Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination.

UNLABELLED: Bacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis during B. subtilis spore germination. Here, we investigated the timing of DPA release and cortex hydrolysis during Clostridium difficile spore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor, cspC, or the cortex hydrolase, sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release during C. difficile spore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediated C. difficile spore germination proceeds through a novel germination pathway. IMPORTANCE: Clostridium difficile infects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease. C. difficile spore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to the Bacillus subtilis germinant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism and C. difficile. We found that C. difficile spore germination proceeds through a novel pathway.

Muricholic acids inhibit Clostridium difficile spore germination and growth.

Infections caused by Clostridium difficile have increased steadily over the past several years. While studies on C. difficile virulence and physiology have been hindered, in the past, by lack of genetic approaches and suitable animal models, newly developed technologies and animal models allow these processes to be studied in detail. One such advance is the generation of a mouse-model of C. difficile infection. The development of this system is a major step forward in analyzing the genetic requirements for colonization and infection. While important, it is equally as important in understanding what differences exist between mice and humans. One of these differences is the natural bile acid composition. Bile acid-mediated spore germination is an important step in C. difficile colonization. Mice produce several different bile acids that are not found in humans. These muricholic acids have the potential to impact C. difficile spore germination. Here we find that the three muricholic acids (α-muricholic acid, ß-muricholic acid and ω-muricholic acid) inhibit C. difficile spore germination and can impact the growth of vegetative cells. These results highlight an important difference between humans and mice and may have an impact on C. difficile virulence in the mouse-model of C. difficile infection.

Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection.

Clostridium difficile spores must germinate in vivo to become actively growing bacteria in order to produce the toxins that are necessary for disease. C. difficile spores germinate in vitro in response to certain bile acids and glycine. In other sporulating bacteria, proteins embedded within the inner membrane of the spore sense the presence of germinants and trigger the release of Ca⁺⁺-dipicolinic acid (Ca⁺⁺-DPA) from the spore core and subsequent hydrolysis of the spore cortex, a specialized peptidoglycan. Based upon homology searches of known germinant receptors from other spore-forming bacteria, C. difficile likely uses unique mechanisms to recognize germinants. Here, we identify the germination-specific protease, CspC, as the C. difficile bile acid germinant receptor and show that bile acid-mediated germination is important for establishing C. difficile disease in the hamster model of infection. These results highlight the importance of bile acids in triggering in vivo germination and provide the first description of a C. difficile spore germinant receptor. Blocking the interaction of bile acids with the C. difficile spore may represent an attractive target for novel therapeutics.

Site-directed mutations in the lanthipeptide mutacin 1140.

The oral bacterium Streptococcus mutans, strain JH1140, produces the antibiotic mutacin 1140. Mutacin 1140 belongs to a group of antibiotics called lanthipeptides. More specifically, mutacin 1140 is related to the epidermin type A(I) lanthipeptides. Mutagenesis experiments of this group of lanthipeptides have been primarily restricted to the posttranslationally modified meso-lanthionine and 3-methyllanthionine residues. Site-directed mutagenesis of the core peptide of mutacin 1140 was performed using the suicide vector pVA891. Substitutions of the N-terminal residue, the charged residue in the hinge region, and residues in ring A and intertwined rings C and D were investigated. A truncation and insertion of residues in ring A and intertwined rings C and D were also performed to determine whether or not they would alter the antimicrobial activity of the producing strain. Bioassays revealed that five of 14 mutants studied had improved antimicrobial activity against the indicator strain Micrococcus luteus ATCC 10240. MICs against Streptococcus mutans UA159, Streptococcus pneumoniae ATCC 27336, Staphylococcus aureus ATCC 25923, Clostridium difficile UK1, and Micrococcus luteus ATCC 10240 were determined for three mutacin 1140 variants that had the most significant increases in bioactivity in the M. luteus bioassay. This mutagenesis study of the epidermin group of lanthipeptides shows that antimicrobial activity can be significantly improved.

Both fidaxomicin and vancomycin inhibit outgrowth of Clostridium difficile spores.

Fidaxomicin (FDX) is approved to treat Clostridium difficile-associated diarrhea and is superior to vancomycin in providing a sustained clinical response (cure without recurrence in the subsequent 25 days). The mechanism(s) behind the low recurrence rate of FDX-treated patients could be multifactorial. Here, we tested effects of FDX, its metabolite OP-1118, and vancomycin on spore germination and determined that none affected the initiation of spore germination but all inhibited outgrowth of vegetative cells from germinated spores.

Validation of quantitative polymerase chain reaction assays for measuring cytokine expression in equine macrophages.

The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. A set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1alpha, IL-1beta, IL-6, IL-8 and TNF-alpha were validated using QPCR primers and probes which were generated for the equine IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha and 18S genes. Amplification efficiency, intra-assay and inter-assay variation were determined using 10-fold dilutions of plasmid for each gene. Under these conditions the amplification efficiencies of the primers and probes ranged from 99% to 101%. The mean coefficient of variation (CV) across five sets of plasmid DNA for both intra-assay and inter-assay variation was 0.63% (range 0.2% to 1.8%). Amplification efficiency was also determined using 2-fold dilutions of cDNA and under these conditions amplification efficiency ranged from 83% to 95%. The specificity of amplification was confirmed by DNA sequencing of reaction products. The QPCR assays were also evaluated using three sets of cDNA from equine monocyte derived macrophages (EMDM) stimulated for 1 h with lipopolysaccharide (LPS). The general trend was the same for all three samples with IL-1alpha showing the greatest induction and IL-6 the lowest induction. The range of cytokine induction was greater than has previously been reported with values ranging from 12-fold to 30,000-fold. We present a set of QPCR primers and probes that are suitable for quantitation of expression of a set of equine cytokines. The primers and probes have been rigorously analyzed, and we demonstrate that they are specific for the desired genes, have a high amplification efficiency and the assays are highly reproducible.