Construction of Mycoplasma hyopneumoniae P97 Null Mutants.

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia, a world-wide problem in the pig industry. This disease is characterized by a dry, non-productive cough, labored breathing, and pneumonia. Despite years of research, vaccines are marginally effective, and none fully protect pigs in a production environment. A better understanding of the host-pathogen interactions of the M. hyopneumoniae-pig disease, which are complex and involve both host and pathogen components, is required. Among the surface proteins involved in virulence are members of two gene families called P97 and P102. These proteins are the adhesins directing attachment of the organism to the swine respiratory epithelium. P97 is the major ciliary binding adhesin and has been studied extensively. Monoclonal antibodies that block its binding to swine cilia have contributed extensively to its characterization. In this study we use recombination to construct null mutants of P97 in M. hyopneumoniae and characterize the resulting mutants in terms of loss of protein by immunoblot using monoclonal antibodies, ability to bind purified swine cilia, and adherence to PK15 cells. Various approaches to recombination with this fastidious mycoplasma were tested including intact plasmid DNA, single-stranded DNA, and linear DNA with and without a heterologous RecA protein. Our results indicate that recombination can be used to generate site-specific mutants in M. hyopneumoniae. P97 mutants are deficient in cilia binding and PK15 cell adherence, and lack the characteristic banding pattern seen in immunoblots developed with the anti-P97 monoclonal antibody.

Use of signature-tagged mutagenesis to identify genes associated with colonization of sheep by E. coli O157:H7.

Outbreaks of Escherichia coli O157:H7 in the United States due to contaminated foods are a public health issue and a continuing problem. The major reservoir for these organisms is the gastrointestinal tract of ruminants where they are a member of the resident microbiota. Several factors that contribute to the colonization of cattle have been identified, but a systematic screen of genes that might contribute to the colonization and persistence phenotype in mature ruminants has not been reported. Using a sheep model of persistence, signature tagged mutagenesis (STM) was used to screen 1326 mutants for a persistence-negative phenotype of E. coli O157:H7. We identified 9 genes by STM that appeared to be required for colonization and/or survival in sheep. Three of the genes had functions associated with central metabolism (thiK, ftrA and nrdB), one was involved with LPS formation (wbdP), one encodes a non-LEE encoded effector protein (nleB) and one was a methyltransferase encoded on a prophage (Z2389). The remaining three genes did not have homology with any known genes. Six sheep given ΔwbdP and 2 sheep each were given mutants (ΔthiK (Z1745), ΔftrA (Z2164) and Z2389). The ΔwbdP mutant was recovered from the feces of 4/6 sheep at 6 days pi with a mean number of 1.42log10CFU/g feces compared to 4.6log10CFU/g feces for the wild type strain. This difference was significant (P<0.001) over the time course of the experiment (days 6-23). Both ΔthiK and ΔftrA mutants were recovered from 1 of 2 sheep at 9 days PI by enrichment procedures (<50CFU/g feces) whereas mutant Z2389 was not recovered from either animal past 2 days pi. The roles of all of these gene products require further study to determine how the persistence phenotype of a given strain of E. coli O157:H7 interacts with host factors.

Analysis of autoinducer-2 quorum sensing in Yersinia pestis.

The autoinducer-2 (AI-2) quorum-sensing system has been linked to diverse phenotypes and regulatory changes in pathogenic bacteria. In the present study, we performed a molecular and biochemical characterization of the AI-2 system in Yersinia pestis, the causative agent of plague. In strain CO92, the AI-2 signal is produced in a luxS-dependent manner, reaching maximal levels of 2.5 µM in the late logarithmic growth phase, and both wild-type and pigmentation (pgm) mutant strains made equivalent levels of AI-2. Strain CO92 possesses a chromosomal lsr locus encoding factors involved in the binding and import of AI-2, and confirming this assignment, an lsr deletion mutant increased extracellular pools of AI-2. To assess the functional role of AI-2 sensing in Y. pestis, microarray studies were conducted by comparing Δpgm strain R88 to a Δpgm ΔluxS mutant or a quorum-sensing-null Δpgm ΔypeIR ΔyspIR ΔluxS mutant at 37°C. Our data suggest that AI-2 quorum sensing is associated with metabolic activities and oxidative stress genes that may help Y. pestis survive at the host temperature. This was confirmed by observing that the luxS mutant was more sensitive to killing by hydrogen peroxide, suggesting a potential requirement for AI-2 in evasion of oxidative damage. We also show that a large number of membrane protein genes are controlled by LuxS, suggesting a role for quorum sensing in membrane modeling. Altogether, this study provides the first global analysis of AI-2 signaling in Y. pestis and identifies potential roles for the system in controlling genes important to disease.

Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to norepinephrine.

Mycoplasma hyopneumoniae, a component of the porcine respiratory disease complex, colonizes the respiratory tract of swine by binding to the cilia of the bronchial epithelial cells. Mechanisms of pathogenesis are poorly understood for M. hyopneumoniae, but previous work has indicated that it responds to the environmental stressors heat shock, iron deprivation and oxidative compounds. For successful infection, M. hyopneumoniae must effectively resist host responses to the colonization of the respiratory tract. Among these are changes in hormonal levels in the mucosal secretions. Recent work in the stress responses of other bacteria has included the response to the catecholamine norepinephrine. The idea that M. hyopneumoniae can respond to a host hormone, however, is novel and has not previously been demonstrated. To test this, organisms in the early exponential phase of growth were exposed to 100 muM norepinephrine for 4 h, and RNA samples from these cultures were collected and compared to RNA samples from control cultures using two-colour PCR-based M. hyopneumoniae microarrays. The M. hyopneumoniae response included slowed growth and changes in mRNA transcript levels of 84 genes, 53 of which were upregulated in response to norepinephrine. A larger proportion of the genes upregulated than those downregulated were involved with transcription and translation. The downregulated genes were mostly involved with metabolism, which correlated with the reduction in growth of the mycoplasma. Approximately 51 % of the genes were hypothetical with no known function. Thus, in response to norepinephrine, M. hyopneumoniae appears to upregulate protein expression while downregulating general metabolism.

Transcriptome analysis of organisms with food safety relevance.

Transcriptome analysis using microarrays has become a powerful tool to better understand the process of disease and other complex biological processes such as food spoilage and biofilm formation. This review is divided into two basic sections: 1) a short history and description of microarrays and 2) a discussion of studies involving bacterial food safety pathogens that focused on whole genome transcript analysis. Not included are the many studies using microarrays to identify, diagnose, or genetically characterize these organisms. This review focuses on studies involving Escherichia coli O157:H7, Salmonella spp., Campylobacter jejuni, Listeria monocytogenes, and Yersinia enterocolitica. Many of the studies involve altering the growth environment to simulate stress conditions and the use of host-pathogen model systems to explore virulence mechanisms. Few studies use conditions that might be considered unique to the food industry. Exceptions are studies of biofilm-specific transcriptome changes and analysis following pressure treatment. This review should not be considered as a comprehensive review, and where appropriate, species-specific reviews are cited that are more complete.

Real-time PCR assays to address genetic diversity among strains of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is an important cause of pneumonia in pigs around the world, but confirming its presence in (or absence from) pigs can be difficult. Culture for diagnosis is impractical, and seroconversion is often delayed after natural infection, limiting the use of serology. Numerous PCR assays for the detection of M. hyopneumoniae have been developed, targeting several different genes. Recently, genetic diversity among strains of M. hyopneumoniae was demonstrated. The effect of this diversity on the accuracy and sensitivity of the M. hyopneumoniae PCR assays could result in false-negative results in current PCR tests. In this study, a panel of isolates of M. hyopneumoniae, M. flocculare, M. hyorhinis, and M. hyosynoviae were tested with a number of M. hyopneumoniae-specific PCR assays. Some M. hyopneumoniae PCR assays tested did not detect all isolates of M. hyopneumoniae. To increase the efficiency of PCR testing, two new real-time PCR assays that are specific and capable of detecting all of the M. hyopneumoniae isolates used in this study were developed.

Transcriptome changes in Mycoplasma hyopneumoniae during infection.

Mycoplasma hyopneumoniae causes swine pneumonia and contributes significantly to the porcine respiratory disease complex. The mechanisms of pathogenesis are difficult to address, since there is a lack of genetic tools, but microarrays are available and can be used to study transcriptional changes that occur during disease as a way to identify important virulence-related genes. Mycoplasmas were collected from bronchial alveolar lavage samples and compared to broth-grown cells using microarrays. Bronchial alveolar lavage was performed on pigs 28 days postinfection, and mycoplasmas were isolated by differential centrifugation. Mycoplasma RNA-enriched preparations were then obtained from total RNA by subtracting eucaryotic ribosomal and messenger RNAs. Labeled cDNAs were generated with mycoplasma open reading frame-specific primers. Nine biological replicates were analyzed. During lung infection, our analysis indicated that 79 M. hyopneumoniae genes were differentially expressed (P < 0.01), at a false-discovery rate of <2.7%. Of the down-regulated genes, 28 of 46 (61%) lacked an assigned function, in comparison to 21 of 33 (63%) of up-regulated genes. Four down-regulated genes and two up-regulated genes encoded putative lipoproteins. secA (mhp295) (P = 0.003) and two glycerol transport permease genes (potA [mhp380; P = 0.006] and ugpA [mhp381; P = 0.003]) were up-regulated in vivo. Elongation factor EF-G (fusA [mhp083]) (P = 0.002), RNA polymerase beta chain (rpoC [mhp635]) (P = 0.003), adenylate kinase (adk [mhp208]) (P = 0.001), prolyl aminoacyl tRNA synthetase (proS [mhp397]) (P = 0.009), and cysteinyl-tRNA synthetase (cysS [mhp661]) (P < 0.001) were down-regulated in vivo.

Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to hydrogen peroxide.

Mycoplasma hyopneumoniae, the causative agent of swine enzootic pneumonia, colonizes the cilia of swine lungs, causing ciliostasis and cell death. M. hyopneumoniae is a component of the porcine respiratory disease complex (PRDC) and is especially problematic for the finishing swine industry, causing the loss of hundreds of millions of dollars in farm revenues worldwide. For successful infection, M. hyopneumoniae must effectively resist oxidative stresses due to the release of oxidative compounds from neutrophils and macrophages during the host's immune response. However, the mechanism that M. hyopneumoniae uses to avert the host response is still unclear. To gain a better understanding of the transcriptional responses of M. hyopneumoniae under oxidative stress, cultures were grown to early exponential phase and exposed to 0.5% hydrogen peroxide for 15 min. RNA samples from these cultures were collected and compared to RNA samples from control cultures using two-colour PCR-based M. hyopneumoniae microarrays. This study revealed significant downregulation of important glycolytic pathway genes and gene transcription proteins, as well as a protein known to activate oxidative stressor cascades in neutrophils. Sixty-nine per cent of the upregulated genes were hypothetical with no known function. This study has also revealed significantly differentially expressed genes common to other environmental stress responses, indicating that further investigation of universal stress response genes of M. hyopneumoniae is merited.

Array-based genomic comparative hybridization analysis of field strains of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist, although it is clear that M. hyopneumoniae adheres to porcine ciliated epithelium by action of a protein called P97. Previous studies have shown variation in the gene encoding the P97 cilium adhesin in different strains of M. hyopneumoniae, but the extent of genetic variation among field strains across the genome is not known. Since M. hyopneumoniae is a worldwide problem, it is reasonable to expect that a wide range of genetic variability may exist given all of the different breeds and housing conditions. This variation may impact the overall virulence of a single strain. Using microarray technology, this study examined the potential variation of 14 field strains compared to strain 232, on which the array was based. Genomic DNA was obtained, amplified with TempliPhi, and labeled indirectly with Alexa dyes. After genomic hybridization, the arrays were scanned and data were analyzed using a linear statistical model. The results indicated that genetic variation could be detected in all 14 field strains but across different loci, suggesting that variation occurs throughout the genome. Fifty-nine percent of the variable loci were hypothetical genes. Twenty-two percent of the lipoprotein genes showed variation in at least one field strain. A permutation test identified a location in the M. hyopneumoniae genome where there is spatial clustering of variability between the field strains and strain 232.

Transcriptional profiling of Mycoplasma hyopneumoniae during iron depletion using microarrays.

Mycoplasma hyopneumoniae, the causative agent of swine enzootic pneumonia and a major component of the porcine respiratory disease complex, continues to confound swine producers despite control programmes worldwide. The disease is chronic and self-limiting, but the host is subject to immunopathological changes that potentiate respiratory disease associated with other pathogens. The response of M. hyopneumoniae to environmental stress is of interest because of its relevance to virulence mechanisms in other bacterial pathogens. One of these stressors, iron deprivation, is a prominent feature of the host innate immune response, and most certainly impacts growth of mycoplasmas in vivo. To study this, microarray technology was applied to the transcriptome analysis of M. hyopneumoniae during iron deprivation. An array consisting of 632 of the 698 ORFs in the genome was used to compare the mRNA isolated from organisms grown under normal laboratory conditions with that from organisms subjected to iron deprivation with the chelator 2,2'-dipyridyl. This analysis identified 27 genes that were either up- or down-regulated in response to low-iron growth conditions (P<0.01), with an estimated false discovery rate below 10 %. These included genes encoding transport proteins, enzymes involved in energy metabolism, and components of the translation process. Ten of the 27 identified genes had no assigned function. These studies indicate that M. hyopneumoniae can respond to changes in environmental conditions, but the mechanism employed remains unknown.

Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays.

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 degrees C to 42 degrees 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.

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.