M. Luisa Gil

Serologic Response to Cell Wall Mannoproteins and Proteins of Candida albicans

SUMMARY The cell wall of Candida albicans not only is the structure in which many biological functions essential for the fungal cells reside but also is a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both the carbohydrate and protein moieties are able to trigger immune responses. Although cell-mediated immunity is often considered to be the most important line of defense against candidiasis, cell wall protein and glycoprotein components also elicit a potent humoral response from the host that may include some protective antibodies. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to influence profoundly the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins for host ligands. In this review, the various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo are examined. Although a number of proteins have been shown to stimulate an antibody response, for some of these species the response is not universal. On the other hand, some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidasis, particularly the disseminated form. In addition, recent studies have focused on the potential for antibodies to cell wall protein determinants to protect the host against infection. Hence, a better understanding of the humoral response to cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis and (ii) novel prophylactic (vaccination) and therapeutic strategies for the management of this type of infection.

Candida albicans triggers proliferation and differentiation of hematopoietic stem and progenitor cells by a MyD88-dependent signaling.

As TLRs are expressed by hematopoietic stem and progenitor cells, these receptors may play a role in hematopoiesis in response to pathogens during infection. We showed here that inactivated yeasts and hyphae of Candida albicans induce in vitro the proliferation of purified murine hematopoietic stem and progenitor cells (Lin(-)c-Kit(+) Sca-1(+)) as well as their differentiation to lineage positive cells, through a MyD88-dependent pathway. These results indicate that TLR-mediated recognition of C. albicans by hematopoietic stem and progenitor cells may augment the host capability for rapidly replenishing the innate immune system during candidiasis.

Changes in the cell wall glycoprotein composition of Candida albicans associated to the inhibition of germ tube formation by EDTA

Hyphal development in Candida albicans was blocked by EDTA. This effect was not due to a general growth inhibition since the chelator did not affect protein and DNA synthesis. Recovery of mycelial growth was observed when EDTA-grown cells were incubated at 37°C in EDTA-free medium. High-molecular-weight mannoproteins (HMWM) that are mycelium-specific wall components, and particularly a 260-kDa species (HMWM-260), were absent in the wall of cells grown under germination conditions in the presence of EDTA. Synthesis of the HMWM-260 species was not inhibited but its incorporation (secretion) into the wall structure seemed to be blocked in EDTA-treated cells.

Candida albicans TDH3 gene promotes secretion of internal invertase when expressed in Saccharomyces cerevisiae as a glyceraldehyde-3-phosphate dehydrogenase-invertase fusion protein

We have checked the ability of the Candida albicans GAPDH polypeptide, which lacks a conventional N‐terminal signal peptide, to reach the cell wall in Saccharomyces cerevisiae by using an intracellular form of the yeast invertase as a reporter protein. A hybrid TDH3–SUC2 gene containing the C. albicans TDH3 promoter sequences and a coding region encoding a fusion protein formed by the C. albicans GAPDH polypeptide, fused at its C‐terminus with the yeast internal invertase, was constructed in a centromer derivative plasmid and transformed into a Suc− S. cerevisiae strain. Transformants displayed invertase activity measured in intact whole cells, and were able to grow on sucrose as the sole fermentable carbon source. Northern blot analysis with both TDH3 and SUC2 probes detected a single mRNA species of the expected size (about 2.7 kb), and Western immunoblot analysis of cell‐free extracts, using a monoclonal antibody (mAb49) against a C. albicans GAPDH epitope, showed the presence of a 90 kDa polypeptide corresponding to the GAPDH‐invertase fusion protein. This indicates that the TDH3 gene is able to direct part of the encoded gene product to the cell wall, and that any putative motifs for this targeting should be within the GAPDH amino acid sequence. Further analysis, using the same approach, of a panel of seven N‐ and C‐terminal GAPDH truncates revealed that the region required for the cell wall targeting is located within the N‐terminal half of the protein. Copyright

TLRs control hematopoiesis during infection.

Recent research has shown that (i) Toll‐like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to proliferate and differentiate along the myeloid lineage in vitro, and (ii) direct TLR‐mediated stimulation of HSPCs also promotes macrophage differentiation in vivo following infection. These new insights demonstrate that TLR signaling in HSPCs, in addition to other TLR‐dependent mechanisms, can contribute to HSPC expansion and myeloid differentiation after infection. Evidence is, therefore, mounting that direct TLR‐induced programming of hematopoiesis plays a key role in host defense by rapidly replenishing the innate immune system with the cells needed to deal with pathogens.

Candida albicans Induces Selective Development of Macrophages and Monocyte Derived Dendritic Cells by a TLR2 Dependent Signalling

As TLRs are expressed by haematopoietic stem and progenitor cells (HSPCs), these receptors may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that in in vitro defined conditions inactivated yeasts and hyphae of Candida albicans induce HSPCs proliferation and differentiation towards the myeloid lineage by a TLR2/MyD88 dependent pathway. In this work, we showed that C. albicans invasive infection with a low virulence strain results in a rapid expansion of HSPCs (identified as LKS cells: Lin− c-Kit+ Sca-1+ IL-7Rα−), that reach the maximum at day 3 post-infection. This in vivo expansion of LKS cells in TLR2−/− mice was delayed until day 7 post- infection. Candidiasis was, as expected, accompanied by an increase in granulopoiesis and decreased lymphopoiesis in the bone marrow. These changes were more pronounced in TLR2−/− mice correlating with their higher fungal burden. Accordingly, emigration of Ly6Chigh monocytes and neutrophils to spleen was increased in TLR2−/− mice, although the increase in macrophages and inflammatory macrophages was completely dependent on TLR2. Similarly, we detected for the first time, in the spleen of C. albicans infected control mice, a newly generated population of dendritic cells that have the phenotype of monocyte derived dendritic cells (moDCs) that were not generated in TLR2−/− infected mice. In addition, C. albicans signalling through TLR2/MyD88 and Dectin-1 promotes in vitro the differentiation of Lin− cells towards moDCs that secrete TNF-α and are able to kill the microorganism. Therefore, our results indicate that during infection C. albicans can directly stimulate progenitor cells through TLR2 and Dectin-1 to generate newly formed inflammatory macrophages and moDCs that may fulfill an essential role in defense mechanisms against the pathogen.

Killed Candida albicans Yeasts and Hyphae Inhibit Gamma Interferon Release by Murine Natural Killer Cells

ABSTRACT Killed yeasts and hyphae of Candida albicans inhibit gamma interferon secretion by highly purified murine NK cells in response to the Toll-like receptor ligands lipopolysaccharide and zymosan. This effect, which is also observed in the presence of NK-activating cytokines (interleukin-2 [IL-2], IL-12, and IL-15), may represent a novel mechanism of immune evasion that contributes to the virulence of C. albicans.

Retinal microglia are activated by systemic fungal infection

PURPOSE We determined whether systemic fungal infection could cause activation of retinal microglia and, therefore, could be potentially harmful for patients with retinal degenerative diseases. METHODS Activation of retinal microglia was measured in a model of sublethal invasive candidiasis in C57BL/6J mice by confocal immunofluorescence and flow cytometry analysis, using anti-CD11b, anti-Iba1, anti-MHCII, and anti-CD45 antibodies. RESULTS Systemic fungal infection causes activation of retinal microglia, with phenotypic changes in morphology, surface markers expression, and microglial relocation in retinal layers. CONCLUSIONS As an excessive or prolonged microglial activation may lead to chronic inflammation with severe pathological side effects, causing or worsening the course of retinal dystrophies, a systemic infection may represent a risk factor to be considered in patients with ocular neurodegenerative diseases, such as diabetic retinopathy, glaucoma, age-related macular degeneration, or retinitis pigmentosa.

In Vivo and In Vitro Studies on Virulence and Host Responses to Saccharomyces cerevisiae Clinical and Non-Clinical Isolates

We have studied the virulence and host responses to several clinical and non-clinical Saccharomyces cerevisiae isolates: two vaginal isolates (60, 61), one isolate from faeces (20), a brewers yeast isolate used in dietetics (D14), one S. boulardii isolate from a commercial probiotic product, and a reference natural wine yeast (CECT 10431). Hematogenously disseminated infection in a mouse model demonstrated that four isolates (all, except 20 and 10431) were able to colonize preferentially the brain, as well as kidney and spleen, to a lesser extent, of immunocompetent mice. In vitro adhesion assays to epithelial and endothelial cell lines also showed an increased adherence ability of strains 60, 61, D14 and S. boulardii. In vitro cytokine production assays by RAW 264.7 murine macrophages challenged with yeasts showed a relative increased production of TNF- in response to the 20 and 10431 strains; viability of RAW cells after coculture was similar in all cases (2-5% non-viable cells) except for 60 strain (11% non-viable cells). In vitro phagocytosis assays of yeasts by RAW cells showed that two isolates (D14 and particularly S. boulardii) were engulfed less efficiently. These results point out that S. cerevisiae isolates, from both clinical and non-clinical (dietetic and probiotic) origin, may vary in the expression of putative virulence factors contributing to their ability to develop the infectious process.

Different Molecular Forms of Invertase in the slime Variant of Neuospora crassa: Comparison with the Wild-type Strain

SUMMARY: Invertase synthesis, regulation and secretion in the wall-less slime variant of Neurospora crassa was studied. Unlike the wild-type, synthesis of the enzyme was not repressed by glucose. This effect was not related to the os mutation harboured by the slime strain, nor to the phenotypic absence of a cell wall. Three molecular forms of extracellular invertase, which varied in size, were detected in the slime strain. These forms were interconvertible, with the equilibrium in favour of the larger form. Polypeptide analysis of the three separated forms revealed that all contained the same glycoprotein with an M r of 97000. This was completely deglycosylated by treatment with endo-β-N-acetylglucosaminidase H (Endo H) to a polypeptide with an M r of 72000. It was concluded that the three interconvertible forms correspond to the monomeric, dimeric and tetrameric states of the enzyme. Three similar forms of invertase, albeit of slightly different electrophoretic mobility, were found in cell-free extracts, cell walls and spent culture medium of the wild-type strain. After Endo H treatment, analysis showed that these forms contained a polypeptide that was equally reactive against anti-Saccharomyces cerevisiae antibodies, and had the same M r, as the polypeptide produced by the slime strain.