María Martínez-Esparza

Specific recognition of Candida albicans by macrophages requires galectin-3 to discriminate Saccharomyces cerevisiae and needs association with TLR2 for signaling.

Stimulation of cells of the macrophage lineage is a crucial step in the sensing of yeasts by the immune system. Glycans present in both Candida albicans and Saccharomyces cerevisiae cell walls have been shown to act as ligands for different receptors leading to different stimulating pathways, some of which need receptor co-involvement. However, among these ligand-receptor couples, none has been shown to discriminate the pathogenic yeast C. albicans. We explored the role of galectin-3, which binds C. albicans β-1,2 mannosides. These glycans are specifically and prominently expressed at the surface of C. albicans but not on S. cerevisiae. Using a mouse cell line and galectin-3-deleted cells from knockout mice, we demonstrated a specific enhancement of the cellular response to C. albicans compared with S. cerevisiae, which depended on galectin-3 expression. However, galectin-3 was not required for recognition and endocytosis of yeasts. In contrast, using PMA-induced differentiated THP-1, we observed that the presence of TLR2 was required for efficient uptake and endocytosis of both C. albicans and S. cerevisiae. TLR2 and galectin-3, which are expressed at the level of phagosomes containing C. albicans, were shown to be associated in differentiated macrophages after incubation with this sole species. These data suggest that macrophages differently sense C. albicans and S. cerevisiae through a mechanism involving TLR2 and galectin-3, which probably associate for binding of ligands expressing β-1,2 mannosides specific to the C. albicans cell wall surface.

Identification of a New Family of Genes Involved in β-1,2-Mannosylation of Glycans in Pichia pastoris and Candida albicans

Structural studies of cell wall components of the pathogenic yeast Candida albicans have demonstrated the presence of β-1,2-linked oligomannosides in phosphopeptidomannan and phospholipomannan. During C. albicans infection, β-1,2-oligomannosides play an important role in host/pathogen interactions by acting as adhesins and by interfering with the host immune response. Despite the importance of β-1,2-oligomannosides, the genes responsible for their synthesis have not been identified. The main reason is that the reference species Saccharomyces cerevisiae does not synthesize β-linked mannoses. On the other hand, the presence of β-1,2-oligomannosides has been reported in the cell wall of the more genetically tractable C. albicans relative, P. pastoris. Here we present the identification, cloning, and characterization of a novel family of fungal genes involved in β-mannose transfer. Employing in silico analysis, we identified a family of four related new genes in P. pastoris and subsequently nine homologs in C. albicans. Biochemical, immunological, and structural analyses following deletion of four genes in P. pastoris and deletion of four genes acting specifically on C. albicans mannan demonstrated the involvement of these new genes in β-1,2-oligomannoside synthesis. Phenotypic characterization of the strains deleted in β-mannosyltransferase genes (BMTs) allowed us to describe the stepwise activity of Bmtps and acceptor specificity. For C. albicans, despite structural similarities between mannan and phospholipomannan, phospholipomannan β-mannosylation was not affected by any of the CaBMT1–4 deletions. Surprisingly, depletion in mannan major β-1,2-oligomannoside epitopes had little impact on cell wall surface β-1,2-oligomannoside antigenic expression.

A study of CD33 (SIGLEC-3) antigen expression and function on activated human T and NK cells: two isoforms of CD33 are generated by alternative splicing

The expression of CD33, a restricted leukocyte antigen considered specific for myeloid lineage, has been studied extensively on lymphoid cells. We demonstrated that wide subsets of mitogen‐ or alloantigen‐activated human T and natural killer (NK) cells express CD33 at protein and nucleic acid levels. CD33+ and CD33– T and NK cell populations showed identical surface expression of activation markers such as CD25, CD28, CD38, CD45RO, or CD95. Myeloid and lymphoid CD33 cDNA were identical. However, lymphoid CD33 protein had lower molecular weight, suggesting cell type‐specific, post‐translational modifications. Additionally, reverse transcriptase‐polymerase chain reaction and Northern blot analysis showed an unknown CD33 isoform (CD33m) expressed on all CD33+ cell lines or T cell clones tested. CD33m was identical to CD33 (CD33M) in the signal peptide, the immunoglobulin (Ig) domain C2, the transmembrane, and the cytoplasmic regions but lacked the extracellular ligand‐binding variable Ig‐like domain encoded by the second exon. CD33m mRNA was mostly detected on NKL and myeloid cell lines but poorly expressed on B cell lines and T lymphocytes. The CD33m extracellular portion was successfully expressed as a soluble fusion protein on transfected human cells, suggesting a functional role on cell membranes. Cross‐linking of CD33 diminished the cytotoxic activity of NKL cells against K562 and P815 target cells, working as an inhibitory receptor on NK cells. These data demonstrate that CD33 expression is not restricted to the myeloid lineage and could exist as two different splicing variants, which could play an important role in the regulation of human lymphoid and myeloid cells.

Host responses to a versatile commensal: PAMPs and PRRs interplay leading to tolerance or infection by Candida albicans.

The molecular interactions between commensal microorganisms and their host are basically different from those triggered by pathogens since they involve tolerance. When the commensal is genetically equipped to become an opportunistic pathogen, as is the case with Candida albicans, the picture becomes more complex. In this case, the balance between protection and invasion depends on host reactivity to altered microbial expression of ligands interacting with innate immune sensors. Based on experimental evidence obtained with C. albicans, we discuss the different molecular processes involved in the sensing of this important opportunistic human pathogen by a panel of pattern recognition receptors (PRRs) according to the numerous pathogen‐associated molecular patterns (PAMPs) that can be exposed at its surface. Beneficial or deleterious immune responses that either maintain a commensal state or favour damage by the yeast result from this dynamic interplay.

Transforming growth factor-beta1 inhibits basal melanogenesis in B16/F10 mouse melanoma cells by increasing the rate of degradation of tyrosinase and tyrosinase-related protein-1

Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-β1 (TGF-β1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-β1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (Dopa) oxidase activities, to less than 30% of the control values but had no effect on dopachrome tautomerase activity (TRP-2). The inhibition affected to similar extents the Dopa oxidase activity associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This inhibition was noticeable between 1 and 3 h after the addition of the cytokine, and maximal after 6 h of treatment. The decrease in the enzymatic activity was paralleled by a decrease in the abundance of the TRP-1 and tyrosinase proteins. TGF-β1 mediated this effect by increasing the rate of degradation of tyrosinase and TRP-1. Conversely, after 48 h of treatment, the expression of the tyrosinase gene decreased only slightly, while TRP-1 and TRP-2 gene expression was not affected. An increased rate of proteolytic degradation of TRP-1 and tyrosinase seems the main mechanism accounting for the inhibitory effect of TGF-β1 on the melanogenic activity of B16/F10 cells.

The mouse silver locus encodes a single transcript truncated by the silver mutation

In mammals, melanin synthesis occurs mainly in highly differentiated cells, the epidermal melanocytes, where it is restricted to specialized organelles called melanosomes. Among the melanosomal proteins known to participate in the control of melanogenesis, those encoded at the silver locus (Kwon et al. 1991) are obviously important, since their impairment by the silver (si) mutation in mice results in premature graying of the hair owing to loss of follicular melanocytes (Kwon et al. 1991), but their function is disputed. It has been proposed that they are structural matrix proteins of the melanosome (Kobayashi et al. 1994). Other roles have been claimed, either as “stablins”, proteins retarding melanogenesis by stabilization of biosynthetic intermediates, or as 5,6dihydroxyindole-2-carboxylic acid polymerases (Chakraborty et al. 1996). The homologous human SILV or PMEL17 gene (OMIM 155550, GDB 6277709) has been widely studied, since its protein products were proposed as potential markers of human melanoma and as immunotherapy targets. An apparently melanocyte-specific cDNA sequence was originally reported by Kwon et al. (1991; accession no. M77348, name PMEL17, also termed D12S53E). It was mapped to human Chromosome (Chr) 12pter-q21, sufficiently close to the murine silver locus (Chr 10; MGI 98301) to suggest similarity. The gene encodes a type 1 transmembrane protein of 668 amino acids, with a potential signal peptide, and putative membrane anchor domain near the C-terminus. Soon thereafter, other cDNA clones were reported encoding a melanocyte-specific protein almost identical to PMEL17, termed either gp100 (as a 100-kDa glycoprotein; Adema et al. 1994), or ME20M (Maresh et al. 1994; accession no. M32295). These products were identical to each other. The amino acid differences from PMEL17 were one substitution (P274L) and the deletion of a heptapeptide (VPGILLT) located just before the transmembrane region (amino acids 588–594). Partial sequence analysis of genomic DNA indicated that gp100 and PMEL17 transcripts originated from a single gene via alternative splicing (Adema et al. 1994). This was confirmed once the full genomic organization and sequence of the human SILV locus were described (Bailin et al. 1996; Kim et al. 1996). The presence of the VPGILLT heptapeptide in PMEL17 but not in gp100 results from alternative mRNA splicing to two competing 38 splice acceptor sites, whose function and regulation are unknown. Both proteins share all other identifiable domains, including the cytosolic ExxPLL motif proposed as a melanosomal targeting signal (Xu et al. 1997), as well as ten Ser, Pro, and Thr-rich tandem repeats in the melanosomal portion. A murine si-derived cDNA named Pmel17 was also first cloned by Kwon et al. (1995; accession no. U14133). This sequence is called “Pmel17m” here for clarity. Comparison with the human sequences showed 77% nucleotide identity with PMEL17, but three main differences: (i) The central Ser/Thr/Pro-rich region is shorter in the murine protein; (ii) the predicted mouse “Pmel17m” protein lacks the heptapeptide distinguishing human PMEL17 from gp100, and so is actually a gp100 homolog; and (iii) there is a region of about 30 amino acids of low similarity to both human proteins in the C-terminal cytosolic domain. Other cDNAs encoding murine homologs of gp100 have been reported and were called “gp100” (Schreurs et al. 1997; Zhai et al. 1997). These were identical and similar to Pmel17m except that the 38 region of low similarity to the human sequences was missing. This was because in this region the murine gp100 sequence aligns numerically with the human, whereas there are three relative single-nucleotide deletions in Pmel17m, producing a local frameshift in the predicted protein compared with the human protein (Fig. 1, panel A). In the present work we will refer to this second murine gp100 homolog as “gp87” for clarity and in reference to the protein’s lower molecular weight determined by SDS-PAGE followed by Western immunoblotting (Schreurs et al. 1997, and data not shown). The murine silver mutation was described as a single base insertion in this same region, causing a frameshift and extension of the protein by 12 amino acids (Kwon et al. 1995). The predicted mutant protein lacks the putative melanosomal targeting signal, suggesting misrouting of the protein. Interestingly, this insertion occurs exactly at the second position where Pmel17m has a deletion relative to gp87 (Fig. 1, panel A). The reported differences between gp87 and Pmel17m are surprising and difficult to explain. It has not been shown whether these sequences can be expressed simultaneously or are allelic, nor whether any murine homolog of PMEL17, the longer human splicing variant, exists. This lack of knowledge of the exact nature and relationships of the murine silver products is a serious drawback for certain immunotherapy studies. We have, therefore, reexamined both the normal and silver mutant sequences, at the cDNA and genomic levels. In order to detect murine silver transcripts, we first amplified cDNAs from B16-F10 melanoma cells, wild-type at the silver locus. The primers used (SF1/SR1) were common to the reported Pmel17m and gp87 transcripts and were chosen to amplify the Correspondence to: J.C. Garcia-Borron

Transforming growth factor β1 mediates hypopigmentation of B16 mouse melanoma cells by inhibition of melanin formation and melanosome maturation

Transforming growth factor-beta1 (TGFbeta1) downregulates tyrosinase in B16 melanoma cells by decreasing gene expression and the intracellular half-life of the enzyme, but does not block tyrosinase stimulation by alpha-melanocyte stimulating hormone (alphaMSH). In the presence of both agents, the enzymatic activity is intermediate between the one of cells treated with either agent alone. Here we show that TGFbeta1 equally inhibits the melanogenic activities of melan-a melanocytes and B16 melanoma cells, thus validating the B16 model. In both cell types, TGFbeta1 (10(-10) M, 48 h) inhibited to comparable levels tyrosine hydroxylation and melanin formation from L-tyrosine. Thus, the inhibitory effect is exerted mainly at the rate limiting step of the pathway. By means of quantitative image analysis techniques, we also studied the effects of TGFbeta1 and alphaMSH on melanosome number, volume density and maturation degree. alphaMSH (10(-7) M, 48 h) increased 7-fold melanosome volume density, whereas TGFbeta1 by itself had no significant effect. However, melanosomal volume density was intermediate in cells treated with both agents, as compared to control or alphaMSH-treated cells. Moreover, TGFbeta1 blocked the alphaMSH-elicited increase in the number of melanosomes. Control and alphaMSH-treated melanocytes contained more stage I+II premelanosomes and stage IV, fully melanized organelles than partially melanized stage III melanosomes. TGFbeta1 increased the percentage of stage III melanosomes. This trend was even more marked in cells treated with alphaMSH and TGFbeta1. The accumulation of incompletely melanized melanosomes is consistent with the inhibition of melanin formation activity by TGFbeta1 and with its hypopigmenting effect.

Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells

We have tested the usefulness of several commercial anti-CD33 monoclonal antibodies (mAb) to determine the expression and localization of the two CD33 isoforms on several hematopoietic cell lines. The expression of the isoform CD33m, a CD33 transmembrane splice variant lacking the ligand-binding V immunoglobulin (Ig)-like domain, was detected by RT-polymerase chain reaction, western blot, confocal microscopy and flow cytometry on the membrane of several human cell types. CD33m was only detected by the anti-CD33 mAb HIM3-4 on the cell surface, whereas WM53, P67.6, 4D3, HIM3-4, WM54, D3HL60.251 or MY9 detected the CD33M isoform, indicating that HIM3-4 is the only mAb recognizing CD33 C(2) Ig domain. Accordingly, HIM3-4 binding to CD33 did not interfere with the binding of other antibodies against the CD33 V-domain. P67.6 mAb interfered with recognition by the rest of antibodies specific for the V domain. HIM3-4 staining could be increased after the sialidase treatment of all CD33(+) cells. However, this increase was stronger in activated T cells, suggesting a CD33 masking state in this cell population. Confocal microscopy analysis of CD33m HEK 293T-transfected cells revealed that this protein is expressed on the cell membrane and also detected in the Golgi compartment. CD33 is constitutively located outside the lipid raft domains, whereas cross-linked CD33 is highly recruited to this signaling platform. The unique ability of HIM3-4 mAb to detect the masking state of CD33 on different cell lineages makes it a good tool to improve the knowledge of the biological role of this sialic acid-binding Ig-like lectin.

Role of trehalose-6P phosphatase (TPS2) in stress tolerance and resistance to macrophage killing in Candida albicans☆

Disruption of the TPS2 gene encoding the only trehalose-6P phosphatase activity in Candida albicans caused a pleiotropic defective phenotype, maintaining the cell wall integrity and the ability to form chlamydospores. A homozygous tps2Delta/tps2Delta showed reduced growth at high temperatures and a marked sensitivity to heat shock (42 degrees C) and severe oxidative exposure (50mM H(2)O(2)). Reintroduction of the TPS2 gene reversed these alterations. A more detailed study of the antioxidant response showed that exponential tps2Delta null cells displayed an adaptive response to oxidative stress as well as cross-tolerance between temperature and oxidative stress. Differential measurement of trehalose and trehalose-6P, using reliable new HPLC methodology, revealed a significant accumulation of trehalose-6P in tps2Delta cells, which was enhanced after oxidative exposure. In contrast, the level of trehalose-6P in parental cells was virtually undetectable, and oxidative treatment only induced the synthesis of free trehalose. A transitory increase in the expression of TPS2 and TPS1 genes was promoted in wild-type cells in response to acute (50mM) but not gentle (5mM) oxidative exposure. TPS1 and TPS2 oxidative-induced transcriptions were completely absent from the tps2Delta mutant. Exponential blastoconidia from both parental and tps2Delta/tps2Delta strains were completely phagocytosed by murine and human macrophages, triggering a subsequent proinflammatory response manifested by the release of TNF-alpha. Reflecting the lower resistance to oxidative stress displayed by the tps2Delta mutant, intracellular survival in resting and IFN-gamma and LPS-stimulated macrophages was also diminished. Taken together, our results confirm the mainly protective role played by the trehalose biosynthetic pathway in the cellular response to oxidative stress and subsequently in the resistance to phagocytosis in C. albicans, a defensive mechanism in which TPS2 would be involved.

Peritoneal macrophage priming in cirrhosis is related to ERK phosphorylation and IL-6 secretion

Eur J Clin Invest 2010; 41 (1): 8–15