INTRODUCTION

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To aid in the invasion of host tissues, microbial cells possess constitutive and inducible hydrolytic enzymes that destroy or derange constituents of host cell membranes, leading to membrane dysfunction and/or physical disruption (167). Since membranes are made up of lipids and proteins, these biochemicals constitute the target of enzyme attack. Pathogenic fungi (e.g., Candida albicans) secrete enzymes which are considered to be integral to their pathogenesis; these are categorized into two main types, proteinases (72, 73), which hydrolyze peptide bonds, and phospholipases (75), which hydrolyze phospholipids. In this review, we focus on fungal phospholipases. It should be emphasized that this field is evolving rapidly and that most of the data reviewed in this article were published recently. New insights into the biology and contribution of these enzymes to fungal virulence will be forthcoming in the near future.

The term "phospholipases" refers to a heterogeneous group of enzymes that share the ability to hydrolyze one or more ester linkage in glycerophospholipids. Although all phospholipases target phospholipids as substrates, each enzyme has the ability to cleave a specific ester bond (Fig. 1a). Thus, qualifying letters, such as A, B, C, and D, are used to differentiate among phospholipases and to indicate the specific bond targeted in the phospholipid molecule (4). For example, phospholipase A₁ (PLA₁) hydrolyzes the fatty acyl ester bond at the sn-1 position of the glycerol moiety, while phospholipase A₂ (PLA₂) removes the fatty acid at the sn-2 position of this molecule. The action of PLA₁ (EC 3.1.1.32) and PLA₂ (EC 3.1.1.4) results in the accumulation of free fatty acids and 2-acyl lysophospholipid or 1-acyl lysophospholipid, respectively. The fatty acid still linked to the lysophospholipid is, in turn, cleaved by other enzymes termed lysophospholipases (Lyso-PL) (EC 3.1.1.5) (Fig. 1b). Phospholipase C (PLC) (EC 3.1.4.3) hydrolyzes the phosphodiester bond in the phospholipid backbone to yield 1,2-diacylglycerol and, depending on the specific phospholipid species involved, phosphatidylcholine, phosphatidylethanolamine, etc. The second phosphodiester bond is cleaved by phospholipase D (PLD) (EC 3.1.4.4) to yield phosphatidic acid and choline or ethanolamine, again depending on the phospholipid class involved (4).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Sites of action of various phospholipases. (a) A1 and A2, PLA1 and PLA2, respectively; B, PLB; C, PLC; D, PLD. (b) Lyso-PL and Lyso-PL transacylase.

Although phospholipase B (PLB) (synonyms: lysophospholipase, lysophospholipase-transacylase) refers to an enzyme that can remove both sn-1 and sn-2 fatty acids, its nomenclature is confusing (4). This confusion arises because PLB has both hydrolase (fatty acid release) and lysophospholipase-transacylase (LPTA) activities. The hydrolase activity allows the enzyme to cleave fatty acids from both phospholipids (PLB activity) and lysophospholipids (lysophospholipase [Lyso-PL] activity), while the transacylase activity allows the enzyme to produce phospholipid by transferring a free fatty acid to a lysophospholipid (Fig. 1b). The finding that PLB has both hydrolase and acyltransferase activities has been reported for enzymes purified from C. albicans, Penicillium notatum, and Saccharomyces cerevisiae (100, 122, 164, 224, 225). This complex pattern of activity led to difficulties in the nomenclature of the enzyme, with some authors naming it PLB (as in the case of S. cerevisiae and P. notatum) and others naming it LPTA (10, 122, 193). Recent cloning and disruption of genes encoding PLB provided further evidence that this enzyme has both PLB and Lyso-PL activities. Deletion of caPLB1, the gene encoding PLB from C. albicans, showed that both PLB and Lyso-PL activities were reduced in the PLB1-deficient mutant relative to the wild type, confirming that caPLB1 encodes an enzyme responsible for the PLB and Lyso-PL activities (75, 101). Similar findings were reported for PLB deletion in S. cerevisiae (100).

Invasion of host cells by microbes entails penetration and damage of the outer cell envelope. This transmigration process is mediated, most probably, by either physical or enzymatic means or a combination of the two. Phospholipids and proteins represent the major chemical constituents of the host cell envelope. Therefore, enzymes capable of hydrolyzing these chemical classes, such as phospholipases and proteinases, are likely to be involved in the membrane disruption processes that occur during host cell invasion. By cleaving phospholipids, phospholipases destabilize the membrane and cell lysis results (166). Evidence implicating phospholipases in host cell penetration, injury, and lysis by microorganisms has been reported for Rickettsia rickettsii (207), Toxoplasma gondii (160, 161), Entamoeba histolytica (154), and C. albicans (101). Consequently, phospholipases have been included among the virulence factors that damage host cells (166).

Besides fungi, which are the subject of this review, extracellular phospholipases have been implicated as pathogenicity factors for bacteria (Table 1). Since bacterial phospholipases have been the subject of at least two recent reviews (183, 194), to avoid repetition, only a brief description of these enzymes and their role in virulence is covered in this article. Furthermore, including some information about phospholipases from organisms other than fungi will show a global perspective of microbial phospholipases.

Clostridium perfringens. PLC (synonyms: alpha-toxin, lecithinase), which is one of the most active bacterial phospholipases, is 1 of at least 12 different soluble antigens referred to as toxins, produced by Clostridium perfringens, that may be involved in pathogenesis. The findings that clostridial PLC was a potent toxin (109, 110, 123) with hemolytic (162, 195), lethal (196), dermonecrotic (115), vascular permeabilization (186), and platelet-aggregating (134, 185) properties attracted a large number of investigators to study this secreted protein, making it the most extensively studied bacterial toxin. Thus, it is not surprising that extensive reviews have been written about this enzyme (67, 79, 194), its application to the study of cell membranes (3, 5, 48, 126, 159, 229), and its role in virulence (183).

Listeria monocytogenes. Similar to C. perfringens, L. monocytogenes secretes a number of extracellular enzymes including listeriolysin (encoded by hly) and two phospholipases: i) PI-PLC or PLC-A, encoded by plcA, which is an inositol-specific PLC (21), and (ii) PC-PLC or PLC-B, encoded by plcB, which is a broadly active PLC with the ability to hydrolyze most cellular phospholipids (203). Although each phospholipase may contribute to the virulence of L. monocytogenes by itself (21, 119, 203), elegant work by Portnoy and colleagues showed that the two phospholipases play overlapping roles in the pathogenesis of L. monocytogenes (178). In their study, mutants harboring a deletion in each PLC, as well as a double mutant lacking both enzymes, were characterized with regard to virulence. Abolishing PI-PLC and PC-PLC resulted in strains that were 2- and 20-fold less virulent than the parent strain, respectively. In addition, these strains were defective in escape from the vacuole and in cell-to-cell spread, depending on which enzyme was deleted. Interestingly, the mutant lacking both PLCs was 500-fold less virulent in mice and was severely diminished in its ability to escape from the vacuole and to spread from cell to cell (178). These findings are consistent with the two enzymes having overlapping functions throughout the course of infection. Importantly, deleting a single gene resulted in only a modest reduction in virulence, while simultaneous deletion of the two genes in the same strain led to a highly significant decrease in the virulence, suggesting that the two enzymes have a synergistic effect on the ability of the organism to invade host tissues. For more details on entry of L. monocytogenes into phagocytic cells and the role of secreted enzymes, including phospholipases, in the ability of the organism to escape from the vacuole and transmigrate cell-to-cell, the reader is referred to references 144, 183, and 194.

Pseudomonas aeruginosa. Two distinct PLCs are produced by Pseudomonas aeruginosa: PLC-N (nonhemolytic) and PLC-H (hemolytic) (183). The genes (plcN and plcS) encoding these enzymes have been cloned (137). Although the expression and secretion of both enzymes are phosphate regulated, each enzyme has a distinct substrate specificity, with PLC-H hydrolyzing sphingomyelin in addition to phosphatidylcholine (PC), while PLC-N is active in phosphatidylserine and PC degradation (137). This difference in substrate specificity has a bearing on the efficiency with which these enzymes degrade eukaryotic membranes. Ostroff et al. (138) proposed that the two enzymes could work sequentially and synergistically to lyse host cells. PLC-H initiates membrane lysis by hydrolyzing PC and sphingomyelin (the major constituents of the membrane outer leaflet), and this is followed by the action of PLC-N which cleaves phosphatidylserine (the major constituent of the membrane inner leaflet). Furthermore, substrate specificity studies have shown that PLC-H preferentially cleaves phospholipids containing quaternary ammonium groups, such as phosphatidylcholine, which are found primarily in eukaryotic membranes and lung surfactants, but that it has minimal activity toward phospholipids such as phosphatidylethanolamine, which are found in the prokaryotic membrane (14). This selective ability may explain why the invading organism can lyse the host cells without damaging its own membrane.

plcS plcR

Bacillus cereus. Several PLCs, including phosphatidylinositol-specific and PC-preferring enzymes, as well as sphingomyelinase, are produced by Bacillus cereus (80, 95, 198, 199). The genes encoding the three different proteins have been cloned and sequenced (84, 85, 96). PC-preferring and sphingomyelinase-encoding genes form a gene cluster (tandemly located) (59), which is not positioned in close proximity to the gene encoding phosphatidylinositol-specific PLC (59, 96). The PC-preferring enzyme has structural homology to the C. perfringens alpha-toxin (103), while the phosphatidylinositol-specific enzyme has stretches of sequence homology to other eukaryotic phospholipases (97).

Rickettsia. Two species of the genus Rickettsia (Rickettsia rickettsii and Rickettsia prowazekii) were shown to possess PLA that was suggested to mediate host cell lysis (63, 177, 220, 222). Previously, Winkler and Miller (221) reported that rickettsiae enter cells through a mechanism involving a PLA. Their hypothesis was based on the demonstration that host cells exposed to large numbers of R. prowazekii release considerable amounts of lysophospholipids and free fatty acids into the culture medium. Involvement of phospholipases in the penetration and damage of host cells by R. rickettsii was first suggested by Walker et al. (207). More recently, Silverman et al. (177) confirmed these findings and provided suggestive evidence that phospholipase activity associated with internalization of this intracellular parasite lies directly with the infecting organism rather than with the host cell. However, as stated by the authors, definitive evidence that the phospholipase originates from these organisms could be provided only by isolation and cloning of specific rickettsial gene(s) involved in the internalization process (177).

Corynebacterium pseudotuberculosis. Corynebacterium pseudotuberculosis, the agent of caseous lymphadenitis in small ruminants and ulcerative lymphangitis in horses, produces extracellular PLD, which possibly plays a role in the pathogenicity of this and similar bacteria (Corynebacterium ulcerans and Arcanobacterium haemolyticum) (1, 34, 118, 184). The genes encoding the PLD in C. pseudotuberculosis and A. haemolyticum have been cloned and sequenced (118). Targeted mutagenesis of PLD in C. pseudotuberculosis reduced the ability of this bacterium to establish a primary infection or cause chronic abscess formation in regional lymph nodes (117). These results indicate that PLD is a virulence determinant of C. pseudotuberculosis, increasing the persistence and spread of the bacteria within the host (117).

Indirect evidence implicating a calcium dependent-PLA₂ in host cell invasion by T. gondii was obtained by Saffer et al. (160), who showed that incorporation of exogenous PLA₂ from snake venom increases host cell penetration by T. gondii. Furthermore, penetration of host fibroblasts by the parasite was inhibited following preincubation with PLA₂ inhibitors, such as p-bromophenacyl bromide and nordihydroguaiaretic acid, or antisera to this enzyme (160). In another study, the same group extended these findings and demonstrated that treating fibroblasts with fractions of disrupted T. gondii led to accumulation of degradation products of the phospholipids (fatty acids and lysophospholipids). The data suggest that PLA may be associated with T. gondii cells (161). Moreover, fractions of T. gondii that had PLA enzymatic activity also increased host cell penetration (161). Although the findings of Saffer and Schwartzman (161) implicate phospholipases in the process of penetration of host cells by T. gondii, these workers used a crude enzyme preparation, which makes interpretation of the results quite difficult. To unequivocally establish a correlation between PLA and T. gondii virulence and to elucide how the enzyme may influence the penetration process of host cells by this parasite, gene cloning and enzyme purification are required.

E. histolytica, similar to T. gondii, possesses PLA (106, 153). Ravdin and coworkers (106) reported that this ameba has two PLA enzymes: a calcium-dependent protein which is associated with the plasma membrane and is most active at an alkaline pH, and a calcium-independent enzyme that is localized predominantly to soluble subcellular fractions of E. histolytica and is optimally active at an acidic pH. Amebic cytolytic activity, and thereby virulence, is associated with the calcium-dependent PLA. However, as in the case with T. gondii, definitive demonstration of the role of amebic PLA enzymes in the cytolytic event awaits their purification, gene cloning, and disruption.

PHOSPHOLIPASES IN PATHOGENIC FUNGI

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Phospholipases of Candida albicans

The overall incidence of Candida bloodstream infections has increased significantly in the last two decades (9, 140, 143), ranging from a 75% increase in small hospitals to an over 400% increase in some large tertiary-care centers (140). This increase led to a tremendous interest in the study of candidal pathogenesis and strategies for control and prevention of this clinically important fungus. Candidal virulence factors have also attracted interest as a possible means for developing novel therapeutic interventions against candidiasis (33, 52, 139). Such virulence factors include adherence (20, 55, 90), germination (180), extracellular proteinases (71, 72) and phospholipases (75), and phenotypic switching (181, 182).

(i) Candidal phospholipase. The secretion of extracellular phospholipases by C. albicans was first reported in the 1960s by Costa et al. (A. Costa, A. Misefari, and A. Amaro, Abstr. Atti XIV Congr. Naz. Microbiol. Messina, abstr. P35 and P36 1967) and Werner (213) by growing the yeast on solid media containing egg yolk or lecithin and analyzing the lipid breakdown products. Later, phospholipase activity was found in many pathogenic C. albicans strains by using media containing blood serum and sheep erythrocytes (30). The observation that C. albicans secretes phospholipase prompted Pugh and Cawson (150) to develop a lecithin-based cytochemical method to detect this enzyme. In a subsequent study, these authors used this method (149) in conjunction with a chicken chorioallantoic membrane model to evaluate ultrastructural details of candidal invasion and to determine the site of phospholipase production. Invasion was initiated by placing stationary-phase blastospores of C. albicans on the membrane, which stimulated cellular changes in the blastospores. Many of the blastospores developed hyphae with phospholipase activity concentrated at the growing tip. The activity was highest where the hyphae were in direct contact with the membrane (149). In general, only hyphae invaded the membrane successfully. Based on these results, the investigators proposed that extracellular phospholipases were important in the invasion of tissue by C. albicans.

View larger version (124K): [in this window] [in a new window]   FIG. 2.   Production of phospholipase by three different clinical C. albicans isolates. Note the difference in the precipitation zones around the three colonies.

(ii) Production of phospholipase by various candidal species. Early studies in which the egg yolk-based assay was used to evaluate the ability of different Candida species to produce phospholipase showed that only C. albicans and not other species of Candida produce extracellular phospholipase. Samaranayake et al. (168) screened 41 Candida isolates for phospholipase activity by using a plate assay method and found that 79% of the C. albicans strains tested produced extracellular phospholipases whereas no strains of C. tropicalis, C. glabrata, and C. parapsilosis produced the enzymes. The quantity of phospholipase produced by C. albicans varied with the specific isolate and correlated with the site of infection. Blood isolates generally produce much higher levels than do isolates from wounds or urine (147). In contrast, Clancy et al. (C. J. Clancy, M. A. Ghannoun, and M. H. Nguyen, Programs Abstr. 36th Annu. Meet. Infect. Dis. Soc. Am., abstr. 317, 1998) recently showed that non-albicans Candida species produce extracellular phospholipases as determined by both egg yolk-based and colorimetric assays. In this study, 41% of C. glabrata, 50% of C. parapsilosis, 70% of C. tropicalis, 80% of C. lusitaniae, and 100% of C. krusei strains produced detectable phospholipase activity. However, it is important to emphasize that relative to C. albicans, the non-albicans species secreted significantly smaller amounts of phospholipase (for example, C. krusei has approximately 10 times less phospholipase activity [P. Mukherjee and M. Ghannoum, unpublished data]). Although Clancy et al. demonstrated that non-albicans species can secrete phospholipase similar to C. albicans, it is important to point out that the number of candidal isolates tested was limited (n = 51). Therefore, the percentages reported in this study may have been significantly different if a larger number of isolates had been examined. Furthermore, the discrepancy observed by different workers in the phospholipase activity for the non-albicans species may be attributed to strain-to-strain variation or differences in the preparation of the egg yolk agar plates used to detect phospholipase secretion in these species.

(iii) Types of candidal phospholipases. The literature contains contradicting reports on the number and specific types of phospholipase enzymes secreted by C. albicans. Costa et al. (Abstr. Atti XIV Cong. Naz. Microbiol. Messina, abstr. P35 and P36, 1967) reported the secretion of both PLA and PLC by this clinically important yeast. Their results were based on the isolation of palmitic acid and phosphatidylcholine from the proximity of candidal colonies cultured on Sabouraud agar supplemented with serum and sheep erythrocytes. Since this medium is not chemically defined, the sources of the hydrolysis products are uncertain. Banno et al. (10) performed a crude fractionation of the proteins in culture filtrates of C. albicans by using DEAE-Sephadex and then assayed the fractions for phospholipase activity. Their data suggested that C. albicans secreted three types of phospholipases: Lyso-PL, LPTA, and PLB. Takahashi et al. (193) purified two distinct forms of LPTA from culture filtrates of C. albicans. These candidal enzymes differed from mammalian enzymes in amino acid composition; however, the substrate specificities were not determined (193).

Evidence correlating phospholipases and virulence. Although the contribution of phospholipases to the pathogenesis of bacteria and protozoa has been known for some time, investigations into whether these enzymes are associated with fungal virulence have only recently been undertaken. Barrett-Bee et al. (11) was the first to evaluate the role of extracellular candidal phospholipases in virulence by using a murine model of candidiasis. When phospholipase activity was measured in six yeasts (four strains of C. albicans and a single strain each of C. parapsilosis and S. cerevisiae), a correlation was found between phospholipase activity and two potential parameters of pathogenicity. The C. albicans isolates which adhered most strongly to buccal epithelial cells and were most pathogenic in mice had the highest phospholipase activities. Less pathogenic isolates of C. albicans, C. parapsilosis, and S. cerevisiae were less adherent to epithelial cells and less lethal to mice and had lower phospholipase activities (11). Although these findings suggest a correlation between phospholipase activity and candidal virulence, the strains used were not genetically related, and therefore the differences observed in the virulence of the tested strains could be attributed to factors other than phospholipase secretion.

(i) Candidal phospholipases B. (a) caPLB1. The C. albicans PLB1 gene was cloned by using a PCR-based approach relying on degenerate oligonucleotide primers designed on the basis of the amino acid sequences of two peptide fragments obtained from a purified candidal enzyme displaying phospholipase activity (122). Sequence analysis of a 6.7-kb EcoRI-ClaI genomic clone revealed a single open reading frame of 1,818 bp that predicts a preprotein of 605 residues. The size of the candidal PLB1 protein is similar to those reported for other fungal PLBs, which ranged between 612 and 664 amino acids (Table 2). The genomic DNA sequence encodes 17 amino acid residues that are absent from the NH₂ terminus of the mature protein. This stretch of residues represents a possible signal sequence (111, 169). The predicted protein contains seven Asn-X-Ser/Thr motifs (residues 199, 261, 399, 451, 465, 492, and 573) that could potentially be N glycosylated. One possible tyrosine phosphorylation site, Lys-Ser-Asn-Ile-Asp-Val-Ser-Ala-Tyr (residues 369 to 377), was also identified (101). Hydropathy analysis of the predicted protein sequence (98) revealed the presence of a single stretch of hydrophobic amino acids present at the amino terminus (residues 1 to 18). This segment of amino acids most probably functions as a single peptide which targets the protein to the endoplasmic reticulum for subsequent processing and, ultimately, secretion. Comparison of the putative candidal phospholipase with other proteins in the redundant database (BLASTP program) revealed significant homology to known fungal PLBs from S. cerevisiae (45%), P. notatum (42%), Torulaspora delbrueckii (48%), and Schizosaccharomyces pombe (38%) (Fig. 3). This gene, designated caPLB1, was mapped to chromosome 6 (101). Consistent with our findings, Hoover et al. (69a) used degenerate oligonucleotide primers derived from conserved regions of PLB1 genes from S. cerevisiae and other fungi to clone the corresponding candidal homolog. Their results confirm our findings and the identity of caPLB1.

View larger version (106K): [in this window] [in a new window]   FIG. 3.   Sequence alignment of fungal PLBs. The amino acid sequences predicted by caPLB1 and caPLB2 (C. alb.1 and C. alb.2) were aligned with PLBs from S. cerevisiae (S. cer.), P. notatum (P. not.), S. pombe (S. pom.), and T. delbrueckii (T. del.). Shaded areas indicate positions where the amino acids are identical.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Phylogenetic tree analysis of fungal PLBs. S. cerevisiae PLB (GenBank accession no. L 23089), S. cerevisiae SPO1 (P 53541), S. cerevisiae YLM006c (S53035), S. cerevisiae YOL011w (S66693), T. delbrueckii PLB (D 32134), P. notatum PLB (P 39457), S. pombe PLB (Z 99258), N. crassa PLB (AF045575), and C. albicans caPLB1 and caPLB2.

(ii) Candidal phospholipase D. PLD catalyzes the hydrolysis of PC to produce phosphatidic acid and choline (Fig. 1a above). Both mammalian and fungal (S. cerevisiae) genes encoding PLD have been cloned and characterized (130, 206). Mammalian PLD has emerged as one of the key enzymes in intracellular signaling (44), while PLD from S. cerevisiae (encoded by SPO14) is essential for meiosis (41, 158, 206). The absence of meiosis in C. albicans, which, unlike S. cerevisiae, exists as a diploid, indicates that PLD in this clinically important yeast may play other roles than meiosis. McLain and Dolan (116) have recently shown that PLD may be an important regulator of the dimorphic transition. Since this yeast-hypha transformation is thought to be an important virulence determinant in C. albicans (33, 54, 133), cloning and disruption of candidal PLD may contribute to our understanding of the biological role of this phospholipase.

Disruption of phospholipase B₁. Gene disruption (deletion) is frequently used to determine the functionality of a specific gene. This is particularly relevant in assessing the contribution of a given gene to microbial virulence (45). Several workers have disrupted a number of genes to evaluate their contribution to the virulence of C. albicans (37, 61, 74, 170, 228). Most recently, C. albicans strains with deletions of the INT1 gene, which encodes a cell surface protein with similarity to mammalian integrins, were constructed (50). Adhesion, hyphal formation, and virulence were subsequently found to be correlated with the expression of this gene.

Phenotypic characterization of phospholipase B₁-deficient mutants. Phenotypic characterization of the parent and deletion mutants is necessary prior to evaluating the virulence of the isogenic strain pair. Such characterization may provide some measure of assurance that deleting the caPLB1 gene results in only loss of PLB production and not other unrelated phenotypic properties. Comparison of the growth rates and germination capabilities of PLB-deficient mutant with that of the parental strain revealed that disruption of caPLB1 did not affect growth and germination of C. albicans (101), indicating that caPLB1 is not essential for these processes.

Testing of phospholipase B₁-deficient mutants in murine models of candidiasis. Two different models of candidiasis, representing different clinical settings, were used to determine the role of caPLB1 in the virulence of C. albicans: (i) a hematogenous-dissemination murine model (58) and (ii) an oral-intragastric infant mouse model (28).

(i) Hematogenous-dissemination model. Leidich et al. (101) challenged BALB/c mice with 5 × 10⁵ yeast cells of either the parent or the PLB-deficient strains intravenously through the lateral tail vein. Both survival and tissue fungal burden were used to assess the pathogenicity of the infecting strains. Their data showed that all mice infected with parental strain SC5314 succumbed to candidal infection within 9 days. In contrast, 50 and 60% of mice challenged with either the PLB-deficient strain plb1-¹ or plb1-², respectively, were alive at day 15. The mean survival time ± standard deviation for mice infected with parent was 4.4 ± 2.1 days, compared to 12.7 ± 2.7 and 13.3 ± 2.6 days for mice infected with plb1-¹ or plb1-², respectively. Statistical analyses revealed that mice infected with either strain plb1-¹ or plb1-² survived significantly longer than did mice infected with strain SC5314 (P < 0.0001 for both comparisons).

(ii) Oral-intragastric infant-mouse model. Seshan et al. (174) used an oral-intragastric infant-mouse model to examine the effect of caPLB1 on the ability of C. albicans to traverse the gastrointestinal barrier and colonize systemic target organs, such as the kidneys and liver. This model simulates candidal migration across the gastrointestinal tract, one of the major routes for contracting disseminated candidiasis (131). In these experiments, inbred infant mice [crl:CFW (SW) BR] were inoculated intragastrically with 2 × 10⁸ blastospores of either the wild-type or caPLB1-deficient strain. Transmigration of candidal cells across the gastrointestinal tract was monitored microscopically as well as by determining tissue fungal burden of target organs.

Expression of phospholipase B₁ during host tissue invasion. One of the criteria to prove that a particular gene or its product plays an important role in the disease process is to show that it is expressed by the microorganism during the infectious process (45, 167). Both the hematogenous-dissemination (101) and oral-intragastric mouse (174) models for candidiasis were used to determine whether PLB is secreted in vivo.

View larger version (105K): [in this window] [in a new window]   FIG. 5.   Expression of PLB during candidal invasion of gastrointestinal tract of an infant mouse. Inbred infant mice [cr1: CFW (CSW) BR] were inoculated intragastrically with 2 × 108 cells of the phospholipase-producing parental strain (SC5314). The stomach was harvested, and sections were prepared for immunogold microscopy. The candidal cell appears in red, and the stomach tissue appears in greenish-yellow. Immunogold complexes that formed following incubation of the sections with PLB antiserum are shown in blue. Magnification, ×10,000.

C. glabrata is recognized increasingly as an important nosocomial pathogen (2, 89, 92, 146, 156, 201, 210, 214, 218, 219); D. W. Warnock, J. Burke, N. J. Cope, E. M. Johnson, N. A. Von Frauenhofer, and E. W. Williams, Letter, Lancet ii:310, 1988). It ranks third among Candida species in the number of reported cases of candidemia, and in one study, C. glabrata surpassed C. tropicalis to become the most common nonalbicans species isolated (51). C. glabrata is important clinically, not only because of the increase in its frequency but also because it is associated with high complication rates (51) and high mortality rates (7, 12). In one study, the mortality rate of 83% exceeded the observed rates from any other Candida species (92). Therefore, identification of factors that contribute to the virulence of this organism may provide new attractive therapeutic targets. Recently, Clancy et al. (Programs Abstr. 36th Annu. Meet. Infect. Dis. Soc. Am., abstr. 317, 1998) reported on the detection of extracellular phospholipase activity in C. glabrata and provided data implicating it as a virulence factor in patients with candidemia caused by this yeast. In their study, phospholipase activity (as determined by opacity around colonies growing on egg yolk-containing media) was investigated in 51 non-albicans Candida species (C. glabrata, 22; C. parapsilosis, 12; C. tropicalis, 10; C. lusitaniae, 5; C. krusei, 2) recovered from patients in a multicenter study of candidemia. Phospholipase activity was detected in 53% of isolates. An assay involving agar containing specific phospholipid substrates was used to identify the type of phospholipase activity secreted by C. glabrata (64). As with C. albicans, PLB and Lyso-PL accounted for 100% of phospholipase activity. No PLA or PLC activities were detected in supernatants collected from various C. glabrata isolates tested. To determine whether a correlation exists between phospholipase activity and clinical virulence, the authors classified the C. glabrata isolates into two groups: persistent isolates were strains that remained in the blood despite therapy with antifungal agents effective in vitro and despite removal of all intravenous catheters, and nonpersistent isolates were strains that were cleared. Among non-albicans Candida species tested, the association between phospholipase activity and persistent candidemia was strongest for C. glabrata (P = 0.004). Moreover, the median PLB and Lyso-PL activities were higher for persistent isolates (9.5 and 24 pmol/mg, respectively) than for nonpersistent isolates (0 and 5.8 pmol/mg) (P < 0.0001 and P = 0.02, respectively).

To establish the role of PLB in the pathogenesis of C. glabrata, an isogenic strain pair of this yeast which differed only in PLB activity was created (C. J. Clancy, A. Leuin, M. A. Ghannoum, and M. H. Nguyen, Programs Abstr. 36th Annu. Meet. Infect. Dis. Soc. Am., abstr. 316, 1998). Conserved sequences of scPLB1, the gene responsible for PLB activity in S. cerevisiae, were used to design guessmer primers for PCR with DNA from a clinical C. glabrata isolate. A 1,030-bp PCR product that encoded part of a putative protein with 78% homology to scPLB1 protein was generated. To create an isogenic mutant with disruption of the PLB gene, Clancy et al. first created a ura3 auxotroph of the parent C. glabrata strain. S. cerevisiae URA3, which is >80% identical to C. glabrata URA3, was amplified by PCR, BamHI restriction sites were added to either end, and the resulting product was inserted in the unique BamHI site of the 1,030-bp PCR fragment. The disrupted product was liberated from the plasmid by EcoRI digestion and was then used to transform C. glabrata ura3 auxotrophs. The resulting mutants were selected by growth on Ura media. Targeted disruption was confirmed by Southern blot analysis. Colorimetric assay of phospholipase activity revealed >90% elimination of both PLB and Lyso-PL activities in the mutant isolate (URA3⁺ plb) compared to the parent C. glabrata (URA3⁺ plb⁺), confirming that the cloned gene was responsible for the vast majority of PLB and Lyso-PL activities. Comparative in vivo pathogenicity studies are currently being performed to determine the role of the cloned gene in C. glabrata virulence.