F. Chris Minion

The Genome Sequence of Mycoplasma hyopneumoniae Strain 232, the Agent of Swine Mycoplasmosis

We present the complete genome sequence of Mycoplasma hyopneumoniae, an important member of the porcine respiratory disease complex. The genome is composed of 892,758 bp and has an average G+C content of 28.6 mol%. There are 692 predicted protein coding sequences, the average protein size is 388 amino acids, and the mean coding density is 91%. Functions have been assigned to 304 (44%) of the predicted protein coding sequences, while 261 (38%) of the proteins are conserved hypothetical proteins and 127 (18%) are unique hypothetical proteins. There is a single 16S-23S rRNA operon, and there are 30 tRNA coding sequences. The cilium adhesin gene has six paralogs in the genome, only one of which contains the cilium binding site. The companion gene, P102, also has six paralogs. Gene families constitute 26.3% of the total coding sequences, and the largest family is the 34-member ABC transporter family. Protein secretion occurs through a truncated pathway consisting of SecA, SecY, SecD, PrsA, DnaK, Tig, and LepA. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The DnaK-DnaJ-GrpR complex is intact, providing the only control over protein folding. There are several proteases that might serve as virulence factors, and there are 53 coding sequences with prokaryotic lipoprotein lipid attachment sites. Unlike other mycoplasmas, M. hyopneumoniae contains few genes with tandem repeat sequences that could be involved in phase switching or antigenic variation. Thus, it is not clear how M. hyopneumoniae evades the immune response and establishes a chronic infection.

Proteolytic Processing of the Mycoplasma hyopneumoniae Cilium Adhesin

ABSTRACT Mycoplasma hyopneumoniae is an economically significant swine pathogen that colonizes the respiratory ciliated epithelial cells. Cilium adherence is mediated by P97, a surface protein containing a repeating element (R1) that is responsible for binding. Here, we show that the cilium adhesin is proteolytically processed on the surface. Proteomic analysis of strain J proteins identified cleavage products of 22, 28, 66, and 94 kDa. N-terminal sequencing showed that the 66- and 94-kDa proteins possessed identical N termini and that the 66-kDa variant was generated by cleavage of the 28-kDa product from the C terminus. The 22-kDa product represented the N-terminal 195 amino acids of the cilium adhesin preprotein, confirming that the hydrophobic leader signal sequence is not cleaved during translocation across the membrane. Comparative studies of M. hyopneumoniae strain 232 showed that the major cleavage products of the cilium adhesin are similar, although P22 and P28 appear to be processed further in strain 232. Immunoblotting studies using antisera raised against peptide sequences within P22 and P66/P94 indicate that processing is complex, with cleavage occurring at different frequencies within multiple sites, and is strain specific. Immunogold electron microscopy showed that fragments containing the cilium-binding site remained associated with the cell surface whereas cleavage products not containing the R1 element were located elsewhere. Not all secreted proteins undergo multiple cleavage, however, as evidenced by the analysis of the P102 gene product. The ability of M. hyopneumoniae to selectively cleave its secreted proteins provides this pathogen with a remarkable capacity to alter its surface architecture.

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.

Construction of Tn4001 lac derivatives to be used as promoter probe vectors in mycoplasmas

Studies of gene expression in mycoplasmas has been difficult, because the elements involved in gene expression remain relatively undefined. In order to be able to examine these regulatory elements in vivo, derivatives of Tn4001 containing the promoterless Escherichia coli lacZ reporter gene have been constructed. A Tn4001lac derivative, Tn4001.2062.2lac, transforms Mycoplasma gallisepticum and Acholeplasma strain ISM1499. Approximately 3% of the M. gallisepticum and 8% of the Acholeplasma ISM1499 transformants appeared to generate lacZ fusions based on blue colony formation on XGal-containing media. However, placement of lacZ into an IS256 arm of Tn4001 resulted in a derivative that transformed at a frequency about 3-7-fold lower than that of wild-type Tn4001. Another Tn4001lac derivative, Tn4001.2065, possesses a plasmid, as well as lacZ, in the IS256 arm. This construct was designed to permit the direct rescue of adjacent chromosomal sequences that are driving the expression of lacZ contained within the transposon. These constructs should be useful in locating and defining the upstream elements involved in mycoplasma gene expression.

Use of a Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal sampling sites in swine

A number of polymerase chain reaction (PCR)-based diagnostic tests have been developed for Mycoplasma hyopneumoniae, including one from this research group. This report presents further development, optimization, and standardization of a nested PCR test. Detection sensitivity was 1 fg of M. hyopneumoniae chromosomal DNA (approximately 1 organism). This exceeded the sensitivity of or compared favorably with other published PCR tests. Polymerase chain reaction primers to porcine β2-microglobulin were included as internal controls for amplifiable chromosomal DNA from porcine samples. To standardize the test, a number of samples from experimentally infected pigs, including nasal, tonsil, tracheobronchial swabs, lung tissue, bronchial alveolar lavage (BAL) fluid, and tracheobronchial brush samples, were examined by PCR. Samples obtained from BAL fluid and tracheobronchial sites were most predictive of infection, whereas nasal swabs and lung tissue were not reliable indicators of experimentally induced infection. In conclusion, the nested PCR developed for this study was found to be a highly sensitive and specific diagnostic tool for M. hyopneumoniae, but the enhanced sensitivity may be unnecessary if the proper sites are sampled.

Characterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which Mycoplasma hyopneumoniae Regulates Surface Topography

ABSTRACT Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50P146, P40P146, and P85P146 that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence 672ATEF↓QQ677, consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1P146-F3P146 that mimic P50P146, P40P146, and P85P146 were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3P146 generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. IMPORTANCE Vaccines used to control Mycoplasma hyopneumoniae infection provide only partial protection. Proteins of the P97/P102 families are highly expressed, functionally redundant molecules that are substrates of endoproteases that generate multifunctional adhesin fragments on the cell surface. We show that P146 displays a chimeric structure consisting of an N terminus, which shares sequence identity with P97, and novel central and C-terminal regions. P146 is endoproteolytically processed at multiple sites, generating at least nine fragments on the surface of M. hyopneumoniae. Dominant cleavage events occurred at S/T-X-F↓X-D/E-like sites generating P50P146, P40P146, and P85P146. Recombinant proteins designed to mimic the major cleavage fragments bind porcine cilia, heparin, and plasminogen. P146 undergoes endoproteolytic processing events at multiple sites and with differential processing efficiency, generating combinatorial diversity on the surface of M. hyopneumoniae. Vaccines used to control Mycoplasma hyopneumoniae infection provide only partial protection. Proteins of the P97/P102 families are highly expressed, functionally redundant molecules that are substrates of endoproteases that generate multifunctional adhesin fragments on the cell surface. We show that P146 displays a chimeric structure consisting of an N terminus, which shares sequence identity with P97, and novel central and C-terminal regions. P146 is endoproteolytically processed at multiple sites, generating at least nine fragments on the surface of M. hyopneumoniae. Dominant cleavage events occurred at S/T-X-F↓X-D/E-like sites generating P50P146, P40P146, and P85P146. Recombinant proteins designed to mimic the major cleavage fragments bind porcine cilia, heparin, and plasminogen. P146 undergoes endoproteolytic processing events at multiple sites and with differential processing efficiency, generating combinatorial diversity on the surface of M. hyopneumoniae.

Transcriptional Profiling of Mycoplasma hyopneumoniae during Heat Shock Using Microarrays

ABSTRACT Bacterial pathogens undergo stress during host colonization and disease processes. These stresses result in changes in gene expression to compensate for potentially lethal environments developed in the host during disease. Mycoplasma hyopneumoniae colonizes the swine epithelium and causes a pneumonia that predisposes the host to enhanced disease from other pathogens. How M. hyopneumoniae responds to changing environments in the respiratory tract during disease progression is not known. In fact, little is known concerning the capabilities of mycoplasmas to respond to changing growth environments. With limited genes, mycoplasmas are thought to possess only a few mechanisms for gene regulation. A microarray consisting of 632 of the 698 open reading frames of M. hyopneumoniae was constructed and used to study gene expression differences during a temperature shift from 37°C to 42°C, a temperature swing that might be encountered during disease. To enhance sensitivity, a unique hexamer primer set was employed for generating cDNA from only mRNA species. Our analysis identified 91 genes that had significant transcriptional differences in response to heat shock conditions (P < 0.01) with an estimated false-discovery rate of 4 percent. Thirty-three genes had a change threshold of 1.5-fold or greater. Many of the heat shock proteins previously characterized in other bacteria were identified as significant in this study as well. A proportion of the identified genes (54 of 91) currently have no assigned function.

Genome-Wide Transposon Mutagenesis Reveals a Role for pO157 Genes in Biofilm Development in Escherichia coli O157:H7 EDL933

ABSTRACT Enterohemorrhagic Escherichia coli O157:H7, a world-wide human food-borne pathogen, causes mild to severe diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The ability of this pathogen to persist in the environment contributes to its dissemination to a wide range of foods and food processing surfaces. Biofilms are thought to be involved in persistence, but the process of biofilm formation is complex and poorly understood in E. coli O157:H7. To better understand the genetics of this process, a mini-Tn5 transposon insertion library was constructed in strain EDL933 and screened for biofilm-negative mutants using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm negative, and transposon insertions were located in 51 distinct genes/intergenic regions that must be involved either directly or indirectly in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Thirty-six genes were unique to this study, including genes on the virulence plasmid pO157. The type V secreted autotransporter serine protease EspP and the enterohemolysin translocator EhxD were found to be directly involved in biofilm formation. In addition, EhxD and EspP were also important for adherence to T84 intestinal epithelial cells, suggesting a role for these genes in tissue interactions in vivo.

Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia pestis antigens.

The design of biodegradable polymeric delivery systems based on polyanhydrides that would provide for improved structural integrity of Yersinia pestis antigens was the main goal of this study. Accordingly, the full-length Y. pestis fusion protein (F1-V) or a recombinant Y. pestis fusion protein (F1(B2T1)-V10) was encapsulated and released from microparticles based on 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic acid (SA) copolymers and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH copolymers fabricated by cryogenic atomization. An enzyme-linked immunosorbent assay was used to measure changes in the antigenicity of the released proteins. The recombinant F1(B2T1)-V10 was unstable upon release from the hydrophobic CPH:SA microparticles, but maintained its structure and antigenicity in the amphiphilic CPTEG:CPH system. The full-length F1-V was stably released by both CPH:SA and CPTEG:CPH microparticles. In order to determine the effect of the anhydride monomers on the protein structure, changes in the primary, secondary, and tertiary structure, as well as the antigenicity of both Y. pestis antigens, were measured after incubation in the presence of saturated solutions of SA, CPH, and CPTEG anhydride monomers. The results indicated that the amphiphilic environment provided by the CPTEG monomer was important to preserve the structure and antigenicity of both proteins. These studies offer an approach by which a thorough understanding of the mechanisms governing antigenic instability can be elucidated in order to optimize the in vivo performance of biodegradable delivery devices as protein carriers and/or vaccine adjuvants.

Mhp493 (P216) is a proteolytically processed, cilium and heparin binding protein of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose‐dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface‐accessible, heparin‐binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2‐D immunoblotting studies and TiO2 chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide 90VSELpSFR96 (position 94 in P85) was identified by MALDI‐MS/MS. Polyhistidine fusion proteins (F1P216, F2P216, F3P216) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1P216, F2P216 and F3P216 adhered to and entered porcine kidney epithelial‐like (PK15) cell monolayers. Microtitre plate‐based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae. Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium‐binding domain.