Cek1 regulates ß(1,3)-glucan exposure through calcineurin effectors in Candida albicans.

In order to successfully induce disease, the fungal pathogen Candida albicans regulates exposure of antigens like the cell wall polysaccharide ß(1,3)-glucan to the host immune system. C. albicans covers (masks) ß(1,3)-glucan with a layer of mannosylated glycoproteins, which aids in immune system evasion by acting as a barrier to recognition by host pattern recognition receptors. Consequently, enhanced ß(1,3)-glucan exposure (unmasking) makes fungal cells more visible to host immune cells and facilitates more robust fungal clearance. However, an understanding of how C. albicans regulates its exposure levels of ß(1,3)-glucan is needed to leverage this phenotype. Signal transduction pathways and their corresponding effector genes mediating these changes are only beginning to be defined. Here, we report that the phosphatase calcineurin mediates unmasking of ß(1,3)-glucan in response to inputs from the Cek1 MAPK pathway and in response to caspofungin exposure. In contrast, calcineurin reduces ß-glucan exposure in response to high levels of extracellular calcium. Thus, depending on the input, calcineurin acts as a switchboard to regulate ß(1,3)-glucan exposure levels. By leveraging these differential ß(1,3)-glucan exposure phenotypes, we identified two novel effector genes in the calcineurin regulon, FGR41 and C1_11990W_A, that encode putative cell wall proteins and mediate masking/unmasking. Loss of either effector caused unmasking and attenuated virulence during systemic infection in mice. Furthermore, immunosuppression restored the colonization decrease seen in mice infected with the fgr41Δ/Δ mutant to wild-type levels, demonstrating a reliance on the host immune system for virulence attenuation. Thus, calcineurin and its downstream regulon are general regulators of unmasking.

Activation of Cph1 causes ß(1,3)-glucan unmasking in Candida albicans and attenuates virulence in mice in a neutrophil-dependent manner.

Masking the immunogenic cell wall epitope ß(1,3)-glucan under an outer layer of mannosylated glycoproteins is an important virulence factor deployed by Candida albicans during infection. Consequently, increased ß(1,3)-glucan exposure (unmasking) reveals C. albicans to the host's immune system and attenuates its virulence. We have previously shown that activation of the Cek1 MAPK pathway via expression of a hyperactive allele of an upstream kinase (STE11ΔN467) induced unmasking. It also increased survival of mice in a murine disseminated candidiasis model and attenuated kidney fungal burden by ≥33 fold. In this communication, we utilized cyclophosphamide-induced immunosuppression to test if the clearance of the unmasked STE11ΔN467 mutant was dependent on the host immune system. Suppression of the immune response by cyclophosphamide reduced the attenuation in fungal burden caused by the STE11ΔN467 allele. Moreover, specific depletion of neutrophils via 1A8 antibody treatment also reduced STE11ΔN467-dependent fungal burden attenuation, but to a lesser extent than cyclophosphamide, demonstrating an important role for neutrophils in mediating fungal clearance of unmasked STE11ΔN467 cells. In an effort to understand the mechanism by which Ste11ΔN467 causes unmasking, transcriptomics were used to reveal that several components in the Cek1 MAPK pathway were upregulated, including the transcription factor CPH1 and the cell wall sensor DFI1. In this report we show that a cph1ΔΔ mutation restored ß(1,3)-glucan exposure to wild-type levels in the STE11ΔN467 strain, confirming that Cph1 is the transcription factor mediating Ste11ΔN467-induced unmasking. Furthermore, Cph1 is shown to induce a positive feedback loop that increases Cek1 activation. In addition, full unmasking by STE11ΔN467 is dependent on the upstream cell wall sensor DFI1. However, while deletion of DFI1 significantly reduced Ste11ΔN467-induced unmasking, it did not impact activation of the downstream kinase Cek1. Thus, it appears that once stimulated by Ste11ΔN467, Dfi1 activates a parallel signaling pathway that is involved in Ste11ΔN467-induced unmasking.

Pathways That Synthesize Phosphatidylethanolamine Impact Candida albicans Hyphal Length and Cell Wall Composition through Transcriptional and Posttranscriptional Mechanisms.

Candida albicans is a leading cause of systemic bloodstream infections, and synthesis of the phospholipid phosphatidylethanolamine (PE) is required for virulence. The psd1Δ/Δ psd2Δ/Δ mutant, which cannot synthesize PE by the cytidine diphosphate diacylglycerol (CDP-DAG) pathway, is avirulent in the mouse model of systemic candidiasis. Similarly, an ept1Δ/Δ mutant, which cannot produce PE by the Kennedy pathway, exhibits decreased kidney fungal burden in systemically infected mice. Conversely, overexpression of EPT1 results in a hypervirulent phenotype in this model. Thus, mutations that increase PE synthesis increase virulence, and mutations that decrease PE synthesis decrease virulence. However, the mechanism by which virulence is regulated by PE synthesis is only partially understood. RNA sequencing was performed on strains with deficient or excessive PE biosynthesis to elucidate the mechanism. Decreased PE synthesis from loss of EPT1 or PSD1 and PSD2 leads to downregulation of genes that impact mitochondrial function. Losses of PSD1 and PSD2, but not EPT1, cause significant increases in transcription of glycosylation genes, which may reflect the substantial cell wall defects in the psd1Δ/Δ psd2Δ/Δ mutant. These accumulated defects could contribute to the decreased virulence observed for mutants with deficient PE synthesis. In contrast to mutants with decreased PE synthesis, there were no transcriptional differences between the EPT1 overexpression strain and the wild type, indicating that the hypervirulent phenotype is a consequence of posttranscriptional changes. It was found that overexpression of EPT1 causes increased chitin content and increased hyphal length. These phenotypes may help to explain the previously observed hypervirulence in the EPT1 overexpressor.

Exposure of Candida albicans ß (1,3)-glucan is promoted by activation of the Cek1 pathway.

Candida albicans is among the most common causes of human fungal infections and is an important source of mortality. C. albicans is able to diminish its detection by innate immune cells through masking of ß (1,3)-glucan in the inner cell wall with an outer layer of heavily glycosylated mannoproteins (mannan). However, mutations or drugs that disrupt the cell wall can lead to exposure of ß (1,3)-glucan (unmasking) and enhanced detection by innate immune cells through receptors like Dectin-1, the C-type signaling lectin. Previously, our lab showed that the pathway for synthesizing the phospholipid phosphatidylserine (PS) plays a role in ß (1,3)-glucan masking. The homozygous PS synthase knockout mutant, cho1Δ/Δ, exhibits increased exposure of ß (1,3)-glucan. Several Mitogen Activated Protein Kinase (MAPK) pathways and their upstream Rho-type small GTPases are important for regulating cell wall biogenesis and remodeling. In the cho1Δ/Δ mutant, both the Cek1 and Mkc1 MAPKs are constitutively activated, and they act downstream of the small GTPases Cdc42 and Rho1, respectively. In addition, Cdc42 activity is up-regulated in cho1Δ/Δ. Thus, it was hypothesized that activation of Cdc42 or Rho1 and their downstream kinases cause unmasking. Disruption of MKC1 does not decrease unmasking in cho1Δ/Δ, and hyperactivation of Rho1 in wild-type cells increases unmasking and activation of both Cek1 and Mkc1. Moreover, independent hyperactivation of the MAP kinase kinase kinase Ste11 in wild-type cells leads to Cek1 activation and increased ß (1,3)-glucan exposure. Thus, upregulation of the Cek1 MAPK pathway causes unmasking, and may be responsible for unmasking in cho1Δ/Δ.

Candida albicans Cannot Acquire Sufficient Ethanolamine from the Host To Support Virulence in the Absence of De Novo Phosphatidylethanolamine Synthesis.

Candida albicans mutants for phosphatidylserine (PS) synthase (cho1ΔΔ) and PS decarboxylase (psd1ΔΔ psd2ΔΔ) are compromised for virulence in mouse models of systemic infection and oropharyngeal candidiasis (OPC). Both of these enzymes are necessary to synthesize phosphatidylethanolamine (PE) by the de novo pathway, but these mutants are still capable of growth in culture media, as they can import ethanolamine from media to synthesize PE through the Kennedy pathway. Given that the host has ethanolamine in its serum, the exact mechanism by which virulence is lost in these mutants is not clear. There are two competing hypotheses to explain their loss of virulence. (i) PE from the Kennedy pathway cannot substitute for de novo-synthesized PE. (ii) The mutants cannot acquire sufficient ethanolamine from the host to support adequate PE synthesis. These hypotheses can be simultaneously tested if ethanolamine availability is increased for Candida while it is inside the host. We accomplish this by transcomplementation of C. albicans with the Arabidopsis thaliana serine decarboxylase gene (AtSDC), which converts cytoplasmic serine to ethanolamine. Expression of AtSDC in either mutant restores PE synthesis, even in the absence of exogenous ethanolamine. AtSDC also restores virulence to cho1ΔΔ and psd1ΔΔ psd2ΔΔ strains in systemic and OPC infections. Thus, in the absence of de novo PE synthesis, C. albicans cannot acquire sufficient ethanolamine from the host to support virulence. In addition, expression of AtSDC restores PS synthesis in the cho1ΔΔ mutant, which may be due to causing PS decarboxylase to run backwards and convert PE to PS.

Cytomegalovirus MCK-2 controls mobilization and recruitment of myeloid progenitor cells to facilitate dissemination.

Murine cytomegalovirus encodes a secreted, pro-inflammatory chemokine-like protein, MCK-2, that recruits leukocytes and facilitates viral dissemination. We have shown that MCK-2-enhanced recruitment of myelomonocytic leukocytes with an immature phenotype occurs early during infection and is associated with efficient viral dissemination. Expression of MCK-2 drives the mobilization of a population of leukocytes from bone marrow that express myeloid marker Mac-1 (CD11b), intermediate levels of Gr-1 (Ly6 G/C), platelet-endothelial-cell adhesion molecule-1 (PECAM-1, CD31), together with heterogeneous levels of stem-cell antigen-1 (Sca-1, Ly-6 A /E). Recombinant MCK-2 mediates recruitment of this population even in the absence of viral infection. Recruitment of this cell population and viral dissemination via the bloodstream to salivary glands proceeds normally in mice that lack CCR2 and MCP-1 (CCL2), suggesting that recruitment of macrophages is not a requisite component of pathogenesis. Thus, a systemic impact of MCK-2 enhances the normal host response and causes a marked increase in myelomonocytic recruitment with an immature phenotype to initial sites of infection. Mobilization influences levels of virus dissemination via the bloodstream to salivary glands and is dependent on a myelomonocytic cell type other than mature macrophages.

Common lymphoid progenitors rapidly engraft and protect against lethal murine cytomegalovirus infection after hematopoietic stem cell transplantation.

Lymphoid deficiency after allogeneic hematopoietic cell transplantation (HCT) results in increased susceptibility to infection; however, transplantation of mature lymphocytes frequently results in a serious complication known as graft-versus-host disease (GVHD). Here we demonstrate in mice that both congenic as well as allogeneic transplantation of low numbers of highly purified common lymphoid progenitors (CLPs)-a rare population of lymphoid-lineage-committed bone marrow cells-accelerates immune reconstitution after lethal irradiation and rescue with hematopoietic stem cells (HSCs). After congenic transplantation, 3 x 10(3) CLPs protected against murine cytomegalovirus (MCMV) infection at a level roughly equivalent to 107 unfractionated lymph node cells. In the allogeneic model of matched unrelated donor HSC transplantation, cotransplantation of 3 x 10(3) CLPs protected thymus-bearing as well as thymectomized hosts from MCMV infection and attenuated disease severity. Immunohistochemistry in combination with antibody depletion of T and natural killer (NK) cells confirmed that CLP-derived as well as residual host lymphocytes contribute to antiviral protection. Importantly, transplantation of allogeneic CLPs provided a durable antiviral immunity without inducing GVHD. These data support the potential for composing grafts with committed progenitors to reduce susceptibility to viral infection following HCT.