Date Published: November 19, 2018
Publisher: Public Library of Science
Author(s): Qian Shen, Matthew J. Beucler, Stephanie C. Ray, Chad A. Rappleye, Xiaorong Lin.
Copper toxicity and copper limitation can both be effective host defense mechanisms against pathogens. Tolerance of high copper by fungi makes toxicity as a defense mechanism largely ineffective against fungal pathogens. A forward genetic screen for Histoplasma capsulatum mutant yeasts unable to replicate within macrophages showed the Ctr3 copper transporter is required for intramacrophage proliferation. Ctr3 mediates copper uptake and is required for growth in low copper. Transcription of the CTR3 gene is induced by differentiation of H. capsulatum into pathogenic yeasts and by low available copper, but not decreased iron. Low expression of a CTR3 transcriptional reporter by intracellular yeasts implies that phagosomes of non-activated macrophages have moderate copper levels. This is further supported by the replication of Ctr3-deficient yeasts within the phagosome of non-activated macrophages. However, IFN-γ activation of phagocytes causes restriction of phagosomal copper as shown by upregulation of the CTR3 transcriptional reporter and by the failure of Ctr3-deficient yeasts, but not Ctr3 expressing yeasts, to proliferate within these macrophages. Accordingly, in a respiratory model of histoplasmosis, Ctr3-deficient yeasts are fully virulent during phases of the innate immune response but are attenuated after the onset of adaptive immunity. Thus, while technical limitations prevent direct measurement of phagosomal copper concentrations and copper-independent factors can influence gene expression, both the CTR3 promoter induction and the attenuation of Ctr3-deficient yeasts indicate activation of macrophages switches the phagosome from a copper-replete to a copper-depleted environment, forcing H. capsulatum reliance on Ctr3 for copper acquisition.
To successfully infect and colonize a host, pathogens must acquire sufficient nutrients from the host to enable microbe growth and proliferation. These metabolic resources include, but are not limited to, essential metals. The nutrient-limited phagosome represents a particularly challenging environment for intracellular pathogens as mammalian hosts can sequester essential elements such as iron and zinc from pathogens. This has been termed “nutritional immunity” [1,2]. For example, host molecules such as heme, ferritin, transferrin, and lactoferrin make iron largely inaccessible to microbes . However, successful pathogens have developed sophisticated strategies to combat iron limitation. For example, Mycobacterium tuberculosis and the fungal pathogen Histoplasma capsulatum secrete iron-chelating siderophores [3–5]. Accordingly, inability to synthesize siderophores severely impairs intracellular growth [5,6]. In addition, H. capsulatum maintains a slightly acidic intra-phagosomal pH which is sufficient to release iron from host transferrin . Mammalian hosts also restrict available zinc by production of zinc chelating proteins such as S100 family proteins and calprotectin [8,9]. In addition, host zinc transporters (ZIPs) are employed to tightly control zinc levels in different cellular compartments . Host zinc limitation mechanisms are an important aspect of activation of cellular immunity . However, as with iron limitation, some pathogens have evolved efficient mechanisms to counteract zinc sequestration. High affinity transporters expressed by Salmonella species and H. capsulatum (ZnuABC and Zrt2, respectively) enable these pathogens to import zinc in environments with low zinc concentrations [12–15]. Without these zinc transporters Salmonella and H. capsulatum intracellular proliferation is significantly attenuated. Employing an alternative strategy, the fungal pathogen Candida albicans expresses zincophore (Pra1), a zinc-chelating molecule, to scavenge zinc during endothelial invasion .
In order to establish infections and proliferate in macrophages, H. capsulatum yeasts must acquire essential metals within the phagosomal environment. H. capsulatum secretes siderophores and expresses zinc transporters to combat host limitation of iron and zinc, respectively. In this study, we demonstrate that growth in macrophages also imposes challenges on yeasts to maintain copper homeostasis. Specifically, H. capsulatum yeasts rely on the Ctr3 copper transporter to acquire sufficient copper when copper becomes limiting, both in liquid culture and within macrophages. Besides Ctr3, the H. capsulatum genome encodes two additional putative copper transporters (Ctr1 and Ctr2). However, Ctr1 and Ctr2 are not simply redundant with Ctr3; Ctr1 and Ctr2 are not as highly expressed as Ctr3, and they are not sufficient for copper acquisition when phagosomal copper levels become severely limited. Thus, Ctr3 is the primary transporter involved in copper acquisition as part of H. capsulatum’s pathogenesis program.