Candida albicans Hyr1p confers resistance to neutrophil killing and is a potential vaccine target

Abstract

Candida albicans is the most common cause of invasive fungal infections in humans. It is unclear how C. albicans escapes from phagocytic attack and survives in the hostile blood environment during life-threatening systemic infections. Using a conditional overexpression or suppression genetic strategy, we discovered that HYR1 gene reduced phagocytic killing activity of C. albicans in vitro and increased tissue fungal burden in vivo. Concordant with its positive regulation by the transcription factor Bcr1p, autonomous expression of HYR1 complemented the hypersusceptibility to phagocyte-mediated killing of a bcr1 null mutant of C. albicans in vitro. As for C. albicans, heterologous expression of HYR1 in Candida glabrata rendered the organism more resistant to neutrophil killing activity. Vaccination with a recombinant Hyr1p significantly protected mice against hematogenously disseminated candidiasis (P = .001). Finally, anti-rHyr1p polyclonal antibodies enhanced mouse neutrophil killing activity by directly neutralizing rHyr1p effects in vitro. Thus, Hyr1 is an important virulence factor for C. albicans, mediating resistance to phagocyte killing. Hyr1p is a promising target for vaccine or other immunological or small molecule intervention to improve the outcomes of disseminated candidiasis.

Figure 1

Conditional expression of HYR1-enhanced neutrophil and macrophage-mediated killing of Candida albicans.(A) Confirmation of HYR1 conditional expression or suppression in strain CAAH-31 blastospores. Reverse-transcription polymerase chain reaction results of HYR1 that demonstrate overexpression of the gene in medium without doxycycline (−DOX) and lack of expression in medium with DOX (+DOX). EFB1 fragment was coamplified and served as a control. Lack of genomic DNA contamination in complementary DNA preparations was demonstrated by the absence of 919 basepair band containing the intron of EFB1. THE31 was the wild-type control strain. (B) C. albicans strains were grown in yeast peptone dextrose with doxycycline (suppression of HYR1) and without doxycycline (expression of HYR1)at 30C overnight and then cocultured with human neutrophil. (C) C. albicans cocultured with HL-60-derived neutrophil and (D) cocultured with HL-60-derived macrophage.

Figure 2

Expression of Candida albicans HYR1 increased human neutrophil killing resistance in bcr1 null mutant or Candida glabrata.(A, B) Autonomous expression of HYR1 in a bcr1-deficient strain of C. albicans completely complemented the hypersusceptibility of the parent strain to neutrophil killing resistance. C. albicans DAY185 (wild-type), CJN702 (bcr1 null mutant), and CJN698 (BCR1 complemented) CJN114, CJN1153, CJN1222, CJN1259, CJN1276, CJN1281, and CJN1288 (autonomously expressing ALS1, ALS3, HWP1, HYR1, RBT5, CHT2, and ECE1 in a bcr1 null mutant background, respectively) were grown in yeast peptone dextrose overnight at 30C. Data are displayed as median ± interquartile range. * P < .04 the wild-type vs BCR1-complemented or HYR1-complemented. (C) Heterologous expression of C. albicans HYR1 increased C. glabrata resistance to HL-60 derived neutrophil mediated killing. * P < .001.

Figure 3

Detection of HYR1 expression in response to neutrophil in vitro and during disseminated candidiasis. (A) HL-60-derived neutrophil initially inhibited wild-type Candida albicans HYR1 expression. C. albicans SC5314 was cultured in Roswell Park Memorial Institute 1640 medium, plus 10% pooled human serum. (B) Kidneys, livers, lungs, spleens, and brains were harvested 6 or 24 h after intravenous infection with C. albicans SC5314. Nested reverse-transcription polymerase chain reaction was used to detect expression of HYR1. C. albicans EFB1 and mouse housekeeping gene G3PDH were used as a control. Plus signs denote infected mice, and minus signs denote uninfected mice. PMN, polymorphonuclear leukocyte.

Figure 4

(A) HYR1 in vivo expression and its effect on fungal burden. Burden of Candida albicans in livers and spleens of immunocompetent mice (8 mice per group) infected with CAAH-31 (HYR1) or THE31 (control) grown in overexpressing (without doxycycline [−DOX]) or suppressing (with doxycycline [+DOX]) conditions. Infectious inocula were 4.1 × 10⁵and 4.2 × 10⁵for CAAH-31 grown in −DOX and +DOX medium, respectively, whereas infectious inocula were 3.6 × 10⁵and 3.7 × 10⁵for THE31 grown in −DOX and +DOX medium, respectively. Organs were harvested 24 h after infection. (B) Expression of HYR1 using real-time polymerase chain reaction in organs harvested 24 h after infection. The y-axes reflect the lower limits of detection of the assay. Data are displayed as median ± interquartile range. * P < .001 for overexpression vs suppression of HYR1 strain (HYR1-DOX) or control strain.

Figure 5

Indirect immunofluorescence with anti-Hyr1p serum demonstrated surface expression of Hyr1p on Candida albicans hyphae. Hyphal formation was induced by incubating C. albicans in Roswell Park Memorial Institute 1640 medium for 90 min. Cells were stained with either the hyr1 null mutant preabsorbed anti-Hyr1p serum (1:100) or the antiprotein preparation from the empty plasmid clone serum (negative control), followed by staining with Alexa labeled anti-mouse Ab. DIC, differential interference contrast.

Figure 6

Recombinant N-terminus of Hyr1p (rHyr1p-N) protected against murine hematogenously disseminated candidiasis. (A) Survival of mice (8 mice per group) vaccinated with rHyr1p-N mixed with complete or incomplete Freund’s adjuvant and infected by means of the tail vein with 2.2 × 10⁵ blastospores of Candida albicans SC5314. (B) Survival of mice (8 mice per group, except the control group, which had 17 mice) vaccinated with rHyr1p-N or detoxified rHyr1p-N mixed with 0.1% alhydrogel and infected with 7 ×10⁵ blastopsores of Candida albicans 15563. * P = .001 by log-rank test. (C) Effect of vaccinated or control F(ab)¹₂ on blocking mouse neutrophil killing of C. albicans conditionally expressed or suppressed Hyr1. Control denotes assay performed in the absence of either F(ab)¹₂. Data are displayed as median ± interquartile range. * P = .001 by Mann-Whitney test.