Haitham Hussain

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.

A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation, Genetic Competence, and Acid Resistance in Streptococcus mutans

We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in Streptococcus mutans. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis.

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

ABSTRACT The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.

Phage ϕC2 Mediates Transduction of Tn6215, Encoding Erythromycin Resistance, between Clostridium difficile Strains

ABSTRACT In this work, we show that Clostridium difficile phage ϕC2 transduces erm(B), which confers erythromycin resistance, from a donor to a recipient strain at a frequency of 10−6 per PFU. The transductants were lysogenic for ϕC2 and contained the erm(B) gene in a novel transposon, Tn6215. This element is 13,008 bp in length and contains 17 putative open reading frames (ORFs). It could also be transferred at a lower frequency by filter mating. IMPORTANCE Clostridium difficile is a major human pathogen that causes diarrhea that can be persistent and difficult to resolve using antibiotics. C. difficile is potentially zoonotic and has been detected in animals, food, and environmental samples. C. difficile genomes contain large portions of horizontally acquired genetic elements. The conjugative elements have been reasonably well studied, but transduction has not yet been demonstrated. Here, we show for the first time transduction as a mechanism for the transfer of a novel genetic element in C. difficile. Transduction may also be a useful tool for the genetic manipulation of C. difficile. Clostridium difficile is a major human pathogen that causes diarrhea that can be persistent and difficult to resolve using antibiotics. C. difficile is potentially zoonotic and has been detected in animals, food, and environmental samples. C. difficile genomes contain large portions of horizontally acquired genetic elements. The conjugative elements have been reasonably well studied, but transduction has not yet been demonstrated. Here, we show for the first time transduction as a mechanism for the transfer of a novel genetic element in C. difficile. Transduction may also be a useful tool for the genetic manipulation of C. difficile.

Demonstration of Conjugative Transposon (Tn5397)-Mediated Horizontal Gene Transfer between Clostridium difficile and Enterococcus faecalis

ABSTRACT Antibiotic-resistant Enterococcus faecalis and Clostridium difficile are responsible for nosocomial infections in humans, in which they inhabit the same niche. Here, we demonstrate transfer of the conjugative transposon Tn5397 from C. difficile 630 to E. faecalis JH2-2, the first reported gene transfer between these two bacteria. Furthermore, transfer from the E. faecalis EF20A transconjugant to the epidemic ribotype 027 C. difficile strain R20291 was also demonstrated. Tn5397 was shown to use a single specific target site in E. faecalis; it also has specific target sites in C. difficile. These experiments highlight the importance of continual monitoring for emerging resistances in these bacteria.

Behavior and target site selection of conjugative transposon Tn916 in two different strains of toxigenic Clostridium difficile.

ABSTRACT The insertion sites of the conjugative transposon Tn916 in the anaerobic pathogen Clostridium difficile were determined using Illumina Solexa high-throughput DNA sequencing of Tn916 insertion libraries in two different clinical isolates: 630ΔE, an erythromycin-sensitive derivative of 630 (ribotype 012), and the ribotype 027 isolate R20291, which was responsible for a severe outbreak of C. difficile disease. A consensus 15-bp Tn916 insertion sequence was identified which was similar in both strains, although an extended consensus sequence was observed in R20291. A search of the C. difficile 630 genome showed that the Tn916 insertion motif was present 100,987 times, with approximately 63,000 of these motifs located in genes and 35,000 in intergenic regions. To test the usefulness of Tn916 as a mutagen, a functional screen allowed the isolation of a mutant. This mutant contained Tn916 inserted into a gene involved in flagellar biosynthesis.

Pancreatic amylase is an environmental signal for regulation of biofilm formation and host interaction in Campylobacter jejuni.

ABSTRACT Campylobacter jejuni is a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran by C. jejuni and that a secreted protease, Cj0511, is required. Exposure of C. jejuni to pancreatic amylase promotes biofilm formation in vitro, increases interaction with human epithelial cell lines, increases virulence in the Galleria mellonella infection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protects C. jejuni from stress conditions in vitro, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism.

Transposon Mutagenesis in Clostridium difficile

Genetic manipulation of Clostridium difficile is notoriously difficult, currently there is only one reliable method for generating random mutations in the organism and that is to use the conjugative transposon Tn916. Tn916 enters the genome of most strains of C. difficile with no obvious target site preference. In order to use the genome strain C. difficile 630 for transposon mutagenesis a erythromycin-sensitive derivative C. difficile 630Deltaerm was constructed and the Tn916 derivative, Tn916DeltaE, was shown to enter the genome at multiple sites enabling the construction of a Tn916 insertion library.

Plasmids can transfer to Clostridium difficile CD37 and 630Δerm both by a DNase resistant conjugation-like mechanism and a DNase sensitive mechanism

Abstract Broad host range conjugative plasmids that replicate in Escherichia coli have been widely used to mobilise smaller replicons, bearing their cognate origin of transfer (oriT) into a variety of organisms that are less tractable genetically, such as Clostridium (Clostridioides) difficile. In this work we demonstrated that the oriT region of pMTL9301 (derived from RK2) is not required for transfer between E. coli and C. difficile strains 630&Dgr;erm and CD37 and that this oriT‐independent transfer is abolished in the presence of DNase when CD37 is the recipient. Transfer to the 630&Dgr;erm strain is DNase resistant even without an obvious oriT, when E. coli CA434 is used as a donor and is sensitive to DNase when E. coli HB101 is the donor.