Molecular pathogenesis of fungal infections

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Molecular pathogenesis of fungal infections Richard Calderone

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key factor in the pathogenesis of fungal infections caused by Candidu alhicuns and Cryptococcus neofomzans is their ability to colonize host tissues and evade host defenses’,2. Virulence in C. albicans appears not to be dctcrmincd by a single phcnotypic property, but rather depends on an array of factors, including adhcsin molcculcs, proteolytic cnzymcs and morphogenetic adaptation to the host environment. For of C. nfmffwwzmzs, encapsulation cells and production of melanin confcr virulcncc. The use of molecular approaches to elucidate the role of putative virulence factors in thcsc and other fungi was discussed at the rcccnt 12th Congress of the Intcrnational Society for Human and /\nimal IMycoses held in Adclaidc, Australia, 13-l 8 March, 1994. Pathogenesis of superficial pathogens Candida alhicans secreted aspartyl proteinase (SAP) continues to be a focus of study as a virulence factor. Ii. Tsuboi and H. Ogawa (Jutendo University, Tokyo, Japan) cxamincd clinical isolates of Candidu in a straturn corncum medium for the production of SAP, lipase and a--glucosidasc. Of all Candida spccics, C. albicans produced the highest activity of each enzyme. Tom Ray (University of Iowa College of Medicine, Iowa City, IA, USA) provided more direct cvidcncc for a role of SAP in virulence, by showing that production of SAP correlates well with cavitation of corneocytcs and ndhcrcncc to skin, mucosal epithelium and chick allantoic membrane. Confocal imaging has demonstrated the prcscnce of SAP during invasion of the stratum corneum. Furthcrmore, comparing production of SAP in the opaque-white switch phenotypes indicarcs that high pro-

duction of SAP by opaque strains can bc corrclatcd with the greater invasion of the dermis and formation of abscesses of these strains, compared with the non-SAPproducing white phenotype. K. Kramano (University of Indonesia, Jakarta, Indonesia) has found an additional virulence determinant: Rramano has shown that mutants of C. alhicans that lack the antigen 6 epitope adhere less readily to cultured keratinocytes. Additional observations on the SAP of C. alhicans were reported by Nina Agabian and colleagues (University of California at San Francisco, CA, USA) in a symposium on the ‘Pathogenesis of Candidiasis’. This group has identified at least four SAP-encoding genes and has shown that the opaque-white switch phcnotypcs diffcrcntially cxprcss the proteins that they encode. Sera from HIV-positive patients were used to screen for antibody to SAP in Candida-infected patients. The immune response seems to bc directed primarily against SAPS 1 and 3, while SAP 2 appears to bc the predominant form produced by the organism in vitro. In a rclatcd poster session, Bernhard Hube and colleagues (Universitaire Vaudois, Lausannc, Switzerland) reported that C. alhimzs products at least six SAPS in vitro. Production of SAPS 1 and 3 was regulated by the opaque-white switch phcnotype, while SAP 2 was only expressed at a high level in blastoconidia. SAP 5 was only expressed by the hyphal growth form, while SAPS 4 and 6 appeared to be silent or onlv expressed weakly under the conditions studied.

Another prcscntation that focused on using molecular approaches to characterize virulence factors discussed the CL ncofomzans gcnc CM.59. This gent is involved in capsule synthesis (Yun Chang and K.J. Kwon Chung, NIH, Bcthcsda, MD, IJSA). Host-cell-fungal interactions The interaction of C. albicans with host cells was the focus of this symposium. Jean-Marcel Senct and Raymond Robert (UFR des Sciences Xledicalcs ct I’harmaccutiqucs, Angers, France) reported that C. albicans is clcarcd from the blood of infected mice quickly after it has been aggregated by platelets. The platelets bind more cxtcnsivcly to germinated forms of the organism. Binding could be inhibited by antibody to germ tubes or blastospores, but not by anti-mannan antibody. Resting platelets bind to a protein of 4.~ kDa from germ tubes, which is apparently rccognixed by the intcgrin gpIIb/IIa of platclcts. Activated platclcts react with another, as-yet-unidcntificd, Candida protein. Jack Edwards and Scott Filler (Harbor-UCI.A, Torrance, CA, USA) reported that C. alhicans interacts with human endothelial cells. Candida germ tubes appear to be preferentially phagocytoscd by cndothclial cells. Phagocytosis of viable germ tubes damages the endothelial cells and induces the synthesis and release of prostaglandins, mainly prostaglandin IL. Prostaglandins may increase the candidicidal activity of neutrophils against hyphal forms, while minimizing damage to cndothelial cells. Richard Diamond (Boston IJnversity School of Medicine, ,\lA, USA) reported that opsonized hyphac attach to and trigger polymorphonuclcar ncutrophils (P.LINs) via the immu~ioglohulin G Fc

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receptor, while unopsonixed organisms may interact with I’MNs through a (kndida inositol-linked glycoprotcin. Triggering of the biochemical activities of the PMS with opsonized is more rapid hyphae. The lcvcl of phospholipases incrcascs and second-messenger events occur during interactions with hyphac. The production of oxidants by PiMNs rises, but this alone is not sufficient to kill Cirndida. Initially, hyphal ATP and protein synthesis increase after contact with I’MNs; however, after 20 min of PMN-Candida contact, cell-wall degradation and oxidant-induced DNA damage occur. Metabolically inactive organisms seem to be protected from lethal effects. The CR2/CR3 complement receptor of C. alhicans may. act as an adhesin in the recogmtion of host endothclial cells (Richard Calderonc, Georgetown University School of Medicine, Washington DC, USA). A 60 kl)a mannoprotein has been identified that binds both RGD and non-RGDcontaining ligands. Mutants lacking this adhesin are avirulent. The cloning of adhesin-encoding genes has begun, and this should yield information about the role of thcsc proteins in virulence. 1Iumoral responses to C. alhicans were studied in BAI.H/c (Cundiduresistant) and CBA/H (susceptible) mice (John Warmington, Curtin -1Jniversity of Technology, Perth, W. Australia). RALK/c mice clear the organism rapidly and produce a more limited antibody response to antigens, the predominant reactivity being, towards the 48 kl>a enolase of C. alhicuns. The antibody response of CBA/H mice was more heterogeneous, with major reactivities being towards a 7.5 kDa heat-shock protein and another protein of 96 kDa. Morphogenesis and dimorphism III the symposium on ‘1Molecular Biological and Biochemical Aspects of Fungal Dimorphism’, the use of Sczccharonzyccs cerevisiae ELItants in isolating C. alkans gcnc dcscri bed. For homologs was example, SLT2 is essential for the growth of S. cercuisiae at 37°C. Mutants in this gene cannot grow

at this temperature; however, compIcmcntation with a C. albicans library that allowed growth of mutant cells has resulted in the isolation of a Candida gem encoding a microtubule-associated protein kinase that is thought to be essential in the growth pathway (Ccsar Nombela and collcagucs, IJniversidad Complutense, Madrid, Spain). IJsing a similar approach, a transcriptional-activator-encoding gene (ahA) from Aspcrgillus uidulam that is involved in conidiophore development was used to transform dip&d S. ccrcvisiae to a pseudohyphal phenotype (Bill Timberlake 3Ild colleagues, 1Mycol’harmaceuticals, Cambridge, ,MA, IJSA). This observation suggests that pathways regulating conidiogencsis and pscudohyphal growth may bc related. The identification of hyphalspecific genes of C. alhicans was also discussed. Chitin synthases (CHSI-.3), HYK 1 (a hyphal-specific gene), four SAP-cncodmg genes and MAJTf (which encodes a mannosy1 transferasc that is involved in O-linked mannoprotein synthesis) wcrc examined for association with the dimorphic switch. Most of these genes were thought to have only a peripheral role in morphogcnesis. However, HYK 1 and CHS2 would appear to be more tightly associated with (and essential to) the dimorphic switch (Neil Gow and colleagues, University of Aberdeen, IJK). ‘I’he gene PMAl is also thought to be essential to morphogcnesis in C. ahicons (K. Cannon, IJniversit) of Otago, Duncdin, New Zealand). Fungal vaccine developments Vaccines against fungal infections have been sought for several years. At a symposium on ‘New Stratcgics in the Development of Fungal Vaccines’, data from studies on protection against Coccidioides immitis, C. albicnns and Trichophyton WY~z~coswz were reported. F antigen, a soluble protein isolated from the cell wall of C. immitis, triggered an intense cell-mediated response in vitro (Gary Cole and colleagues, University of I’exas, Austin, TX, USA). More detailed study of this protein will determine whether it has potential as a vaccine. IIIII~LI-

nization of calves with ribosomal preparations of 7’. ucrrucosum decreased the duration of infection (Esther Scgal md colleagues, l‘clAviv University, Israel). l’assivc immunization with an antibody raised against the heat-shock protein 90 (Hsp90) of C. albicans protected mice from systemic challenge (Kuth -Matthews, University of .Manchcstcr Medical School, UK). Likewise, an anti-idiotypic antibody raised against a monoclonal antibody that neutralizes the antifungal activity of a l’ichia cznomala killer toxin protected rats from vaginal infection when used immunogcn (Luciano as an I’olonelli, Universita degli Studi di I’arma, Pat-ma, Italy). The importance of T,,l cells in immunoprotection against cryptococcosis (Juncann Murphy and collcagucs, University of Oklahoma School of ,Medicinc, Oklahoma City, OK, USA) and candidiasis (Francesco Bistoni and colleagues, IJniversity of I’crugia, Italy) was reported. containing Sponges cryptococcal culture-filtrate antigcn wcrc implanted into the backs of immunized mice. ‘l’he antigen induced a granulomatous tissue rcsponse, with tumor necrosis factor, interferon y (IFN-y), intcrleukin 2 (II.-2) and IL-S being the prcdominant cytokincs. The role of II.-2 and II-N-y in protection against cryptococcosis was established by the injection of antigen intravenously, which rcduccd both the production of these cytokines and the clearance of the organism from tissues. Likewise, the role of cytokines in protection against candidiasis was established by treating non-hcnler (Candida-susceptible) mice with antibodies against specific cytokines. Anti-IL-4 treatment converted non-healer mice to a healer (less-susccptiblc) phenotype. Furthermore, when given anti-EN-y treatment, healer mice bccamc more susceptible to secondary challcngc with Cundida. ‘I’hus, susceptibility to candidiasis may bc controlled bv the T,, response, and manipula;ion of this response may induct protection. IFN-y also stimulated murinc peritoneal macrophages to kill C. alhicans; the active anti-Cnndidn

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appears to be an component ~1000 kDa nonapproximately cationic pcptidc (K. Kagayn and Y. Fukazawa, Yamanashi Medical College,Japan). Kccombinant IFIX-y also activated murine splenic and peritoneal macrophagcs to kill Histoplasmz capsulatuwz (Dexter 1Toward and colleagues, UC1.A School of .Mcdicinc, 1.0s Angeles, (:I\, USA). ‘I’his cffcct is overcome when synthesis of nitric acid

(NO) by macrophages is inhibited. Howcvcr, other data do not indicate a role for NO in killing H. cu~xzhttm: Other highlights

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ccular approaches to diagnosis and species identification may now offer alternatives to the clinical mycology laboratory in situations where current protocols lack sufficient sensitivity and specificity.

Other highlights of the meeting indiscussions on the mcchanisms and clinical impact of resistancc to antifungals, management of fungal infections and the progress in antifungal therapy. klolcludcd

Programmed cell death: a fundamental protective response to pathogens Gwyn T. Williams

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ltruistic cell death frequently has important advantages .in multicellular organisms. Such programmed death usually takes the form of apoptosis’ in higher animals and plays a very significant part in cmhryonic dcvelopment, selection of the imof mune systcrti’,’ and removal damaged cells, cspccially those with damaged DNA’. In addition, progratnmcd cell death as a rcsponsc to intracellular irifcction has now lxcn observed for a wide range of pathogens and host orgnnisms, and is cmcrging as a particular focus of interest. The suicide of a pathogeninfected cell may be a productive strategy for the survival of the multicellular organism as a whole, if the production of viable pathogenic organisms is reduced or eliminated. Considering the pathogen, also, the death of the ccl1 may often be required, for example, to allow the eventual release of intracellular organisms (reviewed by Zychlinsky ‘), and scvcral intracellular hactcrial infections have indeed 1x33 found to induct apoptosis. S/7&/1~7 fkxrwi” md Lkwdetclla pcrtussis.’ both

induct apoptosis of host macrophagcs. hrdetclla pcrtussis organisms that are deficient in adenylatecyclasc-hacniolysin do not induce apoptosis of the macrophagcs that they infect, suggesting that these bacteria may L~SC this protein to exploit the suicidal rcsponsc of the host cell, allowing the bacteria to cscapc the microbicidal abilities of the host. A further possibility is suppression of the death of the host ccl1 by the pathogen, to prolong the period of multiplication, which would naturally tend to obscure any suicidal rcsponsc of the host. Several examples of these opposing effects arc now known, and the induction or suppression of hostccl1 death is beginning to look like a major battleground in the evolution of host-pathogen relationships”, with the halance of these processes likely to hc crucial to the progress of infection. The apoptosis of mammalian ceils that is induced bv infection with Sindbis (an alpha;irus) illus-

tratcs some of the complexities in the relationship’. Several cell lines arc triggered to undergo apoptosis when infected with the virus, rcsulting eventually in a lyric infection. I lowever, most cells transfccted with the cellular proto-oncogene hcl-2 dcvclop persistent productive infections instead. Similar phenonc11;1have been reported recently with this gene in at least some cells infected with influenza viruses i\ and H (Ref. 10). The M-2 gent was originally isolated from the breakpoint of the chromosomal translocation that is strongly associated with follicular lymphoma, and has subscqucntly been shown to suppress apoptosis induced by divcrsc stimuli -from cytokine withdrawal, through the corticosteroid-induced apoptosis of thymocytes, to apoptosis induced by canccr-chcmotherapeutic drugs, particularly those that cause DNA damage. Although the molecular mechanism by which Bcl-2 exerts its effects, and cvcn its subcellular localization, are still controversial, these observations have provided the first clear example of genetic suppression of npoptosis. Epstein-Harr virus appears to