Clostridium novyi

Clostridium novyi (oedematiens) a Gram-positive, endospore- forming, obligate anaerobic bacteria of the class clostridia. It is ubiquitous, being found in the soil and faeces. It is pathogenic, causing a wide variety of diseases in man and animals. It comes in three types, labelled A, B, and a non-pathogenic type C distinguished by the range of toxins they produce. Some authors include Clostridium haemolyticum as Clostridium novyi type D. C novyi is closely related to Clostridium botulinum types C and D as Yoshimasa Sasaki et al. have demonstrated by 16S rDNA sequence analysis.^[1] The PAGE analysis reported in ref 1 seems to indicate that the differences between these closely related types is a matter of gene expression rather than major genetic differences. For example type C can be induced to produce the lethal alpha-toxin.

Growth in culture proceeds through 3 stages: Initial growth wherein no toxin is produced; vigorous growth wherein toxin is produced; and spore formation wherein endospores are formed and toxin production decreases. It is suggested that type C may be type B that forms spores more readily so does not go through the toxin-production stage.

Isolating and identifying C novyi is difficult due to its extreme anaerobic nature. Commercial kits may not be adequate.^[2][3]

It is also fastidious and difficult to culture, requiring the presence of thiols.^[4]

Toxins

The toxins are designated by Greek letters. The toxins normally produced by the various types are shown in table 1^[5]

Table 1
C novyi type	Toxins
A	alpha, gamma, delta, epsilon
B	alpha, beta, zeta
C	gamma

The alpha-toxin of Clostridium botulinum types C and D, is similar to the C novyi beta-toxin. The A and B toxins of Clostridium difficile show homology with the alpha-toxin of C novyi as does the lethal toxin of clostridium sordellii.^[6]

Alpha toxin

The alpha-toxin is characterised as lethal and necrotizing.

The type A alpha-toxin is oedematising.^[7] It acts by causing morphological changes to all cell types especially endothelial cells by inhibition of signal transduction pathways,^[8] resulting in the breakdown of cytoskeletal structures.^[9] The cells of the microvascular system become spherical and the attachments to neighbouring cells are reduced to thin strings. This results in leakage from the capillaries, leading to oedema. The threshold concentration for this action to occur is 5 ng/ml (5 parts per billion) with 50% of cells rounded at 50 ng/ml.

The duodenum is particularly sensitive to the toxin. Injection into dogs resulted in extreme oedema of the submucosal tissues of the duodenum while leaving the stomach uninjured. Injection into the eye resulted in lesions similar to flame haemorrhages found in diabetic retinopathy.^[7]

The toxin is a large 250-kDa protein the active part of which is the NH₂-terminal 551 amino acid fragment.^[10] Alpha-toxins are glycosyltransferases, modifying and thereby inactivating different members of the Rho and Ras subfamily of small GTP-binding proteins.^[11][12][13] C novyi type A alpha-toxin is unique in using UDP-N-acetylglucosamine rather than UDP-glucose as a substrate.^[14]

Beta-Toxin

The beta-toxin is characterised as haemolytic, necrotizing lecithinase.

Gamma-Toxin

The gamma-toxin is characterised as haemolytic, lecithinase.

Delta-Toxin

The delta-toxin is characterised as oxygen labile haemolysin.

Epsilon-Toxin

The epsilon-toxin is characterised as lecithino-vitelin and thought to be responsible for the pearly layer found in cultures.

Zeta-Toxin

The zeta-toxin is characterised as haemolysin.

Human diseases

The type and severity of the disease caused depends on penetration of the tissues. The epithelium of the alimentary tract, in general, provides an effective barrier to penetration. However, spores may escape from the gut and lodge in any part of the body and result in spontaneous infection should local anaerobic conditions occur.

Tissue penetration

Wound infection by C novyi and many other clostridium species cause gas gangrene^[15] Spontaneous infection is mostly associated with predisposing factors of hematologic or colorectal malignancies and with diabetes mellitus,^[16] although Gram-negative organisms, including Escherichia coli, may lead to a gas gangrene-like syndrome in diabetic patients. This presents with cellulitis and crepitus, and may be mistaken for gas gangrene.^[17] Spontaneous, nontraumatic, or intrinsic infections from a bowel source have been increasingly reported recently.^[18]

C novyi has been implicated in mortality among injecting illegal drug users.^[19][20]

Epithelial infections

Symptoms are often non-specific including, colitis, oedematous duodenitis, and fever with somnolence.

Testing is problematical with figures presented by McLauchlin and Brazier [cited above] suggesting a false negative rate of about 40% under ideal conditions. Only positive results may be regarded as reliable. In the absence of a positive test, C. novyi type A may be inferred from characterisation by clinical observation, table 2.

Table 2
Observation	Comment
Oedema	Especially if extreme with rapid onset. In view of the sensitivity of the duodenum to the alpha-toxin, oedematous duodenum is always suspect.
Anaerobic	Infection occurs at an anaerobic site such as the gut or salivary gland. It may also occur at a site temporarily made anaerobic by occlusion and maintained in this state by oedema.
Gram positive	If penicillin causes remission of oedema then a Gram positive organism is the causative agent.

Chronic infection leading to leaky capillaries may also cause retinal haemorrhages and oedema in the lower extremities leading to necrosis and gangrene. Leaky nephrons may compromise the ability of kidneys to concentrate urine leading to frequent urination and dehydration.

Animal diseases

Gas gangrene: Infectious necrotic hepatitis (Black disease)^[21]

Clostridium novyi-NT - Potential Therapeutic Uses in Cancers

In general, solid tumors are characterized by hypoxic areas in the tumor core. This is due to irregular and insufficient tumor vessel growth and heavy metabolic demands of the surrounding tumor cells.

Much of the core within a tumor core is necrotic, however some live tumor cells reside there – often in a quiescent state. These cells are often quite resistant to standard treatments such as radiotherapy (which relies heavily on DNA damage in actively reproducing cancer cells from radiation-induced oxygen-based free radical species) and chemotherapy which has poor access to the poorly perfused tumor core and a weak effect on non-dividing quiescent cells. As a result, cells in the hypoxic tumor core often survive treatment and become a source for subsequent cancer recurrence and spread.

Clostridium novyi-NT is a genetically modified form of Clostridium novyi that lacks a major toxin. Because C. novyi-NT is a strict anaerobe; it grows selectively in hypoxic tumor cores; elsewhere, it tends to exist as inactive spores. C. novyi-NT activates and effectively infects and lyses tumor cells in hypoxic tumor cores.

Early work on use of strict anaerobes in tumors goes back several decades. Strongly lytic, infective bacteria tended to be the most effective (however, most earlier research was abandoned due to the risk of toxicity from release of toxins).

One major limitation on the use of C. novyi-NT or other strict anaerobes in cancer treatment is that it tends to affect only the hypoxic tumor core, leaving the active cancer cells in the well-perfused tumor rim alive and intact.

It is thus not surprising that this has led to attempts to combine C. novyi-NT with traditional chemotherapy and/or with radiotherapy (both of which tend to be preferentially effective within the well-perfused tumor rim).

A variety of other clever approaches are under continuing investigation, these include :

• "RAIT" (radioactive immunotherapy) – Radioactive monoclonal antibodies have been used as a means of targeting radiotherapy to tumors using common tumor antigens such as CEA). This approach could be used to target antigenic epitopes on C. novyi-NT itself, using the tumor-localized vegetative forms to deliver radiation to specifically tumor cells thus sparing more healthy tissue. This could be viewed as a form of molecular brachytherapy).
• Prodrug converting enzymes can be produced by further genetic modification of C. novyi-NT causing the activation of chemotherapeutic prodrugs at the tumor site.
• Anti-cancer drugs may be packaged in liposomes and then specifically released at the tumor site by tumor-localized C. novyi-NT bacteria, improving the effectiveness and safety of the therapy. This approach exposes the tumors to a six times greater concentration of chemotherapy compared to the liposomal drug alone, without increasing the levels of chemotherapy in healthy tissue. Drug release from the liposomes is mediated by an enzyme secreted by C. novyi-NT called liposomase.^[22]
• Other chemotherapy delivery technologies using minicells may be used to more specifically deliver chemotheraputic agents to the site of the remaining tumor rim. This could be achieved for example through the conjugation of bispecific antibodies targeted to epitopes on C. novyi-NT. Minicells are a very promising technology in themselves that use bispecific antibodies to dramatically increase the delivery specificity of chemotheraputic drugs by several orders of magnitude, potentially allowing effective chemotheraputic dosages that are hundreds of times the current tolerated systemic levels. Nevertheless, minicells are limited by perfusion access to tumor cores – so combination with C. novyi-NT may provide an excellent complement.
• Various genetic modifications to C. novyi-NT seek to further stimulate local inflammation and immune response to boost the immunogenicity of the tumor rim. Many of these approaches secrete immunomodulators/cytokines; others try to use siRNA or other approaches to further shut down tumor cells.
• The hypoxic core can be made temporarily wider by use of drugs like dolastatin, or by temporarily reducing oxygenation. This then allows C. novyi-NT to lyse more of the tumor.
• Most approaches have used single administrations of C. novyi-NT, but it may be useful to give repeated injections to promote an immune response in the area of the active bacteria (the former tumor core and adjacent rim) to create a "bystander effect" on the nearby tumor cells (e.g., boosting the bystander immune response to the tumor cells).^[23] It may also help slow relapses by colonizing metastases early after they became large enough to have a significantly sized hypoxic core.

In summary, C. novyi-NT is a promising new component to the treatment of solid tumors - effectively targeting the hypoxic tumor cores that were a source of ongoing treatment resistance and recurrence. It is likely that additional modalities will be needed to treat the well-perfused tumor rims.

Further reading

• Mengesha, Asferd; Dubois, Ludwig; Paesmans, Kim; Wouters, Brad; Lambin, Philippe; Theys, Jan (2009). "Clostridia in Anti-tumour Therapy". In Brüggemann, Holger; Gottschalk, Gerhard. Clostridia: Molecular Biology in the Post-genomic Era. Norfolk, England: Caister Academic Press. pp. 199–214. ISBN 978-1-904455-38-7. 
• Bettegowda C, Dang LH, Abrams R et al. (December 2003). "Overcoming the hypoxic barrier to radiation therapy with anaerobic bacteria". Proceedings of the National Academy of Sciences of the United States of America 100 (25): 15083–8. doi:10.1073/pnas.2036598100. PMC 299912. PMID 14657371. 
• Groot AJ, Mengesha A, van der Wall E, van Diest PJ, Theys J, Vooijs M (December 2007). "Functional antibodies produced by oncolytic clostridia". Biochemical and Biophysical Research Communications 364 (4): 985–9. doi:10.1016/j.bbrc.2007.10.126. PMID 17971292. 
• Dang LH, Bettegowda C, Huso DL, Kinzler KW, Vogelstein B (December 2001). "Combination bacteriolytic therapy for the treatment of experimental tumors". Proceedings of the National Academy of Sciences of the United States of America 98 (26): 15155–60. doi:10.1073/pnas.251543698. PMC 64999. PMID 11724950. 
• St Jean AT, Zhang M, Forbes NS (October 2008). "Bacterial Therapies: Completing the Cancer Treatment Toolbox". Current Opinion in Biotechnology 19 (5): 511–7. doi:10.1016/j.copbio.2008.08.004. PMC 2600537. PMID 18760353. 

References