Neil Fairweather

The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome.

We determined the complete genome sequence of Clostridium difficile strain 630, a virulent and multidrug-resistant strain. Our analysis indicates that a large proportion (11%) of the genome consists of mobile genetic elements, mainly in the form of conjugative transposons. These mobile elements are putatively responsible for the acquisition by C. difficile of an extensive array of genes involved in antimicrobial resistance, virulence, host interaction and the production of surface structures. The metabolic capabilities encoded in the genome show multiple adaptations for survival and growth within the gut environment. The extreme genome variability was confirmed by whole-genome microarray analysis; it may reflect the organisms niche in the gut and should provide information on the evolution of virulence in this organism.

Antibiotic Treatment of Clostridium difficile Carrier Mice Triggers a Supershedder State, Spore-Mediated Transmission, and Severe Disease in Immunocompromised Hosts

ABSTRACT Clostridium difficile persists in hospitals by exploiting an infection cycle that is dependent on humans shedding highly resistant and infectious spores. Here we show that human virulent C. difficile can asymptomatically colonize the intestines of immunocompetent mice, establishing a carrier state that persists for many months. C. difficile carrier mice consistently shed low levels of spores but, surprisingly, do not transmit infection to cohabiting mice. However, antibiotic treatment of carriers triggers a highly contagious supershedder state, characterized by a dramatic reduction in the intestinal microbiota species diversity, C. difficile overgrowth, and excretion of high levels of spores. Stopping antibiotic treatment normally leads to recovery of the intestinal microbiota species diversity and suppresses C. difficile levels, although some mice persist in the supershedding state for extended periods. Spore-mediated transmission to immunocompetent mice treated with antibiotics results in self-limiting mucosal inflammation of the large intestine. In contrast, transmission to mice whose innate immune responses are compromised (Myd88−/−) leads to a severe intestinal disease that is often fatal. Thus, mice can be used to investigate distinct stages of the C. difficile infection cycle and can serve as a valuable surrogate for studying the spore-mediated transmission and interactions between C. difficile and the host and its microbiota, and the results obtained should guide infection control measures.

Binding of Clostridium difficile Surface Layer Proteins to Gastrointestinal Tissues

ABSTRACT Clostridium difficile is the etiological agent of antibiotic-associated diarrhea, a potentially serious condition frequently affecting elderly hospitalized patients. While tissue damage is primarily induced by two toxins, the mechanism of gut colonization, and particularly the role of bacterial adherence to the mucosa, remains to be clarified. Previous studies have shown binding of C. difficile whole cells to cultured cell lines and suggested the existence of multiple adhesins, only one of which has been molecularly characterized. In this paper, we have investigated tissue binding of C. difficile surface layer proteins (SLPs), which are the predominant outer surface components and are encoded by the slpA gene. The adherence of C. difficile to HEp-2 cells was studied by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis, which showed that antibodies to the high-molecular-weight (MW) SLP inhibited adherence. Immunohistochemical analysis of human gastrointestinal tissue sections revealed strong binding both to the surface epithelium lining the digestive cavities and to the subjacent lamina propria, while glands were negative. A similar pattern was observed in the mouse. By using purified recombinant SLPs, we show that binding is largely mediated by the high-MW SLP. By Western blotting analysis, we have identified two potential ligands of the C. difficile SLPs, one of which may be specific to the gut. By using purified extracellular matrix components immobilized on nitrocellulose, we also show SLP binding to collagen I, thrombospondin, and vitronectin, but not to collagen IV, fibronectin, or laminin. These results raise the possibility that the SLPs play a role both in the initial colonization of the gut by C. difficile and in the subsequent inflammatory reaction.

Molecular characterization of the surface layer proteins from Clostridium difficile

Many bacteria express a surface‐exposed proteinaceous layer, termed the S‐layer, which forms a regular two‐dimensional array visible by electron microscopy. Clostridium difficile is unusual in expressing two S‐layer proteins (SLPs), which are of varying size in a number of strains. In an approach combining molecular biology with mass spectrometric sequencing strategies, we have identified the structural gene (slpA) for the S‐layer from three strains of C. difficile. Both proteins are derived from a common precursor, and processing involves the removal of a signal peptide and a second cleavage to release the two mature SLPs. To our knowledge, this is the first example in which two SLPs have been shown to derive from a single gene product through post‐translational processing, rather than from the expression of separate genes. The higher molecular weight (MW) SLP is highly conserved among the three strains, whereas the lower MW SLP shows considerable sequence diversity, reflecting the results from Western blotting. The high‐MW SLP shows weak homology to N‐acetyl muramoyl‐l‐alanine amidase from Bacillus subtilis, and both the native SLP from C. difficile and a recombinant protein expressed in Escherichia coli were found to display amidase activity by zymography. The high‐MW SLPs showed evidence of glycosylation, whereas the lower MW proteins did not. A family of genes with sequence homology to the amidase domain of the high‐MW SLP was identified in the C. difficile strain 630 genome, some of which are located in the same region of the genome as slpA and were shown by reverse transcription–polymerase chain reaction (RT–PCR) analysis to be transcribed.

Bacterial Spores as Vaccine Vehicles

ABSTRACT For the first time, bacterial spores have been evaluated as vaccine vehicles. Bacillus subtilis spores displaying the tetanus toxin fragment C (TTFC) antigen were used for oral and intranasal immunization and were shown to generate mucosal and systemic responses in a murine model. TTFC-specific immunoglobulin G titers in serum (determined by enzyme-linked immunosorbent assay) reached significant levels 33 days after oral dosing, while responses against the spore coat proteins were relatively low. Tetanus antitoxin levels were sufficient to protect against an otherwise lethal challenge of tetanus toxin (20 50% lethal doses). The robustness and long-term storage properties of bacterial spores, coupled with simplified genetic manipulation and cost-effective manufacturing, make them particularly attractive vehicles for oral and intranasal vaccination.

Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties

Acellular whooping cough vaccines are based on pertussis toxoid but their effectiveness may be increased by the addition of other Bordetella pertussis antigens. We expressed the immunogenic outer membrane protein pertactin (P69) from B. pertussis to high levels in multi-copy transformants of the industrial yeast Pichia pastoris. In high-density fermentations, engineered P. pastoris yielded greater than 3 g of the protein per litre of culture. Purified recombinant pertactin was able to stimulate the incomplete protection afforded by toxoid to the level of the whole-cell vaccine, as shown by the Kendrick test, supporting its inclusion in future acellular vaccines.

Structural insights into the molecular organization of the S-layer from Clostridium difficile.

Clostridium difficile expresses a surface layer (S‐layer) which coats the surface of the bacterium and acts as an adhesin facilitating interaction of the bacterium with host enteric cells. The S‐layer contains a high‐molecular‐weight S‐layer protein (HMW SLP) and its low‐molecular‐weight partner protein (LMW SLP). We show that these proteins form a tightly associated non‐covalent complex, the H/L complex, and we identify the regions of both proteins responsible for complex formation. The 2.4 Å X‐ray crystal structure of a truncated derivative of the LMW SLP reveals two domains. Domain 1 has a two‐layer sandwich architecture while domain 2, predicted to orientate towards the external environment, contains a novel fold. Small‐angle X‐ray scattering analysis of the H/L complex shows an elongated molecule, with the two SLPs arranged ‘end‐to‐end’ interacting with each other through a small contact area. Alignment of LMW SLPs, which exhibit high sequence diversity, reveals a core of conserved residues that could reflect functional conservation, while allowing for immune evasion through sequence variation. These structures are the first described for the S‐layer of a bacterial pathogen, and provide insights into the assembly and biogenesis of the S‐layer.

Stable expression of foreign antigens from the chromosome of Salmonella typhimurium vaccine strains.

A simple and versatile system has been developed using a new cloning vector which can serve as a vehicle for integrating DNA fragments, which direct the expression of heterologous antigens, into the aroC gene on the Salmonella chromosome. The system is based on Escherichia coli plasmid vectors which contain the DNA fragment, cloned from the chromosome of S. typhimurium C5, which encodes the aroC gene. The aroC gene was modified using synthetic oligodeoxyribonucleotides so that it contained several unique restriction sites into which DNA, directing the expression of heterologous antigens, could be cloned. DNA was integrated into the S. typhimurium chromosome at aroC by transferring the vectors into S. typhimurium polA mutants and allowing homologous recombination to occur between the cloned and chromosomal aroC genes. The vectors were used to integrate nucleotide sequences into the S. typhimurium chromosome which directed the expression of tetanus toxin fragment C and the Treponema pallidum lipoprotein. The expression of both antigens was detected by Western blotting.

Construction and characterization of Bordetella pertussis mutants lacking the vir-regulated P.69 outer membrane protein

The Bordetella pertussis P.69 protein is an immunogen with vaccine potential. The role of this protein in pathogenesis is unclear; it has been associated with the toxic adenylate cyclase and adhesion to eukaryotic cells. For further analysis of the role of P.69 in the biology of B. pertussis, we have constructed strains which specifically lack P.69.

Phospholipase C and haemolytic activities of Clostridium perfringens alpha-toxin cloned in Escherichia coli: sequence and homology with a Baciilus cereus phospholipase C

The Clostridium perfringens alpha‐toxin (phospholipase C) gene (cpa) has been cloned and expressed in Escherichia coli. The biological activities of the cloned gene product have been analysed and the complete nucleotide sequence of the cpa gene has been determined. The cloned cpa gene product, which is exported to the periplasm in E. coli, possesses both phospholipase C and haemolytic activities. Haemolysis is not apparent when cell extracts are incubated with isotonic suspensions of sheep erythrocytes, but can be detected and quantified readily when dilutions of the same extracts are placed in wells in sheep‐blood agar plates. Like other sequenced clostridial genes, the cpa gene has a high AT content (66.4%), exhibits a strong bias for using codons with A or T in the wobble position, and the 350 base pairs upstream from the gene have a significantly higher AT content (79.5%) than the coding region. The cpa gene encodes a 398 amino acid polypeptide with a deduced molecular weight of 45481D. This is very similar to the estimated molecular weight (Mr) of the cpa primary gene product expressed in an in vitro transcription‐translation system (Mr 46000), but larger than the cpa gene product detected in E. coli minicells, E. coli whole cells or in C. perfringens cells (Mr 43000), suggesting post‐translational processing. The 28 N‐terminal residues of the deduced alpha‐toxin sequence possess the consensus features of a signal peptide and may be removed during secretion. The deduced alpha‐toxin sequence shares significant structural homology with the phosphatidylcholine‐preferring phospholipase C of Bacillus cereus.