Coordination of Candida albicans Invasion and Infection Functions by Phosphoglycerol Phosphatase Rhr2

Pathogens 2015, 4, 573-589; doi:10.3390/pathogens4030573 OPEN ACCESS pathogens ISSN 2076-0817 www.mdpi.com/journal/pathogens Article Coordination of Candida albicans Invasion and Infection Functions by Phosphoglycerol Phosphatase Rhr2 Jigar V. Desai 1,†, Shaoji Cheng 2, Tammy Ying 1, M. Hong Nguyen 3, Cornelius J. Clancy 4,5, Frederick Lanni 6 and Aaron P. Mitchell 7,* 1 2 3 4 5 6 7 † Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave (MI- 289), Pittsburgh, PA 15213, USA; E-Mails: or (J.V.D.); (T.Y.) Department of Medicine, Infectious Disease Division, University of Pittsburgh, 3550 Terrace Street, Scaife S869, Pittsburgh, PA 15261, USA; E-Mail: Department of Medicine, Infectious Disease Division, University of Pittsburgh, 3550 Fifth Ave, Suite 872 Scaife Hall, Pittsburgh, PA 15261, USA; E-Mail: Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA; E-Mail: Infectious Diseases Section, VA Pittsburgh Healthcare System, 3550 Fifth Ave, Suite 867 Scaife Hall, Pittsburgh, PA 15261, USA Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave (MI- 294C), Pittsburgh, PA 15213, USA; E-Mail: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave (MI- 200B), Pittsburgh, PA 15213, USA Current Address: National Institutes of Health, Bldg 10, Room 11C116, 10 Center Drive, Bethesda, MD 20814, USA. * Author to whom correspondence should be addressed; E-Mail: ; Tel.: +1-412-268-5844; Fax: +1-412-268-7129. Academic Editor: Lawrence S. Young Received: 13 June 2015 / Accepted: 13 July 2015 / Published: 24 July 2015 Abstract: The Candida albicans RHR2 gene, which specifies a glycerol biosynthetic enzyme, is required for biofilm formation in vitro and in vivo. Prior studies indicate that RHR2 is ultimately required for expression of adhesin genes, such as ALS1. In fact, RHR2 is unnecessary for biofilm formation when ALS1 is overexpressed from an RHR2-independent Pathogens 2015, 4 574 promoter. Here, we describe two additional biological processes that depend upon RHR2: invasion into an abiotic substrate and pathogenicity in an abdominal infection model. We report here that abiotic substrate invasion occurs concomitantly with biofilm formation, and a screen of transcription factor mutants indicates that biofilm and hyphal formation ability correlates with invasion ability. However, analysis presented here of the rhr2Δ/Δ mutant separates biofilm formation and invasion. We found that an rhr2Δ/Δ mutant forms a biofilm upon overexpression of the adhesin gene ALS1 or the transcription factor genes BRG1 or UME6. However, the biofilm-forming strains do not invade the substrate. These results indicate that RHR2 has an adhesin-independent role in substrate invasion, and mathematical modeling argues that RHR2 is required to generate turgor. Previous studies have shown that abdominal infection by C. albicans has two aspects: infection of abdominal organs and persistence in abscesses. We report here that an rhr2Δ/Δ mutant is defective in both of these infection phenotypes. We find here that overexpression of ALS1 in the mutant restores infection of organs, but does not improve persistence in abscesses. Therefore, RHR2 has an adhesin-independent role in abdominal infection, just as it does in substrate invasion. This report suggests that RHR2, through glycerol synthesis, coordinates adherence with host- or substrate-interaction activities that enable proliferation of the C. albicans population. Keywords: Candida albicans; adherence; invasion; biofilm; intra-abdominal candidiasis; glycerol; turgor; infection; adhesin 1. Introduction Infection often begins with adherence. Microbes attach to host cells, tissues, or implanted devices, and then engage in pathogenic interactions such as invasion or inflammation. For many infectious microbes, the mechanisms that coordinate attachment and pathogenesis are well characterized. For example, in the case of enteropathogenic and enterohaemorrhagic Escherichia coli, the injected bacterial Tir protein mediates both bacterial attachment and host cytoskeletal rearrangements that ultimately lead to pathological effects [1]. In the case of Helicobacter pylori, contact with gastric epithelial cells induces formation of pili that translocate pathogenic effectors into host cells [2]. The pathogens Yersinia pestis and Candida albicans express invasin proteins that promote both attachment to and entry into host cells [3,4]. Because mechanisms that coordinate adherence and attachment are linchpins of infection, they provide insight into pathogenicity functions, and also present potential therapeutic targets. Our focus is the fungus C. albicans, which is responsible for diverse mucosal and disseminated infections [5]. This organism can grow as ovoid yeast cells, cylindrical hyphal cells, and other cell types [6,7]. Hyphae express numerous surface adhesins that mediate adherence to host cells and abiotic surfaces [8–10]. Adherence thus establishes a foothold for invasion and biofilm formation. Hyphae from biofilms, on either mucosal or abiotic substrates, invade the underlying surface [11]. There are two modes of hyphal invasion. The first mechanism depends upon host cell functions: C. albicans hyphae induce their own endocytosis via the surface invasins Als3 and Ssa1 [9,12,13]. These invasins stimulate the actin-mediated endocytic pathway through interaction with E-cadherin and EGFR/HER2 on the Pathogens 2015, 4 575 epithelial surface [13,14]. The second mechanism is host cell-independent: C. albicans hyphae may invade a cell or substrate through exertion of force. This mechanism has been revealed through analysis of hyphal growth behaviors on abiotic surfaces [11,15]. Force-mediated invasion has been well studied in the plant pathogenic fungi Magnaporthe oryzae and Colletotrichum graminicola [16–19]. These fungi form a special melanized cell structure, the appresorium, which initiates invasion after adherence to the host surface. The appresorium generates enormous turgor via glycerol accumulation that drives a penetration peg through an underlying leaf surface [16]. It seems reasonable that C. albicans might also rely upon glycerol accumulation to generate turgor. Glycerol has a prominent role in C. albicans biofilm formation, as first shown by Bonhomme and colleagues [20]. They found that the glycerol biosynthetic gene RHR2, which specifies 3-phosphoglycerol phosphatase, is up-regulated in biofilm cells compared to planktonic cells. This gene expression relationship reflects function, because they found that an rhr2Δ/Δ deletion mutant is defective in biofilm formation in vitro. Direct measurements have confirmed that C. albicans biofilm cells accumulate higher levels of glycerol than planktonic cells [21,22]. We confirmed the studies of Bonhomme and colleagues, and extended the observations to show that RHR2 is required for biofilm formation in vivo in a catheter (...truncated)