The genome sequence of Clostridium tetani, the causative agent of tetanus disease

Abstract

Tetanus disease is one of the most dramatic and globally prevalent diseases of humans and vertebrate animals, and has been reported for over 24 centuries. The manifestation of the disease, spastic paralysis, is caused by the second most poisonous substance known, the tetanus toxin, with a human lethal dose of approximately 1 ng/kg. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid; nevertheless, according to the World Health Organization, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The causative agent of tetanus disease is Clostridium tetani, an anaerobic spore-forming bacterium, whose natural habitat is soil, dust, and intestinal tracts of various animals. Here we report the complete genome sequence of toxigenic C. tetani E88, a variant of strain Massachusetts. The genome consists of a 2,799,250-bp chromosome encoding 2,372 ORFs. The tetanus toxin and a collagenase are encoded on a 74,082-bp plasmid, containing 61 ORFs. Additional virulence-related factors could be identified, such as an array of surface-layer and adhesion proteins (35 ORFs), some of them unique to C. tetani. Comparative genomics with the genomes of Clostridium perfringens, the causative agent of gas gangrene, and Clostridium acetobutylicum, a nonpathogenic solvent producer, revealed a remarkable capacity of C. tetani: The organism can rely on an extensive sodium ion bioenergetics. Additional candidate genes involved in the establishment and maintenance of a pathogenic lifestyle of C. tetani are presented.

Figure 1

Circular maps of the chromosome and the plasmid pE88 of C. tetani. The coding sequence of the chromosome is shown in blue or green, depending on strand orientation. ORFs of C. tetani that have homologous proteins in C. perfringens (15) but not in C. acetobutylicum (16) are shown in orange. ORFs of C. tetani, which are not present in the two other mentioned clostridial genomes, are shown in pink. The plasmid map shows ORFs color-coded according to their assigned functions. The gene of the tetanus toxin, tetX (CTP60), and the gene encoding a collagenase, colT (CTP33), are highlighted in red. Numbers at the inner ring refer to genes mentioned in the text. Most inner rings of both maps show the G+C content variation (higher values outward).

Figure 2

Comparison of clostridial collagenases. Segment comparison of ColG and ColH of C. histolyticum (21), ColA of C. perfringens (22), ColB of C. botulinum (Sanger Centre, C. botulinum sequencing project; online access: ftp://ftp.sanger.ac.uk/pub/pathogens/cb/), and ColT of C. tetani. Bacterial collagenases consist of three different segments (21). Segment 1 represents the catalytic domain containing the consensus motif for zinc proteases, HEXXH. Segment 2 contains the so-called PKD domain (PF00801) of unknown function. The name refers to the polycystic kidney disease protein PKD1, in which the domain was detected in multiple copies. Segment 3 is thought to be the collagen-binding domain.

Figure 3

Occurrence of homologous ORFs in C. tetani, C. perfringens, and C. acetobutylicum. Clostridial “backbone” ORFs, white; ORFs in C. tetani with homologues in C. perfringens but not in C. acetobutylicum, orange; C. tetani ORFs present in C. acetobutylicum but absent in C. perfringens, red; C. tetani ORFs not present in C. perfringens and C. acetobutylicum, pink. The size of the circles is proportional to the number of ORFs in each organism.

Figure 4

Role of Na⁺ in amino acid fermentations by C. tetani. The color code is as in Fig. 3. Information on the NADH:ubiquinone oxidoreductase/Rhodobacter-specific nitrogen fixation electron transport system is published as Fig. 7 in supporting information on the PNAS web site. The substrate specificity of many Na⁺-dependent symporters (tagged with *) remains obscure (31). V-type ATPase, vacuolar-type ATPase; V-type Ppase, vacuolar-type pyrophosphatase; NatAB, an ATP-binding cassette transporter (32); Fd, ferredoxin; NorM, a Na⁺-translocating multidrug transporter characterized in Vibrio parahaemolyticus (33); SMR, small multidrug resistance family; NhaC and NhaP, one-subunit secondary H⁺/Na⁺ antiporters; MRP/SHA, this seven-subunit H⁺/Na⁺ antiporter with similarities to hydrophobic components of the respiratory Complex I has been characterized in Bacillus subtilis and Staphylococcus aureus (34).