A Novel Bacteriophage with Broad Host Range against Clostridioides difficile Ribotype 078 Supports SlpA as the Likely Phage Receptor.

Bacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ΦCD08011, ΦCD418, ΦCD1801, and ΦCD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ΦCD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ΦCD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI. IMPORTANCE Research into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridioides difficile infection (CDI) is in its infancy, where an optimal "one size fits all" phage cocktail is yet to be derived. The pursuit thus far has aimed to find phages with the broadest possible host range. However, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad host range activity are yet to be discovered. In this study, we isolate four novel myoviruses, including ΦCD1801, which exerts the broadest host range activity toward RT 078 reported in the literature. Through the application of ΦCD1801 to phage-binding assays, we provide data to support the prior notion that SlpA represents the likely phage receptor on the bacterial cell surface. Our finding directs research attention toward the isolation of phages with activity toward strains possessing defined S-layer cassette types.

Phosphorylation and functionality of CdtR in Clostridium difficile.

The production of TcdA, TcdB and CDT in Clostridium difficile PCR ribotype 027, is regulated by the two-component system response regulator CdtR. Despite this, little is known about the signal transduction pathway leading to the activation of CdtR. In this study, we generated R20291ΔPalocΔcdtR model strains expressing CdtR phospho-variants in which our predicted phospho-accepting Asp, Asp61 was mutated for Ala or Glu. The constructs were assessed for their ability to restore CDT production. Dephospho-CdtR-Asp61Ala was completely non-functional and mirrored the cdtR-deletion mutant, whilst phospho-CdtR-Asp61Glu was functional, possessing 38-52% of wild-type activity. Taken together, these data suggest that CdtR is activated by phosphorylation of Asp61. The same principles were applied to assess the function of PCR ribotype 078-derived CdtR, which was shown to be non-functional owing to polymorphisms present within its coding gene. Conversely, polymorphisms present within its promoter region, provide significantly enhanced promoter activity compared with its PCR ribotype 027 counterpart. To ensure our data were representative for each ribotype, we determined that the cdtR nucleotide sequence was conserved in a small library of eight PCR ribotype 027 clinical isolates and nineteen PCR ribotype 078 isolates from clinical and animal origin.

Effect of antibiotic treatment on the formation of non-spore Clostridium difficile persister-like cells.

Background: The spore is the virulence factor identified to be involved in the recurrence of the disease caused by Clostridium difficile. Objectives: To demonstrate that lethal antibiotic concentrations induce the appearance of C. difficile persister-like non-spore cells. Methods: C. difficile and derivative spo0A mutant strains were tested for their susceptibility to antibiotics, as determined using an agar dilution method. Persister-cell generation was determined for all strains using up to 10â€Š× the MIC of every antibiotic for up to 6 days. Results: Using up to 10â€Š× the MIC of every antibiotic, we were able to induce the appearance of persister-like behaviour since biphasic killing curves could be observed in response to treatment antibiotics. Conclusions: To the best of our knowledge, this work provides, for the first time, experimental evidence of the appearance of C. difficile persister-like cells, opening a new research avenue in the pathogenesis of this nosocomial pathogen.

Development of Clostridium difficile R20291ΔPaLoc model strains and in vitro methodologies reveals CdtR is required for the production of CDT to cytotoxic levels.

Assessing the regulation of Clostridium difficile transferase (CDT), is complicated by the presence of a Pathogenicity locus (PaLoc) which encodes Toxins A and B. Here we developed R20291ΔPaLoc model strains and cell-based assays to quantify CDT-mediated virulence. Their application demonstrated that the transcriptional regulator, CdtR, was required for CDT-mediated cytotoxicity.

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo.

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration.

Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT).

Carboxypeptidase G2 (CP) is a bacterial enzyme, which is targeted to tumours by an antitumour antibody for local prodrug activation in antibody-directed enzyme prodrug therapy (ADEPT). Repeated cycles of ADEPT are desirable but are hampered by human antibody response to CP (HACA). To address this, we aimed to identify and modify clinically important immunogenic sites on MFECP, a recombinant fusion protein of CP with MFE-23, a single chain Fv (scFv) antibody. A discontinuous conformational epitope at the C-terminus of the CP previously identified by the CM79 scFv antibody (CM79-identified epitope) was chosen for study. Modification of MFECP was achieved by mutations of the CM79-identified epitope or by addition of a hexahistidine tag (His-tag) to the C-terminus of MFECP, which forms part of the epitope. Murine immunisation experiments with modified MFECP showed no significant antibody response to the CM79-identified epitope compared to A5CP, an unmodified version of CP chemically conjugated to an F(ab)(2) antibody. Success of modification was also demonstrated in humans because patients treated with His-tagged MFECP had a significantly reduced antibody response to the CM79-identified epitope, compared to patients given A5CP. Moreover, the polyclonal antibody response to CP was delayed in both mice and patients given modified MFECP. This increases the prospect of repeated treatment with ADEPT for effective cancer treatment.

Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis.

A major obstacle in cancer gene therapy is selective tumor delivery. Previous studies have suggested that genetically engineered anaerobes of the genus Clostridium might be gene therapy vectors because of their ability to proliferate selectively in the hypoxic/necrotic regions common to solid tumors. However, the tumor colonization efficiency of the strain previously used was insufficient to produce any antitumor effect. Here we describe for the first time the successful transformation of C. sporogenes, a clostridial strain with the highest reported tumor colonization efficiency, with the E. coli cytosine deaminase (CD) gene and show that systemically injected spores of these bacteria express CD only in the tumor. This enzyme can convert the nontoxic prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU). Furthermore, systemic delivery of 5-FC into mice previously injected with CD-transformed spores of C. sporogenes produced greater antitumor effect than maximally tolerated doses of 5-FU. Since most human solid tumors have hypoxic and necrotic areas this vector system has considerable promise for tumor-selective gene therapy.

Recombinant expression analysis of natural and synthetic bovine alpha-casein in Escherichia coli.

As a prelude to developing a yeast-based fermentation process for the production of phenylalanine-free alpha-casein as a foodstuff for patients suffering from phenylketonuria, we cloned the gene encoding bovine alpha-casein. We synthesised a modified gene sequence encoding the same, but devoid of phenylalanine codons and with a codon bias similar to that of naturally occurring highly expressed genes in Saccharomyces cerevisiae. The results show that both gene sequences are readily expressed in Escherichia coli when cloned in an E. coli bacteriophage T7 promoter-driven plasmid vector. In this host, the natural and synthetic casein proteins were produced at levels equating to 18.0% and 7.6% of the cell's soluble protein, respectively.

Development of a transformation and gene reporter system for group II, non-proteolytic Clostridium botulinum type B strains.

Non-proteolytic, Group II strains of Clostridium botulinum are of particular concern to the food industry because of their ability to survive and grow in REPFEDs (refrigerated processed foods of extended durability). Their analysis would benefit from the availability of a gene transfer system. In the present study we have been able, for the first time, to demonstrate transformation in a representative Group II strain, ATCC 25765. Initial attempts to transform ATCC 25765 with existing clostridial cloning vectors (pMTL540E and pMTL500E) were, however, prevented by a restriction barrier. Through a combination of classical and molecular approaches we were able to show that strain ATCC 25765 possesses a restriction endonuclease (Cbol) and a methylase activity (M. Cbol) which have the same specificity as Mspl and M.Mspl, respectively. Cbol cleaves the palindrome 5'-CCGG-3' to generate a 3'-GC sticky end, whilst M.Cbol specifically methylates the external C residue. An E. coli host was generated which expressed a Bacillus subtilis methylase enzyme (M.BsuF1) with equivalent specificity to M.Cbol. Plasmids (pMTL540E and pMTL500E) prepared in this strain were subsequently shown to be capable of transforming ATCC 25765. The highest frequencies (0.8 X 10(4) transformants per microg of DNA) were obtained when cells were cultivated in media supplemented with 1% (w/v) glycine, and when the electroporation was undertaken at 10 kV/cm, 25 microF and at 400 ohms. Having developed an effective transformation procedure, we went on to construct reporter cassettes based on the Thermanaerobacterium sulfurigenes lacZ and the Vibrio fischeri luxAB genes. Using the former, and promoter regions isolated from the botulinum toxin genes, we have obtained preliminary evidence that reporter genes may be used to evaluate the physiological factors that affect toxin production in the food environment.

Genetically modified clostridium for gene therapy of tumors.

Many murine and human tumors contain hypoxic or necrotic regions in which the oxygen tension is abnormally low. For example,>50% of primary tumors of the breast, cervix, and head and neck contain areas that are hypoxic. Because hypoxic regions are not present in normal tissue, this provides the potential for selectively targeting gene therapy to tumor cells.

Molecular analysis of the malR gene of Clostridium butyricum NCIMB 7423, a member of the LacI-GalR family of repressor proteins.

Deletion of a region of DNA 5' to a previously characterised malQ gene of Clostridium butyricum resulted in increased production of the enzyme activity encoded by malQ, 4-alpha-glucanotransferase. Nucleotide sequence analysis revealed the presence of an open reading frame capable of encoding a protein of 335 amino acids. This protein was found to share 33% amino acid sequence identity with the Bacillus subtilis CcpA (catabolite control protein) repressor, 28% identity with the Streptomyces coelicolor MalR repressor, and 30%, 25%, and 21% amino acid identity with the Escherichia coli repressors GalR, LacI and MalI, respectively. The amino-terminal domain was predicted to be able to form a helix-turn-helix structure, and shared highest similarity with the equivalent functional domain from the E. coli LacI repressor. Interruption of malR by the generation of a frameshift mutation led to a 10-fold increase in MalQ activity. These data suggest that the identified open reading frame encodes a repressor of the C. butyricum malQ gene, and of the adjacent malP gene. The gene has, therefore, been designated malR, and its encoded gene product MalR.

A gene system for glucitol transport and metabolism in Clostridium beijerinckii NCIMB 8052.

The gutD gene of Clostridium beijerinckii NCIMB 8052 encoding glucitol 6-phosphate dehydrogenase was cloned on a 5.7-kbp chromosomal DNA fragment by complementing an Escherichia coli gutD mutant strain and selecting for growth on glucitol. Five open reading frames (ORFs) in the order gutA1 gutA2 orfX gutB gutD were identified in a 4.0-kbp region of the cloned DNA. The deduced products of four of these ORFs were homologous to components of the glucitol phosphotransferase system (PTS) and glucitol 6-phosphate dehydrogenase from E. coli, while the remaining ORF (orfX) encoded an enzyme which had similarities to members of a family of transaldolases. A strain in which gutD was inactivated by targeted integration lacked glucitol 6-phosphate dehydrogenase activity. The gutA1 and gutA2 genes encoded two polypeptides forming enzyme IIBC of the glucitol PTS comprising three domains in the order CBC. Domain IIA of the glucitol PTS was encoded by gutB. Glucitol phosphorylation assays in which soluble and membrane fractions of cells grown on glucose (which did not synthesize the glucitol PTS) or cells grown on glucitol were used confirmed that there is a separate, soluble, glucitol-specific PTS component, which is the product of the gutB gene. The gut genes were regulated at the level of transcription and were induced in the presence of glucitol. Cells grown in the presence of glucose and glucitol utilized glucose preferentially. Following depletion of glucose, the glucitol PTS and glucitol 6-phosphate dehydrogenase were synthesized, and glucitol was removed from the culture medium. RNA analysis showed that the gut genes were not expressed until glucose was depleted.

Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment.

A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.

Genetic characterisation of the botulinum toxin complex of Clostridium botulinum strain NCTC 2916.

An 8 kb segment of the Clostridium botulinum NCTC 2916 genome 5' to the type A botulinum neurotoxin gene has been sequenced revealing five open reading frames. Four encode components (HA70, HA17, HA34 and NTNH/A) of the progenitor toxin complex. The product of the fifth, OrfX, possesses a putative C-terminal helix-turn-helix motif, exhibits homology with known regulatory proteins (including MsmR from Streptococcus mutans, UviA from C. perfringens and Orftxe1 located upstream of the C. difficile toxin B gene) and is also found within the vicinity of genes encoding tetanus toxin and types B, C, D and G botulinum toxins. Primer extension and Northern blotting analysis demonstrates that the genes are expressed as two divergent operons [HA34, HA17, HA70] and [NTNH/A, type A toxin gene], with the OrfX gene expressed singly. Immediately adjacent to the transcriptional start sites of the HA34 and NTNH/A genes are two highly conserved motifs (5'-ATTTTagGTTTACAAAA-3' and 5'-ATGTTATATgTaA-3'), separated by 12 bp, that span the putative -35 and -10 promoter regions. Homologous sequences occur in the equivalent position relative to the genes at type C botulinum toxin gene and the tetanus toxin gene loci. It is likely that these sequence motifs, together with OrfX, are involved in the co-ordinate expression of the genes encoding the various components of the botulinum toxin complex in groups I, III and IV C. botulinum strains and in that of the tetanus toxin gene.

In vitro and in vivo characterisation of a recombinant carboxypeptidase G2::anti-CEA scFv fusion protein.

BACKGROUND: There is considerable interest in the specific targeting of therapeutic agents to cancer cells. Of particular promise is a technique known as Antibody-Directed Enzyme Prodrug Therapy (ADEPT). In this approach an enzyme is targeted to the tumour by its conjugation to a tumour specific-antibody tumour. After allowing sufficient time for the conjugate to localise at the tumour and clear from the circulatory system, a relatively non-toxic prodrug is administered. This prodrug is converted to a highly cytotoxic drug by the action of the targeted enzyme localised at the tumour site. OBJECTIVES: To construct gene fusions between the pseudomonad carboxypeptidase G2 (CPG2) gene and DNA encoding MFE-23 (an anti-carcinoembryonic antigen (CEA) single-chain Fv (scFv) molecule), derived from a phage display library. To overexpress the resultant gene fusions in Escherichia coli, and assess the in vitro and in vivo properties of the purified fusion proteins. STUDY DESIGN: To introduce unique cloning restriction sites into the 5'-end of the CPG2 gene by site-directed mutagenesis to facilitate fusion to the 3'-end of the gene encoding MFE-23 (constructs with or without a flexible (Gly4Ser)3 linker-encoding sequence were designed). To overexpress the resultant gene fusions under transcriptional control of the lac promoter and to direct the fusion proteins produced to the periplasmic space of E. coli through translational coupling to the pelB signal peptide. RESULTS: Biologically active recombinant CPG2::MFE-23 scFv fusion proteins were produced in E. coli and shown to possess enzyme and anti-CEA activity. Affinity chromatography followed by size exclusion gel filtration yielded approximately 0.7-1.4 mg/l from shake flask culture. The fusion protein in which the enzyme and antibody moieties were joined by a linker peptide was shown to be effectively localised in nude mice bearing human colon tumour xenografts, giving favourable tumour to blood ratios. CONCLUSION: MFE-23 scFv serves as an ideal candidate for the antibody arm of a bacterially expressed fusion protein with CPG2. The biological properties of this recombinant protein suggest that it may be employed for tumour specific prodrug activation. However, further assessment of its stability and pharmokinetics is required if genetic fusion is to be considered as an alternative to chemical conjugation.

Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia.

Certain species of anaerobic bacteria have been shown to localise and germinate specifically in the hypoxic regions of tumours, resulting in tumour lysis. We propose an innovative approach to cancer gene therapy in which genetically engineered anaerobic bacteria of the genus Clostridium are used to achieve tumour-specific gene delivery. Our strategy involves enzyme/prodrug therapy, in which the Escherichia coli enzyme cytosine deaminase is used to convert the non-toxic prodrug 5-fluorocytosine to the active chemotherapeutic agent 5-fluorouracil. The E. coli gene encoding cytosine deaminase has been cloned into a clostridial expression vector and transformed into Clostridium beijerinckii, resulting in constitutive expression of cytosine deaminase and significant levels of active enzyme in the bacterial medium. When added to an in vitro clonogenic survival assay, supernatant from clostridia expressing cytosine deaminase increased the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine approximately 500-fold. This high level of prodrug activation, combined with the specificity of clostridia for hypoxic regions of tumours, indicates a potential use in cancer gene therapy.

Expression of the bacterial nitroreductase enzyme in mammalian cells renders them selectively sensitive to killing by the prodrug CB1954.

A recombinant retrovirus encoding E. coli nitroreductase (NTR) was used to infect mammalian cells. NIH3T3 cells expressing NTR were killed by the prodrug CB1954, which NTR converts to a bifunctional alkylating agent. Admixed, unmodified NIH3T3 cells could also be killed. In contrast to the Herpes simplex virus (HSV) thymidine kinase (TK)/ganciclovir(GCV) enzyme/prodrug system, NTR/CB1954 cell killing was effective in non-cycling cells. Co-operative killing was observed when cells expressing both NTR and TK were treated with a combination of CB1954 and GCV. NTR expression in human melanoma, ovarian carcinoma or mesothelioma cells also rendered them sensitive to CB1954 killing. These data suggest that delivery of the NTR gene to human tumours, followed by treatment with CB1954, may provide a novel tumour gene therapy approach.

Chemotherapeutic tumour targeting using clostridial spores.

The toxicity associated with conventional cancer chemotherapy is primarily due to a lack of specificity for tumour cells. In contrast, intravenously injected clostridial spores exhibit a remarkable specificity for tumours. This is because, following their administration, clostridial spores become exclusively localised to, and germinate in, the hypoxic/necrotic tissue of tumours. This unique property could be exploited to deliver therapeutic agents to tumours. In particular, genetic engineering could be used to endow a suitable clostridial host with the capacity to produce an enzyme within the tumour which can metabolise a systemically introduced, non-toxic prodrug into a toxic metabolite. The feasibility of this strategy (clostridial-directed enzyme prodrug therapy, CDEPT) has been demonstrated by cloning the Escherichia coli B gene encoding nitroreductase (an enzyme which converts the prodrug CB1954 to a highly toxic bifunctional alkylating agent) into a clostridial expression vector and introducing the resultant plasmid into Clostridium beijerinckii (formerly C. acetobutylicum) NCIMB 8052. The gene was efficiently expressed, with recombinant nitroreductase representing 8% of the cell soluble protein. Following the intravenous injection of the recombinant spores into mice, tumour lysates have been shown, by Western blots, to contain the E. coli-derived enzyme.