In vitro activity of the lipopeptide PAL-Lys-Lys-NH2, alone and in combination with antifungal agents, against clinical isolates of Candida spp

Candida albicans is known to be the organism most often associated with serious fungal infection, but other Candida spp. are emerging as clinical pathogens associated with opportunistic infections. Among antimycotic treatments, increasing attention is currently given to anti-infective drugs based upon naturally occurring peptides, such as the short lipopeptide palmitoyl PAL-Lys-Lys-NH2 (PAL). The aim of this study is to evaluate the activity of this peptide compared to the traditional antifungal agents Fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) on Candida spp. 24 clinical isolates of Candida spp. were tested against PAL_, FLU, AMB and CAS using in vitro susceptibility tests, time killing and checkerboard assay. All of the drugs studied showed good activity against clinical isolates of candida; in particular CAS and AMB which have MICs value lower than PAL and FLU. Moreover we observed synergistic interactions for PAL/FLU (81.25%), PAL/AMB (75%) and particularly for PAL/CAS (87.5). We think that our results are interesting since synergy between PAL and CAS might be useful in clinic trails to treat invasive fungal infections.     

Voriconazole, Combined with Amphotericin B, in the Treatment for Pulmonary Cryptococcosis Caused by C. neoformans (Serotype A) in Mice with Severe Combined Immunodeficiency (SCID)

Cryptococcosis is a subacute or chronic systemic mycosis with a cosmopolitan nature, caused by yeast of the genus Cryptococcus neoformans. The model of systemic cryptococcosis in mice with severe combined immunodeficiency (SCID) is useful for immunological and therapeutic study of the disease in immunodeficient hosts. Amphotericin B, fluconazole and flucytosine are the drugs most commonly used to treat cryptococcosis. Voriconazole is a triazole with high bioavailability, large distribution volume, and excellent penetration of the central nervous system. The objective of this study was to evaluate treatment with amphotericin B (AMB), voriconazole (VRC), and AMB, used in combination with VRC, of experimental pulmonary cryptococcosis in a murine model (SCID). The animals were inoculated intravenously (iv) with a solution containing 3.0 × 10(5) viable cells of C. neoformans ATCC 90112, (serotype A). Treatments were performed with amphotericin B (1.5 mg/kg/day), voriconazole (40.0 mg/kg/day) and AMB (1.5 mg/kg/day) combined with VRC (40.0 mg/kg/day); began 1 day after the initial infection; were daily; and lasted 15 days. Evaluations were performed using analysis of the survival curve and isolation of yeast in the lung tissue. There was a significant increase in survival in groups treated with AMB combined with VRC, compared with the untreated group and groups receiving other treatments (P < 0.05). In the group treated only with VRC and AMB combined with VRC, there was a significant reduction (P < 0.05) in the isolation of C. neoformans in lung tissue. Amphotericin B combined with voriconazole may be an effective alternative to increasing survival and may reduce yeast in the lung tissue of mice with pulmonary cryptococcosis and SCID.     

Facilitated diffusion of glucosamine-6-phosphate synthase inhibitors enhances their antifungal activity

N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) and 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) are strong inhibitors of the essential fungal enzyme, glucosamine-6-phosphate synthase, but their antifungal activity is poor, due to slow penetration of these agents through the cytoplasmic membrane. In the present studies we have exploited the possibility of enhancement of ADGP and FMDP antifungal activity by improving their transport properties. It has been found that membrane-permeabilising polyene macrolides amphotericin B (AMB) and its N-methyl-N-fructosyl methyl ester derivative (MF-AME), at subinhibitory concentrations, facilitate diffusion of ADGP through the fungal cell membrane, thus allowing a decrease of its minimal inhibitory concentration (MIC). Synergistic effects have been observed for combinations of ADGP with AMB or MF-AME. Fractional inhibitory concentration (FIC) indexes, determined against a number of Candida spp., have been in the 0.18-0.81 range. Weak antifungal synergistic effects have been found for combinations of FMDP with AMB or MF-AME. ADGP can be easily encapsulated into unilamellar lipid vesicles. Liposomal preparations of ADGP demonstrated stronger antifungal activity against some fungal strains than free ADGP.     

Validating the in vitro antimicrobial activity of Artemisia afra in polyherbal combinations to treat respiratory infections

Artemisia afra is one of the most widely used medicinal plants in African traditional medicine and is commonly administered in polyherbal combinations to treat respiratory infections. Focussing on plant volatiles, the aim of this study was to provide scientific evidence for the antimicrobial activity of A. afra (principle plant) in combination with essential oils from three medicinal aromatic plants; Agathosma betulina, Eucalyptus globulus and Osmitopsis asteriscoides. In vitro minimum inhibitory concentration (MIC) assays were undertaken on four pathogens (Enterococcus faecalis ATCC 29212, Moraxella catarrhalis ATCC 23246, Klebsiella pneumoniae NCTC 9633 and Cryptococcus neoformans ATCC 90112) to determine antimicrobial efficacy of the oils and their combinations. The fractional inhibitory concentration (FIC) and isobolograms were used to interpret pharmacodynamic interactions such as synergy, antagonism or additive profiles. The antimicrobial activity of the individual oils mostly displayed moderate activity. Predominantly, additive interactions were noted. The most prominent synergistic interaction (FIC value of 0.5) was observed when A. afra was combined with O. asteriscoides in the 8:2 ratio (eight parts A. afra with two parts O. asteriscoides) against C. neoformans. No antagonistic interactions were evident.     

Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology

Cryptococcus neoformans is an encapsulated fungus that causes cryptococcosis. Central nervous system infection is the most common clinical presentation followed by pulmonary, skin and eye manifestations. Cryptococcosis is primarily treated with amphotericin B (AMB), fluconazole (FLC) and itraconazole (ITC). In the present work, we evaluated the in vitro effect of terbinafine (TRB), an antifungal not commonly used to treat cryptococcosis. We specifically examined the effects of TRB, either alone or in conjunction with AMB, FLC and ITC, on clinical C. neoformans isolates, including some isolates resistant to AMB and ITC. Broth microdilution assays showed that TRB was the most effective drug in vitro. Antifungal combinations demonstrated synergism of TRB with AMB, FLC and ITC. The drug concentrations used for the combination formulations were as much as 32 and 16-fold lower than the minimum inhibitory concentration (MIC) values of FLC and AMB alone, respectively. In addition, calcofluor white staining revealed the presence of true septa in hyphae structures that were generated after drug treatment. Ultrastructural analyses demonstrated several alterations in response to drug treatment, such as cell wall alterations, plasma membrane detachment, presence of several cytoplasmic vacuoles and mitochondrial swelling. Therefore, we believe that the use of TRB alone or in combination with AMB and azoles should be explored as an alternative treatment for cryptococcosis patients who do not respond to standard therapies.     

In Vitro Interaction of Itraconazole with Amphotericin B,Caspofungin, and Terbinafine Against Clinical Isolates of <Emphasis Type="Italic">Trichosporon asahii</Emphasis>

Treatment of disseminated trichosporosis is still challenging. Itraconazole is a widely used broad-spectrum antifungal drug. In vitro interactions of itraconazole (ICZ) with amphotericin B (AMB), caspofungin (CAS), and terbinafine (TBF) against 18 clinical isolates of Trichosporon asahii were assessed by chequerboard microdilution method. ICZ combined with CAS showed the highest percentage of synergistic effect (72.2%), followed by ICZ/AMB (11%) and ICZ/TBF (11%) combination. Antagonistic effect was not observed. This study demonstrates that itraconazole can enhance the antifungal activity of CAS against T. asahii, suggesting that this combination may be a potential strategy for treating disseminated trichosporosis.     

In Vitro Interactions of Amphotericin B Combined with Non-antifungal Agents Against Rhodotorula mucilaginosa Strains

Rhodotorula species are emerging as opportunistic pathogens, causing catheter-associated fungemia in patients with compromised immunity. R. mucilaginosa is considered the most common species involved in human infections. Correct identification and susceptibility testing of Rhodotorula isolates recovered from the blood stream or central nervous system are essential to determine the best management of this unusual infection. The antifungal susceptibility tests showed that Rhodotorula was susceptible to low concentrations of amphotericin B (AMB) but was less susceptible to voriconazole. Combinations of AMB plus several non-antifungal medications were evaluated against 35 susceptible (Rm AMB-S) and resistant (Rm AMB-R) clinical Rhodotorula isolates using the broth microdilution checkerboard technique. We showed that in vitro exposure to increasing concentrations of AMB changed the susceptibility profile to these strains, which were named the Rm AMB-R group. The most synergistic interactions were AMB?+?simvastatin, followed by AMB?+?amlodipine and AMB?+?warfarin. Synergism and antagonism were observed in both groups for the combination AMB?+?cyclosporine A. AMB combined with a fluoroquinolone (AMB?+?levofloxacin) also demonstrated antagonism for the Rm AMB-S strains, but a high percentage of synergistic interactions was observed for the Rm AMB-R group. A combination drug approach can provide a different strategy to treat infections caused by AMB-resistant R. mucilaginosa.

     

In vitro inhibitory activity of sertraline against clinical isolates of Sporothrix schenckii

BackgroundSertraline (SRT) is an antidepressant that has proven its activity in vitro against Cryptococcus, Coccidioides, Trichosporon and other fungi. Disseminated sporotrichosis, although rare, has a high mortality and its treatment is difficult and prolonged, often relying in combining two or more antifungals.AimsIn our study we evaluate the antifungal activity of SRT, alone and in combination with itraconazole (ITC), voriconazole (VRC) and amphotericin B (AMB), against 15 clinical isolates of Sporothrix schenckii.MethodsWe used the broth microdilution method as described by the CLSI to test the susceptibility to antifungals, and the checkerboard microdilution method to evaluate drug interactions.ResultsThe minimum inhibitory concentration (MIC) with SRT was in the range of 4–8 μg/ml, while for AMB, VRC and ITC were 0.5–4 μg/ml, 0.5–8 μg/ml and 0.125–2 μg/ml, respectively. In addition, SRT showed synergy with ITC in one strain, mainly additivity with VRC, and indifference with AMB in others.ConclusionsThe MIC values with SRT for the isolates studied show the potential role of this drug as an adjuvant in the treatment of sporotrichosis, especially in disseminated or complicated cases.     

Investigation into in vitro anti-leishmanial combinations of calcium channel blockers and current anti-leishmanial drugs

The need for drug combinations to treat visceral leishmaniasis (VL) arose because of resistance to antimonials, the toxicity of current treatments and the length of the course of therapy. Calcium channel blockers (CCBs) have shown anti-leishmanial activity; therefore their use in combination with standard drugs could provide new alternatives for the treatment of VL. In this work, in vitro isobolograms of Leishmania (Leishmania) chagasi using promastigotes or intracellular amastigotes were utilised to identify the interactions between five CCBs and the standard drugs pentamidine, amphotericin B and glucantime. The drug interactions were assessed with a fixed ratio isobologram method and the fractional inhibitory concentrations (FICs), sum of FICs (ΣFICs) and the overall mean ΣFIC were calculated for each combination. Graphical isobologram analysis showed that the combination of nimodipine and glucantime was the most promising in amastigotes with an overall mean ΣFIC value of 0.79. Interactions between CCBs and the anti-leishmanial drugs were classified as indifferent according to the overall mean ΣFIC and the isobologram graphic analysis.     

Comparative Analysis of the Vitek 2 Antifungal Susceptibility System and E-test with the CLSI M27-A3 Broth Microdilution Method for Susceptibility Testing of Indian Clinical Isolates of Cryptococcus neoformans

The emergence of antifungal resistance among Cryptococcus neoformans isolates is a matter of great concern. The Clinical and Laboratory Standards Institute (CLSI) broth microdilution reference method (BMD) for antifungal susceptibility testing of C. neoformans is tedious and time-consuming. Consequently, there is a greater need for a reproducible in vitro susceptibility testing method for use in clinical microbiology laboratories. By random amplified polymorphic DNA analysis, the 62 Indian clinical isolates were characterized as Cryptococcus neoformans var. grubii. We evaluated the susceptibilities of these isolates for amphotericin B (AMB) and fluconazole (FLC) by two commercial techniques, i.e., Vitek 2 and E-test against the CLSI M27-A3 BMD. The essential agreement (EA) between the Vitek 2 and E-test with the reference procedure for FLC was similar (82.2%). For AMB, EA of 92 and 76% was obtained with E-test and Vitek 2. Excellent categorical agreement (CA) (98.3% and 100% by Vitek 2 and E-test, respectively) was obtained for AMB. The CA for FLC was 81 and 77.4% by Vitek 2 and E-test. We conclude that both E-test and Vitek 2 system have acceptable levels of accuracy for susceptibility testing of both the drugs. Both of them could identify fluconazole-resistant strains. Vitek 2 could be used for testing susceptibility of voriconazole and 5-flucytosine also at the same time.     

Phospholipase D2 mediates signaling by ATPase class I type 8B membrane 1

Functional defects in ATPase class I type 8B membrane 1 (ATP8B1 or familial intrahepatic cholestasis 1, FIC1) lead to cholestasis by mechanism(s) that are not fully understood. One proposed pathophysiology involves aberrant signaling to the bile acid sensor, the farnesoid X receptor (FXR), via protein kinase C ζ (PKCζ). The following cell line-based studies investigated whether phospholipase D2 may transduce a signal from FIC1 to FXR. PLD2 gain of function led to activation of the bile salt export pump (BSEP) promoter, a well-characterized FXR response. BSEP activation by PLD2 could be blocked by abrogating either PKCζ or FXR signaling. PLD2 loss of function led to a reduction in BSEP promoter activity. In addition, a variety of proteins that are activated by FXR, including BSEP, were reduced in HepG2 cells treated with PLD2 siRNA. Similar effects were observed in freshly isolated human hepatocytes. Activation of BSEP by FIC1 gain of function was blocked when PLD2 but not PLD1 was silenced. Overexpression of wild-type but not Byler mutant FIC1 led to an increase in membrane associated PLD activity. An intermediate level of activation of PLD activity was induced when a benign recurrent intrahepatic cholestasis FIC1 mutant construct was expressed. These studies show that FIC1 signals to FXR via a signaling pathway including PLD2 and PKCζ.     

Activities of therapeutic agents against Naegleria fowleri in vitro and in a mouse model of primary amebic meningoencephalitis

Inhalation of water contaminated with Naegleria fowleri may lead to a potentially fatal infection of the central nervous system known as primary amebic meningoencephalitis (PAM). Amphotericin B (AMB), an antifungal drug, is the only agent with established clinical efficacy in the treatment of PAM, though therapy with this drug is not always effective and has been associated with adverse effects on the kidneys and other organs. We investigated the activity of various therapeutic agents against N. fowleri in an attempt to identify other useful agents for treating PAM. Several of these agents exhibited in vitro activity against the Lee (M67) strain of N. fowleri. The minimum inhibitory concentrations of these agents were 0.1 microg/ml (ketoconazole), 1 microg/ml (liposomal AMB), and 10 microg/ml (minocycline, quinupristin-dalfopristin, and trifluoperazine). Other agents had a minimum inhibitory concentration > 10 microg/ml (linezolid) or > 100 microg/ml (rifampin). In a mouse model of PAM, none of the untreated control mice survived, whereas the survival of treated animals was 50% (quinupristin-dalfopristin), 30% (ketoconazole and liposomal AMB), 20% (trifluoperazine), and 10% (linezolid and minocycline). Further studies are needed to ascertain whether these agents have synergistic activity with AMB in vitro and in vivo.     

Induction of phenylalanine ammonia-lyase and increase in phenolics in lettuce leaves in relation to the development of russet spotting caused by ethylene

Russet spotting (RS), consisting of numerous small brown spots on the midrib of head lettuce (Lactuca sativa), is a physiological disorder induced by exposure to ethylene. In leaves suffering RS, the increase in spotting was accompanied by a parallel increase in the amount of phenolic compounds. Of these, chlorogenic acid and isochlorogenic acid were identified. Ethylene induced high phenylalanine ammonia-lyase (PAL) activity and RS formation in the susceptible cultivar Salinas, but not in the resistant cultivar Calmar. In the absence of ethylene neither significant PAL induction nor RS occurred. No correlation was found between the increase in polyphenol oxidase or peroxidase and the development of RS. The increase in PAL activity, however, was closely correlated with the development of RS. The increase in PAL activity preceded the development of RS, and the extent of RS was directly related to the level of PAL. Three temperatures (0.5, 5.5, and 12.5 C) were compared on the basis of their influence on both RS and PAL induction. At the lowest temperature (0.5 C) neither PAL induction nor RS occurred to a significant extent. At the highest temperature (12.5 C) an initial rapid increase in PAL activity and an earlier development of spotting were observed, but subsequently there was a decrease in both PAL activity and the rate of development of RS. At the medium temperature (5.5 C) both PAL activity and RS increased progresively with time. The decline of PAL activity at a higher temperature might be attributed to inactivation of the enzyme. Thus, a temperature favorable for induction of PAL activity by ethylene was also favorable for RS. These observations indicate a close interrelationship between the induction of PAL activity and the development of RS in response to ethylene, and suggest a causal relationship between the two events. PAL serves as a useful biochemical marker for the RS reaction.     

Effects of glyphosate on phenolic metabolism in yellow nutsedge leaves

The action of the herbicide glyphosate [N-(phosphonomethyl)-glycine] on phenolic metabolism and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity was investigated in yellow nutsedge (Cyperus esculentus L.). Glyphosate caused significant increases in the amount of total soluble hydroxyphenolics in the three fractions studied (neutral, acid and residual). Qualitative and quantitative differences in relation to these fractions and the amount of applied glyphosate were observed. Most of the phenolic compounds which increased after glyphosate treatment were benzoic acids (gentisic. p -OH-benzoic, salicylic and vanillic). Gentisic acid showed the greatest increase in neutral and acid fractions, being twenty- and four-fold, respectively, of the amount found in the control. PAL activity was not affected by the lowest doses of glyphosate (10−⁴and 10−³ M) , but a dramatic decrease in PAL activity was observed after 10−² M treatment. These findings, together with the low levels of cinnamic acids measured in treated yellow nutsedge plants, suggest that PAL activity is only marginally involved in glyphosate action. Since the herbicidal action probably takes place at 5-enol-pyruvylshikimate-3-P synthase (EC 2.5.1.19), an alternative pathway to PAL in phenolic biosynthesis should be activated yielding benzoic acids.     

Induction of phenylalanine ammonia-lyase (PAL) in germinating lettuce seeds (Lactuca sativa)

Dry lettuce seeds (achenes of Lactuca sativa L. cv. Grand Rapids) contain no detectable phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity. Enzyme activity could be detected in these seeds within 4 h of imbibition under white light. The specific activity of PAL increased rapidly during the next 12–16 h of imbibition. Far-red light completely suppressed germination as well as the development of PAL. Gibberellic acid (GA₃, 0.1 m M ), although effective in causing almost 100% germination in dark, did not induce proportionate increases in PAL. Seed germination as well as PAL activity were substantially inhibited by cis -4-cyclohexene-l, 2-dicarboximide (CHDC, 1.0 m M ) both in light and dark. Both GA₃ and benzyladenine (BA, 0.1 m M ) retarded radicle elongation in light. Concomitantly, a decrease in PAL activity was observed. Benzyladenine was able to reverse the effects of CHDC on germination but PAL activity was still highly reduced, probably due to the inhibitory effects of BA on elongation of the radicle. More than 95% of the extractable PAL was found to be present in the radicle. When seeds incubated in white light for 10 h were transferred to FR, further increases in PAL activity as well as the growth of the radicle were severely inhibited. It is suggested that the induction of PAL in light-sensitive lettuce seeds is coincidental with the germination of seeds, and the amount of PAL per germinated seed is related to the extent of elongation of the embryonic axes.     

Enzyme activity and shikonin production in Lithospermum erythrorhizon cell cultures

The activities of the biosynthetic enzmes phenylalanine ammonia lyase (PAL) and 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) were measured in cells transferred from growth to production medium in a two-stage batch culture. It was found that both these enzymes showed transient increases, PAL (three- to fourfold) and HMGR (two- to four-fold), at or near the point of exhaustion of nitrogen source (NO(3)). Production of shikonin derivatives also started at this time. The addition of excess nitrate to the medium shortly before nitrate exhaustion (days 6-8) markedly reduced the final product yield (by 70-80%) while addition of excess nitrate in the later stationary growth phase (days 14-16) had no significant effect. When the production rate of shikonin derivatives was correlated with PAL activity, it was observed that production rate is very low (less than 1 mg/L . day) at low levels of PAL activity (below 0.1 unit/mg protein). Once a threshold level of PAL activity (about 0.15 unit/mg protein) is reached, the biosynthetic rate of shikonin derivatives increases. Such a relationship could not be deduced for HMGR activity. It was concluded that the production of shikonin derivatives may be limited at the phenylalanine deaminating step at low levels of PAL activity.     

In Vitro Synergistic Activities of Essential Oils and Surfactants in Combination with Cosmetic Preservatives Against Pseudomonas aeruginosa and Staphylococcus aureus

The aim of this study is to evaluate possible synergistic antimicrobial interactions between common cosmetic preservatives and selected essential oils or surfactants. The antimicrobial efficacy of six essential oils, three surfactants and five preservatives against Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 43387 was assessed by a broth micro-dilution assay. MICs for individual and combined antimicrobials were determined and then transformed to fractional inhibitory concentration (FIC) indexes. All essential oils exhibited antibacterial activity; among surfactants, bacteria resulted most susceptible to the cationic agent. Synergy was observed when essential oils of eucalyptus and mint were combined with methylparaben against P. aeruginosa, while essential oils of mint, oregano and sage combined with propylparaben and imidazolidinyl urea acted against S. aureus. Many binary mixtures of preservatives and surfactants produced synergistic activity with the most effective interactions involving the cationic and amphoteric compounds under study. FIC indexes demonstrated synergistic effects when preservatives were combined with either essential oils or surfactants against both bacterial strains. These results highlight the potential usefulness of essential oils and surfactants to enhance the activities of conventional biocides. This kind of study should contribute to the selection and optimization of preservative systems for cosmetic preparations.     

Identification by high-performance liquid chromatography of tyrosine ammonia-lyase activity in purified fractions of Phaseolus vulgaris phenylalanine ammonia-lyase

Activities of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) were assessed at each stage of a three-step purification of PAL. Assays were performed by high-performance liquid chromatographic (HPLC) separation and ultraviolet detection of reaction products. Use of HPLC permitted assay of low activities of PAL and TAL for periods up to approximately four and two days, respectively. HPLC also facilitated the accurate quantitation of the product of the TAL reaction, trans-p-coumaric acid, which was observed to isomerize readily under experimental conditions. PAL and TAL were associated throughout the purification procedure, with TAL activity at 0.6–1.3% of PAL activity. It was concluded that, contrary to previous reports, TAL and PAL activities are mediated by the same enzyme, or else by chromatographically very similar enzymes.     

In Vitro Activity of Berberine Alone and in Combination with Antifungal Drugs Against Planktonic Forms and Biofilms of <Emphasis Type="Italic">Trichosporon Asahii</Emphasis>

Trichosporon asahii (T. asahii) is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients, with high mortality rates up to 80% despite treated with antifungal drugs. The biofilms-forming ability of T. asahii on indwelling medical devices may account for the resistance to antifungal drugs. Berberine (BBR) has been demonstrated to have antifungal activity and synergistic effects in combination with antifungal drugs against pathogenic fungi. In the present study, the in vitro activities of BBR alone or combined with fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), caspofungin (CAS) and amphotericin B (AMB) against planktonic forms and biofilms of 21 clinical T. asahii isolates were evaluated using checkerboard microdilution method and XTT reduction assay, respectively. The fractional inhibitory concentration index (FICI) was used to interpret drug interactions. BBR alone did not exhibit significant antifungal activities against both T. asahii planktonic cells (MICs, 32 → 128 μg/ml) and T. asahii biofilms (SMICs, >128 μg/ml). However, BBR exhibited synergistic effects against T. asahii planktonic cells in combination with AMB, FLC and CAS (FICI ≤ 0.5) and exhibited synergistic effects against T. asahii biofilms in combination with AMB and CAS (FICI ≤ 0.5). BBR/ITC and BBR/VRC combinations yielded mainly indifferent interactions against T. asahii planktonic cells. BBR/FLC, BBR/ITC and BBR/VRC combinations also yielded indifferent interactions against T. asahii biofilms. Our study highlights the therapeutic potential of BBR to be used as an antifungal synergist in combination with antifungal drugs against T. asahii infections, especially BBR/AMB combination. Further in vivo studies are needed to validate our findings.     

Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells

Pathogenic yeast Cryptococcus neoformans causes devastating cryptococcal meningoencephalitis. Our previous studies demonstrated that C. neoformans hyaluronic acid was required for invasion into human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In this report, we demonstrate that C. neoformans hyaluronic acid interacts with CD44 on HBMEC. Our results suggest that HBMEC CD44 is a primary receptor during C. neoformans infection, based on the following observations. First, anti-CD44 neutralizing antibody treatment was able to significantly reduce C. neoformans association with HBMEC. Second, C. neoformans association was considerably impaired using either CD44-knock-down HBMEC or C. neoformans hyaluronic acid-deficient strains. Third, overexpression of CD44 in HBMEC increased their association activity towards C. neoformans. Fourth, confocal microscopic images showed that CD44 was enriched at and around the C. neoformans association sites. Fifth, upon C. neoformans and HBMEC engagement, a subpopulation of CD44 and actin translocated to the host membrane rafts. Our results highlight the interactions between C. neoformans hyaluronic acid and host CD44 and the dynamic results of these interactions, which may represent events during the adhesion and entry of C. neoformans at HBMEC membrane rafts.