Substrate dependent homeostatic control of c-di-AMP synthase (MsDisA) and hydrolase (MsPDE) from Mycobacterium smegmatis

In bacteria, cyclic-di-nucleotide based second messengers regulate various physiological processes including the stress response. For the past few decades, cyclic diadenosine monophosphate (c-di-AMP) is emerging as a crucial second messenger in bacterial world. It being an essential molecule, is implicated in fatty acid metabolism, antibiotic resistance, biofilm formation, virulence and activates the cytosolic pathway of innate immunity in host cell. The level of c-di-AMP is maintained within the cell by the action of two opposing enzymes, namely diadenylate cyclases and phosphodiesterases. However, such kind of c-di-AMP modulation remains to be explored in Mycobacterium smegmatis. Here, we systematically investigate the c-di-AMP synthase (MsDisA) and a hydrolase (MsPDE) from M. smegmatis at different pHs and osmolytic conditions. Our biochemical assays showed that the MsDisA activity is enhanced during the alkaline stress and c-di-AMP is readily produced without any intermediates. At pH 9.4, the MsDisA promoter activity increases significantly, further strengthening this observation. However, under physiological conditions, the activity of MsDisA is moderate with the formation of intermediates. We also observe that the size of MsDisA is significantly increased upon incubation with substrate. To further get deep insights into the structural characteristics, we report a 3.8 Å cryo-EM structure of the MsDisA protein, distinct from the earlier reported structure of DisA from Thermotoga maritima. The domain mutant experiments prove that the N-terminal minimal region can form a functional octamer. Thus, our results reveal how mycobacterial c-di-AMP is biochemically and structurally regulated in response to different environments. Keywords: Mycobacteria, second messengers, stress response, Cyclic-di-AMP, MsDisA, MsPDE, Cryo-EM

Characterization of the Human TARDBP Gene Promoter

The expression of TDP-43, the main component of neuronal intracellular inclusions across a broad spectrum of ALS and FTD disorders, is developmentally regulated and studies in vivo have shown that TDP-43 overexpression can be toxic, even before observation of pathological aggregates. Starting from these observations, the regulation of its expression at transcriptional level might represent a further key element for the pathogenesis of neurodegenerative diseases. Therefore, we have characterized the human TARDBP promoter, in order to study the transcriptional mechanisms of expression. Mapping of cis-acting elements by luciferase assays in different cells outlined that the activity of the promoter seems to be higher in cell lines of neuronal origin. We have identified the first 400 nucleotides upstream from the transcription start site as the minimal region with a significant transcription activity. In addition, we tested the effects of two SNPs found in the the promoter region of ALS patients and observed no significant effect on transcription levels. Then, TDP-43 overexpression did not affect significantly the activity of its promoter, suggesting that TDP-43 does not influence its own transcription. Finally, the presence of the 5'UTR sequence and of intron-1 splicing seem to impact positively on TDP-43 expression at transcriptional level.

New Insights into Potocki-Shaffer Syndrome: Report of Two Novel Cases and Literature Review

Potocki-Shaffer syndrome (PSS) is a rare non-recurrent contiguous gene deletion syndrome involving chromosome 11p11.2. Current literature implies a minimal region with haploinsufficiency of three genes, ALX4 (parietal foramina), EXT2 (multiple exostoses), and PHF21A (craniofacial anomalies, and intellectual disability). The rest of the PSS phenotype is still not associated with a specific gene. We report a systematic review of the literature and included two novel cases. Because deletions are highly variable in size, we defined three groups of patients considering the PSS-genes involved. We found 23 full PSS cases (ALX4, EXT2, and PHF21A), 14 cases with EXT2-ALX4, and three with PHF21A only. Among the latter, we describe a novel male child showing developmental delay, café-au-lait spots, liner postnatal overgrowth and West-like epileptic encephalopathy. We suggest PSS cases may have epileptic spasms early in life, and PHF21A is likely to be the causative gene. Given their subtle presentation these may be overlooked and if left untreated could lead to a severe type or deterioration in the developmental plateau. If our hypothesis is correct, a timely therapy may ameliorate PSS phenotype and improve patients’ outcomes. Our analysis also shows PHF21A is a candidate for the overgrowth phenotype.

A partial pathogenicity chromosome in Fusarium oxysporum is sufficient to cause disease and can be horizontally transferred

During host colonization, plant pathogenic fungi secrete proteins, called effectors, to facilitate infection. Collectively, effectors may defeat the plant immune system, but usually not all effectors are equally important for infecting a particular host plant. In Fusarium oxysporum f.sp. lycopersici, all known effector genes – also called SIX genes – are located on a single accessory chromosome which is required for pathogenicity and can also be horizontally transferred to another strain. To narrow down the minimal region required for virulence, we selected partial pathogenicity chromosome deletion strains by fluorescence-assisted cell sorting of a strain in which the two arms of the pathogenicity chromosome were labelled with GFP and RFP, respectively. By testing the virulence of these deletion mutants, we show that the complete long arm and part of the short arm of the pathogenicity chromosome are not required for virulence. In addition, we demonstrate that smaller versions of the pathogenicity chromosome can also be transferred to a non-pathogenic strain and they are sufficient to turn the non-pathogen into a pathogen. Surprisingly, originally non-pathogenic strains that had received a smaller version of the pathogenicity chromosome were much more aggressive than recipients with a complete pathogenicity chromosome. Whole genome sequencing analysis revealed that partial deletions of the pathogenicity chromosome occurred mainly close to repeats, and that spontaneous duplication of sequences in accessory regions is frequent both in chromosome deletion strains and in horizontal transfer (recipient) strains.Author SummaryFungal genomes can often be divided into a core genome, which is essential for growth, and an accessory genome which is dispensable. The accessory genome in fungi can be beneficial under some conditions. For example, in some plant-pathogenic fungi, virulence genes are present in the accessory genome, which enable these fungi to cause disease on certain hosts. In Fusarium oxysporum f.sp. lycopersici, which infects tomato, all host-specific virulence genes are located on a single accessory chromosome. This ‘pathogenicity chromosome’ can be horizontally transferred between strains. Here, we found that many suspected virulence genes are in fact not required for virulence because strains without a large part of the pathogenicity chromosome, including these genes, showed no reduced virulence. In addition, we demonstrate that partial pathogenicity chromosomes can be horizontally transferred to a non-pathogen. Surprisingly, originally non-pathogenic strains that had received a partial pathogenicity chromosome were more virulent than strains that had received the complete pathogenicity chromosome.

Novel antimicrobial peptide discovery using machine learning and biophysical selection of minimal bacteriocin domains

Bacteriocins are ribosomally produced antimicrobial peptides that represent an untapped source of promising antibiotic alternatives. However, inherent challenges in isolation and identification of natural bacteriocins in substantial yield have limited their potential use as viable antimicrobial compounds. In this study, we have developed an overall pipeline for bacteriocin-derived compound design and testing that combines sequence-free prediction of bacteriocins using a machine-learning algorithm and a simple biophysical trait filter to generate minimal 20 amino acid peptide candidates that can be readily synthesized and evaluated for activity. We generated 28,895 total 20-mer peptides and scored them for charge, α-helicity, and hydrophobic moment, allowing us to identify putative peptide sequences with the highest potential for interaction and activity against bacterial membranes. Of those, we selected sixteen sequences for synthesis and further study, and evaluated their antimicrobial, cytotoxicity, and hemolytic activities. We show that bacteriocin-based peptides with the overall highest scores for our biophysical parameters exhibited significant antimicrobial activity against E. coli and P. aeruginosa. Our combined method incorporates machine learning and biophysical-based minimal region determination, to create an original approach to rapidly discover novel bacteriocin candidates amenable to rapid synthesis and evaluation for therapeutic use.

Chemical Gating of the Mechanosensitive Piezo1 Channel by Asymmetric Binding of its Agonist Yoda1

Piezo proteins are homotrimeric ion channels that play major roles in normal and pathological mechanotransduction signaling in mammalian organisms. Their pharmacological control hence represents a potential therapeutic avenue. Yoda1, a Piezo1-selective small molecule agonist, is the only known selective Piezo modulator. How Yoda1 selectively interacts with Piezo1 and opens its pore is unknown. Here, by engineering and characterizing chimeras, we identified a minimal region responsible for Yoda1 binding. This region is located at the interface between the pore and the putative mechanosensory domains in each subunit. By characterizing hybrid channels containing Yoda1-insensitive and Yoda1-sensitive monomers, we demonstrate that the presence of only one Yoda1-sensitive Piezo1 subunit is sufficient for chemical activation, implicating that the asymmetric binding of Yoda1 to a single subunit enables channel opening. These findings shed light onto the gating mechanisms of Piezo channels and will pave the way for the rationale design of new Piezo channels modulators.