Systemic metabolic depletion of gut microbiome undermines responsiveness to melanoma immunotherapy.

Immunotherapy has proven to be a boon for patients battling metastatic melanoma, significantly improving their clinical condition and overall quality of life. A compelling link between the composition of the gut microbiome and the efficacy of immunotherapy has been established in both animal models and human patients. However, the precise biological mechanisms by which gut microbes influence treatment outcomes remain poorly understood. Using a robust dataset of 680 fecal metagenomes from melanoma patients, a detailed catalog of metagenome-assembled genomes (MAGs) was constructed to explore the compositional and functional properties of the gut microbiome. Our study uncovered significant findings that deepen the understanding of the intricate relationship between gut microbes and the efficacy of melanoma immunotherapy. In particular, we discovered the specific metagenomic profile of patients with favorable treatment outcomes, characterized by a prevalence of MAGs with increased overall metabolic potential and proficiency in polysaccharide utilization, along with those responsible for cobalamin and amino acid production. Furthermore, our investigation of the biosynthetic pathways of short-chain fatty acids, known for their immunomodulatory role, revealed a differential abundance of these pathways among the specific MAGs. Among others, the cobalamin-dependent Wood-Ljungdahl pathway of acetate synthesis was directly associated with responsiveness to melanoma immunotherapy.

PpCas9 from Pasteurella pneumotropica - a compact Type II-C Cas9 ortholog active in human cells.

CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17 and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.

Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA.

Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5'-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17-19 downstream of PAM in the non-target DNA strand, and after the 22nd position of target strand, producing 3-5 nucleotide-long 5'-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6-8nt 5'-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.

DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 Type II-C system.

Type II CRISPR-Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

Reiterative Synthesis by the Ribosome and Recognition of the N-Terminal Formyl Group by Biosynthetic Machinery Contribute to Evolutionary Conservation of the Length of Antibiotic Microcin C Peptide Precursor.

Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichiacoli cells bearing a plasmid-borne mcc gene cluster. Most MccA precursors, encoded by validated mcc operons from diverse bacteria, are 7 amino acids long, but the significance of this precursor length conservation has remained unclear. Here, we created derivatives of E. colimcc operons encoding longer precursors and studied their synthesis and bioactivities. We found that increasing the precursor length to 11 amino acids and beyond strongly decreased antibiotic production. We found this decrease to depend on several parameters. First, reiterative synthesis of the MccA peptide by the ribosome was decreased at longer mccA open reading frames, leading to less efficient competition with other messenger RNAs. Second, the presence of a formyl group at the N-terminal methionine of the heptameric peptide had a strong stimulatory effect on adenylation by the MccB enzyme. No such formyl group stimulation was observed for longer peptides. Finally, the presence of the N-terminal formyl on the heptapeptide adenylate stimulated bioactivity, most likely at the uptake stage. Together, these factors should contribute to optimal activity of McC-like compounds as 7-amino-acid peptide moieties and suggest convergent evolution of several steps of the antibiotic biosynthesis pathway and their adjustment to sensitive cell uptake machinery to create a potent drug.IMPORTANCEEscherichia coli microcin C (McC) is a representative member of peptide-nucleotide antibiotics produced by diverse microorganisms. The vast majority of biosynthetic gene clusters responsible for McC-like compound production encode 7-amino-acid-long precursor peptides, which are C-terminally modified by dedicated biosynthetic enzymes with a nucleotide moiety to produce a bioactive compound. In contrast, the sequences of McC-like compound precursor peptides are not conserved. Here, we studied the consequences of E. coli McC precursor peptide length increase on antibiotic production and activity. We show that increasing the precursor peptide length strongly decreases McC production by affecting multiple biosynthetic steps, suggesting that the McC biosynthesis system has evolved under significant functional constraints to maintain the precursor peptide length.

Efficient in vivo synthesis of lasso peptide pseudomycoidin proceeds in the absence of both the leader and the leader peptidase.

Bacterial lasso peptides are made from linear ribosomally synthesized precursors by specific cleavage at the leader-core junction site of the precursor by a dedicated protease recognizing the leader, followed by cyclisation of the newly formed N-terminus of the core part with a side chain of the internal aspartic or glutamic residue catalyzed by a macrolactam synthetase. The resulting structure has a tail that is threaded and fixed inside the cycle formed. Here, we characterize a new lasso peptide, pseudomycoidin, encoded by Bacillus pseudomycoides DSM 12442. The most surprising and unique feature of pseudomycoidin is that it can be produced in vivo from the ribosomally synthesized core part by a macrolactam synthetase, in the absence of the leader protease. The minimalism of the pseudomycoidin synthesis system makes it a powerful model to generate pseudomycoidin-based lasso-peptide libraries and to study the poorly understood process of lasso formation. We detected two additional pseudomycoidin modifications: phosphorylation of a terminal residue that was previously observed in another lasso peptide, followed by glycosylation, which was not observed heretofore. We speculate that these bulky C-terminal modifications may help maintain the threaded lasso topology of the compound synthesized by the macrolactam synthetase.