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Interspecies hydrogen transfer in anoxic ecosystems is essential for the complete microbial breakdown of organic matter to methane. Acetogenic bacteria are key players in anaerobic food webs and have been considered as prime candidates for hydrogen cycling. We have tested this hypothesis by mutational analysis of the hydrogenase in the model acetogen Acetobacterium woodii. Hydrogenase-deletion mutants no longer grew on H2 + CO2 or organic substrates such as fructose, lactate, or ethanol. Heterotrophic growth could be restored by addition of molecular hydrogen to the culture, indicating that hydrogen is an intermediate in heterotrophic growth. Indeed, hydrogen production from fructose was detected in a stirred-tank reactor. The mutant grew well on organic substrates plus caffeate, an alternative electron acceptor that does not require molecular hydrogen but NADH as reductant. These data are consistent with the notion that molecular hydrogen is produced from organic substrates and then used as reductant for CO2 reduction. Surprisingly, hydrogen cycling in A. woodii is different from the known modes of interspecies or intraspecies hydrogen cycling. Our data are consistent with a novel type of hydrogen cycling that connects an oxidative and reductive metabolic module in one bacterial cell, "intracellular syntrophy."
Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar‐based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H2‐dependent CO2 reductase and furthermore belong to different classes of ‘Rnf’‐ and ‘Ech‐acetogens’. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood–Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g−1 h−1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO‐based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.
The impact of 2-desaza-annomontine on processes of inflammation and its resolution in leukocytes
(2024)
This present study investigated the effects of the b-carboline derivative C81, also called 2-desaza-annomontine, on the inflammatory response and resolution processes in vivo and in vitro. The study focused on leukocytes and on the elucidation of the underlying pharmacological mode of action. C81 potently reduced the inflammatory response in an imiquimod-induced psoriasis mouse model and additionally resolved the inflammation more quickly. In a CNV model, C81 significantly decreased the microglial infiltration in the inflamed laser lesion in vivo. In vitro experiments revealed that C81 inhibits the migration of macrophages and leukocyte-endothelial cell interaction by reducing the activation of integrins on leukocytes, in particular LFA-1, without affecting the total protein level on the cell surface.
Further experiments revealed that C81 inhibited the expression of EPAC-1, required for Rap1 activation. Consequently, C81 reduced the LPS/PMA-induced Rap1 activity, which is responsible for integrin activation. Moreover, C81 potently reduced the LPS-induced formation of pro-inflammatory mediators, including cytokines and eicosanoids, in macrophages. The C81-derived inhibition of eicosanoid release was surprisingly potent, probably due to the C81-evoked inhibition of cPLA2 expression, resulting in less liberated arachidonic acid, the precursor for eicosanoids. At the same time, C81 only delayed COX-2 expression, but completely diminished LPS-induced mPGES-1 expression. In addition to the potent anti-inflammatory effects in vitro, C81-derived impact was complemented with promising pro-resolving effects. Hence, C81 significantly induced neutrophil apoptosis without affecting the cell viability of other leukocytes, such as macrophages. Accordingly, the caspase 3 activity in neutrophils increased upon C81 treatment. The underlying mechanism altered by C81 was the expression of the anti-apoptotic mediator Mcl-1, which is required for the survival of neutrophils, but not macrophages. Furthermore, neutrophils treated with C81 were significantly better efferocytosed by macrophages. Analyzes of the pharmacological mode of action of C81 revealed DYRK1B as the key target kinase in inflammatory processes in leukocytes. Of note, experiments with pharmacological inhibition of DYRK1B by C81 or the ‘selective’ DYRK1B inhibitor AZ-DYRK1B-33, could not completely exclude the involvement of the CLKs and other DYRKs. Therefore, DYRK1B knockdown and overexpression experiments were conducted to elucidate the impact of DYRK1B alone. Pharmacological inhibition of DYRK1B and DYRK1B knockdown phenocopied the inhibitory effect of C81 on leukocyte adhesion. In parallel, DYRK1B overexpression mitigated the C81-evoked effect, supporting the hypothesis that C81 inhibits DYRK1B to mediate its effects on leukocytes. Furthermore, DYRK1B inhibition and DYRK1B knockdown resulted in depletion of STAT3 phosphorylation. In addition, C81-evoked STAT3 inhibition was again mitigated by DYRK1B overexpression, suggesting a link or even an interaction between DYRK1B and STAT3. Indeed, direct interaction between DYRK1B and STAT3 was confirmed by a NanoBRET assay. Importantly, in vitro experiments demonstrated, that C81 did not affect LPS recognition mechanisms, investigated by TLR-4 and CD14 expression, and other important inflammatory signaling pathways. Although C81 inhibited the regeneration of IkBa, this had no effect on the translocation of the NFkB subunit p65. Furthermore, C81 did not alter the activation of MAPK pathways, including p38, JNK and ERK. As a result, the focus was on the potent inhibition of LPS-nduced STAT3 activation mediated by DYRK1B, which was shown to be IL-6 independent. Indeed, direct STAT3 inhibition by Stattic phenocopied all tested C81-derived effects on leukocytes, including migration, adhesion, pro-inflammatory cytokine expression, eicosanoid formation and cell type specific induction of neutrophil apoptosis. The underlying mechanisms altered by Stattic in terms of migration/adhesion and lipid mediator formation were the same as for C81. STAT3 inhibition led to decreased EPAC1 expression and accordingly to reduced Rap1 activation. In addition, inhibited STAT3 phosphorylation resulted in reduced cPLA2 expression and also in attenuated mPGES-1 expression.
Finally, the C81-derived depleted Mcl-1 expression was linked to reduced STAT3 inhibition. As C81 abolished STAT3 phosphorylation, less STAT3 was translocated into the nucleus upon LPS stimulation and less STAT3 enrichment at the MCL1 promoter was observed, leading to reduced gene expression and consequently protein levels.
In summary, using the natural product-derived compound C81, the DYRK1B/STAT3 axis was identified as a novel key regulator of inflammatory processes in human leukocytes. This present study revealed that interfering with the DYRK1B-STAT3 signaling can address essential cell functions including leukocyte extravasation, pro-inflammatory mediator release, neutrophil apoptosis and efferocytosis (Figure 1). Furthermore, two different mouse models demonstrated the in vivo relevance of this signaling axis and highlight DYRK1B as an important kinase modulating inflammation and resolution.