Selective Capture Of Transcribed Sequences Research Articles

Establishment of Streptococcus suis Biofilm Infection Model In Vivo and Comparative Analysis of Gene Expression Profiles between In Vivo and In Vitro Biofilms.

Streptococcus suis is a zoonotic pathogen that continuously threatens animal husbandry and public health worldwide. Studies have shown that S. suis can cause persistent infection by forming biofilms. In this study, a model of S. suis biofilm-related infection was successfully constructed for the first time by simulating the natural infection of S. suis, and biofilm of S. suis in vivo was successfully observed in the lung tissue of infected pigs by a variety of detection methods. Subsequently, selective capture of transcribed sequences (SCOTS) was used to identify genes expressed by S. suis in vivo biofilms. Sixty-nine genes were captured in in vivo biofilms formed by S. suis for the first time by SCOTS; they were mainly involved in metabolism, cell replication, and division, transport, signal transduction, cell wall, etc. Genes related to S. suis in vitro biofilm formation were also identified by SCOTS and RNA sequencing. Approximately half of the genes captured by SCOTS in the in vivo and in vitro biofilms were found to be different. In summary, our study provides powerful clues for future exploration of the mechanisms of S. suis biofilm formation. IMPORTANCE Streptococcus suis is considered an important zoonotic pathogen, and persistent infection caused by biofilm is currently considered to be the reason why S. suis is difficult to control in swine. However, to date, a model of the biofilm of S. suis in vivo has not been successfully constructed. Here, we successfully detected biofilms of S. suis in vivo in lung tissues of piglets infected with S. suis. Selective capture of transcribed sequences and the transcriptome were used to obtain gene profiles of S. suis in vivo and in vitro biofilms, and the results showed large differences between them. Such data are of importance for future experimental studies exploring the mechanism of biofilm formation by S. suis in vivo.

Comparative study of Salmonella enterica serovar Enteritidis genes expressed within avian and murine macrophages via selective capture of transcribed sequences (SCOTS).

Salmonella enterica serovar Enteritidis (SE) is a communicable zoonotic bacterium. Macrophages are essential for Salmonella survival, transmission, and infection. In this study, selective capture of transcribed sequences (SCOTS) was used to screen genes preferentially expressed by SE during contact with macrophages from different hosts. We found 57 predicted genes and 52 genes expressed by SE during interaction with avian HD-11 and murine RAW264.7 cells, respectively. These expressed genes were involved in virulence, metabolism, stress response, transport, regulation, and other functions. Although genes related to survival or metabolic pathways were needed during SE infection, different gene expression profiles of SE occurred in the two macrophage cell lines. qRT-PCR results confirmed that most screened genes were upregulated during infection in contrast to the observation during in vitro cultivation, with different expression levels in infected avian macrophages at 2-h and 7-h post-infection. In addition, in vitro and in vivo competition assays confirmed that SEN3610 (a putative deoR family regulator) and rfaQ (related to LPS synthesis) were closely related to SE virulence in both mice and chickens. Three putative transcriptional regulators, SEN2967, SEN4299, and rtcR, were related to SE colonization in mice, while the ycaM mutation caused decreased infection and survival of SE in HD-11 cells without influencing virulence in mice or chicken. Genes showing differential expression between SE-infected avian and murine macrophages indicate specific pathogen adaptation to enable infection of various hosts.

Identification of Aeromonas hydrophila Genes Preferentially Expressed after Phagocytosis by Tetrahymena and Involvement of Methionine Sulfoxide Reductases.

Free-living protozoa affect the survival and virulence evolution of pathogens in the environment. In this study, we explored the fate of Aeromonas hydrophila when co-cultured with the bacteriovorous ciliate Tetrahymena thermophila and investigated bacterial gene expression associated with the co-culture. Virulent A. hydrophila strains were found to have ability to evade digestion in the vacuoles of this protozoan. In A. hydrophila, a total of 116 genes were identified as up-regulated following co-culture with T. thermophila by selective capture of transcribed sequences (SCOTS) and comparative dot-blot analysis. A large proportion of these genes (42/116) play a role in metabolism, and some of the genes have previously been characterized as required for bacterial survival and replication within macrophages. Then, we inactivated the genes encoding methionine sulfoxide reductases, msrA, and msrB, in A. hydrophila. Compared to the wild-type, the mutants ΔmsrA and ΔmsrAB displayed significantly reduced resistance to predation by T. thermophila, and 50% lethal dose (LD50) determinations in zebrafish demonstrated that both mutants were highly attenuated. This study forms a solid foundation for the study of mechanisms and implications of bacterial defenses.

Comparative Analysis of Xenorhabdus koppenhoeferi Gene Expression during Symbiotic Persistence in the Host Nematode.

Species of Xenorhabdus and Photorhabdus bacteria form mutualistic associations with Steinernema and Heterorhabditis nematodes, respectively and serve as model systems for studying microbe-animal symbioses. Here, we profiled gene expression of Xenorhabdus koppenhoeferi during their symbiotic persistence in the newly formed infective juveniles of the host nematode Steinernema scarabaei through the selective capture of transcribed sequences (SCOTS). The obtained gene expression profile was then compared with other nematode-bacteria partnerships represented by Steinernema carpocapsae—Xenorhabdus nematophila and Heterorhabditis bacteriophora—Photorhabdus temperata. A total of 29 distinct genes were identified to be up-regulated and 53 were down-regulated in X. koppenhoeferi while in S. scarabaei infective juveniles. Of the identified genes, 8 of the up-regulated and 14 of the down-regulated genes were similarly expressed in X. nematophila during persistence in its host nematode S. carpocapsae. However, only one from each of these up- and down-regulated genes was common to the mutualistic partnership between the bacterium P. temperata and the nematode H. bacteriophora. Interactive network analysis of the shared genes between X. koppenhoeferi and X. nematophila demonstrated that the up-regulated genes were mainly involved in bacterial survival and the down-regulated genes were more related to bacterial virulence and active growth. Disruption of two selected genes pta (coding phosphotransacetylase) and acnB (coding aconitate hydratase) in X. nematophila with shared expression signature with X. koppenhoeferi confirmed that these genes are important for bacterial persistence in the nematode host. The results of our comparative analyses show that the two Xenorhabdus species share a little more than a quarter of the transcriptional mechanisms during persistence in their nematode hosts but these features are quite different from those used by P. temperata bacteria in their nematode host H. bacteriophora.

Selective capture of transcribed sequences in the functional gene analysis of microbial pathogens.

Selective capture of transcribed sequences (SCOTS) is an effective method to identify bacterial genes differentially expressed during different biological processes, including pathogenic interactions with a host species. The method can be used to elucidate molecular mechanisms driving and maintaining such interactions. The method is a powerful genetic tool that overcomes limitations found in other methods, by working with small amounts of mRNA and allowing for the separation of bacterial cDNA from host cDNA. It has been increasingly used in the discovery of genes involved in the bacterium-host interaction. In this review, we briefly introduce the SCOTS method, outline the technical advances offered in the method, and focus on the method's applications in several microbial pathogens.

Identification of Genes Preferentially Expressed by Highly Virulent Piscine Streptococcus agalactiae upon Interaction with Macrophages

Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood–brain barrier (BBB). The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS) was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB.

Identification of genes transcribed by Streptococcus equi ssp. zooepidemicus in infected porcine lung

Streptococcus equi ssp. zooepidemicus (SEZ) is a Gram-positive bacterium responsible for respiratory tract infection, septicemia, meningitis, endocarditis and arthritis in swine and humans. However, the expression and regulation of SEZ genes during an infection in vivo are poorly understood. In this study, we focused on the identification of SEZ genes preferentially expressed in vivo during infection in pigs. This study identified 45 SEZ genes that were upregulated in infected porcine lung tissues using the selective capture of transcribed sequences (SCOTS) technique and comparative dot-blot analysis, followed by quantitative RT-PCR validation. The identified genes were characterized into 6 functional categories: metabolism, cell wall-associated, stress response, transporters, regulators and unknown functions. Our study successfully identified multiple genes, which can deepen our understanding about SEZ pathogenesis and infer probable virulence factors. It will promote the development of novel vaccines and therapies about this pathogen for further study.

Selective Capture of Transcribed Sequences: A Promising Approach for Investigating Bacterium-Insect Interactions.

Bacterial interactions with eukaryotic hosts are complex processes which vary from pathogenic to mutualistic. Identification of bacterial genes differentially expressed in the host, promises to unravel molecular mechanisms driving and maintaining such interactions. Several techniques have been developed in the past 20 years to investigate bacterial gene expression within their hosts. The most commonly used techniques include in-vivo expression technology, signature-tagged mutagenesis, differential fluorescence induction, and cDNA microarrays. However, the limitations of these techniques in analyzing bacterial in-vivo gene expression indicate the need to develop alternative tools. With many advantages over the other methods for analyzing bacterial in-vivo gene expression, selective capture of transcribed sequences (SCOTS) technique has the prospect of becoming an elegant tool for discovery of genes involved in the bacterium-host interaction. Here, we summarize the advances in SCOTS technique, including its current and potential applications in bacterial gene expression studies under a variety of conditions from in-vitro to in-vivo and from mammals to insects.

Global changes in gene expression by the opportunistic pathogen Burkholderia cenocepacia in response to internalization by murine macrophages

BackgroundBurkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in patients with cystic fibrosis. The bacterium survives within macrophages by interfering with endocytic trafficking and delaying the maturation of the B. cenocepacia-containing phagosome. We hypothesize that B. cenocepacia undergoes changes in gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation.ResultsWe examined gene expression by intracellular B. cenocepacia using selective capture of transcribed sequences (SCOTS) combined with microarray analysis. We identified 767 genes with significantly different levels of expression by intracellular bacteria, of which 330 showed increased expression and 437 showed decreased expression. Affected genes represented all aspects of cellular life including information storage and processing, cellular processes and signaling, and metabolism. In general, intracellular gene expression demonstrated a pattern of environmental sensing, bacterial response, and metabolic adaptation to the phagosomal environment. Deletion of various SCOTS-identified genes affected bacterial entry into macrophages and intracellular replication. We also show that intracellular B. cenocepacia is cytotoxic towards the macrophage host, and capable of spread to neighboring cells, a role dependent on SCOTS-identified genes. In particular, genes involved in bacterial motility, cobalamin biosynthesis, the type VI secretion system, and membrane modification contributed greatly to macrophage entry and subsequent intracellular behavior of B. cenocepacia.ConclusionsB. cenocepacia enters macrophages, adapts to the phagosomal environment, replicates within a modified phagosome, and exhibits cytotoxicity towards the host cells. The analysis of the transcriptomic response of intracellular B. cenocepacia reveals that metabolic adaptation to a new niche plays a major role in the survival of B. cenocepacia in macrophages. This adaptive response does not require the expression of any specific virulence-associated factor, which is consistent with the opportunistic nature of this microorganism. Further investigation into the remaining SCOTS-identified genes will provide a more complete picture of the adaptive response of B. cenocepacia to the host cell environment.

In Vivo Expression of Salmonella enterica Serotype Typhi Genes in the Blood of Patients with Typhoid Fever in Bangladesh

Background Salmonella enterica serotype Typhi is the cause of typhoid fever. It is a human-restricted pathogen, and few data exist on S. Typhi gene expression in humans.Methodology/Principal FindingsWe applied an RNA capture and amplification technique, Selective Capture of Transcribed Sequences (SCOTS), and microarray hybridization to identify S. Typhi transcripts expressed in the blood of five humans infected with S. Typhi in Bangladesh. In total, we detected the expression of mRNAs for 2,046 S. Typhi genes (44% of the S. Typhi genome) in human blood; expression of 912 genes was detected in all 5 patients, and expression of 1,100 genes was detected in 4 or more patients. Identified transcripts were associated with the virulence-associated PhoP regulon, Salmonella pathogenicity islands, the use of alternative carbon and energy sources, synthesis and transport of iron, thiamine, and biotin, and resistance to antimicrobial peptides and oxidative stress. The most highly represented group were genes currently annotated as encoding proteins designated as hypothetical, unknown, or unclassified. Of the 2,046 detected transcripts, 1,320 (29% of the S. Typhi genome) had significantly different levels of detection in human blood compared to in vitro cultures; detection of 141 transcripts was significantly different in all 5 patients, and detection of 331 transcripts varied in at least 4 patients. These mRNAs encode proteins of unknown function, those involved in energy metabolism, transport and binding, cell envelope, cellular processes, and pathogenesis. We confirmed increased expression of a subset of identified mRNAs by quantitative-PCR.Conclusions/SignificanceWe report the first characterization of bacterial transcriptional profiles in the blood of patients with typhoid fever. S. Typhi is an important global pathogen whose restricted host range has greatly inhibited laboratory studies. Our results suggest that S. Typhi uses a largely uncharacterized genetic repertoire to survive within cells and utilize alternate energy sources during infection.

Analysis of Salmonella enterica Serotype Paratyphi A Gene Expression in the Blood of Bacteremic Patients in Bangladesh

Background Salmonella enterica serotype Paratyphi A is a human-restricted cause of paratyphoid fever, accounting for up to a fifth of all cases of enteric fever in Asia.Methodology/Principal FindingsIn this work, we applied an RNA analysis method, Selective Capture of Transcribed Sequences (SCOTS), and cDNA hybridization-microarray technology to identify S. Paratyphi A transcripts expressed by bacteria in the blood of three patients in Bangladesh. In total, we detected 1,798 S. Paratyphi A mRNAs expressed in the blood of infected humans (43.9% of the ORFeome). Of these, we identified 868 in at least two patients, and 315 in all three patients. S. Paratyphi A transcripts identified in at least two patients encode proteins involved in energy metabolism, nutrient and iron acquisition, vitamin biosynthesis, stress responses, oxidative stress resistance, and pathogenesis. A number of detected transcripts are expressed from PhoP and SlyA-regulated genes associated with intra-macrophage survival, genes contained within Salmonella Pathogenicity Islands (SPIs) 1–4, 6, 10, 13, and 16, as well as RpoS-regulated genes. The largest category of identified transcripts is that of encoding proteins with unknown function. When comparing levels of bacterial mRNA using in vivo samples collected from infected patients to samples from in vitro grown organisms, we found significant differences for 347, 391, and 456 S. Paratyphi A transcripts in each of three individual patients (approximately 9.7% of the ORFeome). Of these, expression of 194 transcripts (4.7% of ORFs) was concordant in two or more patients, and 41 in all patients. Genes encoding these transcripts are contained within SPI-1, 3, 6 and 10, PhoP-regulated genes, involved in energy metabolism, nutrient acquisition, drug resistance, or uncharacterized genes. Using quantitative RT-PCR, we confirmed increased gene expression in vivo for a subset of these genes.Conclusion/SignificanceTo our knowledge, we describe the first microarray-based transcriptional analysis of a pathogen in the blood of naturally infected humans.

Differential gene expression in bacterial symbionts from loliginid squids demonstrates variation between mutualistic and environmental niches.

Luminescent bacteria (gamma-Proteobacteria: Vibrionaceae) are found in complex bilobed light organs of both sepiolid and loliginid squids (Mollusca: Cephalopoda). Despite the existence of multiple strain colonization between Vibrio bacteria and loliginid squids, specificity at the genus level still exists and may influence interactions between symbiotic and free-living stages of the symbiont. The environmentally transmitted behaviour of Vibrio symbionts bestows a certain degree of recognition that exists prior and subsequent to the colonization process. Therefore, we identified bacterial genes required for successful colonization of loliginid light organs by examining transcripts solely expressed in either the light organ or free-living stages. Selective capture of transcribed sequences (SCOTS) was used to differentiate genes expressed by the same bacterium when thriving in two different environments (i.e. loliginid light organs and seawater). Genes specific for squid light organs included vulnibactin synthetase, outer membrane protein W and dihydroxy dehydratase, which have been associated with the maintenance of bacterial host associations in other systems. In contrast, genes that were solely expressed in the free-living condition consisted of transcripts recognized as important factors for bacterial survival in the environment. These transcripts included genes for methyl accepting chemotaxis proteins, arginine decarboxylase and chitinase. These results provide valuable information regarding mechanisms determining specificity, establishment, and maintenance of bacteria-squid associations.

Identification of Streptococcus suis serotype 2 genes preferentially expressed in the natural host

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen for swine and humans. Previous research about the mechanism of SS2 infection was largely established on in vitro or ex vivo models. In this study, we focused on the identification of SS2 genes preferentially expressed in vivo during natural infection in pigs. Eighty SS2 genes were identified to be up-regulated in the porcine brains and lungs by selective capture of transcribed sequences (SCOTS) and comparative dot blot analysis, followed by quantitative RT-PCR validation. These genes could be classified into 5 functional categories: metabolism, cell wall associated proteins, transporters, cell replication, and function unknown. Some of these genes may contribute to the survival and pathogenesis of SS2 in the host via the following strategies. First, SS2 evades the host innate immune clearance through modifying its metabolism and cell wall composition as indicated by the up-regulation of the corresponding gene ldh and pbp2A, respectively. Secondly, SS2 adapts to the in vivo conditions by inducing the expression of the two-component signal transduction system VicKR which may function on the target genes such as pcsB involved in stress response and cell wall biosynthesis. Thirdly, SS2 enhances its virulence in vivo by up-regulating the virulence genes, such as sly, pdgA, ssp, gidA, gcp and hp1311. Further study of these in vivo up-regulated genes will contribute to understanding the in vivo survival mechanism and pathogenesis of SS2.

Identification ofStreptococcus suisgenes preferentially expressed under iron starvation by selective capture of transcribed sequences

Streptococcus suis serotype 2 is a zoonotic Gram-positive bacterium responsible for arthritis, meningitis, pneumonia and septicemia in swine and humans. Little information about the regulation of iron on its gene expression had been reported. In this study, 63 S. suis genes upregulated under an iron-restricted condition were identified using selective capture of transcribed sequences (SCUTS) technique: 23 genes involved in metabolism, 22 genes responsible for the replication and genetic information proceeding of the bacteria, eight genes relative to the construction of the cell wall, five ATP-binding cassette transporters, four transcriptional regulators and one uncharacterized gene conserved among streptococcal species. To adapt to the stress, S. suis modulated its physiological activities, which were validated by the upregulation of RelA (a crucial enzyme in stringent response), ArcA (a component of the arginine deiminase system catalyzing the conversion of arginine to ornithine) and CpdB (a cell surface protein that is a substrate of sortase A). All of them were reported to be virulence factors in S. suis or other bacteria. Besides, together with the results of quantitative reverse transcriptase PCR, we found that several homologous genes (fur, fhuGBDA operons) associated with iron uptake as reported in other bacteria were also upregulated under an iron-restricted condition in S. suis.

Identification of genes transcribed by Haemophilus parasuis in necrotic porcine lung through the selective capture of transcribed sequences (SCOTS)

Haemophilus parasuis is the aetiological agent of Glässer's disease, which has received more attention in the past decade due to the increasing economic losses in the pig industry worldwide. Little is known about the mechanisms by which H. parasuis survives in the host. In this study, selective capture of transcribed sequences (SCOTS) was used to identify H. parasuis genes upregulated in necrotic porcine lung 7 days post infection. Thirty-eight genes were identified that were upregulated during infection of the lung tissue of pigs, compared with growth in culture medium. In two examples chosen gene expression was not confined to the lungs, there being variation between tissues. The data support biofilm formation being an important mode of growth for colonization and/or persistence. Results from the in vitro studies suggest that, as for other pathogens, iron and oxygen restriction and heat stress are important environmental signals to regulate gene expression. This study has identified genes of H. parasuis that are upregulated during infection of porcine lung tissue as compared with in vitro growth conditions.

Transcriptional analysis of diverse strains Mycobacterium avium subspecies paratuberculosis in primary bovine monocyte derived macrophages

In this study we analyzed the macrophage-induced gene expression of three diverse genotypes of Mycobacterium avium subsp. paratuberculosis (MAP). Using selective capture of transcribed sequences (SCOTS) on three genotypically diverse MAP isolates from cattle, human, and sheep exposed to primary bovine monocyte derived macrophages for 48 h and 120 h we created and sequenced six cDNA libraries. Sequence annotations revealed that the cattle isolate up-regulated 27 and 241 genes; the human isolate up-regulated 22 and 53 genes, and the sheep isolate up-regulated 35 and 358 genes, at the two time points respectively. Thirteen to thirty-three percent of the genes identified did not have any annotated function. Despite variations in the genes identified, the patterns of expression fell into overlapping cellular functions as inferred by pathway analysis. For example, 10–12% of the genes expressed by all three strains at each time point were associated with cell-wall biosynthesis. All three strains of MAP studied up-regulated genes in pathways that combat oxidative stress, metabolic and nutritional starvation, and cell survival. Taken together, this comparative transcriptional analysis suggests that diverse MAP genotypes respond with similar modus operandi for survival in the host.

Identification of Haemophilus ducreyi genes expressed during human infection

To identify Haemophilus ducreyi transcripts that are expressed during human infection, we used selective capture of transcribed sequences (SCOTS) with RNA isolated from pustules obtained from three volunteers infected with H. ducreyi, and with RNA isolated from broth-grown bacteria used to infect volunteers. With SCOTS, competitive hybridization of tissue-derived and broth-derived sequences identifies genes that may be preferentially expressed in vivo. Among the three tissue specimens, we identified 531 genes expressed in vivo. Southern blot analysis of 60 genes from each tissue showed that 87 % of the identified genes hybridized better with cDNA derived from tissue specimens than with cDNA derived from broth-grown bacteria. RT-PCR on nine additional pustules confirmed in vivo expression of 10 of 11 selected genes in other volunteers. Of the 531 genes, 139 were identified in at least two volunteers. These 139 genes fell into several functional categories, including biosynthesis and metabolism, regulation, and cellular processes, such as transcription, translation, cell division, DNA replication and repair, and transport. Detection of genes involved in anaerobic and aerobic respiration indicated that H. ducreyi likely encounters both microenvironments within the pustule. Other genes detected suggest an increase in DNA damage and stress in vivo. Genes involved in virulence in other bacterial pathogens and 32 genes encoding hypothetical proteins were identified, and may represent novel virulence factors. We identified three genes, lspA1, lspA2 and tadA, known to be required for virulence in humans. This is the first study to broadly define transcripts expressed by H. ducreyi in humans.

Construction and Characterization of Avian Pathogenic Escherichia coli Mutants with iro and/or tsh Gene Mutation

Iro system and temperature-sensitive hemagglutinin (Tsh) genes were identified by suppression subtractive hybridization (SSH) and selective capture of transcribed sequences (SCOTS) in avian pathogenic Escherichia coli (APEC) isolates. To get more insights in the distribution and the occurrence of the iroC and tsh genes, 243 avian E. coli isolates were examined for the presences of these genes. Amongst 243 avian E. coli isolates, iroC gene was present in 84.4% strains (205/243). Of the 205 iroC-positive isolates, iroC gene was found in 184 (89.8%), 18 (8.8%) and 3 (1.5%) isolates with high, intermediate and low pathogenicity, respectively. Of the 167 tsh-positive isolates, tsh gene was detected in 146 (87.4%), 21 (12.6%) and 0 (0%) isolates with high, intermediate and low pathogenicity, respectively. Amongst tsh-positive isolates, 89.5 to 100% of the highly pathogenic isolates of O1, O2 or O78 serogroups had the tsh gene, while 53.3% of the highly pathogenic isolates of non-O1, O2 and O78 serogroups had the tsh gene ( P<0.01). Suicide vectors for deletion of the iroBCDEN or tsh genes were constructed as follows. The 715-bp fragment of iroB and 603-bp fragment of the iroN were amplified by PCR, respectively. Both these two fragments together with EGFP gene were cloned into pUC18, termed pUC18-iroBNEGFP. A resultant suicide vector combined with the iroB-EGFP-iroN fragment was obtained and named pMEG375-iroBNEGFP. Similarly, both the 685-bp fragment of tshF and the 692-bp fragment of the tshR together with gentamycin gene were cloned into pUC18, resulting in pUC18-tshFRGm, and a resultant suicide vector combined with the tshF-Gm-tshR fragment was named pMEG375-tshFRGm. Mutant derivatives of strain E037 were generated by allelic replacements and were named E037 (Δiro), E037 (Δtsh) and E037 (ΔiroΔtsh). The 50% lethal dose (LD50) of E037, E037 (Δiro), E037 (Δtsh) and E037 (ΔiroΔtsh) in commercial day-old chickens experimentally inoculated via intratrachea were determined as 10 5.6, 10 8.4, 10 9.0 and 10 9.5CFU, respectively. In the chicken challenge model, the mutants were evaluated on the individual role of these genes for virulence and persistence in chickens. The result indicated that Iro system and Tsh were important in the pathogenicity of APEC.

Use of Selective Capture of Transcribed Sequences To Identify Genes Preferentially Expressed byStreptococcus suisupon Interaction with Porcine Brain Microvascular Endothelial Cells

By using the selective capture of transcribed sequences (SCOTS) approach, we identified 28 genes preferentially expressed by the major swine pathogen and zoonotic agent Streptococcus suis upon interaction with porcine brain microvascular endothelial cells. Several of these genes may be considered new S. suis candidate virulence factors. Results from this study demonstrate the suitability of SCOTS for the elucidation of gene expression in streptococcal species and may contribute to a better understanding of the pathogenesis of S. suis infections.

Selective capture of transcribed sequences (SCOTS) of Actinobacillus pleuropneumoniae in the chronic stage of disease reveals an HlyX-regulated autotransporter protein

Actinobacillus pleuropneumoniae, an important respiratory pathogen in swine, is able to persist in host tissues for extended periods of time. In the study presented here, selective capture of transcribed sequences (SCOTS) analysis was used to identify genes expressed by A. pleuropneumoniae in the chronic stage of the disease (21 days post infection). After isolation and reverse transcription of RNA from infected lungs as well as from culture-grown A. pleuropneumoniae, transcribed A. pleuropneumoniae sequences were captured from infected lung tissue and subjected to a subtractive hybridization procedure of lung-derived against culture-derived A. pleuropneumoniae cDNA. Twenty-nine of the thirty-six genes that were identified as in vivo-expressed are involved in transport or metabolic processes. We identified a surface-associated putative 104 kDa subtilisin-like autotransporter serine protease, designated AasP, which has not been described in A. pleuropneumoniae to date. The gene was shown to be present in all 15 A. pleuropneumoniae serotypes. It is transcribed in porcine lung tissue on days 7 and 21 post infection. Under anaerobic conditions in vitro, its expression depends on the global anaerobic regulator HlyX. To our knowledge, this is the first report of an autotransporter protein being regulated by a global anaerobic regulator.