CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have become ubiquitous approaches to control gene expression in bacteria due to their simple design and effectiveness. By regulating transcription of a target gene(s), CRISPRi/a can dynamically engineer cellular metabolism, implement transcriptional regulation circuitry, or elucidate genotype-phenotype relationships from smaller targeted libraries up to whole genome-wide libraries. While CRISPRi/a has been primarily established in the model bacteria Escherichia coli and Bacillus subtilis, a growing numbering of studies have demonstrated the extension of these tools to other species of bacteria (here broadly referred to as non-model bacteria). In this mini-review, we discuss the challenges that contribute to the slower creation of CRISPRi/a tools in diverse, non-model bacteria and summarize the current state of these approaches across bacterial phyla. We find that despite the potential difficulties in establishing novel CRISPRi/a in non-model microbes, over 190 recent examples across eight bacterial phyla have been reported in the literature. Most studies have focused on tool development or used these CRISPRi/a approaches to interrogate gene function, with fewer examples applying CRISPRi/a gene regulation for metabolic engineering or high-throughput screens and selections. To date, most CRISPRi/a reports have been developed for common strains of non-model bacterial species, suggesting barriers remain to establish these genetic tools in undomesticated bacteria. More efficient and generalizable methods will help realize the immense potential of programmable CRISPR-based transcriptional control in diverse bacteria.

/pdf/crispr-based-approaches-for-gene-regulation-in-non-model-1g8leggm.pdf

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

Nicotinamide adenine dinucleotide (NAD+) serves as a critical cofactor in cellular metabolism and redox reactions. Bacterial pathways rely on NAD+ participation, where its stability and concentration govern essential homeostasis and functions. This review delves into the role and metabolic regulation of NAD+ in bacteria, highlighting its influence on physiology and virulence. Notably, we explore enzymes linked to NAD+ metabolism as antibacterial drug targets and vaccine candidates. Moreover, we scrutinize NAD+'s medical potential, offering insights for its application in biomedicine. This comprehensive assessment informs future research directions in the dynamic realm of NAD+ and its biomedical significance. 

From bacteria to biomedicine: Developing therapies exploiting NAD+ metabolism

INTRODUCTION
In fragment-based drug design, fragment linking is a popular strategy where two fragments binding to different sub-pockets of a target are linked together. This attractive method remains challenging especially due to the design of ideal linkers.


AREAS COVERED
The authors review the types of linkers and chemical reactions commonly used to the synthesis of linkers, including those utilized in protein-templated fragment self-assembly, where fragments are directly linked in the presence of the protein. Finally, they detail computational workflows and software including generative models that have been developed for fragment linking.


EXPERT OPINION
The authors believe that fragment linking offers key advantages for compound design, particularly for the design of bivalent inhibitors linking two distinct pockets of the same or different subunits. On the other hand, more studies are needed to increase the potential of protein-templated approaches in FBDD. Important computational tools such as structure-based de novo software are emerging to select suitable linkers. Fragment linking will undoubtedly benefit from developments in computational approaches and machine learning models.

Linkers in fragment-based drug design: an overview of the literature.

Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes the synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knock down NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus . Furthermore, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence-associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection. 

NAD kinase promotes Staphylococcus aureus pathogenesis by supporting production of virulence factors and protective enzymes

Complementary to hydrophobic five membered ring β-amino acids (eg ACPC), β-sugar amino acids (β-SAAs) have found increasing application as hydrophilic building blocks of foldamers and α/β chimeric peptides Fmoc-protected β-SAAs [eg Fmoc-RibAFU(ip)-OH] are indeed useful Lego elements, ready to use for SPPS The removal of 1,2-OH isopropylidene protecting group increasing the hydrophilicity of such SAA is presented here We first used N3-RibAFU(ip)-OH model compound to optimize mild deprotection conditions The formation of the 1,2-OH free product N3-RibAFU-OH and its methyl glycoside methyl ester, N3-RibAFU(Me)-OMe were monitored by RP-HPLC and found that either 50% TFA or 8 eqv Amberlite IR-120 H+ resin in MeOH are optimal reagents for the effective deprotection These conditions were then successfully applied for the synthesis of chimeric oligopeptide: -GG-X-GG- [X=RibAFU(ip)] We found the established conditions to be effective and-at the same time-sufficiently mild to remove 1,2-O-isopropylidene protection and thus, it is proposed to be used in the synthesis of oligo- and polypeptides of complex sequence combination

/pdf/synthesis-of-chimera-oligopeptide-including-furanoid-b-sugar-zxy2tw5v32.pdf

Synthesis of chimera oligopeptide including furanoid β-sugar amino acid derivatives with free OHs: mild but successful removal of the 1,2-O-isopropylidene from the building block.

Antibiotic resistance is a worldwide threat due to the decreasing supply of new antimicrobials. Novel targets and innovative strategies are urgently needed to generate pathbreaking drug compounds. NAD kinase (NADK) is essential for growth in most bacteria, as it supports critical metabolic pathways. Here, we report the discovery of a new class of antibacterials that targets bacterial NADK. We generated a series of small synthetic adenine derivatives to screen those harboring promising substituents in order to guide efficient fragment linking. This led to NKI1, a new lead compound inhibiting NADK that showed in vitro bactericidal activity against Staphylococcus aureus. In a murine model of infection, NKI1 restricted survival of the bacteria, including methicillin-resistant S. aureus. Collectively, these findings identify bacterial NADK as a potential drug target and NKI1 as a lead compound in the treatment of staphylococcal infections.

/pdf/from-substrate-to-fragments-to-inhibitor-activein-29ietixc1n.pdf

From substrate to fragments to inhibitor activein vivoagainstStaphylococcus aureus.

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From substrate to fragments to inhibitor activein vivoagainstStaphylococcus aureus.