The relationship between yeast cell size and cell division in Candida albicans

The mean size and percentage of budded and unbudded cells of Candida albicans grown in batch culture over a wide range of doubling times have been measured. Cell volume decreased with increased doubling time and a nonlinear approach to an asymptotic minimum was observed. When cells were separated by age according to bud scars, each age showed a similar decrease. During each cell division cycle, size increased slowly during both budded and unbudded periods so that each generation was significantly larger than the preceding. There was no difference in size between the parent portion of budded cells and unbudded cells of the same age. Time-lapse photomicroscopy of cells growing on solid medium showed that cells divide asymmetrically with larger parents having a shorter subsequent cycle time than the smaller daughter, although the time utilized for bud formation was similar. When cells were shifted from a medium supporting a low growth rate and small size to a medium supporting a faster growth rate and larger size, both budded and unbudded cells increased significantly in size. As the doubling time increased, both the budded and unbudded portions of parental and daughter cycles increased.     

Importance of the Candida albicans cell wall during commensalism and infection

An imbalance of the normal microbial flora, breakage of epithelial barriers or dysfunction of the immune system favour the transition of the human pathogenic yeast Candida albicans from a commensal to a pathogen. C. albicans has evolved to be adapted as a commensal on mucosal surfaces. As a commensal it has also acquired attributes, which are necessary to avoid or overcome the host defence mechanisms. The human host has also co-evolved to recognize and eliminate potential fungal invaders. Many of the fungal genes that have been the focus of this co-evolutionary process encode cell wall components. In this review, we will discuss the transition from commensalism to pathogenesis, the key players of the fungal cell surface that are important for this transition, the role of the morphology and the mechanisms of host recognition and response.     

Homology,disruption and phenotypic analysis of CaGS Candida albicans gene induced during macrophage infection

During macrophage infection Candida albicans expresses differentially several genes whose functions are associated with its survival strategy. Among others, we have isolated CaGS gene, which is homologous to SNF3, a glucose sensor of Saccharomyces cerevisiae. To elucidate its potential role during infection, CaGS has been disrupted and the resulting phenotype analyzed on different solid media. The null mutant lost the ability to form hyphae on a medium with low glucose concentration and serum. Furthermore, this mutant does not disrupt macrophage in in vitro infections. We believe that this putative glucose sensor is involved in hyphal development during macrophage infection.     

Homology, disruption and phenotypic analysis of CaGS Candida albicans gene induced during macrophage infection

During macrophage infection Candida albicans expresses differentially several genes whose functions are associated with its survival strategy. Among others, we have isolated CaGS gene, which is homologous to SNF3, a glucose sensor of Saccharomyces cerevisiae. To elucidate its potential role during infection, CaGS has been disrupted and the resulting phenotype analyzed on different solid media. The null mutant lost the ability to form hyphae on a medium with low glucose concentration and serum. Furthermore, this mutant does not disrupt macrophage in in vitro infections. We believe that this putative glucose sensor is involved in hyphal development during macrophage infection.     

Candida albicans isolates with different genomic backgrounds display a differential response to macrophage infection

Few human pathogens possess the ability exhibited by Candida albicans to colonize and cause symptomatic infections at different body sites. The host immune system is the major factor determining whether this opportunistic yeast behaves as a commensal or as a pathogen, since C. albicans strains appear capable of expressing similar virulence factors in response to specific body-district cues. This report provides evidence showing that C. albicans isolates with diverse genomic backgrounds (b and c karyotypes) differently modulate their pathogenic potential when assayed in cocultures with human monocytic derived macrophages (THP-1 cells). Striking differences were observed in the ability to undergo bud-hypha transition, a relevant C. albicans virulence factor, between b and c karyotypes (P<0.0001) upon their internalization by macrophages. All c types were able to develop hyphal forms, resist intracellular killing, replicate, and escape from macrophages. The b type isolates, which were shown to be more efficiently ingested by THP-1 cells than the c type strains (P=0.013), were susceptible to intracellular killing and predominantly found as blastoconidia inside macrophages. Despite their different intracellular disposition, both b and c type isolates were equally able to undergo morphogenesis and to express NRG1 and HWP1 genes, markers of the bud-hypha transition program, during in vitro propagation. Since macrophages play a critical role in the host resistance to C. albicans, the different response of b and c isolates to macrophage infection suggests that the c type strains are better suited to behave as a more virulent strain cluster.     

Loss of cell wall mannosylphosphate in Candida albicans does not influence macrophage recognition

The outer layer of the cell wall of the human pathogenic fungus Candida albicans is enriched with heavily mannosylated glycoproteins that are the immediate point of contact between the fungus and cells of the host, including phagocytes. Previous work had identified components of the acid-labile fraction of N-linked mannan, comprising beta-1,2-linked mannose residues attached via a phosphodiester bond, as potential ligands for macrophage receptors and modulators of macrophage function. We therefore isolated and disrupted the CaMNN4 gene, which is required for mannosyl phosphate transfer and hence the attachment of beta-1,2 mannose oligosaccharides to the acid-labile N-mannan side chains. With the mannosylphosphate eliminated, the mnn4Delta null mutant was unable to bind the charged cationic dye Alcian Blue and was devoid of acid-labile beta-1,2-linked oligomannosaccharides. The mnn4Delta mutant was unaffected in cell growth and morphogenesis in vitro and in virulence in a murine model of systemic C. albicans infection. The null mutant was also not affected in its interaction with macrophages. Mannosylphosphate is therefore not required for macrophage interactions or for virulence of C. albicans.     

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.     

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。     

Influence of aging on murine neutrophil and macrophage function against Candida albicans

Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1beta, and tumor necrosis factor-alpha and chemokines (MIP-2) by purified (CD11b(+)) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.     

The Candida albicans vacuole is required for differentiation and efficient macrophage killing

Yeast-hypha differentiation is believed to be necessary for the normal progression of Candida albicans infections. The emergence and extension of a germ tube from a parental yeast cell are accompanied by dynamic changes in vacuole size and morphology. Although vacuolar function is required during this process, it is unclear if it is vacuolar expansion or some other vacuolar function that is important. We previously described a C. albicans vps11Delta mutant which lacked a recognizable vacuole compartment and with defects in multiple vacuolar functions. These include sensitivities to stress, reduced proteolytic activities, and severe defects in filamentation. Herein we utilize a partially functional VPS11 allele (vps11hr) to help define which vacuolar functions are required for differentiation and which influence interaction with macrophages. Mutant strains harboring this allele are not osmotically or temperature sensitive and have normal levels of secreted aspartyl protease and carboxypeptidase Y activity but have a fragmented vacuole morphology. Moreover, this mutant is defective in filamentation, suggesting that the major role the vacuole plays in yeast-hypha differentiation may relate directly to its morphology. The results of this study support the hypothesis that vacuole expansion is required during germ tube emergence. Both vps11 mutants were severely attenuated in their ability to kill a macrophage cell line. The viability of the vps11delta mutant was significantly reduced during macrophage interaction compared to that in the control strains, while the vps11hr mutant was unaffected. This implies some vacuolar functions are required for Candida survival within the macrophage, while additional vacuolar functions are required to inflict injury on the macrophage.     

抗系统性白念珠菌感染的先天性免疫

白念珠菌是引起浅部、深部真菌感染常见的病原菌.先天免疫反应在宿主抗系统性白念珠菌感染中起主导作用.介导宿主抗念珠菌感染的先天性免疫包括一系列真菌识别受体及免疫效应细胞.宿主对系统性白念珠菌感染的免疫反应是决定患者预后的关键.本文就宿主抗系统性白念珠菌感染的先天性免疫机制进行综述.     

Candida albicans actively modulates intracellular membrane trafficking in mouse macrophage phagosomes

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 ( efg1 Δ /cph1 Δ). 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 efg1 Δ /cph1 Δ mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO.     

Cell division cycle 20 promotes cell proliferation and invasion and inhibits apoptosis in osteosarcoma cells

Cdc20 (cell division cycle 20 homologue) has been reported to exhibit an oncogenic role in human tumorigenesis. However, the function of Cdc20 in osteosarcoma (OS) has not been investigated. In the current study, we aim to explore the role of Cdc20 in human OS cells. Multiple approaches were used to measure cell growth, apoptosis, cell cycle, migration and invasion in OS cells after depletion of Cdc20 or overexpression of Cdc20. We found that down-regulation of Cdc20 inhibited cell growth, induced apoptosis and triggered cell cycle arrest in OS cells. Moreover, Cdc20 down-regulation let to inhibition of cell migration and invasion in OS cells. Consistently, overexpression of Cdc20 in OS cells promoted cell growth, inhibited apoptosis, enhanced cell migration and invasion. Mechanistically, our Western blotting results showed that overexpression of Cdc20 reduced the expression of Bim and p21, whereas depletion of Cdc20 upregulated Bim and p21 levels in OS cells. Altogether, our findings demonstrated that Cdc20 exerts its oncogenic role partly due to regulation of Bim and p21 in OS cells, suggesting that targeting Cdc20 could be useful for the treatment of OS.     

Innate versus adaptive immunity in Candida albicans infection

Candida albicans is a common opportunistic pathogen, causing both superficial and systemic infection. Clinical observations indicate that mucocutaneous infections are commonly associated with defective cell-mediated immune responses, whereas systemic infection is more frequently seen in patients with deficiencies in neutrophil number or function. Analysis of mechanisms of host resistance against gastrointestinal and oral infection in mouse models has demonstrated an absolute dependence on CD4(+) T cells, although clearance also involves phagocytic cells. Both IL-12 and TNF-alpha appear to be important mediators, but mouse strain-dependent variations in susceptibility to infection may be related to T-cell enhancement of production of phagocytic cells by the bone marrow. In murine systemic infection, the role of innate and adaptive responses is less well defined. Studies in immunodeficient and T-cell-depleted mice suggest that clearance of the yeast may be predominantly a function of the innate response, whereas the adaptive response may either limit tissue damage or have the potential to cause immunopathology, depending on the host genetic context in which the infection takes place.     

Morphogenesis and cell cycle progression in Candida albicans

Candida albicans, an opportunistic human pathogen, displays three modes of growth: yeast, pseudohyphae and true hyphae, all of which differ both in morphology and in aspects of cell cycle progression. In particular, in hyphal cells, polarized growth becomes uncoupled from other cell cycle events. Yeast or pseudohyphae that undergo a cell cycle delay also exhibit polarized growth, independent of cell cycle progression. The Spitzenk?rper, an organelle composed of vesicles associated with hyphal tips, directs continuous hyphal elongation in filamentous fungal species and also in C. albicans hyphae. A polarisome mediates cell cycle dependent growth in yeast and pseudohyphae. Regulation of morphogenesis and cell cycle progression is dependent upon specific cyclins, all of which affect morphogenesis and some of which function specifically in yeast or hyphal cells. Future work will probably focus on the cell cycle checkpoints involved in connecting morphogenesis to cell cycle progression.