Molecular biology, genetics and biotechnologyComparative genomic analysis of a neurotoxigenic Clostridium species using partial genome sequence: Phylogenetic analysis of a few conserved proteins involved in cellular processes and metabolism
Introduction
Clostridial organisms produce botulinum neurotoxin and tetanus neurotoxin, which are generally regarded as the two most potent toxic substances of biological origin [1]. Neurotoxigenic clostridia have attracted considerable attention during the past decades. These neurotoxins have gained increasing significance as potential biological toxin warfare (BTW) agents and are generally more lethal than microbial pathogens, quickly causing incapacitation or death within minutes or hours [2]. Since the introduction of potent vaccine during World War II, cases of tetanus disease have been only sporadic in industrial countries. However, the disease, and in particular maternal and neonatal tetanus, is still an important cause of death due to insufficient immunization [3]. Neonatal tetanus is considered endemic to 90 developing countries and resulted in 248,000 deaths in 1997 (World Health Organization; www.who.int/vaccine-diseases/NeonatalTetanus.shtml). In 1989, the worldwide public community made a commitment to the elimination of neonatal tetanus which has resulted in substantial decrease in the number of reported cases [4]. However, tetanus as whole continues to cause about 213,000–293,000 deaths worldwide each year, predominantly in low-income and middle-income countries. Tetanus is characterized by muscle rigidity and painful muscle spasms caused by tetanus toxin's blockade of inhibitory neurons that normally oppose and modulate the action of excitatory motor neurons. The muscle rigidity usually begins in the masseter muscles, resulting in trismus (lockjaw). As disease severity increases, muscle rigidity extends throughout the body progressing to spontaneous longlasting excruciating spasms of many muscle groups. Death occurs due to respiratory failure or autonomic dysfunction resulting in haemodynamic and cardiac arrest.
The mode of action of lethal neurotoxins produced by clostridia is now well understood [1], [3], [5], [6]. There are seven serotypes (A through G) of botulinum neurotoxins (BoNTs), produced by bacteria of the genus Clostridium, largely by Clostridium botulinum species, but BoNTs are also reported from other members of this heterogeneous group [7], [8]. Together with tetanus neurotoxin (TeNT) produced by Clostridium tetani, the BoNTs makeup the CNT family. TeNT shares ∼65% homology and ∼35% identity with the BoNT serotypes [9]. It has been proposed that these toxins arose from a single ancestral form referred to as ancestral-NT [10] that arguably originated in a species distinct from C. botulinum and C. tetani. What is more intriguing though is the subsequent divergence and heterogeneity of C. botulinum as against an apparently monophyletic Clostridium tetani group. Lack of multiple serotypes for C. tetani cannot be considered an indicator of its genetic homogeneity, since genealogical trees derived from BoNT antigenic types (A through G) show marked discordance with those depicting natural relationships inferred from 16S rRNA and phenotypic clusters. The genus Clostridium is indeed not a monophylectic group but actually consists of more than 20 genera and probably embraces several families [11].
Until the publication of complete genome sequence of strain E88, genetic information on C. tetani was mainly restricted to nucleotide sequences of tetanus toxin TeTx and of its direct transcriptional activator TetR, both of which are encoded on a plasmid [12], [13]. It is surprising that unlike botulinum, variations in the neurotoxin genes have not been reported among C. tetani strains in spite of the fact that they share similar habitats and are capable of thriving in the environment and colonizing host niche. Further, it is not clear why divergence of ancestral-NT in C. botulinum group I, II, and III organisms was much more significant than the tetanus toxin, though NTs partitioned in clostridial subpopulations early in course of its evolution.
During the last few years, whole genome sequence data is accumulating for several strains of various bacterial species, increasing out understanding of the intra-species diversity [14], [15]. It is now believed that sequencing a single strain is not sufficient to fully cover the genomic structure of bacterial pathogen. Some bacterial pathogens have been shown to posses ‘open pan-genome’ (e.g. group B Streptococcus) with remarkable heterogeneity while others are shown to be largely homogenous with a ‘closed pan-genome’ [16], [17]. Knowledge about the microevolution within a pathogenic bacterial species is especially important from the perspective of designing strategies for protection against them. For example protection against a candidate vaccine shall provide sufficient strain coverage while therapeutic targets should be screened against the panel of strains preferably from diverse sources [18]. Not many strains of C. tetani are genetically described, though reports of such kind are many from C. botulinum strains. It is counterintuitive to believe that strains of a bacterium that occupies a dual niche (host and the soil environment) will be physiologically and genetically coherent especially when its counterpart with similar pathogenicity profile displays remarkable phenotypic and genotypic heterogeneity. This could possibly be due to lack of characterization data from several strains of C. tetani, which has not drawn attention of many investigators; there is only one serotype of TeNT as against seven serotypes of BoNTs. Comparison of gene sets from other isolates with E88 strain may provide valuable insight into the biology of this medically important bacterial pathogen.
We have isolated in our laboratory a Clostridium species, producing a neurotoxin which was neutralized by botulinum antitoxin (A + B + E) as well as tetanus antitoxin when tested by mouse protection bioassay. The toxin genes were sequenced and formed a tight cluster with respective components of TeNT [19]. The close phylogenetic affinities of this strain at 16S rDNA and toxin gene level, with C. tetani E88 indicated it to be a strain of C. tetani. Here we report a comparison of this strain at the genomic level by random sampling and sequencing of a small proportion of the genome.
Section snippets
Bacterial strain and culture conditions
A C. tetani strain isolated previously from decaying fish sample was used for the studies that we have now designated as strain drde. Cells were grown in trypticase peptone yeast extract glucose (TPYG) broth containing pancreatic digest of casein, 50 g; peptone, 5 g; yeast extract, 20 g; glucose, 4 g; sodium thioglycollate, 1g; NaCl, 2g; soluble starch, 1g; cycloserine, 250 mg; sulphamethoxazole, 76 mg and trimethoprim, 4 mg per litre. Growth was carried out anaerobically in TPYG broth at 37 °C
Results
C. tetani drde was isolated from intestine of decaying fish by the method earlier described [21]. The strain exhibited closest similarity (99%) with C. tetani at 16S rRNA level and produced a neurotoxin that was neutralized by botulinum antitoxin (A + B + E) as well as tetanus antitoxin when tested by mouse protection assay [19]. This strain also exhibited phenotypic differences both, with C. botulinum and C. tetani type strains especially with respect to the utilization of carbon sources (data
Discussion
Although we know for a number of bacterial species that particular strains or clones are more pathogenic or virulent than others, currently for risk assessment all members of a species are considered as equal; possibly due to lack of hard data, or out of precaution. For future we wish hazard characterization to include more precise data on variability in virulence between strains.
Over the last several years a number of studies on genetic variability of bacterial pathogens have been conducted
Conclusions
C. tetani drde appears to be an environmental strain of C. tetani, closely related to the clinical strain E88 at genetic level. The phenotypic differences observed for this strain has possibly arisen by alterations of genes involved in metabolic functions. There appears to be further loss of transposable elements in C. tetani drde when compared with strain E88, further supporting the view that C. tetani genome is stable. Phylogenetic affinities of different genes varied in terms of tree
Acknowledgments
We thank Dr. R. Vijayaraghawan, Director, DRDE, Gwalior for providing all facilities and support required for this study. The work has been funded by Defence Research and Development Organization, Government of India.
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