SR-like RNA-binding protein Slr1 affects Candida albicans filamentation and virulence

Abstract

Candida albicans causes both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of both Saccharomyces cerevisiae and Aspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence of C. albicans. BLAST searches with S. cerevisiae SR-like protein Npl3 (ScNpl3) identified two C. albicans proteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, another SR-like RNA-binding protein with no close S. cerevisiae ortholog. Whereas ScNpl3 was critical for growth, deletion of NPL3 in C. albicans resulted in few phenotypic changes. In contrast, the slr1Δ/Δ mutant had a reduced growth rate in vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cells in vitro. Mice infected intravenously with the slr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type or slr1Δ/Δ mutant complemented with SLR1 (slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization of slr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected with slr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cells in vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin on slr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influences C. albicans growth, filamentation, host cell interactions, and virulence.

Fig 1

C. albicans proteins similar to S. cerevisiae and S. pombe SR-like proteins. In domain comparison of two SR-like proteins in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, RNA recognition motifs (RRM) and arginine-rich (R-rich) domains, as well as nonconserved N- and C-terminal domains, are shown. Asterisks indicate locations of SR/RS dipeptides. The second domain of the C. albicans Npl3-like protein (RRM′) has sequence similarity with the other two atypical RRMs in ScNpl3 and SpSrp2 but is not recognized as an RRM by protein signature recognition searches (InterProScan). GenBank accession numbers for these proteins are as follows: NP_010720.1 (ScNpl3), XP_715038.1 (CaNpl3), NP_594570.1 (SpSrp2), CAA22007.1 (CaSlr1), and NP_596398.1 (SpSrp1).

Fig 2

Growth defects associated with SLR1 deletion in C. albicans. (A) Generation times for indicated strains (5 to 10 replicates) were calculated from 24-h growth curves as described in Materials and Methods. Complete genotypes are presented in Table 1. WT, wild type. (B) Two dilutions (10× = 5 × 10⁴ cells; 1× = 5 × 10³ cells) of strains were grown at 30°C for 1 day on YPD without or with 0.02% SDS or 20 μM calcofluor white (CW). Relevant genotypes of deletion strains tested (2 left spots) are denoted on the left; the wild-type gene reintroduced into each deletion strain to create reconstituted strains (2 right spots) is denoted on the right.

Fig 3

Absence of Slr1 decreases filamentation on solid medium. Stationary-phase cells (10⁷) with the indicated genotypes were spotted onto plates containing 5% serum (A) or onto RPMI agar plates (B) and grown for 6 days at 37°C.

Fig 4

Cells lacking Slr1 can form hyphae in liquid RPMI. Overnight YPD cultures of cells with the indicated genotypes were diluted to 10⁵ cells/ml in RPMI and incubated on gelatin-coated coverslips for 1.5 (A) or 3 (B) h at 37°C and 5% CO₂. Paraformaldehyde-fixed cells were imaged by differential interference contrast (DIC) microscopy. Scale bars = 5 μm.

Fig 5

Deletion of SLR1 decreases cell damage by C. albicans in vitro. FaDu oral epithelial cells (A) or human umbilical vein endothelial cells (B) were preloaded with ⁵¹Cr and incubated for 3 h at 37°C with the following C. albicans strains: the wild type (WT1, DAY185; WT2, AMC81), the npl3Δ/Δ mutant (AMC18), the npl3Δ/Δ+NPL3 strain (AMC22), the slr1Δ/Δ mutant (AMC82), the slr1Δ/Δ+SLR1 strain (AMC104), the npl3Δ/Δslr1Δ/Δ mutant (AMC83), the npl3Δ/Δ slr1Δ/Δ+SLR1 strain (AMC111), and the npl3Δ/Δ+NPL3 slr1Δ/Δ strain (AMC113). Results are the means ± SD of data from 3 experiments, each performed in triplicate. *, P < 0.01 compared to results for the wild type.

Fig 6

Effects of deletion of SLR1 on C. albicans virulence in the mouse model of OPC. Immunocompromised mice were infected orally with the wild type (AMC81), the slr1Δ/Δ mutant (AMC82), or the slr1Δ/Δ mutant complemented with SLR1 (AMC104). The oral fungal burden was determined after 5 days of infection for 6 or 7 mice per strain. Each symbol represents the results for an individual mouse, and the horizontal lines indicate the median values. *, P = 0.02 compared to results for the wild-type strain.

Fig 7

Deletion of SLR1 affects C. albicans virulence, filamentation, and intrakidney localization during disseminated candidiasis. (A) Survival of mice injected intravenously with the wild type (AMC81), the slr1Δ/Δ mutant (AMC82), or the slr1Δ/Δ mutant complemented with SLR1 (AMC104). Each strain was injected into 8 mice, and host survival was monitored over a 3-week period. *, P < 0.0001 versus results for other strains. (B) Histopathology of kidney sections after 4 days of infection with the indicated strains. Sections were stained with periodic acid-Schiff stain. Scale bar = 20 μm.

Fig 8

Deletion of SLR1 increases brain tropism of C. albicans. (A) Deletion of SLR1 increases fungal burden in the brain. Brain fungal burdens of mice 4 days after intravenous inoculation with the wild-type (AMC81), slr1Δ/Δ (AMC82), or slr1Δ/Δ+SLR1 (AMC104) strain are shown. Each symbol represents the results for an individual mouse, and the horizontal lines indicate the median values. *, P < 0.005 compared to results for the wild-type and reconstituted strains. (B and C) The strains in panel A were tested for invasion of HBMECs and HUVECs after 3 h of incubation (B) and for their capacity to cause HBMEC damage after 16 h of infection (C). Results are the means ± SD for 3 experiments, each performed in triplicate. *, P ≤ 0.02 versus results for the wild-type and reconstituted strains. (D) Level of surface expression of Als3 for the indicated strains as assessed by flow cytometry. Abbreviations: orgs, oganisms; HPF, high-powered field.