Proline catabolism is a key factor facilitating Candida albicans pathogenicity.

Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.

Correction: Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence of Candida albicans.

[This corrects the article DOI: 10.1371/journal.ppat.1008328.].

Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence of Candida albicans.

Candida albicans cells depend on the energy derived from amino acid catabolism to induce and sustain hyphal growth inside phagosomes of engulfing macrophages. The concomitant deamination of amino acids is thought to neutralize the acidic microenvironment of phagosomes, a presumed requisite for survival and initiation of hyphal growth. Here, in contrast to an existing model, we show that mitochondrial-localized NAD+-dependent glutamate dehydrogenase (GDH2) catalyzing the deamination of glutamate to α-ketoglutarate, and not the cytosolic urea amidolyase (DUR1,2), accounts for the observed alkalization of media when amino acids are the sole sources of carbon and nitrogen. C. albicans strains lacking GDH2 (gdh2-/-) are viable and do not extrude ammonia on amino acid-based media. Environmental alkalization does not occur under conditions of high glucose (2%), a finding attributable to glucose-repression of GDH2 expression and mitochondrial function. Consistently, inhibition of oxidative phosphorylation or mitochondrial translation by antimycin A or chloramphenicol, respectively, prevents alkalization. GDH2 expression and mitochondrial function are derepressed as glucose levels are lowered from 2% (~110 mM) to 0.2% (~11 mM), or when glycerol is used as primary carbon source. Using time-lapse microscopy, we document that gdh2-/- cells survive, filament and escape from primary murine macrophages at rates indistinguishable from wildtype. In intact hosts, such as in fly and murine models of systemic candidiasis, gdh2-/- mutants are as virulent as wildtype. Thus, although Gdh2 has a critical role in central nitrogen metabolism, Gdh2-catalyzed deamination of glutamate is surprisingly dispensable for escape from macrophages and virulence. Consistently, using the pH-sensitive dye (pHrodo), we observed no significant difference between wildtype and gdh2-/- mutants in phagosomal pH modulation. Following engulfment of fungal cells, the phagosomal compartment is rapidly acidified and hyphal growth initiates and sustained under consistently acidic conditions within phagosomes. Together, our results demonstrate that amino acid-dependent alkalization is not essential for hyphal growth, survival in macrophages and hosts. An accurate understanding of the microenvironment within macrophage phagosomes and the metabolic events underlying the survival of phagocytized C. albicans cells and their escape are critical to understanding the host-pathogen interactions that ultimately determine the pathogenic outcome.

Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans.

Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence.

Apoptotic ß-cells induce macrophage reprogramming under diabetic conditions.

Type 2 diabetes mellitus (T2DM) occurs when insulin-producing pancreatic ß-cells fail to secrete sufficient insulin to compensate for insulin resistance. As T2DM progresses, apoptotic ß-cells need to be removed by macrophages through efferocytosis that is anti-inflammatory by nature. Paradoxically, infiltrating macrophages are a main source of inflammatory cytokines that leads to T2DM. It is unclear how apoptotic ß-cells impact macrophage function. We show under diabetic conditions, phagocytosis of apoptotic ß-cells causes lysosomal permeabilization and generates reactive oxygen species that lead to inflammasome activation and cytokine secretion in macrophages. Efferocytosis-induced lipid accumulation transforms islet macrophages into foam cell-like outside the context of atherosclerosis. Our study suggests that whereas macrophages normally play a protective anti-inflammatory role, the increasing demand of clearing apoptotic cells may trigger them to undergo proinflammatory reprogramming as T2DM progresses. This shift in the balance between opposing macrophage inflammatory responses could contribute to chronic inflammation involved in metabolic diseases. Our study highlights the importance of preserving macrophage lysosomal function as a therapeutic intervention for diabetes progression.

Multifunctional in vivo imaging of pancreatic islets during diabetes development.

Pancreatic islet dysfunction leading to insufficient glucose-stimulated insulin secretion triggers the clinical onset of diabetes. How islet dysfunction develops is not well understood at the cellular level, partly owing to the lack of approaches to study single islets longitudinally in vivo Here, we present a noninvasive, high-resolution system to quantitatively image real-time glucose metabolism from single islets in vivo, currently not available with any other method. In addition, this multifunctional system simultaneously reports islet function, proliferation, vasculature and macrophage infiltration in vivo from the same set of images. Applying our method to a longitudinal high-fat diet study revealed changes in islet function as well as alternations in islet microenvironment. More importantly, this label-free system enabled us to image real-time glucose metabolism directly from single human islets in vivo for the first time, opening the door to noninvasive longitudinal in vivo studies of healthy and diabetic human islets.

Clinical Results of Carpal Tunnel Release Using Ultrasound Guidance in Over 100 Patients at Two to Six Years.

Purpose: The purpose of this study was to determine the clinical results of carpal tunnel release using ultrasound guidance (CTR-US) at a minimum of 2 years postprocedure. Methods: The study consisted of 102 patients (162 hands) treated with CTR-US by the same physician between June 2017 and October 2020 for whom minimum 2-year follow-up data were available. Questionnaires were sent to gather long-term information, with additional phone calls for clarification if needed. Outcomes included Boston Carpal Tunnel Questionnaire symptom severity (BCTQ-SSS) and functional status (BCTQ-FSS) scores; Quick Disabilities of the Arm, Shoulder, and Hand (QuickDASH) scores; global satisfaction scores; and subsequent surgeries. Results: The 102 patients included 68 females and 34 males with a mean age of 56.9 years at the time of surgery. Fifty-five (53.9%) patients had simultaneous bilateral procedures, 42 (41.2%) had unilateral procedures, and 5 (4.9%) had staged bilateral procedures. Significant improvements in BCTQ-SSS, BCTQ-FSS, and QuickDASH scores persisted at a mean final follow-up of 46 months (range 2-6 years). At final follow-up, 91.2% of patients reported satisfaction with the procedure. No outcomes were significantly different between those treated with simultaneous bilateral versus unilateral procedures. No revision surgeries were reported. Conclusions: CTR-US is a safe and effective procedure that results in significant improvements that persist up to 6 years postprocedure. Long-term results of simultaneous bilateral and unilateral procedures are similar. Type of study/level of evidence: Therapeutic IV.

Regulation of adipocyte differentiation and gene expression-crosstalk between TGFß and wnt signaling pathways.

Obesity results in reduced differentiation potential of adipocytes leading to adipose tissue insulin resistance. Elevated proinflammatory cytokines from adipose tissue in obesity, such as TNFα have been implicated in the reduced adipocyte differentiation. Other mediators of reduced adipocyte differentiation include TGFß and wnt proteins. Although some overlap exists in the signaling cascades of the wnt and TGFß pathways it is unknown if TGFß or wnt proteins reciprocally induce the expression of each other to maximize their biological effects in adipocytes. Therefore, we investigated the possible involvement of TGFß signaling in wnt induced gene expression and vice versa in 3T3-L1 adipocyte. Effect of TGFß and Wnt pathways on differentiation was studied in preadipocytes induced to differentiate in the presence of Wnt3a or TGFß1 and their inhibitors (FZ8-CRD and SB431542, respectively). Regulation of intracellular signaling and gene expression was also studied in mature adipocytes. Our results show that both TGFß1 and Wnt3a lead to increased accumulation of ß-catenin, phosphorylation of AKT and p44/42 MAPK. However, differences were found in the pattern of gene expression induced by the two proteins suggesting that distinct, but complex, signaling pathways are activated by TGFß and wnt proteins to independently regulate adipocyte function.

Stigmasterol prevents glucolipotoxicity induced defects in glucose-stimulated insulin secretion.

Type 2 diabetes results from defects in both insulin sensitivity and insulin secretion. Elevated cholesterol content within pancreatic ß-cells has been shown to reduce ß-cell function and increase ß-cell apoptosis. Hyperglycemia and dyslipidemia contribute to glucolipotoxicity that leads to type 2 diabetes. Here we examined the capacity of glucolipotoxicity to induce free cholesterol accumulation in human pancreatic islets and the INS-1 insulinoma cell line. Glucolipotoxicity treatment increased free cholesterol in ß-cells, which was accompanied by increased reactive oxygen species (ROS) production and decreased insulin secretion. Addition of AAPH, a free radical generator, was able to increase filipin staining indicating a link between ROS production and increased cholesterol in ß-cells. We also showed the ability of stigmasterol, a common food-derived phytosterol with anti-atherosclerotic potential, to prevent the increase in both free cholesterol and ROS levels induced by glucolipotoxicity in INS-1 cells. Stigmasterol addition also inhibited early apoptosis, increased total insulin, promoted actin reorganization, and improved insulin secretion in cells exposed to glucolipotoxicity. Overall, these data indicate cholesterol accumulation as an underlying mechanism for glucolipotoxicity-induced defects in insulin secretion and stigmasterol treatment as a potential strategy to protect ß-cell function during diabetes progression.

Biglycan deletion alters adiponectin expression in murine adipose tissue and 3T3-L1 adipocytes.

Obesity promotes increased secretion of a number of inflammatory factors from adipose tissue. These factors include cytokines and very lately, extracellular matrix components (ECM). Biglycan, a small leucine rich proteoglycan ECM protein, is up-regulated in obesity and has recently been recognized as a pro-inflammatory molecule. However, it is unknown whether biglycan contributes to adipose tissue dysfunction. In the present study, we characterized biglycan expression in various adipose depots in wild-type mice fed a low fat diet (LFD) or obesity-inducing high fat diet (HFD). High fat feeding induced biglycan mRNA expression in multiple adipose depots. Adiponectin is an adipokine with anti-inflammatory and insulin sensitizing effects. Due to the importance of adiponectin, we examined the effect of biglycan on adiponectin expression. Comparison of adiponectin expression in biglycan knockout (bgn(-/0)) and wild-type (bgn(+/0)) reveals higher adiponectin mRNA and protein in epididymal white adipose tissue in bgn(-/0) mice, as well higher serum concentration of adiponectin, and lower serum insulin concentration. On the contrary, knockdown of biglycan in 3T3-L1 adipocytes led to decreased expression and secretion of adiponectin. Furthermore, treatment of 3T3-L1 adipocytes with conditioned medium from biglycan treated macrophages resulted in an increase in adiponectin mRNA expression. These data suggest a link between biglycan and adiponectin expression. However, the difference in the pattern of regulation between in vivo and in vitro settings reveals the complexity of this relationship.

Adipose tissue biglycan as a potential anti-inflammatory target of sodium salicylate in mice fed a high fat diet.

BACKGROUND: Inflammation in adipose tissue (AT) during obesity causes impaired AT function. Although multiple extracellular matrix (ECM) proteins are expressed in AT their potential role in adipose tissue inflammation is unclear. Biglycan, a pro-inflammatory ECM gene, is highly enriched in adipose tissue. However, whether it is correlated with adipose tissue inflammation is unknown. We provide evidence in support of a strong association between biglycan expression and inflammatory status of adipose tissue. METHODS: C57BL6 mice were fed either a control (10% fat calories) or a high fat diet (HFD) (60% fat calories) for 8 weeks. Adipose tissue was analyzed for the expression of biglycan, IL-6 and TNFα. Biglycan knockout or wild type were also fed a high fat diet for 8 weeks and the expression of inflammatory genes in the mesenteric adipose tissue was examined. To test anti-inflammatory treatment on biglycan expression, a group of mice were fed either the low fat or high fat diet for eight weeks supplemented with either saline or sodium salicylate @ 25mg/100ml in their drinking water. RESULTS: Mice on HFD had an increase in ECM genes (BGN and COL1A1), inflammatory genes (IL-6 and TNFα) in both the subcutaneous and epididymal depots. However, correlation analysis only shows a positive correlation between biglycan, IL-6 and TNFα expression. In addition, lower expression of IL-6 and CD68 was found in the mesenteric adipose tissue of biglycan knockout mice compared to the wild type. Sodium salicylate treatment reduced subcutaneous adipose tissue expression of BGN, COL1A1, and COL6A1 and a concurrent downregulation of TNFα and IL-6 and TLR4 expression. Salicylate also lowered the serum TGFß1 levels. CONCLUSION: Biglycan expression correlates with adipose tissue inflammation, especially in the subcutaneous depot compared to the epididymal depot. This is supported by the greater effect of sodium salicylate in attenuating both inflammatory and ECM gene expression the subcutaneous adipose depot compared to the epididymal depot. These results show that inflammatory state may explain the induction of biglycan, and perhaps, other ECM genes in adipose tissue.