Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo.

Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 μM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT

Genetic Organisation, Mobility and Predicted Functions of Genes on Integrated, Mobile Genetic Elements in Sequenced Strains of Clostridium difficile

Background: Clostridium difficile is the leading cause of hospital-associated diarrhoea in the US and Europe. Recently the incidence of C. difficile-associated disease has risen dramatically and concomitantly with the emergence of 'hypervirulent' strains associated with more severe disease and increased mortality. C. difficile contains numerous mobile genetic elements, resulting in the potential for a highly plastic genome. In the first sequenced strain, 630, there is one proven conjugative transposon (CTn), Tn5397, and six putative CTns (CTn1, CTn2 and CTn4-7), of which, CTn4 and CTn5 were capable of excision. In the second sequenced strain, R20291, two further CTns were described.Results: CTn1, CTn2 CTn4, CTn5 and CTn7 were shown to excise from the genome of strain 630 and transfer to strain CD37. A putative CTn from R20291, misleadingly termed a phage island previously, was shown to excise and to contain three putative mobilisable transposons, one of which was capable of excision. In silico probing of C. difficile genome sequences with recombinase gene fragments identified new putative conjugative and mobilisable transposons related to the elements in strains 630 and R20291. CTn5-like elements were described occupying different insertion sites in different strains, CTn1-like elements that have lost the ability to excise in some ribotype 027 strains were described and one strain was shown to contain CTn5-like and CTn7-like elements arranged in tandem. Additionally, using bioinformatics, we updated previous gene annotations and predicted novel functions for the accessory gene products on these new elements.Conclusions: The genomes of the C. difficile strains examined contain highly related CTns suggesting recent horizontal gene transfer. Several elements were capable of excision and conjugative transfer. The presence of antibiotic resistance genes and genes predicted to promote adaptation to the intestinal environment suggests that CTns play a role in the interaction of C. difficile with its human host

Metabolic engineering of Clostridium autoethanogenum for selective alcohol production

Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols

The role of the restriction-modification system of Clostridium pasteurianum on its electro-transformation

Dissertação de mestrado em BioengenhariaClostridium pasteurianum is a Gram-positive and anaerobic bacterium with a great biotechnological potential. It is one of the few microorganisms capable of hydrolyzing glycerol to produce solvents as ethanol and butanol, which have a wide applicability in the market as biofuels. The development of a genetic system for this microorganism would increase its application opportunities since gene overexpression or inactivation could improve their solventogenic characteristics. Its genetic information is already known but this organism has a particular resistance to transformation. This resistance can be explained by a very efficient restriction system that does not allow the entrance of non-methylated DNA or DNA with a methylation pattern different from it. Therefore, foreign DNA must be correctly methylated prior to transformation. For this purpose, a specific methyltransferase is needed to transfer methyl groups to a certain nucleotide of a specific sequence. The goal of this thesis was to create a genetic system in C. pasteurianum that allows genome modification and foreign protein expression, ultimately improving C. pasteurianum DSM 525 transformation. Preliminary simple electro-transformations in which the parameters to make competent cells and the electroporation conditions were altered, did not result in positive results. Being aware of the possibility of a restriction system presence in this organism, experiments with M.MspI methylated DNA were performed, however they demonstrated the inability of this methyltransferase to improve the microorganism transformation. The presence of restriction enzymes was confirmed when a characterization of the restriction system of C. pasteurianum was performed using MspI methylated and non-methylated DNA. The presence of a discrete digestion pattern was detected, and M.MspI methylation could not protect the foreign DNA from C. pasteurianum restriction action. The polyamine spermidine, with known affinity for negatively charged DNA, showed to be efficient against C. pasteurianum crude extract digestion action, however not sufficiently to facilitate this microorganism electrotransformation. By accessing the genome information, the Restriction/Modification (R/M) systems of this microorganism were analyzed. The GATC type IIP R/M system was chosen in order to verify the restriction and methylation enzymes activity with the same target sequence. Three genes, one REase (DpnII) and two MTases (Dam and MdpnII) were cloned in pETduet-1, followed by overproduction in BL21 (DE3). The codon usage of the host and original organism were not compatible, and the protein production in tRNAs provider strains was tested. Protein production was detected, however was not possible to re-confirm their presence. The common protein folding problems were analyzed using a disulfide bond enhancer strain. Nevertheless, the production problem may not be related to this, since no different protein over-production was detected. Restriction reactions with the REase BstUI and C. pasteurianum crude extract, using DNA methylated by M.SssI (m5CG), were developed and showed that the REase responsible for hindering foreign DNA entering C. pasteurianum recognizes the sequence 5'-CGCG- 3'. In a second analysis of the C. pasteurianum genome a methyltransferase-encoding gene was identified that may be involved in methylating the sequence 5'-CGCG- 3'. The in silico analysis was performed and its codon usage was also improved to be compatible with E. coli. In this work, the reasons for C. pasteurianum’s recalcitrance to transformation were identified, the knowledge about its R/M systems was extended, and a proposal to efficiently transform this bacterium was provided.Clostridium pasteurianum DSM 525 é uma bactéria Gram-positiva anaeróbia com um elevado potencial biotecnológico. Este é um dos poucos microrganismos capaz de hidrolisar glicerol para produzir solventes como etanol e butanol, que têm uma grande aplicabilidade no mercado. O desenvolvimento de um sistema genético para este organismo permitiria aumentar as suas oportunidades de aplicação sendo que a sobre-expressão ou inativação de um determinado gene pode melhorar as suas características solventogénicas. A sua informação genética já é conhecida, mas este microrganismo apresenta uma particular resistência à transformação. Esta resistência pode ser explicada pela presença de um eficiente sistema de restrição que não permite a entrada de DNA não metilado ou DNA metilado de forma diferente da própria bactéria. Desta forma, o DNA estranho deve ser corretamente metilado antes da transformação. Para que isto seja possível é necessária a presença de uma metilase específica para transferir grupos metilo para um determinado nucleótido de uma sequência específica. O objetivo desta tese foi criar um sistema genético em C. pasteurianum que permitisse modificações no genoma e a expressão de proteínas heterólogas, ou seja, que permitisse melhorar a transformação de C. pasteurianum DSM 525. Foram realizadas transformações preliminares simples com parâmetros que diferem na forma de obter células competentes e nas condições de eletroporação, contudo os resultados obtidos não foram positivos. Tendo conhecimento da possibilidade da presença de um sistema de restrição neste organismo, foram realizadas experiências com DNA metilado pela enzima M.MspI, sendo que estas demonstraram a incapacidade da metiltransferase para melhorar a transformação deste microrganismo. Foi confirmada a presença de enzimas de restrição aquando da caracterização do sistema de restrição de C. pasteurianum usando DNA não metilado ou metilado pela enzima M.MspI. Foi detetada a presença de um padrão de digestão distinto, verificando-se que a enzima M.MspI não consegue proteger o DNA estranho da ação de restrição de C. pasteurianum. A poliamina espermidina, com conhecida afinidade por DNA negativamente carregado, mostrou ser eficiente contra a ação de digestão do extrato cru de C. pasteurianum, contudo não o suficiente para facilitar a electrotransformação deste microrganismo. Tendo acesso ao genoma, foi então analisado o sistema de Restrição e Modificação (R/M) deste microrganismo. Foi escolhido o sistema R/M tipo IIP GATC para verificar a atividade de enzimas de restrição e metilação com a mesma sequência de reconhecimento. Foram clonados três genes no vetor pETduet-1, uma enzima de restrição (REase – DpnII) e duas metiltransferases (MTases – Dam and Mdpn), seguindo-se a produção em BL21 (DE3). O conjunto de codões usados pelo hospedeiro e pelo organismo de origem não eram compatíveis, foi então testada a produção proteica em estirpes fornecedoras de tRNAs. Foi observada produção proteica contudo não foi possível re-avaliar a sua presença. Foram analisados problemas de enrolamento (do inglês folding), usando uma estirpe que facilita a formação de pontes dissulfito. No entanto, o problema na produção não deve estar associado ao enrolamento proteico sendo que não foi detectada produção proteica nestas condições. Foram desenvolvidas reações de restrição com a REase BstUI e extrato cru de C. pasteurianum usando DNA metilado pela enzima M.SssI (m5CG) e foi mostrado que a REase responsável pelo impedimento da entrada de DNA em C. pasteurianum reconhece a sequência 5'-CGCG- 3'. Numa segunda análise ao genoma de C. pasteurianum DSM 525 foi identificado um gene que codifica uma metiltransferase que pode estar envolvida na metilação da sequência 5'-CGCG- 3'. Foi feita a análise in silico e o tipo de codões usados foi melhorado para ser compatível com E. coli. Neste trabalho, foram identificadas as razões para a resistência deste microrganismo à transformação, foi consolidado o conhecimento sobre o seu sistema de R/M e foi proposta uma metodologia para transformar de forma eficiente esta bactéria

Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824.

Background: Consolidated bioprocessing (CBP) is reliant on the simultaneous enzyme production, saccharification of biomass, and fermentation of released sugars into valuable products such as butanol. Clostridial species that produce butanol are, however, unable to grow on crystalline cellulose. In contrast, those saccharolytic species that produce predominantly ethanol, such as Clostridium thermocellum and Clostridium cellulolyticum, degrade crystalline cellulose with high efficiency due to their possession of a multienzyme complex termed the cellulosome. This has led to studies directed at endowing butanol-producing species with the genetic potential to produce a cellulosome, albeit by localising the necessary transgenes to unstable autonomous plasmids. Here we have explored the potential of our previously described Allele-Coupled Exchange (ACE) technology for creating strains of the butanol producing species Clostridium acetobutylicum in which the genes encoding the various cellulosome components are stably integrated into the genome. Results: We used BioBrick2 (BB2) standardised parts to assemble a range of synthetic genes encoding C. thermocellum cellulosomal scaffoldin proteins (CipA variants) and glycoside hydrolases (GHs, Cel8A, Cel9B, Cel48S and Cel9K) as well as synthetic cellulosomal operons that direct the synthesis of Cel8A, Cel9B and a truncated form of CipA. All synthetic genes and operons were integrated into the C. acetobutylicum genome using the recently developed ACE technology. Heterologous protein expression levels and mini-cellulosome self-assembly were assayed by western blot and native PAGE analysis. Conclusions: We demonstrate the successful expression, secretion and self-assembly of cellulosomal subunits by the recombinant C. acetobutylicum strains, providing a platform for the construction of novel cellulosomes. © 2013 Kovács et al.; licensee BioMed Central Ltd

A novel arabinose-inducible genetic operation system developed for Clostridium cellulolyticum

<p> Background: Clostridium cellulolyticum and other cellulolytic Clostridium strains are natural producers of lignocellulosic biofuels and chemicals via the consolidated bioprocessing (CBP) route, and systems metabolic engineering is indispensable to meet the cost-efficient demands of industry. Several genetic tools have been developed for Clostridium strains, and an efficient and stringent inducible genetic operation system is still required for the precise regulation of the target gene function.</p

Horizontal gene transfer converts non-toxigenic Clostridium difficile strains into toxin producers.

Clostridium difficile is a major nosocomial pathogen and the main causative agent of antibiotic-associated diarrhoea. The organism produces two potent toxins, A and B, which are its major virulence factors. These are chromosomally encoded on a region termed the pathogenicity locus (PaLoc), which also contains regulatory genes, and is absent in non-toxigenic strains. Here we show that the PaLoc can be transferred from the toxin-producing strain, 630Δerm, to three non-toxigenic strains of different ribotypes. One of the transconjugants is shown by cytotoxicity assay to produce toxin B at a similar level to the donor strain, demonstrating that a toxigenic C. difficile strain is capable of converting a non-toxigenic strain to a toxin producer by horizontal gene transfer. This has implications for the treatment of C. difficile infections, as non-toxigenic strains are being tested as treatments in clinical trials

Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum

Clostridium acetobutylicum is an industrially important Gram-positive organism which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum

Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291

Citation: Girinathan, B. P., Monot, M., Boyle, D., McAllister, K. N., Sorg, J. A., Dupuy, B., & Govind, R. (2017). Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291. Msphere, 2(1), 14. doi:10.1128/mSphere.00383-16Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 are linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291