Proteasome Activity Is Required for Anthrax Lethal Toxin To Kill Macrophages

Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines such as RAW 264.7. LF is translocated by PA into the cytosol of target cells, where it acts as a metalloprotease to cleave mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins. In this study, we show that proteasome inhibitors such as acetyl-Leu-Leu-norleucinal, MG132, and lactacystin efficiently block LeTx cytotoxicity, whereas other protease inhibitors do not. The inhibitor concentrations that block LF cytotoxicity are similar to those that inhibit the proteasome-dependent IkappaB-alpha degradation induced by lipopolysaccharide. The inhibitors did not interfere with the proteolytic cleavage of MEK1 in LeTx-treated cells, indicating that they do not directly block the proteolytic activity of LF. However, the proteasome inhibitors did prevent ATP depletion, an early effect of LeTx. No overall activation of the proteasome by LeTx was detected, as shown by the cleavage of fluorogenic substrates of the proteasome. All of these results suggest that the proteasome mediates a toxic process initiated by LF in the cell cytosol. This process probably involves degradation of unidentified molecules that are essential for macrophage homeostasis. Moreover, this proteasome-dependent process is an early step in LeTx intoxication, but it is downstream of the cleavage by LF of MEK1 or other putative substrates.     

Antifungal activity of cerebrospinal fluid against Cryptococcus neoformans and Candida species.

The effect of human cerebrospinal fluid (CSF) on the growth of Cryptococcus neoformans and Candida species was tested in RPMI-1640. CSF alone was highly fungistatic for both yeasts and inhibited growth in a concentration-dependent manner. Unlike human serum, CSF did not collaborate with fluconazole for killing C. neoformans. Molecular sieve fractionation of CSF on a G-200 Sephadex column yielded a highly antifungal fraction with a molecular weight around 66 kDa. On SDS-PAGE this fraction migrated as a major and a minor band corresponding to the mobility of bovine serum albumin. These novel findings suggest that CSF contains a factor(s) that provides resistance to the growth of C. neoformans or Candida species.     

Growth of Cryptococcus neoformans Within Human Macrophages In Vitro

Macrophages cultured from human peripheral blood monocytes were infected with Cryptococcus neoformans in vitro. Although C. neoformans were actively ingested, there was no detectable intracellular killing by macrophages. Over 2 days or more, intracellular fungi grew more rapidly than a corresponding inoculum of extracellular cryptococci growing in tissue culture medium containing human serum. Macrophages were induced to develop into cells which appeared to be activated by morphological and phagocytic criteria. However, these activated cells did not acquire an ability to kill or inhibit intracellular growth of C. neoformans. There were no detectable differences between macrophages from normal subjects and those from cryptococcosis patients.     

Cryptococcus neoformans Resides in an Acidic Phagolysosome of Human Macrophages

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2′,7′-difluorofluorescein (Oregon Green). These probes have pK_as of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 μM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.     

Antifungal susceptibility of Cryptococcus neoformans to amphotericin B and fluconazole

Cryptococcus neoformans has emerged as an important opportunistic fungal pathogen in immunocompromised individuals. The therapeutic options of C. neoformans an opportunistic fungal pathogen include flucytosine, amphotericin B, and azole agents. However in the present scenario, emergence of resistance has been reported, hence this study was undertaken to evaluate antifungal susceptibility pattern of C. neoformans isolates from this southern part of India. Ten isolates of C. neoformans were tested against Amp B and fluconazole, of which 7 were susceptible to both and a single isolate of C. neoformans var gatti was resistant to both with MIC of 32mg/ml and 64mg/ml respectively.     

Human neutrophil-mediated nonoxidative antifungal activity against Cryptococcus neoformans

It has long been appreciated that polymorphonuclear leukocytes (PMN) kill Cryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing of C. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation of C. neoformans with these fractions for 18 h resulted in percent inhibition of growth of 67.4 +/- 3.4, 84.6 +/- 4.4, and 29.2 +/- 10.5 (mean +/- standard error, n = 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C(4) column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules.     

Role of Dendritic Cells and Alveolar Macrophages in Regulating Early Host Defense against Pulmonary Infection with Cryptococcus neoformans

Successful pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires a T1 adaptive immune response. This response takes up to 3 weeks to fully develop. The role of the initial, innate immune response against the organism is uncertain. In this study, an established model of diphtheria toxin-mediated depletion of resident pulmonary dendritic cells (DC) and alveolar macrophages (AM) was used to assess the contribution of these cells to the initial host response against cryptococcal infection. The results demonstrate that depletion of DC and AM one day prior to infection results in rapid clinical deterioration and death of mice within 6 days postinfection; this effect was not observed in infected groups of control mice not depleted of DC and AM. Depletion did not alter the microbial burden or total leukocyte recruitment in the lung. Mortality (in mice depleted of DC and AM) was associated with increased neutrophil and B-cell accumulation accompanied by histopathologic evidence of suppurative neutrophilic bronchopneumonia, cyst formation, and alveolar damage. Collectively, these data define an important role for DC and AM in regulating the initial innate immune response following pulmonary infection with C. neoformans. These findings provide important insight into the cellular mechanisms which coordinate early host defense against an invasive fungal pathogen in the lung.Cryptococcus neoformans, an opportunistic fungal pathogen acquired through inhalation, causes significant morbidity and mortality primarily in patients with impairments in host defense, including those with AIDS, those with lymphoid or hematological malignancies, or those receiving immunosuppressive therapy secondary to autoimmune disease or organ transplantation (31, 33, 60). The development of a T1 antigen-specific immune response characterized by gamma interferon production and classical activation of macrophages is required to eradicate the organism (4, 8, 21, 23, 24, 28). This adaptive immune response takes 2 to 3 weeks to develop and coincides with the CCR2-mediated recruitment of additional pulmonary dendritic cells (DC) and T cells to the lung (51, 66, 67). The role of initial, innate immune responses against the organism (prior to the development of adaptive immunity) is not well understood.Resident lung phagocytic cells, primarily DC and alveolar macrophages (AM), are likely the first immune cells exposed to C. neoformans upon inhalation of the organism into the lung. Both DC and AM express lectin receptors, including macrophage mannose receptor and DC-specific non-ICAM3 grabbing nonintergrin (DC-SIGN) (14, 15), which bind C. neoformans glycoantigens, including mannoproteins (42, 55). DC and AM phagocytose the organism in vitro and in vivo (29, 34, 63, 77, 78), and phagocytosis (and/or exposure to soluble glycoantigens or cryptococcal DNA) is associated with cytokine and chemokine production (5, 29, 40, 48, 49, 55, 61) and yeast lysis (77). It is unclear whether phagocytosis by resident DC and AM contributes to early clearance and/or the later development of adaptive immunity.DC represent an important interface between innate and adaptive immunity (reviewed in references 25, 53, 59, and 62). DC-cryptococcal interactions alter DC antigen-presenting functions and modulate resultant T-cell responses in vitro (10, 19, 55, 71). Following cryptococcal infection in vivo, DC migrate to thoracic lymph nodes (4, 52, 67). Thereafter, newly recruited DC colocalize with T cells within bronchovascular infiltrates in the lung (51). This is associated with interleukin-12 and gamma interferon production, yet direct evidence that DC-T-cell interactions modulate anticryptococcal responses in vivo remains sparse.A well-described murine model of in vivo DC depletion has been used to evaluate the contribution of DC to the development of dynamic, antigen-specific immune responses against a variety of antigens and microbial pathogens (16, 26, 27, 37, 56, 57, 69). Depletion results from the administration of diphtheria toxin (DT) to transgenic (Tg) mice in which the DT receptor (DTR) has been linked to the CD11c promoter. DT administration transiently depletes tissue DC (which express CD11c) for up to 96 h. This model has helped in assessment of the role of pulmonary DC in mediating adaptive immune responses against inhaled antigen (ovalbumin) and influenza virus (16, 69). In these studies, DT administration was associated with transient depletion of AM (which also express CD11c). The effect of CD11c cell depletion on innate immune responses has not been assessed (or reported) in these studies.In the current study, this established model of DT-mediated depletion of resident DC and AM was used to assess the in vivo contribution of these cells to the initial host response against cryptococcal infection. This objective is clinically relevant, as studies modulating DC numbers are in development for the treatment of patients with asthma (30, 32), autoimmunity (65, 68, 74), organ transplantation (12, 39, 43), and cancer (3, 11, 73). It is unknown whether manipulating the number of tissue DC will alter the innate or adaptive antifungal host defense in these patients. Our results demonstrate that DC and AM are critical regulators of the initial immune response against C. neoformans within the lung. Early mortality in DC- and AM-depleted mice precluded our ability to assess the role of these cells in the development of adaptive immunity.     

Bivalency Is Required for Anticapsular Monoclonal Antibodies To Optimally Suppress Activation of the Alternative Complement Pathway by the Cryptococcus neoformans Capsule

Encapsulated cells of Cryptococcus neoformans are potent activators of the alternative complement pathway. Previous studies found that monoclonal antibodies (MAbs) specific for the major capsular polysaccharide, termed glucuronoxylomannan (GXM), can markedly suppress the ability of the capsule to accumulate C3 from normal human serum via the alternative pathway. The present study examined the abilities of F(ab)₂ and Fab fragments of three MAbs (MAbs 439, 3C2, and 471) to mediate the suppressive effect. The results showed that F(ab)₂ fragments of all three MAbs suppressed activation and binding of C3 via the alternative pathway in a manner similar to that of intact antibodies. In contrast, Fab fragments of MAb 439 and MAb 3C2 showed no suppressive activity, and Fab fragments of MAb 471 were markedly reduced in suppressive activity. Indeed, there was an earlier accumulation of C3 on encapsulated cryptococci in the presence of the Fab fragments. Study of subclass switch families of MAb 439 and MAb 471 found that MAbs of an immunoglobulin G (IgG) subclass with increased flexibility in the hinge region (IgG2b) had less suppressive activity than MAbs of IgG subclasses with less flexibility (IgG1 or IgG2a). Taken together, these results indicate that cross-linking of the capsular matrix is an essential component in suppression of the alternative complement pathway by anti-GXM MAbs.The capsule of the pathogenic yeast Cryptococcus neoformans is a powerful activator of the alternative complement pathway (8, 16). Incubation of encapsulated cryptococci in normal human serum (NHS) leads to deposition of 10⁷ to 10⁸ molecules of C3 onto the typical yeast cell (18, 38); the capsule itself is the site for C3 binding (19, 21). Such activation and binding of C3 is due solely to the action of the alternative pathway (20, 21, 37). Binding of C3 via the alternative pathway in NHS is characterized by a delay of approximately 4 to 6 min before bound C3 is readily detectable (20).The major component of the cryptococcal capsule is the high-molecular-weight polysaccharide glucuronoxylomannan (GXM). Several anti-GXM monoclonal antibodies (MAbs) have been shown to provide a measure of protection in a murine model of cryptococcosis (9, 24, 30). In the accompanying report, we have examined the ability of anti-GXM MAbs to initiate the classical pathway, leading to accelerated deposition of C3 onto the yeast (17). These studies showed that most anti-GXM MAbs promote early deposition of C3 fragments into the capsule. However, depending on the epitope specificity of the MAb, some anti-GXM MAbs markedly reduced the apparent rate of amplification of bound C3, with the net result that fewer C3 molecules bound to the cell over a 20- to 30-min incubation period than would have bound in the absence of the antibody. When classical pathway initiation was blocked by the use of EGTA to chelate Ca²⁺ (12, 28), antibodies with the suppressive epitope specificity almost completely blocked the normal alternative pathway activation and binding of C3 that would have occurred in the absence of the MAbs.The ability of an antibody to block antibody-independent activation of the alternative pathway is without an obvious parallel in the literature. There are several potential mechanisms for antibody-mediated suppression. First, the antibody could bind to and occlude specific sites on the capsule that might be preferred acceptors for metastable C3b. Second, the capsule could contain specific domains that regulate the ability of the capsule to activate the alternative pathway. Antibodies specific for such regulatory domains could influence the ability of the cell to activate the alternative pathway. Finally, multivalent antibody could cross-link the capsule in a manner that prevents effective amplification. For example, binding of a multivalent antibody could reduce the ability of metastable C3b to diffuse from sites of C3 convertase activity. We have reasoned that the first two mechanisms for antibody-induced suppression of C3 binding would be mediated by intact antibody, F(ab)₂ fragments of the antibody, and Fab fragments. In contrast, inhibition that is dependent on cross-linking of the capsular matrix would be mediated by intact antibodies and F(ab)₂ fragments but not by Fab fragments.The objective of our study was to examine three anticapsular MAbs that suppress alternative pathway-dependent C3 binding. The suppressive activities of intact antibodies, F(ab)₂ fragments, and Fab fragments were compared. The results showed that intact antibodies and F(ab)₂ fragments of the antibodies suppressed accumulation of C3 fragments on the capsule. In contrast, Fab fragments of the suppressive antibodies showed markedly reduced or no ability to block alternative pathway activation by the capsule; indeed, Fab fragments derived from suppressive antibodies accelerated activation and binding of C3 via the alternative pathway.     

Fbp1-Mediated Ubiquitin-Proteasome Pathway Controls Cryptococcus neoformans Virulence by Regulating Fungal Intracellular Growth in Macrophages

Cryptococcus neoformans is a human fungal pathogen that often causes lung and brain infections in immunocompromised patients, with a high fatality rate. Our previous results showed that an F-box protein, Fbp1, is essential for Cryptococcus virulence independent of the classical virulence factors, suggesting a novel virulence control mechanism. In this study, we show that Fbp1 is part of the ubiquitin-proteasome system, and we further investigated the mechanism of Fbp1 function during infection. Time course studies revealed that the fbp1Δ mutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection. The fbp1Δ mutant cannot disseminate to other organs following pulmonary infection in the murine inhalation model of cryptococcosis but still causes brain infection in a murine intravenous injection model, suggesting that the block of dissemination of the fbp1Δ mutant is due to its inability to leave the lung. The fbp1Δ mutant showed a defect in intracellular proliferation after phagocytosis in a Cryptococcus-macrophage interaction assay, which likely contributes to its virulence attenuation. To elucidate the molecular basis of the SCF(Fbp1) E3 ligase function, we analyzed potential Fbp1 substrates based on proteomic approaches combined with phenotypic analysis. One substrate, the inositol phosphosphingolipid-phospholipase C1 (Isc1), is required for fungal survival inside macrophage cells, which is consistent with the role of Fbp1 in regulating Cryptococcus-macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis.     

Interleukin-12 Is Essential for a Protective Th1 Response in Mice Infected with Cryptococcus neoformans

To analyze the roles of interleukin-12 (IL-12) and the IL-12-dependent Th1 response in resistance to Cryptococcus neoformans, we have established a chronic infection model in wild-type mice and in mice with targeted disruptions of the genes for the IL-12p35 and IL-12p40 subunits (IL-12p35^−/− and IL-12p40^−/− mice, respectively) as well as in mice with a targeted disruption of the IL-4 gene. Long-term application of exogenous IL-12 prevented death of infected wild-type mice for the entire period of the experiment (up to 180 days) but did not resolve the infection. Infected IL-12p35^−/− and IL-12p40^−/− mice died significantly earlier than infected wild-type mice, whereas infection of IL-4-deficient mice led to prolonged survival. Interestingly, infected IL-12p40^−/− mice died earlier and developed higher organ burdens than IL-12p35^−/− mice, which, for the first time in an infection model, suggests a protective role of the IL-12p40 subunit independent of the IL-12 heterodimer. The fungal organ burdens of IL-4-deficient mice and IL-12-treated wild-type mice were significantly reduced compared to those of untreated wild-type mice and IL-12-deficient mice. Histopathological analysis revealed reduction of the number of granulomatous lesions following treatment with IL-12. Susceptibility of both IL-12p35^−/− and IL-12p40^−/− mice was associated with marginal production of gamma interferon and elevated levels of IL-4 from CD4⁺ T cells, which indicates Th2 polarization in the absence of IL-12, whereas wild-type mice developed a Th1 response. Taken together, our data emphasize the essential role of IL-12 for protective Th1 responses against C. neoformans.     

Extensive genetic diversity within the Dutch clinical Cryptococcus neoformans population

A set of 300 Dutch Cryptococcus neoformans isolates, obtained from 237 patients during 1977 to 2007, was investigated by determining the mating type, serotype, and AFLP and microsatellite genotype and susceptibility to seven antifungal compounds. Almost half of the studied cases were from HIV-infected patients, followed by a patient group of individuals with other underlying diseases and immunocompetent individuals. The majority of the isolates were mating type α and serotype A, followed by αD isolates and other minor categories. The most frequently observed genotype was AFLP1, distantly followed by AFLP2 and AFLP3. Microsatellite typing revealed a high genetic diversity among serotype A isolates but a lower diversity within the serotype D set of isolates. One patient was infected by multiple AFLP genotypes. Fluconazole and flucytosine had the highest geometric mean MICs of 2.9 and 3.5 μg/ml, respectively, while amphotericin B (0.24 μg/ml), itraconazole (0.08 μg/ml), voriconazole (0.07 μg/ml), posaconazole (0.06 μg/ml), and isavuconazole (0.03 μg/ml) had much lower geometric mean MICs. One isolate had a high flucytosine MIC (>64 μg/ml), while decreased susceptibility (≥16 μg/ml) for flucytosine and fluconazole was found in 9 and 10 C. neoformans isolates, respectively.     

Cytokine Signaling Regulates the Outcome of Intracellular Macrophage Parasitism by Cryptococcus neoformans

The pathogenic yeast Cryptococcus neoformans and C. gattii commonly cause severe infections of the central nervous system in patients with impaired immunity but also increasingly in immunocompetent individuals. Cryptococcus is phagocytosed by macrophages but can then survive and proliferate within the phagosomes of these infected host cells. Moreover, Cryptococcus is able to escape into the extracellular environment via a recently discovered nonlytic mechanism (termed expulsion or extrusion). Although it is well established that the host''s cytokine profile dramatically affects the outcome of cryptococcal disease, the molecular basis for this effect is unclear. Here, we report a systematic analysis of the influence of Th1, Th2, and Th17 cytokines on the outcome of the interaction between macrophages and cryptococci. We show that Th1 and Th17 cytokines activate, whereas Th2 cytokines inhibit, anticryptococcal functions. Intracellular yeast proliferation and cryptococcal expulsion rates were significantly lower after treatment with the Th1 cytokines gamma interferon and tumor necrosis factor alpha and the Th17 cytokine interleukin-17 (IL-17). Interestingly, however, the Th2 cytokines IL-4 and IL-13 significantly increased intracellular yeast proliferation while reducing the occurrence of pathogen expulsion. These results help explain the observed poor prognosis associated with the Th2 cytokine profile (e.g., in human immunodeficiency virus-infected patients).The two encapsulated yeast species Cryptococcus neoformans (serotypes A and D) and C. gattii (serotypes B and C), the causative agents of cryptococcosis, can cause life-threatening infections of the central nervous system (e.g., meningoencephalitis) (9).Initial infection with Cryptococcus is believed to occur via the inhalation of airborne propagules and the subsequent colonization of the respiratory tract (9). In mouse and rat model systems, C. neoformans is internalized by alveolar macrophages shortly after inhalation (17, 22). Furthermore, C. neoformans phagocytosis by mouse, rat, guinea pig, and human macrophages in vitro has been demonstrated repeatedly (8, 16, 37, 49) and is triggered by direct recognition of the yeast or by receptor-mediated recognition via complement or antibodies (38). However, Cryptococcus seems to have developed a unique method to manipulate host macrophages. After phagocytosis, C. neoformans can survive and proliferate within these infected host cells, eventually leading to macrophage lysis (2, 15, 17, 18, 33, 50). Moreover, a novel expulsive mechanism by which the yeast can exit macrophages without killing the host cell, thus avoiding a local inflammatory response, has recently been described (3, 34).Results from restriction fragment length polymorphism analyses suggest that initial infection with Cryptococcus often occurs in early childhood and can be followed by a long latent phase in immunocompetent individuals (21). However, C. neoformans is generally capable of disseminating to other organs within the human body and shows a predilection for the central nervous system, where it can lead to life-threatening meningitis and meningoencephalitis (27). Although the molecular basis of latency and expulsion is not known, the so-called Trojan Horse model suggests that replication in and eventual expulsion from macrophages may offer a potential explanation for how C. neoformans stays latent and spreads within the host without triggering immediate immune responses (11, 46). An improved understanding of the interaction between macrophages and Cryptococcus is therefore critical for the development of more effective therapies.In healthy hosts, the cryptococcal infection is usually self-limiting, suggesting effective clearance or maintenance in a latent state by phagocytic cells. The outcome of cryptococcosis depends on the immune status of the infected individual and the cytokine pattern generated in response to the pathogen. Although it is well established that the host''s cytokine profile dramatically affects the outcome of cryptococcal disease, the molecular basis for this effect is unclear. Both Th1 and Th2 cytokines are involved in protection against C. neoformans, but whereas Th1-associated cytokines are essential for natural immunity, Th2-associated immunity is not protective in mice (6, 24, 25). Increased expression of Th1 cytokines, such as tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), results in improved fungal control (19, 29, 36, 53), while IFN-γ knockout mice show increased fungal burdens (4). In 2007, Müller et al. (39) showed a significant role for the Th17 response and the proinflammatory cytokine interleukin-17 (IL-17) in modulating the survival of Cryptococcus-infected mice. In contrast, Th2 cytokines such as IL-4 and IL-13 reduce the host''s ability to deal with C. neoformans in vivo (7, 14, 28, 39).Despite these observations from animal models, little work on the in vitro effects of Th1, Th17, and Th2 cytokines on the interaction between macrophages and Cryptococcus has been done. Here, we report a systematic study of cytokine influence on macrophage-Cryptococcus interactions for a representative selection of Cryptococcus strains. Our results demonstrate that Th1- and Th17-stimulated macrophages are significantly better at phagocytosing cryptococci and at controlling the intracellular proliferation of this pathogen than Th2-stimulated cells. In contrast, Th2-activated macrophages show a significantly lower rate of cryptococcal expulsion than Th1- or Th17-activated cells. Together, these data help explain the susceptibility phenotype associated with Th2 cytokine profiles in vivo.     

Evidence That Cryptococcus neoformans Is Melanized in Pigeon Excreta: Implications for Pathogenesis

Structures similar to the melanin "ghosts" of melanized cryptococcal cells were isolated from pigeon excreta contaminated with Cryptococcus neoformans, and their growth in pigeon excreta supported melanization. The results suggest that environmental C. neoformans cells are melanized and imply that initial infection may involve exposure to melanized cells.     

Isolation of the Third Capsule-Associated Gene, CAP60, Required for Virulence in Cryptococcus neoformans

A polysaccharide capsule is one of the most important virulence factors for the pathogenic fungus Cryptococcus neoformans. We previously characterized two capsule-associated genes, CAP59 and CAP64. To further dissect the molecular mechanism of capsule synthesis, 16 acapsular mutants induced by 4-nitroquinoline-1-oxide were obtained. The acapsular phenotype of one of these mutants was complemented. The cloned gene was designated CAP60, and deletion of this newly described capsule-associated gene resulted in an acapsular phenotype. The proposed 67-kDa Cap60p contains 592 amino acids and appears to have a putative transmembrane domain close to the N terminus. DNA sequence analysis revealed that CAP60 has similarity to CAP59 at the center portion of its coding regions. Contour-clamped homogeneous electric field blot analysis suggested that these two genes are on the same chromosome. CAP60 and CAP59, however, could not be functionally substituted for each other by direct complementation or by domain swap experiments. In addition, CAP60 is closely linked to a gene which is similar to a cellulose growth-specific gene of Agaricus bisporus, CEL1. Immunogold electron microscopy studies of the epitope-tagged CAP60 gene revealed that Cap60p was primarily localized to the nuclear membrane. Animal model studies indicated that CAP60 is essential for virulence. Thus, CAP60 is required for both capsule formation and virulence.     

Antigen-Induced Protective and Nonprotective Cell-Mediated Immune Components against Cryptococcus neoformans

Mice immunized with two different cryptococcal antigen preparations, one a soluble culture filtrate antigen (CneF) in complete Freund’s adjuvant (CFA) and the other heat-killed Cryptococcus neoformans cells (HKC), develop two different profiles of activated T cells. CneF-CFA induces CD4⁺ T cells responsible for delayed-type hypersensitivity (DTH) reactivity and for amplification of the anticryptococcal DTH response, whereas HKC induce CD4⁺ and CD8⁺ T cells involved in anticryptococcal DTH reactivity and activated T cells which directly kill C. neoformans cells. The main purpose of this study was to assess the level of protection afforded by each of the two different T-cell profiles against challenge with viable C. neoformans cells, thereby identifying which activated T-cell profile provides better protection. CBA/J mice immunized with CneF-CFA had significantly better protective responses, based on better clearance of C. neoformans from tissues, on longer survival times, and on fewer and smaller lesions in the brain, than HKC-immunized mice or control mice similarly infected with C. neoformans. Both immunization protocols induced an anticryptococcal DTH response, but neither induced serum antibodies to glucuronoxylmannan, so the protection observed in the CneF-CFA immunized mice was due to the activated T-cell profile induced by that protocol. HKC-immunized mice, which displayed no greater protection than controls, did not have the amplifier cells. Based on our findings, we propose that the protective anticryptococcal T cells are the CD4⁺ T cells which have been shown to be responsible for DTH reactivity and/or the CD4⁺ T cells which amplify the DTH response and which have been previously shown to produce high levels of gamma interferon and interleukin 2. Our results imply that there are protective and nonprotective cell-mediated immune responses and highlight the complexity of the immune response to C. neoformans antigens.     

Cryptococcus neoformans var. grubii: Separate Varietal Status for Cryptococcus neoformans Serotype A Isolates

Cryptococcus neoformans var. neoformans presently includes isolates which have been determined by the immunologic reactivity of their capsular polysaccharides to be serotype A and those which have been determined to be serotype D. However, recent analyses of the URA5 sequences and DNA fingerprinting patterns suggest significant genetic differences between the two serotypes. Therefore, we propose to recognize these genotypic distinctions, as well as previously reported phenotypic differences, by restricting C. neoformans var. neoformans to isolates which are serotype D and describing a new variety, C. neoformans var. grubii, for serotype A isolates.     

Humoral response against Cryptococcus neoformans mannoprotein antigens in HIV-infected patients

Twenty-four sera from healthy donors, 18 from HIV-positive patients (< 200 CD4+/mm3) and 18 sera collected before and during cryptococcosis from HIV-positive patients were analysed for the presence of humoral response to C. neoformans mannoproteins. Our results show that samples from healthy subjects and from HIV-positive patients had one of three antibody response profiles: (i) presence of reactive antibodies against both 105 and 80 kilodalton mannoproteins; (ii) presence of reactive antibodies against one of the two mannoproteins; or (iii) absence of reactive antibodies. Importantly the percentage of unreactive sera increased 6-fold in HIV-positive patients and more than 10-fold in patients with cryptococcosis. In addition, in the latter patients no variation of humoral response before and during cryptococcosis was observed. These results suggest that HIV-positive patients show a marked difficulty in mounting or maintaining antibody response to mannoprotein and this could contribute to predisposition to cryptococcosis.     

Cleavage of Escherichia coli Cytotoxic Necrotizing Factor 1 Is Required for Full Biologic Activity

Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by pathogenic Escherichia coli strains. CNF1 constitutively activates small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. The toxin is taken up into mammalian cells by receptor-mediated endocytosis and is delivered from late endosomes into the cytosol. Here, we show that an approximately 55-kDa fragment of CNF1, which contains the catalytic domain and an additional part of the toxin, is present in the cytosol. The processing of this fragment requires an acidic pH and insertion of the toxin into the endosomal membrane. We define the cleavage site region as the region located between amino acids 532 and 544 of CNF1. The data provide insight into the complex mechanism of uptake of bacterial toxins into mammalian cells.Cytotoxic necrotizing factor 1 (CNF1) is produced by pathogenic Escherichia coli strains which cause urinary tract infections and neonatal meningitis (4, 10). CNF1 belongs to a family of Rho GTPase-modifying proteins which deamidate the small GTPases at glutamine 63 or 61, leading to their constitutive activation (6, 20). Other members of this toxin family are the E. coli toxins CNF2 and CNF3, which have more than 70% identity to CNF1, and CNF_Y produced by Yersinia pseudotuberculosis, which has about 60% identity to CNF1 (15, 17, 18). Whereas CNF1 modifies RhoA, Rac, and Cdc42 in HeLa cells, recombinant CNF_Y selectively activates RhoA (8). All CNFs are 115-kDa single-chain AB toxins with an N-terminal receptor-binding domain and a C-terminal catalytic domain, which contains the deamidase activity (3, 12). The two domains are separated by a putative translocation domain with two hydrophobic helices involved in membrane translocation (for reviews, see references 9 and 13).CNF1 has been shown to enter cells by receptor-mediated endocytosis, which is independent of clathrin and of sphingolipid-cholesterol-rich membrane microdomains (lipid rafts), including caveolae (5). From late endosomes the toxin enters the cytosol in an acidic pH-dependent manner (1). It was speculated that the toxin reaches the cytosol as an uncleaved protein, because CNF1 could be translocated directly through the plasma membrane by an acidic pulse apparently without a requirement for proteolytic cleavage (19). Using a monoclonal antibody that binds to the catalytic domain of CNF1, we detected an approximately 55-kDa fragment of CNF1 in the cytosolic fraction of intoxicated cells. We show that the appearance of this fragment in the cytosol is dependent on the acidic pH in late endosomes and insertion of the translocation loop into the membrane. We narrow the cleavage site region to a stretch of 13 amino acids (amino acids 532 to 544).