Clostridium sporogenes

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1. Classification

a. Higher order taxa

Domain Bacteria; Phylum Firmicutes; Class Clostridia; Order Clostridiales; Family Clostridaceae; Genus Clostridium

2. Description and significance

Clostridium sporogenes is a Gram-positive, rod shaped bacteria that exhibits spore production and flagellar motility (ABIS). C. sporogenes can be found in a variety of places including the following: soil, sediment in both marine and freshwater environments, preserved meat and dairy products, fecal matter, snake venom, and infections in domestic animals and humans (ABIS). C. sporogenes is phenotypically similar to other members of its genus C. difficile and C. botulinum; however, while C. botulinum produces the neurotoxin botulinum, which can cause disease in human, C. sporogenes is classified as a harmless biosafety level I organism by the American Type Culture Collection (Kubiak, 2015). This classification of C. sporogenes makes it a candidate for use in the medical field; specifically, the ability of C. sporogenes’ spores to only release its pathogens in a tumor-specific vector will be beneficial in cancer treatments aiming to reduce damage to non-cancerous cells within the host (Theys, 2006).

3. Genome structure

The Clostridium sporogenes DSM 795 genome, which is the first strain of C. sporogenes ever isolated, consists of a single circular chromosome 4.1 Mega-base pairs in length with an overall GC content of 27.81% (Poehlein, 2015) A total of 3,832 genes are encoded; however, only 3,744 genes have been identified as protein coding via computational analysis (i.e. they contain an open reading frame [ORF] of 300 or more bases within the gene sequence), while 80 genes have been identified to encode RNAs (10 rRNA genes and 70 tRNA genes). Six different genes that encode selenocysteine-containing proteins are located within the genome.

Clostridium botulinum strain A consists of a circular chromosome 3.9 Mega-base pairs in length with an overall GC content of 28.2%. A plasmid of 16,344 base pairs is also found within the genome (Bradbury, 2012). 16S rDNA sequence analysis shows a 99.7% sequence similarity between Clostridium sporogenes and Clostridium botulinum strain A. Similarity between C. sporogenes and C. botulinum is furthered by the 2,016 orthologous genes shared between the two.

4. Cell structure

Clostridium sporogenes is a bacterium of the genus Clostridium. Cells are Gram-positive and thus have a thick peptidoglycan layer containing teichoic acid surrounding the outer membrane of the cell. Typically cells take the shape of singular and/or chained rods (Poehlein, 2015). Like other members of the Clostridium genus, C. sporogenes produce endospores as a mechanism to survive unfavorable environmental conditions, thus making the bacterium difficult to kill (Brunt et al). An important distinction between C. sporogenes and its close relative C. botulinum is that C. sporogenes does not produce botulinum neurotoxins.

5. Metabolic processes

C. sporogenes are obligate anaerobes, thus they can neither utilize nor survive in the presence of oxygen. The bacterium survives via the fermentation of amino acids, also known as Stickland fermentation. In the reaction an electron donor amino acid is oxidized into a carboxylic acid one carbon shorter than the original amino acid, while an electron acceptor amino acid is reduced into carboxylic acid the same length as the previous amino acid (Nisman, 1954). This mechanism allows C. sporogenes to avoid the use of [H+] ions as electron acceptors. While nutrition is more commonly supplied by amino acid fermentation, glucose fermentation can be utilized as an additional source of nutrition. C. sporogenes is considered a neutrophile, thus having an optimal pH range of about 6.0 (acidic) – 7.6 (basic).

6. Ecology

Clostridium sporogenes is commonly isolated from soil, marine and fresh lake water sediment, preserved meat and dairy product, human intestines as well as human infections (Bergey, 2009). Some strains are capable of producing bacteriocin-like substances that can inhibit other C. sporogenes strains (Betz, 1964). In the gut microflora of human, C. sporogenes converts tryptophan into indole and subsequently indole-3-propionic acid (IPA), a potent antioxidant in human body (Karbownik, 2001) that being investigated as a possible treatment for Alzheimer's disease (Bendheim, 2002).

7. Pathology

Unlike Clostridium botulinum, C. sporogenes does not carry plasmids that are responsible for neurotoxicity (Weickert, 1986). C. sporogenes toxins can induce hemorrhage in rabbit but not in other animals (Hara, 1997). Although isolated from many infections, the role of C. sporogenes as pathogen has been not yet determined. However, the highly proteolytic nature of C. sporogenes is thought to possibly act as adjuvant for opportunistic pathogens in human and animal infections (Bergy, 2009).

8. Medical Applications

Due to its anaerobic nature and sporulation, C. sporogenes colonizes specific hypoxic areas of solid tumor when intravenously delivered into tumor-transplanted mice (Minton, 2001). Combined administration of C. sporogenes with enzyme for antitumor prodrug cleavage is currently being investigated as a promising and low-toxicity treatment in cancer therapy (Liu, Lambin).

9. Current Research

Spores of C. sporogenes target the hypoxic core of a tumor and can cause the tumor to lyse as a reaction to simple reproduction and expansion of the bacteria. C. sporogenes can be effectively transformed with a plasmid expressing prodrug-converting enzyme (PCE) that cleaves nontoxic, pre-administered prodrug CB1954 into its cytotoxic form which lyses solid tumor at the hypoxic core (Heap, 2014). The experiment was repeated 3 times over the period of 70 days, and no microenvironmental shifts were observed in tumor area (Heap, 2014). The same specificity was demonstrated in the transformation through conjugation of C sporogenes with cytosine deaminase (CD) plasmid, whose product can cleave the pre-administered 5-flourocytosine (5-FC) into the cytotoxic 5-flourouracil (5-FU) for tumor lysis (Liu, 2002). However, due to high risk of infection and toxicity after the spore germinated in tumor-bearing animal (Dang et al, 2001), bacterial treatment is not widely accepted. Researcher have attempted to overcome this limitation by heat-inactivating C.sporogenes to produce its non-viable derivative before administration to a tumor culture. Non-viable C.sporogenes have demonstrated an ability to inhibit colorectal cancer cells in vivo (Bhave et al, 2015).

10. References

ABIS Encyclopedia. “Clostridium sporogenes.” ABIS Encyclopedia. Regnum Prokaryote, n.d. http://www.tgw1916.net/Clostridium/sporogenes.html. Web.

Bendheim PE, et al. “Development of indole-3-propionic acid (OXIGON) for Alzheimer's disease”. Journal of Molecular Neuroscience. 2002;19:213–217.

Bergey, D. H., William B. Whitman, Paul De Vos, George M. Garrity, and D. Jones. "Genus I. Clostridium." Bergey's Manual of Systematic Bacteriology. 9th ed. Vol. 3. New York: Springer, 2009. 817. Print.

Betz, J. V., and K. E. Anderson. 1964. Isolation and characterization of bacteriophages active on Clostridium sporogenes. J. Bacteriol. 87:408-415.

Bhave, Madhura Satish et al. “Effect of Heat-Inactivated Clostridium Sporogenes and Its Conditioned Media on 3-Dimensional Colorectal Cancer Cell Models.” Scientific Reports 5 (2015): 15681. PMC. Web.

Brunt, Jason et al. “Functional Characterisation of Germinant Receptors in Clostridium Botulinum and Clostridium Sporogenes Presents Novel Insights into Spore Germination Systems.” Ed. Abraham L. Sonenshein. PLoS Pathogens 10.9 (2014): e1004382. PMC.

Dang, Long H. et al. “Combination Bacteriolytic Therapy for the Treatment of Experimental Tumors.” Proceedings of the National Academy of Sciences of the United States of America 98.26 (2001): 15155–15160. PMC. Web.

Hara, K, Y., A. Ogura, Y. Noguchi, and S. Kumagai. "Characteristics of Toxicity and Haemorrhagic Toxin Produced by Clostridium Sporogenes in Various Animals and Cultured Cells." Journal of Medical Microbiology 46.4 (1997): 270-75. Web.

Heap, John T. et al. “Spores of Clostridium Engineered for Clinical Efficacy and Safety Cause Regression and Cure of Tumors in Vivo.” Oncotarget 5.7 (2014): 1761–1769. Print.

Karbownik M, et al. “Indole-3-propionic acid, a melatonin-related molecule, protects hepatic microsomal membranes from iron-induced oxidative damage: Relevance to cancer reduction”. Journal of Cell Biochemistry. 2001;81:507–513.

Kubiak, Aleksandra M. et al. “Complete Genome Sequence of the Nonpathogenic Soil-Dwelling Bacterium Clostridium Sporogenes Strain NCIMB 10696.” Genome Announcements 3.4 (2015): e00942–15. PMC.

Lambin, P., J. Theys, W. Landuyt, P. Rijken, A. Van Der Kogel, E. Van Der Schueren, R. Hodgkiss, J. Fowler, S. Nuyts, E. De Bruijn, L. Van Mellaert, and J. Anné. "Colonisation Of Clostridium In the Body Is Restricted to Hypoxic and Necrotic Areas of Tumours." Anaerobe 4.4 (1998): 183-88.

Liu, S-C, Np Minton, Aj Giaccia, and Jm Brown. "Anticancer Efficacy of Systemically Delivered Anaerobic Bacteria as Gene Therapy Vectors Targeting Tumor Hypoxia/necrosis." Gene Therapy Gene Ther 9.4 (2002): 291-96.

Minton, Nigel P., J. Martin Brown, Philippe Lambin, and Jozef Anné. "Clostridia in Cancer Therapy." Biotechnology and Medical Applications Clostridia (2001): 251-70. Web

Poehlein, Anja et al. “Genome Sequence of Clostridium Sporogenes DSM 795T, an Amino Acid-Degrading, Nontoxic Surrogate of Neurotoxin-ProducingClostridium Botulinum.” Standards in Genomic Sciences 10 (2015): 40. PMC. Web.

Theys, J et al. “Repeated Cycles of Clostridium-Directed Enzyme Prodrug Therapy Result in Sustained Antitumour Effects in Vivo.” British Journal of Cancer 95.9 (2006): 1212–1219. PMC.

Weickert, M J, G H Chambliss, and H Sugiyama. “Production of Toxin by Clostridium Botulinum Type A Strains Cured by Plasmids.” Applied and Environmental Microbiology 51.1 (1986): 52–56. Print.

Wikoff, William R. et al. “Metabolomics Analysis Reveals Large Effects of Gut Microflora on Mammalian Blood Metabolites.” Proceedings of the National Academy of Sciences of the United States of America 106.10 (2009): 3698–3703. PMC. Web.