Identification of Genes Transcribed by<i>Actinobacillus pleuropneumoniae</i>in Necrotic Porcine Lung Tissue by Using Selective Capture of Transcribed Sequences

Baltes, Nina; Gerlach, Gerald-F.

doi:10.1128/iai.72.11.6711-6716.2004

Cited by 40 publications

(68 citation statements)

References 45 publications

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“…As we had shown in previous studies, genes containing putative HlyX binding motifs are expressed by A. pleuropneumoniae under the influence of bronchoalveolar lavage fluid from infected pigs, and deletion of these genes can cause attenuation of the organism (2,15). In addition, an analysis of genes expressed in necrotic porcine lung tissue revealed the expression of DMSO reductase and several other genes involved in anaerobic metabolism under these conditions (1). This repeated isolation of presumably HlyX-regulated genes led to the hypothesis that deletion of this global anaerobic regulator would inhibit colonization and/or persistence in A. pleuropneumoniae if it was indeed involved in the regulation of the genes we identified.…”

Section: Discussionsupporting

confidence: 60%

“…We have shown previously that A. pleuropneumoniae genes upregulated under anaerobic conditions in culture are not only upregulated in sequestered necrotic lung tissue where anoxic conditions are to be expected (1) but also upon the supplementation of culture medium with bronchoalveolar lavage fluid from A. pleuropneumoniae-infected pigs, which mimics conditions as they occur on respiratory epithelium (1)(2)(3)15). Further, we have shown that isogenic mutants lacking aspartate ammonia lyase (aspartase) activity or both dimethyl sulfoxide (DMSO) reductase and aspartase activity were reduced in virulence and in their ability to persist on unaltered respiratory epithelium (2,15).…”

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Deletion of the Anaerobic Regulator HlyX Causes Reduced Colonization and Persistence ofActinobacillus pleuropneumoniaein the Porcine Respiratory Tract

et al. 2005

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Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to persist on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated lung sequesters. We have demonstrated previously that putative HlyX-regulated genes, coding for dimethyl sulfoxide (DMSO) reductase and aspartate ammonia lyase, are upregulated during infection and that deletions in these genes result in attenuation of the organism. The study presented here investigates the role of HlyX, the fumarate nitrate reductase regulator (FNR) homologue of A. pleuropneumoniae. By constructing an isogenic A. pleuropneumoniae hlyX mutant, the HlyX protein is shown to be responsible for upregulated expression of both DMSO reductase and aspartate ammonia lyase (AspA) under anaerobic conditions. In a challenge experiment the A. pleuropneumoniae hlyX mutant is shown to be highly attenuated, unable to persist in healthy lung epithelium and tonsils, and impaired in survival inside sequestered lung tissue. Further, using an A. pleuropneumoniae strain carrying the luxAB genes as transcriptional fusion to aspA on the chromosome, the airway antioxidant glutathione was identified as one factor potentially responsible for inducing HlyX-dependent gene expression of A. pleuropneumoniae in epithelial lining fluid.Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia (10), is able to persist in host tissues, such as tonsillar crypts and sequestered necrotic lung, where the oxygen supply is scarce. The resulting carrier animals are the major source of infection for previously A. pleuropneumoniae-free herds (10) and, therefore, unraveling the mechanisms of persistence is highly relevant to effective vaccination and control of the infection.In Escherichia coli a number of genes expressed under anaerobic conditions are regulated by the global regulator FNR (for fumarate nitrate reductase regulator) (24). An A. pleuropneumoniae FNR homologue, HlyX, has been found to induce hemolytic activity in E. coli under anoxic conditions and to be able to complement E. coli fnr deletions (18, 26). Like FNR, HlyX contains four iron-sulfur clusters responsible for the DNA-binding ability of the protein (12).In A. pleuropneumoniae, anaerobically regulated genes appear to play a role in virulence and persistence. We have shown previously that A. pleuropneumoniae genes upregulated under anaerobic conditions in culture are not only upregulated in sequestered necrotic lung tissue where anoxic conditions are to be expected (1) but also upon the supplementation of culture medium with bronchoalveolar lavage fluid from A. pleuropneumoniae-infected pigs, which mimics conditions as they occur on respiratory epithelium (1-3, 15). Further, we have shown that isogenic mutants lacking aspartate ammonia lyase (aspartase) activity or both dimethyl sulfoxide (DMSO) reductase and aspartase activity were reduced in virulence and in their ability to persist on unaltered respiratory epithelium (2, 15).Since both the DMSO reductase (d...

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Deletion of the Anaerobic Regulator HlyX Causes Reduced Colonization and Persistence ofActinobacillus pleuropneumoniaein the Porcine Respiratory Tract

et al. 2005

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“…However, other putative adhesins have also been described, such as type IV fimbriae expressed under specific conditions in different serotypes (63), a 60-kDa collagen-binding protein (14), a 55-kDa outer membrane protein (61), and an autotransporter protein (4).…”

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“…In this study, many genes involved in iron acquisition were shown to be upregulated, while genes involved mainly in energy metabolism were downregulated. In vivo studies based on selective capture of transcribed sequences, in vivo expression technology, or signature-tagged mutagenesis (3,4,22,31,53) have allowed the detection of adhesin and toxin genes and of genes involved in metabolism, stress, regulation, and transport.…”

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Host-Pathogen Interactions ofActinobacillus pleuropneumoniaewith Porcine Lung and Tracheal Epithelial Cells

et al. 2009

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Host-pathogen interactions are of great importance in understanding the pathogenesis of infectious microorganisms. We developed in vitro models to study the host-pathogen interactions of porcine respiratory tract pathogens using two immortalized epithelial cell lines, namely, the newborn pig trachea (NPTr) and St. Jude porcine lung (SJPL) cell lines. We first studied the interactions of Actinobacillus pleuropneumoniae, an important swine pathogen, using these models. Under conditions where cytotoxicity was absent or low, we showed that A. pleuropneumoniae adheres to both cell lines, stimulating the induction of NF-B. The NPTr cells consequently secrete interleukin 8, while the SJPL cells do not, since they are deprived of the NF-B p65 subunit. Cell death ultimately occurs by necrosis, not apoptosis. The transcriptomic profile of A. pleuropneumoniae was determined after contact with the porcine lung epithelial cells by using DNA microarrays. Genes such as tadB and rcpA, members of a putative adhesin locus, and a gene whose product has high homology to the Hsf autotransporter adhesin of Haemophilus influenzae were upregulated, as were the genes pgaBC, involved in biofilm biosynthesis, while capsular polysaccharide-associated genes were downregulated. The in vitro models also proved to be efficient with other swine pathogens, such as Actinobacillus suis, Haemophilus parasuis, and Pasteurella multocida. Our results demonstrate that interactions of A. pleuropneumoniae with host epithelial cells seem to involve complex cross talk which results in regulation of various bacterial genes, including some coding for putative adhesins. Furthermore, our data demonstrate the potential of these in vitro models in studying the host-pathogen interactions of other porcine respiratory tract pathogens.

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“…Selective capture of transcribed sequences (SCOTS) has been used to identify genes expressed in vivo by bacterial pathogens such as Mycobacterium (13,14), Salmonella (15)(16)(17), Actinobacillus (18), Helicobacter pylori (19), and avian pathogenic Escherichia coli (20). In this report, we demonstrate that SCOTS can be used to obtain high-quality transcript profiles from intracellular bacteria such as Typhi within human macrophages, and that the use of microarrays and SCOTS-cDNA hybridization provides an effective means to elucidate the global bacterial expression profile from infected host cells.…”

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Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences

Faucher

Porwollik²,

Dozois³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

145

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The cDNA obtained by selective capture of transcribed sequences is a complex mixture that can be used in conjunction with microarrays to determine global gene expression by a pathogen during infection. We used this method to study genes expressed by Salmonella enterica serovar Typhi, the etiological agent of typhoid fever, within human macrophages. Global expression profiles of Typhi grown in vitro and within macrophages at different time points were obtained and compared. Known virulence factors, such as the SPI-1-and SPI-2-encoded type III secretion systems, were found to be expressed as predicted during infection by Salmonella, which validated our data. Typhi inside macrophages showed increased expression of genes encoding resistance to antimicrobial peptides, used the glyoxylate bypass for fatty acid utilization, and did not induce the SOS response or the oxidative stress response. Genes coding for the flagellar apparatus, chemotaxis, and iron transport systems were down-regulated in vivo. Many cDNAs corresponding to genes with unknown functions were up-regulated inside human macrophages and will be important to consider for future studies to elucidate the intracellular lifestyle of this human-specific pathogen. Real-time quantitative PCR was consistent with the microarray results. The combined use of selective capture of transcribed sequences and microarrays is an effective way to determine the bacterial transcriptome in vivo and could be used to investigate transcriptional profiles of other bacterial pathogens without the need to recover many nanograms of bacterial mRNA from host and without increasing the multiplicity of infection beyond what is seen in nature.microarrays ͉ in vivo bacterial gene expression ͉ host-pathogen interaction

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Identification of Genes Transcribed byActinobacillus pleuropneumoniaein Necrotic Porcine Lung Tissue by Using Selective Capture of Transcribed Sequences

Cited by 40 publications

References 45 publications

Deletion of the Anaerobic Regulator HlyX Causes Reduced Colonization and Persistence ofActinobacillus pleuropneumoniaein the Porcine Respiratory Tract

Deletion of the Anaerobic Regulator HlyX Causes Reduced Colonization and Persistence ofActinobacillus pleuropneumoniaein the Porcine Respiratory Tract

Host-Pathogen Interactions ofActinobacillus pleuropneumoniaewith Porcine Lung and Tracheal Epithelial Cells

Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences

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