Rosalía Diez-Orejas

Integrated Proteomics and Genomics Strategies Bring New Insight into Candida albicans Response upon Macrophage Interaction

The interaction of Candida albicans with macrophages is considered a crucial step in the development of an adequate immune response in systemic candidiasis. An in vitro model of phagocytosis that includes a differential staining procedure to discriminate between internalized and non-internalized yeast was developed. Upon optimization of a protocol to obtain an enriched population of ingested yeasts, a thorough genomics and proteomics analysis was carried out on these cells. Both proteins and mRNA were obtained from the same sample and analyzed in parallel. The combination of two-dimensional PAGE with MS revealed a total of 132 differentially expressed yeast protein species upon macrophage interaction. Among these species, 67 unique proteins were identified. This is the first time that a proteomics approach has been used to study C. albicans-macrophage interaction. We provide evidence of a rapid protein response of the fungus to adapt to the new environment inside the phagosome by changing the expression of proteins belonging to different pathways. The clear down-regulation of the carbon-compound metabolism, plus the up-regulation of lipid, fatty acid, glyoxylate, and tricarboxylic acid cycles, indicates that yeast shifts to a starvation mode. There is an important activation of the degradation and detoxification protein machinery. The complementary genomics approach led to the detection of specific pathways related to the virulence of Candida. Network analyses allowed us to generate a hypothetical model of Candida cell death after macrophage interaction, highlighting the interconnection between actin cytoskeleton, mitochondria, and autophagy in the regulation of apoptosis. In conclusion, the combination of genomics, proteomics, and network analyses is a powerful strategy to better understand the complex host-pathogen interactions.

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 (efg1Δ/cph1Δ). 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 Candidas 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 efg1Δ/cph1Δ mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO.

Phenotypic characterization of a Candida albicans strain deficient in its major exoglucanase

Both alleles of the XOG1 gene of Candida albicans, which encodes a protein with exoglucanase activity, were sequentially disrupted. Enzymic analysis of either cell extracts or culture supernatants of disrupted strains revealed that this gene is responsible for the major exoglucanase activity in C. albicans, although residual exoglucanase activity could still be detected. xog1 null mutants showed similar growth rates in both rich and minimal liquid medium as compared to the wild-type strain, indicating that the enzyme is not essential for C. albicans growth. In addition, no differences were observed between wild-type and xog1 null mutants with respect to their ability to undergo dimorphic transition. However, small but repeatable differences were found between the wild-type and the null mutant with respect to susceptibility to chitin and glucan synthesis inhibitors. Using a murine model of experimental infection, no significant differences in virulence were observed. The xog1 null strain is thus a suitable recipient for studying Candida gene expression using the exoglucanase as a reporter gene.

Immunoproteomic analysis of the protective response obtained from vaccination with Candida albicans ecm33 cell wall mutant in mice.

Systemic candidiasis remains a major cause of disease and death, particularly among immunocompromised patients. The cell wall of Candida albicans defines the interface between host and pathogen and surface proteins are major elicitors of host immune responses during candidiasis. The C. albicans ecm33 mutant (RML2U) presents an altered cell wall, which entails an increase in the outermost protein layer. Vaccination of BALB/c mice with RML2U mutant protected them from a subsequent lethal infection with virulent strain SC5314 in a systemic candidiasis model. Using immunoproteomics (2‐DE followed by Immunoblotting) we detected 29 immunoreactive proteins specifically recognized by antibodies from vaccinated mice sera, six of which are described as immunogenic for the first time (Gnd1p, Cit1p, Rpl10Ep, Yst1p, Cys4p, Efb1p). Furthermore, identification of wild type and mutant cell surface proteome (surfome), confirmed us that the mutant surfome presented a larger number of proteins than the wild type. Interestingly, proteins exclusively identified in the mutant surfome (Met6p, Eft2p, Tkl1p, Rpl10Ep, Atp1p, Atp2p) were also detected as immunogenic, supporting the idea that their surface location enhances their immunoprotective capacity.

Estradiol impairs the Th17 immune response against Candida albicans

Candida albicans is a commensal opportunistic pathogen that is also a member of gastrointestinal and reproductive tract microbiota. Exogenous factors, such as oral contraceptives, hormone replacement therapy, and estradiol, may affect susceptibility to Candida infection, although the mechanisms involved in this process have not been elucidated. We used a systemic candidiasis model to investigate how estradiol confers susceptibility to infection. We report that estradiol increases mouse susceptibility to systemic candidiasis, as in vivo and ex vivo estradiol‐treated DCs were less efficient at up‐regulating antigen‐presenting machinery, pathogen killing, migration, IL‐23 production, and triggering of the Th17 immune response. Based on these results, we propose that estradiol impairs DC function, thus explaining the increased susceptibility to infection during estrus.

Combined Inactivation of the Candida albicans GPR1 and TPS2 Genes Results in Avirulence in a Mouse Model for Systemic Infection

ABSTRACT Inhibition of the biosynthesis of trehalose, a well-known stress protectant in pathogens, is an interesting approach for antifungal or antibacterial therapy. Deletion of TPS2, encoding trehalose-6-phosphate (T6P) phosphatase, results in strongly reduced virulence of Candida albicans due to accumulation of T6P instead of trehalose in response to stress. To further aggravate the deregulation in the pathogen, we have additionally deleted the GPR1 gene, encoding the nutrient receptor that activates the cyclic AMP-protein kinase A signaling pathway, which negatively regulates trehalose accumulation in yeasts. A gpr1 mutant is strongly affected in morphogenesis on solid media as well as in vivo in a mouse model but has only a slightly decreased virulence. The gpr1 tps2 double mutant, on the other hand, is completely avirulent in a mouse model for systemic infection. This strain accumulates very high T6P levels under stress conditions and has a growth defect at higher temperatures. We also show that a tps2 mutant is more sensitive to being killed by macrophages than the wild type or the gpr1 mutant. A double mutant has susceptibility similar to that of the single tps2 mutant. For morphogenesis on solid media, on the other hand, the gpr1 tps2 mutant shows a phenotype similar to that of the single gpr1 mutant. Taken together these results show that there is synergism between Gpr1 and Tps2 and that their combined inactivation results in complete avirulence. Combination therapy targeting both proteins may prove highly effective against pathogenic fungi with increased resistance to the currently used antifungal drugs.

Two different NO-dependent mechanisms account for the low virulence of a non-mycelial morphological mutant of Candida albicans

Abstract We have previously described the low virulence of a Candida albicans morphological mutant: 92′. We have now used this strain to examine the role of phagocytes in its pathogenesis. Our results show that C. albicans 92′ cannot evade innate host macrophage defence mechanisms as efficiently as the parental strain. In addition to the high susceptibility to phagocytosis by peritoneal macrophages, the NO produced by macrophages is a very important element in the low virulence of this agerminative mutant, a thesis supported by in vivo and in vitro experiments. Whereas the parental strain was able to inhibit macrophage NO production, the mutant was quite inefficient at reducing NO production by macrophages. In addition, the mutant showed high sensitivity to a NO generator. Treatment of mice with aminoguanidine (a preferred inducible NO synthase inhibitor) caused 90% mortality in 92′ systemic infection, thus supporting a role for NO in the low virulence of this strain. Our data show that both the low inhibitory effect of 92′ on macrophage NO production and the higher sensitivity to NO underlie the low virulence of this strain.

The Importance of the Phagocytes' Innate Response in Resolution of the Infection Induced by a Low Virulent Candida albicans Mutant

We have reported that a Candida albicans mkc1Δ/mkc1Δ mutant, deleted in the Mkc1p mitogen‐activated protein kinase, an essential element of the cell integrity signalling pathway, has reduced virulence in a murine model of systemic infection. We analyse here the immunological basis for this feature in view of its failure to vaccinate. Firstly, the influence of the Th response was analysed by infecting different knockout mice, revealing the importance of interferon‐γ in the resolution of mkc1 systemic infection. Secondly, the role of innate immunity was studied. The infection of neutropenic mice revealed that the candidacidal activity of neutrophils is crucial during the first 3 days of infection for the mutant strain. Macrophages played a critical role in the clearance of infection. Although a similar anti‐Candida activity was found for both fungal strains with naïve macrophages, activated macrophages discriminated between both strains. In vitro experiments revealed that the mutant strain displayed a greater susceptibility to nitric oxide (NO), a reduced inhibitory effect on macrophage NO production and an increased capacity of macrophage stimulation by cell wall extracts. The importance of NO in systemic infection with the mutant strain was confirmed by the strong increase in the susceptibility of aminoguanidine (an iNOs inhibitor)‐treated mice.

Candida albicans–macrophage interactions: genomic and proteomic insights

Candida albicans infection is a significant cause of morbidity and mortality in immunocompromised patients. In vivo and in vitro models have been developed to study both the fungal and the mammalian immune responses. Phagocytic cells (i.e., macrophages) play a key role in innate immunity against C. albicans by capturing, killing and processing the pathogen for presentation to T cells. The use of microarray technology to study global fungal transcriptional changes after interaction with different host cells has revealed how C. albicans adapts to its environment. Proteomic tools complement molecular approaches and computational methods enable the formulation of relevant biological hypotheses. Therefore, the combination of genomics, proteomics and bioinformatics tools (i.e., network analyses) is a powerful strategy to better understand the biological situation of the fungus inside macrophages; part of the fungal population is killed while a significantly high percentage survives.

Molecular analysis of an HLA-DP mutant cell line selected for its resistance to killing by HLA-DPw2-specific T-cell clones

A collection of HLA-DP mutants was generated, using ICR 191 as the mutagenic agent and resistance to lysis mediated by HLA-DPw2 allospecific cytotoxic T lymphocytes (CTLs) as the selection criterion. These mutants were derived from the HLA haploid lymphoblastoid cell line 45.1. Loss of HLA-DPw2 surface expression accounted for the lack of HLA-DPw2 CTL recognition in all the mutants. However, one of them, 45.EM19, binds to DPw2-specific monoclonal antibodies (mAb) after cell permeabilization. HLA-DPA1 and DPB1 mRNA expression studies permitted the classification of the mutants in four categories: 1) DPA1-negative, DPB1-positive; 2) DPA1-positive, DPB1-negative; 3) DPA1- and DPB1-negative, and 4) DPA1- and DPB1-positive mutants. Mutant 45.EM19 is included in the last group. The cloning and sequencing of the full-length DPA1 (DPA1*0103) and DPB1 (DPB1*02012) cDNAs from this mutant showed no changes in the DPA1 sequence compared to the wild-type sequence. However, a frame-shift mutation in the DPB1 gene exon coding for the transmembrane region was detected. The insertion of a guanine nucleotide provokes an extension of the open reading frame, increasing the length of the C-terminal domain and changing the hydropathicity pattern of the transmembrane domain. This change should be responsible for the phenotype of the 45.EM19 mutant.