mBio最新文献

{"title":"Functional assessment of the glycoproteins of a novel Hendra virus variant reveals contrasting fusogenic capacities of the receptor-binding and fusion glycoproteins.","authors":"Andrew Z Ma, Yao Yu Yeo, Jean F Lee, Colin M Kim, Shahrzad Ezzatpour, Carolina Menchaca, Viraj Upadhye, Edward J Annand, John-Sebastian Eden, Raina K Plowright, Alison J Peel, David W Buchholz, Hector C Aguilar","doi":"10.1128/mbio.03482-23","DOIUrl":"https://doi.org/10.1128/mbio.03482-23","url":null,"abstract":"<p><p>A novel Hendra virus (HeV) genotype (HeV genotype 2 [HeV-g2]) was recently isolated from a deceased horse, revealing high-sequence conservation and antigenic similarities with the prototypic strain, HeV-g1. As the receptor-binding (G) and fusion (F) glycoproteins of HeV are essential for mediating viral entry, functional characterization of emerging HeV genotypic variants is key to understanding viral entry mechanisms and broader virus-host co-evolution. We first confirmed that HeV-g2 and HeV-g1 glycoproteins share a close phylogenetic relationship, underscoring HeV-g2's relevance to global health. Our <i>in vitro</i> data showed that HeV-g2 glycoproteins induced cell-cell fusion in human cells, shared receptor tropism with HeV-g1, and cross-reacted with antibodies raised against HeV-g1. Despite these similarities, HeV-g2 glycoproteins yielded reduced syncytia formation compared to HeV-g1. By expressing heterotypic combinations of HeV-g2, HeV-g1, and Nipah virus (NiV) glycoproteins, we found that while HeV-g2 G had strong fusion-promoting abilities, HeV-g2 F consistently displayed hypofusogenic properties. These fusion phenotypes were more closely associated with those observed in the related NiV. Further investigation using HeV-g1 and HeV-g2 glycoprotein chimeras revealed that multiple domains may play roles in modulating these fusion phenotypes. Altogether, our findings may establish intrinsic fusogenic capacities of viral glycoproteins as a potential driver behind the emergence of new henipaviral variants.</p><p><strong>Importance: </strong>HeV is a zoonotic pathogen that causes severe disease across various mammalian hosts, including horses and humans. The identification of unrecognized HeV variants, such as HeV-g2, highlights the need to investigate mechanisms that may drive their evolution, transmission, and pathogenicity. Our study reveals that HeV-g2 and HeV-g1 glycoproteins are highly conserved in identity, function, and receptor tropism, yet they differ in their abilities to induce the formation of multinucleated cells (syncytia), which is a potential marker of viral pathogenesis. By using heterotypic combinations of HeV-g2 with either HeV-g1 or NiV glycoproteins, as well as chimeric HeV-g1/HeV-g2 glycoproteins, we demonstrate that the differences in syncytial formation can be attributed to the intrinsic fusogenic capacities of each glycoprotein. Our data indicate that HeV-g2 glycoproteins have fusion phenotypes closely related to those of NiV and that fusion promotion may be a crucial factor driving the emergence of new henipaviral variants.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0348223"},"PeriodicalIF":5.1,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864784","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Shared mechanisms of enhanced plasmid maintenance and antibiotic tolerance mediated by the VapBC toxin:antitoxin system.","authors":"Sarah Hollingshead, Gareth McVicker, Maria R Nielsen, YuGeng Zhang, Giulia Pilla, Rebekah A Jones, Jonathan C Thomas, Sarah E H Johansen, Rachel M Exley, Ditlev E Brodersen, Christoph M Tang","doi":"10.1128/mbio.02616-24","DOIUrl":"https://doi.org/10.1128/mbio.02616-24","url":null,"abstract":"<p><p>Toxin:antitoxin (TA) systems are widespread in bacteria and were first identified as plasmid addiction systems that kill bacteria lacking a TA-encoding plasmid following cell division. TA systems have also been implicated in bacterial persistence and antibiotic tolerance, which can be precursors of antibiotic resistance. Here, we identified a clinical isolate of <i>Shigella sonnei</i> (CS14) with a remarkably stable pINV virulence plasmid; pINV is usually frequently lost from <i>S. sonnei</i>, but plasmid loss was not detected from CS14. We found that the plasmid in CS14 is stabilized by a single nucleotide polymorphism (SNP) in its <i>vapBC</i> TA system. VapBC TA systems are the most common Type II TA system in bacteria, and consist of a VapB antitoxin and VapC PIN domain-containing toxin. The plasmid stabilizing SNP leads to a Q12L substitution in the DNA-binding domain of VapB, which reduces VapBC binding to its own promoter, impairing <i>vapBC</i> autorepression. However, VapB^L12C mediates high-level plasmid stabilization because VapB^L12 is more prone to degradation by Lon than wild-type VapB; this liberates VapC to efficiently kill bacteria that no longer contain a plasmid. Of note, mutations that confer tolerance to antibiotics in <i>Escherichia coli</i> also map to the DNA-binding domain of VapBC encoded by the chromosomally integrated F plasmid. We demonstrate that the tolerance mutations also enhance plasmid stabilization by the same mechanism as VapB^L12. Our findings highlight the links between plasmid maintenance and antibiotic tolerance, both of which can promote the development of antimicrobial resistance.</p><p><strong>Importance: </strong>Our work addresses two processes, the maintenance of plasmids and antibiotic tolerance; both contribute to the development of antimicrobial resistance in bacteria that cause human disease. Here, we found a single nucleotide change in the vapBC toxin:antitoxin system that stabilizes the large virulence plasmid of <i>Shigella sonnei</i>. The mutation is in the vapB antitoxin gene and makes the antitoxin more likely to be degraded, releasing the VapC toxin to efficiently kill cells without the plasmid (and thus unable to produce more antitoxin as an antidote). We found that vapBC mutations in <i>E. coli</i> that lead to antibiotic tolerance (a precursor to resistance) also operate by the same mechanism (<i>i.e.</i>, generating VapB that is prone to cleavage); free VapC during tolerance will arrest bacterial growth and prevent susceptibility to antibiotics. This work shows the mechanistic links between plasmid maintenance and tolerance, and has applications in biotech and in the design and evaluation of vaccines against shigellosis.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0261624"},"PeriodicalIF":5.1,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864817","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The palmitoyl-CoA ligase Fum16 is part of a <i>Fusarium verticillioides</i> fumonisin subcluster involved in self-protection.","authors":"Fabio Gherlone, Katarina Jojić, Ying Huang, Sandra Hoefgen, Vito Valiante, Slavica Janevska","doi":"10.1128/mbio.02681-24","DOIUrl":"https://doi.org/10.1128/mbio.02681-24","url":null,"abstract":"<p><p><i>Fusarium verticillioides</i> produces the mycotoxin fumonisin B₁ (FB₁), which disrupts sphingolipid biosynthesis by inhibiting ceramide synthase and affects the health of plants, animals, and humans. The means by which <i>F. verticillioides</i> protects itself from its own mycotoxin are not completely understood. Some fumonisin (<i>FUM</i>) cluster genes do not contribute to the biosynthesis of the compound, but their function has remained enigmatic. Recently, we showed that <i>FUM17</i>, <i>FUM18,</i> and <i>FUM19</i> encode two ceramide synthases and an ATP-binding cassette transporter, respectively, which play a role in antagonizing the toxicity mediated by FB₁. In the present work, we uncovered functions of two adjacent genes, <i>FUM15</i> and <i>FUM16</i>. Using homologous and heterologous expression systems, in <i>F. verticillioides</i> and <i>Saccharomyces cerevisiae</i>, respectively, we provide evidence that both contribute to protection against FB₁. Our data indicate a potential role for the P450 monooxygenase Fum15 in the modification and detoxification of FB₁ since the deletion and overexpression of the respective gene affected extracellular FB₁ levels in both hosts. Furthermore, relative quantification of ceramide intermediates and an <i>in vitro</i> enzyme assay revealed that Fum16 is a functional palmitoyl-CoA ligase. It co-localizes together with the ceramide synthase Fum18 to the endoplasmic reticulum, where they contribute to sphingolipid biosynthesis. Thereby, <i>FUM15-19</i> constitute a subcluster within the <i>FUM</i> biosynthetic gene cluster dedicated to the fungal self-protection against FB₁.IMPORTANCEThe study identifies a five-gene <i>FUM</i> subcluster (<i>FUM15-19</i>) in <i>Fusarium verticillioides</i> involved in self-protection against FB₁. <i>FUM16</i> (palmitoyl-CoA ligase), <i>FUM17,</i> and <i>FUM18</i> (ceramide synthases) enzymatically supplement ceramide biosynthesis, while <i>FUM19</i> (ATP-binding cassette transporter) acts as a repressor of the <i>FUM</i> cluster. The evolutionary conservation of <i>FUM15</i> (P450 monooxygenase) in <i>Fusarium</i> and <i>Aspergillus FUM</i> clusters is discussed, and its effect on extracellular FB₁ levels in both native (<i>F. verticillioides</i>) and heterologous (<i>Saccharomyces cerevisiae</i>) hosts is highlighted. These findings enhance our understanding of mycotoxin self-protection mechanisms and could inform strategies for predicting biological activity of unknown secondary metabolites, managing mycotoxin contamination, and developing resistant crop cultivars.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0268124"},"PeriodicalIF":5.1,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864821","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Acinetobacter baumannii</i> represses type VI secretion system through a manganese-dependent small RNA-mediated regulation.","authors":"Somok Bhowmik, Avik Pathak, Shivam Pandey, Kuldip Devnath, Abhiroop Sett, Nishant Jyoti, Timsy Bhando, Jawed Akhter, Saurabh Chugh, Ramandeep Singh, Tarun Kumar Sharma, Ranjana Pathania","doi":"10.1128/mbio.03025-24","DOIUrl":"https://doi.org/10.1128/mbio.03025-24","url":null,"abstract":"<p><p>Type VI secretion system (T6SS) is utilized by many Gram-negative bacteria to eliminate competing bacterial species and manipulate host cells. <i>Acinetobacter baumannii</i> ATCC 17978 utilizes T6SS at the expense of losing pAB3 plasmid to induce contact-dependent killing of competitor microbes, resulting in the loss of antibiotic resistance carried by pAB3. However, the regulatory network associated with T6SS in <i>A. baumannii</i> remains poorly understood. Here, we identified an Mn²⁺-dependent post-transcriptional regulation of T6SS mediated by a bonafide small RNA, AbsR28. <i>A. baumannii</i> utilizes MumT, an Mn²⁺-uptake inner membrane transporter, for the uptake of extracellular Mn²⁺ during oxidative stress. We demonstrate that the abundance of intracellular Mn²⁺ enables complementary base pairing of AbsR28-<i>tssM</i> mRNA (that translates to TssM, one of the vital inner membrane components of T6SS), inducing RNase E-mediated degradation of <i>tssM</i> mRNA and resulting in T6SS repression. Thus, AbsR28 mediates a crosstalk between MumT and T6SS in <i>A. baumannii</i>.IMPORTANCESmall RNAs (sRNAs) are identified as critical components within the bacterial regulatory networks involved in fine regulation of virulence-associated factors. The sRNA-mediated regulation of type VI secretion system (T6SS) in <i>Acinetobacter baumannii</i> was unchartered. Previously, it was demonstrated that <i>A. baumannii</i> ATCC 17978 cells switch from T6- to T6+ phenotype, resulting in the loss of antibiotic resistance conferred by plasmid pAB3. Furthermore, the derivatives of pAB3 found in recent clinical isolates of <i>A. baumannii</i> harbor expanded antibiotic resistance genes and multiple determinants for virulence factors. Hence, the loss of this plasmid for T6SS activity renders <i>A. baumannii</i> T6+ cells susceptible to antibiotics and compromises their virulence. Our findings show how <i>A. baumannii</i> tends to inactivate T6SS through an sRNA-mediated regulation that relies on Mn²⁺ and retains pAB3 during infection to retain antibiotic resistance genes carried on the plasmid.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0302524"},"PeriodicalIF":5.1,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864779","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Accurate phenotype-to-genotype mapping of high-diversity yeast libraries by heat-shock-electroporation (HEEL).","authors":"Marcus Wäneskog, Emma Elise Hoch-Schneider, Shilpa Garg, Christian Kronborg Cantalapiedra, Elena Schäfer, Michael Krogh Jensen, Emil Damgaard Jensen","doi":"10.1128/mbio.03197-24","DOIUrl":"https://doi.org/10.1128/mbio.03197-24","url":null,"abstract":"<p><p>High-throughput DNA transformation techniques are invaluable when generating high-diversity mutant libraries, a cornerstone of successful protein engineering. However, transformation efficiencies have a direct correlation with the probability of introducing multiple DNA molecules into each cell, although reliable library screenings require cells that contain a single unique genotype. Thus, transformation methods that yield a high multiplicity of transformations are unsuitable for high-diversity library screenings. Here, we describe an innovative yeast library transformation method that is both simple and highly efficient. Our dual heat-shock and electroporation approach (HEEL) creates high-quality DNA libraries by increasing the fraction of mono-transformed yeast cells from 20% to over 70% of all transformed cells, thus allowing for near-perfect phenotype-to-genotype associations. HEEL also allows more than 10⁷ yeast cells per reaction to be transformed with a circular plasmid molecule, which corresponds to an almost 100-fold improvement compared with current yeast transformation methods. To further refine our library screening approach, we integrated an automated yeast genotyping workflow with a dual-barcode design that employs both a single nucleotide polymorphism and a high-diversity region. This design allows for robust identification and quantification of unique genotypes within a heterogeneous population using standard Sanger sequencing. Our findings demonstrate that the longstanding trade-off between the size and quality of transformed yeast libraries can be overcome. By employing the HEEL method, large DNA libraries can be transformed into yeast with high-efficiency, while maintaining high library quality, essential for successful mutant screenings. This advancement holds significant promise for the fields of molecular biology and protein engineering.IMPORTANCEWith the recent expansion of artificial intelligence in the field of synthetic biology, there has never been a greater need for high-quality data and reliable measurements of phenotype-to-genotype relationships. However, one major obstacle to creating accurate computer-based models is the current abundance of low-quality phenotypic measurements originating from numerous high-throughput but low-resolution assays. Rather than increasing the quantity of measurements, new studies should aim to generate as accurate measurements as possible. The HEEL methodology presented here aims to address this issue by minimizing the problem of multi-plasmid uptake during high-throughput yeast DNA transformations, which leads to the creation of heterogeneous cellular genotypes. HEEL should enable highly accurate phenotype-to-genotype measurements going forward, which could be used to construct better computer-based models.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0319724"},"PeriodicalIF":5.1,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864781","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mono-allelic epigenetic regulation of polycistronic transcription initiation by RNA polymerase II in <i>Trypanosoma brucei</i>.","authors":"Rudo Kieft, Laura Cliffe, Haidong Yan, Robert J Schmitz, Stephen L Hajduk, Robert Sabatini","doi":"10.1128/mbio.02328-24","DOIUrl":"https://doi.org/10.1128/mbio.02328-24","url":null,"abstract":"<p><p>Unique for a eukaryote, protein-coding genes in trypanosomes are arranged in polycistronic transcription units (PTUs). This genome arrangement has led to a model where Pol II transcription of PTUs is unregulated and changes in gene expression are entirely post-transcriptional. <i>Trypanosoma brucei brucei</i> is unable to infect humans because of its susceptibility to an innate immune complex, trypanosome lytic factor (TLF) in the circulation of humans. The initial step in TLF-mediated lysis of <i>T.b.brucei</i> requires high affinity haptoglobin/hemoglobin receptor (HpHbR) binding. Here, we demonstrate that by <i>in vitro</i> selection with TLF, resistance is obtained in a stepwise process correlating with loss of HpHbR expression at an allelic level. RNA-seq, Pol II ChIP, and run-on analysis indicate HpHbR silencing is at the transcriptional level, where loss of Pol II binding at the promoter region specifically shuts down transcription of the HpHbR-containing gene cluster and the adjacent opposing gene cluster. Reversible transcriptional silencing of the divergent PTUs correlates with DNA base J modification of the shared promoter region. Base J function in establishing transcriptional silencing, rather than maintenance, is suggested by the maintenance of PTU silencing following the inhibition of J-biosynthesis and subsequent loss of the modified DNA base. Therefore, we show that epigenetic mechanisms exist to regulate gene expression via Pol II transcription initiation of gene clusters in a mono-allelic fashion. These findings suggest epigenetic chromatin-based regulation of gene expression is deeply conserved among eukaryotes, including early divergent eukaryotes that rely on polycistronic transcription.IMPORTANCEThe single-cell parasite <i>Trypanosoma brucei</i> causes lethal diseases in both humans and livestock. <i>T. brucei</i> undergoes multiple developmental changes to adapt in different environments during its digenetic life cycle. With protein-coding genes organized as polycistronic transcription and apparent absence of promoter-mediated regulation of transcription initiation, it is believed that developmental gene regulation in trypanosomes is essentially post-transcriptional. In this study, we found reversible Pol II transcriptional silencing of two adjacent polycistronic gene arrays that correlate with the novel DNA base J modification of the shared promoter region. Our findings support epigenetic regulation of Pol II transcription initiation as a viable mechanism of gene expression control in <i>T. brucei</i>. This has implications for our understanding how trypanosomes utilize polycistronic genome organization to regulate gene expression during its life cycle.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0232824"},"PeriodicalIF":5.1,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864725","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Progression of <i>ampC</i> amplification during <i>de novo</i> amoxicillin resistance development in <i>E. coli</i>.","authors":"Luyuan Nong, Martijs Jonker, Wim de Leeuw, Meike T Wortel, Benno Ter Kuile","doi":"10.1128/mbio.02982-24","DOIUrl":"https://doi.org/10.1128/mbio.02982-24","url":null,"abstract":"<p><p>Beta-lactam antibiotics are the most applied antimicrobials in human and veterinarian health care. Hence, beta-lactam resistance is a major health problem. Gene amplification of AmpC beta-lactamase is a main contributor to <i>de novo</i> β-lactam resistance in <i>Escherichia coli</i>. However, the time course of amplification and the accompanying DNA mutations are unclear. Here, we study the progression of <i>ampC</i> amplification and <i>ampC</i> promoter mutations during the evolution of resistance induced by stepwise increasing amoxicillin concentrations. <i>AmpC</i> promoter mutations occurred by day 2, while the approximately eight-fold amplification occurred after more than 6 days of amoxicillin exposure. The combination of the amplification and the promoter mutations increased the <i>ampC</i> mRNA level by an average factor of 200 after 22 days. An IS<i>1</i> insertion is identified in the amplification junction after resistance induction in the wild type (WT) and the <i>ampC</i> gene complementation strain (CompA), but not in ∆<i>ampC</i>, suggesting that the amplification depends on mobile genetic element transposition. In order to elucidate the correlation between gene mutations and <i>ampC</i> amplification, the DNA mutations acquired during resistance evolution by the WT, ∆<i>ampC</i>, and CompA were analyzed. Compared to evolved ∆<i>ampC</i>, several resistance-causing mutations are absent in evolved WT, while more mutations accumulated in stress response. The amoxicillin-resistant ∆<i>ampC</i> did not show amplification of the fragment around the original <i>ampC</i> position but exhibited a large duplication or triplication at another position, suggesting the essential role of the duplicated genes in resistance development.IMPORTANCEAmoxicillin is the most used antimicrobial against bacterial infections. DNA fragments containing <i>ampC</i> are amplified upon prolonged and stepwise increasing exposure to amoxicillin, causing resistance. These <i>ampC</i>-containing fragments have been identified in extended-spectrum beta-lactamase plasmids, which are considered the main cause of beta-lactam resistance. In this study, we document the time course of two important factors for <i>ampC</i> transcription enhancement, <i>ampC</i> amplification and <i>ampC</i> promoter mutations, during <i>de novo</i> amoxicillin resistance evolution. We propose that the transposon IS<i>1</i> contributes to the amplification <i>ampC</i> region, that the sigma factor 70 regulates <i>ampC</i> overexpression, and that these combined form the backbone of a putative mechanism for <i>ampC</i> amplification.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0298224"},"PeriodicalIF":5.1,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864814","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification and characterization of the functional tetrameric UDP-glucose pyrophosphorylase from <i>Klebsiella pneumoniae</i>.","authors":"Isabel Ramón Roth, Pavel Kats, Timm Fiebig, Françoise Routier, Roman Fedorov, Larissa Dirr, Jana I Führing","doi":"10.1128/mbio.02071-24","DOIUrl":"https://doi.org/10.1128/mbio.02071-24","url":null,"abstract":"<p><p>In all kingdoms of life, the enzyme uridine diphosphate-glucose pyrophosphorylase (UGP) occupies a central role in metabolism, as its reaction product uridine diphosphate-glucose (UDP-Glc) is involved in various crucial cellular processes. Pathogens, including fungi, parasites, and bacteria, depend on UGP for the synthesis of virulence factors; in particular, various bacterial species utilize UDP-Glc and its derivatives for the synthesis of lipopolysaccharides, capsular polysaccharides, and biofilm exopolysaccharides. UGPs have, therefore, gained attention as anti-bacterial drug target candidates, prompting us to study their structure-function relationships to provide a basis for the rational development of specific inhibitors. UGP function is tied to its oligomeric state, and the majority of bacterial homologs have been described as tetramers encoded by the <i>galU</i> gene. Uniquely, enterobacterial species harbor a second gene, <i>galF</i>, encoding a protein with high homology to UGP, whose function is somewhat controversial. Here, we show that the <i>galF</i> gene of the opportunistic pathogen <i>Klebsiella pneumoniae</i> encodes a dimeric protein that has lost UGP activity, likely due to a combination of active site mutations and an inability to tetramerize, whereas the functional <i>K. pneumoniae</i> UGP, encoded by <i>galU</i>, is an active tetramer. Our AlphaFold-assisted structure-function relationship studies underline that tetramerization is essential for bacterial UGP function and is facilitated by a common mechanism utilizing conserved key residues. Targeting the respective molecular interfaces, which are absent in human UGP, could provide a means of selectively inhibiting the bacterial virulence factor UGP and potentially rendering pathogenic species avirulent.IMPORTANCEThe enzyme uridine diphosphate-glucose pyrophosphorylase (UGP) is important for the virulence of bacterial pathogens and, therefore, a potential drug target. In this study, we identify the gene encoding the functional UGP in <i>Klebsiella pneumoniae</i>, a bacterium notoriously causing severe antibiotic-resistant infections in humans, and reveal structural and functional features that may aid in the development of new antibiotics.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0207124"},"PeriodicalIF":5.1,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864719","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Temperate phage-antibiotic synergy is widespread-extending to <i>Pseudomonas</i>-but varies by phage, host strain, and antibiotic pairing.","authors":"Rabia Fatima, Alexander P Hynes","doi":"10.1128/mbio.02559-24","DOIUrl":"https://doi.org/10.1128/mbio.02559-24","url":null,"abstract":"<p><p>Bacteriophages (phages) are bacterial-specific viruses that can be used alone or with antibiotics to reduce bacterial load. Most phages are unsuitable for therapy because they are \"temperate\" and can integrate into the host genome, forming a lysogen that is protected from subsequent phage infections. However, integrated phages can be awakened by stressors such as antibiotics. Supported by this interaction, here we explore the potential use of combined temperate phage and antibiotic against the multi-drug-resistant pathogen, <i>Pseudomonas aeruginosa</i>. In all, thirty-nine temperate phages were isolated from clinical strains, and a subset was screened for synergy with six antibiotics (ciprofloxacin, levofloxacin, meropenem, piperacillin, tobramycin, and polymyxin B), using checkerboard assays. Interestingly, our screen identified phages that can synergize with each antibiotic, despite their widely differing targets; however, these are highly phage-antibiotic and phage-host pairing specific. Screening across multiple clinical strains reveals that temperate phages can reduce the antibiotic minimum inhibitory concentration up to 32-fold, even in a resistant isolate, functionally re-sensitizing the bacterium to the antibiotic. Meropenem and tobramycin did not reduce the frequency of lysogens, suggesting a mechanism of action independent of the temperate nature of the phages. By contrast, ciprofloxacin and piperacillin were able to reduce the frequency of lysogeny, the former by inducing phages-as previously reported in <i>E. coli</i>. Curiously, synergy with piperacillin reduced lysogen survivors, but not by inducing the phages, suggesting an alternative mechanism for biasing the phage lysis-lysogeny equilibrium. Overall, our findings indicate that temperate phages can act as adjuvants in clinically relevant pathogens, even in the presence of antibiotic resistance, thereby drastically expanding their therapeutic potential.</p><p><strong>Importance: </strong>The recent discovery that otherwise therapeutically unusable temperate phages can potentiate the activity of antibiotics, resulting in a potent synergy, has only been tested in <i>E. coli</i>, and with a single model phage. Here, working with clinical isolates of <i>Pseudomonas</i> and phages from these isolates, we highlight the broad applicability of this synergy-across a variety of mechanisms but also highlight the limitations of predicting the phage, host, and antibiotic combinations that will synergize.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0255924"},"PeriodicalIF":5.1,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864819","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A relay race of ESCRT-III paralogs drives cell division in a hyperthermophilic archaeon.","authors":"Junfeng Liu, Mickaël Lelek, Yunfeng Yang, Audrey Salles, Christophe Zimmer, Yulong Shen, Mart Krupovic","doi":"10.1128/mbio.00991-24","DOIUrl":"https://doi.org/10.1128/mbio.00991-24","url":null,"abstract":"<p><p>Cell division is a fundamental process ensuring the perpetuation of all cellular life forms. Archaea of the order Sulfolobales divide using a simpler version of the eukaryotic endosomal sorting complexes required for transport (ESCRT) machinery, composed of three ESCRT-III homologs (ESCRT-III, -III-1, and -III-2), AAA+ ATPase Vps4 and an archaea-specific component CdvA. Here, we clarify how these components act sequentially to drive the division of the hyperthermophilic archaeon <i>Saccharolobus islandicus</i>. Our data suggest that ESCRT-III plays an active role during the early stage of membrane constriction during cytokinesis, whereas ESCRT-III-1 and ESCRT-III-2 are indispensable for the \"pre-late\" and \"late\" stages of cytokinesis, respectively. In the <i>escrt-III-1</i> deletion strain, the division is blocked when the mid-cell constriction reaches ~30% of the initial cell diameter (\"pre-late\" stage), yielding \"chain-like\" cellular aggregates. Depletion of ESCRT-III-2 leads to the accumulation of cells connected through narrow membrane bridges (\"late\" stage), consistent with the key role of this protein in the final membrane abscission. We used 3D-single molecule localization microscopy to image ESCRT-III rings of different diameters and show that the decrease in the ESCRT-III ring diameter and membrane constriction are inconsistent with a mechanism exclusively based on spiraling of the ESCRT-III filaments. By contrast, the cone-shaped assemblies of ESCRT-III-1 and ESCRT-III-2 are consistent with spiral formation, highlighting the distinct roles of the three ESCRT-III proteins during the cytokinesis. We propose the \"relay race\" model, whereby the cytokinesis is achieved through a sequential and concerted action of different ESCRT machinery components.</p><p><strong>Importance: </strong>Two major cytokinesis mechanisms, rooted in contractile FtsZ and endosomal sorting complexes required for transport (ESCRT) rings, respectively, have emerged in the course of evolution. Whereas bacteria rely on the FtsZ-based mechanism, different lineages of archaea use either of the two systems, and eukaryotes have inherited the ESCRT-based cell division machinery from their archaeal ancestors. The mechanism of ESCRT-based cell division in archaea remains poorly understood and mechanistic studies on different archaeal model systems are essential to unravel the natural history of the ESCRT machinery. Here we investigate the interplay between three major ESCRT-III homologs during the division of a hyperthermophilic archaeon <i>Saccharolobus islandicus</i> and propose the \"relay race\" model of cytokinesis.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0099124"},"PeriodicalIF":5.1,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854704","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}