Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores

<p>Abstract</p> <p>Background</p> <p>The bacterial endospore (spore) has recently been proposed as a new surface display system. Antigens and enzymes have been successfully exposed on the surface layers of the <it>Bacillus subtilis </it>spore, but only in a few cases the efficiency of expression and the effective surface display and have been determined. We used this heterologous expression system to produce the A subunit of the urease of the animal pathogen <it>Helicobater acinonychis</it>. Ureases are multi-subunit enzymes with a central role in the virulence of various bacterial pathogens and necessary for colonization of the gastric mucosa by the human pathogen <it>H. pylori</it>. The urease subunit UreA has been recognized as a major antigen, able to induce high levels of protection against challenge infections.</p> <p>Results</p> <p>We expressed UreA from <it>H. acinonychis </it>on the <it>B. subtilis </it>spore coat by using three different spore coat proteins as carriers and compared the efficiency of surface expression and surface display obtained with the three carriers. A combination of western-, dot-blot and immunofluorescence microscopy allowed us to conclude that, when fused to CotB, UreA is displayed on the spore surface (ca. 1 × 10³recombinant molecules per spore), whereas when fused to CotC, although most efficiently expressed (7-15 × 10³recombinant molecules per spore) and located in the coat layer, it is not displayed on the surface. Experiments with CotG gave results similar to those with CotC, but the CotG-UreA recombinant protein appeared to be partially processed.</p> <p>Conclusion</p> <p>UreA was efficiently expressed on the spore coat of <it>B. subtilis </it>when fused to CotB, CotC or CotG. Of these three coat proteins CotC allows the highest efficiency of expression, whereas CotB is the most appropriate for the display of heterologous proteins on the spore surface.</p

Hypoxia and tissue destruction in pulmonary TB.

BACKGROUND: It is unknown whether lesions in human TB are hypoxic or whether this influences disease pathology. Human TB is characterised by extensive lung destruction driven by host matrix metalloproteinases (MMPs), particularly collagenases such as matrix metalloproteinase-1 (MMP-1). METHODS: We investigated tissue hypoxia in five patients with PET imaging using the tracer [18F]-fluoromisonidazole ([18F]FMISO) and by immunohistochemistry. We studied the regulation of MMP secretion in primary human cell culture model systems in normoxia, hypoxia, chemical hypoxia and by small interfering RNA (siRNA) inhibition. RESULTS: [18F]FMISO accumulated in regions of TB consolidation and around pulmonary cavities, demonstrating for the first time severe tissue hypoxia in man. Patlak analysis of dynamic PET data showed heterogeneous levels of hypoxia within and between patients. In Mycobacterium tuberculosis (M.tb)-infected human macrophages, hypoxia (1% pO2) upregulated MMP-1 gene expression 170-fold, driving secretion and caseinolytic activity. Dimethyloxalyl glycine (DMOG), a small molecule inhibitor which stabilises the transcription factor hypoxia-inducible factor (HIF)-1α, similarly upregulated MMP-1. Hypoxia did not affect mycobacterial replication. Hypoxia increased MMP-1 expression in primary respiratory epithelial cells via intercellular networks regulated by TB. HIF-1α and NF-κB regulated increased MMP-1 activity in hypoxia. Furthermore, M.tb infection drove HIF-1α accumulation even in normoxia. In human TB lung biopsies, epithelioid macrophages and multinucleate giant cells express HIF-1α. HIF-1α blockade, including by targeted siRNA, inhibited TB-driven MMP-1 gene expression and secretion. CONCLUSIONS: Human TB lesions are severely hypoxic and M.tb drives HIF-1α accumulation, synergistically increasing collagenase activity which will lead to lung destruction and cavitation.Medical Research CouncilThis is the final version of the article. It first appeared from the British Medical Journal via https://doi.org/10.1136/thoraxjnl-2015-20740

Transcriptional analysis of temporal gene expression in germinating Clostridium difficile 630 endospores.

Clostridium difficile is the leading cause of hospital acquired diarrhoea in industrialised countries. Under conditions that are not favourable for growth, the pathogen produces metabolically dormant endospores via asymmetric cell division. These are extremely resistant to both chemical and physical stress and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants. Spores are the primary infective agent and must germinate to allow for vegetative cell growth and toxin production. While spore germination in Bacillus is well understood, little is known about C. difficile germination and outgrowth. Here we use genome-wide transcriptional analysis to elucidate the temporal gene expression patterns in C. difficile 630 endospore germination. We have optimized methods for large scale production and purification of spores. The germination characteristics of purified spores have been characterized and RNA extraction protocols have been optimized. Gene expression was highly dynamic during germination and outgrowth, and was found to involve a large number of genes. Using this genome-wide, microarray approach we have identified 511 genes that are significantly up- or down-regulated during C. difficile germination (p≤0.01). A number of functional groups of genes appeared to be co-regulated. These included transport, protein synthesis and secretion, motility and chemotaxis as well as cell wall biogenesis. These data give insight into how C. difficile re-establishes its metabolism, re-builds the basic structures of the vegetative cell and resumes growth

Whole-genome analysis of sporulation and germination in Clostridium difficile

Clostridium difficile is a Gram-positive, obligate anaerobe and a leading cause of hospital-acquired diarrhoea in humans. Under conditions that are not favourable for growth, C. difficile triggers sporulation, producing metabolically dormant endospores through asymmetric cell division. These are regarded as the principal infective stage in the C. difficile life cycle, but must germinate to allow for vegetative cell growth and toxin production, representing an attractive target for intervention. A detailed understanding of sporulation and germination could thus have direct applications for disease prevention. While sporulation and germination in the model sporeformer Bacillus subtilis is well understood, little is known about these events in C. difficile. Therefore, the primary aim of this project was to provide a genome-wide overview of sporulation and germination in C. difficile and to identify genes that are essential in these processes using a combination of molecular microbiology, microscopy, transcriptomics and next-generation sequencing. The first part of this thesis is devoted to the characterisation of C. difficile sporulation and germination dynamics, and is followed by a transcriptional analysis of temporal gene expression in germinating spores. A functional analysis of the 511 genes identified as differentially regulated during germination is provided and the results validated for a number of selected genes. One gene in particular, encoding a membrane associated cell wall protein Cwp7, is examined in more detail. In the second part of this thesis I describe the construction of the first comprehensive transposon mutant library which is then used to identify genes that are essential for sporulation and/or germination using Transposon-Directed Insertion Site Sequncing (TraDIS). The results of this study are validated by constructing in-frame deletions in four selected genes followed by a thorough analysis of the resulting phenotypes. Two genes: CD0125 encoding a homologue of B. subtilis SpoIIQ and CD3567 encoding a putative cell wall hydrolase are shown to be involved in spore formation while CD0106 encoding a homologues of B. subtilis CwlD is shown to be involved in germination.Open Acces

Influence of Process Parameters on the Resistivity of 3D Printed Electrically Conductive Structures

With recent developments in conductive composites, new possibilities emerged for 3D printed conductive structures. Complementary to a vast number of publications on materials properties, here we investigate the influence of printing parameters on the resistance of 3D printed structures. The influence of printing temperature on the resistance is significant, with too low value (210 &deg;C) leading to nozzle clogging, while increasing the temperature by 20 &deg;C above the recommended printing settings decreases resistivity by 15%, but causing degradation of the polymer matrix. The limitations of the FDM technique, related to the dimension accuracy emerging from the layer-by-layer printing approach, greatly influence the samples&rsquo; cross-section, causing irregular resistivity values for different layer heights. For samples with layer thickness lower than 0.2 mm, regardless of the nozzle diameter (0.5&ndash;1 mm), high resistance is attributed to the quality of samples. But for a 1 mm nozzle, we observe stabilized values or resistance for 0.3 to 1 mm layer height. Comparing resistance values and layer height generated from the slicer software, we observe a direct correlation&mdash;for a larger height of the sample resistance value decrease. Presented modifications in printing parameters can affect the final resistance by 50%. Controlling several parameters simultaneously poses a great challenge for designing high-efficiency structural electronics

RNAi-mediated depletion of histone H1 proteins results in a moderate reduction in growth rates.

<p><b>A.</b> Growth curves of parental BF <i>T. brucei</i> or two independent BF <i>T. brucei</i> RYT3-H1 clones in the presence (+) or absence (−) of tetracycline to induce histone H1 RNAi. The mean of three experiments is shown with the standard deviation indicated with error bars. In each case, the cell number was multiplied by the dilution factor to obtain a value for cumulative cell growth. <b>B.</b> Western blot analysis of Tris-tricine gels showing knockdown of histone H1 in two independent BF <i>T. brucei</i> RYT3-H1 histone H1 RNAi clones compared with the parental (P) cell line. Histone H1 RNAi was induced with tetracycline for the time indicated in hours (h). BiP is shown as a loading control. <b>C.</b> Experiment similar to as shown in panel A. performed in PF <i>T. brucei</i> cells. <b>D.</b> A similar experiment as shown in panel B. performed using PF <i>T. brucei</i> cells. Histone H1 RNAi was induced with tetracycline for the time indicated in days (d).</p

Depletion of histone H1 results in an increase in VSG switching.

<p><b>A.</b> The frequency of generation of ganciclovir resistant (GCVR) trypanosomes per generation (GCVR tryps/gen) in parental (Par) <i>T. brucei</i> and 221pGFPhyTK-H1 cells in the presence of histone H1 RNAi for the time indicated in hours (h). Bars indicate the mean of independent experiments [parental n = 3; H1 RNAi cells (0 h and 48 h histone H1 RNAi), n = 5]. The standard deviation is indicated with error bars. A higher rate of generation of GCVR clones as a measure for VSG switching was observed in uninduced <i>T. brucei</i> 221pGFPhyTK-H1 cells (containing the histone H1 RNAi construct) compared with parental cells (statistical significance **, P<0.01). However, there was a statistically significant increase in this frequency after the induction of histone H1 RNAi for 48 hours (***, P<0.001). <b>B.</b> Mechanism of VSG switching in clones from seven independent cultures after the induction of histone H1 RNAi for 48 hours (n = 64) (purple bars) or in four independent cultures generated in the absence of histone H1 RNAi (n = 33) (blue bars). The genotypes and phenotypes of the clones were determined using microscopy and PCR, allowing determination of the VSG switching mechanism used. The percentage of clones that had switched using each mechanism is plotted. VSG switch mechanisms are as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003010#ppat-1003010-g008" target="_blank">Figure 8</a>, as well as cells which appeared to have mutated the TK gene (TK mut.) as determined by their resistance to GCV and continued expression of VSG221. <b>C.</b> Frequency of generation of GCVR clones per generation (freq/gen) in parental (Par) and 221pGFPhyTK-H1 cells grown in the absence (0 h) or presence of histone H1 RNAi for 48 hours (48 h). These experiments were conducted in the presence of puromycin to select for DNA rearrangements in the vicinity of the telomeric <i>VSG</i>. Bars indicate the mean of three independent experiments with standard deviation indicated with error bars. As shown in panel A., cells containing the H1 RNAi construct show a statistically significant increase in the frequency of ganciclovir resistant cells (statistical significance *, P = 0.024). This frequency does not further increase after depletion of histone H1 for 48 hours (statistical significance of increase compared with parental *, P = 0.014). <b>D.</b> Increased <i>VSG</i> switching mediated by gene conversion (GC) in cells depleted for histone H1. VSG switching was monitored in <i>T. brucei</i> 221pGFPhyTK-H1 cells in the absence (0 h) or presence of H1 RNAi for 48 hours. VSG switching mechanisms were determined in clones derived from at least three independent cultures for each experiment. All GCVR clones were generated in the presence of puromycin to select for VSG switches mediated by DNA rearrangements at the telomere of the active ES. VSG switching mechanisms were determined using immunofluorescence microscopy and PCR as in panel B. Using parental cells (Par) or uninduced histone H1 RNAi cells (0 h) ∼90% of the obtained clones (Par n = 19; H1 RNAi 0 h n = 21) continued to express VSG221, indicating that they are TK mutants (TK mut.). However, when histone H1 RNAi had been induced for 48 hours, less than half of the generated clones were TK mutants, and ∼33% had switched through VSG gene conversion (VSG GC). Clones that had switched through telomere exchange (Telo XO) were also observed using this assay.</p

Strategy for determination of VSG switching frequencies after blocking histone H1 synthesis.

<p><b>A.</b> Diagram of the BF <i>T. brucei</i> 221pGFPhyTK-H1 cell line used for analysis of VSG switching after histone H1 knock-down. The large red box indicates a cell, with a construct containing the puromycin (Pur) resistance gene and <i>eGFP</i> integrated immediately behind the active <i>VSG221</i> ES promoter (indicated with a flag). A construct containing a fusion protein for hygromycin resistance and thymidine kinase (HYGTK) activity is integrated at the telomeric end of the ES between characteristic 70 bp repeat sequences (70 bp) and the telomeric <i>VSG221</i> gene. A silent ES with an unknown <i>VSG</i> (X) is indicated below, as well as chromosome internal silent <i>VSG</i>s (Y) located in tandem arrays. A construct allowing transcription of histone H1 RNAi (H1) from opposing T7 promoters (arrows) as well as a phleomycin (Phleo) gene transcribed from an rDNA promoter (black flag) has also been introduced. <b>B.</b> Schematic of VSG switching mechanisms detectable in our assay, adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003010#ppat.1003010-Aitcheson1" target="_blank">[66]</a>. Switching the active <i>VSG</i> can be mediated through a <i>VSG</i> gene conversion (GC), telomere exchange (XO), or <i>in situ</i> activation of another ES (<i>in situ</i>). In addition, VSG switch events resulting in loss of the <i>VSG221</i> ES by either gene conversion (ES GC) or a deletion event after an <i>in situ</i> switch (<i>in situ</i>+ES del) were identified. Each type of VSG switch event, in addition to mutations in the TK gene itself (not shown), result in resistance to ganciclovir (GCV). Presence of the single copy <i>VSG221</i> gene is indicated below (DNA). In addition, expression of the GFP or VSG221 protein are indicated. The schematic is labeled as indicated in panel A.</p

Nuclear localization of histone H1 in <i>T. brucei</i>.

<p>BF <i>T. brucei</i> HNI 221+ and PF <i>T. brucei</i> 221BsrDsRed cells were fixed and analysed by immunofluorescence microscopy using anti-histone H1 antibodies (αH1). The monoclonal L1C6 antibody was used to visualise the nucleolus, and a differential interference contrast (DIC) image is shown. Scale bar is 5 µm.</p

Distribution of histone H1 in the <i>T. brucei</i> genome.

<p><b>A.</b> Schematic of the BF <i>T. brucei</i> HNI 221+ cell line used for ChIP experiments indicated with a large red box. <i>VSG</i> expression sites (ESs) containing the hygromycin (Hyg) or neomycin (Neo) resistance genes, as well as the telomeric <i>VSG221</i> (221) and <i>VSGVO2</i> (VO2) genes are indicated. The ES promoters are indicated with flags, and transcription of the active <i>VSG221</i> ES with an arrow. <b>B.</b> Representative slot blots of chromatin immunoprecipitation (ChIP) samples showing the presence of histone H3 or histone H1 on characteristic 50 bp repeat sequences found flanking ESs, or 177 bp repeats comprising the bulk of the <i>T. brucei</i> minichromosomes. Experiments were performed with no antibody (No ab) or pre-immune serum (Pre-imm.) from the rabbit used to produce the histone H1 antibody as negative controls (−). For each immunoprecipitated sample, 10% of the ChIP material was loaded on a slot blot and compared to 0.1% of the total input. <b>C.</b> Quantitation of material immunoprecipitated (% IP) using anti-histone H3 (H3) or anti-histone H1 (H1) in the slot blots shown in panel B. Bars show the mean of three experiments with standard deviation indicated with error bars. Two negative controls (−) were used: no antibody, or the pre-immune serum. <b>D.</b> Distribution of histone H1 within the genome of BF <i>T. brucei</i> as determined using qPCR analysis of ChIP material. The bars indicate the amount immunoprecipitated (% IP) using the anti-histone H1 antibody (H1) or pre-immune serum (Pre-imm.) as a control, with the standard deviation in three experiments indicated with error bars. Statistically significant (P<0.01) amounts of histone H1 were immunoprecipitated in all regions with the exception of the rDNA promoter, 18S rRNA, hygro and <i>VSG221</i> genes (Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003010#ppat.1003010.s003" target="_blank">Fig. S3</a>). The RNA polymerase II (Pol II) transcribed regions analysed are the actin, γ-tubulin (γ-tub), RNA polymerase I large subunit (pol I), <i>URA3</i>, paraflagellar rod protein B (PFR) and spliced leader (SL) gene loci. The SL intergenic region (int.), promoter region (pro.), or the SL gene itself (SL) are indicated. The ribosomal DNA (rDNA) regions analysed include the rDNA intergenic region (int.), promoter (pro.) or the 18S rDNA gene (18S). The EP procyclin locus analysed includes the region upstream of the EP promoter (up.), the promoter (pro.), or the EP procyclin gene (EP). Higher levels of histone H1 were immunoprecipitated upstream of the promoters compared with at the promoter regions themselves, with the statistical significance indicated with asterisks (** indicates P<0.01, and **** indicates P<0.0001). ES sequences analysed include a region immediately upstream of the ES promoter (up.) as well as at the promoter itself (pro.). These primer pairs can be expected to recognise most if not all ESs. Sequences specific for the active <i>VSG221</i> ES include the hygromycin resistance gene (Hygro) as well as <i>VSG221.</i> Sequences present in the silent <i>VSGVO2</i> ES include the neomycin resistance gene (Neo) and <i>VSGVO2</i>. Note that the <i>VSGVO2</i> primers detect both the telomeric ES located <i>VSGVO2</i> gene, as well as the chromosome-internal copy of <i>VSGVO2</i>. <i>VSG118</i> is found in the silent <i>VSG</i> basic copy arrays.</p