IRUCAA@TDC : Csa2, a member of the Rbt5 protein family, is involved in the utilization of iron from human hemoglobin during Candida albicans hyphal growth

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Csa2, a member of the Rbt5 protein family, is involved in the utilization of iron from human hemoglobin during Candida albicans hyphal growth Author(s)

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Okamoto-Shibayama, K; Kikuchi, Y; Kokubu, E; Sato, Y; Ishihara, K

Journal FEMS yeast research, 14(4): 674-677 URL http://hdl.handle.net/10130/3926

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This is a pre-copyedited, author-produced PDF of an article accepted for publication in FEMS yeast research following peer review. The version of record FEMS Yeast Res. 2014 Jun;14(4):674-7.

Okamoto-Shibayama K. et al is available online at: http://dx.doi.org/10.1111/1567-1364.12160.

FEMS Yeast Research

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Csa2, a member of the Rbt5 protein family, is involved in the utilization of iron from human hemoglobin during Candida albicans hyphal growth

Running Head: Csa2 in iron utilization for C. albicans hyphal growth

Kazuko Okamoto-Shibayama1, Yuichiro Kikuchi1, Eitoyo Kokubu1, Yutaka Sato2, and Kazuyuki Ishihara1

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Department of Microbiology; and 2Biochemistry, Tokyo Dental College

Department of Microbiology, Tokyo Dental College, 2-1-4 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan

Tel: +81-3-6380-9578, Fax: +81-3-6380-3752 E-mail: [email protected]

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Csa2 is a member of both the Candida albicans Rbt5 protein family and the CFEM (Common

in Fungal Extracellular Membranes) protein superfamily. CFEM proteins are characterized by

an internal domain containing eight equally spaced cysteine residues. Csa2 is involved in iron

uptake from hemoglobin and heme proteins; however, its precise role is unclear. Here, we

provide quantitative evidence of the involvement of Csa2 in the utilization of iron from

human hemoglobin during C. albicans hyphal growth. The ability of the hyphal form of the

wild-type, a homozygote csa2Δ mutant, and a complemented strain of C. albicans to utilize

hemoglobin as an iron source under iron-restricted conditions was examined through growth

studies and a crystal violet-staining assay. Hemoglobin-binding activity was assessed

indirectly by using a hemoglobin-sensitized tube method. Although hyphal growth of the

wild-type and csa2Δ/Δ::CSA2 strains was completely recovered when a high concentration of

human hemoglobin was added to the iron-restricted culture medium, the recovery of the csa2Δ/Δ mutant was significantly diminished. Furthermore, hemoglobin binding was impaired in the csa2Δ/Δ mutant compared with the wild-type and csa2Δ/Δ::CSA2 strains,

revealing that Csa2 is involved in the utilization of hemoglobin as an iron source by the

hyphal form of C. albicans.

Keywords: Candida albicans; hemoglobin; hyphal growth; iron utilization; Rbt5 protein

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Candida albicans is a major fungal pathogen that causes mucosal and systemic infections in

immunocompromised hosts. A key factor in the virulence of C. albicans is its ability to

switch from a yeast to a hyphal form while inside a host. This switch has been implicated in

the pathogenesis of systemic C. albicans infections, because mutants with defective hyphal growth show reduced virulence (Lo et al., 1997; Jacobsen et al., 2012). The hyphal form of C. albicans is able to use hemoglobin as a source of iron, suggesting that this is an important

factor in the switch to the hyphal form and in injury to the host. Because hemoglobin

promotes true hyphal morphogenesis, hemoglobin utilization is considered a virulence factor (Tanaka et al., 1997; Pendrak & Roberts, 2007).

Candida albicans has three main systems for acquiring iron: a reductive system, a

siderophore uptake system, and a heme iron uptake system. The reductive pathway involves

the release of iron from transferrin or ferritin or the exploitation of free iron in the

environment. The glycosylphosphatidylinositol (GPI)-anchored cell-wall protein Als3 is a

receptor for ferritin in the host environment (Almeida et al., 2009). Candida albicans does

not secrete siderophores, which are high-affinity iron chelators, to scavenge iron, but instead

it takes up siderophores synthesized by other microorganisms via the Sit1 transporter

(Heymann et al., 2002). Independent of these two iron uptake systems, C. albicans is also

capable of taking up iron from hemoglobin and hemeproteins through a process of erythrocyte lysis, hemoglobin binding, heme extraction, and endocytosis (Manns et al., 1994;

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Weissman & Kornitzer, 2004). It has been suggested that the members of the C. albicans

heme-receptor protein family that possess the CFEM (Common in Several Fungal

Extracellular Membranes) domain (i.e., Csa1, Csa2 and Pga7 [Rbt6], Rbt5, and Rbt51

[Pga10]) are involved in this third system of iron uptake (Weissman & Kornitzer, 2004).

Unlike the other CFEM-containing proteins, which are attached to both the cell wall and the

plasma membrane, the small, secretory protein Csa2 does not possess a GPI anchor (Sosinska et al., 2008; Weissman et al., 2008; Sorgo et al., 2010, 2011). Transcriptional studies have indicated that several cell wall proteins are regulated by iron availability, and that the

increased expression of CFEM-containing proteins in conditions of iron starvation supports their involvement in iron acquisition (Lan et al., 2004; Chen et al., 2011). The precise roles of these heme-binding proteins in virulence have not yet been well described; in particular, little

is known about the function of Csa2 in heme-iron uptake. Therefore, we aimed to clarify the

involvement of Csa2 in iron acquisition during hyphal growth in C. albicans.

A csa2Δ/Δ mutant, previously described as Δorf19.3117, and a triple-auxotrophic strain,

BWP17, complemented with plasmid CIp30, which was used as the wild-type control in all

experiments, were kindly provided by Professor B. Hube of Hans Knoell Institute, Germany

(Zakikhany et al., 2007). A csa2Δ/Δ::CSA2 complemented strain was constructed by

introducing the wild-type allele of the CSA2 gene into the RP10 locus of the csa2Δ/Δ mutant

5 reaction and DNA sequencing analyses.

Iron-restricted culture conditions were used as described (Weissman et al., 2008). Yeast cells

were cultured at 30°C in YPD medium (1% yeast extract, 2% peptone, and 2% glucose per

liter) supplemented with 1 mM ferrozine (an iron chelator) to elicit iron starvation. To induce

switching to the hyphal form, yeast cells (1  104 cells/mL) were transferred to RPMI 1640

medium in which free iron had been removed by the addition of 100 g/mL of apotransferrin,

and then cultured for 16 to 24 h under 5% CO2 at 37°C.

Preliminary experiments showed that the growth of all three strains in RPMI 1640 medium

was similar. All three strains also exhibited similar dose-dependent growth inhibition by

chelating free iron, which is essential for growth, when incubated with a series of

apotransferrin dilutions in RPMI 1640 medium. Growth was totally inhibited by the addition

of 100 g/mL apotransferrin; therefore, this concentration of apotransferrin was used to

chelate the free iron in the culture medium. Hyphal growth morphology was confirmed

microscopically and growth was assessed by means of a crystal violet (CV)-staining assay.

Viable Candida hyphal cells were quantified by measuring the photometric absorbance of the

Candida-bound CV extract at a wavelength of 590 nm (Abe et al., 1994).

Figure 1(a) shows the inhibitory effects of apotransferrin on hyphal growth and the rescue of

hyphal growth by FeCl3. All three strains exhibited a similar growth pattern when incubated

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csa2Δ/Δ::CSA2 complemented strain was completely rescued by the addition of FeCl3, but

only partially so in the csa2Δ/Δ mutant, indicating that Csa2 may play a role in

non-hemoglobin iron utilization.

Next, we examined whether C. albicans was able to utilize hemoglobin as an iron source

under iron-restricted conditions and whether the addition of human hemoglobin to the

environment was able to rescue impaired hyphal growth (Fig. 1b). With the addition of

human hemoglobin, hyphal growth was restored in the wild-type and csa2Δ/Δ::CSA2

complemented strain but only partially so in the csa2Δ/Δ mutant. The csa2Δ/Δ mutant grew at

approximately one-third the rate of the wild-type strain when a high concentration of human

hemoglobin was the sole source of iron. These results indicate that Csa2 is involved in the

utilization of human hemoglobin as a source of iron. Interestingly, deletion of CSA2 did not

fully attenuate growth, implying that additional genes and mechanisms are involved in the

exploitation of environmental iron.

In a hemoglobin binding assay, to quantify the adherent C. albicans, we used

hemoglobin-sensitized tubes that were prepared by incubation overnight at 37°C with human

hemoglobin in Ca2+- and Mg2+-free phosphate-buffered saline, as described previously

(Tanaka et al., 1997). We observed attenuation of the capacity of the csa2Δ/Δ mutant to bind

human hemoglobin compared with that of wild-type C. albicans (Fig. 1c).

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activity of the C. albicans culture supernatant. Culture supernatants from the three strains of

C. albicans were mixed with human erythrocytes and incubated for 12 to 72 h at 37°C, and

the hemolytic activity was measured by the absorption at 405 nm (Tanaka et al., 1997). No

significant differences in hemolytic activity between the culture supernatant of wild-type C.

albicans and that of the csa2Δ/Δ mutant were observed(data not shown), indicating that Csa2

was not involved in hemolysis.

Taken together, our results show that Csa2 is involved in iron utilization and hemoglobin

binding, and that Csa2 binds to hemoglobin before presenting it to other heme-binding

proteins.

Although the functions of the proteins in the Rbt5 family that possess the conserved CFEM

are largely unknown, they are suspected to play important roles in fungal pathogenesis

(Kulkarni et al., 2003). Sorgo et al. (2013) used mass spectrometry to characterize the

dynamics of the response of the C. albicans cell-wall proteome to iron starvation, and in

particular the remarkable changes in the levels of the GPI-modified members of the Rbt5

family; their results suggest that the CFEM domain is responsible for the heme-binding

properties of these proteins. Furthermore, secretome analyses have shown that the level of

Csa2 increases under conditions of iron starvation (Sorgo et al., 2013). Interestingly, in

addition to using hemoglobin as an iron source, C. albicans also uses hemoglobin as a

8 matrix proteins (Pendrak & Roberts, 2007).

Further studies are required to delineate the molecular basis of iron uptake by C. albicans.

Our results suggest that members of the Rbt5 protein family collaborate in iron acquisition

and that Csa2 may bind to hemoglobin before presenting it to other heme-binding proteins.

Our group is currently attempting to characterize the involvement of Csa2 in iron acquisition

and virulence. In conclusion, our findings suggest that Csa2 is involved in the utilization of

iron from human hemoglobin during hyphal growth in C. albicans.

Acknowledgments. We are grateful to Professor Bernhard Hube (Department of Microbial

Pathogenicity Mechanisms, Hans Knoell Institute, Jena, Germany) for providing us with the

C. albicans strains and plasmid. This work was supported by a Japan Society for the

Promotion of Science grant-in-aid for scientific research (Kakenhi 21791797) and a grant

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Figure legend

Fig. 1. Involvement of Csa2 in iron utilization during hyphal growth in Candida albicans.

(a) Inhibitory effects of transferrin on C. albicans hyphal growth and the restoration of hyphal

growth by the addition of FeCl3. Candida albicans (1 × 104 cells/mL in RPMI 1640 medium

containing 100 g/mL apotransferrin) was cultured with various concentrations of FeCl3

(0.01 to 1000 g/mL). Hyphal growth was measured by means of a crystal violet

(CV)-staining assay. Three independent experiments were performed in duplicate. Data are

presented as mean ± S.D. wt, wild-type. (b) Rescue of C. albicans hyphal growth by the

addition of human hemoglobin. Candida albicans (1 × 104 cells/mL in RPMI 1640 medium

containing 100 g/mL apotransferrin) was cultured with various concentrations of human

hemoglobin (0.1 to 1000 g/mL). Hyphal growth was measured by means of a CV-staining

assay. Three independent experiments were performed in duplicate. Data are presented as

mean ± S.D. Student’st-test, *P < 0.001 vs. wild-type (wt). (c) Binding activity of C. albicans

to human hemoglobin. Candida albicans (1 × 104 cells/mL) was cultured in human

hemoglobin–sensitized tubes containing RPMI 1640 medium for 24 h. Student’st-test, *P <

13 0 0.1 1 10 100 1000 0.1 0.2 0.3 0.4 0.5 0.6 0.7 FeCl3 (g/mL) Hyphal g rowth (OD 590 ) Fig. 1(a) wt, apotransferrin (-) wt, apotoransferrin (+) csa2/, apotransferrin (-) csa2/, apotransferrin (+)

csa2/::CSA2, apotransferrin (-) csa2/::CSA2, apotransferrin (+)

0 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 wt csa2















14 wt  csa2 ::C SA2  /  csa 2 0.0 0.1 0.2 0.3 0.4 0.5  B in d in g a c ti vi ty ( OD 590 ) Fig. 1 (c)