Biology subjects

Wakade, R. S.

Publications and source records attributed to Wakade, R. S..

3 recordsLinked to original sources

bioRxiv2022

Candida albicans filamentation does not require the cAMP-PKA pathway in vivo

Candida albicans is one of the most prevalent human fungal pathogens. Its ability to transition between budding yeast and filamentous morphological forms (pseudohyphae and hyphae) is tightly associated with its pathogenesis. Based on in vitro studies, the cAMP-Protein Kinase A (PKA) pathway is a key regulator of C. albicans morphogenesis. Using an intravital imaging approach, we investigated the role of the cAMP-PKA pathway during infection. Consistent with their roles in vitro, the downstream effectors of the cAMP-PKA pathway Efg1 and Nrg1 function, respectively, as an activator and a repressor of in vivo filamentation. Surprisingly, strains lacking the adenylyl cyclase, CYR1, showed only slightly reduced filamentation in vivo despite being completely unable to filament in RPMI+10% serum at 37{degrees}C. Consistent with these findings, deletion of the catalytic subunits of PKA (Tpk1 and Tpk2), either singly or in combination, generated strains that also filamented in vivo but not in vitro. In vivo transcription profiling of C. albicans isolated from both ear and kidney tissue showed that the expression of a set of 184 environmentally responsive correlated well with in vitro filamentation (R2 0.62-0.68) genes. This concordance suggests that the in vivo and in vitro transcriptional responses are similar but that the upstream regulatory mechanisms are distinct. As such, these data emphatically emphasize that C. albicans filamentation is a complex phenotype that occurs in different environments through an intricate network of distinct regulatory mechanisms. ImportanceThe fungus Candida albicans causes a wide range of disease in humans from common diaper rash to life-threatening infections in patients with compromised immune systems. As such, the mechanisms for its ability to cause disease are of wide interest. An intensely studied virulence property of C. albicans is its ability to switch from a round yeast form to filament-like forms (hyphae and pseudohyphae). Surprisingly, we have found that a key signaling pathway that regulates this transition in vitro, the protein kinase A pathway, is not required for filamentation during infection of the host. Our work not only demonstrates that the regulation of filamentation depends upon the specific environment C. albicans inhabits but also underscores the importance of studying these mechanisms during infection.

microbiology↗

bioRxiv2021

In vivo complex haploinsufficiency-based genetic analysis identifies a transcription factor circuit regulating Candida albicans oropharyngeal infection and epithelial cell endocytosis

Oropharyngeal candidiasis (OPC) is a common infection that complicates a wide range of medical conditions which can cause either mild or severe disease depending on the patient. The pathobiology of OPC shares many features with candidal biofilms of abiotic surfaces. The transcriptional regulation of C. albicans formation of biofilms on abiotic surfaces has been extensively characterized and involves six key transcription factors (Efg1, Ndt80, Rob1, Bcr1, Brg1, and Tec1). To determine whether this same in vitro biofilm transcriptional regulatory network played a role in OPC, we have carried out the first systematic genetic interaction analysis in a mouse model of C. albicans infection. Whereas all six transcription factors are required for in vitro biofilm formation, only three homozygous deletion mutants (tec1{Delta}{Delta}, bcr1{Delta}{Delta}, and rob1{Delta}{Delta}) and one heterozygous mutant (tec1{Delta}/TEC1) have reduced infectivity in a mouse model of OPC, indicating the network is more robust in vivo than in vitro. Although single mutants (heterozygous or homozygous) of BRG1 and EFG1 have no effect on fungal burden, the double heterozygous and homozygous mutants have dramatically reduced infectivity, indicating a critical genetic interaction between these two transcription factors. Using epistasis analysis, we have formulated a genetic circuit [EFG1+BRG1][->]TEC1[->]BCR1 that is required for OPC infectivity and oral epithelial cell endocytosis. Surprisingly, we also found transcription factor mutants with in vitro defects in filamentation such as efg1{Delta}{Delta} and brg1{Delta}{Delta} filament during oral infection and that decreased filamentation did not correlate with decreased infectivity. Taken together, these data indicate that key in vitro biofilm transcription factors are involved in OPC but that the network characteristics and functional connections are remodeled significantly during interactions with tissues. Author SummaryThe pathology of oral candidiasis has features of biofilm formation, a well-studied process in vitro. Based on that analogy, we hypothesized that network of transcription factors that regulates in vitro biofilm formation might have similarities and differences in during oral infection. To test this, we employed the first systematic genetic interaction analysis of C. albicans in a mouse model of oropharyngeal infection. This revealed that the six regulators involved in in vitro biofilm formation played roles in vivo but that the functional connections between factors were quite distinct. Surprisingly, we also found that, while many of the factors are required for filamentation in vitro, none of the transcription factor deletion mutants was deficient for this key virulence trait in vivo. These observations clearly demonstrate that C. albicans regulates key aspects of its biology differently in vitro and in vivo.

microbiology↗

bioRxiv2021

Intravital imaging of Candida albicans identifies differential in vitro and in vivo filamentation phenotypes for transcription factor deletion mutants

Candida albicans is an important cause of human fungal infections. A widely studied virulence trait of C. albicans is its ability to undergo filamentation to hyphae and pseudohyphae. Although yeast, pseudohyphae and hyphae are present in pathological samples of infected mammalian tissue, it has been challenging to characterize the role of regulatory networks and specific genes during in vivo filamentation. In addition, the phenotypic heterogeneity of C. albicans clinical isolates is becoming increasingly recognized and correlating this heterogeneity with pathogenesis remains an important goal. Here, we describe the use of an intravital imaging approach to characterize C. albicans filamentation in a mammalian model of infection by taking advantage of the translucence of mouse pinna (ears). Using this model, we have found that the in vitro and in vivo filamentation phenotypes of different C. albicans isolates can vary significantly, particularly when in vivo filamentation is compared to solid agar-based assays. We also show that the well-characterized transcriptional regulators Efg1 and Brg1 appear to play important roles both in vivo and in vitro. In contrast, Ume6 is much more important in vitro than in vivo. Finally, strains that are dependent on Bcr1 for in vitro filamentation are able to form filaments in vivo. This intravital imaging approach provides a new approach to the systematic characterization of this important virulence trait during mammalian infection. Our initial studies provide support for the notion that the regulation and initiation of C. albicans filamentation in vivo is distinct from in vitro induction. ImportanceCandida albicans is one of the most common causes of fungal infections in humans. C. albicans undergoes a transition from a round yeast form to a filamentous form during infection which is critical for its ability to cause disease. Although this transition has been studied in the laboratory for years, methods to do so in an animal model of infection have been limited. We have developed a microscopy method to visualize fluorescently labeled C. albicans undergoing this transition in the subcutaneous tissue of mice. Our studies indicate that the regulation of C. albicans filamentation during infection is distinct from that observed in laboratory conditions.

microbiology↗