Influence of Molecular Sizes of Cryptococcus neoformans Capsular Polysaccharide on Phagocytosis

The role of capsular polysaccharides (CPS) of Cryptococcus neoformans in phagocytosis by murine alveolar macrophages was investigated in four strains of C. neoformans serotype A, YC-11, YC-5, YC-27 and YC-13. Phagocytosis rates increased markedly after adding 10% mouse serum, compared to fetal calf serum. The reverse relation between capsular thickness of C. neoformans and phagocytosis by alveolar macrophages was observed except in YC-27, which had thin capsules and high virulence. The phagocytosis rate in mice serum was 17.3% in YC-11 (capsule thickness 2.8-3.5 μm), 39.8% in YC-5 (capsule size 0.8-1.5 μm), 20.3% in YC-27 (capsule size 0.6-1.1 μm), and 62.8% in YC-13 (capsule not detected microscopically). The CPS of YC-11, YC-5, and YC-27 analyzed by gel-filtration using CL-2B showed high molecular fractions near the void volume. However, the CPS of YC-13 showed only low molecular fractions. The widely eluted CPS of YC-11 was separated into 3 fractions and each fraction was added in the phagocytosis assay of YC-13. Phagocytosis was markedly suppressed particularly by the addition of a higher molecular fraction. These results suggest that phagocytosis of C. neoformans by alveolar macrophages is influenced by the molecular sizes of the CPS.     

Un vivo depletion of murine CD8 positive T cells impairs survival during infection with a highly virulent strain ofCryptococcus neoformans

Cell-mediated immunity plays an important but incompletely understood role in host defense againstCryptococcus neoformans. Because of their multiple capacities as cytokine-secreting cells, cytotoxic cells, and antigen-specific suppressor cells, CD8 positive T lymphocytes could potentially either enhance or impair host defense againstC. neoformans. To determine whether CD8 T cells enhance or inhibit host defence during an infection with a highly virulent strain ofC. neoformans, we examined the effect of in vivo CD8 cell depletion on suNival and on the number of organisms in mice infected by either the intratracheal or intravenous routes. Adequacy of depletion was confirmed both phenotypically and functionally. Regardless of the route of infection, we found that survival of mice depleted of CD8 T cells was significantly reduced compared to undepleted mice. Surprisingly, however, CD8 depletion did not alter organism burden measured by quantitative CFU assay in mice infected by either route. These data demonstrate that CD8 positive T cells participate in the immune response to a highly virulent strain ofC. neoformans. By contrast to minimally virulent isolates that do not cause a life threatening infection, the immune response to a highly virulent isolate does not alter the burden of organisms, but does enhance host defense as it is necessary for the optimal survival of infected mice.Abbreviations ³H-TdR ³H-thmidine - CFU colony forming units - FITC Fluorescein isothiocyanate - MLR mixed lymphocyte reaction - PBS phosphate buffered saline     

In vivo and in vitro studies with an atypical,rhinotropic isolate of Cryptococcus neoformans

An atypical isolate of Cryptococcus neoformans was investigated because of its consistent and reproducible production of gross nasal pathology following i.v. injection in Swiss albino mice. Dose response to graded concentrations ranging from 1×l0²–1×l0⁷ cells/mouse yielded an LD₅₀ of 1.4×10³ cells/mouse for the atypical rhinotropic strain H140 which was significantly less virulent (p<0.01) than our reference strain of Cryptococcus neoformans. There was no significant difference in mortality following the injection of in vitro vs. in vivo passed inoculum. As early as two weeks after inoculation, this strain produced gross nasal enlargement to approximately 2–3 × normal dimensions with granulomatous and ulcerated lesions. The LD₆₀ resulted in the greatest percentage of nasal involvement (85%). C. neoformans was demonstrated by culture and histopathology in the noses, brains, lungs, livers and kidneys. A temperature selection was indicated by findings of a lower temperature minimum for subcultures isolated from the noses relative to those isolated from the brain, and by the fact that the most densely populated organs following intraperitoneal injection were the testes. This route of inoculation resulted in cutaneous nasal involvement in a manner analogous to that following i.v. injection. The atypical isolate was unable to assimilate trehalose or raffinose but otherwise was entirely consistent with identification as C. neoformans and produced characteristic CNS and general organ system disease in addition to the rhinotropic cutaneous manifestations. The model characterized here in normal mice may be of value in studies of fungal dermotropism.     

Interleukin-10 but not Transforming Growth Factor beta inhibits murine activated macrophages Paracoccidioides brasiliensis killing: Effect on H2O2 and NO production

Paracoccidioidomycosis is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis (P. brasiliensis). Most often, this mycosis runs as a chronic progressive course affecting preferentially the lungs. In vitro fungicidal activity against a high virulent strain of P. brasiliensis by murine peritoneal macrophages preactivated with IFN-γ or TNF-α is high and correlates with increased NO and H₂O₂ production. Within this context, the purpose of this work was to study the role of suppressor cytokines, such as IL-10 and TGF-β, in this process. Incubation of either IFN-γ or TNF-α with IL-10 inhibits fungicidal activity of these cells. However, TGF-β had no effect on fungicidal activity of IFN-γ or TNF-α-activated macrophages. The suppression of fungicidal activity by IL-10 correlated with the inhibition of NO and H₂O₂ production supporting the involvement of these metabolites in P. brasiliensis killing. These results suggest that IL-10 production in vivo could represent an evasion mechanism of the fungus to avoid host immune response.     

Galleria mellonella Model Identifies Highly Virulent Strains among All Major Molecular Types of Cryptococcus gattii

Cryptococcosis is mainly caused by Cryptococcus neoformans. However, the number of cases due to C. gattii is increasing, affecting mainly immunocompetent hosts. C. gattii is divided into four major molecular types, VGI to VGIV, which differ in their host range, epidemiology, antifungal susceptibility and geographic distribution. Besides studies on the Vancouver Island outbreak strains, which showed that the subtype VGIIa is highly virulent compared to the subtype VGIIb, little is known about the virulence of the other major molecular types. To elucidate the virulence potential of the major molecular types of C. gattii, Galleria mellonella larvae were inoculated with ten globally selected strains per molecular type. Survival rates were recorded and known virulence factors were studied. One VGII, one VGIII and one VGIV strain were more virulent (p <0.05) than the highly virulent Vancouver Island outbreak strain VGIIa (CDCR265), 11 (four VGI, two VGII, four VGIII and one VGIV) had similar virulence (p >0.05), 21 (five VGI, five VGII, four VGIII and seven VGIV) were less virulent (p <0.05) while one strain of each molecular type were avirulent. Cell and capsule size of all strains increased markedly during larvae infection (p <0.001). No differences in growth rate at 37°C were observed. Melanin synthesis was directly related with the level of virulence: more virulent strains produced more melanin than less virulent strains (p <0.05). The results indicate that all C. gattii major molecular types exhibit a range of virulence, with some strains having the potential to be more virulent. The study highlights the necessity to further investigate the genetic background of more and less virulent strains in order to recognize critical features, other than the known virulence factors (capsule, melanin and growth at mammalian body temperature), that maybe crucial for the development and progression of cryptococcosis.     

小檗碱抗新生隐球菌活性和作用机制

【目的】研究小檗碱对新生隐球菌的抗菌活性及其作用机制。【方法】采用微量肉汤稀释法测定小檗碱对新生隐球菌标准菌株和临床分离菌株的最小抑菌浓度,通过棋盘法测定小檗碱与氟康唑、两性霉素B的协同作用,测定小檗碱对隐球菌重要毒力因子的表达,以及对巨噬细胞和隐球菌互作的影响,采用隐球菌感染大蜡螟模型测定小檗碱的体内杀菌活性。【结果】小檗碱是一种杀真菌化合物,在测试的菌株中,最小抑菌浓度(minimum inhibitory concentration, MIC)范围为8-16μg/mL。亚致死剂量小檗碱能够抑制隐球菌荚膜大小、产黑色素能力和有性生殖能力,并能增强巨噬细胞的杀菌能力。锌指转录因子Nrg1介导了上述重要的过程。在隐球菌感染动物模型中,小檗碱能够延长感染大蜡螟的存活时间。【结论】小檗碱在体内外具有优异的抗隐球菌活性,有望作为抗隐球菌药物开发的起始化合物。     

Comparative profiles of intramacrophage behavior of Mycobacterium tuberculosis and Mycobacterium avium complex with different levels of virulence

Mycobacterium tuberculosis (MTB) and M. avium complex (MAC) strains with different levels of virulence in mice were examined for profiles of interaction with murine peritoneal macrophages (Mphis). Their growth rates in Mphis were in these orders: H37Ra strain (attenuated) > H37Rv strain (virulent) for MTB, and N-260 strain (moderate virulence) > MAC N-444 strain (low virulence) for MAC. MTB but not MAC caused the necrotic death of host Mphis in terms of increased release of lactate dehydrogenase from infected Mphis. The MTB H37Ra strain induced a greater production of reactive nitrogen intermediates (RNI) by Mphis than the MTB H37Rv strain did. However, this phenomenon was not observed with MAC, implying less important roles of RNI in the expression of Mphi antimicrobial activity against MAC organisms.     

Cryptococcus neoformans: A central nervous system isolate from an AIDS patient that is rhinotropic in a normal mouse model

A strain of Cryptococcus neoformans that was isolated from the cerebrospinal fluid of a human diagnosed as having acquired immunodeficiency syndrome (AIDS), and that produced cutaneous lesions in experimentally infected, normal mice is described. Although no unusual cutaneous manifestations were noted in the patient's records, this isolate of C. neoformans proved to be dermotropic when injected intravenously into CD-1 mice. The LD₅₀ at 28 days post infection ranged from 3.6–7.5×10⁵ cells per mouse, and in vitro growth rate studies demonstrated that this isolate grew well at 35 °C and at 37 °C, but did not grow at 40 °C and higher. This isolate was rhinotropic producing large granulomatous lesions in the nasal tissues. Other cutaneous tissues affected were the periocular tissues, ears, feet and tail, although the granulomas were nodular in structure and less necrotic than the nasal lesions. The brain, lungs, liver, kidneys and spleen also were culture positive for C. neoformans. Histopathologically, each affected tissue examined had large densities of yeast cells and a chronic, granulomatous host response. Animals surviving the infection appeared to develop a commensal-type relationship with the infective yeast. This is the first report of an isolate of C. neoformans from an AIDS patient that has caused cutaneous manifestations in an animal model. The model described in this report may be useful for elucidating pathogenic mechanisms of cryptococcosis, particularly cutaneous manifestations of the disease.     

Pathogenesis of Cryptococcus neoformans is Associated with Quantitative Differences in Multiple Virulence Factors

Two isolates of Cryptococcus neoformans were previously described as being highly divergent in their level of capsule synthesis in vivo and in their virulence for mice. The highly virulent isolate (NU-2) produced more capsule than a weakly virulent isolate (184A) in vitro under tissue culture conditions and in vivo. This investigation was done to determine if there were differences between the two isolates in other factors that might also contribute to virulence. Growth rate was not a factor as NU-2 grew more slowly than 184A. Based on PCR fingerprinting the two isolates were genetically different providing an opportunity to examine differences in multiple virulence traits. Quantitative analysis revealed that NU-2 expressed significantly more melanin and mannitol than did 184A. Although the isolates expressed the same capsular chemotype, NU-2 produced an additional structure reporter group (SRG)under tissue culture conditions that was not present when grown in glucose salts/urea/basal medium (GSU).Capsular polysaccharide SRGs of 184A were unaffected by shifting the growth conditions from GSU to tissue culture conditions. Our results suggest that pathogenesis of a C. neoformans strain is dictated by the quantitative expression of the strain's combined virulence traits. Regulators of the expression of these genes may be playing key roles in virulence.This revised version was published online in October 2005 with corrections to the Cover Date.     

Quantitative evaluation of cryptococcal pathogenesis and antifungal drugs using a silkworm infection model with Cryptococcus neoformans

Aims: To develop an in vivo system that could quantitatively evaluate the therapeutic effects of antifungal drugs using a silkworm infection model with Cryptococcus neoformans. Methods and Results: Silkworms reared at 37°C died after an injection of viable serotype A C. neoformans fungus into the haemolymph. The serotype A C. neoformans, which is known to have higher mammal pathogenicity than the serotype D, was also more virulent against the silkworm. Furthermore, the deletion mutants of genes gpa1, pka1 and cna1, which are genes known to be necessary for the pathogenesis in mammals, showed an increase in the number of fungal cells necessary to kill half of the silkworm population (LD₅₀ value). Antifungal drugs, amphotericin B, flucytosine, fluconazole and ketoconazole, showed therapeutic effects in silkworms infected with C. neoformans. However, amphotericin B was not therapeutically effective when injected into the silkworm intestine, comparable to the fact that amphotericin B is not absorbed by the intestine in mammals. Conclusions: The silkworm–C. neoformans infection model is useful for evaluating the therapeutic effects of antifungal drugs. Significance and Impact of the Study: The silkworm infection model has various advantages for screening antifungal drug candidates. We can also elucidate the cryptococcal pathogenesis and evaluate the in vivo pharmacokinetics and toxicity of each drug.     

Studies on organ specificity in experimental murine cryptococcosis

The chief histopathological features found in patients with cryptococcosis are both a cystic (gelatinous) lesion and a granulomatous reaction. These two tissue reactions are definitely different from each other, because a cyst is not accompanied with a significant cellular response, while a granuloma is formed as a result of various cell reactions. Therefore, it is very interesting that these two types of lesion can be observed in the same patient or in the same animal infected with Cryptococcus neoformans. From our previous paper (II) the authors reach such a thought that two steps may be required for the granuloma formation against C. neoformans infection: first, of phagocytosis by sessile macrophages of C. neoformans and second is related to T-cell function. This experiment was done to verify that the granulomatous response against C. neoformans infection might occur easily in the organs rich in sessile macrophages as compared with those poor in them and a polysaccharide capsule surrounding cryptococci may have effects to inhibit a migration of polymorphonuclear leucocytes or monocytes toward C. neoformans. C. neoformans strain RIB 12 (serological type A, mating type α) was used in this experiment. After a culture of a brain heart infusion glucose agar slant at 37 C for 3 days, yeast cells of the strain were harvested, and suspended in 1/15 M(p_H7.4) sterile phosphate buffered saline solution. Infective inoculum was prepared by adjusting the number of the yeast cells to 10⁵, 10⁶ or 5×10⁶/0.2 ml in a hemacytometer. Fourty-two male mice strain ddY were divided into 3 groups consisting of 14 each and one group was allotted to one of the cell suspensions. Each mouse was inoculated with 0.2 ml of the cell suspension into a tail vein and one mouse from each group was sacrificed at adequate intervals. At necropsies the brain, thymus, lungs, heart, liver, kidneys, spleen, pancreas, mesenteric lymph nodes, a part of the small intestine, testes and fat tissue were removed. From these organs histopathological sections, stained with HE or by PAS, were prepared. To investigate effects of a polysaccharide capsule to a migration of polymorphonuclear leucocytes or monocytes, double infections with C. neoformans and Aspergillus fumigatus, and an observation by the ‘Agar-Implantation method’ were done. As results, granulomata were formed easily in the organs rich in macrophages or lymphocytes such as the liver, spleen, lymph nodes, thymus, lungs, small intestine and fat tissue. On the contrary, in organs poor in the macrophages such as the brain, heart, pancreas, kidneys, adrenal glands and testes, the chief histopathological feature was a cyst formation containing numerous yeast cells. In the double infection, two types of lesions such as cysts and abscesses were observed in the sections of the brain. The former occurred against C. neoformans infection and the latter, against A. fumigatus infection. Even though a cyst was very close to an abscess, polymorphonuclear leucocytes or monocytes were never induced to C. neoformans. In the observation using the ‘Agar-Implantation method’, a severe cellular infiltration occurred to a perfect (teleomorphic) state of C. neoformans and very weak response, to yeast cells with a polysaccharide capsule. The difference may be due to the existence of the capsule, because a perfect state of C. neoformans is not surrounded by it.     

Cryptococcus neoformans Hyperfilamentous Strain Is Hypervirulent in a Murine Model of Cryptococcal Meningoencephalitis

Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated.     

A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals

The pathogenic yeast Cryptococcus neoformans causes cryptococcosis, a life-threatening fungal disease. C. neoformans has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of C. neoformans strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as HVA1 (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of HVA1 was negatively correlated with virulence and was reduced in vitro and in vivo in both mouse- and Galleria-passaged strains of C. neoformans. Phenotypic analysis in hva1Δ and hva1Δ+HVA1 strains revealed no significant differences in established virulence factors. Mice infected intravenously with the hva1Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the hva1Δ+HVA1 strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the hva1Δ strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism.     

Amphotericin B synergy testing by the FCST

The flow cytofluorometric susceptibility test (FCST) was incorporated into two in vitro synergy assays of amphotericin B-drug combinations. The FCST checkerboard assay and the FCST concentration-effect assay were developed to detect subtle modulation of amphotericin B fungicidal effects onCandida albicans andCryptococcus neoformans. Amphotericin B × cyclosporine A was a synergistic fungicidal combination against bothC. albicans andC. neoformans. Amphotericin B×gentamicin and amphotericin B×ketoconazole were synergistic combinations againstC. neoformans. In all cases tested, the synergy was effective when 0.50–0.62 g amphotericin B/ml was used with>-0.50 g of the other drug/ml. The fungicidal effect of 1.00 g amphotericin B/ml overwhelmed the synergistic effects. A number of other drug combinations were additive, autonomous, or antagonistic.     

苏北地区规模化猪场产肠毒素大肠杆菌的分离鉴定及灭活疫苗效果评估

[目的]产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起仔猪腹泻的重要病原菌,本研究通过调查苏北地区规模化猪场ETEC的流行情况,分析其生物学特性,研制具有免疫保护效果的优势血清型菌株的灭活疫苗,以期对苏北地区ETEC的防控提供参考。[方法]从苏北地区规模化猪场采集3-30日龄的仔猪新鲜粪样、肛拭子及小肠组织样,分离出ETEC,对分离菌株进行血清型鉴定、耐药性测定、小鼠致病力测定;最后通过动物免疫试验研究优势血清型菌株灭活疫苗对小鼠的免疫保护效果。[结果]从21个规模化猪场采集病料562份,通过PCR鉴定及测序得到141株ETEC;血清凝集试验鉴定出85株菌的O抗原血清型,其中08、0101和0128为优势血清型,占定型菌株的61.2%(52/85),其他血清型包括09、03、020、0148、0149等;分析141株ETEC对14种常见抗生素的耐药情况,得出分离株对新霉素、红霉素、四环素、庆大霉素、强力霉素、阿莫西林、甲氧苄啶/磺胺甲恶唑高度耐药,耐药率均高达80%以上;对恩诺沙星敏感性较高,敏感率达50.4%(71/141);对多粘菌素B和头孢噻肟中介耐药,占比分别为66%(93/141)和51.8%(73/141);多重耐药现象严重,其中10重耐药的菌株占比最大,为19%(27/141);小鼠攻毒试验测得08血清型强毒株YC-6的半数致死量(median lethal dose,LD50)为1.4×10^7 CFU/只,最低致死量(minimum lethal dose,MLD)为3×10^7 CFU/只;08血清型强毒株YC-6和0101血清型强毒株LYG-3制备的单价灭活疫苗对小鼠的保护率均达到100%,因此利用08血清型强毒株YC-6和0101血清型强毒株LYG-3研制二价灭活疫苗,结果显示该二价疫苗对感染不同血清型ETEC小鼠的保护率在83%以上。[结论]本研究通过对苏北地区ETEC的流行病学调查,得出其优势血清型,并研制出针对对优势血清型免疫保护效果较好的二价灭活疫苗,给临床ETEC的监测和防控提供参考。     

Enhanced activity of antifungal drugs using natural phenolics against yeast strains of Candida and Cryptococcus

Aims: Determine whether certain, natural phenolic compounds enhance activity of commercial antifungal drugs against yeast strains of Candida and Cryptococcus neoformans. Methods and Results: Twelve natural phenolics were examined for fungicidal activity against nine reference strains of Candida and one of C. neoformans. Six compounds were selected for synergistic enhancement of antifungal drugs, amphotericin B (AMB), fluconazole (FLU) and itraconazole (ITR). Matrix assays of phenolic and drug combinations conducted against one reference strain, each, of Candida albicans and C. neoformans showed cinnamic and benzoic acids, thymol, and 2,3‐ and 2,5‐dihydroxybenzaldehydes (‐DBA) had synergistic interactions depending upon drug and yeast strain. 2,5‐DBA was synergistic with almost all drug and strain combinations. Thymol was synergistic with all drugs against Ca. albicans and with AMB in C. neoformans. Combinations of benzoic acid or thymol with ITR showed highest synergistic activity. Of 36 combinations of natural product and drug tested, none were antagonistic. Conclusions: Relatively nontoxic natural products can synergistically enhance antifungal drug activity, in vitro. Significance and Impact of the Study: This is a proof‐of‐concept, having clinical implications. Natural chemosensitizing agents could lower dosages needed for effective chemotherapy of invasive mycoses. Further studies against clinical yeast strains and use of animal models are warranted.     

Factors affecting experimental infection with Cryptococcus neoformans in mice with special reference to an endotoxic substance of C. neoformans

A close correlation was observed between body weight and length of the survival time of mice inoculated intravenously (i.v.) with Cryptococcus neoformans (p<0.001). An endotoxic substance of C. neoformans (Cr-ET) increased the susceptibility of mice to i.v. infection of C. neoformans only when more than 50 g of Cr-ET was injected i.v. 24 hours before infection. Intraperitoneal (i.p.) administration of dimethyl sulfoxide which is found to enhance bacterial infection did not enhance death rate of mice infected i.p. with C. neoformans.     

Heterologous expression of a gene of Magnaporthe oryzae chrysovirus 1 strain A disrupts growth of the human pathogenic fungus Cryptococcus neoformans

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1‐A) is the causal agent of growth repression and attenuated virulence (hypovirulence) of the rice blast fungus, M. oryzae. We have previously reported that heterologous expression of MoCV1‐A ORF4 in Saccharomyces cerevisiae results in growth defects, a large central vacuole and other cytological changes. In this study, the effects of open reading frame (ORF) 4 expression in Cryptococcus neoformans, a human pathogenic fungus responsible for severe opportunistic infection, were investigated. Cells expressing the ORF4 gene in C. neoformans showed remarkably enlarged vacuoles, nuclear diffusion and a reduced growth rate. In addition, expression of ORF4 apparently suppressed formation of the capsule that surrounds the entire cell wall, which is one of the most important components of expression of virulence. After 5‐fluoroorotic acid treatment of ORF4‐expressing cells to remove the plasmid carrying the ORF4 gene, the resultant plasmid‐free cells recovered normal morphology and growth, indicating that heterologous expression of the MoCV1‐A ORF4 gene induces negative effects in C. neoformans. These data suggest that the ORF4 product is a candidate for a pharmaceutical protein to control disease caused by C. neoformans.     

Combination Efficacy of Voriconazole and Amphotericin B in the Experimental Disease in Immunodeficient Mice Caused by Fluconazole-resistant <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

The therapeutic efficacy of amphotericin B and voriconazole alone and in combination with one another were evaluated in immunodeficient mice (BALB/c-SCID) infected with a fluconazole-resistant strain of Cryptococcus neoformans var. grubii. The animals were infected intravenously with 3 × 10⁵ cells and intraperitoneally treated with amphotericin B (1.5 mg/kg/day) in combination with voriconazole (40 mg/kg/days). Treatment began 1 day after inoculation and continued for 7 and 15 days post-inoculation. The treatments were evaluated by survival curves and yeast quantification (CFUs) in brain and lung tissues. Treatments for 15 days significantly promoted the survival of the animals compared to the control groups. Our results indicated that amphotericin B was effective in assuring longest-term survival of infected animals, but these animals still harbored the highest CFU of C. neoformans in lungs and brain at the end of the experiment. Voriconazole was not as effective alone, but in combination with amphotericin B, it prolonged survival for the second-longest time period and provided the lowest colonization of target organs by the fungus. None of the treatments were effective in complete eradication of the fungus in mice lungs and brain at the end of the experiment.