The conserved cAMP-dependent protein kinase (PKA) plays critical roles in the regulation of morphological transitions and virulence in the human fungal pathogen Candida albicans. It has long been thought that the PKA catalytic subunit is essential for cell viability in this fungus. Paradoxically, the single adenylyl cyclase-encoding gene, CYR1, which is required for the production of cAMP in C. albicans, is not essential for cell growth. Here, a double mutant of TPK1 and TPK2 (tpk2/tpk2 tpk1/tpk1, t2t1), which encode two isoforms of the PKA catalytic subunit was successfully generated, suggesting that this subunit is not essential for cell viability. Inactivation of the PKA catalytic subunit blocked filamentation and dramatically attenuated white-to-opaque switching, but promoted sexual mating. Comparative transcriptomic analyses demonstrated that the t2t1 and cyr1/cyr1 mutants exhibited similar global gene expression profiles. Compared with the WT strain, the general transcriptional activity and metabolism were significantly decreased in both the t2t1 and cyr1/cyr1 mutants. Using combined phosphoproteomic and bioinformatic analyses, we identified 181 potential PKA phosphorylation targets, which represent 148 unique proteins involved in a wide spectrum of biological processes. The study sheds new insights into the global regulatory features of the cAMP/PKA pathway in C. albicans.

The conserved cAMP-dependent protein kinase (PKA) is composed of the regulatory and catalytic subunits and acts as the central component of the cAMP signaling pathway. In the human fungal pathogen Candida albicans, the PKA regulatory subunit Bcy1 plays a critical role in the regulation of cell differentiation and death. It has long been considered that Bcy1 is essential for cell viability in C. albicans. In the current study, surprisingly, we found that Bcy1 is not required for cell growth, and we successfully generated a bcy1/bcy1 null mutant in C. albicans. Deletion of BCY1 leads to multiple cellular morphologies and promotes the development of filaments. Filamentous and smooth colonies are two typical morphological types of the bcy1/bcy1 mutant, which can undergo spontaneous switching between the two types. Cells of filamentous colonies grow better on a number of different culture media and have a higher survival rate than cells of smooth colonies. In addition, deletion of BCY1 significantly increased the frequency of white-to-opaque switching on N-acetylglucosamine (GlcNAc)-containing medium. The bcy1/bcy1 null mutant generated herein provides the field a new resource to study the biological functions of the cAMP signaling pathway in C. albicans.

Phenotypic switching is a strategy by which microbial organisms adapt to environmental changes. The human fungal pathogens, Candida albicans and Candida tropicalis, are closely related species and capable of undergoing morphological transitions. C. albicans primarily exists in human or warm-blooded animals as a commensal, whereas C. tropicalis not only exists as a commensal but also is widely distributed in the environment. In this study, we describe the environmental and genetic regulatory mechanisms of white-opaque switching in C. tropicalis, which is associated with virulence and sexual mating. A comparative study with C. albicans demonstrated that C. tropicalis responds to environmental stimuli, such as elevated CO levels and pH changes, in opposite manners. An acidic pH and elevated CO levels promote the opaque phenotype in C. albicans but have an opposite effect in C. tropicalis, whereas alkaline pH conditions facilitate white-to-opaque switching and sexual mating in C. tropicalis. The conserved Rim101-mediated pH sensing and Ras1-cAMP/PKA signaling pathways are involved in this regulation. By screening an overexpression library of transcription factors, we identified 26 white-opaque regulators, including WOR1, AHR1, EFG1, CUP9, BCR1 and SFL2. Transcriptional analysis indicated that the pH sensing and Ras1-cAMP/PKA signaling pathways and transcriptional regulators coordinately regulate white-to-opaque switching.

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Abstract Background: Endometritis adversely affects the ability of cattle to reproduce, and significantly reduces milk production. Consequently, it has great influence on the economic value of dairy cows. The endometrium is mainly composed of epithelial and stromal cells and they produce the first immune response to invading pathogens. Epithelial cells are the first cellular barrier through which bacteria enter the uterine endometrium. However, most of the epithelial cells are disrupted and stromal cells are exposed to an inflammatory environment when endometritis occurs, especially postpartum. A loss of the protective epithelium allows bacteria or toxins to access the underlying stromal cells. The activation of Toll-like receptor（TLRs）on stromal cells induces increased production of cytokines and chemokines. Understanding the genome-wide characterization of the bovine endometritis will be beneficial for prevention and treatment of endometritis. In this study, whole-transcriptomic gene changes in bovine stromal cells treated with LPS were compared with those treated with PBS (control group) and were analyzed by RNA sequencing (RNA-seq). Results: Compared with the control group, a total of 366 differentially expressed genes (DEGs) were identified in LPS-induced group (234 upregulated and 132 downregulated genes), with an adjusted P-value＜0.05 by DESeq. Gene ontology (GO) enrichment analysis revealed DEGs were most enriched in lymphocyte activation, interleukin-1 receptor binding, regulation of cell activation, and lymphocyte-activated interleukin-12 production. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed DEGs were most enriched in TNF signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction, nucleotide-binding oligomerization domain-like (NOD-like) receptor signaling pathway, NF-κB signaling pathway, and chemokine signaling pathway.Conclusion: The results of this study unraveled endometrial stromal cells transcriptome profile alterations in bovine affected by LPS which may have a significant effect on the eliminating or reducing inflammation by comprehending molecular mechanisms and authenticating unique genes related to endometritis.

Bovine endometrial stromal cells (bESCs) are exposed to a complex environment of bacteria and viruses due to the rupture of epithelial cells after delivery. Inflammatory responses are elicited by the activation of host pattern recognition receptors through pathogen-related molecules such as lipopolysaccharides (LPS) on the cell membrane. Forsythoside A (FTA) is a major active constituent of Forsythia suspensa (Thunb.) Vahl. is a flowering plant widely employed as a traditional Chinese herbal medicine to treat various inflammatory diseases such as nephritis, eye swelling, scabies, ulcers, and mastitis; however, the molecular mechanisms underlying its therapeutic effects on bovine endometritis are still unclear. The aim of this study was to explore the role of miRNA and the mechanisms underlying the protective activity of FTA on the inflammation of bovine endometrial stromal cells induced by LPS. Based on previous research, we isolated and cultured bESCs in vitro and categorized them into LPS and LPS+FTA groups with three replicates. Upon reaching 80% confluence, the bESCs were treated with 0.5 μg/mL of LPS or 0.5 μg/mL of LPS + 100 μg/mL of FTA. We, then, performed high-throughput sequencing (RNA-Seq) to investigate the effects of FTA on LPS-stimulated primary bESCs and their underlying mechanisms. We identified 167 miRNAs differentially expressed in the LPS groups; 72 miRNAs were up-regulated, and 95 were down-regulated. Gene ontology enrichment analysis revealed that differentially expressed microRNA (DEGs) were most enriched during the cellular metabolic process; they were mostly located intracellularly and participated in protein, enzyme, and ion binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the DEGs were most enriched in the mitogen-activated protein kinase, tumor necrosis factor, and Interleukin-17 signaling pathways. These results reveal the complex molecular mechanism involved in the FTA and provide a basis for future studies of bovine endometritis treatment with traditional Chinese medicine monomer.

Endometritis adversely affects the ability of cattle to reproduce and significantly reduces milk production. The is mainly composed of epithelial and stromal cells, and they produce the first immune response to invading pathogens. However, most of the epithelial cells are disrupted, and stromal cells are exposed to an inflammatory environment when endometritis occurs, especially postpartum. Many bacteria and toxins start attacking stromal cell due to loss of epithelium, which stimulates Toll-like receptor (TLRs) on stromal cells and causes upregulated expression of cytokines. Understanding the genome-wide characterization of bovine endometritis will be beneficial for prevention and treatment of endometritis. In this study, whole-transcriptomic gene changes in bovine endometrial stromal cells (BESCs) treated with LPS were compared with those treated with PBS (control group) and were analyzed by RNA sequencing. Compared with the control group, a total of 366 differentially expressed genes (DEGs) were identified in the LPS-induced group (234 upregulated and 132 downregulated genes), with an adjusted P < 0.05 by DESeq. Gene Ontology (GO) enrichment analysis revealed that DEGs were most enriched in interleukin-1 receptor binding, regulation of cell activation, and lymphocyte-activated interleukin-12 production. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed DEGs were most enriched in the TNF signaling pathway, Toll-like receptor signaling pathway, cytokine–cytokine receptor interaction, NF-κB signaling pathway, and chemokine signaling pathway. The results of this study unraveled BESCs affected with LPS transcriptome profile alterations, which may have a significant effect on treatment inflammation by comprehending molecular mechanisms and authenticating unique genes related to endometritis.

Abstract Background: Endometritis adversely affects the ability of cattle to reproduce, and significantly reduces milk production. Consequently, it has great influence on the economic value of dairy cows. The endometrium is mainly composed of epithelial and stromal cells and they produce the first immune response to invading pathogens. Epithelial cells are the first cellular barrier through which bacteria enter the uterine endometrium. However, most of the epithelial cells are disrupted and stromal cells are exposed to an inflammatory environment when endometritis occurs, especially postpartum. Inflammation in endometrial stromal cells is thus activated, immune-related cytokines and signaling pathways are activated. This indicates that endometritis is becoming more and more serious. However, inflammatory response in bovine endometrial stromal cells is yet to be studied in detail. Understanding the genome-wide characterization of the bovine endometritis will be beneficial for prevention and treatment of endometritis. In this study, whole-transcriptomic gene changes in bovine stromal cells treated with LPS were compared with those treated with PBS (control group) and were analyzed by RNA sequencing (RNA-seq). Results: Compared with the control group, a total of 366 differentially expressed genes (DEGs) were identified in LPS-induced group (234 upregulated and 132 downregulated genes), with an adjusted P -value＜0.05 by DESeq. Gene ontology (GO) enrichment analysis revealed DEGs were most enriched in lymphocyte activation, interleukin-1 receptor binding, regulation of cell activation, and lymphocyte-activated interleukin-12 production. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed DEGs were most enriched in TNF signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction, nucleotide-binding oligomerization domain-like (NOD-like) receptor signaling pathway, NF-κB signaling pathway, and chemokine signaling pathway. Conclusion: Our results proved and expanded findings of previous studies on bovine endometritis. These results will be useful to propose new therapeutic targets and eliminate or reduce inflammation by comprehending molecular mechanisms and authenticating unique genes related to endometritis.