A pinboard by
Sushna Maharjan

Associate Professor, Chitwan Medical College


Elimination of fungal infection depends on host defense mechanism by phagocytic cells.

Macrophages and neutrophils together may play a role in host defense against fungal infection; however neither yeast nor filamentous forms affect the presence of neutrophils and macrophages. Thus, it indicates both yeast and filamentous forms may have pathogenic effects.


Functional genomic analysis of Candida glabrata-macrophage interaction: role of chromatin remodeling in virulence.

Abstract: Fungal septicemia is an increasingly common complication of immunocompromised patients worldwide. Candida species are the leading cause of invasive mycoses with Candida glabrata being the second most frequently isolated Candida species from Intensive Care Unit patients. Despite its clinical importance, very little is known about the mechanisms that C. glabrata employs to survive the antimicrobial and immune response of the mammalian host. Here, to decipher the interaction of C. glabrata with the host immune cells, we have screened a library of 18,350 C. glabrata Tn7 insertion mutants for reduced survival in human THP-1 macrophages via signature-tagged mutagenesis approach. A total of 56 genes, belonging to diverse biological processes including chromatin organization and golgi vesicle transport, were identified which are required for survival and/or replication of C. glabrata in macrophages. We report for the first time that C. glabrata wild-type cells respond to the intracellular milieu of macrophage by modifying their chromatin structure and chromatin resistance to micrococcal nuclease digestion, altered epigenetic signature, decreased protein acetylation and increased cellular lysine deacetylase activity are the hall-marks of macrophage-internalized C. glabrata cells. Consistent with this, mutants defective in chromatin organization (Cgrsc3-aΔ, Cgrsc3-bΔ, Cgrsc3-aΔbΔ, Cgrtt109Δ) and DNA damage repair (Cgrtt107Δ, Cgsgs1Δ) showed attenuated virulence in the murine model of disseminated candidiasis. Further, genome-wide transcriptional profiling analysis on THP-1 macrophage-internalized yeasts revealed deregulation of energy metabolism in Cgrsc3-aΔ and Cgrtt109Δ mutants. Collectively, our findings establish chromatin remodeling as a central regulator of survival strategies which facilitates a reprogramming of cellular energy metabolism in macrophage-internalized C. glabrata cells and provide protection against DNA damage.

Pub.: 24 Aug '12, Pinned: 06 Mar '18

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

Abstract: 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.

Pub.: 24 Nov '07, Pinned: 06 Mar '18

Candida albicans morphogenesis is not required for macrophage interleukin 1β production.

Abstract: The interaction of Candida albicans with macrophages induces the production of interleukin 1β (IL-1β) through inflammasome activation in a process that is required for host survival. C. albicans hypha formation has been linked to IL-1β production, but the question of whether hyphae are sufficient to trigger IL-1β production has not been examined directly. To address this question, a C. albicans library of 165 transcription factor deletion mutants was screened for strains with altered IL-1β production by lipopolysaccharide (LPS)-primed J774 cells, a murine macrophage-like cell line. Eight mutants with decreased and two mutants with increased IL-1β secretion were identified. In addition, 12 mutants with previously identified morphology deficits were found to induce IL-1β secretion to levels similar to those of the wild type. Examination of the morphology of both low and normal IL-1β-inducing mutants in macrophages revealed that two mutants (upc2Δ/upc2Δ and ahr1Δ/Δ mutants) were indistinguishable from the wild type with respect to morphology yet induced low levels of IL-1β; conversely, the ndt80Δ/Δ mutant was deficient for hypha formation but induced levels of IL-1β similar to those of the wild type. Transcription factor mutants deficient for IL-1β secretion also caused markedly lower levels of macrophage lysis. Similarly, the ability of a mutant to cause macrophage lysis was independent of its ability to form hyphae. Taken together, our observations indicate that the physical formation of hyphae is not sufficient to trigger IL-1β secretion or macrophage lysis and suggest that other mechanisms, such as pyroptosis, a caspase-1-dependent response to intracellular pathogens, may play a role in the interaction of macrophages with C. albicans.The ability of Candida albicans to transition from yeast to filamentous cells plays an important and complex role in pathogenesis. Recent results from a number of investigators indicate that the host responds to yeast and hyphal C. albicans differently. For example, a C. albicans mutant unable to form hyphae also fails to induce interleukin 1β (IL-1β) secretion from macrophages. We have identified C. albicans transcription factor mutants that have decreased IL-1β secretion but retain the ability to form hyphae in response to macrophages. In addition, these mutants cause significantly less macrophage lysis. These observations indicate that the physical presence of the hyphal structure in the macrophage is not sufficient to trigger IL-1β secretion nor does it cause physical lysis of the cell. Our data indicate that characteristics of hyphae separate from its physical morphology are responsible for triggering the release of IL-1β release and causing macrophage lysis. Since these observations are inconsistent with some current models, alternative mechanisms for the interaction of C. albicans with macrophages must be considered.

Pub.: 28 Dec '12, Pinned: 06 Mar '18

Candida albicans actively modulates intracellular membrane trafficking in mouse macrophage phagosomes.

Abstract: The intracellular trafficking/survival strategies of the opportunistic human pathogen Candida albicans are poorly understood. Here we investigated the infection of RAW264.7 macrophages with a virulent wild-type (WT) filamentous C. albicans strain and a hyphal signalling-defective mutant (efg1Delta/cph1Delta). A comparative analysis of the acquisition by phagosomes of actin, and of early/late endocytic organelles markers of the different fungal strains was performed and related to Candida's survival inside macrophages. Our results show that both fungal strains have evolved a similar mechanism to subvert the 'lysosomal' system, as seen by the inhibition of the phagosome fusion with compartments enriched in the lysobisphosphatidic acid and the vATPase, and thereby the acquisition of a low pH from the outset of infection. Besides, the virulent WT strain displayed additional specific survival strategies to prevent its targeting to compartmentsdisplaying late endosomal/lysosomal features, such as induction of active recycling out of phagosomes of the lysosomal membrane protein LAMP-1, the lysosomal protease cathepsin D and preinternalized colloidal gold. Finally, both virulent and efg1Delta/cph1Delta mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO.

Pub.: 13 Jan '09, Pinned: 06 Mar '18

Immune response to fungal infections.

Abstract: The immune mechanisms of defence against fungal infections are numerous, and range from protective mechanisms that were present early in evolution (innate immunity) to sophisticated adaptive mechanisms that are induced specifically during infection and disease (adaptive immunity). The first-line innate mechanism is the presence of physical barriers in the form of skin and mucous membranes, which is complemented by cell membranes, cellular receptors and humoral factors. There has been a debate about the relative contribution of humoral and cellular immunity to host defence against fungal infections. For a long time it was considered that cell-mediated immunity (CMI) was important, but humoral immunity had little or no role. However, it is accepted now that CMI is the main mechanism of defence, but that certain types of antibody response are protective. In general, Th1-type CMI is required for clearance of a fungal infection, while Th2 immunity usually results in susceptibility to infection. Aspergillosis, which is a disease caused by the fungus Aspergillus, has been the subject of many studies, including details of the immune response. Attempts to relate aspergillosis to some form of immunosuppression in animals, as is the case with humans, have not been successful to date. The defence against Aspergillus is based on recognition of the pathogen, a rapidly deployed and highly effective innate effector phase, and a delayed but robust adaptive effector phase. Candida albicans, part of the normal microbial flora associated with mucous surfaces, can be present as congenital candidiasis or as acquired defects of cell-mediated immunity. Resistance to this yeast is associated with Th1 CMI, whereas Th2 immunity is associated with susceptibility to systemic infection. Dermatophytes produce skin alterations in humans and other animals, and the essential role of the CMI response is to destroy the fungi and produce an immunoprotective status against re-infection. The resolution of the disease is associated with a delayed hypersensitive response. There are many effective veterinary vaccines against dermatophytoses. Malassezia pachydermatis is an opportunistic yeast that needs predisposing factors to cause disease, often related to an atopic status in the animal. Two species can be differentiated within the genus Cryptococcus with immunologic consequences: C. neoformans infects predominantly immunocompromised hosts, and C. gattii infects non-immunocompromised hosts. Pneumocystis is a fungus that infects only immunosupressed individuals, inducing a host defence mechanism similar to that induced by other fungal pathogens, such as Aspergillus.

Pub.: 21 Jun '08, Pinned: 06 Mar '18

Enhanced killing of Candida albicans by human macrophages adherent to type 1 collagen matrices via induction of phagolysosomal fusion.

Abstract: Candida albicans, a component of the normal flora of the alimentary tract and mucocutaneous membranes, is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. As little is known about the regulation of monocyte/macrophage anti-Candida activity, we sought to determine if fungicidal activity might be regulated by extracellular matrix proteins to which monocytes/macrophages are adherent in vivo. Compared to monocyte/macrophages that adhered to plastic, human monocytes and monocyte-derived macrophages that adhered to type 1 collagen matrices, but not to fibronectin, vitronectin, or laminin, demonstrated a significant increase in candidacidal activity. The enhancement of monocyte fungicidal activity was maintained over a 4-h period, whereas macrophage fungicidal activity was maximum at 1 h. Although adherence of monocytes and macrophages to collagen matrices concomitantly enhanced the production of superoxide anion, only the fungicidal activity of collagen-adherent monocytes was partially blocked by superoxide dismutase and catalase. Remarkably, we found that only 10% of the phagosomes in C. albicans-infected macrophages that adhered to plastic fused with lysosomes. In contrast, 80% of yeast-containing phagosomes of collagen-adherent macrophages fused with lysosomes. These data suggest that nonoxidative mechanisms are critical for human macrophage anti-Candida activity and that C. albicans pathogenicity is mediated, in part, by its ability to inhibit phagolysosomal fusion in macrophages.

Pub.: 25 Jan '05, Pinned: 06 Mar '18

Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA.

Abstract: Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest that it does not play a major role in killing this fungus. Instead, NETs may have a fungistatic effect and may prevent further spreading.

Pub.: 06 May '10, Pinned: 06 Mar '18

Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects.

Abstract: Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response.

Pub.: 01 Jan '16, Pinned: 06 Mar '18

Phagocyte responses towards Aspergillus fumigatus.

Abstract: The saprophytic fungus Aspergillus fumigatus is a mold which is ubiquitously present in the environment. It produces large numbers of spores, called conidia that we constantly inhale with the breathing air. Healthy individuals normally do not suffer from true fungal infections with this pathogen. A normally robust resistance against Aspergillus is based on the presence of a very effective immunological defense system in the vertebrate body. Inhaled conidia are first encountered by lung-resident alveolar macrophages and then by neutrophil granulocytes. Both cell types are able to effectively ingest and destroy the fungus. Although some responses of the adaptive immune system develop, the key protection is mediated by innate immunity. The importance of phagocytes for defense against aspergillosis is also supported by large numbers of animal studies. Despite the production of aggressive chemicals that can extracellularly destroy fungal pathogens, the main effector mechanism of the innate immune system is phagocytosis. Very recently, the production of extracellular neutrophil extracellular traps (NETs) consisting of nuclear DNA has been added to the armamentarium that innate immune cells use against infection with Aspergillus. Phagocyte responses to Aspergillus are very broad, and a number of new observations have added to this complexity in recent years. To summarize established and newer findings, we will give an overview on current knowledge of the phagocyte system for the protection against Aspergillus.

Pub.: 17 May '11, Pinned: 06 Mar '18

Evaluation of the relationship between fungal infection, neutrophil leukocytes and macrophages in cervicovaginal smears: Light microscopic examination.

Abstract: Right after opportunistic fungi become pathogenic, they face immune system cells including macrophages and neutrophil leukocytes. Although the relationship between fungi and immune cells are being widely studied by using animal models and culture techniques, cervicovaginal smears have not been used to evaluate this interaction yet.The aim of this study was to investigate the interactions between fungal infection, macrophages and neutrophil leukocytes in cervicovaginal smear.Papanicolaou-stained cervicovaginal smears from 2307 women, aged between 18 and 73 years, were examined by light microscopy. Periodic acid-Schiff stain was also used to confirm the presence of fungal cell walls.Fungal infections were detected in 239 of 2307 patients (10.4%), and these cases were taken as the study group. Cases without any infectious agents (n = 1800, 78%) were considered as the control group. When the study and control groups were statistically compared in view of macrophages and neutrophil leukocytes, a significant relationship between presence of fungal infection, macrophages and neutrophil leukocytes was detected (P < 0.05). Furthermore, macrophages and neutrophil leukocytes were found to work against the fungal infection together (P < 0.05). Additionally, when the relationship between the existence of yeast or filamentous forms and these immune cells were evaluated, a significant correlation was not found (P > 0.05).Our findings indicate that macrophages and neutrophils may play a determining role in host defense against fungal infection together, but neither yeast nor filamentous forms affect the presence of neutrophil leukocytes and macrophages. As a result of this, both yeast and filamentous forms may have pathogenic effects.

Pub.: 01 Aug '15, Pinned: 06 Mar '18

The presence of eosinophil leucocytes in cervicovaginal smears with Actinomyces-like organisms: Light microscopic examination.

Abstract: Actinomyces species are part of mucosal surfaces of oral cavity, gastrointestinal and genital tracts. When these mucosal surfaces disrupt, Actinomyces become pathogen and cause infection. Eosinophil leucocytes participate in host defense against helminthic infestation and they generally play a role in asthma and allergy. However, the role of eosinophil leucocytes in host defense against bacteria is conflicting.To determine whether there is a relationship between Actinomyces-like organisms (ALOs) and eosinophil leucocytes at light microscopic level.Cervicovaginal samples obtained from 200 patients were examined by both Pap smear microscopy and anaerobic culturing. Since the results obtained by these methods were not concordant for diagnosis of genital Actinomyces, 6 of 200 patients (3%) diagnosed with ALOs by Pap smear microscopy became the study group. Patients without any infectious agents (n=134) were the control group. Statistical analyses were conducted with χ(2) test using SPSS program.The study and control groups were compared statistically in view of the presence of eosinophil leucocytes and it was found that there was a significant correlation between the presence of ALOs and eosinophil leucocytes (P<0.05). Abundant polymorphonuclear leucocytes (PMNLs) and macrophages were also detected in the study group.This study implies that eosinophil leucocytes might have a role in host defense against Actinomyces in addition to PMNLs and macrophages.

Pub.: 18 Jan '13, Pinned: 06 Mar '18