Effect of various antibiotics on gastrointestinal colonization and dissemination by Candida albicans

Mice were treated orally with various antibiotics to determine which members of the indigenous intestinal microflora normally suppress Candida albicans colonization and dissemination from the gastrointestinal (GI) tract. The mice were given penicillin, clindamycin, vancomycin, erythromycin, or gentamicin for 3 days, and then challenged orally with C. albicans. Penicillin, clindamycin, and vancomycin, but not gentamicin or erythromycin, decreased the total anaerobic bacterial populations in the animals ceca, and increased the enteric bacilli population levels. All three of the former antibiotics allowed C. albicans to proliferate in the gut and, subsequently, disseminate from the GI tract to visceral organs. The ability of C. albicans to associate with intestinal mucosal surfaces was also tested. It was found that antibiotics which reduced anaerobic population levels, but not enteric bacilli or aerobes, also predisposed animals to mucosal association by C. albicans. It is suggested that the strictly anaerobic bacterial populations which predominate in the gut ecosystem are responsible for the inhibition of C. albicans adhesion, colonization and dissemination from the GI tract.     

Fungemia in acute leukemia patients: a single institution's experience

A retrospective study was conducted in 22 episodes of candidemia in 445 patients with acute leukemia (AL) (incidence 4.9%) observed at our Institution between February 1996 and November 2003 to evaluate the variables related to the onset and outcome of infection. Of 22 patients, 20 (90.9%) had refractory/relapsed AL. C. albicans was responsible for 7/22 of the fungemia cases (32%) and C. non albicans for 15/22 (68%). The median absolute neutrophil count was 0.1 x10(9)/L (range 0.04-0.3) at the time of the candidemia diagnosis. The fungemia responded to antifungal therapy in 15/22 (68.1%) patients; among patients with a positive outcome of the fungemia, in 14/15 (93.3%) the neutrophil count recovered within 7 days after the diagnosis of candidemia (p < 0.05). The mortality rate due to candidemia was 31.9% (1/7: 14.2% and 6/15: 40% in the C. albicans and C. non albicans groups, respectively). In our experience the determinants of a positive outcome of fungemia were infection by the C. albicans species and recovery of the neutrophil count.     

Chlamydospore-like cells of Candida albicans in the gastrointestinal tract of infected, immunocompromised mice

We have demonstrated in a previously described murine model of gastrointestinal (GI) and systemic candidiasis that the antifungal angent cilofungin was efficacious in clearing infection of body organs when administered subcutaneously by infusion, but permitted large numbers of Candida albicans in the GI tract to persist. Yeast and hyphae in these animals were associated primarily with the stratified squamous epithelium of the stomach. Administration of immunocompromising drugs (cyclophosphamide plus cortisone acetate) to animals with persistent GI infection resulted in relapse of systemic candidiasis. Histological examination of the gastric mucosa revealed invasive hyphal elements and yeast as well as multiple chlamydospore-like cells. Comparative histochemical and electron-microscopic examinations of these latter cells produced in host tissue and chlamydospores formed in vitro were conducted. The results suggested that similarities in wall and cytoplasmic composition and ultrastructure exist between these in vivo and in vitro produced C. albicans cells. Exposure of C. albicans to cyclophosphamide during in vitro growth resulted in stimulation of chlamydospore production. No significant effect of cortisone acetate on C. albicans morphogenesis was detected. The murine model used in this study permits investigation of the formation of chlamydospore-like cells of C. albicans during early stages of fungal invasion of cyclophosphamide-treated mice, and of the possible influence of these cells on immunological response of the host to persistent candidiasis of the GI tract.     

Horizontal transmission of Candida albicans and evidence of a vaccine response in mice colonized with the fungus

Disseminated candidiasis is the third leading nosocomial blood stream infection in the United States and is often fatal. We previously showed that disseminated candidiasis was preventable in normal mice by immunization with either a glycopeptide or a peptide synthetic vaccine, both of which were Candida albicans cell wall derived. A weakness of these studies is that, unlike humans, mice do not have a C. albicans GI flora and they lack Candida serum antibodies. We examined the influence of C. albicans GI tract colonization and serum antibodies on mouse vaccination responses to the peptide, Fba, derived from fructose bisphosphate aldolase which has cytosolic and cell wall distributions in the fungus. We evaluated the effect of live C. albicans in drinking water and antimicrobial agents on establishment of Candida colonization of the mouse GI tract. Body mass, C. albicans in feces, and fungal-specific serum antibodies were monitored longitudinally. Unexpectedly, C. albicans colonization occurred in mice that received only antibiotics in their drinking water, provided that the mice were housed in the same room as intentionally colonized mice. The fungal strain in unintentionally colonized mice appeared identical to the strain used for intentional GI-tract colonization. This is the first report of horizontal transmission and spontaneous C. albicans colonization in mice. Importantly, many Candida-colonized mice developed serum fungal-specific antibodies. Despite the GI-tract colonization and presence of serum antibodies, the animals made antibodies in response to the Fba immunogen. This mouse model has potential for elucidating C. albicans horizontal transmission and for exploring factors that induce host defense against disseminated candidiasis. Furthermore, a combined protracted GI-tract colonization with Candida and the possibility of serum antibody responses to the presence of the fungus makes this an attractive mouse model for testing the efficacy of vaccines designed to prevent human disseminated candidiasis.     

Murine Models of Candida Gastrointestinal Colonization and Dissemination

Ninety-five percent of infectious agents enter through exposed mucosal surfaces, such as the respiratory and gastrointestinal (GI) tracts. The human GI tract is colonized with trillions of commensal microbes, including numerous Candida spp. Some commensal microbes in the GI tract can cause serious human infections under specific circumstances, typically involving changes in the gut environment and/or host immune conditions. Therefore, utilizing animal models of fungal GI colonization and dissemination can lead to significant insights into the complex pathophysiology of transformation from a commensal organism to a pathogen and host-pathogen interactions. This paper will review the methodologic approaches used for modeling GI colonization versus dissemination, the insights learned from these models, and finally, possible future directions using these animal modeling systems.     

Live imaging of disseminated candidiasis in zebrafish reveals role of phagocyte oxidase in limiting filamentous growth

Candida albicans is a human commensal and a clinically important fungal pathogen that grows in both yeast and hyphal forms during human infection. Although Candida can cause cutaneous and mucosal disease, systemic infections cause the greatest mortality in hospitals. Candidemia occurs primarily in immunocompromised patients, for whom the innate immune system plays a paramount role in immunity. We have developed a novel transparent vertebrate model of candidemia to probe the molecular nature of Candida-innate immune system interactions in an intact host. Our zebrafish infection model results in a lethal disseminated disease that shares important traits with disseminated candidiasis in mammals, including dimorphic fungal growth, dependence on hyphal growth for virulence, and dependence on the phagocyte NADPH oxidase for immunity. Dual imaging of fluorescently marked immune cells and fungi revealed that phagocytosed yeast cells can remain viable and even divide within macrophages without germinating. Similarly, although we observed apparently killed yeast cells within neutrophils, most yeast cells within these innate immune cells were viable. Exploiting this model, we combined intravital imaging with gene knockdown to show for the first time that NADPH oxidase is required for regulation of C. albicans filamentation in vivo. The transparent and easily manipulated larval zebrafish model promises to provide a unique tool for dissecting the molecular basis of phagocyte NADPH oxidase-mediated limitation of filamentous growth in vivo.     

Murine model of concurrent oral and vaginal Candida albicans colonization to study epithelial host-pathogen interactions

We report the creation of a new low-estrogen murine model of concurrent oral and vaginal C. albicans colonization that resembles human candidal carriage at both mucosal sites. Weekly estrogen administration of 5 microg intramuscular and subcutaneously was optimal for enhancement of oral colonization and was essential for vaginal colonization. In BALB/c mice, a number of C. albicans clinical isolates (n=3) colonized both oral and/or vaginal sites, but only strain 529L colonized 100% of mice persistently for over 5 weeks. Laboratory strains SC5314 and NCPF 3153 did not colonize the model; however, NCPF 3156 showed vaginal colonization up to week 5. Prior passaging through mice enhanced subsequent colonization of SC5314. Intranasal immunization with a C. albicans virulence antigen (secreted aspartyl proteinase 2) significantly reduced or abolished the fungal burden orally and vaginally by week 2 and 7. Our concurrent model of mucosal colonization reduces the numbers of experimental mice by half, can be used to assess potential vaccine candidates, and permits the detailed analysis of host-fungal interactions during the natural state of Candida colonization.     

Differential Interaction of the Two Related Fungal Species Candida albicans and Candida dubliniensis with Human Neutrophils

Candida albicans, the most common facultative human pathogenic fungus is of major medical importance, whereas the closely related species Candida dubliniensis is less virulent and rarely causes life-threatening, systemic infections. Little is known, however, about the reasons for this difference in pathogenicity, and especially on the interactions of C. dubliniensis with the human immune system. Because innate immunity and, in particular, neutrophil granulocytes play a major role in host antifungal defense, we studied the responses of human neutrophils to clinical isolates of both C. albicans and C. dubliniensis. C. dubliniensis was found to support neutrophil migration and fungal cell uptake to a greater extent in comparison with C. albicans, whereas inducing less neutrophil damage and extracellular trap formation. The production of antimicrobial reactive oxygen species, myeloperoxidase, and lactoferrin, as well as the inflammatory chemokine IL-8 by neutrophils was increased when stimulated with C. dubliniensis as compared with C. albicans. However, most of the analyzed macrophage-derived inflammatory and regulatory cytokines and chemokines, such as IL-1α, IL-1β, IL-1ra, TNF-α, IL-10, G-CSF, and GM-CSF, were less induced by C. dubliniensis. Similarly, the amounts of the antifungal immunity-related IL-17A produced by PBMCs was significantly lower when challenged with C. dubliniensis than with C. albicans. These data indicate that C. dubliniensis triggers stronger early neutrophil responses than C. albicans, thus providing insight into the differential virulence of these two closely related fungal species, and suggest that this is, in part, due to their differential capacity to form hyphae.     

Candida albicans colonization and dissemination from the murine gastrointestinal tract: the influence of morphology and Th17 immunity

The ability of Candida albicans to cause disease is associated with its capacity to undergo morphological transition between yeast and filamentous forms, but the role of morphology in colonization and dissemination from the gastrointestinal (GI) tract remains poorly defined. To explore this, we made use of wild‐type and morphological mutants of C. albicans in an established model of GI tract colonization, induced following antibiotic treatment of mice. Our data reveal that GI tract colonization favours the yeast form of C. albicans, that there is constitutive low level systemic dissemination in colonized mice that occurs irrespective of fungal morphology, and that colonization is not controlled by Th17 immunity in otherwise immunocompetent animals. These data provide new insights into the mechanisms of pathogenesis and commensalism of C. albicans, and have implications for our understanding of human disease.     

The Hsp90 Co-Chaperone Sgt1 Governs Candida albicans Morphogenesis and Drug Resistance

The molecular chaperone Hsp90 orchestrates regulatory circuitry governing fungal morphogenesis, biofilm development, drug resistance, and virulence. Hsp90 functions in concert with co-chaperones to regulate stability and activation of client proteins, many of which are signal transducers. Here, we characterize the first Hsp90 co-chaperone in the leading human fungal pathogen, Candida albicans. We demonstrate that Sgt1 physically interacts with Hsp90, and that it governs C. albicans morphogenesis and drug resistance. Genetic depletion of Sgt1 phenocopies depletion of Hsp90, inducing yeast to filament morphogenesis and invasive growth. Sgt1 governs these traits by bridging two morphogenetic regulators: Hsp90 and the adenylyl cyclase of the cAMP-PKA signaling cascade, Cyr1. Sgt1 physically interacts with Cyr1, and depletion of either Sgt1 or Hsp90 activates cAMP-PKA signaling, revealing the elusive link between Hsp90 and the PKA signaling cascade. Sgt1 also mediates tolerance and resistance to the two most widely deployed classes of antifungal drugs, azoles and echinocandins. Depletion of Sgt1 abrogates basal tolerance and acquired resistance to azoles, which target the cell membrane. Depletion of Sgt1 also abrogates tolerance and resistance to echinocandins, which target the cell wall, and renders echinocandins fungicidal. Though Sgt1 and Hsp90 have a conserved impact on drug resistance, the underlying mechanisms are distinct. Depletion of Hsp90 destabilizes the client protein calcineurin, thereby blocking crucial responses to drug-induced stress; in contrast, depletion of Sgt1 does not destabilize calcineurin, but blocks calcineurin activation in response to drug-induced stress. Sgt1 influences not only morphogenesis and drug resistance, but also virulence, as genetic depletion of C. albicans Sgt1 leads to reduced kidney fungal burden in a murine model of systemic infection. Thus, our characterization of the first Hsp90 co-chaperone in a fungal pathogen establishes C. albicans Sgt1 as a global regulator of morphogenesis and drug resistance, providing a new target for treatment of life-threatening fungal infections.     

A novel role for the NLRC4 inflammasome in mucosal defenses against the fungal pathogen Candida albicans

Candida sp. are opportunistic fungal pathogens that colonize the skin and oral cavity and, when overgrown under permissive conditions, cause inflammation and disease. Previously, we identified a central role for the NLRP3 inflammasome in regulating IL-1β production and resistance to dissemination from oral infection with Candida albicans. Here we show that mucosal expression of NLRP3 and NLRC4 is induced by Candida infection, and up-regulation of these molecules is impaired in NLRP3 and NLRC4 deficient mice. Additionally, we reveal a role for the NLRC4 inflammasome in anti-fungal defenses. NLRC4 is important for control of mucosal Candida infection and impacts inflammatory cell recruitment to infected tissues, as well as protects against systemic dissemination of infection. Deficiency in either NLRC4 or NLRP3 results in severely attenuated pro-inflammatory and antimicrobial peptide responses in the oral cavity. Using bone marrow chimeric mouse models, we show that, in contrast to NLRP3 which limits the severity of infection when present in either the hematopoietic or stromal compartments, NLRC4 plays an important role in limiting mucosal candidiasis when functioning at the level of the mucosal stroma. Collectively, these studies reveal the tissue specific roles of the NLRP3 and NLRC4 inflammasome in innate immune responses against mucosal Candida infection.     

Neutrophil Fc gamma RI as target for immunotherapy of invasive candidiasis

Invasive candidiasis represents a life-threatening disease for immunocompromised patients. This study focused on new immunotherapeutic approaches for systemic Candida albicans infections in a human FcgammaRI-transgenic mouse model. FcgammaRI (CD64) is a potent immunoactivating receptor on phagocytic and dendritic cells. In vivo targeting of C. albicans toward neutrophil-FcgammaRI by bispecific Abs and G-CSF effectively protected FcgammaRI-transgenic mice from lethal candidiasis. Nontransgenic mice were not protected, and treatment with bispecific Ab or G-CSF alone did not reduce mortality. Furthermore, infected FcgammaRI-transgenic mice developed high titers of anti-C. albicans IgG, and survival was extended on secondary infection without further treatment. These findings document the capacity of FcgammaRI to initiate potent anti-C. albicans immunity and support the development of FcgammaRI-directed immunotherapy of invasive fungal disease.     

IL-13 attenuates gastrointestinal candidiasis in normal and immunodeficient RAG-2(-/-) mice via peroxisome proliferator-activated receptor-gamma activation

We recently demonstrated that in vitro peroxisome proliferator-activated receptor-gamma (PPARgamma) activation of mouse peritoneal macrophages by IL-13 or PPARgamma ligands promotes uptake and killing of Candida albicans through mannose receptor overexpression. In this study, we demonstrate that i.p. treatment of immunocompetent and immunodeficient (RAG-2(-/-)) mice with natural and synthetic PPARgamma-specific ligands or with IL-13 decreases C. albicans colonization of the gastrointestinal (GI) tract 8 days following oral infection with the yeast. We also showed that Candida GI infection triggers macrophage recruitment in cecum mucosa. These mucosal macrophages, as well as peritoneal macrophages, overexpress the mannose receptor after IL-13 and rosiglitazone treatments. The treatments promote macrophage activation against C. albicans as suggested by the increased ability of peritoneal macrophages to phagocyte C. albicans and to produce reactive oxygen intermediates after yeast challenge. These effects on C. albicans GI infection and on macrophage activation are suppressed by treatment of mice with GW9662, a selective PPARgamma antagonist, and are reduced in PPARgamma(+/-) mice. Overall, these data demonstrate that IL-13 or PPARgamma ligands attenuate C. albicans infection of the GI tract through PPARgamma activation and hence suggest that PPARgamma ligands may be of therapeutic value in esophageal and GI candidiasis in immunocompromised patients.     

Antifungal type 1 responses are upregulated in IL-10-deficient mice

C57BL/6 mice are highly resistant to infections caused by Candida albicans and Aspergillus fumigatus. To elucidate the role of IL-10 produced by C57BL/6 mice during these infections, parameters of infection and immunity to it were evaluated in IL-10-deficient and wild-type mice with disseminated or gastrointestinal candidiasis or invasive pulmonary aspergillosis. Unlike parasitic protozoan infection, C. albicans or A. fumigatus infection did not induce significant acute toxicity in IL-10-deficient mice, who, instead, showed reduced fungal burden and fungal-associated inflammatory responses. The increased resistance to infections as compared to wild-type mice was associated with upregulation of innate and acquired antifungal Th1 responses, such as a dramatically higher production of IL-12, nitric oxide (NO) and TNF-alpha as well as IFN-gamma by CD4+ T cells. Pharmacological inhibition of NO production greatly reduced resistance to gastrointestinal candidiasis, thus pointing to the importance of IL-10-dependent NO regulation at mucosal sites in fungal infections. These results are reminiscent of those obtained in genetically susceptible mice, in which IL-10 administration increased, and IL-10 neutralization decreased, susceptibility to C. albicans and A. fumigatus infections. Collectively, these observations indicate that the absence of IL-10 augments innate and acquired antifungal immunity by upregulating type 1 cytokine responses. The resulting protective Th1 responses lead to a prompt reduction of fungal growth, thus preventing tissue destruction and lethal levels of proinflammatory cytokines.     

Induction of ERK-kinase signalling triggers morphotype-specific killing of Candida albicans filaments by human neutrophils

Candida albicans is among the most important fungal pathogens in humans. Morphological plasticity has been linked to its pathogenic potential as filamentous forms are associated with tissue invasion and infection. Here we show that human neutrophils discriminate between yeasts and filaments of C. albicans . Whereas filaments induced targeted motility, resulting in the establishment of close contact between neutrophils and fungal cells, yeast forms were largely ignored during coincubation. In transwell assays, C. albicans filaments induced significantly higher migratory activity in neutrophils than yeasts. Neutrophil motility based on actin rearrangement was essential for killing of C. albicans filaments but not involved in killing of yeast forms. Using inhibitors for MAP-kinase cascades, it was shown that recognition of C. albicans filaments by neutrophils is mediated via the MEK/ERK cascade and independent of JNK or p38 activation. Inhibition of the ERK signalling pathway abolished neutrophil migration induced by C. albicans filaments and selectively impaired the ability to kill this morphotype. These data show that invasive filamentous forms of C. albicans trigger a morphotype-specific activation of neutrophils, which is strongly dependent on neutrophil motility. Therefore, human neutrophils are capable of sensing C. albicans invasion and initiating an appropriate early immune response.     

Production of pyruvate by Candida albicans: proposed role in virulence

Proposed herein is a mechanism for virulence by Candida albicans based upon this organism's ability to produce high levels of pyruvate, potentially resulting in localized tissue ketosis and undermining the normal defensive function of neutrophil myeloperoxidase. Neutrophils, a key component of our innate defense against microbial infections, seem to play a particularly important role protecting us against fungal agents such as C. albicans. In this regard, it is myeloperoxidase which is central to many of the antimicrobial properties of neutrophils. We have previously shown that metabolic ketones inactivate myeloperoxidase and impair phagocytosis. Thus, production of pyruvate by C. albicans may indeed be a significant virulence factor.     

Protection of mice from lethal endogenous Candida albicans infection by immunization with Candida membrane antigen

The protective effects of immunization with Candida membrane antigen (CMA) on a systemic infection originating from intestinally colonized Candida albicans were examined. The colonization of orally inoculated C. albicans in the intestinal tract was established in BALB/c mice that had been concomitantly treated with oral doses of antibacterial drugs. In these animals, a systemic dissemination of C. albicans with fatal outcome was induced by a repeated dosing of prednisolone. In this endogenous infection model, the effects of immunization by CMA on the infection were examined. CMA-immunized mice showed a longer lifespan than unimmunized mice. The protective effect of CMA immunization in immunosuppressed mice was also measured by a decrease in body weight loss after treatment with prednisolone and in the number of viable Candida cells in the target organs, the kidneys and livers. However, the CFU of C. albicans in the intestinal tract was not significantly lowered. These results suggest that CMA immunization inhibited the dissemination of systemic Candida infection from the intestinal tract induced by treatment with prednisolone.