachea from measurements of each tracheal section, and average mucosal widths were used in subsequent statistical analyses to determine the relevance of differences in mucosal thickness between inoculated groups.
The genomic DNA of each recovered mutant culture was extracted, and PCR was performed using a primer set containing the conserved forward primer (SG857) and a mutant-specific reverse primer to confirm the identities. Amplicons were visualized by UV transillumination of an ethidium bromide-stained 0.8% agarose gel. If the potential virulence mutant was not recovered at the same rate as wild-type Rlow and demonstrated reduced histopathology compared to wild-type lesion scores, then it was considered to have a virulence deficiency.
Homogenate preparation. Mycoplasma cells were suspended in 1 ml of 40% glycerol, 60% Tris hydrochloride buffer (pH 7.4) (9). The suspension was diluted to 1 mg of protein per ml with Tris hydrocholoride buffer (pH 7.4) containing 1 mM EDTA and 1 mM 2-mercaptoethanol. Three cycles of freeze-thaw and passage through a syringe with an 18-gauge needle were used to homogenize the samples. Total protein was determined by the Bradford assay (2).
Pyruvate dehydrogenase complex activity assay. The pyruvate dehydrogenase complex (PDHC) activity was assayed by a radiochemical method using [1-14C] pyruvic acid-sodium salt (Amersham, Piscataway, NJ) as a substrate. Mycoplasma homogenates containing 100 μg of protein were placed in disposable test tubes in an ice bath and brought to 200 μl with 0.01 M Tris (pH 7.4) containing 1 mM EDTA and 1 mM 2-mercaptoethanol. A 10-μl solution containing coenzyme A (CoA), NAD+, and thiamine pyrophosphate (2 mM each) in Tris buffer was added to each test tube and then mixed. Finally, 20 nmol 1-14C-pyruvic acid was added and mixed. The negative control preparation was immediately inactivated with 15 μl of 50% trichloroacetic acid. The test tubes were stoppered with 1.5-ml microcentrifuge tubes with their bottoms cut off containing paper wicks soaked with 50 μl of 2.5 M NaOH to absorb the evolving CO2. The reaction mixtures were incubated at 37°C and stopped at 15-, 30-, 45-, and 60-min intervals by the addition of 15 μl of 50% trichloroacetic acid. Each time point assessment was performed in duplicate. The tubes were again stoppered and incubated at 37°C in an orbital shaking incubator at 200 rpm for 2 h to collect all evolved CO2. Radioactivity was measured by counting the contents of each tube in 5 ml ScintiSafe (Fisher Chemicals, Fairlawn, NJ) liquid scintillation cocktail in a Beckman LS 3801 scintillation counter (Beckman Coulter, Fullerton, CA). The activity of PDHC was determined by the average decrease of radiolabeled pyruvic acid in the test tube due to decarboxylation, creating 14CO2 that was released from the reaction mixture and collected in the wicks (9).
Statistical analysis. Analysis of variance using the f test was used to determine if there were significant differences between the mean lesion scores of chicken groups. When differences were found, a mean separation analysis using Duncan's multiple-range test was performed. The PDHC activity was analyzed by the paired Student t test to determine significant differences between enzyme activities. Analyses were performed with the statistics program SAS version 8.01 (SAS Institute, Cary, NC).
Sequence analysis. TMHMM (26), PSIPRED (22, 30), MEMSAT2 (23), and PredictProtein (37) were used to predict protein topology. Sequence homology searches were performed using the PROSITE (11), Pfam (3), PRINTS (1), and BLOCKS (16) databases. Amino acid sequence homologies were considered significant if they possessed 30% identity or similarity.
RESULTS
Identification of Tn4001mod mutants in mixed culture. Insertion sites were determined by sequencing using the outward primer SG857 (Table 2). The precise location of Tn4001mod in each of the transformants was identified by comparison with the annotated M. gallisepticum strain R (Table 1). In addition to identifying the transposition site, disruption of function of a coding DNA sequence (CDS) by transposon insertion was determined using principles previously described by Hutchison et al. (21). A CDS was considered disrupted if Tn4001mod inserted downstream of nucleotide 9 and within the first 80% of the 5'-most end of the gene. Based on these criteria, a CDS disruption is denoted in Table 1 as "yes" or "no." Unique reverse primers were designed based on genomic sequence flanking the transposon insertion site of each transformant; they are used for the PCR amplification of a unique mutant identifier (Table 2). Reverse primers were synthesized for each Tn4001mod mutant and paired with the outward primer SG857 in PCR. The resultant PCR profile (Fig. 1, top) generated amplicons at the expected sizes for all 27 transformants, thus confirming the specificity of this assay for the identification of individual transposon mutants in a mixed culture.
In vivo virulence assay. Based on the total output population PCR profiles from the tissues tested in three chickens, transformant Mg 7 was not recovered from the in vivo virulence assay, whereas the remaining 26 mutants were recovered at least once from other sample sites (Table 3). Since Mg 7 was not found in any of the assayed chicken tissues, it was classified as a potential virulence mutant. Figure 1, bottom, illustrates one output population PCR profile.
Transposon insertion stability. After 30 passages in nonselective media, the Mg 7 insertion was found to be stably maintained. The rate of transposon loss in the Mg 7 population was negligible.
Growth curve analysis. Mg 7 did not demonstrate any growth deficiency compared to wild-type Rlow over 54 hours of growth.
Potential virulence mutant assay. Histopathologic evaluation of tracheas from chickens challenged with Mg 7 showed a range of responses, from no significant lesions to mild diffuse lymphocytic infiltrates (LS = 0 to 1) without attenuation or deciliation of the epithelium (Fig. 2A). Chickens challenged with Rlow demonstrated minimal to moderate mucosal thickening (LS = 0.5 to 2) resulting from multifocal to diffuse lymphocytic and histiocytic infiltrates accompanied by heterophils and flattening and attenuation of the epithelium (Fig. 2B). Chickens in the PBS control group had no significant lesions (Fig. 2C). Examination of the tracheal mucosal width revealed a significant difference between the Rlow (60.4 μm)-challenged birds and the Mg 7 (40.6 μm) and PBS (44.6 μm) challenge groups.
Regarding the air sacs, birds challenged with Mg 7 had minimal to mild multifocal lymphofollicular infiltrates (LS = 0 to 1) amounting to minimal airsacculitis (Fig. 3A). Histopathological evaluation of birds challenged with Rlow revealed mild to moderate thickening of the air sacs resulting from mild to moderate lymphofollicular infiltration accompanied in some cases by luminal heterophilic exudates (LS = 1 to 2.5) (Fig. 3B). Chickens in the PBS control group had no significant lesions (Fig. 3C). Overall, in comparison to chickens from the Rlow challenge group, Mg 7 challenges demonstrated a marked reduction in lymphohistiocytic infiltrates, air sac exudates, and heterophil recruitment.
No significant differences were found between chickens challenged with Mg 7 and those challenged with PBS in either the trachea or air sac. Rlow-challenged birds exhibited a significant difference in air sac lesion scores compared to either Mg 7 or PBS challenges (Table 4). Birds in the Rlow challenge group developed airsacculitis and had extensive luminal exudates. Rlow was recovered from 20 out of 24 (83%) tissues sampled, while Mg 7 was recovered from 3 (12.5%) of the 24 cultures from the tissues sampled in this study. An Mg 7 isolate was recovered from the air sac of one bird, the trachea of another, and the lungs of the third. The presence of Mg 7 in the recovered cultures was confirmed by PCR. None of the challenge groups exhibited significantly different tracheal-lesion scores (P > 0.05).
Mg 7 characteristics and sequence analysis. Tn4001mod mutant Mg 7 has a transposon insertion in the dihydrolipoamide dehydrogenase CDS (lpd). The Tn4001mod insertion site is between amino acids 222 and 223, preventing the expression of over 50% of the carboxyl-terminal portion of the 467-amino-acid protein (Fig. 4). Structural-homology searches of Lpd in other sequenced mycoplasma and nonmycoplasma species, such as Mycoplasma pneumoniae, Mycoplasma hyopneumoniae, Mycoplasma genitalium, Mycoplasma penetrans, Mycoplasma pulmonis, Pseudomonas fluorescens, Azotobacter vinelandii, and Neisseria meningitidis (4, 7, 13, 20, 27, 28, 29, 38), revealed that this disulfide oxidoreductase has a redox-active site and a NAD/FAD binding domain within the intracellular amino-terminal portion of the polypeptide sequence. It also has a highly conserved dimerization domain located in its intracellular carboxyl-terminal portion. This dimerization domain has been extensively characterized in P. fluorescens, A. vinelandii, and N. meningitidis; the conformational stability of this enzyme is maintained only while dimerized to another Lpd molecule (4). Through the further proteomic analysis of the other two components of the PDHC, it is clear that dimerization of this enzyme is also important to the overall integrity of the PDHC, since it interacts with the moieties of the complex only in that polypeptide region. Protein topology analysis identified two putative transmembrane domains flanking the transposon insertion site in the extracellular domain (Fig. 4).
Pyruvate dehydrogenase complex activity assay. The first reaction in the PDHC is the decarboxylation of 1 mole of pyruvate, producing 1 mole of carbon dioxide. As the decarboxylation proceeds, radiolabeled CO2 is produced from the 14C-pyruvic acid and released from the reaction mixture. After 15 minutes, both Rlow and Mg 7 demonstrated similar rates of catabolic activity. The PDHC activity reached a plateau in the Mg 7 mutant at the 30-minute time point and was significantly different from Rlow catabolic activity at time points 45 and 60 (Fig. 5).
DISCUSSION
The definition of what constitutes a virulence-associated determinant is not limited to toxins and cytadhesins, but also includes proteases, regulatory proteins, stress response proteins, transport proteins, and proteins involved in metabolism. A virulence-associated determinant is therefore defined as any factor that confers a selective advantage on the pathogen, allowing it to colonize the host, persist, propagate, and cause disease.
The SSM assay allows the comprehensive screening of the genome for CDSs that may play roles in pathogenicity. Due to the random insertion pattern of transposon mutagenesis, it is likely that there will be disruptions of genes involved in pathogenesis. At least 30% of the CDSs in the M. gallisepticum genome sequence are annotated to code for polypeptides of unknown function. Emphasis has focused on the characterization of cytadhesins, such as GapA and CrmA (32, 34, 35), and hemagglutinins, such as the VlhA family of lipoproteins (33), but CDSs of unknown function must be thoroughly investigated as to their potential roles in pathogenicity. The ability to screen a large number of transposon mutants in one challenge experiment greatly accelerates the identification of novel virulence determinants among putative CDSs.
This study has described the identification of Lpd as an important protein in host colonization and pathogenesis. Histopathologic findings demonstrated differences in the severity of lesions caused by Mg 7 compared to Rlow, which were most clearly illustrated by the differences in the lesion scores of air sacs. The lesion scores of air sacs from Mg 7-challenged birds were significantly lower than those of Rlow-challenged chickens and lacked evidence of intense chronic inflammatory responses, such as the presence of lymphofollicular aggregates and epithelial attenuation.
Traditionally, the Lpd protein would be considered a metabolic factor, and therefore, its role in pathogenicity might never have been investigated. It is also important to note that the transposon insertion site in Mg 7 was stable when assayed, and therefore, no secondary transposon-induced mutations occurred during the chicken challenge. The in vitro growth rate of Mg 7 resembled that of wild-type M. gallisepticum, but the lack of the complete enzyme complex greatly affected its ability to colonize and survive within the host.
The Lpd protein is an essential functional subunit of the multienzyme PDHC, with a predicted molecular mass of 51 kDa. The PDHC is normally comprised of multiple copies of the ternary complex of Lpd, dihydrolipoamide transacetylase, and pyruvate dehydrogenase enzymes and is involved in the oxidation of pyruvate, the end product of glycolysis, to acetyl-CoA by the following reaction: pyruvate + CoA + NAD+ acetyl-CoA + CO2 + NADH + H.
The mutation in lpd interrupts the cyclical nature of the enzyme complex, rendering it inoperative after the reaction occurs once. The dihydrolipoamide dehydrogenase enzyme permits the continued function of the pathway by maintaining the correct redox state of the enzymes involved in the bioconversion. The PDHC activity assay confirmed that the protein homogenate from Mg 7 containing the incomplete complex failed to maintain activity, whereas the Rlow homogenate functioned properly.
Acetate is one of the end products of the fermentative metabolism carried out by M. gallisepticum. Through the combined actions of phosphate acetyltransferase and acetate kinase, 1 mole of ATP is generated during the conversion of acetyl-CoA to acetate (36). Without the complete PDHC, acetyl-CoA is not produced at wild-type levels, and the cell loses the ability to make a mole of ATP from the metabolic intermediate, leading to a possible intracellular energy shortage. The reduction in ATP could lower the cumulative activity of the ATP binding cassette transporters upon which the bacterium relies heavily to acquire many of the precursors necessary for its viability. While this feature is easily masked when Mg 7 is grown in the nutrient-rich Hayflick's medium, the deficiency may affect the microorganism's ability to acquire the precursor molecules and cofactors needed to persist and adapt to its host environment.
In a recent report, Haemophilus influenzae type B was shown to be attenuated by a transposon insertion in lpdA, the dihydrolipoamide dehydrogenase gene homolog. When the H. influenzae LpdA mutant was used to challenge 5-day-old infant rats intraperitoneally, it was unrecoverable compared to the wild-type recovery rate (18).
Despite screening of an additional 1,000 M. gallisepticum transposon mutants, a second clone with a mutation in lpd has not been found. Due to the limited molecular tools available for the study of mycoplasma genetics to date, complementation and expression of the wild-type lpd gene via insertion with a second transposon into Mg 7 has been unsuccessful. Although the possibility remains that an undetected second-site mutation may have contributed to the Mg 7 phenotype, we have established that the lpd gene is disrupted and the biologic function impaired. This, coupled with the similar finding in H. influenzae, supports the potential for this to be a valid virulence-associated factor. Further studies are in progress to assess Mg 7 as a vaccine candidate.
ACKNOWLEDGMENTS
We thank Vickie Weidig for technical assistance with necropsy and Ione Jackman and Sallyane Gemme for histologic preparations. Helpful discussions with Meghan May and the technical assistance of Martha Gladd and Xiaofen Liao are also acknowledged.
This work was supported by USDA grant 58-1940-0-007 to S.J.G. and by the Center of Excellence for Vaccine Research (CEVR).
Present address: TIGR, 9712 Medical Center Drive, Rockville, MD 20850.
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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.
INTRODUCTION
Mycoplasma gallisepticum is the primary etiologic agent of the chronic respiratory disease complex in chickens and infectious sinusitis in turkeys. Primary inflammatory responses in the respiratory tract associated with infection are sinusitis, tracheitis, bronchitis, and airsacculitis. Colonization of the respiratory tract leads to ciliostasis and deciliation of the tracheal epithelium, allowing secondary infection by other bacterial and viral pathogens, such as Newcastle disease virus, infectious bronchitis virus, and Escherichia coli (24). Common signs of M. gallisepticum infection include nasal discharge, tracheal rales, weight loss, and decreased egg production. Mycoplasma gallisepticum is highly contagious in commercial chicken and turkey flocks, spreading horizontally in populations through aerosol, dust, and feathers and vertically transmitted through eggs (8, 10, 24, 40). Its economic impact on the poultry industry is significant, leading to millions of dollars of loss each year. Efforts to improve disease prevention and control programs require increased research to identify novel virulence-associated determinants. These determinants may provide the basis for new and promising vaccines or targets for other antimicrobial therapies (41).
In 1995, a global virulence determinant identification technique called signature-tagged mutagenesis was developed in the laboratory of David Holden. Using a pool of uniquely tagged transposons for random mutagenesis, Hensel et al. (17) were able to screen a large mutant population for organisms demonstrating attenuated virulence through negative selection in vivo. This approach allows a comprehensive screening of bacterial genomes for the identification of genes encoding novel virulence determinants. We developed a modification of this technique, termed signature sequence mutagenesis (SSM), and utilized it for the discovery of novel virulence-associated genes in M. gallisepticum. The genome of virulent M. gallisepticum strain R has recently been sequenced and annotated (33) and is the cornerstone of this SSM technique. Signature sequence mutagenesis avoids one of the primary problems of signature-tagged mutagenesis, i.e., the issue of cross-hybridizing transposon tags (19). The Southern hybridization step is replaced with primer sets consisting of a common forward primer annealing at the end of the transposon and a mutant-specific reverse primer annealing in the genomic sequence flanking the transposon. SSM allows the screening of multiple mutants in one animal challenge experiment, thus minimizing the number of animals used. Here, we describe the application of SSM to identify virulence-associated determinants in M. gallisepticum and specifically characterize the dihydrolipoamide dehydrogenase gene (lpd) as a virulence-associated gene.
MATERIALS AND METHODS
Organisms and culture conditions. M. gallisepticum strain R low (Rlow) (passage 3 after in vivo reisolation) was cultured at 37°C in Hayflick's medium (10% horse serum and 5% yeast extract) (8) or Hayflick's agar with 1% Noble agar (Difco, Franklin Lakes, NJ). Gentamicin sulfate (150 μg/ml in broth and 100 μg/ml in agar) was added to the media for the propagation of the Tn4001mod mutants. E. coli DH5 carrying pISM2062 (25) was propagated in Luria broth and on plates (10 g tryptone, 5 g yeast extract, and 5 g of NaCl per liter) with ampicillin (100 μg/ml).
Nucleic acid extraction. The genomic DNA of each Tn4001mod mutant was extracted from 30 ml of mid-logarithmic-phase culture using the Easy-DNA extraction kit (Invitrogen, Carlsbad, Calif.). DNA concentrations were determined using spectrophotometric-absorbance measurements at 260 nm; purities were verified by absorbance ratios at 260 nm and 280 nm. Plasmid DNA was extracted using the QIAGEN Plasmid Mini kit protocol following the manufacturer's protocol (QIAGEN Inc., Santa Clarita, Calif.).
M. gallisepticum Rlow transformation. M. gallisepticum Rlow was transformed by electroporation (Bio-Rad Gene Pulser, Hercules, Calif.) with the suicide plasmid pISM2062 (a gift of F. Chris Minion, Iowa State University). This plasmid carries the gram-positive transposon Tn4001mod, which contains the gentamicin resistance gene (5). M. gallisepticum cultures were grown to mid-logarithmic phase at 37°C in 5 ml of Hayflick's broth. The cells were harvested by centrifugation at 12,000 x g for 10 min at room temperature and then resuspended in 1 ml of electroporation buffer (8 mM HEPES, 272 mM sucrose, pH 7.4). After an additional centrifugation at 12,000 x g for 10 min, 60 μl electroporation buffer and 10 μg pISM2062 plasmid DNA were added, and the mixture was incubated on ice for 15 min. The cell-DNA suspension was transferred to a chilled 0.2-cm-gap electroporation cuvette and immediately pulsed (2.5 kV; 25 μF; 100 ). One milliliter of cold Hayflick's broth was added, and the mixture was incubated at room temperature for 10 min; the cells were then incubated at 37°C for 2 to 3 h (6, 12, 14). Fifty microliters of the transformation mixture was diluted 1:10, and then 200 μl of that dilution was spread onto Hayflick's agar plates with gentamicin (100 μg/ml). The plates were incubated at 37°C for 7 to 10 days. Single visible colonies were picked and then placed in 500 μl Hayflick's broth with gentamicin and grown to mid-logarithmic phase. These M. gallisepticum transformants were titered and then stored at –80°C as stock cultures.
Transposon insertion site identification. The Tn4001mod M. gallisepticum mutant genomic DNA was extracted and sequenced using the outward primer SG857, annealing 27 bp upstream from the end of the transposon. The primer was designed to utilize the unique BamHI-SmaI cut site located in one of the two insertion sequences (IS256) of the transposon.
Linear sequencing with BigDye Terminator Mix (PE-Applied Biosystems) was used to identify the site of transposon insertion for each Tn4001mod mutant. Each sequencing reaction mixture contained 16 μl BigDye Terminator Mix, 30 pmol of primer, and 2 to 3 μg genomic DNA in a total volume of 32 μl. The following sequencing conditions were used: initial denaturation at 95°C for 5 min, followed by 45 cycles at 95°C for 30 s, 55°C for 20 s, and 60°C for 4 min (15). Excess dye terminators were removed with AutoSeq G-50 spin columns (Amersham Biosciences, Piscataway, NJ), and the samples were vacuum dried. Sequencing was performed by the University of Connecticut Biotechnology Center. Using Artemis v. 5 (Sanger Centre, United Kingdom) and Vector NTI 8 (Invitrogen, Carlsbad, Calif.), the genomic-sequencing results from the mutants were compared to the sequenced and annotated genome of M. gallisepticum strain R (33).
Determining transposon insertion stability. Sterile 1.5-ml microcentrifuge tubes containing 0.5 ml Hayflick's broth were inoculated with 30 μl of Tn4001mod mutant stock and incubated at 37°C until growth was evident. These mutant cultures were passed 30 times in nonselective broth, and on every 10th passage, 30 μl of the culture was transferred and grown in 30 ml of Hayflick's broth with 150 μg/ml gentamicin for genomic-DNA extraction and transposon insertion site sequencing as described above. The resultant sequence was compared to the known insertion site sequence, and any transposon movement was documented. To determine the rate of transposon loss, 30 μl of each mutant was used to inoculate 0.5 ml of nonselective broth, and every day for 14 days, the cultures were passed once. After the 14th passage, the culture was serially diluted and duplicate plated on nonselective Hayflick's plates and those containing 100 μg/ml gentamicin. After incubation at 37°C, the CFU were quantified.
Growth curve analysis of the Tn4001mod mutants. Sterile 250-ml Erlenmeyer flasks containing 69 ml of Hayflick's broth were inoculated simultaneously with 1 ml of equal numbers of pretitered (based on previous optical-density values at 620 nm [OD620]) M. gallisepticum Rlow and each of the individual mutant stock cultures. The cells were incubated at 37°C in a 250-rpm orbital shaking incubator and were harvested at 2, 4, 6, 8, 10, 12, 14, 16, 28, 32, 40, 49, and 54 hours postinoculation. The growth was measured spectrophotometrically by assessing the OD620. All values were graphed semilogarithmically as OD620 values versus time. All growth curve analyses were performed in duplicate.
Design of the unique mutant identifier. The identification of the transposon insertion sites within the M. gallisepticum genome permitted the design of unique reverse primers for each Tn4001mod mutant. These reverse primers were used with the forward primer SG857 and each respective genomic DNA template to produce an amplicon of known size. Each amplicon ranged in size from 300 to 650 bp and represented a unique mutant identifier that distinguished one clone from another.
Animals. Female 4-week-old White Leghorn specific-pathogen-free chickens (SPAFAS, North Franklin, CT) were used in the challenge experiments. The birds were tagged upon arrival and placed in HEPA-filtered isolators (Controlled Isolator Systems, Pittsburgh, PA). After a 1-week period of acclimation (in accordance with approved Institutional Animal Care and Use Committees protocol), all challenge experiments were initiated.
In vivo virulence assay. Three chickens were used in the mutant pool assay. The chickens were challenged with a mixed population of 27 Rlow Tn4001mod mutants (input population) at 1.5 x 106 CFU of each unique transformant. One milliliter of pretitered stock aliquots of each mutant was treated as follows: 100 μl of each mutant culture was transferred to a common 15-ml plug-sealed tube, and the resultant mixed culture was pelleted by centrifugation at 12,000 x g and 4°C for 10 min. The remaining 900 μl of culture in each aliquot was centrifuged as stated above, decanted, and stored at –80°C as a template to produce an input population PCR profile for comparison to the output population PCR profile generated after mutant recovery. The pellet of the mixed culture was then thoroughly resuspended in 100 μl of Hayflick's medium, transferred to a 1.5-ml microcentrifuge tube, and placed on ice in preparation for the challenge. The above procedure was repeated for each chicken in the study. The chickens were infected by intratracheal inoculation using a P200 pipetter carrying 100-μl suspensions of the organism.
Two weeks postchallenge, the chickens were humanely killed by cervical dislocation and immediately necropsied, and samples of the tracheas and air sacs were removed for isolation of M. gallisepticum Tn4001mod mutants. Tissue samples and tracheal swabs were used to inoculate 30 ml of Hayflick's medium containing 150 μg/ml gentamicin, vortexed, and incubated at 37°C for 8 h. The cultures were then passed through 0.45-μm filters and transferred to new plug-sealed tubes, and incubation was continued. Cultures that were acidic (yellow) after being filtered were adjusted to pH 7.4 by the addition of 10 N NaOH, and incubation was continued at 37°C until growth was evident. The genomic DNA of each culture was extracted, and PCR was performed to determine the presence or absence of individual mutants in the output population by using primer sets containing a conserved forward primer (SG857) and a reverse primer specific for each transposon mutant (Table 2). Amplicons were visualized in an ethidium bromide-stained 0.8% agarose gel. The PCR profile of this output population was compared to the input population profile, and those mutants not present in the output population were considered potential virulence mutants.
Assessment of potential virulence mutants. Sixteen chickens were used in this assay. The chickens were divided into three groups, with two consisting of six chickens each (groups 1 and 2) and the third group consisting of four chickens (group 3). Group 1 chickens were challenged with 1.5 x 107 CFU of Rlow. The chickens in group 2 received 1.5 x 107 CFU of the individual mutant, and group 3 was given 1x phosphate-buffered saline (PBS) as a negative control. All groups were challenged on day 0 and day 2.
Two weeks postchallenge, the chickens were humanely killed by cervical dislocation and immediately necropsied, and samples of tracheas and air sacs were taken for M. gallisepticum recovery, if present. Tissue samples from groups 1 and 3 were placed in Hayflick's medium, while those from group 2 were grown in gentamicin selective medium. Using sterile instruments, samples of left and right thoracic and abdominal air sacs were removed and either placed in specific culture media (as detailed above) or fixed in 10% neutral buffered formalin for histopathological evaluation. The tracheas were divided into cranial, middle, and caudal thirds; cross-sections 3 to 5 mm in width were fixed in 10% neutral buffered formalin for histopathologic evaluation; and the remaining segments were placed in specific culture media. Formalin-fixed tissue samples were routinely processed, embedded in paraffin blocks, sectioned at 4 μm, and stained with hematoxylin and eosin according to standard histological protocols (39). Histopathological evaluations were performed in a blinded fashion and were based on criteria established by Nunoya et al. (31) and implemented in previous histopathologic studies of M. gallisepticum lesions in the trachea (34) and air sac (32). Lesion scores (LS) were assigned separately to trachea and air sacs.
The LS table adapted to this study to grade tracheal lesions was defined as follows: 0, no significant findings; 0.5, minimal multifocal lymphocytic or lymphofollicular infiltrates amounting to one to three discrete foci; 1, mild mucosal thickening resulting from either mild diffuse lymphocytic infiltrates or multifocal lymphocytic or lymphofollicular infiltrates amounting to four or more discrete foci without edema or heterophils; 2, moderate mucosal thickening resulting from multifocal to diffuse lymphocytic and histiocytic infiltrates with or without lymphofollicular infiltrates, intraepithelial and lamina proprial infiltrates of heterophils, and luminal exudates; and 3, severe mucosal thickening resulting from diffuse infiltrates of lymphocytes, histiocytes, and heterophils with flattening and attenuation of epithelium and luminal exudates. Lesions that had features intermediate in intensity between two scoring values were assigned midrange scores, e.g., 1.5 or 2.5.
The LS table adapted for scoring air sacs was defined as follows: 0, no significant findings; 0.5, minimal multifocal lymphocytic infiltrates with one to three discrete, small, widely separated foci; 1, mild stromal thickening due to either multifocal lymphocytic or lymphofollicular infiltrates amounting to four or more discrete foci or mild diffuse lymphocytic infiltrates without edema or heterophils; 1.5, lesions comparable to 1 with mild heterophilic infiltrates; 2, moderate stromal thickening resulting from multifocal to diffuse lymphocytic, histiocytic, and heterophilic infiltrates that included lymphofollicular aggregates and intraepithelial heterophils; 2.5, lesions comparable to 2 plus significant luminal heterophilic exudates; 3, severe stromal thickening resulting from multifocal to diffuse lymphocytic, histiocytic, and heterophilic infiltrates that might include epithelial attenuation or hyperplasia and luminal heterophilic or granulomatous exudates. In addition to qualitative scoring as described above, the tracheal mucosal width was measured using an ocular micrometer. The widths of the mucosa at four equidistant points along the circumference of each tracheal section were measured using the base of the cilia or the adluminal surface of deciliated or flattened epithelial cells as one boundary and the beginning of the collagenous stroma of the lamina propria-tunica submucosa as the opposite boundary. An average mucosal width was generated for each trachea from measurements of each tracheal section, and average mucosal widths were used in subsequent statistical analyses to determine the relevance of differences in mucosal thickness between inoculated groups.
The genomic DNA of each recovered mutant culture was extracted, and PCR was performed using a primer set containing the conserved forward primer (SG857) and a mutant-specific reverse primer to confirm the identities. Amplicons were visualized by UV transillumination of an ethidium bromide-stained 0.8% agarose gel. If the potential virulence mutant was not recovered at the same rate as wild-type Rlow and demonstrated reduced histopathology compared to wild-type lesion scores, then it was considered to have a virulence deficiency.
Homogenate preparation. Mycoplasma cells were suspended in 1 ml of 40% glycerol, 60% Tris hydrochloride buffer (pH 7.4) (9). The suspension was diluted to 1 mg of protein per ml with Tris hydrocholoride buffer (pH 7.4) containing 1 mM EDTA and 1 mM 2-mercaptoethanol. Three cycles of freeze-thaw and passage through a syringe with an 18-gauge needle were used to homogenize the samples. Total protein was determined by the Bradford assay (2).
Pyruvate dehydrogenase complex activity assay. The pyruvate dehydrogenase complex (PDHC) activity was assayed by a radiochemical method using [1-14C] pyruvic acid-sodium salt (Amersham, Piscataway, NJ) as a substrate. Mycoplasma homogenates containing 100 μg of protein were placed in disposable test tubes in an ice bath and brought to 200 μl with 0.01 M Tris (pH 7.4) containing 1 mM EDTA and 1 mM 2-mercaptoethanol. A 10-μl solution containing coenzyme A (CoA), NAD+, and thiamine pyrophosphate (2 mM each) in Tris buffer was added to each test tube and then mixed. Finally, 20 nmol 1-14C-pyruvic acid was added and mixed. The negative control preparation was immediately inactivated with 15 μl of 50% trichloroacetic acid. The test tubes were stoppered with 1.5-ml microcentrifuge tubes with their bottoms cut off containing paper wicks soaked with 50 μl of 2.5 M NaOH to absorb the evolving CO2. The reaction mixtures were incubated at 37°C and stopped at 15-, 30-, 45-, and 60-min intervals by the addition of 15 μl of 50% trichloroacetic acid. Each time point assessment was performed in duplicate. The tubes were again stoppered and incubated at 37°C in an orbital shaking incubator at 200 rpm for 2 h to collect all evolved CO2. Radioactivity was measured by counting the contents of each tube in 5 ml ScintiSafe (Fisher Chemicals, Fairlawn, NJ) liquid scintillation cocktail in a Beckman LS 3801 scintillation counter (Beckman Coulter, Fullerton, CA). The activity of PDHC was determined by the average decrease of radiolabeled pyruvic acid in the test tube due to decarboxylation, creating 14CO2 that was released from the reaction mixture and collected in the wicks (9).
Statistical analysis. Analysis of variance using the f test was used to determine if there were significant differences between the mean lesion scores of chicken groups. When differences were found, a mean separation analysis using Duncan's multiple-range test was performed. The PDHC activity was analyzed by the paired Student t test to determine significant differences between enzyme activities. Analyses were performed with the statistics program SAS version 8.01 (SAS Institute, Cary, NC).
Sequence analysis. TMHMM (26), PSIPRED (22, 30), MEMSAT2 (23), and PredictProtein (37) were used to predict protein topology. Sequence homology searches were performed using the PROSITE (11), Pfam (3), PRINTS (1), and BLOCKS (16) databases. Amino acid sequence homologies were considered significant if they possessed 30% identity or similarity.
RESULTS
Identification of Tn4001mod mutants in mixed culture. Insertion sites were determined by sequencing using the outward primer SG857 (Table 2). The precise location of Tn4001mod in each of the transformants was identified by comparison with the annotated M. gallisepticum strain R (Table 1). In addition to identifying the transposition site, disruption of function of a coding DNA sequence (CDS) by transposon insertion was determined using principles previously described by Hutchison et al. (21). A CDS was considered disrupted if Tn4001mod inserted downstream of nucleotide 9 and within the first 80% of the 5'-most end of the gene. Based on these criteria, a CDS disruption is denoted in Table 1 as "yes" or "no." Unique reverse primers were designed based on genomic sequence flanking the transposon insertion site of each transformant; they are used for the PCR amplification of a unique mutant identifier (Table 2). Reverse primers were synthesized for each Tn4001mod mutant and paired with the outward primer SG857 in PCR. The resultant PCR profile (Fig. 1, top) generated amplicons at the expected sizes for all 27 transformants, thus confirming the specificity of this assay for the identification of individual transposon mutants in a mixed culture.
In vivo virulence assay. Based on the total output population PCR profiles from the tissues tested in three chickens, transformant Mg 7 was not recovered from the in vivo virulence assay, whereas the remaining 26 mutants were recovered at least once from other sample sites (Table 3). Since Mg 7 was not found in any of the assayed chicken tissues, it was classified as a potential virulence mutant. Figure 1, bottom, illustrates one output population PCR profile.
Transposon insertion stability. After 30 passages in nonselective media, the Mg 7 insertion was found to be stably maintained. The rate of transposon loss in the Mg 7 population was negligible.
Growth curve analysis. Mg 7 did not demonstrate any growth deficiency compared to wild-type Rlow over 54 hours of growth.
Potential virulence mutant assay. Histopathologic evaluation of tracheas from chickens challenged with Mg 7 showed a range of responses, from no significant lesions to mild diffuse lymphocytic infiltrates (LS = 0 to 1) without attenuation or deciliation of the epithelium (Fig. 2A). Chickens challenged with Rlow demonstrated minimal to moderate mucosal thickening (LS = 0.5 to 2) resulting from multifocal to diffuse lymphocytic and histiocytic infiltrates accompanied by heterophils and flattening and attenuation of the epithelium (Fig. 2B). Chickens in the PBS control group had no significant lesions (Fig. 2C). Examination of the tracheal mucosal width revealed a significant difference between the Rlow (60.4 μm)-challenged birds and the Mg 7 (40.6 μm) and PBS (44.6 μm) challenge groups.
Regarding the air sacs, birds challenged with Mg 7 had minimal to mild multifocal lymphofollicular infiltrates (LS = 0 to 1) amounting to minimal airsacculitis (Fig. 3A). Histopathological evaluation of birds challenged with Rlow revealed mild to moderate thickening of the air sacs resulting from mild to moderate lymphofollicular infiltration accompanied in some cases by luminal heterophilic exudates (LS = 1 to 2.5) (Fig. 3B). Chickens in the PBS control group had no significant lesions (Fig. 3C). Overall, in comparison to chickens from the Rlow challenge group, Mg 7 challenges demonstrated a marked reduction in lymphohistiocytic infiltrates, air sac exudates, and heterophil recruitment.
No significant differences were found between chickens challenged with Mg 7 and those challenged with PBS in either the trachea or air sac. Rlow-challenged birds exhibited a significant difference in air sac lesion scores compared to either Mg 7 or PBS challenges (Table 4). Birds in the Rlow challenge group developed airsacculitis and had extensive luminal exudates. Rlow was recovered from 20 out of 24 (83%) tissues sampled, while Mg 7 was recovered from 3 (12.5%) of the 24 cultures from the tissues sampled in this study. An Mg 7 isolate was recovered from the air sac of one bird, the trachea of another, and the lungs of the third. The presence of Mg 7 in the recovered cultures was confirmed by PCR. None of the challenge groups exhibited significantly different tracheal-lesion scores (P > 0.05).
Mg 7 characteristics and sequence analysis. Tn4001mod mutant Mg 7 has a transposon insertion in the dihydrolipoamide dehydrogenase CDS (lpd). The Tn4001mod insertion site is between amino acids 222 and 223, preventing the expression of over 50% of the carboxyl-terminal portion of the 467-amino-acid protein (Fig. 4). Structural-homology searches of Lpd in other sequenced mycoplasma and nonmycoplasma species, such as Mycoplasma pneumoniae, Mycoplasma hyopneumoniae, Mycoplasma genitalium, Mycoplasma penetrans, Mycoplasma pulmonis, Pseudomonas fluorescens, Azotobacter vinelandii, and Neisseria meningitidis (4, 7, 13, 20, 27, 28, 29, 38), revealed that this disulfide oxidoreductase has a redox-active site and a NAD/FAD binding domain within the intracellular amino-terminal portion of the polypeptide sequence. It also has a highly conserved dimerization domain located in its intracellular carboxyl-terminal portion. This dimerization domain has been extensively characterized in P. fluorescens, A. vinelandii, and N. meningitidis; the conformational stability of this enzyme is maintained only while dimerized to another Lpd molecule (4). Through the further proteomic analysis of the other two components of the PDHC, it is clear that dimerization of this enzyme is also important to the overall integrity of the PDHC, since it interacts with the moieties of the complex only in that polypeptide region. Protein topology analysis identified two putative transmembrane domains flanking the transposon insertion site in the extracellular domain (Fig. 4).
Pyruvate dehydrogenase complex activity assay. The first reaction in the PDHC is the decarboxylation of 1 mole of pyruvate, producing 1 mole of carbon dioxide. As the decarboxylation proceeds, radiolabeled CO2 is produced from the 14C-pyruvic acid and released from the reaction mixture. After 15 minutes, both Rlow and Mg 7 demonstrated similar rates of catabolic activity. The PDHC activity reached a plateau in the Mg 7 mutant at the 30-minute time point and was significantly different from Rlow catabolic activity at time points 45 and 60 (Fig. 5).
DISCUSSION
The definition of what constitutes a virulence-associated determinant is not limited to toxins and cytadhesins, but also includes proteases, regulatory proteins, stress response proteins, transport proteins, and proteins involved in metabolism. A virulence-associated determinant is therefore defined as any factor that confers a selective advantage on the pathogen, allowing it to colonize the host, persist, propagate, and cause disease.
The SSM assay allows the comprehensive screening of the genome for CDSs that may play roles in pathogenicity. Due to the random insertion pattern of transposon mutagenesis, it is likely that there will be disruptions of genes involved in pathogenesis. At least 30% of the CDSs in the M. gallisepticum genome sequence are annotated to code for polypeptides of unknown function. Emphasis has focused on the characterization of cytadhesins, such as GapA and CrmA (32, 34, 35), and hemagglutinins, such as the VlhA family of lipoproteins (33), but CDSs of unknown function must be thoroughly investigated as to their potential roles in pathogenicity. The ability to screen a large number of transposon mutants in one challenge experiment greatly accelerates the identification of novel virulence determinants among putative CDSs.
This study has described the identification of Lpd as an important protein in host colonization and pathogenesis. Histopathologic findings demonstrated differences in the severity of lesions caused by Mg 7 compared to Rlow, which were most clearly illustrated by the differences in the lesion scores of air sacs. The lesion scores of air sacs from Mg 7-challenged birds were significantly lower than those of Rlow-challenged chickens and lacked evidence of intense chronic inflammatory responses, such as the presence of lymphofollicular aggregates and epithelial attenuation.
Traditionally, the Lpd protein would be considered a metabolic factor, and therefore, its role in pathogenicity might never have been investigated. It is also important to note that the transposon insertion site in Mg 7 was stable when assayed, and therefore, no secondary transposon-induced mutations occurred during the chicken challenge. The in vitro growth rate of Mg 7 resembled that of wild-type M. gallisepticum, but the lack of the complete enzyme complex greatly affected its ability to colonize and survive within the host.
The Lpd protein is an essential functional subunit of the multienzyme PDHC, with a predicted molecular mass of 51 kDa. The PDHC is normally comprised of multiple copies of the ternary complex of Lpd, dihydrolipoamide transacetylase, and pyruvate dehydrogenase enzymes and is involved in the oxidation of pyruvate, the end product of glycolysis, to acetyl-CoA by the following reaction: pyruvate + CoA + NAD+ acetyl-CoA + CO2 + NADH + H.
The mutation in lpd interrupts the cyclical nature of the enzyme complex, rendering it inoperative after the reaction occurs once. The dihydrolipoamide dehydrogenase enzyme permits the continued function of the pathway by maintaining the correct redox state of the enzymes involved in the bioconversion. The PDHC activity assay confirmed that the protein homogenate from Mg 7 containing the incomplete complex failed to maintain activity, whereas the Rlow homogenate functioned properly.
Acetate is one of the end products of the fermentative metabolism carried out by M. gallisepticum. Through the combined actions of phosphate acetyltransferase and acetate kinase, 1 mole of ATP is generated during the conversion of acetyl-CoA to acetate (36). Without the complete PDHC, acetyl-CoA is not produced at wild-type levels, and the cell loses the ability to make a mole of ATP from the metabolic intermediate, leading to a possible intracellular energy shortage. The reduction in ATP could lower the cumulative activity of the ATP binding cassette transporters upon which the bacterium relies heavily to acquire many of the precursors necessary for its viability. While this feature is easily masked when Mg 7 is grown in the nutrient-rich Hayflick's medium, the deficiency may affect the microorganism's ability to acquire the precursor molecules and cofactors needed to persist and adapt to its host environment.
In a recent report, Haemophilus influenzae type B was shown to be attenuated by a transposon insertion in lpdA, the dihydrolipoamide dehydrogenase gene homolog. When the H. influenzae LpdA mutant was used to challenge 5-day-old infant rats intraperitoneally, it was unrecoverable compared to the wild-type recovery rate (18).
Despite screening of an additional 1,000 M. gallisepticum transposon mutants, a second clone with a mutation in lpd has not been found. Due to the limited molecular tools available for the study of mycoplasma genetics to date, complementation and expression of the wild-type lpd gene via insertion with a second transposon into Mg 7 has been unsuccessful. Although the possibility remains that an undetected second-site mutation may have contributed to the Mg 7 phenotype, we have established that the lpd gene is disrupted and the biologic function impaired. This, coupled with the similar finding in H. influenzae, supports the potential for this to be a valid virulence-associated factor. Further studies are in progress to assess Mg 7 as a vaccine candidate.
ACKNOWLEDGMENTS
We thank Vickie Weidig for technical assistance with necropsy and Ione Jackman and Sallyane Gemme for histologic preparations. Helpful discussions with Meghan May and the technical assistance of Martha Gladd and Xiaofen Liao are also acknowledged.
This work was supported by USDA grant 58-1940-0-007 to S.J.G. and by the Center of Excellence for Vaccine Research (CEVR).
Present address: TIGR, 9712 Medical Center Drive, Rockville, MD 20850.
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