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A family of secreted pathogenesis-related proteins in Candida albicans

ABSTRACT: 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.

ORGANISM(S): Candida albicans SC5314 Candida albicans

PROVIDER: GSE28212 | GEO | 2013/03/25

SECONDARY ACCESSION(S): PRJNA139659

REPOSITORIES: GEO

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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:Polymicrobial biofilms are of large medical importance, but little is known about their physiology and the underlying interspecies interactions. Here we studied two human pathogens, the opportunistic fungus Candida albicans and the caries promoting bacterium Streptococcus mutans. Both species formed biofilms in monoculture, with C. albicans growing mainly in the virulence-associated hyphae form, and S. mutans forming a thick layer of extracellular polymeric substances (EPS). Biofilm growth was enhanced in dual-species biofilms, which reached twice the biomass of monospecies biofilms and higher cell numbers of both S. mutans and C. albicans. EPS production by S. mutans was strongly suppressed in dual-species biofilms. Virulence traits of S. mutans, e.g. genetic competence, biofilm formation and bacteriocin synthesis are controlled by quorum sensing through activation of the alternative sigma factor SigX. SigX is induced by the pheromones CSP (competence stimulating factor) or XIP (sigX inducing peptide). Strong induction of sigX was observed in dual species biofilms indicated by fluorescence of a reporter strain for the sigX promoter, S. mutans PcomX-gfp, as well as by qRT-PCR of comX. The peak of sigX expression occurred after 10 h of biofilm growth. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion mutants for the comC and comS genes encoding the precursors of CSP and XIP, respectively, were constructed. Conditioned media from mixed biofilms with S. mutans DcomS were unable to induce sigX in the reporter strain, while deletion of comC had no effect. These data show that synthesis of XIP was induced in S. mutans by coculture with C. albicans. Transcriptome analysis of S. mutans in single and mixed biofilms confirmed strong induction of comS, sigX, and the downstream late competence genes in dual-species biofilms. Among the late competence genes, fratricins were discovered for the first time. The comCDE operon and bacteriocin related genes were also induced, but much weaker. Genes related to oxidative stress, chaperones and glycosyltransferase genes required for EPS synthesis from sucrose were down-regulated, while glycogen synthesis genes were up-regulated, indicating that S. mutans was protected from oxidative stress and provided with excess sugar for storage polymer synthesis in mixed biofilms. The data show that in dual-species biofilms, C. albicans improves growth of S. mutans, suppresses its EPS formation and induces the complete quorum sensing signalling system, thus fundamentally changing the virulence properties of the caries pathogen, including its potential interactions with other members of the polymicrobial dental plaque community.

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A family of secreted pathogenesis-related proteins in Candida albicans

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