Bacterial phospholipases and pathogenesis

Microbes and Infection, 1, 1999, 1103−1112 © 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

Review

Bacterial phospholipases and pathogenesis Deborah H. Schmiela*, Virginia L. Millera,b a

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA b Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA

ABSTRACT – Phospholipases are produced from a diverse group of bacterial pathogens causing very different diseases. In some cases, secreted phospholipases appear to be the major cause of pathophysiological effects. Yet in other cases, phospholipases are key virulence factors, contributing to bacterial survival or dissemination without causing tissue destruction. Perhaps the most intriguing aspect of phospholipases as virulence factors is their potential to interfere with cellular signaling cascades and to modulate the host immune response. © 1999 Éditions scientifiques et médicales Elsevier SAS bacteria / phospholipase / pathogenesis

1. Background Phospholipases are a heterologous group of proteins produced by bacteria and their eukaryotic hosts. In eukaryotic cells, phospholipases perform the mundane task of phospholipid turnover in addition to their role in signal transduction; similar enzymatic activities involved in phospholipid turnover for membrane maintenance also release precursors of lipid second messengers. Some mammalian cells also produce secreted phospholipases which are present in extracellular fluids including serum and synovial fluid; as part of the inflammatory response, secreted phospholipase A2 (PLA2, see below) are produced and secreted in response to cytokines during inflammatory events [1]. The secreted PLA2 have also been implicated in the progression of damaging inflammatory responses, septic shock, acute lung injury, and other inflammatory disorders (reviewed in [1]). Bacteria produce membrane-associated phospholipases that are thought to function primarily in membrane maintenance, but their function has not been definitively established. Escherichia coli mutants lacking a phospholipase have been isolated, suggesting these enzymes are not essential for viability [2]. Various bacterial genera also produce secreted phospholipases and lipases. In general, secreted phospholipases are thought to function in phosphate acquisition, carbon source acquisition, and in some cases as virulence factors for pathogenic species.

* Correspondence and reprints Mailing address: Department of Molecular Microbiology, Washington University School of Medicine, Campus Box 8230, 660 S. Euclid Ave., St. Louis, MO 63110–1093, USA Microbes and Infection 1999, 1103-1112

The amphipathic nature of phospholipids creates obstacles for the enzymes as the substrates are assembled into bilayers or even micelles, and are not present in significant amounts as single soluble substrates. It should be noted that the amphipathic phospholipid substrates are defined by their polar head group, of which the most common in mammalian cells are phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) (figure 1). These designations actually define a group of molecules with differing degrees of saturation and length of the two fatty acyl groups; only a limited range of degrees of saturation and potential acyl chain lengths are found in biological systems. Both the acyl chains and the phospho-head groups influence the substrate specificity of phospholipases, some enzymes being much more restricted than others. Because the phospholipid composition varies with the source of the membrane, i.e., bacterial versus eukaryotes, the substrate specificity of a particular phospholipase can suggest or eliminate potential target membranes. In contrast to mammalian cells, E. coli membranes are primarily composed of PE with some phosphatidylglycerol, cardiolipin (double phospholipid joined at the phosphate by glycerol) and trace amounts of PS. Furthermore, only a subset of the products of phospholipase activity are direct precursors for second messengers in mammalian cells, i.e., the fatty acid arachidonic acid is the precursor for eicosanoid biosynthesis. Therefore, given some knowledge of the substrate specificity of a particular phospholipase, some inference regarding its function can be made. Two major divisions of phospholipase activities can be defined by the site of cleavage, whether the cleavage is in the hydrophobic diacylglycerol moiety (PLA) or in the polar head group of the amphipathic phospholipid (PLC 1103

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Figure 1. Structures of common mammalian phospholipids and the signaling pathways induced by the second messengers released by phospholipases. Portions of phospholipid molecules are color coded: polar head groups in green, glycerol backbone in blue and fatty acids in red and purple. Diacylglycerol encompasses all of the phospholipid except the green polar head group, and likewise the lysophospholipid encompasses everything but the fatty acid shown in red. Arrows do not necessarily indicate a direct induction.

and PLD; figure 1). PLAs hydrolyze a fatty acid from the glycerol backbone (leaving a lysophospholipid) in a reaction similar to lipases hydrolyzing fatty acids from triacylglycerol lipids. In fact, a few enzymes have significant PLA activity as well as triacylglycerol lipase activity; therefore these enzymes hydrolyze either hydrophobic or amphipathic substrates with reasonable efficiency [3]. PLAs can be further defined by their positional specificity, preference for the acyl group attached to position 1 or 2 of the glycerol backbone, PLA1 and PLA2, respectively; PLBs have both PLA1 and PLA2 activity, i.e., little or no positional specificity. The other types of phospholipases cleave on either side of the head group phosphate. Cleavage by PLC releases the phospho-head group (e.g., choline phosphate, inositol phosphate, and inositol triphosphate, figure 1) and diacylglycerol, whereas PLD cleaves on the other side of the phosphate producing phosphatidic acid and the head group (e.g., choline and inositol, figure 1). Phospholipases of all types have various substrate specificities with respect to the polar head group and also with respect to the length and saturation of the fatty acyl groups (more so for PLAs). Some phospholipases are very specific, hydrolyzing a particular phospholipid with greater effi1104

ciency. Nevertheless, hydrolysis by any phospholipase type releases products that destabilize membranes; if sufficiently active, bacterial phospholipases may initiate further degradation of the phospholipids by other cellular enzymes including phospholipases.

2. Roles of bacterial phospholipases in disease Phospholipases are considered virulence factors for bacterial species which cause disparate disease syndromes, from infections causing massive tissue destruction, such as gas gangrene and the skin and lung infections of Pseudomonas aeruginosa to food-borne listeriosis (table I). Previous reviews have focused on the bestcharacterized bacterial phospholipase virulence factors which are mostly PLC [4–6]. Rather than reiterate what has already been thoroughly and expertly reviewed, only two examples of PLC virulence factors are described in detail below so that some description of the less understood PLA factors could be included. We wished to increase the scope of the review in terms of the types of Microbes and Infection 1999, 1103-1112

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Table I. Bacterial phospholipases implicated in pathogenesis. Species Bacillus cereus Campylobacter coli Clostridium perfringens

Phospholipase type PLC PLA2 PLC (α toxin)

Corynebacterium pseudotuberculosis

PLD

Helicobacter pylori

PLA2

Listeria monocytogenes

PlcA PlcB

Mycobacterium tuberculosis Pseudomonas aeruginosa

PLC (PLD) PLC

Rickettsia prowazekii

PLA

Salmonella newport

PLA

Staphylococcus aureus Vibrio parahaemolyticus Vibrio cholerae Yersinia enterocolitica

PLC (β toxin)

PLA

Cytokines/ eicosanoidsa

Properties

Hemolytic with sphingomyelinase Hemolysis, null mutant nonhemolytic IL-8, TNF-α PAF Cytolysis and tissue destruction, leukocyte killing; purified α toxin induces many features of septic shock and tissue necrosis in animals, induces arachidonic acid cascade Purified PLD is dermonecrotic and lethal; null mutant no or limited abscess formation at inoculum causing 100% abscesses with wild type; interferes with neutrophil chemotaxis and increases vascular permeability With other exoenzymes destroys protective gastric mucus; lysophospholipid product cytolytic and acts as mucin surfactant Specific digestion of endosomal membranes Synergy with listeriolysin O; double mutant attenuated in animal model different phenotypes, yet overlap of function Genes identified, hemolytic when expressed in E. coli IL-8, thromoboxanes, Tissue destruction, hemolysis; purified PLC leukotrienes reproduces many of the pathologies seen in infected burn patients and fleecerot in sheep Hemolysis, production of lysophospholipids; proposed to induce phagocytosis, endosomal escape, and host cell lysis Injection purified PLA induces same histopathologic changes in ligated ileal loops as bacteria IL-1β Hemolytic, cytotoxic for monocytes and epithelial cells; null mutant causes less damage in animal models; pure PLC causes inflammation in keratitis model Hemolysis

PLA? PLA

Cytotoxic for intestinal cell line; mutant demonstrated no difference in fluid influx into ligated ileal loops compared to wild type Mutant reduced colonization of tissues and caused less inflammation and necrosis than wild type in infected mouse tissues

References [52] [47] [14, 15, 19, 22, 23]

[34, 36, 37]

[45] [9, 10, 11, 30]

[53, 54] [13, 20, 55, 56] [38, 39, 41] [49] [57–59]

[46] [48] [50]

a

Published evidence for the induction of cytokines or eicosanoids by the phospholipase.

phospholipases covered, but not slight the contribution that other PLCs (especially from Pseudomonas) have made to understanding the roles of bacterial phospholipases in disease. In the disease process phospholipases have been implicated in a number of roles, which are considered below (figure 2). Even though the importance of some bacterial phospholipases in pathogenesis is certain and the enzyme has been characterized, it is clear that the exact mechanism of the phospholipase action in vivo has not been definitively determined. Historically, the first indication that bacterial phospholipases were virulence factors was the realization that some bacterial toxins were in fact secreted phospholipases, for example the Clostridium perfringens α toxin [7] and Staphylococcus aureus β toxin [8]. Microbes and Infection 1999, 1103-1112

In general, phospholipase toxicity has been linked to cytolytic activity and is presumed to be directly due to phospholipase activity upon membrane phospholipids and membrane destruction (figure 2). Cytolysis is one of the more common characteristics attributed to bacterial phospholipase virulence factors (table I). Whether cytolysis results from the accumulation of membrane destabilizing products or by the wholesale destruction of membrane phospholipids, it can be caused directly by a very active bacterial phospholipase with broad specificity or in concert with host degradative enzymes induced by the bacterial phospholipase. Naturally, the cytolytic activity varies greatly amongst bacterial phospholipases. The cytolytic properties of the phospholipase itself are dependent on the ability to interact directly with and hydrolyze phospholip1105

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Figure 2. Drawing depicting the roles of bacterial phospholipases during infection. The imaginary bacterial phospholipase is shown performing most of the well-established as well as proposed functions ascribed to all the bacterial phospholipases discussed. From the upper left corner, the bacteria depicted as green rods secrete a phospholipase which aids degradation and penetration of the mucus layer (H. pylori and P. aeruginosa). Once the bacteria reach the epithelial surface phospholipase action triggers engulfment by an epithelial cell, a nonprofessional phagocyte (Rickettsia?). Phospholipase activity then promotes escape from the vacuole or phagosome in the right-hand epithelial cell and the macrophage below (L. monocytogenes) and promotes cytolysis in the left-hand epithelial cell. Cytolysis can be triggered from within or without (C. perfringens and many others). Bacteria which have escaped the macrophage phagosome are being carried from the site of infection by the migrating macrophage (L. monocytogenes). Effects associated with the blood vessel, bottom right, are depicted in the absence of bacteria because they are mostly indirect effects attributed to eicosanoid or cytokine induction (excepting hemolysis). The vessel is drawn partly dilated and partly constricted because different eicosanoids have opposite vasoactive properties. When blood leukocytes are induced to migrate into tissues, the vessel generally dilates, reducing blood velocity, and several different leukocyte adhesion receptors are sequentially induced in the endothelial cells. Weaker interactions promote transient binding, and the leukocyte rolls along the endothelial wall. Subsequent strong binding stops the rolling and allows the leukocyte to pass between endothelial cells. The migrating leukocytes follow the chemokine gradient to reach infected tissues (C. perfringens and C. pseudotuberculosis). The bottom left depicts an abnormal state where overexpression of adhesion integrins on the endothelium promotes sustained leukocyte adherence; degranulation of the static leukocytes could then cause damage to the vessel (C. perfringens). 1106

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ids in the membrane. Thus the enzymatic activity determined using solublized substrates is not indicative of the cytolytic potential of a particular phospholipase if it interacts poorly with membranes. Frequently, the lytic activity is measured as hemolysis for convenience, regardless of the likelihood for a particular pathogen to cause systemic infection or bacterial phospholipase to enter the bloodstream. Furthermore, different eukaryotic membranes are composed of different phospholipids, even the two leaflets (inner and outer) of a particular membrane may have very distinct phospholipid composition. For instance, the plasma membrane of most eukaryotic cells contains predominantly PC and sphingomyelin in the outer leaflet and PI, PE, and PS in the inner leaflet. Therefore, an extracellular phospholipase would not be expected to lyse a target cell unless the outer leaflet phospholipids were efficiently hydrolyzed substrates of the enzyme. Of course, this does not exclude possible cooperativity between several phospholipases or between a phospholipase and another type of ‘lysin’ (hemolysin or cytolysin) molecule as has been proposed for Listeria monocytogenes [9]. Specificity for phospholipids in a particular membrane type or leaflet has had some interesting implications. For in addition to eukaryotic cell cytolysis, bacterial phospholipases have been implicated in the very specific destruction of endosomal or phagosomal membranes (see below and [9, 11, 12]). This releases the bacterium into the nutrient-rich host cell cytoplasm and is one method of escape from possible annihilation by the lysosome (figure 2). As some of the more active cytolytic phospholipases are linked to diseases characterized by tissue destruction, one could speculate that destruction is solely due to lysis of individual cells comprising the tissues. However, some of the best-studied examples of phospholipase virulence factors implicated in destructive infections, for example the two P. aeruginosa PLC and the C. perfringens α toxin (see below), have also demonstrated phospholipasedependent induction of immunomodulatory cytokines that stimulate the inflammatory response, including interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and platelet activating factor (PAF) (table I) [13-15]. Both IL-8 and PAF are chemotactic for leukocytes and activate neutrophils, whereas TNF-α is an inducer of the local inflammatory response, activating the vascular endothelium and increasing vascular permeability (figure 2). Consequently, in these examples tissue destruction has been proposed to be partially due to the overstimulation of the inflammatory response indirectly caused by the phospholipase. Furthermore, these bacterial PLCs are proposed to trigger the same signal transduction pathways as the mammalian cytoplasmic PLC (figure 1 and [16]). Hydrolysis of phospholipids by PLC leads to the production of diacylglycerol, a lipid second messenger. Diacylglycerol has been shown to activate protein kinase C (PKC), which is known to modulate a variety of cellular processes and growth [16] including activation of neutrophils and macrophages [17]. Treatment with exogenous bacterial PLC was shown to induce tissue-destructive matrix metalloproteinase secretion from human epithelial cells probably by PKC induction via diacylglycerol [18]. In turn, PKC activates cytosolic PLA2 and PLC serving as positive feedback, Microbes and Infection 1999, 1103-1112

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thus activation of PKC would increase the total phospholipase activity in the cell. In addition, C. perfringens α toxin [19], and the hemolytic P. aeruginosa PLC [20] were shown to stimulate the arachidonic acid cascade probably by further hydrolysis of diacylglycerol by endogenous cytoplasmic enzymes. Hydrolysis of diacylglycerol could release arachidonic acid, the precursor for the eicosanoids, lipid second messengers which include prostaglandins, thromboxanes, and leukotrienes [21]. Prostaglandins and thromboxanes are vasoactive and modulate platelet activation locally [21]. Leukotrienes are important mediators of acute inflammation affecting neutrophil migration, superoxide generation, and degranulation. Interestingly, hydrolysis of phospholipids by PLA2 releases arachidonic acid directly because it is most commonly found in position 2 of mammalian phospholipids. Though bacterial PLAs have been implicated as virulence factors, the potential for stimulation of the arachidonic acid cascade has not been examined.

3. C. perfringens α toxin plays an essential role in the pathogenesis of gas gangrene Perhaps the best-established, destructive phospholipase virulence factor is the α toxin of C. perfringens, the most cytotoxic bacterial PLC characterized thus far [4]. Though one of several toxins secreted by C. perfringens, the α toxin is considered to be the predominant lethal factor elaborated by C. perfringens type A [4, 22, 23]. Prior immunization with α toxoid prevented discernible tissue damage and protected against lethal challenge by C. perfringens [24]. An α toxin null mutant strain was found to be considerably less virulent in a mouse myonecrosis model for gas gangrene [22]. Mice infected with the mutant strain had minimal muscle destruction, inflammation, and no necrosis or death, in marked contrast to the mice infected with the α toxin-positive parent strain which exhibited severe muscle destruction, inflammation, necrosis, and 70% mortality within 18 h. Furthermore, injection of purified α toxin was shown to reproduce many of the symptoms of C. perfringens induced shock in rabbits; α toxin indirectly induced cytokines and directly inhibited myocardial contractility, thus decreasing arterial pressure [23]. The precise mode of action of the α toxin is not yet certain, but the destructive effects were initially thought to be solely due to cytolysis. However, it has become apparent that indirect effects resulting from the products of PLC activity contribute to tissue destruction. Incubation with purified α toxin was demonstrated to induce expression of cytokines, including the proinflammatory TNF-α PAF [15], and the neutrophil chemoattractant IL-8 [14]. In addition, α toxin activity was shown to trigger the arachidonic acid cascade which leads to cytokine release and inflammation. A striking absence of leukocytes within gangrenous lesions has been noted, yet leukocytes accumulate on the periphery. In contrast, migration of leukocytes into lesions has been observed after infection with the α toxin null mutant [15]. Recent findings suggest that α 1107

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toxin contributes to leukocyte killing and interferes with leukocyte migration proximal to the infection [15]. Stevens et al. [15] suggest that α toxin and perfringolysin O increase endothelial expression of leukocyte adhesion integrins such that the leukocytes stay adhered to endothelial surfaces rather than migrating into the tissues normally. Within the endothelium, stimulation by bacterial products or cytokines would lead to degranulation and release of damaging compounds into the vessels; reduced blood perfusion and oxygen levels due to vascular damage then could promote the anaerobic conditions ideal for growth of Clostridia (figure 2).

4. Two PLCs are essential for the intracellular lifestyle of L. monocytogenes In contrast to the destructiveness of α toxin, the two L. monocytogenes PLCs have a more subtle role in the infectious process yet are key virulence factors for the intracellular pathogen. L. monocytogenes is a facultative intracellular pathogen which is capable of invading and growing in the cytoplasm of a wide variety of cell types, including macrophages, fibroblasts, and epithelial cells. Once listeriae have invaded a cell, the bacteria can spread from cell to cell, enabling them to form plaques in tissue culture cell monolayers. One phospholipase was originally identified in a screen for small plaque mutants, indicating a reduced ability to spread from cell to cell [25]; this secreted PLC, encoded by plcA, is quite specific for the substrate PI [26]. The other phospholipase, PlcB, is active on a wide range of substrates, PE, PS, sphingomyelin and especially PC, and is weakly hemolytic [27, 28]; mutations in plcB also result in strains that form small plaques in cell culture [29]. Null mutants of each PLC were tested in the mouse: the PlcA mutant had only a two- to fourfold increase in the LD50 whereas the PlcB mutant had a 10- to 20-fold increase (the double mutant showed a synergistic effect ≈ 200- to 500-fold increase) [9, 30]. These results suggest at least partial redundancy of function for the two PLCs and the absolute requirement for PLC activity in L. pathogenesis. Examination of the behavior of these null mutants in cell culture led to the proposal of an elegant model for listeriae escape from the phagosome and cell-to-cell spread. L. monocytogenes escapes into the host cell cytoplasm, polymerizes actin propelling listeriae around the cytoplasm, and pushes out cellular projections which can be engulfed by neighboring cells, leaving the newly translocated bacterium surrounded by a double vacuolar membrane (for review see [10–12]). Mutation of plcA resulted in a significant decrease in escape of the L. monocytogenes from the phagocytic vacuole and a slight defect in cell-to-cell spread. The null mutation in plcA also reduced survival in primary murine macrophages, suggesting that PlcA is required for escape from the macrophage phagosome [9]. In contrast, the plcB mutants escape the phagosome efficiently (though there is an additive effect in mutants lacking both PLCs), but are restricted in their cell-to-cell spread as measured by the small plaque size. 1108

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Therefore, a model was proposed that predominantly PlcA in combination with listeriolysin O, a proteinaceous cytolysin similar to perfringolysin O, promotes the degradation of the primary phagosome membrane [9]. This vacuolar disruption frees the listeriae into the cytoplasm without compromising the integrity of the cytoplasmic membrane, so that listeriae can replicate within an otherwise healthy host cell. After cell-to-cell spread, predominantly PlcB (with PlcA) probably aided by listeriolysin O promote the degradation of the double membrane surrounding the bacterium, starting with the inner leaflet of the cytoplasmic membrane of the first host cell. Thus, listeriae are released into the cytoplasm of the neighboring cell without compromising the host’s cell integrity. In this case, the synergy between a bacterial phospholipase PlcA and the cytolysin listeriolysin O is clear. What has not been determined is whether the dissolution of membranes is solely due to the bacterial proteins or whether host phospholipases are also induced. From their cytoplasmic location, listeriae are perfectly positioned to secrete the PLCs where they can release diacylglycerol and other lipid second messengers directly into the cytoplasm. Within the mammalian cell cytoplasm, lipid second messengers, including diacylglycerol, are normal parts of signal transduction pathways that lead to modulation of cellular functions. Fairly recent reports have implicated both listerial PLCs in elevated ceramide levels (product of PLC hydrolysis of sphingomyelin) in cultured endothelial cells with consequent upregulation of leukocyte adhesion molecules via NFjB [31]. The products of the listerial PLC diacylglycerol and ceramide [30] were proposed to be second messengers in pathways leading to NFjB activation, i.e., direct bacterial interference in cellular signal transduction pathways [32]. One would expect such changes to potentiate the immune response, not confer an advantage to the pathogen. However, Schwarzer et al., [31] proposed that increased migration of listeriae-infected monocytes might spread the infection (figure 2). Certainly, future studies might address this question and whether other second messenger products contribute to immune modulation and the pathogenesis of L. monocytogenes.

5. The role of the PLD of Corynebacterium pseudotuberculosis in caseous lymphadenitis Though the mechanism of disease progression by C. pseudotuberculosis in sheep and goats that leads to caseous lymphadenitis is not well understood, the importance of the PLD exotoxin is clear. Generally, the infection starts through a break in the skin, bacteria multiply at this site, the inflammatory response is triggered, forming a primary abscess, and the infection disseminates via the lymph to infect lymph nodes, which also become abscessed (occasionally infection disseminates via blood to visceral organs) [33]. C. pseudotuberculosis PLD is predominantly found in the secreted fraction; it degrades sphingomyelin and lysophosphatidylcholine (product of PLA hydrolysis of PC). Purified PLD is dermonecrotic and Microbes and Infection 1999, 1103-1112

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lethal when injected into animals [34]. However, in culture the secreted C. pseudotuberculosis PLD alone cannot cause hemolysis. It seems probable that C. pseudotuberculosis PLD activity induces host enzymes that cumulatively cause cytolysis, but how exactly the exotoxin exerts its toxic effects is not known. PLD-toxoid vaccines give significant protection against caseous lymphadenitis in sheep, supporting the role of PLD in tissue destruction either directly or indirectly [35]. C. pseudotuberculosis is thought to survive and multiply within neutrophils and macrophages; thus the effects of PLD might be manifested when released intracellularly rather than extracellularly. Indeed, PLD interferes with neutrophil chemotaxis and can be lethal for neutrophils especially when introduced into the cell [36]. Experimental infections comparing a PLD null mutant with the parent C. pseudotuberculosis strain determined that infection with 2 logs greater inoculum (108 CFU) of the mutant failed to cause abscesses when all the animals infected with the parent strain (106 CFU) had abscesses [37]. Moreover, at higher doses (108 or 109 CFU) the mutant caused limited abscess formation at the site of inoculation and was unable to cause abscesses in the draining lymph nodes. In total, these results suggest that PLD promotes survival and dissemination of C. pseudotuberculosis in vivo and supports the significance of the role which PLD plays in disease. Lastly, data suggest that PLD increases vascular permeability, perhaps enhancing spread of the infection through tissues to the lymphatics [34]. The mechanism by which PLD affects neutrophils and alters vascular permeability is not understood at this time, though it is tempting to infer that PLD activity might indirectly induce release of lipid second messengers which modulate these effects.

6. The phospholipase A of Rickettsia sp. Though PLA activity has been identified in a number of pathogenic bacterial genera, they have not been as well studied as the Clostridium and Listeria PLCs discussed above. However, evidence suggests that PLA activity has a significant role in pathogenesis of Rickettsia (especially R. prowazekii), and this PLA is proposed to serve several different functions in vivo. Rickettsia are obligate intracellular bacterial parasites which infect the vascular endothelium; rickettsiae multiply within the host cell cytoplasm, eventually filling and rupturing the host cell. PLA activity was initially linked to hemolysis by R. prowazekii [38]. Examination of the interaction between R. prowazekii and fibroblast cell lines demonstrated that PLA activity released fatty acids from fibroblast cell membrane phospholipids [39, 40]. Superinfection with Rickettsia species or inhibition of rickettsial uptake with cytochalasin B leads to lysis of the fibroblasts, suggesting that PLA activity might be part of the bacterial entry process. Entry of Rickettsia has been termed ‘induced phagocytosis,’ as it requires metabolically active rickettsiae and host cells. Therefore, PLA activity was proposed to trigger internalization of attached R. prowazekii as the host cell attempts to maintain its integrity by internalizing damaged Microbes and Infection 1999, 1103-1112

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plasma membrane [39]. Rickettsial PLA activity was detected throughout growth within the cell, prompting the suggestion that PLA activity could also promote escape from the phagosome into the cytoplasm as well as rupture the cytoplasmic membrane once the replicating rickettsiae had exhausted the host cell. Thus hemolysis was later suggested to be an abortive attempt of the Rickettsia species to gain entry to the erythrocyte and probably is of little relevance in disease [41]. Though the PLA has not yet been purified nor the gene identified, the preponderance of evidence supports the idea that the PLA is rickettsial in origin rather than induced from the host cell. This does not exclude the possibility that the activity of the rickettsial PLA also induces host phospholipases and lysophopholipases which contribute to membrane destruction. R. prowazekii is the causative agent of epidemic typhus in which mortality is generally a result of peripheral vascular collapse probably due to increased vascular permeability [42]. The mouse typhus model demonstrates similar vascular changes when live rickettsiae are inoculated intravascularly, yet searches for a rickettsial toxin have yielded nothing. However, incubation with live rickettsiae resulted in increased secretion of prostaglandins from endothelial cells [43], the usual target cells in rickettsial infection. Increase of prostaglandin secretion correlated with hemolytic activity and was reduced upon addition of a PLA inhibitor. During infection, rickettsiae also encounter polymorphonuclear leukocytes, which upon coincubation with R. prowazekii, secreted prostaglandins and leukotrienes [44]. As mediators of inflammation prostaglandins and leukotrienes are vasoactive; therefore, it is attractive to propose that induction of cytokine release from infected cells leads to the systemic effects characteristic of rickettsial typhus. Because many other bacterial components are known to trigger inflammation, these results are merely suggestive. However, intracellular rickettsial PLA activity hydrolyzing host membrane phospholipids could directly stimulate the arachidonic acid cascade by the release of the precursor leading to the production of proinflammatory eicosanoids.

7. Phospholipases of Helicobacter pylori Destruction of the essential protective mucus layer is a hallmark of gastric H. pylori infections. Gastric mucus is composed of glycoprotein polymer, proteins, lipids, and phospholipids dispersed throughout. In vitro experiments have demonstrated that the H. pylori PLA and PLC with proteinases and lipases degrade gastric mucus, exposing the gastric mucosa to further bacterial assault [45]. This idea of phospholipase activity contributing to mucus degradation is not unique, since the two PLCs of P. aeruginosa are similarly thought to degrade the protective lung mucus layer which is enriched in PC [6]. Furthermore, the lysophospholipid product of PLA has cytolytic properties that were implicated in damage to the gastric epithelium. Thus, the role of H. pylori PLA and PLC in pathogenesis has been generally accepted. Unfortunately, a suitable animal model to assess the potential for immune response modulation during infection is currently lacking – nor have 1109

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defined phospholipase mutant strains been examined in any model system.

8. Phospholipase A produced by other pathogenic bacteria The bacterial PLAs have not been as well studied, nor is their role in disease as well established as the bacterial PLCs (reviewed in [4–6]). However, their potential for a role in pathogenesis in terms of the disruption of host membranes as well as increasing eicosanoid synthesis is apparent. Some bacterial PLAs cause cytolysis or hemolysis as do some of the important PLC virulence factors. The PLA from Vibrio parahaemolyticus [46] and Campylobacter coli [47] have hemolytic activity in vitro, yet these PLAs have not been tested in animal models. Although Fiore et al., [48] have tested a defined phospholipase negative mutant of Vibrio cholerae in the rabbit ligated ileal loop model, the mutant was found to induce similar amounts of fluid accumulation as the parent strain. In the case of the R. prowazekii hemolytic PLA, studies using the mouse typhus model have not examined the effects of purified PLA or a defined null mutant. Furthermore, those studies also suffer from the limitations of the animal model; the rickettsiae do not multiply within mice; rather they cause a rapid systemic toxicity. Certainly, limitations of the bacterial system and/or lack of a really good animal model has contributed to the lack of information about the importance and effects of bacterial PLA in disease. However, preliminary evidence derived from studies in animal models suggests that several bacterial PLAs are important in disease progression. Injection of either virulent Salmonella newport or purified S. newport PLA into rabbit ligated ileal loops induced similar levels of fluid accumulation, desquamation, and mononuclear cell infiltration, suggesting that the PLA might be the key inducer of the physiological effects [49]. Moreover, inoculation of mice with a PLA null mutant Yersinia enterocolitica resulted in less bacterial colonization of the Peyer’s patches and mesenteric lymph nodes than in mice infected with the parent strain; and even mouse tissues infected with the mutant strain showed less progression of inflammation to necrosis than in tissues infected with the parent strain [50]. The implication yet to be proven in these latter two cases is that the bacterial PLA induces a greater inflammatory response. However, these latter two bacterial systems are both amenable to genetic manipulation in addition to being good animal models for disease. Thus the mechanisms by which these bacterial PLAs contribute to disease can be more closely examined and those systems can serve as paradigms for the role bacterial PLA play as virulence factors. Furthermore, the question of whether the PLA might stimulate the arachidonic acid cascade could be addressed using these systems.

9. Conclusions The roles that bacterial phospholipases play in disease is quite varied – from triggering bacterial entry, endosomal 1110

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lysis, and cytolysis to modulating the local immune response and stimulating cytokine secretion. Though phospholipases may be of the same type e.g., PLC, their roles in virulence can be quite different. Certainly, substrate specificity and activity can account for these differences, but also the location where the phospholipase is released, for instance intra- or extracellular, may well influence its effects. In addition to examining the role of more phospholipases with defined null mutations in animal and cellular model systems, eventually it would be interesting to swap phospholipases of similar types between two pathogens. (Similarly, Bacillus subtilis expressing listeriolysin O was able to escape from the macrophage phagosome and grow within the cytoplasm [51].) For instance, could α toxin of C. perfringens replace the PLC of L. monocytogenes, or would it destroy the host cell, robbing the intracellular listeriae of their niche? Alternatively, are phospholipases so exquisitely designed for their task in terms of specificity, activity, and regulation that phospholipases which perform different tasks in different locations are unable to functionally complement one another? Immune modulation seems to be a fairly common trait amongst those phospholipase virulence factors which have been more thoroughly studied. Perhaps this effect is not altogether surprising if one considers that addition of an exogenous phospholipase activity (i.e., bacterial) is likely to upset the delicate control designed to stimulate an adequate, but not overly damaging, immune response. Considering the tendency for amplification of phospholipase signals by induction of other host phospholipases, it seems likely that immune modulatory activity would be a general consequence of bacterial phospholipase activity. How the type of phospholipase, its substrate specificity, and site of action, etc. affect the likelihood or degree of any immunomodulatory effects, requires more information and can not really be commented on at this point. These types of questions eventually will be addressed using cellular or animal models and asking refined questions with specific inhibitors of signaling pathways of the inflammatory response and cytokine induction. Initially, the data seem to suggest that the phospholipases are stimulating the immune response; ostensibly this effect would interfere with, rather than enhance virulence. Perhaps some damage to the host tissues resulting from the immune response promotes deeper tissue penetration, and the edema from the inflammatory response could further bacterial spread. Moreover, misregulation of the immune response could hamper clearance of the infection, as the induction of integrins by C. perfringens α toxin has been proposed to increase binding of lymphocytes to the endothelium, interfering with lymphocyte migration into the tissues [15]. If damage to the vascular endothelium results from lymphocyte stimulation, reduction in tissue perfusion then could promote growth of anaerobic clostridia. Furthermore, increasing lymphocyte migration into tissues for those bacteria capable of growing within macrophages or neutrophils could actually serve to spread the infection. This hypothesis has been proposed for L. monocytogenes [31]. These are very interesting hypotheses which merit further investigation. In general, the Microbes and Infection 1999, 1103-1112

Bacterial phospholipases and pathogenesis

question of how immune modulation by phospholipases might be an advantage to pathogens remains to be answered.

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