Role of RNPP and Agr quorum sensing in the regulation of stationary phase phenotypes in Clostridium acetobutylicum

The Gram-positive, endospore forming, strictly anaerobic bacterium Clostridium acetobutylicum is well known for its ability to ferment sugars to organic acids and solvents, most notably butanol. In batch culture, following an initial phase of acid production, the organism undergoes a metabolic and physiological shift to solvent production, granulose accumulation and initiation of endospore formation, usually associated with entry into stationary phase. The regulatory control of this shift and the associated phenotypes is poorly understood and has previously been linked to the master regulator Spo0A, but also the RNPP-like quorum sensing regulators Ca_c0957 and Ca_c0958 and Agr quorum sensing. The main objective of this study was to provide a better understanding of these systems and their interactions with respect to shift-associated phenotypes. The role of Ca_c0957 and Ca_c0958 as master regulators of stationary phase phenotypes were confirmed through in-frame deletion and chromosomal complementation and both were shown to be essential for solvent formation and sporulation. While previously assumed to act primarily through control of Spo0A phosphorylation, this study demonstrated that some Spo0A activity remained in the absence of Ca_c0957 and served to independently control granulose accumulation. Inactivation of Ca_c0082, a LanC-like peptide cyclase controlled by Ca_c0957, produced a granulose-negative phenotype with reduced sporulation but capable of producing the Agr signalling peptide, thus resembling a Spo0A mutant. C-terminally tagged Ca_c0957 and Ca_c0958 proteins were constructed and confirmed to be active, allowing for their future structural characterisation, even though initial purification attempts remained unsuccessful. Agr signalling, controlled by Ca_c0957/Ca_c0958, was shown to be more promiscuous than previously thought, with several synthetic peptides displaying biological activity, including one representing the putative Clostridium roseum AgrD signal. Agr signalling influenced sporulation primarily through granulose accumulation, as granulose-deficient mutants were shown to produce fewer spores but generate solvents earlier than the wild type, especially under glucose limitation

The Lanthipeptide Synthetase-like Protein CA_C0082 Is an Effector of Agr Quorum Sensing in Clostridium acetobutylicum

Lanthipeptide synthetases are present in all domains of life. They catalyze a crucial step during lanthipeptide biosynthesis by introducing thioether linkages during posttranslational peptide modification. Lanthipeptides have a wide range of functions, including antimicrobial and morphogenetic activities. Intriguingly, several Clostridium species contain lanthipeptide synthetase-like genes of the class II (lanM) family but lack other components of the lanthipeptide biosynthetic machinery. In all instances, these genes are located immediately downstream of putative agr quorum sensing operons. The physiological role and mode of action of the encoded LanM-like proteins remain uncertain as they lack conserved catalytic residues. Here we show for the industrial organism Clostridium acetobutylicum that the LanM-like protein CA_C0082 is not required for the production of active AgrD-derived signaling peptide but nevertheless acts as an effector of Agr quorum sensing. Expression of CA_C0082 was shown to be controlled by the Agr system and is a prerequisite for granulose (storage polymer) formation. The accumulation of granulose, in turn, was shown to be required for maximal spore formation but also to reduce early solvent formation. CA_C0082 and its putative homologs appear to be closely associated with Agr systems predicted to employ signaling peptides with six-membered ring structures and may represent a new subfamily of LanM-like proteins. This is the first time their contribution to bacterial Agr signaling has been described

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Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol

Chirally pure (R)-1,3-butanediol ((R)-1,3-BDO) is a valuable intermediate for the production of fragrances, pheromones, insecticides and antibiotics. Biotechnological production results in superior enantiomeric excess over chemical production and is therefore the preferred production route. In this study (R)-1,3-BDO was produced in the industrially important whole cell biocatalyst Clostridium saccharoperbutylacetonicum through expression of the enantio-specific phaB gene from Cupriavidus necator. The heterologous pathway was optimised in three ways: at the transcriptional level choosing strongly expressed promoters and comparing plasmid borne with chromosomal gene expression, at the translational level by optimising the codon usage of the gene to fit the inherent codon adaptation index of C. saccharoperbutylacetonicum, and at the enzyme level by introducing point mutations which led to increased enzymatic activity. The resulting whole cell catalyst produced up to 20 mM (1.8 g/l) (R)-1,3-BDO in non-optimised batch fermentation which is a promising starting position for economical production of this chiral chemical

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Role of RNPP and Agr quorum sensing in the regulation of stationary phase phenotypes in Clostridium acetobutylicum

The Gram-positive, endospore forming, strictly anaerobic bacterium Clostridium acetobutylicum is well known for its ability to ferment sugars to organic acids and solvents, most notably butanol. In batch culture, following an initial phase of acid production, the organism undergoes a metabolic and physiological shift to solvent production, granulose accumulation and initiation of endospore formation, usually associated with entry into stationary phase. The regulatory control of this shift and the associated phenotypes is poorly understood and has previously been linked to the master regulator Spo0A, but also the RNPP-like quorum sensing regulators Ca_c0957 and Ca_c0958 and Agr quorum sensing. The main objective of this study was to provide a better understanding of these systems and their interactions with respect to shift-associated phenotypes. The role of Ca_c0957 and Ca_c0958 as master regulators of stationary phase phenotypes were confirmed through in-frame deletion and chromosomal complementation and both were shown to be essential for solvent formation and sporulation. While previously assumed to act primarily through control of Spo0A phosphorylation, this study demonstrated that some Spo0A activity remained in the absence of Ca_c0957 and served to independently control granulose accumulation. Inactivation of Ca_c0082, a LanC-like peptide cyclase controlled by Ca_c0957, produced a granulose-negative phenotype with reduced sporulation but capable of producing the Agr signalling peptide, thus resembling a Spo0A mutant. C-terminally tagged Ca_c0957 and Ca_c0958 proteins were constructed and confirmed to be active, allowing for their future structural characterisation, even though initial purification attempts remained unsuccessful. Agr signalling, controlled by Ca_c0957/Ca_c0958, was shown to be more promiscuous than previously thought, with several synthetic peptides displaying biological activity, including one representing the putative Clostridium roseum AgrD signal. Agr signalling influenced sporulation primarily through granulose accumulation, as granulose-deficient mutants were shown to produce fewer spores but generate solvents earlier than the wild type, especially under glucose limitation

Local immune responses and systemic cytokine responses in zoster: relationship to the development of postherpetic neuralgia

Varicella zoster virus (VZV) causes varicella (chickenpox) as the primary infection and zoster (shingles) on reactivation from latency, often many years later. One of the most common and most severe sequela of zoster is postherpetic neuralgia (PHN). Apart from age, factors which predispose towards PHN are unknown. In the present study, the concentration of a variety of Th1 and Th2 cytokines in the serum of 30 zoster patients at the time of the acute disease were correlated with the subsequent development of PHN in nine of these patients, but no association was found. In addition, although some cytokines such as IFN-γ, IL-6 and IL-8 were slightly raised in the zoster group compared with a group of normal healthy subjects of a similar age distribution, these differences only verged on significance. Antibody titres to VZV were raised in the zoster group compared with the controls but these did not differ between the patients who developed PHN and those who did not. Biopsies of zoster lesions were collected from nine patients. There were significantly fewer infiltrating lymphocytes in the lesions of the three patients who subsequently developed PHN compared with the six who did not, although the expression of the neuropeptide, substance P, did not differ between the two groups. It is possible that the poor inflammatory response at the time of the acute zoster may result in less effective containment of the VZV and more damage in the dermatome, thus contributing to the persistence of the neuralgia