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1.
Mol Microbiol ; 121(5): 850-864, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38323722

RESUMO

The diarrheal disease cholera is caused by the versatile and responsive bacterium Vibrio cholerae, which is capable of adapting to environmental changes. Among others, the alternative sigma factor RpoS activates response pathways, including regulation of motility- and chemotaxis-related genes under nutrient-poor conditions in V. cholerae. Although RpoS has been well characterised, links between RpoS and other regulatory networks remain unclear. In this study, we identified the ArcAB two-component system to control rpoS transcription and RpoS protein stability in V. cholerae. In a manner similar to that seen in Escherichia coli, the ArcB kinase not only activates the response regulator ArcA but also RssB, the anti-sigma factor of RpoS. Our results demonstrated that, in V. cholerae, RssB is phosphorylated by ArcB, which subsequently activates RpoS proteolysis. Furthermore, ArcA acts as a repressor of rpoS transcription. Additionally, we determined that the cysteine residue at position 180 of ArcB is crucial for signal recognition and activity. Thus, our findings provide evidence linking RpoS response to the anoxic redox control system ArcAB in V. cholerae.


Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Fator sigma , Vibrio cholerae , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Fator sigma/metabolismo , Fator sigma/genética , Fosforilação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Quimiotaxia/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Redes Reguladoras de Genes
2.
Biochim Biophys Acta Gen Subj ; 1867(9): 130427, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37454915

RESUMO

BACKGROUND & AIMS: Transport functions of albumin are of clinical and pharmacological interest and are determined by albumin's properties like posttranslational modifications or bound ligands. Both are affected in pathological conditions and in therapeutic grade albumin solutions. The term effective albumin concentration was introduced as a measure of functionally intact albumin. Our aim was to evaluate the impact of ligands and modifications with different approaches as a measure of effective albumin. APPROACH & RESULTS: We used a spin labelled fatty acid and dansylsarcosine to characterize binding properties of albumin i) prepared from plasma of patients and healthy control donors, ii) measured directly out of plasma, iii) research grade albumin, iv) in vitro modified albumin, and v) therapeutic infusion solutions before and after removal of stabilizers. Bilirubin is the main determinant for binding function in patients' albumin. In in vitro prepared albumin bound fatty acids correlated with impaired binding. Human nonmercaptalbumin1, not human nonmercaptalbumin2, showed reduced binding properties. Binding and transport function of therapeutic albumin was severely impaired and restored by filtration. Glycation of research grade albumin had no effect on the binding of dansylsarcosine and only a minor effect on fatty acid binding. CONCLUSIONS: Our results suggest that effective albumin -in terms of binding properties- is primarily determined by bound ligands and only to a minor extent by posttranslational modifications. Characterizing albumin directly from plasma better reflects the physiological situation whereas in the case of therapeutic grade albumin stabilizers should be removed to make its binding properties accessible.


Assuntos
Albuminas , Ácidos Graxos , Humanos , Ligantes , Albuminas/metabolismo , Compostos de Dansil/química , Compostos de Dansil/metabolismo
3.
Int J Med Microbiol ; 312(4): 151555, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35483107

RESUMO

Cholera is a life-threatening diarrheal disease caused by the human pathogenic bacterium Vibrio cholerae. Regulatory elements are essential for bacterial transition between the natural aquatic environment and the human host. One of them is the alternative sigma factor RpoS and its anti-sigma factor RssB. Regulation principles seem to be conserved among RpoS/RssB interaction modes between V. cholerae and Enterobacteriaceae species, however the associated input and output pathways seem different. In Escherichia coli, RpoS/RssB is important for the activation of an emergency program to increase persistence and survival. Whereas, it activates motility and chemotaxis in V. cholerae, used strategically to escape from starvation conditions. We characterised a starvation-induced interaction model showing a negative feedback loop between RpoS and RssB expression. We showed by genotypic and phenotypic analysis that rssB influences motility, growth behaviour, colonization fitness, and post-infectious survival. Furthermore, we found that RssB itself is a substrate for proteolysis and a critical Asp mutation was identified and characterised to influence rssB phenotypes and their interaction with RpoS. In summary, we present novel information about the regulatory interaction between RpoS and RssB being active under in vivo colonization conditions and mark an extension to the feedback regulation circuit, showing that RssB is a substrate for proteolysis.


Assuntos
Proteínas de Escherichia coli , Vibrio cholerae , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Vibrio cholerae/metabolismo
4.
Metabolites ; 11(11)2021 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-34822420

RESUMO

Magnesium-deficiency is implicated in many metabolic disorders, e.g., type 2 diabetes and metabolic syndrome, representing risk factors for non-alcoholic fatty liver disease (NAFLD). This study aims to investigate the contribution of magnesium-restriction to the development of NAFLD. Magnesium-deficiency was induced in C57BL/6 mice by feeding a magnesium-deficient-diet. Metabolic markers as well as markers of inflammation and liver function were assessed. Furthermore, liver tissue was examined histopathologically and compared with specimens from high-fat-diet fed and control mice. Finally, the hepatic inflammatory response was quantified by determining hepatic IL-6, TNFα, and MCP-1. Magnesium-restriction resulted in at least a 2-fold significant reduction of serum magnesium levels compared to the high-fat-diet fed and control mice, whereas the hepatic magnesium content was decreased due to high-fat-diet feeding. No changes in metabolic markers in magnesium-restricted mice were observed, while the cholesterol content was elevated in high-fat-diet fed mice. Magnesium-restricted mice additionally featured inflammation and enlarged hepatocytes in liver histology. Furthermore, magnesium-restricted and high-fat-diet fed mice exhibited elevated hepatic TNFα levels compared to control mice. Accordingly, our data suggest that magnesium is involved in hepatic inflammatory processes and hepatocyte enlargement, key histological features of human NAFLD, and may therefore contribute to development and progression of the disease.

5.
Commun Biol ; 4(1): 731, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127764

RESUMO

Human serum albumin (HSA) constitutes the primary transporter of fatty acids, bilirubin, and other plasma compounds. The binding, transport, and release of its cargos strongly depend on albumin conformation, which is affected by bound ligands induced by physiological and pathological conditions. HSA is both highly oxidized and heavily loaded with fatty acids and bilirubin in chronic liver disease. By employing small-angle X-ray scattering we show that HSA from the plasma of chronic liver disease patients undergoes a distinct opening compared to healthy donors. The extent of HSA opening correlates with clinically relevant variables, such as the model of end-stage liver disease score, bilirubin, and fatty acid levels. Although the mild oxidation of HSA in vitro does not alter overall structure, the alteration of patients' HSA correlates with its redox state. This study connects clinical data with structural visualization of albumin dynamicity in solution and underlines the functional importance of albumin's inherent flexibility.


Assuntos
Hepatopatias/metabolismo , Albumina Sérica/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Bilirrubina/sangue , Estudos de Casos e Controles , Doença Hepática Terminal/metabolismo , Ácidos Graxos/sangue , Humanos , Hepatopatias/sangue , Masculino , Pessoa de Meia-Idade , Oxirredução , Estrutura Terciária de Proteína , Adulto Jovem
6.
PLoS One ; 11(5): e0155163, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27167736

RESUMO

Although non-alcoholic and alcoholic fatty liver disease have been intensively studied, concerning pathophysiological mechanisms are still incompletely understood. This may be due to the use of different animal models and resulting model-associated variation. Therefore, this study aimed to compare three frequently used wild type mouse strains in their susceptibility to develop diet-induced features of non-alcoholic/alcoholic fatty liver disease. Fatty liver disease associated clinical, biochemical, and histological features in C57BL/6, CD-1, and 129Sv WT mice were induced by (i) high-fat diet feeding, (ii) ethanol feeding only, and (iii) the combination of high-fat diet and ethanol feeding. Hepatic and subcutaneous adipose lipid profiles were compared in CD-1 and 129Sv mice. Additionally hepatic fatty acid composition was determined in 129Sv mice. In C57BL/6 mice dietary regimens resulted in heterogeneous hepatic responses, ranging from pronounced steatosis and inflammation to a lack of any features of fatty liver disease. Liver-related serum biochemistry showed high deviations within the regimen groups. CD-1 mice did not exhibit significant changes in metabolic and liver markers and developed no significant steatosis or inflammation as a response to dietary regimens. Although 129Sv mice showed no weight gain, this strain achieved most consistent features of fatty liver disease, apparent from concentration alterations of liver-related serum biochemistry as well as moderate steatosis and inflammation as a result of all dietary regimens. Furthermore, the hepatic lipid profile as well as the fatty acid composition of 129Sv mice were considerably altered, upon feeding the different dietary regimens. Accordingly, diet-induced non-alcoholic/alcoholic fatty liver disease is most consistently promoted in 129Sv mice compared to C57BL/6 and CD-1 mice. As a conclusion, this study demonstrates the importance of genetic background of used mouse strains for modeling diet-induced non-alcoholic/alcoholic fatty liver disease.


Assuntos
Gorduras na Dieta/administração & dosagem , Suscetibilidade a Doenças , Etanol/administração & dosagem , Fígado Gorduroso Alcoólico/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Gordura Subcutânea/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Biomarcadores/metabolismo , Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Ácidos Graxos não Esterificados/metabolismo , Fígado Gorduroso Alcoólico/etiologia , Fígado Gorduroso Alcoólico/genética , Fígado Gorduroso Alcoólico/patologia , Fígado/patologia , Testes de Função Hepática , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Especificidade da Espécie , Gordura Subcutânea/patologia , Triglicerídeos/metabolismo , Aumento de Peso
7.
PLoS One ; 7(10): e47756, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23144706

RESUMO

Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dissulfetos/metabolismo , Fatores de Transcrição/metabolismo , Vibrio cholerae/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cólera/genética , Cólera/microbiologia , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dissulfetos/química , Regulação Bacteriana da Expressão Gênica , Immunoblotting , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Periplasma/metabolismo , Porinas/genética , Porinas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Multimerização Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/química , Fatores de Transcrição/genética , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Virulência/genética
8.
Mol Microbiol ; 82(4): 1015-37, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22032623

RESUMO

Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae.


Assuntos
Biofilmes/crescimento & desenvolvimento , DNA Bacteriano/metabolismo , Desoxirribonucleases/metabolismo , Vibrio cholerae/enzimologia , Vibrio cholerae/fisiologia , Aderência Bacteriana , Matriz Extracelular/química , Matriz Extracelular/metabolismo
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