Construction and analysis of chromosomal Clostridium difficile mutants

Abstract

SummaryClostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence but our ability to study the molecular mechanisms underlying the pathogenesis of C. difficile-associated disease has been limited because of a lack of tools for its genetic manipulation. We have now developed a reproducible method for the targeted insertional inactivation of chromosomal C. difficile genes. The approach relies on the observation that an Escherichia coli-Clostridium perfringens shuttle vector is unstable in C. difficile and can be used as a form of conditional lethal vector to deliver gene constructs to the chromosome. We have used this methodology to insertionally inactivate two putative response regulator genes, rgaR and rgbR, which encode proteins with similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic analysis demonstrated that the C. difficile RgaR protein positively regulated four genes, including a putative agrBD operon. The RgaR protein was also purified and shown to bind specifically to sites that contained two consensus VirR boxes located just upstream of the putative promoters of these genes. The development of this methodology will significantly enhance our ability to use molecular approaches to develop a greater understanding of the ability of C. difficile to cause disease.

Clostridium difficile glutamate dehydrogenase is a secreted enzyme that confers resistance to H2O2

“…C. difficile strains R20291 (Stabler et al, 2009), CD646 (Keel et al, 2007), JIR8094 (O'Connor et al, 2006) and the gluD mutants (Table 1) were grown anaerobically (10 % H 2 , 10 % CO 2 and 80 % N 2 ) in TY (tryptose yeast extract) broth or TY agar as described previously (Dupuy & Sonenshein, 1998). Escherichia coli strain S17-1 (Teng et al, 1998) used for conjugation was cultured aerobically in LB and supplemented with chloramphenicol (30 mg ml 21 ) or ampicillin (100 mg ml 21 ), when needed.…”

The role of toxin A and toxin B in Clostridium difficile infection

“…However, perhaps more likely is that there is one or more key differences between the strains of C. difficile studied. Although both strains are erythromycin-sensitive derivatives of strain 630 10, 11 , they were isolated independently through serial sub-culture 8,12 . Therefore, either strain could have acquired one or more secondary mutations, which may affect the action of either one or both of the toxins.…”

Both, toxin A and toxin B, are important inClostridium difficileinfection.

“…Interestingly, although the data were not presented, it was reported to have undergone the same specific deletion of ermB as 630Δerm. 20,28 It is our hypothesis that during repeated subculture, ancillary mutations have arisen which have impacted on the virulence potential of one or other of the two strains in the presence of different toxin gene alleles. Crude phenotypic analyses support this view, as preliminary sideby-side comparisons of the 630Δerm and 630E have shown clear phenotypic differences.…”

“…The creation of insertional mutants by the former approach was achieved using either replication deficient 19 or defective 20 plasmids which are inserted into the chromosome via a single crossover recombination event. As such an event results in duplication of a region of DNA on either side of the inserted plasmid, the mutants generated are predicted to be unstable, as a subsequent recombination event between the duplicated DNA will result in plasmid excision.…”

“…Indeed, such instability has been observed in a number of studies to date. [20][21][22] In the second approach, a system was developed that capitalised on the ability to retarget the specificity of a mobile group II intron to virtually any desired chromosomal locus. Pioneered by the Lambowitz laboratory, this system was marketed by Sigma Aldrich under the brand-name 'TargeTron'.…”