Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, UK
Correspondence
Richard W. Titball
rtitball{at}dstl.gov.uk
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Introduction |
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Clostridium perfringens and disease |
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Gas gangrene is also a disease which occurs in the civilian community, and the elderly, diabetics and accident victims are all especially susceptible to the disease. It is difficult to get good estimates of the incidence of disease, but bearing in mind that the proportion of the elderly and diabetics is increasing, it seems likely that the incidence of gas gangrene will increase in future years.
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The pathogenesis of gas gangrene |
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Although all strains of C. perfringens produce -toxin (it is actually diagnostic for C. perfringens), type A strains produce this protein in especially large amounts. Since the 1940s it had been suspected that
-toxin was a major virulence determinant of gas gangrene caused by C. perfringens (MacFarlane, 1955
; MacLennan & MacFarlane, 1944
). However, many of the studies to investigate the properties of the toxin used protein which was purified from C. perfringens culture supernatant fluid. This approach is not without its limitations. Firstly, the purification of the toxin is not a simple task, and secondly, it is extremely difficult to remove all traces of the other (minor) toxins. This is of especial concern because the minor toxins are not necessarily produced in small quantities, nor do they necessarily have low toxicities. Rather, they are termed minor toxins because they are not used to type C. perfringens strains. Therefore, it is difficult to be certain that the properties previously ascribed to
-toxin are not really due to any one of the minor toxins.
A major step forwards in resolving the question of the role of -toxin in disease came from Julian Rood's laboratory in the mid 1990s. An allelic replacement mutant in a virulent type A strain was constructed and tested in the murine model of gas gangrene (Awad et al., 1995
). The mutant showed almost complete loss of virulence. In addition, the characteristic signs of gas gangrene in the mouse (foot swelling, foot blackening and muscle necrosis) were almost completely absent in mice challenged with the
-toxin mutant (Awad et al., 1995
). The reintroduction of the gene encoding
-toxin into the mutant restored the virulence properties of the wild-type, fulfilling molecular Koch's postulates. Overall this work confirmed for the first time that
-toxin is the major virulence determinant in gas gangrene.
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The effect of ![]() |
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The -toxin is a phospholipase C and is active towards phospholipids which are in micellar or monodispersed forms. This activity can be measured relatively simply in the laboratory. However, most phospholipid in the host is present in cell membranes, rather than in micellar or monodispersed forms. Incubation of cells with suitably high concentrations of
-toxin does result in cell lysis as a consequence of extensive damage to the membrane, and this can easily be measured. One simple assay involves measuring the lysis of erythrocytes (Titball et al., 1989
). However, such crude indicators of the interaction with host cells may not fully indicate the nature of the interaction with
-toxin. For example, sublytic quantities of the toxin have been shown to activate the arachidonic acid cascade in a range of cell types (Fujii & Sakurai, 1989
; Gustafson & Tagesson, 1990
). The end-products of this cascade include prostaglandins, thromboxanes and leukotrienes (Samuelsson, 1983
). These compounds play important roles in regulating inflammatory processes. However, the production of thromboxanes is known to be associated with platelet aggregation. The aggregation of platelets does occur after the administration of
-toxin (Fujii et al., 1986
; Ohsaka et al., 1978
; Sugahara et al., 1977
), and this may well be due to the activation of the arachidonic acid cascade in these cells. The formation of platelet aggregates occludes blood vessels (Bryant et al., 2000b
) and appears to be responsible for the steep decline in blood flow to tissues (Bryant et al., 2000a
). It is possible to envisage the situation where
-toxin diffuses away from the initial site of infection into adjacent healthy tissues, and the resultant reduction in blood supply to these tissues then provides the appropriate conditions for the spread of the infection into these tissues.
The spread of the infection in this way is also dependent on the suppression of the host inflammatory response. Remarkably, as long ago as 1917 two military surgeons who had dealt with cases of gas gangrene on the battlefield noted that leucocytes are generally conspicuous by their absence in the muscular tissue involved (McNee & Dunn, 1917). More recently, in mice, it has been shown that tissues surrounding the site of challenge with wild-type C. perfringens are devoid of neutrophils whereas after challenge with an
-toxin mutant the expected influx of phagocytes occurs (Awad et al., 1995
). Additional studies have shown that after the administration of
-toxin, neutrophils accumulate along the walls of blood vessels rather than migrating into tissue spaces (Bryant et al., 2000a
). Collectively, these observations clearly point towards a role for
-toxin in modulating the host immune response.
Damage to the circulatory system and the consequential reduction in blood supply to tissues might also enhance the susceptibility of host cells to -toxin in a completely unexpected way. Mammalian cells grown under low-oxygen or low-glucose conditions become deficient in UDP-glucose, and UDP-glucose deficiency has been reported in the skeletal muscle of diabetic animals (Flores-Diaz et al., 1998
). A mutant fibroblast cell line, deficient in UDP-glucose, has been shown to be 105-fold more sensitive to
-toxin (Flores-Diaz et al., 1998
). However, It is not clear at this stage why UDP-glucose-depleted cells show this enhanced susceptibility to the toxin. Whatever the molecular mechanism, the conditions found in the muscle tissues of some diabetics or in muscle tissues following a traumatic injury might enhance the effect of the toxin on host cells.
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The molecular basis of toxicity |
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Therefore the available evidence indicates that the amino-terminal domain of -toxin contains the phospholipase C active site and the carboxy-terminal domain allows binding of the toxin to membrane phospholipids. Calcium ions play a key role in binding of the toxin to membranes, and also appear to be associated with structural changes which open the active site and allow the side chains of Trp214 and Phe334 to become surface exposed. This information allows a model to be constructed (Naylor et al., 1998
) explaining how
-toxin interacts with membrane phospholipids (Fig. 2
). The protein becomes partially inserted into the outer leaflet of the membrane, bringing the active site into juxtaposition with the phospholipid head group. Calcium ions are able to form bridges with the phospholipid phosphate group, whilst the hydrophobic side chains of Trp214 and Phe334 become inserted into the hydrophobic core of the membrane.
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The development of a vaccine |
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It is possible that the -toxoid will be valuable for the prevention of other diseases in domesticated livestock. For example, there is evidence that
-toxin plays a key role in the pathogenesis of some enteric diseases of calves and piglets (Cygan, 1997
; Ginter et al., 1996
; Wierup, 2001
). There is some evidence that these isolates produce a form of
-toxin with increased resistance to proteolysis (Ginter et al., 1996
). This might be consistent with the site of production of the toxin in the gut. Again, the extent of immunological cross-reactivity of
-toxin from these strains with
-toxin from gas gangrene isolates is not known.
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Other bacterial phospholipases and virulence |
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For other pathogens, phospholipases can play very different roles in different diseases. For example, mucosal surfaces are generally coated with a phospholipid-rich surfactant, and there is an obvious possible role for these enzymes in the degradation of the surfactant layer, allowing access to the underlying tissues. Surfactant degradation has been demonstrated in vitro (Holm et al., 1991) but has yet to be demonstrated in vivo during disease. Nevertheless, a role for Helicobacter pylori and Pseudomonas aeruginosa phospholipases in the colonization of the stomach and respiratory tract, respectively, has been proposed (Langton & Cesareo, 1992
; Saiman et al., 1992
; Titball, 1999
). In the former case it may be significant that bismuth salts, which were in the past often used to treat stomach ulcers, are potent inhibitors of the H. pylori phospholipase (Ottlecz et al., 1993
).
In addition to the C. perfringens -toxin, there are a number of bacterial phospholipases which have been directly implicated in promoting the growth of pathogens in vivo. For example, the phospholipase D of Corynebacterium pseudotuberculosis increases vascular permeability and is known to be a major virulence determinant in cases of lymphadenitis/lymphangitis in ruminants (Hodgson et al., 1992
; McNamara et al., 1994
). Listeria monocytogenes produces two phospholipases C, PLC-A, which is active against phosphatidylinositol, and PLC-B, which preferentially hydrolyses phosphatidylcholine. These enzymes have overlapping functions, and a
plcA
plcB double mutant was 500-fold attenuated in a mouse model of disease (Smith et al., 1995
). These phospholipases appear to promote escape of the bacteria from the phagosome and allow cell-to-cell spread (Smith et al., 1995
).
It is studies with the P. aeruginosa phospholipases which have revealed some of the most unexpected roles of these enzymes in the pathogenesis of disease. The P. aeruginosa PLC-H phospholipase C appears to play a role in disease of the respiratory tract (Granström et al., 1984; Saiman et al., 1992
). Like C. perfringens
-toxin, PLC-H appears to be able to modulate the metabolism of mammalian cells by activating the arachidonic acid cascade (König et al., 1997
). Additionally PLC-H is able to convert phosphatidylcholine into choline-betaine, which accumulates in the bacterial cell and acts as an osmoprotectant (Shortridge et al., 1992
). The bacterium might rely on this pathway for protection against the high osmotic strength environment in the lung.
A different role in disease has been proposed for the P. aeruginosa PLC-B phospholipase, which appears to play a role in the generation of diacyglycerol-based compounds which act as a chemoattractant (Barker et al., 2004). It is possible that this compound attracts bacteria to preferential sites in the respiratory tract. Yet another P. aeruginosa phospholipase, ExoU, is one of the effector proteins of the type III secretion system. After delivery into the host cell, the enzyme appears to cause damage to the membranes of different organelles and leads to necrotic cell death (Sato & Frank, 2004
).
Other bacterial phospholipases have either been shown to play a role in virulence or are strongly implicated in disease, but their precise function is yet to be determined. For example, inactivation of the cell-surface phospholipases of Yersinia pseudotuberculosis and Yersinia enterocolitica results in partial attenuation (Darwin & Miller, 1999; Karlyshev et al., 2001
). There appears to be multiple redundancy of the Mycobacterium tuberculosis phospholipases C, and triple or quadruple mutants show a 10-fold reduction in the colonization of mouse lungs in the later stages of disease (Raynaud et al., 2002
). It is possible that these enzymes play a role in the establishment of chronic disease.
Some phospholipases actually appear to play a role in reducing the virulence of pathogens a phospholipase (-toxin) mutant of Staphylococcus aureus colonized tissues at a lower level than the wild-type in the murine model of mastitis (Bramley et al., 1989
). Others appear to play roles in the colonization of non-mammalian hosts P. aeruginosa PLC-H plays a role in disease of plants (Rahme et al., 1995
) whilst the Yersinia pestis phospholipase D is essential for the colonization of flea vectors (Hinnebusch et al., 2002
).
Phospholipases are true multifunctional virulence determinants, playing a range of roles in the pathogenesis of disease. Additional enzymes which play a role in virulence will probably be identified in the future. It seems likely that some of these enzymes will interact with host cells using mechanisms similar to those identified for C. perfringens -toxin. For others, the molecular basis of activity is yet to be determined.
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ACKNOWLEDGEMENTS |
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Colworth Prize Lecture 2005
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