Human gut microbiota senses its environment and responds by releasing metabolites, some of which are key regulators of human health and disease. In this study, we characterize gut-associated bacteria in their ability to decarboxylate levodopa to dopamine via tyrosine decarboxylases. Bacterial tyrosine decarboxylases efficiently convert levodopa to dopamine, even in the presence of tyrosine, a competitive substrate, or inhibitors of human decarboxylase. In situ levels of levodopa are compromised by high abundance of gut bacterial tyrosine decarboxylase in patients with Parkinson’s disease. Finally, the higher relative abundance of bacterial tyrosine decarboxylases at the site of levodopa absorption, proximal small intestine, had a significant impact on levels of levodopa in the plasma of rats. Our results highlight the role of microbial metabolism in drug availability, and specifically, that abundance of bacterial tyrosine decarboxylase in the proximal small intestine can explain the increased dosage regimen of levodopa treatment in Parkinson’s disease patients.
Genome‐wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae. However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn‐seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high‐content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi‐based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp‐proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP‐dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.
This study was conducted to investigate individual metabolic and endocrine adaptation to lactation under conditions of identical housing and feeding conditions in high-yielding dairy cows. Forty-five cows were studied on a research farm under standardized but practical conditions. From wk 2 before calving until wk 14 postpartum, blood samples were collected at weekly intervals and assayed for blood chemistry and various metabolites and hormones. Body weight, BCS, and backfat thickness were also recorded weekly. Milk yield, milk composition, and feed intake and energy balance were accordingly measured during the postpartum phase. The animals were retrospectively classified according to their plasma concentration of beta-hydroxybutyrate (BHB): cows in which a BHB threshold of 1 mM was exceeded at least once during the experiment were classified as BHB positive (BHB+); cows with BHB values consistently below this threshold were classified as BHB negative (BHB -). Using this classification, differences for NEFA and glucose concentrations were observed, but the mean calculated energy balance did not differ between the groups during the experimental period (-22.2 MJ of NE(1)/d +/- 4.7 for BHB+ and -18.9 MJ of NE(1)/d +/- 4.9 for BHB-). In BHB+ cows, the peripartum decrease (P < 0.05) of BW, BCS, and backfat thickness was more pronounced than in BHB- cows. Mean milk yields did not differ between groups. However, BHB+ cows had greater milk fat and lesser milk protein contents (P < 0.05), resulting in a greater (P < 0.05) fat:protein ratio than in BHB- cows. Thus, to some extent, cows were able to compensate for the negative energy balance by adjustments in performance. Milk acetone concentrations followed BHB concentrations in blood. Insulin-like growth factor-I and leptin concentrations were greater (P < 0.05) in BHB- cows during the time of observation than in the BHB+ cows. Comparing the reproductive variables recorded (first increase of progesterone, first service conception rate, number of services per conception, interval from calving to first AI, interval from first AI to conception, and days open) between the 2 groups yielded no significant differences. Our findings imply that despite comparable energy balance, there is considerable individual variation of the adaptive ability of cows during early lactation based on a variety of metabolic and endocrine variables.
Bacterial cell division is mediated by a multi-protein machine known as the “divisome”, which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.
Increasing evidence is supporting the hypothesis of ␣-synuclein pathology spreading from the gut to the brain although the exact etiology of Parkinson's disease (PD) is unknown. Furthermore, it has been proposed that inflammation, via the gastrointestinal tract, potentially through infections, may contribute to ␣-synuclein pathogenesis, and thus to the risk of developing PD. Recently, many studies have shown that PD patients have an altered microbiota composition compared to healthy controls. Inflammation in the gut might drive microbiota alterations or vice versa. Many studies focused on the detection of biomarkers of the etiology, onset, or progression of PD however also report metabolites from bacterial origin. These metabolites might reflect the bacterial composition and as well play an important role in immune homeostasis, ultimately affecting the progression of PD. Besides the bacterial metabolites, pharmacological treatment of PD might play a crucial role during the progression and thus treatment of the disease on the immune system. This review aims to establish a link between the microbial composition with the observed alterations of bacterial metabolites and their impact on the immune system, which could have influential effect in onset, progression and etiology of PD.
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