Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis

Abstract

Background: Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis.

Objective: We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections.

Methods: PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated.

Results: We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ-induced gene expression, but we found impaired responses to IFN-γ restimulation.

Conclusion: Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ-mediated inflammation.

Fig 1

Patient 1. A, Extensive right upper lung, mediastinal, and pleural involvement with C immitis. B, MRI showing an intramedullary spinal cord lesion (arrow) with edema and cord compression.

Fig 2

STAT1 mutants. A, STAT1 coding region. TAD, Transactivation domain. Mutations were associated with (blue arrows) disseminated coccidioidomycosis and (green arrows) disseminated histoplasmosis. L706S was the dominant negative mutation. B, U3A cells transfected with STAT1 mutants, WT or empty vector, and WB with anti-STAT1 and anti-tubulin antibodies.

Fig 3

STAT1 mutants lead to delayed dephosphorylation and enhanced luciferase GAS-induced activity. A, Dephosphorylation in EBV-B cells from patients and control subjects (Nm) stimulated with IFN-γ for the indicated periods. B, Dephosphorylation assayed by means of flow cytometry in U3A cells. Results are representative of at least 3 independent experiments. MFI, Mean fluorescence intensity; NS, nonstimulated. C, Transcriptional responses to IFN-γ and IFN-α in U3A cells transfected with STAT1 mutant constructs and when cotransfected with WT STAT1. D, U3A cells transfected with L706S showed no negative effect on the mutants A267V and E353K. Data show the mean fold increase relative to the WT nonstimulated specimens from a total of 5 experiments. *P < .05 when compared with stimulated WT, respectively. **P < .01 compared with L706S.

Fig 3

STAT1 mutants lead to delayed dephosphorylation and enhanced luciferase GAS-induced activity. A, Dephosphorylation in EBV-B cells from patients and control subjects (Nm) stimulated with IFN-γ for the indicated periods. B, Dephosphorylation assayed by means of flow cytometry in U3A cells. Results are representative of at least 3 independent experiments. MFI, Mean fluorescence intensity; NS, nonstimulated. C, Transcriptional responses to IFN-γ and IFN-α in U3A cells transfected with STAT1 mutant constructs and when cotransfected with WT STAT1. D, U3A cells transfected with L706S showed no negative effect on the mutants A267V and E353K. Data show the mean fold increase relative to the WT nonstimulated specimens from a total of 5 experiments. *P < .05 when compared with stimulated WT, respectively. **P < .01 compared with L706S.

Fig 4

Increased STAT1/PIAS1 association. A, After interferon stimulation, cell lysates from EBV-B cells of patients and control subjects (Nm) were IP:WB anti-STAT1: anti-PIAS1 antibody. B, EBV-B cells treated with SAMe were evaluated as above. Blots are representative of 3 independent experiments for each condition. Numbers are band densities normalized to the nonstimulated samples (NS).

Fig 5

Gene expression: restimulation experiments in transfected U3A cells. Cells were stimulated or not (NS) with IFN-γ, washed, and restimulated (IFN-γ/IFN-γ) or not (IFN-γ rest). Expression of IFN-γ (CXCL9 [A] and CXCL10 [B]) target genes was evaluated. Results are means ± SDs of 3 independent experiments. *P < .05 compared with IFN-γ rest.

Fig 5

Gene expression: restimulation experiments in transfected U3A cells. Cells were stimulated or not (NS) with IFN-γ, washed, and restimulated (IFN-γ/IFN-γ) or not (IFN-γ rest). Expression of IFN-γ (CXCL9 [A] and CXCL10 [B]) target genes was evaluated. Results are means ± SDs of 3 independent experiments. *P < .05 compared with IFN-γ rest.

Fig 6

Reduction of PIAS1 modulates the IFN-γ–induced gene response. Evaluation of gene expression in U3A cells cotransfected with WT or mutant STAT1 and with siRNA directed against PIAS1. Cells were treated as described in Fig5. Restimulated cells =IFN-γ/IFN-γ or PIAS siRNA IFN-γ/IFN-γ. Results are means ± SDs of 3 independent experiments. NS, Nonstimulated. *P < .05 when compared with IFN-γ rest. A, WT and E353K with and without PIAS siRNA. B, WT, A267V, and R274G with and without PIAS siRNA.

Fig 6

Reduction of PIAS1 modulates the IFN-γ–induced gene response. Evaluation of gene expression in U3A cells cotransfected with WT or mutant STAT1 and with siRNA directed against PIAS1. Cells were treated as described in Fig5. Restimulated cells =IFN-γ/IFN-γ or PIAS siRNA IFN-γ/IFN-γ. Results are means ± SDs of 3 independent experiments. NS, Nonstimulated. *P < .05 when compared with IFN-γ rest. A, WT and E353K with and without PIAS siRNA. B, WT, A267V, and R274G with and without PIAS siRNA.