Transcriptomic profiling of Clostridium difficile grown under microaerophillic conditions

Transcriptomic profiling of Clostridium difficile grown under microaerophillic conditions Abstract Clostridium difficile (Cd) is an anaerobic, spore-forming bacterium capable of colonizing the gastrointestinal tract of humans. Colonization usually occurs following antibiotic-induced disruption of the host microbiota, which also leads to an increase in oxygen within the gastrointestinal tract. We sought to understand how Cd responds to this microaerophilic condition that is likely experienced within the host. Transcriptome profiling showed differential regulation of genes involved in sugar metabolism, pyruvate metabolism and stress responses. These data provide insight into potential mechanisms of Cd adaptation to the host environment and should lead to the elucidation unknown mechanisms of Cd oxygen resistance and pathogenesis. Clostridium difficile, microaerophillic, transcriptomics INTRODUCTION Clostridium difficile (Cd) is an anaerobic, Gram-positive, spore-forming bacterium capable of colonizing the mammalian gastrointestinal tract following disruption of the microbiota, typically after antibiotic treatment (Leffler and Lamont 2015). Cd infection (CDI) is the leading cause of gastroenteritis- associated death in the USA with almost 500,000 infections and 29,000 deaths annually, with healthcare costs exceeding $1 billion (Lessa et al.2015). CDI has historically been a nosocomial infection; however, more recently there has been an increase in community-acquired infections. CDI is transmitted by the aerotolerant and metabolically dormant spore form of Cd. Cd spores are ubiquitous in the environment and can be found anywhere from hospital surfaces to recreational waters (Hensgens et al.2012; Eyre et al.2013). These spores, which are also acid, antibiotic and heat resistant, enter the host via the fecal-oral route (Leffler and Lamont 2015). Following ingestion, the spores germinate in response to host bile salts present in the gasterointestinal tract (Sorg and Sonenshein 2008). Germination of spores leads to outgrowth and eventual rapid division of vegetative Cd bacilli, which produce virulence factors, including toxins, that cause the pathology associated with CDI. The primary risk factor for CDI is the disruption of the normal microbiota of the gastrointestinal tract, typically due to antibiotic treatment. A healthy gastrointestinal tract with a diverse microbiota maintains low oxygen levels under normal circumstances (Kelly et al.2015). However, there is a gradient of oxygen near the surface of the intestinal epithelium as demonstrated by GFP fluorescence (Marteyn et al.2010). Additionally, the level of oxygen can increase during antibiotic treatment (Kelly et al.2015; Rivera-Chávez et al.2016). One proposed mechanism behind the increase in oxygen levels is the antibiotic-mediated depletion of commensal bacteria that produce butyrate, a short-chain fatty acid that reacts with oxygen in the epithelium (Rivera-Chávez et al.2016). Furthermore, the colon of a germ-free mouse has a higher oxygen concentration than conventional mice, demonstrating a role for commensal bacteria in maintaining low levels of oxygen (Kelly et al.2015). Here, we used transcriptome profiling to search for oxygen responsive expression patterns that could lead to the identification of putative mechanisms of oxygen resistance and pathogenesis in Cd. These data provide a greater understanding of how Cd responds to low levels of oxygen, which might be encountered within the host environment. MATERIALS AND METHODS Bacterial cultivation and growth conditions All experiments described here were performed with Cd strain 630. Cd was grown in an anaerobic chamber or hypoxic chamber (Coy Laboratory Products, MI) as indicated. Anaerobic conditions were monitored with oxygen sensors and maintained at below 0.002%. The hypoxic chamber is capable of stable oxygen levels down to approximately 0.5% ± 0.1%. Cd was cultured in BHIS (brain–heart infusion broth supplemented with 0.5% yeast extract and 0.1% cysteine) for all experiments. Media was equilibrated in the desired growth environment for a minimum of 48 h prior to initiation of each experiment. Overnight cultures were grown anaerobically at 28°C then back diluted 1:10 and allowed to grow for an additional hour at 37°C to ensure growth experiments were initiated with actively growing bacteria. These cultures were then used to inoculate cultures under the indicated conditions. Each culture was inoculated with Cd 630 at a starting OD600 of 0.05. For growth curves shown, Cd was grown in the indicated oxygen environment at 37°C with shaking (250 rpm) to ensure uniform oxygenation of cultures. RNA isolation and analysis RNA isolations were performed as previously described (Carlson et al.2009, 2015). Briefly, at indicated timepoints, cultures were passed over a 0.2 mM filter. Bacteria were resuspended from the filter with ice-cold nuclease-free water. Lysis buffer (2% SDS, 10 mM EDTA, 200 mM NaCl) was immediately added to the resuspended bacteria, and the mixture was placed in a boiling water bath for 3 min. All steps prior to placing the sample in the boiling water bath were performed within either an anaerobic or hypoxic chamber. Nucleic acid was subsequently isolated through two hot phenol extractions (65°C) followed by phenol:chloroform and chloroform extractions (22°C). The isolated nucleic acids were then precipitated overnight at −20°C following the addition of 0.1 volume ammonium acetate and 2.5 volumes of ethanol. The remaining steps including DNase treatment of the RNA samples, microarray sample preparation and microarray hybridization were performed as described previously (Carlson et al.2009, 2015). The microarrays used were custom designed Cd Agilent arrays. Microarray data analysis Analysis of microarray data was performed as previously described (Carlson et al.2009, 2015). Tables S1 and S2 (Supporting Information) report both the J5 value and fold change for all genes with a significant change in transcription levels between conditions. Quantitative PCR All quantitative PCR (qPCR) experiments were performed as previously described (Carlson et al.2009; Carlson et al.2015). Cd630_04380 was used as the internal reference, as its expression was determined to remain unchanged between growth conditions tested (not shown). RESULTS AND DISCUSSION Although often considered a strict anaerobic environment, levels of oxygen in the gastrointestinal tract range from 2% to 7% oxygen (He et al.1999). Since Cd is typically considered a strictly anaerobic bacterium, and therefore highly sensitive to oxygen, we sought to understand how Cd responds to low levels of oxygen. To assess the ability of Cd to grow under microaerophillic conditions, cultures were grown under a range of oxygen concentrations in a modified hypoxic chamber. Cd was grown in BHIS broth under 0%, 1%, 2%, 3% or 5% oxygen (Fig. 1a). While the bacterium was unable to grow in the presence of 3% or 5% oxygen, significant growth was observed under 1% and 2% oxygen (Fig. 1a, blue and red lines). Subsequent experiments were performed under 2% oxygen due to both the biological relevance of this concentration and the attenuated growth of Cd under these conditions (Fig. 1a). RNA was isolated following 4.5 h of growth under either 0% or 2% oxygen and used to assess gene expression changes induced by the presence of oxygen. Figure 1. View largeDownload slide Growth of C. difficile in low oxygen conditions. (A) Growth was monitored over time by measuring the change in OD600 at the following oxygen conditions; green—0, blue–1%, red–2%, purple—3%, light brown—5%. (B) Growth experiments used for RNA isolation. Quadruplicate cultures were grown in either 2% oxygen (red lines) or under anaerobic (green lines) conditions as indicated. RNA was harvested at 4.5 h after inoculation (arrow). Figure 1. View largeDownload slide Growth of C. difficile in low oxygen conditions. (A) Growth was monitored over time by measuring the change in OD600 at the following oxygen conditions; green—0, blue–1%, red–2%, purple—3%, light brown—5%. (B) Growth experiments used for RNA isolation. Quadruplicate cultures were grown in either 2% oxygen (red lines) or under anaerobic (green lines) conditions as indicated. RNA was harvested at 4.5 h after inoculation (arrow). RNA isolated from three independent cultures was used to assess gene expression changes on a custom Agilent Cd microarray. Statistical significance was determined by J5-score and fold change (Patel and Lyons-Weiler 2004). Following 4.5 h of exposure to oxygen, the expression of 198 genes was significantly altered (109 induced and 89 repressed; Tables S1 and S2, Supporting Information). Table 1 shows the 25 genes that were most significantly induced during growth in 2% oxygen. All genes that exhibited significant changes in gene expression were analyzed using GenePattern software (Golub et al.1999) for hierarchical clustering (Fig. 2a). Several genes identified as significantly changed were then chosen for confirmation by qPCR. Genes that were both induced and repressed were chosen for this assessment. Expression patterns observed for these genes were consistent with the microarray data (Fig. 2b). Figure 2. View largeDownload slide Validation of microarray data. Gene expression changes were measured by qPCR at 4.5 h. Four independent RNA samples were tested for expression of specific genes using quantitative real-time PCR. The results of qPCR are represented with white bars (n = 4), while corresponding values from the microarray experiments are represented with black bars (n = 3). Data are presented as mean ± SEM of log2 transformed fold change where fold change is the ratio of expression in 2% oxygen compared to 0% oxygen. Figure 2. View largeDownload slide Validation of microarray data. Gene expression changes were measured by qPCR at 4.5 h. Four independent RNA samples were tested for expression of specific genes using quantitative real-time PCR. The results of qPCR are represented with white bars (n = 4), while corresponding values from the microarray experiments are represented with black bars (n = 3). Data are presented as mean ± SEM of log2 transformed fold change where fold change is the ratio of expression in 2% oxygen compared to 0% oxygen. Table 1. Twenty-five genes most induced during growth in 2% oxygen. Locus tag  J5  F.C.  Abbreviation  Description  CD630_22700  9.08  6.53  fruK  Fructose 1-phosphate kinase  CD630_22690  8.15  5.86  fruABC  PTS system fructose-specific transporter subunit IIABC  CD630_29471  7.83  5.63  –  Hypothetical protein CD630_29471  CD630_23270  7.76  5.58  –  PTS system fructose/mannitol family transporter subunit IIA  CD630_09240  7.45  5.36  –  Hypothetical protein CD630_09240  CD630_29380  7.33  5.27  –  Hypothetical protein CD630_29380  CD630_29320  7.32  5.27  –  Hypothetical protein CD630_29320  CD630_29300  7.31  5.26  –  Anti-repressor protein  CD630_29410  7.28  5.23  –  Single-strand DNA-binding protein  CD630_29400  7.23  5.20  –  Hypothetical protein CD630_29400  CD630_29390  7.16  5.15  –  Hypothetical protein CD630_29390  CD630_29490  7.13  5.13  –  Transcriptional regulator  CD630_29480  7.10  5.11  –  Hypothetical protein CD630_29480  CD630_26000  7.10  5.11  cstA  Carbon starvation protein, CstA  CD630_09210  6.81  4.90  –  Hypothetical protein CD630_09210  CD630_29330  6.79  4.88  –  Hypothetical protein CD630_29330  CD630_03010  6.73  4.84  rbsA  ABC transporter ribose-specific ATP-binding protein  CD630_29360  6.67  4.80  –  Hypothetical protein CD630_29360  CD630_29350  6.54  4.70  –  Hypothetical protein CD630_29350  CD630_29340  6.50  4.67  –  Hypothetical protein CD630_29340  CD630_09200  6.47  4.65  –  Hypothetical protein CD630_09200  CD630_29420  6.41  4.61  –  Resolvase/integrase  CD630_29310  6.36  4.57  –  Endodeoxyribonuclease RusA-like  CD630_02990  6.35  4.57  rbsK  Ribokinase, pfkB family  CD630_03000  6.15  4.42  rbsB  ABC transporter ribose-specific extracellular solute-binding protein  Locus tag  J5  F.C.  Abbreviation  Description  CD630_22700  9.08  6.53  fruK  Fructose 1-phosphate kinase  CD630_22690  8.15  5.86  fruABC  PTS system fructose-specific transporter subunit IIABC  CD630_29471  7.83  5.63  –  Hypothetical protein CD630_29471  CD630_23270  7.76  5.58  –  PTS system fructose/mannitol family transporter subunit IIA  CD630_09240  7.45  5.36  –  Hypothetical protein CD630_09240  CD630_29380  7.33  5.27  –  Hypothetical protein CD630_29380  CD630_29320  7.32  5.27  –  Hypothetical protein CD630_29320  CD630_29300  7.31  5.26  –  Anti-repressor protein  CD630_29410  7.28  5.23  –  Single-strand DNA-binding protein  CD630_29400  7.23  5.20  –  Hypothetical protein CD630_29400  CD630_29390  7.16  5.15  –  Hypothetical protein CD630_29390  CD630_29490  7.13  5.13  –  Transcriptional regulator  CD630_29480  7.10  5.11  –  Hypothetical protein CD630_29480  CD630_26000  7.10  5.11  cstA  Carbon starvation protein, CstA  CD630_09210  6.81  4.90  –  Hypothetical protein CD630_09210  CD630_29330  6.79  4.88  –  Hypothetical protein CD630_29330  CD630_03010  6.73  4.84  rbsA  ABC transporter ribose-specific ATP-binding protein  CD630_29360  6.67  4.80  –  Hypothetical protein CD630_29360  CD630_29350  6.54  4.70  –  Hypothetical protein CD630_29350  CD630_29340  6.50  4.67  –  Hypothetical protein CD630_29340  CD630_09200  6.47  4.65  –  Hypothetical protein CD630_09200  CD630_29420  6.41  4.61  –  Resolvase/integrase  CD630_29310  6.36  4.57  –  Endodeoxyribonuclease RusA-like  CD630_02990  6.35  4.57  rbsK  Ribokinase, pfkB family  CD630_03000  6.15  4.42  rbsB  ABC transporter ribose-specific extracellular solute-binding protein  View Large Table 1. Twenty-five genes most induced during growth in 2% oxygen. Locus tag  J5  F.C.  Abbreviation  Description  CD630_22700  9.08  6.53  fruK  Fructose 1-phosphate kinase  CD630_22690  8.15  5.86  fruABC  PTS system fructose-specific transporter subunit IIABC  CD630_29471  7.83  5.63  –  Hypothetical protein CD630_29471  CD630_23270  7.76  5.58  –  PTS system fructose/mannitol family transporter subunit IIA  CD630_09240  7.45  5.36  –  Hypothetical protein CD630_09240  CD630_29380  7.33  5.27  –  Hypothetical protein CD630_29380  CD630_29320  7.32  5.27  –  Hypothetical protein CD630_29320  CD630_29300  7.31  5.26  –  Anti-repressor protein  CD630_29410  7.28  5.23  –  Single-strand DNA-binding protein  CD630_29400  7.23  5.20  –  Hypothetical protein CD630_29400  CD630_29390  7.16  5.15  –  Hypothetical protein CD630_29390  CD630_29490  7.13  5.13  –  Transcriptional regulator  CD630_29480  7.10  5.11  –  Hypothetical protein CD630_29480  CD630_26000  7.10  5.11  cstA  Carbon starvation protein, CstA  CD630_09210  6.81  4.90  –  Hypothetical protein CD630_09210  CD630_29330  6.79  4.88  –  Hypothetical protein CD630_29330  CD630_03010  6.73  4.84  rbsA  ABC transporter ribose-specific ATP-binding protein  CD630_29360  6.67  4.80  –  Hypothetical protein CD630_29360  CD630_29350  6.54  4.70  –  Hypothetical protein CD630_29350  CD630_29340  6.50  4.67  –  Hypothetical protein CD630_29340  CD630_09200  6.47  4.65  –  Hypothetical protein CD630_09200  CD630_29420  6.41  4.61  –  Resolvase/integrase  CD630_29310  6.36  4.57  –  Endodeoxyribonuclease RusA-like  CD630_02990  6.35  4.57  rbsK  Ribokinase, pfkB family  CD630_03000  6.15  4.42  rbsB  ABC transporter ribose-specific extracellular solute-binding protein  Locus tag  J5  F.C.  Abbreviation  Description  CD630_22700  9.08  6.53  fruK  Fructose 1-phosphate kinase  CD630_22690  8.15  5.86  fruABC  PTS system fructose-specific transporter subunit IIABC  CD630_29471  7.83  5.63  –  Hypothetical protein CD630_29471  CD630_23270  7.76  5.58  –  PTS system fructose/mannitol family transporter subunit IIA  CD630_09240  7.45  5.36  –  Hypothetical protein CD630_09240  CD630_29380  7.33  5.27  –  Hypothetical protein CD630_29380  CD630_29320  7.32  5.27  –  Hypothetical protein CD630_29320  CD630_29300  7.31  5.26  –  Anti-repressor protein  CD630_29410  7.28  5.23  –  Single-strand DNA-binding protein  CD630_29400  7.23  5.20  –  Hypothetical protein CD630_29400  CD630_29390  7.16  5.15  –  Hypothetical protein CD630_29390  CD630_29490  7.13  5.13  –  Transcriptional regulator  CD630_29480  7.10  5.11  –  Hypothetical protein CD630_29480  CD630_26000  7.10  5.11  cstA  Carbon starvation protein, CstA  CD630_09210  6.81  4.90  –  Hypothetical protein CD630_09210  CD630_29330  6.79  4.88  –  Hypothetical protein CD630_29330  CD630_03010  6.73  4.84  rbsA  ABC transporter ribose-specific ATP-binding protein  CD630_29360  6.67  4.80  –  Hypothetical protein CD630_29360  CD630_29350  6.54  4.70  –  Hypothetical protein CD630_29350  CD630_29340  6.50  4.67  –  Hypothetical protein CD630_29340  CD630_09200  6.47  4.65  –  Hypothetical protein CD630_09200  CD630_29420  6.41  4.61  –  Resolvase/integrase  CD630_29310  6.36  4.57  –  Endodeoxyribonuclease RusA-like  CD630_02990  6.35  4.57  rbsK  Ribokinase, pfkB family  CD630_03000  6.15  4.42  rbsB  ABC transporter ribose-specific extracellular solute-binding protein  View Large Genes with annotated functions in various metabolic processes, especially sugar and amino acid metabolism, exhibited the greatest expression changes during growth in oxygen. These data appear to show a shift in carbohydrate utilization during growth in the presence of 2% oxygen. Interestingly, induction of genes for uptake of ribose, fructose, mannose and lactose was observed along with repression of genes involved in beta-glucoside and glucose import. A previous study examining gene expression profiles of Cd during murine infection found induction of fructose-specific PTS systems and repression of those associated with glucose, identical to the results seen here (Jenior et al.2017). The carbohydrate composition is altered in cecal contents from Cd-susceptible mice compared to Cd-resistant mice (Theriot et al.2014). However, given the large number of significantly altered transporters seen in this study and the number of carbohydrate transporters encoded in the Cd genome, it is unclear how these data correlate to those of our current study. Overall, our data suggest a shift in carbohydrate metabolism when Cd is grown in the presence of oxygen, and these data may be relevant to in vivo growth. Several genes involved in energy generation during anaerobic growth were altered when exposed to 2% oxygen. These changes suggest that stress-induced adaptations to microaerophilic conditions are occurring; however, no obvious superoxide stress responses were induced. Some genes with Fe-S clusters, including acnB and nrdDG, were induced by oxygen exposure, which would be expected to inactivate these proteins. Escherichia coli encodes two aconitates, AcnA and AcnB, which function as post-transcriptional regulars in the Fe-S inactivated apo form and contribute to expression of superoxide stress responses (Tang et al.2002). AcnB is hypothesized to be the first level of stress response yielding to AcnA and SoxRS regulation as superoxide stress increases (Tang et al.2002). Perhaps AcnB also acts as an initial defense in Cd, and other superoxide responses would be altered with continued oxidative stress. In addition, superoxide stress can induce DNA damage. The class III ribonucleotide reductases, nrdD and nrdG, are involved in DNA repair and synthesis genes under anaerobic conditions (Garriga et al.1996). These genes were induced in the presence of 2% oxygen, though it is unclear if these proteins are functional in microaerophilic conditions. The genome of Cd strain 630 has many mobile genetic elements, including two prophages (Sebaihia et al.2006). Twenty-two genes encoding for one of the prophages were induced in low-oxygen conditions. Antibiotic treatment can induce the replication of these Cd prophages and enhance prophage mobility in vitro and in vivo (Meessen-Pinard, Sekulovic and Fortier 2012). Given these data, it is likely that prophage gene expression seen here is the result of a stress response in Cd, rather than a specific response to oxygen. One caveat of this study is that the two conditions tested differ greatly in bacterial growth rates and some of the observed changes may be due to this difference rather than oxygen exposure. Genes involved in sporulation were identified as significantly repressed during growth in 2% oxygen. Although it is clear that growth in oxygen leads to a stress response in Cd, sporulation does not appear to be specifically controlled by exposure to oxygen. Likewise, expression of toxin genes was identified as repressed during growth in oxygenated conditions. As both sporulation and toxin genes are expressed late during the growth cycle of Cd (Saujet et al.2011), these observed differences are more likely due to the attenuated growth seen in Cd during growth in 2% oxygen (Fig. 2b) than a result of oxygen exposure specifically. Overall, these data show that there are significant changes in the Cd transcriptome when Cd is grown in 2% oxygen in comparison to anaerobic conditions. Most of these changes are in metabolic systems and potential stress responses. The data presented here show potential targets for genetic manipulation and understanding how Cd survives in the low levels of oxygen present in the human gastrointestinal tract. AVAILABILITY OF SUPPORTING DATA Microarray data have been deposited in the NCBI GEO database under accession number GSE109175. FUNDING This work was supported by the Intramural Research Program of the Center for Biologics Evaluation and Research, Food and Drug Administration. This project was supported in part by an appointment to the Research Fellowship Program at the OVRR/CBER, US Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and Food and Drug Administration. 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Transcriptomic profiling of Clostridium difficile grown under microaerophillic conditions

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Abstract

Abstract Clostridium difficile (Cd) is an anaerobic, spore-forming bacterium capable of colonizing the gastrointestinal tract of humans. Colonization usually occurs following antibiotic-induced disruption of the host microbiota, which also leads to an increase in oxygen within the gastrointestinal tract. We sought to understand how Cd responds to this microaerophilic condition that is likely experienced within the host. Transcriptome profiling showed differential regulation of genes involved in sugar metabolism, pyruvate metabolism and stress responses. These data provide insight into potential mechanisms of Cd adaptation to the host environment and should lead to the elucidation unknown mechanisms of Cd oxygen resistance and pathogenesis. Clostridium difficile, microaerophillic, transcriptomics INTRODUCTION Clostridium difficile (Cd) is an anaerobic, Gram-positive, spore-forming bacterium capable of colonizing the mammalian gastrointestinal tract following disruption of the microbiota, typically after antibiotic treatment (Leffler and Lamont 2015). Cd infection (CDI) is the leading cause of gastroenteritis- associated death in the USA with almost 500,000 infections and 29,000 deaths annually, with healthcare costs exceeding $1 billion (Lessa et al.2015). CDI has historically been a nosocomial infection; however, more recently there has been an increase in community-acquired infections. CDI is transmitted by the aerotolerant and metabolically dormant spore form of Cd. Cd spores are ubiquitous in the environment and can be found anywhere from hospital surfaces to recreational waters (Hensgens et al.2012; Eyre et al.2013). These spores, which are also acid, antibiotic and heat resistant, enter the host via the fecal-oral route (Leffler and Lamont 2015). Following ingestion, the spores germinate in response to host bile salts present in the gasterointestinal tract (Sorg and Sonenshein 2008). Germination of spores leads to outgrowth and eventual rapid division of vegetative Cd bacilli, which produce virulence factors, including toxins, that cause the pathology associated with CDI. The primary risk factor for CDI is the disruption of the normal microbiota of the gastrointestinal tract, typically due to antibiotic treatment. A healthy gastrointestinal tract with a diverse microbiota maintains low oxygen levels under normal circumstances (Kelly et al.2015). However, there is a gradient of oxygen near the surface of the intestinal epithelium as demonstrated by GFP fluorescence (Marteyn et al.2010). Additionally, the level of oxygen can increase during antibiotic treatment (Kelly et al.2015; Rivera-Chávez et al.2016). One proposed mechanism behind the increase in oxygen levels is the antibiotic-mediated depletion of commensal bacteria that produce butyrate, a short-chain fatty acid that reacts with oxygen in the epithelium (Rivera-Chávez et al.2016). Furthermore, the colon of a germ-free mouse has a higher oxygen concentration than conventional mice, demonstrating a role for commensal bacteria in maintaining low levels of oxygen (Kelly et al.2015). Here, we used transcriptome profiling to search for oxygen responsive expression patterns that could lead to the identification of putative mechanisms of oxygen resistance and pathogenesis in Cd. These data provide a greater understanding of how Cd responds to low levels of oxygen, which might be encountered within the host environment. MATERIALS AND METHODS Bacterial cultivation and growth conditions All experiments described here were performed with Cd strain 630. Cd was grown in an anaerobic chamber or hypoxic chamber (Coy Laboratory Products, MI) as indicated. Anaerobic conditions were monitored with oxygen sensors and maintained at below 0.002%. The hypoxic chamber is capable of stable oxygen levels down to approximately 0.5% ± 0.1%. Cd was cultured in BHIS (brain–heart infusion broth supplemented with 0.5% yeast extract and 0.1% cysteine) for all experiments. Media was equilibrated in the desired growth environment for a minimum of 48 h prior to initiation of each experiment. Overnight cultures were grown anaerobically at 28°C then back diluted 1:10 and allowed to grow for an additional hour at 37°C to ensure growth experiments were initiated with actively growing bacteria. These cultures were then used to inoculate cultures under the indicated conditions. Each culture was inoculated with Cd 630 at a starting OD600 of 0.05. For growth curves shown, Cd was grown in the indicated oxygen environment at 37°C with shaking (250 rpm) to ensure uniform oxygenation of cultures. RNA isolation and analysis RNA isolations were performed as previously described (Carlson et al.2009, 2015). Briefly, at indicated timepoints, cultures were passed over a 0.2 mM filter. Bacteria were resuspended from the filter with ice-cold nuclease-free water. Lysis buffer (2% SDS, 10 mM EDTA, 200 mM NaCl) was immediately added to the resuspended bacteria, and the mixture was placed in a boiling water bath for 3 min. All steps prior to placing the sample in the boiling water bath were performed within either an anaerobic or hypoxic chamber. Nucleic acid was subsequently isolated through two hot phenol extractions (65°C) followed by phenol:chloroform and chloroform extractions (22°C). The isolated nucleic acids were then precipitated overnight at −20°C following the addition of 0.1 volume ammonium acetate and 2.5 volumes of ethanol. The remaining steps including DNase treatment of the RNA samples, microarray sample preparation and microarray hybridization were performed as described previously (Carlson et al.2009, 2015). The microarrays used were custom designed Cd Agilent arrays. Microarray data analysis Analysis of microarray data was performed as previously described (Carlson et al.2009, 2015). Tables S1 and S2 (Supporting Information) report both the J5 value and fold change for all genes with a significant change in transcription levels between conditions. Quantitative PCR All quantitative PCR (qPCR) experiments were performed as previously described (Carlson et al.2009; Carlson et al.2015). Cd630_04380 was used as the internal reference, as its expression was determined to remain unchanged between growth conditions tested (not shown). RESULTS AND DISCUSSION Although often considered a strict anaerobic environment, levels of oxygen in the gastrointestinal tract range from 2% to 7% oxygen (He et al.1999). Since Cd is typically considered a strictly anaerobic bacterium, and therefore highly sensitive to oxygen, we sought to understand how Cd responds to low levels of oxygen. To assess the ability of Cd to grow under microaerophillic conditions, cultures were grown under a range of oxygen concentrations in a modified hypoxic chamber. Cd was grown in BHIS broth under 0%, 1%, 2%, 3% or 5% oxygen (Fig. 1a). While the bacterium was unable to grow in the presence of 3% or 5% oxygen, significant growth was observed under 1% and 2% oxygen (Fig. 1a, blue and red lines). Subsequent experiments were performed under 2% oxygen due to both the biological relevance of this concentration and the attenuated growth of Cd under these conditions (Fig. 1a). RNA was isolated following 4.5 h of growth under either 0% or 2% oxygen and used to assess gene expression changes induced by the presence of oxygen. Figure 1. View largeDownload slide Growth of C. difficile in low oxygen conditions. (A) Growth was monitored over time by measuring the change in OD600 at the following oxygen conditions; green—0, blue–1%, red–2%, purple—3%, light brown—5%. (B) Growth experiments used for RNA isolation. Quadruplicate cultures were grown in either 2% oxygen (red lines) or under anaerobic (green lines) conditions as indicated. RNA was harvested at 4.5 h after inoculation (arrow). Figure 1. View largeDownload slide Growth of C. difficile in low oxygen conditions. (A) Growth was monitored over time by measuring the change in OD600 at the following oxygen conditions; green—0, blue–1%, red–2%, purple—3%, light brown—5%. (B) Growth experiments used for RNA isolation. Quadruplicate cultures were grown in either 2% oxygen (red lines) or under anaerobic (green lines) conditions as indicated. RNA was harvested at 4.5 h after inoculation (arrow). RNA isolated from three independent cultures was used to assess gene expression changes on a custom Agilent Cd microarray. Statistical significance was determined by J5-score and fold change (Patel and Lyons-Weiler 2004). Following 4.5 h of exposure to oxygen, the expression of 198 genes was significantly altered (109 induced and 89 repressed; Tables S1 and S2, Supporting Information). Table 1 shows the 25 genes that were most significantly induced during growth in 2% oxygen. All genes that exhibited significant changes in gene expression were analyzed using GenePattern software (Golub et al.1999) for hierarchical clustering (Fig. 2a). Several genes identified as significantly changed were then chosen for confirmation by qPCR. Genes that were both induced and repressed were chosen for this assessment. Expression patterns observed for these genes were consistent with the microarray data (Fig. 2b). Figure 2. View largeDownload slide Validation of microarray data. Gene expression changes were measured by qPCR at 4.5 h. Four independent RNA samples were tested for expression of specific genes using quantitative real-time PCR. The results of qPCR are represented with white bars (n = 4), while corresponding values from the microarray experiments are represented with black bars (n = 3). Data are presented as mean ± SEM of log2 transformed fold change where fold change is the ratio of expression in 2% oxygen compared to 0% oxygen. Figure 2. View largeDownload slide Validation of microarray data. Gene expression changes were measured by qPCR at 4.5 h. Four independent RNA samples were tested for expression of specific genes using quantitative real-time PCR. The results of qPCR are represented with white bars (n = 4), while corresponding values from the microarray experiments are represented with black bars (n = 3). Data are presented as mean ± SEM of log2 transformed fold change where fold change is the ratio of expression in 2% oxygen compared to 0% oxygen. Table 1. Twenty-five genes most induced during growth in 2% oxygen. Locus tag  J5  F.C.  Abbreviation  Description  CD630_22700  9.08  6.53  fruK  Fructose 1-phosphate kinase  CD630_22690  8.15  5.86  fruABC  PTS system fructose-specific transporter subunit IIABC  CD630_29471  7.83  5.63  –  Hypothetical protein CD630_29471  CD630_23270  7.76  5.58  –  PTS system fructose/mannitol family transporter subunit IIA  CD630_09240  7.45  5.36  –  Hypothetical protein CD630_09240  CD630_29380  7.33  5.27  –  Hypothetical protein CD630_29380  CD630_29320  7.32  5.27  –  Hypothetical protein CD630_29320  CD630_29300  7.31  5.26  –  Anti-repressor protein  CD630_29410  7.28  5.23  –  Single-strand DNA-binding protein  CD630_29400  7.23  5.20  –  Hypothetical protein CD630_29400  CD630_29390  7.16  5.15  –  Hypothetical protein CD630_29390  CD630_29490  7.13  5.13  –  Transcriptional regulator  CD630_29480  7.10  5.11  –  Hypothetical protein CD630_29480  CD630_26000  7.10  5.11  cstA  Carbon starvation protein, CstA  CD630_09210  6.81  4.90  –  Hypothetical protein CD630_09210  CD630_29330  6.79  4.88  –  Hypothetical protein CD630_29330  CD630_03010  6.73  4.84  rbsA  ABC transporter ribose-specific ATP-binding protein  CD630_29360  6.67  4.80  –  Hypothetical protein CD630_29360  CD630_29350  6.54  4.70  –  Hypothetical protein CD630_29350  CD630_29340  6.50  4.67  –  Hypothetical protein CD630_29340  CD630_09200  6.47  4.65  –  Hypothetical protein CD630_09200  CD630_29420  6.41  4.61  –  Resolvase/integrase  CD630_29310  6.36  4.57  –  Endodeoxyribonuclease RusA-like  CD630_02990  6.35  4.57  rbsK  Ribokinase, pfkB family  CD630_03000  6.15  4.42  rbsB  ABC transporter ribose-specific extracellular solute-binding protein  Locus tag  J5  F.C.  Abbreviation  Description  CD630_22700  9.08  6.53  fruK  Fructose 1-phosphate kinase  CD630_22690  8.15  5.86  fruABC  PTS system fructose-specific transporter subunit IIABC  CD630_29471  7.83  5.63  –  Hypothetical protein CD630_29471  CD630_23270  7.76  5.58  –  PTS system fructose/mannitol family transporter subunit IIA  CD630_09240  7.45  5.36  –  Hypothetical protein CD630_09240  CD630_29380  7.33  5.27  –  Hypothetical protein CD630_29380  CD630_29320  7.32  5.27  –  Hypothetical protein CD630_29320  CD630_29300  7.31  5.26  –  Anti-repressor protein  CD630_29410  7.28  5.23  –  Single-strand DNA-binding protein  CD630_29400  7.23  5.20  –  Hypothetical protein CD630_29400  CD630_29390  7.16  5.15  –  Hypothetical protein CD630_29390  CD630_29490  7.13  5.13  –  Transcriptional regulator  CD630_29480  7.10  5.11  –  Hypothetical protein CD630_29480  CD630_26000  7.10  5.11  cstA  Carbon starvation protein, CstA  CD630_09210  6.81  4.90  –  Hypothetical protein CD630_09210  CD630_29330  6.79  4.88  –  Hypothetical protein CD630_29330  CD630_03010  6.73  4.84  rbsA  ABC transporter ribose-specific ATP-binding protein  CD630_29360  6.67  4.80  –  Hypothetical protein CD630_29360  CD630_29350  6.54  4.70  –  Hypothetical protein CD630_29350  CD630_29340  6.50  4.67  –  Hypothetical protein CD630_29340  CD630_09200  6.47  4.65  –  Hypothetical protein CD630_09200  CD630_29420  6.41  4.61  –  Resolvase/integrase  CD630_29310  6.36  4.57  –  Endodeoxyribonuclease RusA-like  CD630_02990  6.35  4.57  rbsK  Ribokinase, pfkB family  CD630_03000  6.15  4.42  rbsB  ABC transporter ribose-specific extracellular solute-binding protein  View Large Table 1. Twenty-five genes most induced during growth in 2% oxygen. Locus tag  J5  F.C.  Abbreviation  Description  CD630_22700  9.08  6.53  fruK  Fructose 1-phosphate kinase  CD630_22690  8.15  5.86  fruABC  PTS system fructose-specific transporter subunit IIABC  CD630_29471  7.83  5.63  –  Hypothetical protein CD630_29471  CD630_23270  7.76  5.58  –  PTS system fructose/mannitol family transporter subunit IIA  CD630_09240  7.45  5.36  –  Hypothetical protein CD630_09240  CD630_29380  7.33  5.27  –  Hypothetical protein CD630_29380  CD630_29320  7.32  5.27  –  Hypothetical protein CD630_29320  CD630_29300  7.31  5.26  –  Anti-repressor protein  CD630_29410  7.28  5.23  –  Single-strand DNA-binding protein  CD630_29400  7.23  5.20  –  Hypothetical protein CD630_29400  CD630_29390  7.16  5.15  –  Hypothetical protein CD630_29390  CD630_29490  7.13  5.13  –  Transcriptional regulator  CD630_29480  7.10  5.11  –  Hypothetical protein CD630_29480  CD630_26000  7.10  5.11  cstA  Carbon starvation protein, CstA  CD630_09210  6.81  4.90  –  Hypothetical protein CD630_09210  CD630_29330  6.79  4.88  –  Hypothetical protein CD630_29330  CD630_03010  6.73  4.84  rbsA  ABC transporter ribose-specific ATP-binding protein  CD630_29360  6.67  4.80  –  Hypothetical protein CD630_29360  CD630_29350  6.54  4.70  –  Hypothetical protein CD630_29350  CD630_29340  6.50  4.67  –  Hypothetical protein CD630_29340  CD630_09200  6.47  4.65  –  Hypothetical protein CD630_09200  CD630_29420  6.41  4.61  –  Resolvase/integrase  CD630_29310  6.36  4.57  –  Endodeoxyribonuclease RusA-like  CD630_02990  6.35  4.57  rbsK  Ribokinase, pfkB family  CD630_03000  6.15  4.42  rbsB  ABC transporter ribose-specific extracellular solute-binding protein  Locus tag  J5  F.C.  Abbreviation  Description  CD630_22700  9.08  6.53  fruK  Fructose 1-phosphate kinase  CD630_22690  8.15  5.86  fruABC  PTS system fructose-specific transporter subunit IIABC  CD630_29471  7.83  5.63  –  Hypothetical protein CD630_29471  CD630_23270  7.76  5.58  –  PTS system fructose/mannitol family transporter subunit IIA  CD630_09240  7.45  5.36  –  Hypothetical protein CD630_09240  CD630_29380  7.33  5.27  –  Hypothetical protein CD630_29380  CD630_29320  7.32  5.27  –  Hypothetical protein CD630_29320  CD630_29300  7.31  5.26  –  Anti-repressor protein  CD630_29410  7.28  5.23  –  Single-strand DNA-binding protein  CD630_29400  7.23  5.20  –  Hypothetical protein CD630_29400  CD630_29390  7.16  5.15  –  Hypothetical protein CD630_29390  CD630_29490  7.13  5.13  –  Transcriptional regulator  CD630_29480  7.10  5.11  –  Hypothetical protein CD630_29480  CD630_26000  7.10  5.11  cstA  Carbon starvation protein, CstA  CD630_09210  6.81  4.90  –  Hypothetical protein CD630_09210  CD630_29330  6.79  4.88  –  Hypothetical protein CD630_29330  CD630_03010  6.73  4.84  rbsA  ABC transporter ribose-specific ATP-binding protein  CD630_29360  6.67  4.80  –  Hypothetical protein CD630_29360  CD630_29350  6.54  4.70  –  Hypothetical protein CD630_29350  CD630_29340  6.50  4.67  –  Hypothetical protein CD630_29340  CD630_09200  6.47  4.65  –  Hypothetical protein CD630_09200  CD630_29420  6.41  4.61  –  Resolvase/integrase  CD630_29310  6.36  4.57  –  Endodeoxyribonuclease RusA-like  CD630_02990  6.35  4.57  rbsK  Ribokinase, pfkB family  CD630_03000  6.15  4.42  rbsB  ABC transporter ribose-specific extracellular solute-binding protein  View Large Genes with annotated functions in various metabolic processes, especially sugar and amino acid metabolism, exhibited the greatest expression changes during growth in oxygen. These data appear to show a shift in carbohydrate utilization during growth in the presence of 2% oxygen. Interestingly, induction of genes for uptake of ribose, fructose, mannose and lactose was observed along with repression of genes involved in beta-glucoside and glucose import. A previous study examining gene expression profiles of Cd during murine infection found induction of fructose-specific PTS systems and repression of those associated with glucose, identical to the results seen here (Jenior et al.2017). The carbohydrate composition is altered in cecal contents from Cd-susceptible mice compared to Cd-resistant mice (Theriot et al.2014). However, given the large number of significantly altered transporters seen in this study and the number of carbohydrate transporters encoded in the Cd genome, it is unclear how these data correlate to those of our current study. Overall, our data suggest a shift in carbohydrate metabolism when Cd is grown in the presence of oxygen, and these data may be relevant to in vivo growth. Several genes involved in energy generation during anaerobic growth were altered when exposed to 2% oxygen. These changes suggest that stress-induced adaptations to microaerophilic conditions are occurring; however, no obvious superoxide stress responses were induced. Some genes with Fe-S clusters, including acnB and nrdDG, were induced by oxygen exposure, which would be expected to inactivate these proteins. Escherichia coli encodes two aconitates, AcnA and AcnB, which function as post-transcriptional regulars in the Fe-S inactivated apo form and contribute to expression of superoxide stress responses (Tang et al.2002). AcnB is hypothesized to be the first level of stress response yielding to AcnA and SoxRS regulation as superoxide stress increases (Tang et al.2002). Perhaps AcnB also acts as an initial defense in Cd, and other superoxide responses would be altered with continued oxidative stress. In addition, superoxide stress can induce DNA damage. The class III ribonucleotide reductases, nrdD and nrdG, are involved in DNA repair and synthesis genes under anaerobic conditions (Garriga et al.1996). These genes were induced in the presence of 2% oxygen, though it is unclear if these proteins are functional in microaerophilic conditions. The genome of Cd strain 630 has many mobile genetic elements, including two prophages (Sebaihia et al.2006). Twenty-two genes encoding for one of the prophages were induced in low-oxygen conditions. Antibiotic treatment can induce the replication of these Cd prophages and enhance prophage mobility in vitro and in vivo (Meessen-Pinard, Sekulovic and Fortier 2012). Given these data, it is likely that prophage gene expression seen here is the result of a stress response in Cd, rather than a specific response to oxygen. One caveat of this study is that the two conditions tested differ greatly in bacterial growth rates and some of the observed changes may be due to this difference rather than oxygen exposure. Genes involved in sporulation were identified as significantly repressed during growth in 2% oxygen. Although it is clear that growth in oxygen leads to a stress response in Cd, sporulation does not appear to be specifically controlled by exposure to oxygen. Likewise, expression of toxin genes was identified as repressed during growth in oxygenated conditions. As both sporulation and toxin genes are expressed late during the growth cycle of Cd (Saujet et al.2011), these observed differences are more likely due to the attenuated growth seen in Cd during growth in 2% oxygen (Fig. 2b) than a result of oxygen exposure specifically. Overall, these data show that there are significant changes in the Cd transcriptome when Cd is grown in 2% oxygen in comparison to anaerobic conditions. Most of these changes are in metabolic systems and potential stress responses. The data presented here show potential targets for genetic manipulation and understanding how Cd survives in the low levels of oxygen present in the human gastrointestinal tract. AVAILABILITY OF SUPPORTING DATA Microarray data have been deposited in the NCBI GEO database under accession number GSE109175. FUNDING This work was supported by the Intramural Research Program of the Center for Biologics Evaluation and Research, Food and Drug Administration. This project was supported in part by an appointment to the Research Fellowship Program at the OVRR/CBER, US Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and Food and Drug Administration. 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Pathogens and DiseaseOxford University Press

Published: Mar 1, 2018

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