In silico analysis of mycobacteriophage Ms6

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2017Isolated in 1989, Ms6 is a renowned temperate mycobacteriophage that earned a spot as model of mycobacteriophage-induced lysis. Despite the studies on the lytic operon, the remainder of the genome of Ms6 was kept mostly unexplored. A modern next generation sequencing, followed by a complete genome annotation, enlightens several aspects concerning this phage. The genome is composed of linear double stranded DNA, with 54252 bp of length and a GC content of 61,5%. The closest homologues of Ms6 are Dlane and Shauna1. The similarities between Ms6 and these and many other F1 clustered phages allow its allocation to this group although 11 ORFs present a first BLASTp hit that is not a gene product of a F1 phage. A total of 105 ORFs were identified, of which 43 were given a putative function according to a combination between location in the genome, homology with previously characterized proteins, existence of conserved motifs or structural similarities. The integrase gene divides the genome in two genomic arms, the left and the right. Within the left arm of the genome, it is possible to distinguish genes with structural roles, such as the head and the tail assembly genes and genes required for processes like packaging and lysis. The right arm is less conserved than its left counterpart and comprises ORFs involved either in DNA modification, like exonucleases or methylases, or in phage regulation, for example the WhiB factor or proteins with helix-turn-helix DNA binding motifs. Besides the pin gene and its phage resistance properties, other interesting features include a possible virion associated lysin and a protein that might encourage homologous recombination.Isolado em 1989, o Ms6 é um micobacteriófago temperado pertencente à família Siphoviridae e que infecta Mycobacterium smegmatis. Apesar dos estudos já efectuados sobre a integração e sobre a lise das micobactérias induzida pelo Ms6, o restante genoma do Ms6 permaneceu maioritariamente por explorar. Através de Next Generation Sequencing, seguido de uma completa anotação do genoma, foi possível esclarecer alguns aspetos relativos a este fago. O genoma é composto por uma dupla cadeia linear de DNA, contendo 54252 bp e um conteúdo em GC de 61,5%. Os fagos mais semelhantes ao Ms6 são o Dlane e o Shauna1. A homologia entre o Ms6 e estes fagos, bem como com muitos outros fagos do subcluster F1, permitem a sua inclusão neste grupo, apesar de 11 ORFs apresentarem um primeiro resultado de BLASTp que não corresponde a proteínas de fagos F1. Foram identificadas 105 ORFs e foi possível atribuir uma função a 43 das mesmas, por combinação de informação proveniente da sua localização no genoma, homologia com proteínas previamente caracterizadas e presença de motivos conservados ou analogias estruturais. A gene que codifica para a integrase divide o genoma em dois ramos, o esquerdo e o direito. Dentro do ramo esquerdo do genoma, é possível distinguir genes com um papel estrutural, tais como os que codificam proteínas da cápside ou da cauda, e genes necessários a processos como o empacotamento do DNA ou a lise da célula hospedeira. O ramo direito é menos conservado e contém genes envolvidos na modificação do DNA, tais como os que codificam exonucleases ou metilases, ou na regulação do fago, codificando o factor WhiB ou proteínas com domínios de ligação ao DNA do tipo HTH. Além do gene pin, cujo produto confere resistência à superinfeção, outros elementos interessantes incluem uma possível lisina associada ao virião e uma proteína que pode estimular a recombinação homóloga

Identification of drivers of mycobacterial resistance to peptidoglycan synthesis inhibitors

Beta-lactams have been excluded from tuberculosis therapy due to the intrinsic resistance of Mycobacterium tuberculosis (Mtb) to this antibiotic class, usually attributed to a potent beta-lactamase, BlaC, and to an unusually complex cell wall. In this pathogen, the peptidoglycan is cross-linked by penicillin-binding proteins (PBPs) and L,D-transpeptidases, the latter resistant to inhibition by most beta-lactams. However, recent studies have shown encouraging results of beta-lactam/beta-lactamase inhibitor combinations in clinical strains. Additional research on the mechanisms of action and resistance to these antibiotics and other inhibitors of peptidoglycan synthesis, such as the glycopeptides, is crucial to ascertain their place in alternative regimens against drug-resistant strains. Within this scope, we applied selective pressure to generate mutants resistant to amoxicillin, meropenem or vancomycin in Mtb H37Rv or Mycolicibacterium smegmatis (Msm) mc2-155. These were phenotypically characterized, and whole-genome sequencing was performed. Mutations in promising targets or orthologue genes were inspected in Mtb clinical strains to establish potential associations between altered susceptibility to beta-lactams and the presence of key genomic signatures. The obtained isolates had substantial increases in the minimum inhibitory concentration of the selection antibiotic, and beta-lactam cross-resistance was detected in Mtb. Mutations in L,D-transpeptidases and major PBPs, canonical targets, or BlaC were not found. The transcriptional regulator PhoP (Rv0757) emerged as a common denominator for Mtb resistance to both amoxicillin and meropenem, while Rv2864c, a lipoprotein with PBP activity, appears to be specifically involved in decreased susceptibility to the carbapenem. Nonetheless, the mutational pattern detected in meropenem-resistant mutants was different from the yielded by amoxicillin-or vancomycin-selected isolates, suggesting that distinct pathways may participate in increased resistance to peptidoglycan inhibitors, including at the level of beta-lactam subclasses. Cross-resistance between beta-lactams and antimycobacterials was mostly unnoticed, and Msm meropenem-resistant mutants from parental strains with previous resistance to isoniazid or ethambutol were isolated at a lower frequency. Although cell-associated nitrocefin hydrolysis was increased in some of the isolates, our findings suggest that traditional assumptions of Mtb resistance relying largely in beta-lactamase activity and impaired access of hydrophilic molecules through lipid-rich outer layers should be challenged. Moreover, the therapeutical potential of the identified Mtb targets should be explored.This work was supported by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID), Switzerland, through Research Grant 2018, and by Fundação para a Ciência e a Tecnologia (FCT), Portugal, through research project PTDC/BIA-MIC/31233/2017, awarded to MJC. FO (SFRH/BD/136853/2018) and CS (2021.05446.BD) are recipients of PhD fellowships from FCT.info:eu-repo/semantics/publishedVersio

The mycobacteriophage ms6 lysb n-terminus displays peptidoglycan binding affinity

Funding Information: Funding: This work was supported in part by Fundação para a Ciência e Tecnologia (FCT-MCES, Portugal) Grant PTDC/IMI-MIC/0694/2012 to MP and PTDC/BIA-MIC/30746/2017 to SF. AG (SFRH/BD/87685/2012) was a recipient PhD fellowship from FCT-MCES, Portugal. Funding Information: This work was supported in part by Funda??o para a Ci?ncia e Tecnologia (FCT-MCES, Portugal) Grant PTDC/IMI-MIC/0694/2012 to MP and PTDC/BIA-MIC/30746/2017 to SF. AG (SFRH/BD/87685/2012) was a recipient PhD fellowship from FCT-MCES, Portugal. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.publishersversionpublishe

Uncovering Beta-Lactam Susceptibility Patterns in Clinical Isolates of Mycobacterium tuberculosis through Whole-Genome Sequencing

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9431576/The increasing threat of drug resistance and a stagnated pipeline of novel therapeutics endanger the eradication of tuberculosis. Beta-lactams constitute promising additions to the current therapeutic arsenal and two carbapenems are included in group C of medicines recommended by the WHO for use in longer multidrug-resistant tuberculosis regimens. However, the determinants underlining diverse Mycobacterium tuberculosis phenotypes to beta-lactams remain largely undefined. To decipher these, we present a proof-of-concept study based on a large-scale beta-lactam susceptibility screening for 172 M. tuberculosis clinical isolates from Portugal, including 72 antimycobacterial drug-resistant strains. MICs were determined for multiple beta-lactams and strains were subjected to whole-genome sequencing to identify core-genome single-nucleotide variant-based profiles. Global and cell wall-targeted approaches were then followed to detect putative drivers of beta-lactam response. We found that drug-resistant strains were more susceptible to beta-lactams, but significant differences were not observed between distinct drug-resistance profiles. Sublineage 4.3.4.2 strains were significantly more susceptible to beta-lactams, while the contrary was observed for Beijing and 4.1.2.1 sublineages. While mutations in beta-lactamase or cell wall biosynthesis genes were uncommon, a rise in beta-lactam MICs was detected in parallel with the accumulation of mutations in peptidoglycan cross-linking or cell division genes. Finally, we exposed that putative beta-lactam resistance markers occurred in genes for which relevant roles in cell wall processes have been ascribed, such as rpfC or pknA. Genetic studies to validate the relevance of the identified mutations for beta-lactam susceptibility and further improvement of the phenotype-genotype associations are needed in the future. IMPORTANCE Associations between differential M. tuberculosis beta-lactam phenotypes and preexisting antimycobacterial drug resistance, strain sublineage, or specific mutational patterns were established. Importantly, we reveal that highly drug-resistant isolates of sublineage 4.3.4.2 have an increased susceptibility to beta-lactams compared with other strains. Thus, directing beta-lactams to treat infections by specific M. tuberculosis strains and refraining its use from others emerges as a potentially important strategy to avoid resistance development. Individual mutations in blaC or genes encoding canonical beta-lactam targets, such as peptidoglycan transpeptidases, are infrequent and do not greatly impact the MICs of potent carbapenem plus clavulanic acid combinations. An improved understanding of the global effect of cumulative mutations in relevant gene sets for peptidoglycan and cell division processes on beta-lactam susceptibility is also provided.This work was supported by Fundação para a Ciência e Tecnologia (PTDC/BIA-MIC/31233/2017 to M.J.C, SFRH/BD/136853/2018 to F.O. and 2021.05446.BD to C.S.) and by the European Society of Clinical Microbiology and Infectious Diseases (Research Grant 2018 to M.J.C.).info:eu-repo/semantics/publishedVersio

WGS-based dual strategy for the identification of key targets to enhance beta-lactam activity in mycobacterium tuberculosis

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CRISPRi-mediated characterization of novel anti-tuberculosis targets: Mycobacterial peptidoglycan modifications promote beta-lactam resistance and intracellular survival

The lack of effective therapeutics against emerging multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) prompts the identification of novel anti-tuberculosis targets. The essential nature of the peptidoglycan (PG) layer of the mycobacterial cell wall, which features several distinctive modifications, such as the N-glycolylation of muramic acid and the amidation of D-iso-glutamate, makes it a target of particular interest. To understand their role in susceptibility to beta-lactams and in the modulation of host-pathogen interactions, the genes encoding the enzymes responsible for these PG modifications (namH and murT/gatD, respectively) were silenced in the model organism Mycobacterium smegmatis using CRISPR interference (CRISPRi). Although beta-lactams are not included in TB-therapy, their combination with beta-lactamase inhibitors is a prospective strategy to treat MDR-TB. To uncover synergistic effects between the action of beta-lactams and the depletion of these PG modifications, knockdown mutants were also constructed in strains lacking the major beta-lactamase of M. smegmatis BlaS, PM965 (M. smegmatis ΔblaS1) and PM979 (M. smegmatis ΔblaS1 ΔnamH). The phenotyping assays affirmed the essentiality of the amidation of D-iso-glutamate to the survival of mycobacteria, as opposed to the N-glycolylation of muramic acid. The qRT-PCR assays confirmed the successful repression of the target genes, along with few polar effects and differential knockdown level depending on PAM strength and target site. Both PG modifications were found to contribute to beta-lactam resistance. While the amidation of D-iso-glutamate impacted cefotaxime and isoniazid resistance, the N-glycolylation of muramic acid substantially promoted resistance to the tested beta-lactams. Their simultaneous depletion provoked synergistic reductions in beta-lactam MICs. Moreover, the depletion of these PG modifications promoted a significantly faster bacilli killing by J774 macrophages. Whole-genome sequencing revealed that these PG modifications are highly conserved in a set of 172 clinical strains of Mtb, demonstrating their potential as therapeutic targets against TB. Our results support the development of new therapeutic agents targeting these distinctive mycobacterial PG modifications