Project description:Analyzing culture supernatants of yeast and hyphal cells of Candida albicans by mass spectrometry, we found two close homologues of pathogenesis-related (PR-) 1 proteins, Rbe1p and Rbt4p, in the secretome of this human pathogen. By sequence homology, we assigned three yet not characterized open reading frames, ORF19.6200, ORF19.2787 and ORF19.2336, in addition to Rbe1p and Rbt4p to a novel family of proteins. Correspondent with our secretome analysis RBE1 was expressed in blastospores and opaque cells, whereas transcription was down-regulated in hyphae. On the contrary, RBT4 was up-regulated in hyphae and down-regulated in opaque cells. Remarkably, transcription of RBT4 and RBE1 was each up-regulated in blastospores of M-bM-^HM-^Frbe1 or hyphae of M-bM-^HM-^Frbt4 deletion strains, respectively, indicating a compensatory function of both proteins. In a M-bM-^HM-^Frbe1/M-bM-^HM-^Frbt4 double deletion strain, genome-wide transcriptional analysis showed differential transcription of a limited set of genes that are also implicated in virulence and oxidative stress response. In this context, deletion of RBE1 or RBT4 in a clinical C. albicans isolate resulted in a moderate but significant attenuation in virulence in a mouse model for disseminated candidiasis. However, a synergistic effect was observed in the M-bM-^HM-^Frbe1/M-bM-^HM-^Frbt4 double deletion strain, where virulence was strongly affected. Furthermore, the double deletion strain showed increased sensitivity to attack by polymorphonuclear leukocytes (neutrophils). Therfore, our data suggest that the crucial contribution of both C. albicans pathogenesis-related proteins for in vivo virulence results at least partially from reduced survival in phagocytes. Experiments were performed under blastospore (YPD) as well as hyphae (alpha-MEM)-inducing conditions. In total, three biological replicates were performed for each condition. All experiments were performed as dye swaps. Thus, in total six arrays have been hybridzed for each comparison (alpha-MEM and YPD, respectively). Hybridization experiments included a reference strain (C. albicans SC5314) and a double deletion strain (C. albicans MRC27). The array included one technical replicate of each probe.
Project description:Analyzing culture supernatants of yeast and hyphal cells of Candida albicans by mass spectrometry, we found two close homologues of pathogenesis-related (PR-) 1 proteins, Rbe1p and Rbt4p, in the secretome of this human pathogen. By sequence homology, we assigned three yet not characterized open reading frames, ORF19.6200, ORF19.2787 and ORF19.2336, in addition to Rbe1p and Rbt4p to a novel family of proteins. Correspondent with our secretome analysis RBE1 was expressed in blastospores and opaque cells, whereas transcription was down-regulated in hyphae. On the contrary, RBT4 was up-regulated in hyphae and down-regulated in opaque cells. Remarkably, transcription of RBT4 and RBE1 was each up-regulated in blastospores of ∆rbe1 or hyphae of ∆rbt4 deletion strains, respectively, indicating a compensatory function of both proteins. In a ∆rbe1/∆rbt4 double deletion strain, genome-wide transcriptional analysis showed differential transcription of a limited set of genes that are also implicated in virulence and oxidative stress response. In this context, deletion of RBE1 or RBT4 in a clinical C. albicans isolate resulted in a moderate but significant attenuation in virulence in a mouse model for disseminated candidiasis. However, a synergistic effect was observed in the ∆rbe1/∆rbt4 double deletion strain, where virulence was strongly affected. Furthermore, the double deletion strain showed increased sensitivity to attack by polymorphonuclear leukocytes (neutrophils). Therfore, our data suggest that the crucial contribution of both C. albicans pathogenesis-related proteins for in vivo virulence results at least partially from reduced survival in phagocytes.
