Critical role of Bcr1-dependent adhesins in C. albicans biofilm formation in vitro and in vivo

Abstract

The fungal pathogen Candida albicans is frequently associated with catheter-based infections because of its ability to form resilient biofilms. Prior studies have shown that the transcription factor Bcr1 governs biofilm formation in an in vitro catheter model. However, the mechanistic role of the Bcr1 pathway and its relationship to biofilm formation in vivo are unknown. Our studies of biofilm formation in vitro indicate that the surface protein Als3, a known adhesin, is a key target under Bcr1 control. We show that an als3/als3 mutant is biofilm-defective in vitro, and that ALS3 overexpression rescues the biofilm defect of the bcr1/bcr1 mutant. We extend these findings with an in vivo venous catheter model. The bcr1/bcr1 mutant is unable to populate the catheter surface, though its virulence suggests that it has no growth defect in vivo. ALS3 overexpression rescues the bcr1/bcr1 biofilm defect in vivo, thus arguing that Als3 is a pivotal Bcr1 target in this setting. Surprisingly, the als3/als3 mutant forms a biofilm in vivo, and we suggest that additional Bcr1 targets compensate for the Als3 defect in vivo. Indeed, overexpression of Bcr1 targets ALS1, ECE1, and HWP1 partially restores biofilm formation in a bcr1/bcr1 mutant background in vitro, though these genes are not required for biofilm formation in vitro. Our findings demonstrate that the Bcr1 pathway functions in vivo to promote biofilm formation, and that Als3-mediated adherence is a fundamental property under Bcr1 control. Known adhesins Als1 and Hwp1 also contribute to biofilm formation, as does the novel protein Ece1.

Figure 1. Effect of Increased BCR1 Expression on Adherence and Hyphal Morphogenesis

Strains were grown under in vitro biofilm assay conditions for 60 h and photographed (A–F) or grown in Spider suspension cultures and examined by phase contrast microscopy at ×400 magnification (G–I). For the biofilms assays, turbid medium with all cells free-floating in the medium rather than attached to the silicone substrate indicates a biofilm-negative phenotype; clear medium with the silicone substrate completely covered with cells indicates a biofilm-positive phenotype. Relevant genotypes are given above each panel for strains CJN1015 (reference strain + TEF1) (A, G), CJN1060 (bcr1/bcr1 + TEF1) (B), CJN1052 (tec1/tec1 + TEF1) (C, H), CJN1039 (reference strain + TEF1-BCR1) (D), CJN1011 (bcr1/bcr1 + TEF1-BCR1) (E), and CJN1035 (tec1/tec1 + TEF1-BCR1) (F, I).

Figure 2. Biofilm Dry Mass Determinations

Biofilm dry mass determinations were made in quadruplicate after 60 h growth under standard biofilm conditions, as detailed in Materials and Methods. Reference strains DAY185 (shown) and CAI4-URA3 (not shown) gave similar results. Strains are grouped for convenience of comparison. Strain Set A contains CJN896 (tec1/tec1), CJN1052 (tec1/tec1 + TEF1), CJN1035 (tec1/tec1 + TEF1-BCR1), CJN1023 (tec1/tec1 + pTEC1), CJN702 (bcr1/bcr1), CJN1060 (bcr1/bcr1 + TEF1), CJN1011 (bcr1/bcr1 + TEF1-BCR1), CJN698 (bcr1/bcr1 + pBCR1), respectively. Strain Set B contains FJS2 (hyr1/hyr1), FJS6 (ece1/ece1), FJS5 (cht2/cht2), FJS8 (rbt5/rbt5), CAYC2YF1U (als1/als1), CAH7-1A1E2 (hwp1/hwp1), CAYF178U (als3/als3), CAQTP178U (als3/als3 + pALS3), respectively. Strain Set C contains CJN1153 (bcr1/bcr1 + TEF1-ALS3), CJN1144 (bcr1/bcr1 + TEF1-ALS1), CJN1288 (bcr1/bcr1 + TEF1-ECE1), CJN1222 (bcr1/bcr1 + TEF1-HWP1), CJN1281 (bcr1/bcr1 + TEF1-CHT2), CJN1259 (bcr1/bcr1 + TEF1-HYR1), CJN1276 (bcr1/bcr1 + TEF1-RBT5), respectively.

Figure 3. Effect of Increased BCR1 Expression on Target Gene RNA Levels

RNA prepared from mid-log phase Spider medium cultures was used to prepare Northern blots or in RT-PCR assays, as indicated. Northern blots were probed for the transcripts indicated along the left side, and PhosphorImager exposures are shown. RT-PCR assays for ALS1, ALS3, and TEF1 were conducted on serial 2-fold dilutions of cDNA preparations and fractionated on agarose gels; only the last two dilutions are shown. TEF1 transcript levels were used as an expression control. Strains included DAY185 (reference strain) (sample 1), CJN1011 (bcr1/bcr1 + TEF1-BCR1) (sample 2), CJN1015 (reference strain + TEF1) (sample 3), CJN1035 (tec1/tec1 + TEF1-BCR1) (sample 4), CJN1039 (reference strain + TEF1-BCR1) (sample 5), CJN1052 (tec1/tec1 + TEF1) (sample 6), and CJN1060 (bcr1/bcr1 + TEF1) (sample 7).

Figure 4. Biofilm Formation In Vitro by Bcr1 Target Gene Mutants

Strains were grown in our standard biofilm assay and photographed after 60 h. Relevant genotypes are given above each panel and include DAY286 (reference strain), CJN459 (bcr1/bcr1), FJS2 (hyr1/hyr1), CAH7-1A1E2 (hwp1/hwp1), FJS5 (cht2/cht2), FJS6 (ece1/ece1), FJS8 (rbt5/rbt5), FJS10 (ecm331/ecm331), CAYF178U (als3/als3), and CAYC2YF1U (als1/als1). Turbid medium with all cells free-floating in the medium rather than attached to the silicone substrate indicates a biofilm-negative phenotype; clear medium with the silicone substrate completely covered with cells indicates a biofilm-positive phenotype. An uninoculated control is shown in the panel labeled “Blank.”

Figure 5. In Vitro Filamentation and Biofilm Formation by the als3/als3 Mutant

Cells were grown in free-living (planktonic) cultures in Spider medium; filamentation was examined by phase contrast microscopy at ×400 magnification (top panels). Biofilms were grown under standard conditions in Spider medium, and stained with concanavalin A conjugate for CSLM visualization. Artificially colored CSLM depth views, in which blue color represents cells closest to the silicone and red color represents cells farthest from the silicone, are shown in middle panels. For the depth views of reference strain CAI4-URA3 (ALS3/ALS3), blue = 0 μm and red = 800 μm; CAYF178U (als3/als3), blue = 0 μm, red = 80 μm; CAQTP178U (als3/als3 + pALS3), blue = 0 μm, red = 600 μm. CSLM side views are shown in lower panels. For the side views, the scale bars represent 50 μm for CAI4-URA3 (ALS3/ALS3) and CAQTP178U (als3/als3 + pALS3); and 20 μm for CAYF178U (als3/als3).

Figure 6. Overexpression of ALS3 in the bcr1/bcr1 Mutant Restores Substantial Biofilm Formation In Vitro

Biofilms were grown under standard conditions and stained with concanavalin A conjugate for CSLM visualization. The top panels show the visual appearance. The next set of panels show depth views, in which blue color represents cells closest to the silicone and red color represents cells farthest from the silicone. The next set of panels show side views. For the depth views of reference strain DAY185 (BCR1/BCR1), blue = 0 μm and red = 600 μm; CJN1149 (BCR1/BCR1 + TEF1-ALS3), blue = 0 μm and red = 500 μm; CJN702 (bcr1/bcr1), blue = 0 μm and red = 80 μm; CJN1153 (bcr1/bcr1 + TEF1-ALS3), blue = 0 μm and red = 180 μm. For the side views, the scale bars represent 50 μm for DAY185 (BCR1/BCR1), CJN1149 (BCR1/BCR1 + TEF1-ALS3), and CJN1153 (bcr1/bcr1 + TEF1-ALS3); and 20 μm for CJN702 (bcr1/bcr1). The next set of panels show RT-PCR analysis of ALS3 expression of the indicated strains with successive 2-fold dilutions of cDNA from left to right. The bottom panels show RT-PCR of control TEF1 transcript levels.

Figure 7. Bcr1 Requirement for Biofilm Formation In Vivo

Central venous catheters were introduced into rats, inoculated with C. albicans strain DAY185 (BCR1/BCR1) (A–C), CJN 702 (bcr1/bcr1) (D–F), or CJN698 (bcr1/bcr1 + pBCR1) (G–I) and then flushed and incubated [18]. Catheters were the removed and their contents visualized by scanning electron microscopy after 12 h (A, D, G), 24 h (B, E, H), and 48 h (C, F, I).

Figure 8. Role of Als3 in Biofilm Formation In Vivo

Central venous catheters were introduced into rats, inoculated with C. albicans strains CAYF178U (als3/als3) (A), CAQTP178U (als3/als3 + pALS3) (B), or CJN1153 (bcr1/bcr1 + TEF1-ALS3) (C), and then flushed and incubated for 24 h [18]. Catheters were subsequently removed and their contents visualized by scanning electron microscopy.

Figure 9. Role of Bcr1 Target Genes in Biofilm Formation

Bcr1 is required for full expression of adhesins Als3, Als1, and Hwp1 and of novel protein Ece1. Gene mutation and overexpression analyses together prove that Als3 is necessary and sufficient among Bcr1 targets for biofilm formation in vitro. Overexpression analysis indicates that Als1, Hwp1, and Ece1 can also restore biofilm formation in the absence of Bcr1 in vitro. The fact that overexpression suppressors Als3, Als1, and Hwp1 are all known adhesins indicates that adherence is the property through which Bcr1 governs biofilm formation. Bcr1 is required for biofilm formation in vivo, and overexpression of Als3 permits biofilm formation in the absence of Bcr1 in vivo. Thus, Bcr1-dependent adherence is critical for biofilm formation in vivo and in vitro.