Lipid regulators of Pkh2 in Candida albicans, the protein kinase ortholog of mammalian PDK1.

Pkh is the yeast ortholog of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1). Pkh phosphorylates the activation loop of Ypks, Tpks, Sch9 and also phosphorylates the eisosome components Lsp1 and Pil1, which play fundamental roles upstream of diverse signaling pathways, including the cell wall integrity and sphingosine/long-chain base (LCB) signaling pathways. In S. cerevisiae, two isoforms, ScPkh1 and ScPkh2, are required for cell viability, while only one ortholog exists in C. albicans, CaPkh2. In spite of the extensive information gathered on the role of Pkh in the LCB signaling, the yeast Pkh kinases are not known to bind lipids and previous studies did not identify PH domains in Pkh sequences. We now describe that the C-terminal region of CaPkh2 is required for its intrinsic kinase activity. In addition, we found that the C-terminal region of CaPkh2 enables its interaction with structural and signaling lipids. Our results further show that phosphatidylserine, phosphatidic acid, phosphatidylinositol (3,4 and 4,5)-biphosphates, and phosphatidylinositol (3,4,5)-trisphosphate inhibit Pkh activity, whereas sulfatide binds with high affinity but does not affect the intrinsic activity of CaPkh2. Interestingly, we identified that its human ortholog PDK1 also binds to sulfatide. We propose a mechanism by which lipids and dihydrosphingosine regulate CaPkh2 kinase activity by modulating the interaction of the C-terminal region with the kinase domain, while sulfatide-like lipids support localization CaPkh2 mediated by a C-terminal PH domain, without affecting kinase intrinsic activity.

Depletion of yeast PDK1 orthologs triggers a stress-like transcriptional response.

BACKGROUND: Pkh proteins are the PDK1 orthologs in S. cerevisiae. They have redundant and essential activity and are responsible for the phosphorylation of several members of the AGC family of protein kinases. Pkh proteins have been involved in several cellular functions, including cell wall integrity and endocytosis. However the global expression changes caused by their depletion are still unknown. RESULTS: A doxycycline-repressible tetO7 promoter driving the expression of PKH2 in cells carrying deletions of the PKH1 and PKH3 genes allowed us to progressively deplete cells from Pkh proteins when treated with doxycycline. Global gene expression analysis indicate that depletion of Pkh results in the up-regulation of genes involved in the accumulation of glycogen and also of those related to stress responses. Moreover, genes involved in the ion transport were quickly down-regulated when the levels of Pkh decreased. The reduction in the mRNA levels required for protein translation, however, was only observed after longer doxycycline treatment (24 h). We uncovered that Pkh is important for the proper transcriptional response to heat shock, and is mostly required for the effects driven by the transcription factors Hsf1 and Msn2/Msn4, but is not required for down-regulation of the mRNA coding for ribosomal proteins. CONCLUSIONS: By using the tetO7 promoter we elucidated for the first time the transcriptomic changes directly or indirectly caused by progressive depletion of Pkh. Furthermore, this system enabled the characterization of the transcriptional response triggered by heat shock in wild-type and Pkh-depleted cells, showing that about 40 % of the observed expression changes were, to some degree, dependent on Pkh.

Impact of bread diet on intestinal dysbiosis and irritable bowel syndrome symptoms in quiescent ulcerative colitis: A pilot study.

Gut microbiota may be involved in the presence of irritable bowel syndrome (IBS)-like symptomatology in ulcerative colitis (UC) patients in remission. Bread is an important source of dietary fiber, and a potential prebiotic. To assess the effect of a bread baked using traditional elaboration, in comparison with using modern elaboration procedures, in changing the gut microbiota and relieving IBS-like symptoms in patients with quiescent ulcerative colitis. Thirty-one UC patients in remission with IBS-like symptoms were randomly assigned to a dietary intervention with 200 g/d of either treatment or control bread for 8 weeks. Clinical symptomatology was tested using questionnaires and inflammatory parameters. Changes in fecal microbiota composition were assessed by high-throughput sequencing of the 16S rRNA gene. A decrease in IBS-like symptomatology was observed after both the treatment and control bread interventions as reductions in IBS-Symptom Severity Score values (p-value < 0.001) and presence of abdominal pain (p-value < 0.001). The treatment bread suggestively reduced the Firmicutes/Bacteroidetes ratio (p-value = 0.058). In addition, the Firmicutes/Bacteroidetes ratio seemed to be associated with improving IBS-like symptoms as suggested by a slight decrease in patient without abdominal pain (p-value = 0.059). No statistically significant differential abundances were found at any taxonomic level. The intake of a bread baked using traditional elaboration decreased the Firmicutes/Bacteroidetes ratio, which seemed to be associated with improving IBS-like symptoms in quiescent ulcerative colitis patients. These findings suggest that the traditional bread elaboration has a potential prebiotic effect improving gut health (ClinicalTrials.gov ID number of study: NCT05656391).

Exploring the Associations of Inflammatory and Oxidative Stress Biomarkers with Pancreatic Diseases: An Observational and Mendelian Randomisation Study.

Identifying biomarkers linked to pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP) is crucial for early detection, treatment, and prevention. Methods: Association analyses of 10 serological biomarkers involved in cell signalling (IFN-γ, IL-6, IL-8, IL-10), oxidative stress (superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities, total glutathione (GSH), malondialdehyde (MDA) levels), and intestinal permeability proteins (zonulin, I-FABP2) were conducted across PDAC (n = 12), CP (n = 21) and control subjects (n = 23). A Mendelian randomisation (MR) approach was used to assess causality of the identified significant associations in two large genetic cohorts (FinnGen and UK Biobank). Results: Observational results showed a downregulation of SOD and GPx antioxidant enzyme activities in PDAC and CP patients, respectively, and higher MDA levels in CP patients. Logistic regression models revealed significant associations between CP and SOD activity (OR = 0.21, 95% CI [0.05, 0.89], per SD), GPx activity (OR = 0.28, 95% CI [0.10, 0.79], per SD), and MDA levels (OR = 2.05, 95% CI [1.36, 3.08], per SD). MR analyses, however, did not support causality. Conclusions: These findings would not support oxidative stress-related biomarkers as potential targets for pancreatic diseases prevention. Yet, further research is encouraged to assess their viability as non-invasive tools for early diagnosis, particularly in pre-diagnostic CP populations.

A Novel Grape-Derived Prebiotic Selectively Enhances Abundance and Metabolic Activity of Butyrate-Producing Bacteria in Faecal Samples.

Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) patients have different faecal microbiota profiles compared to healthy controls. Prebiotics intake influences intestinal microbiota composition which in turn influence the growth of short-chain fatty acids (SCFA) producing bacteria. This study aimed to evaluate the capacity of Previpect, a new prebiotic obtained from grapes fibre, to balance the dysbiosis found in patients with intestinal disorders. This was achieved through the analysis of specific bacterial markers and SCFA production using an in vitro fermentation system and comparing the obtained results with those obtained with other commercial prebiotics. Fresh faecal samples from patients with IBD (N = 6), IBS (N = 3), and control subjects (N = 6) were used. Previpect showed high fermentative ability enabling the growth of butyrate producing bacteria and increasing SCFA concentration up to 2.5-fold. Previpect is a promising prebiotic which may be used as a therapeutic strategy towards promotion of intestinal microbiota restoration, microbial healing, and as a preventive supplement for healthy individuals.

RAID Prediction: Pilot Study of Fecal Microbial Signature With Capacity to Predict Response to Anti-TNF Treatment.

BACKGROUND AND AIMS: Crohn's disease and ulcerative colitis evolve with alternate outbreaks and remissions of variable duration in both cases. Despite the advances, about 10-30% of patients do not respond to the treatment after the induction period. Besides, between 20% to 50% further patients need an optimization of the dose to respond the treatment. Recent studies have pointed gut microbiota can play a role in the anti-TNF treatment response. This study aimed to define a bacterial signature that could be used to predict the response of patients to anti-TNF treatment. METHODS: There were obtained 38 stool samples from 38 IBD patients before starting anti-TNF treatments: Adalimumab, Golimumab or Infliximab. Patients were differentiated in 2 groups: responders and non-responders to biological treatment. From each sample, DNA was purified and used in a qPCR for the quantification of the 8 microbial markers. RESULTS: In this proof of concept, the predictive ability to identify anti-TNF treatment responders was analyzed. An algorithm consisting in the combination of 4 bacterial markers showed a high capacity to discriminate between responders and non- responders. The algorithm proved high sensitivity and specificity reporting values of 93.33% and 100% respectively, with a positive predictive value of 100% and a negative predictive value of 75% for predicting response to biologic treatment. CONCLUSIONS: A specific bacterial signature could beneficiate patients with inflammatory bowel disease predicting the therapeutic effectiveness of an anti-TNF treatment, leading to a personalized therapy, improving the patients' quality of life, saving costs and gaining time in patient improvement.

This study aimed to define a microbial signature that could be used to predict the response of patients to anti-TNF treatment in inflammatory bowel disease. An algorithm consisting in the combination of 4 bacterial markers showed a high capacity to discriminate between responders and nonresponders.

In vitro Prebiotic Effect of Bread-Making Process in Inflammatory Bowel Disease Microbiome.

Inflammatory bowel disease (IBD), including its two main categories (Crohn's disease and ulcerative colitis), has been linked both to gut microbiota and to diet. Bread is a daily food that has a potential capacity as a prebiotic. Our aim was to evaluate different bread-making processes and their effect on fecal colonic microbiota in IBD patients. The microbial composition of several sourdoughs and dough samples was analyzed by high-throughput sequencing of 16S and 18S rRNA genes. Three types of bread, which followed different bread-making processes, were in vitro digested and incubated with feces from IBD patients. Changes in gut microbiota were assessed by a quantitative polymerase chain reaction using specific bacterial sequence targets. Short-chain fatty acid production was also analyzed by gas chromatography. Lactobacillus sanfranciscensis was the dominant lactic acid bacteria species found in sourdough and bread doughs prepared using sourdough, whereas Saccharomyces cerevisiae was the most dominant yeast in all groups, especially in bread doughs before baking. Differences in microbial composition in raw bread doughs were more related to the type of dough and elaboration than to fermentation time lengths. The analysis of in vitro fecal incubations with bread conditions revealed an increase in most bacterial groups analyzed and short-chain fatty acid production, both in Crohn's disease and ulcerative colitis samples. Most remarkable increases in short-chain fatty acid production mirrored higher abundances of Roseburia species. The potential prebiotic properties observed were mainly obtained when using a high quantity of bread, regardless of bread type. Overall, this study highlights the bacterial dynamics within the bread-making process and the potential prebiotic effect in IBD patients.

New fecal bacterial signature for colorectal cancer screening reduces the fecal immunochemical test false-positive rate in a screening population.

Guidelines recommend routine screening for colorectal cancer (CRC) in asymptomatic adults starting at age 50. The most extensively used noninvasive test for CRC screening is the fecal immunochemical test (FIT), which has an overall sensitivity for CRC of approximately 61.0%-91.0%, which drops to 27.0%-67.0% for advanced adenomas. These figures contain a high false-positive rate and a low positive predictive value. This work aimed to develop a new, noninvasive CRC screening tool based on fecal bacterial markers capable of decreasing FIT false-positive rates in a FIT-positive population. We defined a fecal bacterial signature (RAID-CRC Screen) in a proof-of-concept with 172 FIT-positive individuals and validated the obtained results on an external cohort of 327 FIT-positive subjects. All study participants had joined the national CRC screening program. In the clinical validation of RAID-CRC Screen, a sensitivity of 83.9% and a specificity of 16.3% were obtained for the detection of advanced neoplasm lesions (advanced adenomas and/or CRC). FIT 20 µg/g produced 184 false-positive results. Using RAID-CRC Screen, this value was reduced to 154, thus reducing the false-positive rate by 16.3%. The RAID-CRC Screen test could be implemented in CRC screening programs to allow a significant reduction in the number of colonoscopies performed unnecessarily for FIT-positive participants of CRC screening programs.

Reduction of faecal immunochemical test false-positive results using a signature based on faecal bacterial markers.

BACKGROUND: Colorectal cancer is the second commonest cause of cancer mortality. Some countries are implementing colorectal cancer screening to detect lesions at an early stage using non-invasive tools like the faecal immunochemical test. Despite affordability, this test shows a low sensitivity for precancerous lesions and a low positive predictive value for colorectal cancer, resulting in a high false-positive rate. AIM: To develop a new, non-invasive colorectal cancer screening tool based on bacterial faecal biomarkers, which in combination with the faecal immunochemical test, could allow a reduction in the false-positive rate. This tool is called risk assessment of intestinal disease for colorectal cancer (RAID-CRC). METHODS: We performed both the faecal immunochemical test and the bacterial markers analysis (RAID-CRC test) in stool samples from individuals with normal colonoscopy (167), non-advanced adenomas (88), advanced adenomas (30) and colorectal cancer (48). All the participants showed colorectal cancer-associated symptoms. RESULTS: Performance of the faecal immunochemical test for advanced neoplasia (ie advanced adenoma and colorectal cancer) was determined by using the cut-off value established in Catalonia (20 µg haemoglobin/g of faeces) for a population-based screening approach. Sensitivity and specificity values of 83% and 80%, respectively, and positive and negative predictive values of 56% and 94%, respectively, were obtained. When both the immunological and the biological analysis were combined, the corresponding values were 80% and 90% for sensitivity and specificity, respectively, and 70% and 94% for positive and negative predictive values, respectively, resulting in a 50% reduction of the false-positive rate. CONCLUSIONS: RAID-CRC test allows a substantial reduction in the faecal immunochemical test false-positive results (50%) in a symptomatic population. Further validation is indicated in a colorectal cancer-screening scenario.

Anti-tumour Necrosis Factor Treatment with Adalimumab Induces Changes in the Microbiota of Crohn's Disease.

BACKGROUND: The composition of the intestinal microbiota is altered in Crohn's disease [CD] patients. The objective of this study was to evaluate the qualitative and quantitative changes in the microbiota of CD patients in 3 months of treatment with adalimumab [ADA], and determine whether or not these changes are produced towards the recovery of the normal, healthy-like microbiota. METHODS: The microbiota composition, and the Faecalibacterium prausnitzii / Escherichia coli quantitative relationship as dysbiosis indicator, were studied at baseline [T0], one month [T1], and 3 months [T3] after starting treatment using a polymerase chain reaction-denaturing gradient gel electrophoresis [PCR-DGGE] of 16S rRNA gene fragments and quantitative PCR, respectively, in rectal mucosal biopsies from 15 CD patients and four healthy subjects. RESULTS: T0 and T3 fingerprints were different in all patients; whereas T1 and T3 presented similar patterns. Recovered phylogroups were Firmicutes [79.1%], Bacteroides [12.5%], and Actinobacteria [6.25%]. The prevalence of E. coli decreased during treatment. Relative E. coli loads in CD samples were significantly reduced at every analysed step [T1 and T3] [p < 0.005] whereas no significant changes were observed in relative F. prausnitzii counts. CONCLUSION: Treatment with ADA induces short-term changes in the microbiota composition which seem to parallel the partial recovery of the gut bacterial ecology, with recovery parameters tending to eubiosis recovery. The quantitative determination of dysbiosis-representative bacteria, such as E. coli, may provide a fast and reliable indicator of the healing state of the intestinal mucosa.

PIF-pocket as a target for C. albicans Pkh selective inhibitors.

The phosphoinositide-dependent protein kinase 1, PDK1, is a master kinase that phosphorylates the activation loop of up to 23 AGC kinases. S. cerevisiae has three PDK1 orthologues, Pkh1-3, which also phosphorylate AGC kinases (e.g., Ypk, Tpk, Pkc1, and Sch9). Pkh1 and 2 are redundant proteins involved in multiple essential cellular functions, including endocytosis and cell wall integrity. Based on similarities with the budding yeast, the Pkh of fungal infectious species was postulated as a novel target for antifungals. Here, we found that depletion of Pkh eventually induces oxidative stress and DNA double-strand breaks, leading to programmed cell death. This finding supports Pkh as an antifungal target since pharmacological inhibition of Pkh would lead to the death of yeast cells, the ultimate goal of antifungals. It was therefore of interest to further investigate the possibility to develop Pkh inhibitors with selectivity for Candida Pkh that would not inhibit the human ortholog. Here, we describe C. albicans Pkh2 biochemically, structurally and by using chemical probes in comparison to human PDK1. We found that a regulatory site on the C. albicans Pkh2 catalytic domain, the PIF-pocket, diverges from human PDK1. Indeed, we identified and characterized PS77, a new small allosteric inhibitor directed to the PIF-pocket, which has increased selectivity for C. albicans Pkh2. Together, our results describe novel features of the biology of Pkh and chemical biology approaches that support the validation of Pkh as a drug target for selective antifungals.