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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2006, p. 3519–3528 0066-4804/06/$08.00ϩ0 doi:10.1128/AAC.00545-06Copyright 2006, American Society for Microbiology. All Rights Reserved.
MINIREVIEW
Impact of Melanin on Microbial Virulence and Clinical Resistance Joshua D. Nosanchuk1* and Arturo Casadevall1,2 Department of Medicine, Division of Infectious Diseases,1 and Microbiology and Immunology,2 Albert Einstein College of Medicine, Bronx, New York Melanins are negatively charged, hydrophobic pigments of cumstances is uncertain, such as melanin in the neurons of the high molecular weight (54, 88, 95, 139) that are composed of substantia nigra in the human brain (147, 148).
polymerized phenolic and/or indolic compounds (Fig. 1) (45, In mammals, melanin synthesis is catalyzed by a tyrosinase 128). Melanins are produced by organisms in all biological (114). In contrast, microbes generally synthesize melanin via kingdoms, including a wide variety of pathogenic bacteria, various phenoloxidases (such as tyrosinases, laccases, or cata- fungi, and helminths (reviewed in reference 90). Remarkably cholases) and/or the polyketide synthase pathway (reviewed in little is known about the structures of melanins, despite their reference 138). Melanins generated from 3,4-dihydroxyphenyal- abundance in the global biomass. This is due to the inability of anine (DOPA) by phenoloxidases are referred to as eumela- current biochemical and biophysical techniques to provide a nins, which are generally black or brown. Yellow or reddish definitive chemical structure, because these complex polymers melanins are called pheomelanins and incorporate cysteine are amorphous, insoluble, and not amenable to either solution with DOPA. Brownish melanins derived from homogentisic or crystallographic structural studies. Consequently, our infor- acid by tyrosinases are called pyomelanins (144). Melanins mation on the structure of melanin is derived from the analysis formed from acetate via the polyketide synthase pathway are of their degradation products and spectroscopic analysis of the typically black or brown and are referred to as dihydroxynaph- melanin polymer (128). Characteristically, melanins are dark in color, insoluble in aqueous or organic fluids, resistant to con- Melanin synthesis has been associated with virulence for a centrated acid, and susceptible to bleaching by oxidizing agents variety of pathogenic microbes. Melanin is believed to contrib- (17, 87, 103). Methods for partial chemical degradation of ute to microbial virulence by reducing a pathogen’s suscepti- melanin followed by high-pressure liquid chromatographic mi- bility to killing by host antimicrobial mechanisms and by influ- croanalysis have been developed and are useful for the char- encing the host immune response to infection. Consequently, acterization of specific types of melanin (128, 129). An oper- melanin and melanin synthesis pathways are potential targets ational definition for a pigment as a melanin can be provided for antimicrobial drug discovery. Interestingly, the drug-bind- by electron spin resonance characteristics, since these pig- ing properties of both host and microbial melanins could in- ments uniquely are stable organic free radicals (29).
fluence the outcome of antimicrobial therapy.
Many diverse functions have been attributed to melanins.
This review discusses the impact of melanin production on Melanins can serve as energy transducers and affect cellular microbial survival in the environment and during infection, on integrity (reviewed in reference 48). Melanin is also used for host immune responses, and on the efficacies of antimicrobial sexual display and camouflage. For instance, the coloration in compounds. The capacity for melanin to bind to diverse com- black and red hair arises from melanin (18). An example in pounds can affect the testing of antimicrobial drugs and reduce which melanin is used for camouflage is the release of ink, a the activity of antimicrobial therapy.
suspension of melanin particles, by the cuttlefish (Sepia offici-nalis) in response to danger (34). Melanin plays a major role inthe innate immune system of insects, which synthesize the MELANINS CONFER A SURVIVAL ADVANTAGE TO
polymer to damage and entomb microbial intruders (85, 104).
ENVIRONMENTAL MICROBES
In insects, invading microbes activate a prophenoloxidase inthe hemolymph, resulting in the encasement of the bacterial, Melanin synthesis in free-living microbes is likely to provide protozoal, or fungal pathogen in melanin (78). Melanins in a survival advantage in the environment (Table 1) (117). This melanocytes in skin provide protection against sunlight and are hypothesis is based on the fact that many fungi constitutively also believed to contribute to the resistance of melanoma to synthesize melanin, and even facultative melanotic microbes therapeutic radiation (47). The role of melanin in other cir- like Cryptococcus neoformans are melanized in soils (94). Mel-anin production in C. neoformans is associated with increasedsurvival after ingestion by environmental amoeboid (118) ornematode (84) predators. Environmental predators often pro-duce hydrolytic enzymes to digest microbes, and melanized C. * Corresponding author. Mailing address: Albert Einstein College of neoformans cells are significantly less susceptible to cell wall- Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718)430-3766. Fax: (718) 430-8968. E-mail: nosanchu@aecom.yu.edu.
degrading enzymes than nonmelanized cells (109). Melanin FIG. 1. Chemical structures of pheomelanin (A) and eumelanin (B) oligomers.
production in diverse environmental melanotic molds has been for the growth of black fungi in the highly contaminated Cher- associated with reduced cellular susceptibility to enzymatic nobyl Reactor No. 4 (83). The pigment significantly contributes degradation (reviewed in reference 90). The mechanism of to the ability of C. neoformans to withstand extremes in heat action for resistance to enzymatic hydrolysis is unclear but may and cold (108). In fungal plant pathogens, melanization of involve sequestration of the enzymes on melanin or may occur appressorium allows a cell to maintain integrity while gener- by steric hindrance (54). Additional evidence supporting a ating pressures in excess of 80 bar to facilitate the penetration protective role for melanin is provided by the fact that addition peg’s entry into a plant cell (reviewed in reference 90).
of synthetic melanin to suspensions of Aspergillus nidulans re- Melanins are able to bind to the heavy metals that are sults in significant inhibition of the hydrolytic activity of glu- routinely found in the environment (35, 105, 153). The car- canase-chitinase on the fungus (67).
boxyl, phenolic, hydroxyl, and amine groups on melanin pro- Melanins confer resistance to UV light by absorbing a broad vide numerous potential binding/biosorption sites for metal range of the electromagnetic spectrum and preventing photo- ions (reviewed in reference 35). Melanized C. neoformans cells induced damage (48). Consequently, melanins are used com- are more resistant to killing by silver nitrate, a compound mercially in photoprotective creams and eye glasses. Melanin highly toxic to bacteria and fungi, than nonmelanized cells protects several fungal and bacterial species from UV, solar, or (42). That study demonstrated that melanin chelated the silver gamma radiation (reviewed in reference 90). Increased mela- compound (42). Although other fungal melanins bind to met- nin production is associated with the greater resistance of als (reviewed in reference 35), a protective role for metal pigmented fungi to radiation (127, 149, 150). The protective binding has not been demonstrated in other microbes. How- properties of melanin against radiation injury could account ever, the evidence from C. neoformans suggests that the utilityof metals as antimicrobial drugs against melanin-producingorganisms may be lower than that against non-melanin-pro- TABLE 1. Melanization protects pathogenic fungi from MELANIN PROTECTS MICROBES
FROM HOST DEFENSES
The ability of melanin to protect microbes from host de- fenses is relevant to antimicrobial therapy because the clinical efficacies of some antimicrobial drugs are complemented by host immune defenses. Melanin has been shown to interfere with numerous host defense mechanisms (Table 2). Melanized C. neoformans yeast cells manifest increased resistance to phagocytosis in vitro and in vivo (82, 130). In macrophage-like cell lines, the phagocytic index for melanized Paracoccidioidesbrasiliensis yeast cells was half that for the nonmelanized cells (21). Since melanins are charged polymers (139), their pres- TABLE 2. Melanin production protects pathogenic fungi against dermatitidis do not reduce phagocytosis or protect against oxidative burst or killing by neutrophils (115).
Melanins are highly effective scavengers of free radicals (116) and have electron transfer properties (41). Electrontransfer from free radical species generated in solution to melanin derived from C. neoformans has been demonstrated by electron spin resonance spectroscopy (131); and similar spectra have been generated with melanins from Histoplasma capsulatum, S. schenckii, P. brasiliensis, and Pneumocystis spp.
(reviewed in reference 90). Also, C. neoformans melanin is involved in the reduction of Fe3ϩ to Fe2ϩ (97) and can facil- itate redox cycling through the exportation of electrons to form extracellular Fe2ϩ, which maintains the reducing capacity of extracellular redox buffers (56). Melanized C. neoformans cells are also less susceptible to the toxic effects of microbicidal peptides than nonmelanized cells (26). The mechanism of ac- tion in this case appears to be adsorption of the microbicidal peptide such that it interferes with the peptide reaching its EVIDENCE THAT MELANINS BIND
a Magnitude indicates the maximal percent increase in protection afforded to TO DRUGS IN VITRO
the organism by the production of melanin compared to that afforded to cellsdeficient in melanin.
Isotherm analysis of adsorption of drugs by melanin. Mel-
Represents attachment rather than ingestion of fungal cells.
c As measured by mitochondrial damage rather than numbers of CFU.
anins bind to chemically diverse compounds (62, 70). Thebinding of gentamicin, methotrexate, and chlorpromazine tomelanins has recently been revisited by using isotherm binding equations to characterize the adsorption of the drugs to syn- ence in the cell wall of C. neoformans can alter the fungal cell thetic and Sepia officinalis melanins (14). Although there were surface charge (88), and this may contribute to inhibition of significant variations in adsorption, each drug bound to mela- phagocytosis. Melanization increased the cellular negative nin. More gentamicin than the other drugs was bound by syn- charge by 3 to 33% in nine different encapsulated strains and thetic melanin. By the best-fit Freundlich equation for genta- by 86% in an acapsular strain (88). In addition to reducing micin [q ϭ q (KC)1/n dm3 · gϪ1, where q is the amount ingestion, melanization protects C. neoformans against killing absorbed [mmol · gϪ1], q is the adsorption capacity, K is the by macrophages (130). Similarly, melanin production in Fon- energy of absorption, C is the equilibrium solution concentra- secaea pedrosoi (20), Sporothrix schenckii (107), and Exophiala tion of solute, and the heterogeneity index 1/n is between 0 and spp. (33, 99, 115) enhances resistance to killing by phagocytic 1], the quantity of gentamicin absorbed with synthetic melanin cells. Melanin in pigmented C. neoformans yeast cells can pro- was 0.49 dm3 · gϪ1, whereas 0.061 dm3 · gϪ1 of methotrexate duce complex immunomodulatory effects that contribute to was bound. Equilibrium isotherms for the interaction of gen- virulence by eliciting changes in the host cytokine/chemokine tamicin with melanin revealed diverse interactions between the response (50, 82). In particular, melanized yeast resulted in drug and melanin. In contrast, methotrexate appeared to bind higher levels of interleukin-4 and monocyte chemoattractant to specific homogeneous sites. The data from that study (14) protein 1 and increased the numbers of pulmonary leukocytes demonstrated the significant capacity of melanin to interact early after infection (82). F. pedrosoi melanin also activates with drugs, since its adsorption capacity was comparable to those of other absorbers, like medicinal activated charcoal.
Melanization protects fungi, such as C. neoformans, Aspergil- Scatchard plot analysis of drug binding by melanin. A Scat-
lus spp., and S. schenckii, and bacteria, such as Proteus mirabilis chard plot-type analysis of drug binding to melanin by the use and Burkholderia cepacia, against injury secondary to nitrogen- of radiolabeled compounds has also demonstrated the pres- or oxygen-derived radical attack (reviewed in reference 90). F. ence of heterologous binding sites. There are at least two pedrosoi melanin significantly inhibits nitric oxide production classes of binding sites on synthetic DOPA melanin for the by macrophages, which affects the pathogenesis of chronic aminoglycoside antibiotics gentamicin (141) and kanamycin chromoblastomycosis (10). The melanized F. pedrosoi cells re- (142). For kanamycin, the association constants for the strong duced the production of nitric oxide by approximately 50% and weak binding sites were 3 ϫ 105 MϪ1 and 4 ϫ 103 MϪ1, compared to the amount produced by nonstimulated murine respectively, and 0.64 ␮M kanamycin was required to saturate peritoneal macrophages and, similarly, suppressed nitric oxide the binding sites in 1 mg melanin (142). Scatchard plot-type production in macrophages stimulated by interferon gamma analyses with melanins have also revealed high- and low-affin- and lipopolysaccharide. Interestingly, melanin is not the only ity binding sites for cocaine (61, 102); amphetamines (6, 43); pigment that can modify the effects of oxidative attacks by host donarubicin (120); and the antiarrhythmics quinidine, disopyr- cells, since carotenoids in Staphylococcus aureus similarly func- amide, and metoprolol (16). More recently, high-resolution tion as antioxidants (75). However, carotenoids in Exophiala magic angle spinning nuclear magnetic resonance spectroscopy revealed highly specific melanin-binding sites for iodobenz-amides (11), which can be exploited to diagnose and stagemelanoma by using radiolabeled drug.
Absorption studies with antifungals. Two methods have
been used to establish that melanin binds to amphotericin Band caspofungin. First, the ability of melanin produced by C.
neoformans
and synthetic melanin to bind to these antifungaldrugs was inferred from experiments whereby melanins wereincubated with various compounds and then the antifungalactivity of the solution was determined (52, 124, 125). Forthose studies, melanin particles were removed by centrifuga-tion prior to the testing of the antifungal drug solutions in MICand time-kill studies. Incubation of amphotericin B and caspo-fungin with melanin significantly reduced their antifungalactivities for C. neoformans. In contrast, incubation of itra-conazole, fluconazole, or flucytosine with melanin had no ef-fect on their antifungal activities. One study showed a 16-foldincrease in the MIC of amphotericin B after adsorption of thedrug with 1 ϫ 107 melanin particles derived from pigmented C.
neoformans
prior to MIC testing (52). Furthermore, the in-crease in MIC correlated with the amount of melanin particles added to the antifungal drug solution. Time-kill assays alsodemonstrated that the addition of melanin particles to ampho- FIG. 2. The pathogenic yeast Cryptococcus neoformans. (A) India tericin B or caspofungin significantly reduced their toxicities ink preparation showing a budding C. neoformans yeast cell with alarge polysaccharide capsule surrounding the cell bodies. Bar, 5 ␮m.
for C. neoformans (124). For example, 71% of yeast cells sur- (B) A melanin “ghost,” a melanin particle isolated from C. neoformans vived exposure to 2ϫ the MIC of amphotericin B preincubated grown for 10 days in the presence of L-dopa by serial treatment of the with synthetic melanin, whereas the rate of survival was 8% for yeast with enzymes, denaturant, chloroform, and hot acid. Bar, 2 ␮m.
the cells exposed to amphotericin B not incubated with mela- (C) Transmission electron micrograph of a cross-section of a C. neo-formans “ghost” showing that the particle is formed of concentric nin. Similarly, 79% of yeast cells exposed to caspofungin pre- layers of melanin. Bar, 1 ␮m. (D) Depiction of the melanin granules incubated with melanin survived exposure to drug, whereas the comprising the melanin layers, demonstrating how the packing of the rate of survival was 11% for cells incubated with native caspo- granules results in pores that obstruct the passage of large molecules, fungin. In contrast, incubation of azoles or flucytosine with such as amphotericin B or caspofungin. Obstruction of antifungal melanin did not affect their MICs for C. neoformans or the molecules can occur by virtue of a reduced melanin pore size ormelanin binding. Panel D is based on data from reference 27.
abilities of the drugs to kill C. neoformans.
Antifungal drug binding to fungal melanin was also inferred by the finding that the elemental composition of melanin waschanged after incubation with antifungal drugs. Incubation of of melanin particles arranged in a concentric manner. The amphotericin B or caspofungin with melanin altered the mel- thickness of the layer appears to depend on cell age, such that anin C:N:O ratio, consistent with drug absorption (124). In older cells may be significantly less susceptible to melanin- contrast, no change in the melanin elemental composition was binding antifungal drugs than younger cells. In order for the observed following the incubation of melanin with flucytosine, fungal cells to survive, the polymer layers composed of gran- voriconazole, fluconazole, or itraconazole (124, 125). Since ules cannot completely exclude nutrients. Nuclear magnetic melanin is located in the cell wall, these data suggest and are resonance cryoporometry revealed that melanin ghosts contain consistent with a mechanism of acquired resistance, whereby pores with diameters between 1 and 4 nm, in addition to a fungal melanin binds to amphotericin B and caspofungin and small number of pores with diameters nearly 30 nm. The pore prevents them from reaching their target sites.
size decreases with the age of the yeast cell. Importantly, cryo- Effect of melanin on the porosity of the microbial cell wall.
porometry studies with melanin-binding antibody show that Analysis of the microstructure of cell wall-associated melanin the larger pores appear to be internal to the smaller pores. In in C. neoformans has provided new insights into the potential another study, the porosity of melanized cryptococcal cell walls of this polymer to interfere with antifungal drug activity (27).
was evaluated by elution of graded dextrans, and similar results Cell wall-associated melanin is composed of discrete granules were described (57). These findings suggest that the tight of roughly uniform dimensions (Fig. 2). This is significant, spaces between melanin granules may prevent or slow the because a granular arrangement would significantly increase entry of large drugs, such as amphotericin B (molecular mass, the surface area available for binding to certain types of drugs.
924 g/mol) and caspofungin (molecular mass, 1,093.5 g/mol), Atomic force microscopy and transmission electron micros- into pigmented cells. This may be particularly significant for copy revealed that the melanin particles range in size from 40 amphotericin B, since this drug tends to form large aggregates to Ͼ100 nm, with an average particle diameter of 76 nm, which in solution (68). In contrast, azoles and flucytosine have sig- is similar to the results for mammalian melanin and melanin nificantly smaller molecular masses, and for these compounds, from S. officinalis (27). Transmission electron microscopy re- melanization does not reduce fungal cell susceptibility. Hence, vealed that cell wall melanin is composed of two to five layers melanin in the cell wall may also reduce the susceptibilities to certain drugs by inhibiting their diffusion into the cell body and The interaction of these chemotherapeutics with melanin may provide greater opportunity for the binding of drug by melanin.
be responsible for decreased wound healing and for an unsat-isfactory response of the tumor to the medication. However,many melanin-binding drugs commonly used in clinical prac- BINDING OF COMPOUNDS BY MELANIN
tice, such as beta-blockers, benzodiazepines, and rifamycins, IN HUMANS IN VIVO
bind to melanin in vitro, without apparent adverse effects in The binding of drugs to host melanin can damage certain tissues, and drug-melanin interactions have been implicated in Aminoglycosides are positively charged at physiological pH the pathogenesis of several diseases, such as Parkinson’s dis- and have a relatively high molecular weight, which limits their ease. In Parkinson’s disease there is a loss of pigment in the penetration into tissues (66). Nevertheless, aminoglycoside melanotic dopaminergic neurons in the substantia nigra of the therapy causes significantly more toxicity in albino animals brain. A provocative connection between the drug-binding than in their pigmented counterparts. The administration of properties of melanin and the etiology of Parkinson’s disease aminoglycosides can result in permanent vestibular and audi- came from the observation that heroin contaminated with tory ototoxicity (7). The cochlear melanin content has been 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused correlated to the pigmentation of the host (65). The ototoxic a similar neurological disease in drug users, possibly because effects of aminoglycosides have been shown by electrophysio- melanin concentrated this compound in substantia nigra neu- logical and morphological methods (19, 136, 143). However, rons (46). Chlorpromazine also accumulates in the substantia no difference in outer hair cell degeneration in the organs of nigra (73), and the side effects of chlorpromazine and other Corti was observed between albino and pigmented guinea pigs phenothiazines include extrapyramidal disorders, such as tar- exposed to various concentrations of kanamycin (137). Ami- dive dyskinesia and parkinsonism. In contrast to MPTP, the noglycosides can also cause functional and morphological parkinsonian symptoms secondary to phenothiazines are usu- changes in the retina, particularly when they are administered ally reversible. The specific retention of other drugs in pig- by intravitreal injection. Studies with albino and pigmented mented tissues can damage cells in the skin, eye, and inner ear.
rabbits have shown that ocular pigmentation can partially pro- These interactions are complex and depend on diverse factors, tect the retina from damage due to aminoglycosides (30). Al- such as cysteine content, pH, and ionic interactions (79). As though it is of unclear significance, the binding of aminoglyco- described above for synthetic DOPA melanin, Scatchard plot side by melanin can augment the antibiotic’s inhibitory effects analysis has revealed the heterogeneity of binding sites on on collagen synthesis in human fibroblasts in vitro (141).
melanin for single compounds (69, 102, 122, 123). The confor- The capacity of melanin to bind to aminoglycosides and mation of the compounds may also influence these interac- other antibiotics may have important implications when these tions. Binding is typically reversible, but the retention times drugs are used in intraocular injections (4, 63). The in vitro can be protracted. For example, chloroquine can be detected efficacies of aminoglycosides, tertracyclines, and vancomyin in the melanin of the eye for a year after receipt of a single were significantly reduced following incubation with mela- dose (74), and chloroquine therapy is associated with retinop- nin (5, 38). In fact, the mixing of 100 ␮g/ml of tobramycin athies (49) that can occur long after treatment (151). In addi- with 1,000 ␮g/ml of melanin resulted in an immediate de- tion to chloroquine, severe retinopathies can occur following crease in antibiotic activity of 80% (5). The efficacies of melanin binding by chlorpromazine. Chloroquine also accumu- fluoroquinolones may also be affected, as these drugs are lates in dermal melanocytes and hair follicles (79), where it bound by melanin within the eye (37, 39, 40) and even in can occasionally cause irreversible hearing loss, tinnitus, and hair (140). Although melanin can bind to fluoroquinolones, dizziness (44). Whereas hearing loss due to chloroquine is penicillins, and cephalosporins, no reduction in antibacterial thought to be a result of effects on the eighth cranial nerve, efficacy has been reported after these drugs have been in- quinine can accumulate in melanin in the stria vascularis of the cochlea and cause cellular degeneration (74).
Thioureylenes are selectively incorporated into melanin MELANIN AND THE EFFICACY OF
(71); and certain compounds, such as propylthiouracil, may ANTIMICROBIAL THERAPY
cause a loss or the depigmentation of hair (70). The carcino-genic effects of polycyclic aromatic hydrocarbons may be com- Melanin has been called an “an antifungal resistance factor,” pounded by the presence of melanin, since these compounds given its ability to reduce the susceptibilities of melanized cells have a prolonged retention time in pigmented tissues (106).
to antifungal drugs (52). Notably, there is no evidence for the Herbicides, such as paraquat (which is structurally related to involvement of melanin in drug efflux pumps or in alterations 1-methyl-4-phenylpyridinium [MPPϩ], the neurotoxic metabo- in the synthesis of ergosterols or glucans in fungal cell wall/cell lite of MPTP), avidly bind to melanin and cause parkinsonian symptoms in experimental animals (3). Cocaine and amphet- Antifungal susceptibility testing. In vitro susceptibility mea-
amines are known to bind to melanin (12, 119), and testing of sures the activity of a drug against a microbe, whereas clinical hair for these compounds is used for medical and legal pur- resistance is a lack of efficacy of a drug in vivo. Although in poses. The cytotoxic effects of anthracycline chemotherapeu- vitro resistance often correlates with clinical treatment failure, tics (such as doxorubicin and donorubicin) can be inhibited by in vitro susceptibility does not necessarily predict clinical suc- melanin. For example, the 50% inhibitory concentration of cess. Standard MIC broth macrodilution testing by use of the donorubicin in an in vitro cell-based assay increased from 0.04 M27A protocol for yeasts of the Clinical and Laboratory Stan- to 0.08 ␮M in the presence of melanin from S. officinalis (121).
dards Institute (CLSI; formerly the National Committee of TABLE 3. Melanin production protects pathogenic fungi (124). Melanization was also associated with reduced suscep- tibility to amphotericin B in melanized versus nonmelanized B.
dermatitidis
cells (96). Melanized P. brasiliensis cells were also more resistant to amphotericin B, fluconazole, ketoconazole,itraconazole, and sulfamethoxazole than nonmelanized cells (21). However, melanization can also increase susceptibility to certain drugs. The antipsychotic drug trifluoperazine had greater fungicidal activity against melanized cryptococcal cells than it did against nonmelanized cryptococcal cells when ac-tivity was measured by both CFU determination and flow cy- tometry with propidium iodide staining (135). In contrast,chloroquine, which is highly bound by melanin, had no fungi- cidal effect on either melanized or nonmelanized C. neofor- mans cells (135). Hence, an association between the capacity of melanin to bind to a drug and reduced susceptibility to thatdrug by melanized cells is not apparent for all agents. This a Magnitude indicates the maximal percent increase in protection afforded to implies the existence of mechanisms other than simple absorp- the organism by the production of melanin compared to that afforded to cellsdeficient in melanin.
tion by melanin as an explanation for the differences in theactivities of certain classes of drugs against melanized andnonmelanized cells.
Clinical Laboratory Standards) revealed no differences in sus- The efficacies of antifungals to melanized cells can also ceptibility between melanized and nonmelanized C. neofor- be evaluated by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- mans cells (52, 124). Similarly, no differences in susceptibility [(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) re- to antifungals by MIC assays were measured between albino duction assay (80). Specifically, XTT was used to show that and pigmented cells of E. dermatitidis (101), H. capsulatum melanization protected C. neoformans from amphotericin B (124), or Blastomyces dermatitidis (96). However, the growth of and caspofungin in biofilms. For example, the metabolic activ- melanized C. neoformans yeast cells in medium without a phe- ity of melanized C. neoformans cells in a biofilm exposed to 32 nolic substrate resulted in large defects in the melanin layer of ␮g/ml of amphotericin B was 40%, whereas it was 20% for the parent cells after budding and an absence of melanin in the daughter cells (89). Hence, even if melanized cells are used Impact of melanin binding on antifungal drugs. The finding
initially, the daughter cells lack melanin, and the CLSI meth- that melanin can bind to amphotericin B and caspofungin, in odology does not compare the susceptibilities of melanized combination with observations of the microstructure of mela- and nonmelanized cells. Incorporation of phenolic substrates nin in C. neoformans, suggests a potential explanation for the into the testing medium for a microdilution or a macrodilution difficulty in eradicating C. neoformans with these drugs. Mel- assay has not been possible because these either precipitate or anization of C. neoformans yeast cells occurs in vivo (93), and autopolymerize into melanin (124). Hence, the CLSI protocol the amount of melanin produced increases with time after is not suitable for distinguishing differences in susceptibility infection (31, 95). Although amphotericin B is fungicidal to between melanized and nonmelanized cells.
nonmelanized C. neoformans cells in vitro, amphotericin B In contrast to the broth dilution method, time-kill studies therapy often fails to eradicate the fungus from patients (100, with CFU determinations have revealed differences in the sus- 152). Given the relative resistance of melanized cells to am- ceptibilities of melanized and albino cells to certain antifungal photericin B, the efficacy of this drug may be due to its activity drugs (52, 124, 125) (Table 3). A major difference between against nonmelanized buds and to its facilitation of tissue these assays is that in time-kill studies the microbes are incu- clearance by host defenses through its powerful immunomodu- bated with the antimicrobial drugs for hours rather than days.
lating effects. Caspofungin is active in vitro against nonmela- Thus, the melanin layer of the fungus remains largely intact. C. nized cells (36), but it is ineffective against experimental infec- neoformans is significantly less susceptible to amphotericin B tions with C. neoformans (1). The inefficacy of caspofungin for when the fungus is grown in the presence of L-dopa (133). This C. neoformans in animal studies cannot be explained by its result was recently confirmed by time-kill studies with ampho- inability to inhibit either 1-3-␤-D- or 1-6-␤-D-glucan synthase tericin and caspofungin, which revealed that the activities of (32). Since acapsular strains of C. neoformans are also resistant these drugs against melanized cells were reduced by 55 and to caspofungin, the large polysaccharide capsules that can oc- 7%, respectively, relative to their activities against nonmela- cur in vivo are not inhibiting the drug from engaging the nized cells (52, 124). In contrast, no differences in activity fungus. Hence, for C. neoformans, the clinical resistance to against melanized and nonmelanized C. neoformans cells was caspofungin could be attributed in part to in vivo yeast cell observed for voriconazole, fluconazole, itraconazole, or flucy- melanization. Caspofungin is clinically effective against tosine (124, 125). However, melanized C. neoformans strains Aspergillus spp., a group of fungi that can produce melanin.
exhibited reduced susceptibilities to higher concentrations of However, its efficacy may be due to the fact that hyphae, the tissue-invasive form of this fungus, are not melanized (146).
Time-kill assays similarly revealed that melanized H. capsu- Dematiaceous fungi are darkly pigmented molds that con- latum yeast cells were less susceptible to amphotericin B and stitutively produce melanin during infection and are extremely caspofungin than nonmelanized H. capsulatum yeast cells difficult to treat with antifungal drugs (13). Although the clin- ical relevance is not clear, antifungal susceptibility testing for fact that voriconazole at 0.125 to 0.5 mg/liter can inhibit filamentous fungi has recently been standardized (86). Ampho- conidiation in diverse Aspergillus spp., resulting in white colo- tericin B has good activity against most clinically important nies (126). Ravuconazole, which is structurally similar to vori- dematiaceous fungi in vitro, but clinical resistance is not un- conazole, had similar effects only against Aspergillus fumigatus common. Scedosporium prolificans and Scopulariopsis brumptii and Aspergillus flavus. It is possible that the inhibition of mel- are consistently resistant to amphotericin B in vitro; and occa- anin formation in vivo may contribute to the therapeutic sional resistance to this drug is reported in several other potencies of these triazoles by increasing the susceptibility to species, including Chaetomium spp., Curvularia spp., Phiale- host defense mechanisms. The possibility that certain antifun- monium spp., and Exophiala spp. (81). Echinocandins are not gal agents are less effective against melanotic molds should clinically useful against these fungi. The inefficacies of the especially be considered when clinicians make choices for em- echinocandins against these fungi and the relative resistance of pirical therapy in patients with presumed mycotic diseases.
these fungi to amphotericin B may be associated with thedense production of melanin in these fungi. The broadest in ACKNOWLEDGMENTS
vitro activity against dematiaceous fungi is achieved with azoles J.D.N. and A.C. are supported in part by NIH grant AI52733.
(81). In this regard, we note that azoles are not bound by The electron microscopy images in Fig. 2 are courtesy of Helene Impact of melanin binding on antibacterial agents. The role
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