scholarly journals Clostridium difficile glutamate dehydrogenase is a secreted enzyme that confers resistance to H2O2

Microbiology ◽  
2014 ◽  
Vol 160 (1) ◽  
pp. 47-55 ◽  
Author(s):  
Brintha Prasummanna Girinathan ◽  
Sterling E. Braun ◽  
Revathi Govind
Keyword(s):  
Clostridium Difficile ◽  
Glutamate Dehydrogenase ◽  
Parent Strain ◽  
Diagnostic Tools ◽  
Growth Defect ◽  
Irreversible Reaction ◽  
Yeast Extract Medium ◽  
Stool Samples ◽  
Higher Sensitivity

Clostridium difficile produces an NAD-specific glutamate dehydrogenase (GDH), which converts l-glutamate into α-ketoglutarate through an irreversible reaction. The enzyme GDH is detected in the stool samples of patients with C. difficile‐associated disease and serves as one of the diagnostic tools to detect C. difficile infection (CDI). We demonstrate here that supernatant fluids of C. difficile cultures contain GDH. To understand the role of GDH in the physiology of C. difficile, an isogenic insertional mutant of gluD was created in strain JIR8094. The mutant failed to produce and secrete GDH as shown by Western blot analysis. Various phenotypic assays were performed to understand the importance of GDH in C. difficile physiology. In TY (tryptose yeast extract) medium, the gluD mutant grew slower than the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the growth defect in TY medium. The presence of extracellular GDH may have a functional role in the pathogenesis of CDI. In support of this assumption we found higher sensitivity to H2O2 in the gluD mutant as compared to the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the H2O2 sensitivity.

scholarly journals Carbohydrate Scaffolds for the Study of the Autism-associated Bacterium, Clostridium bolteae

2019 ◽  
Vol 26 (35) ◽  
pp. 6341-6348 ◽  
Author(s):  
Brittany Pequegnat ◽  
Mario A. Monteiro
Keyword(s):  
Autism Spectrum ◽  
Diagnostic Tools ◽  
Gi Tract ◽  
Autism Symptoms ◽  
Number Of Children ◽  
Stool Samples ◽  
Brain Connection ◽  
Surface Carbohydrates ◽  
Clostridium Bolteae

A large number of children in the autism spectrum disorder suffer from gastrointestinal (GI) conditions, such as constipation and diarrhea. Clostridium bolteae is a part of a set of pathogens being regularly detected in the stool samples of hosts affected by GI and autism symptoms. Accompanying studies have pointed out the possibility that such microbes affect behaviour through the production of neurotoxic metabolites in a so-called, gut-brain connection. As an extension of our Clostridium difficile polysaccharide (PS)-based vaccine research, we engaged in the discovery of C. bolteae surface carbohydrates. So far, studies revealed that C. bolteae produces a specific immunogenic PS capsule comprised of disaccharide repeating blocks of mannose (Manp) and rhamnose (Rhap) units: α-D-Manp-(1→[-4)-β-D-Rhap- (1→3)-α-D-Manp-(1→]n. For vaccinology and further immunogenic experiments, a method to produce C. bolteae PS conjugates has been developed, along with the chemical syntheses of the PS non-reducing end linkage, with D-Rha or L-Rha, α-D-Manp-(1→4)-α-D-Rhap- (1→O(CH2)5NH2 and α-D-Manp-(1→4)-α-L-Rhap-(1→O(CH2)5NH2, equipped with an aminopentyl linker at the reducing end for conjugation purposes. The discovery of C. bolteae PS immunogen opens the door to the creation of non-evasive diagnostic tools to evaluate the frequency and role of this microbe in autistic subjects and to a vaccine to reduce colonization levels in the GI tract, thus impeding the concentration of neurotoxins.

Abstract 53: Measurement of Peripheral Blood 18-Oxocortisol as the Diagnostic Method of Primary Aldosteronism and Its Subtypes

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Fumitoshi Satoh ◽  
Ryo Morimoto ◽  
Masataka Kudo ◽  
Yoshitsugu Iwakura ◽  
Yoshikiyo Ono ◽  
...  
Keyword(s):  
Primary Aldosteronism ◽  
Peripheral Blood ◽  
Roc Analysis ◽  
Maximum Level ◽  
Immunohistochemical Analysis ◽  
Diagnostic Tools ◽  
Expert Radiologist ◽  
Aldosterone Producing Adenoma ◽  
Higher Sensitivity

Backgrounds: Primary aldosteronism (PA) is diagnosed and treated by the long steps, such as screening, confirmation testing and subtype diagnosis (computed tomography (CT) scan and adrenal venous sampling (AVS)). AVS has been the only reliable subtype classification method between surgically curable unilateral aldosterone producing adenoma (APA) and bilateral idiopathic hyperaldosteronism (BHA). In spite of increased numbers of specialized centers with superior ability of AVS in the world, unfortunately, this test is costly and requires a dedicated and expert radiologist; therefore it is not generally utilizable in most hospitals. It is understandable that easier diagnostic tools to be substitutes for AVS might be hoped by clinicians. Objective: The aim of the study was to determine the role of peripheral plasma levels of 18-oxo-cortisol (p18oxoF) and 18-hydroxycortisol (p18OHF) in differentiate APA from BHA. Patients: The study included 265 PA patients (113 patients with CT-positive macro APA, 31 patients with CT-negative micro APA and 121 patients with BHA) and 79 patients with essential hypertension (EH). Methods: All of 265 PA patients underwent AVS successfully, and any case with APA was surgically proven and pathologically confirmed including immunohistochemical analysis of steroidogenic enzymes. We measured p18oxoF and p18OHF of all PA patients by high sensitive LC ms/ms. Results: APA patients showed significantly higher p18oxoF and p18OHF than those of BHA patients and EH patients. The ROC analysis of p18oxoF in macro APA versus BHA demonstrated clinically useful discrimination with higher sensitivity of 0.83 and higher specificity of 0.99 by cutoff value of 4.7 (ng/dl), compared to the classification by p18OHF. Practically, 86 (76%) of macro APA patients with p18oxoF above 6.1 (ng/dl), which was the maximum level in BHA patients, might have been able to undergo surgery without AVS. Because the maximum p18oxoF in EH patients was 5.6 (ng/dl), the patients with p18oxoF above 6.1 (ng/dl) after screening of PA could also undergo surgery if unilateral adenoma(s) can be found on CT; thus it means omitting confirmation tests. Conclusions: Peripheral blood 18oxoF measurement can be a clinically useful method for the diagnosis of PA and its subtypes.

scholarly journals Alternative fate of glyoxylate during acetate and hexadecane metabolism in Acinetobacter oleivorans DR1

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chulwoo Park ◽  
Bora Shin ◽  
Woojun Park
Keyword(s):  
Parent Strain ◽  
Isocitrate Lyase ◽  
Alternative Pathway ◽  
Malate Synthase ◽  
Growth Defect ◽  
Rna Seq ◽  
Wild Type ◽  
E Coli ◽  
Malate Synthase G

Abstract The glyoxylate shunt (GS), involving isocitrate lyase (encoded by aceA) and malate synthase G (encoded by glcB), is known to play important roles under several conditions including oxidative stress, antibiotic defense, or certain carbon source metabolism (acetate and fatty acids). Comparative growth analyses of wild type (WT), aceA, and glcB null-strains revealed that aceA, but not glcB, is essential for cells to grow on either acetate (1%) or hexadecane (1%) in Acinetobacter oleivorans DR1. Interestingly. the aceA knockout strain was able to grow slower in 0.1% acetate than the parent strain. Northern Blot analysis showed that the expression of aceA was dependent on the concentration of acetate or H2O2, while glcB was constitutively expressed. Up-regulation of stress response-related genes and down-regulation of main carbon metabolism-participating genes in a ΔaceA mutant, compared to that in the parent strain, suggested that an ΔaceA mutant is susceptible to acetate toxicity, but grows slowly in 0.1% acetate. However, a ΔglcB mutant showed no growth defect in acetate or hexadecane and no susceptibility to H2O2, suggesting the presence of an alternative pathway to eliminate glyoxylate toxicity. A lactate dehydrogenase (LDH, encoded by a ldh) could possibly mediate the conversion from glyoxylate to oxalate based on our RNA-seq profiles. Oxalate production during hexadecane degradation and impaired growth of a ΔldhΔglcB double mutant in both acetate and hexadecane-supplemented media suggested that LDH is a potential detoxifying enzyme for glyoxylate. Our constructed LDH-overexpressing Escherichia coli strain also showed an important role of LDH under lactate, acetate, and glyoxylate metabolisms. The LDH-overexpressing E. coli strain, but not wild type strain, produced oxalate under glyoxylate condition. In conclusion, the GS is a main player, but alternative glyoxylate pathways exist during acetate and hexadecane metabolism in A. oleivorans DR1.

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