Inducible defense mechanism against nitric oxide in Candida albicans

The yeast Candida albicans is an opportunistic pathogen that threatens patients with compromised immune systems. Immune cell defenses against C. albicans are complex but typically involve the production of reactive oxygen species and nitrogen radicals such as nitric oxide (NO) that damage the yeast or inhibit its growth. Whether Candida defends itself against NO and the molecules responsible for this defense have yet to be determined. The defense against NO in various bacteria and the yeast Saccharomyces cerevisiae involves an NO-scavenging flavohemoglobin. The C. albicans genome contains three genes encoding flavohemoglobin-related proteins, CaYHB1, CaYHB4, and CaYHB5. To assess their roles in NO metabolism, we constructed strains lacking each of these genes and demonstrated that just one, CaYHB1, is responsible for NO consumption and detoxification. In C. albicans, NO metabolic activity and CaYHB1 mRNA levels are rapidly induced by NO and NO-generating agents. Loss of CaYHB1 increases the sensitivity of C. albicans to NO-mediated growth inhibition. In mice, infections with Candida strains lacking CaYHB1 still resulted in lethality, but virulence was decreased compared to that in wild-type strains. Thus, C. albicans possesses a rapid, specific, and highly inducible NO defense mechanism involving one of three putative flavohemoglobin genes.     

系统性白色念珠菌感染的免疫应答反应及疫苗研究进展

由于肿瘤化疗药物和免疫抑制剂的广泛应用,使系统性念珠菌感染的发病率明显升高,该病严重地威胁着住院患者的生命。在白色念珠菌感染过程中,特异性抗体、Th细胞和一些非特异性免疫细胞存在着复杂的调节机制。先天性免疫应答和获得性免疫应答机制的研究,对开发出有效的抗真菌疫苗具有重要意义。     

Modulation of neutrophil function in host defense against disseminated Candida albicans infection in mice

Neutrophils (PMNs) constitute the main mechanism of host defense against acute invasive and disseminated candidiasis. Recent studies have demonstrated that tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) play an important role in the recruitment of PMNs at the site of invasive Candida infection. In the absence of either TNFalpha or IL-6, the course of experimental disseminated candidiasis is more severe, due to defective PMN recruitment. Treatment of mice with recombinant G-CSF (rG-CSF) leads to a significantly reduced mortality during disseminated candidiasis. The outgrowth of Candida albicans from the organs of rG-CSF-treated mice is significantly decreased. Treatment with the combination of rG-CSF and fluconazole has an additive effect on the reduction of fungal load in the organs. In subacute or chronic disseminated Candida infection, rG-CSF is less effective, indicating that neutrophil recruitment and activation are crucial in acute, life-threatening candidiasis, whereas other host defense mechanisms control the outcome of less overwhelming invasive Candida infection.     

Murine Defense Mechanism against Candida albicans Infection: II. Opsonization,Phagocytosis, and Intracellular Killing of C. albicans

The phagocytic and intracellular killing activities of normal mouse phagocytes against Candida albicans were studied to elucidate the role of these activities in nonspecific and specific defense mechanisms. In the presence of fresh normal mouse serum, viable C. albicans cells were ingested by mouse peripheral blood leukocytes (PBLs) and peritoneal macrophages (PMPs) at the same rate. Serum-chelation experiments indicated that the factors involved in the alternative complement pathway are opsonins for C. albicans. PBLs killed intracellular C. albicans more effectively than PMPs. Lymphokine-activated PMPs manifested marked intracellular killing activity and the occurrence of increased superoxide anion- and singlet oxygen production, in the absence of increased myeloperoxidase (MPO) production, suggests that the enhanced, MPO-independent, oxidative mechanism may play an important role in the candidacidal activity. Specific rabbit antibodies played no role in the phagocytosis and intracellular killing of C. albicans. These results suggest that PMNs and factors involved in the alternative complement pathway, and lymphokine-activated macrophages play major roles in the protection of mice against C. albicans infection.     

Immune defense mechanisms of Culex quinquefasciatus (Diptera: Culicidae) against Candida albicans infection

Mosquitoes have an efficient defense system against infection. The cellular immune defense mechanism initiated by the mosquito Culex quinquefasciatus infected with the fungus Candida albicans was investigated in this study. Differences in the hemocyte counts in hemolymph perfused from uninoculated, saline-inoculated, and C. albicans-infected mosquitoes were compared using a light microscope. Phagocytosis was also investigated using electron microscopy. Four types of hemocytes were identified in control mosquitoes: prohemocytes (9.8%), plasmatocytes (38.8%), granular cells (44.2%), and oenocytoids (7.3%). Between 3 and 18 h postinoculation the total hemocyte count was significantly higher in infected, compared to uninfected, mosquitoes. Differential hemocyte counts from infected mosquitoes at 3, 6, and 18 h after inoculation showed that the relative proportion of plasmatocytes (48.6, 50.7, 45%) was higher and, concomitantly, the proportion of granular cells was lower (38, 36.8, 35%, respectively). Yeast cells were phagocytosed and limited growth was observed within the plasmatocytes. Melanized nodules were found attached to different insect tissues at 24 to 72 h following infection. These results suggest that phagocytosis, followed by nodule formation, was capable of clearing the hemolymph of yeast cells.     

Studies on defense mechanisms against Candida albicans infection in congenitally athymicnude (nu/nu) mice

The defense mechanisms against Candida albicans infection were studied by using a mouse thigh lesion model in congenitally athymic nude (nu/nu) mice and their normal littermates (nu/+). Nu/nu mice were more resistant to C. albicans infection than nu/+ mice judging from the course of the thigh lesion, the results of CFUs (colony-forming units) of C. albicans in the lesion, and histopathological observations. Histopathological and serological studies revealed that granulocytic cellular infiltration was predominant, and there were few indications of development of cell-mediated immunity to protect Candida infection in Candida-infected nu/nu and nu/+ mice. These results confirmed that lower susceptibility of nu/nu mice to C. albicans infection as compared with nu/ + mice was due to accelerated non-specific defense mechanisms in nu/nu mice, and that cell-mediated or humoral immunity played a minor role in the defense against Candida infection in this experimental model.Furthermore, treatment with high titer of rabbit anti-C. albicans serum was effective to control the number of Candida cells in thigh lesions of BALB/c mice.Above experimental results seem to clearly indicate the great variability of defense manifestation according to the experimental model exployed.     

Suppression of humoral response during the course of Candida albicans infection in mice

This paper aims at demonstrating the non-specific immunosuppression as regards thyme-dependent antigens sheep erythrocytes (SRBC) during the course of Candida albicans systemic infection.Three lots of syngeneic /BALB/c mice, 8–12 weeks of age, were used. The first normal lot was inoculated via the intraperitoneal route with a (SRBC) suspension (4×10⁸ cells ml) in a Hank's balanced saline solution. The primary response of antibodies formed by splenic cells was measured from 4 to 8 days after inoculation using the direct plaque forming cells technique. The second lot was infected by the same route with a suspension of Candida albicans (1×10⁷ cells). Positive retrocultures from the blood and kidneys of these infected mice were obtained. These yeasts cultivated in a Sabouraud medium were harvested after 20 h at 37 °C. Following the same methodology the immune response to SRBC was determined. The serum obtained from infected mice was transferred to a third lot of mice at different intervals during the course of the infection. The immune response to SRBC was done by the direct plaque-forming cells technique. Controls were carried out using normal donors and recipients.A suppression of the immune response was obtained as from the 2nd day of inoculation up to the 28th day. It was not possible to transfer such suppression passively by means of the serum.These results suggest that the systemic infection by Candida albicans induce a non-specific immunosuppression in the organism, already demonstrated in viral infections, bacteria, protozoaria and metazoaria in mammals.In some way, this will contribute to explain the mechanisms of immune response to Candida albicans.     

Outbreak of systemic Candida albicans in intensive care unit caused by cross infection.

The first documented outbreak of systemic candidosis shown to be due to cross infection with a particular strain of Candida albicans is reported. Over nine months in an intensive care unit 13 patients developed definite and one probable systemic candidosis. Twenty five further patients had superficial candidal infections. The strain that caused the outbreak (serotype A, morphotype A1, biotype 0/(1)5 5/7) was responsible for all the cases of systemic candidosis acquired in the intensive care unit, 11 (44%) of the superficial candidal infections in the unit, and 17% of candidal infections outside the unit but in the same hospital. The strain was also isolated from oral swabs taken from four nurses working in the unit and the hands of one of these nurses. Two out of 17 nurses were shown to have acquired the strain on their hands when examined immediately after nursing systemically infected patients. No environmental source could be identified. The strain also showed enhanced survival in handwashing experiments and was relatively resistant to Hibiscrub. Management of patients with systemic candidosis might include measures to prevent cross infection and handwashing with disinfectants that are active against candida.     

Contribution of the antibodies response induced by a low virulent Candida albicans strain in protection against systemic candidiasis

A low virulent Candida albicans mutant, CNC13, deleted in the Mitogen Activated Protein (MAP) kynase HOG1 was used to immunize BALB/c mice. Hog1p is essential for the oxidative stress and hyperosmolarity responses. Several doses and immunization procedures were employed. The protection capacity of the different sera generated was analyzed in a murine model of systemic candidiasis. Using a proteomic approach (two-dimensional gel electrophoresis followed by Western blotting), we were able to distinguish two categories of serum: protective and nonprotective, which showed different titres of total Immunoglobulins (Igs) and IgG2a (analyzed by enzyme-linked immunosorbent assay). The levels of Igs and IgG2a in protective sera were significantly higher compared to nonprotective sera. The pattern of a "nonprotective" profile was composed of enolase (Eno1p), transketolase, heat shock protein and methionine synthase. Only antibodies against enolase are the IgG2a isotype. The pattern of a "protective" sera, on the other hand, was composed of antibodies against the following antigens: several isoforms of Eno1p, pyruvate decarboxylase, pyruvate kynase, a protein of the 40S ribosomal subunit, triosephosphate isomerase, DL-glycerol phosphatase and fructose-bisphosphate aldolase. All these antibodies are the IgG2a isotype. The proteins described in the protective sera might be useful for future vaccine development.     

Candida albicans double-stranded DNA can participate in the host defense against disseminated candidiasis

In the present work, we studied the in vitro immunomodulatory properties of double-stranded Candida albicans DNA and its protective effect in murine disseminated candidiasis. DNA induced the production of TNF-alpha by peritoneal macrophages and splenocytes in vitro through a chloroquine-dependent mechanism. Yeast DNA acted synergistically with IFN-gamma in triggering the secretion of nitric oxide by macrophages and enabled them to stimulate the proliferation of T cells in response to soluble anti-CD3. The effect of DNA on splenocytes is associated with an enhanced synthesis of IFN-gamma, IL-2 and IL-10. In vivo, DNA decreased the mortality and lowered the kidney contamination in mice intraperitoneally inoculated with C. albicans simultaneously with an increase in the specific proliferative response and cytokine production. The present results indicate that C. albicans DNA can provide protection against disseminated infection.     

Effect of N-acetylchitohexaose against Candida albicans infection of tumor-bearing mice

A water-soluble oligosaccharide, N-acetylchitohexaose (NACOS-6), was able to enhance the protective effect against Candida albicans infection in mice during the early phase of tumor-bearing. A significant decrease in the number of C. albicans cells in the kidneys of NACOS-6-treated tumor-bearing mice was observed 8 days after the fungal infection, or 15 days after the tumor transplantation. The candidacidal activity of polymorphonuclear leukocytes from NACOS-6-treated tumor-bearing mice did not differ from that of NACOS-6-untreated tumor-bearing mice. On the other hand, the candidacidal activities of both macrophages and T lymphocytes increased following administration of NACOS-6 in the early phase of tumor-bearing. The culture supernatant of T lymphocytes from NACOS-6-treated tumor-bearing mice also potentiated the candidacidal activity of casein-induced macrophages. An enhancement of natural killer cell activity of splenic lymphocytes obtained from NACOS-6-treated tumor-bearing mice was also observed.     

Mechanisms of antibody-mediated protection against lymphocytic choriomeningitis virus infection: mother-to-baby transfer of humoral protection.

The role of antiviral antibodies in resistance to lymphocytic choriomeningitis virus (LCMV) infection was explored. Immune serum and monoclonal antibodies prevented fatal T-cell-mediated immunopathology following acute LCMV infections. In addition, 10- and 14-day-old mice that received maternally derived anti-LCMV antibodies through nursing were protected from an otherwise lethal LCMV challenge. Detailed investigation of the mechanism(s) by which these antiviral antibodies provided was carried out by using anti-LCMV monoclonal antibodies. Protection correlated directly with the ability of the antibodies to reduce viral titers in the tissues of conventional (K. E. Wright and M. J. Buchmeier, J. Virol. 65:3001-3006, 1991) and nude mice. However, this reduction was not simply a reflection of virus neutralizing activity, since not all antibodies which neutralized in vitro were protective. A correlation was also found between immunoglobulin isotype and protection: all of the protective antibodies were immunoglobulin G2a (IgG2a), while IgG1 antibodies mapping to the same epitopes were not. Protection appeared to be associated with events controlled by the Fc region. Functional F(ab')2 fragments which retained in vitro neutralizing activity were not protective in vivo. Furthermore, this Fc-associated function was not related to complement-mediated cell lysis, since C5-deficient mouse strains were also protected. These results suggest a role for antibody in protection from arenavirus infections and indicate that a distinct immunoglobulin subclass, IgG2a, may be essential for this protection.     

Mechanisms of host defense against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages

We studied the biochemical basis of phagocytosis of Candida albicans, a serious pathogen, and Candida parapsilosis, which is rarely pathogenic, by human monocytes (Mo) and monocyte-derived macrophages (MDM). Optimal phagocytosis of both species by Mo required the presence of extracellular Ca2+ and opsonization through both the classic and alternative complement pathways. Serum-opsonized Candida were ingested equally by Mo and MDM; unopsonized Candida were phagocytosed only by macrophages, and uptake began slowly. This opsonin-independent phagocytosis required Ca2+ and could be blocked by yeast mannan or mannose-BSA conjugate, suggesting a role for the mannose receptor. Opsonized Candida elicited a vigorous increase in the concentration of [Ca2+]i in Mo and MDM, but no Ca2+ transient was detected in MDM stimulated with unopsonized Candida. Pretreatment of MDM with ionomycin to increase [Ca2+]i had no effect on phagocytosis of unopsonized Candida. Addition of 5 mM EGTA completely inhibited changes in [Ca2+]i in Mo and MDM, suggesting that the Ca2+ transient induced by opsonized Candida is due to an influx of extracellular Ca2+. Differences in pathogenicity between the two Candida species could not be explained by differences in any aspect of phagocytosis. Uptake mediated by the macrophage mannose receptor could play a role in clearance of Candida under opsonin-poor conditions.     

Immunomodulation by Candida albicans: crucial role of organ colonization and chronic infection with an attenuated agerminative strain of C. albicans for establishment of anti-infectious protection

Intravenous inoculation of an attenuated agerminative strain of Candida albicans (PCA-2) of low virulence, but not of two other species of Candida of low virulence (C. parapsilosis and C. viswanathii) into CD2F1 mice conferred protection against the highly virulent microbes C. albicans CA-6, Staphylococcus aureus and Aspergillus fumigatus. To provide protection, a definite inoculum size (10(6) cells per mouse) resulting in organ colonization and establishment of a long-lasting chronic infection with PCA-2 was needed. An inoculum of 10(5) cells gave rise to transient kidney colonization whereas inocula greater than 10(6) cells led to acute septicaemia and eventual death. Chronic infection of mice following inoculation of 10(6) PCA-2 cells was accompanied by detectable mannoprotein antigen levels in the serum (30-70 ng ml-1) while specific antibodies did not appear until 14 d after inoculation, at which time low antimannan antibody was present (ELISA titre 1:40-1:80). Chronic infection was characterized by the presence in the kidneys of 2-3 x 10(6) c.f.u. of PCA-2 for at least 40 d after inoculation. Pharmacological modulation of the host through administration of either an anti-Candida drug, amphotericin B, or an immunosuppressive agent, cyclophosphamide, strongly supported the premise that the anti-infectious state conferred by PCA-2 'immunization' correlated with the maintenance of a sufficient number of PCA-2 in vivo. Protection was 'switched on' when 2-3 x 10(5) cells were present in the kidneys. It was maximal at a kidney count of 2-3 x 10(6) c.f.u. of PCA-2, and promptly declined when the number of PCA-2 cells in the kidney fell below 2 x 10(5). Mice chronically infected with PCA-2 had splenic macrophages with pronounced candidacidal activity in vitro. Modulation of the growth of PCA-2 in vivo, which determined activation or deactivation of the protective state, was paralleled by a similar modulation in macrophage activation, showing that in all cases resistance to virulent organisms persisted as long as macrophage activation was present. The results demonstrate that a critical in vivo antigenic load is crucial for the occurrence of resistance to infection and suggests that macrophages could be involved in this protection.     

A novel Th cell epitope of Candida albicans mediates protection from fungal infection

Fungal pathogens are a frequent cause of opportunistic infections. They live as commensals in healthy individuals but can cause disease when the immune status of the host is altered. T lymphocytes play a critical role in pathogen control. However, specific Ags determining the activation and function of antifungal T cells remain largely unknown. By using an immunoproteomic approach, we have identified for the first time, to our knowledge, a natural T cell epitope from Candida albicans. Isolation and sequencing of MHC class II-bound ligands from infected dendritic cells revealed a peptide that was recognized by a major population of all Candida-specific Th cells isolated from infected mice. Importantly, human Th cells also responded to stimulation with the peptide in an HLA-dependent manner but without restriction to any particular HLA class II allele. Immunization of mice with the peptide resulted in a population of epitope-specific Th cells that reacted not only with C. albicans but also with other clinically highly relevant species of Candida including the distantly related Candida glabrata. The extent of the reaction to different Candida species correlated with their degree of phylogenetic relationship to C. albicans. Finally, we show that the newly identified peptide acts as an efficient vaccine when used in combination with an adjuvant inducing IL-17A secretion from peptide-specific T cells. Immunized mice were protected from fatal candidiasis. Together, these results uncover a new immune determinant of the host response against Candida ssp. that could be exploited for the development of antifungal vaccines and immunotherapies.     

Antifungal activity of a Candida albicans GGTase I inhibitor-alanine conjugate. Inhibition of Rho1p prenylation in C. albicans

An alanine conjugate of a Candida albicans geranylgeranyl transferase I inhibitor was synthesized to facilitate its uptake into the fungal cell. The antifungal activity of CaGGTase-Ala conjugate is demonstrated. It is also shown that the CaGGTase-Ala conjugate affects prenylation of endogenous Rho1p, but has no effect on prenylation of endogenous Ras1p.