title: Pho4 Is Essential for Dissemination ofCryptococcus neoformansto the Host Brain by Promoting Phosphate Uptake and Growth at Alkaline pH.
abstract: Phosphate acquisition by fungi is regulated by the phosphate-sensing and acquisition (PHO) signaling pathway.Cryptococcus neoformansdisseminates from the lung to the brain and is the commonest cause of fungal meningitis worldwide. To investigate the contribution of PHO signaling to cryptococcal dissemination, we characterized a transcription factor knockout strain (hlh3Δ/pho4Δ) defective in phosphate acquisition. Despite little similarity with other fungal Pho4 proteins, Hlh3/Pho4 functioned like a typical phosphate-responsive transcription factor in phosphate-deprived cryptococci, accumulating in nuclei and triggering expression of genes involved in phosphate acquisition. Thepho4Δ mutant strain was susceptible to a number of stresses, the effect of which, except for alkaline pH, was alleviated by phosphate supplementation. Even in the presence of phosphate, the PHO pathway was activated in wild-type cryptococci at or above physiological pH, and under these conditions, thepho4Δ mutant had a growth defect and compromised phosphate uptake. Thepho4Δmutant was hypovirulent in a mouse inhalation model, where dissemination to the brain was reduced dramatically, and markedly hypovirulent in an intravenous dissemination model. Thepho4Δmutant was not detected in blood, nor did it proliferate significantly when cultured with peripheral blood monocytes. In conclusion, dissemination of infection and the pathogenesis of meningitis are dependent on cryptococcal phosphate uptake and stress tolerance at alkaline pH, both of which are Pho4 dependent.IMPORTANCECryptococcal meningitis is fatal without treatment and responsible for more than 500,000 deaths annually. To be a successful pathogen,C. neoformansmust obtain an adequate supply of essential nutrients, including phosphate, from various host niches. Phosphate acquisition in fungi is regulated by the PHO signaling cascade, which is activated when intracellular phosphate decreases below a critical level. Induction of phosphate acquisition genes leads to the uptake of free phosphate via transporters. By blocking the PHO pathway using a Pho4 transcription factor mutant (pho4Δmutant), we demonstrate the importance of the pathway for cryptococcal dissemination and the establishment of brain infection in murine models. Specifically, we show that reduced dissemination of thepho4Δmutant to the brain is due to an alkaline pH tolerance defect, as alkaline pH mimics the conditions of phosphate deprivation. The end result is inhibited proliferation in host tissues, particularly in blood.
authors.affiliation: Fungal Pathogenesis Laboratory, Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, NSW, Australia; The Westmead Clinical School, Sydney Medical School, The University of Sydney, Westmead, NSW, Australia.
authors.forename: Sophie
authors.identifier: 0000-0003-3511-457X
authors.initials: S
authors.lastname: Lev
authors.affiliation: Fungal Pathogenesis Laboratory, Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Keren
authors.initials: K
authors.lastname: Kaufman-Francis
authors.affiliation: Fungal Pathogenesis Laboratory, Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, Westmead, NSW, Australia.
authors.forename: Desmarini
authors.initials: D
authors.lastname: Desmarini
authors.affiliation: Fungal Pathogenesis Laboratory, Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Pierre G
authors.initials: PG
authors.lastname: Juillard
authors.affiliation: Fungal Pathogenesis Laboratory, Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Li
authors.affiliation: Immunology and Host Defense Group, Department of Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia; Mycobacterial Research Program, The Centenary Institute, Camperdown, NSW, Australia.
authors.forename: Sebastian A
authors.initials: SA
authors.lastname: Stifter
authors.affiliation: Immunology and Host Defense Group, Department of Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia; Mycobacterial Research Program, The Centenary Institute, Camperdown, NSW, Australia.
authors.forename: Carl G
authors.initials: CG
authors.lastname: Feng
authors.affiliation: Fungal Pathogenesis Laboratory, Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, NSW, Australia; The Westmead Clinical School, Sydney Medical School, The University of Sydney, Westmead, NSW, Australia.
authors.forename: Tania C
authors.initials: TC
authors.lastname: Sorrell
authors.affiliation: Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, NSW, Australia; Vascular Immunology Unit, Department of Pathology, School of Medical Sciences, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Georges E R
authors.initials: GE
authors.lastname: Grau
authors.affiliation: Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea.
authors.forename: Yong-Sun
authors.initials: YS
authors.lastname: Bahn
authors.affiliation: Fungal Pathogenesis Laboratory, Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, NSW, Australia; The Westmead Clinical School, Sydney Medical School, The University of Sydney, Westmead, NSW, Australia.
authors.forename: Julianne T
authors.initials: JT
authors.lastname: Djordjevic
keywords: Cryptococcus neoformans; HLH-type transcription factor; cryptococcal meningitis; cryptococcoma; fungal pathogenesis; intravenous inoculation; murine models of cryptococcosis; phosphate-sensing and acquisition (PHO) pathway; signal transduction pathway