RESUMO
Ciprofloxacin-resistant Salmonella Typhimurium (S. Typhimurium) causes a significant health burden worldwide. A wealth of studies has been published on the contributions of different mechanisms to ciprofloxacin resistance in Salmonella spp. But we still lack a deep understanding of the physiological responses and genetic changes that underlie ciprofloxacin exposure. This study aims to know how phenotypic and genotypic characteristics are impacted by ciprofloxacin exposure, from ciprofloxacin-susceptible to ciprofloxacin-resistant strains in vitro. Here, we investigated the multistep evolution of resistance in replicate populations of S. Typhimurium during 24 days of continuously increasing ciprofloxacin exposure and assessed how ciprofloxacin impacts physiology and genetics. Numerous studies have demonstrated that RamA is a global transcriptional regulator that prominently perturbs the transcriptional landscape of S. Typhimurium, resulting in a ciprofloxacin-resistant phenotype appearing first; the quinolone resistance-determining region mutation site can only be detected later. Comparing the microbial physiological changes and RNA sequencing (RNA-Seq) results of ancestral and selectable mutant strains, the selectable mutant strains had some fitness costs, such as decreased virulence, an increase of biofilm-forming ability, a change of "collateral" sensitivity to other drugs, and inability to utilize galactitol. Importantly, in the ciprofloxacin induced, RamA directly binds and activates the gatR gene responsible for the utilization of galactitol, but RamA deletion strains could not activate gatR. The elevated levels of RamA, which inhibit the galactitol metabolic pathway through the activation of gatR, can lead to a reduction in the growth rate, adhesion, and colonization resistance of S. Typhimurium. This finding is supported by studies conducted in M9 medium as well as in vivo infection models. IMPORTANCE: Treatment of antibiotic resistance can significantly benefit from a deeper understanding of the interactions between drugs and genetics. The physiological responses and genetic mechanisms in antibiotic-exposed bacteria are not well understood. Traditional resistance studies, often retrospective, fail to capture the entire resistance development process and typically exhibit unpredictable dynamics. To explore how clinical isolates of S. Typhimurium respond to ciprofloxacin, we analyzed their adaptive responses. We found that S. Typhimurium RamA-mediated regulation disrupts microbial metabolism under ciprofloxacin exposure, affecting genes in the galactitol metabolic pathways. This disruption facilitates adaptive responses to drug therapy and enhances the efficiency of intracellular survival. A more comprehensive and integrated understanding of these physiological and genetic changes is crucial for improving treatment outcomes.
Assuntos
Antibacterianos , Ciprofloxacina , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium , Ciprofloxacina/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Virulência , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Animais , Camundongos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , MutaçãoRESUMO
Persistent microbial contamination commonly occurs in the environment. However, the characteristics and associated risks remain largely unknown. The coexistence of virulence factor genes (VFGs) and "last-resort" antibiotic resistance genes (LARGs) on human bacterial pathogens (HBPs) are notorious, creating ecological concerns and health risks. Herein, we explored the pathogenicity and antibiotic resistance levels of LARG-harboring HBPs in agricultural soils. Our findings revealed a high distribution level of VFGs and LARGs in soils (an absolute abundance up to 4.7 × 107 gene copies/g soil) by quantitative PCR (qPCR). Furthermore, most isolated LARG-harboring HBPs exhibited a 100 % lethality rate to Galleria mellonella. LARG-carrying plasmids had a low fitness cost to their host bacteria, implying the high adaptation of these plasmids within the HBPs. Most importantly, multiple LARG and VFG plasmid fusion and core genetic arrangements suggested that these LARG/VFG-linked plasmids endowed the stable and persistent horizontal spread of these genes in and/or cross the species and environments. This study not only unveiled high risk, multisource, compliance and stability aspects of environmentally persistent microbial contamination but also illuminated the importance of linking the phenotype-genotype-niche colonization of environmental microbial contamination within "One Health" framework.
Assuntos
Agricultura , Microbiologia do Solo , Plasmídeos/genética , Fatores de Virulência/genética , Resistência Microbiana a Medicamentos/genética , Bactérias/genética , Bactérias/efeitos dos fármacos , Solo/química , Humanos , Antibacterianos/farmacologia , Animais , Farmacorresistência Bacteriana/genéticaRESUMO
The misuse of antibiotics has led to the global spread of drug-resistant bacteria, especially multi-drug-resistant (MDR) ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). These opportunistic bacteria pose a significant threat, in particular within hospitals, where they cause nosocomial infections, leading to substantial morbidity and mortality. To comprehensively explore ESKAPE pathogenesis, virulence, host immune response, diagnostics, and therapeutics, researchers increasingly rely on necessitate suitable animal infection models. However, no single model can fully replicate all aspects of infectious diseases. Notably when studying opportunistic pathogens in immunocompetent hosts, rapid clearance by the host immune system can limit the expression of characteristic disease symptoms. In this study, we examine the critical role of animal infection models in understanding ESKAPE pathogens, addressing limitations and research gaps. We discuss applications and highlight key considerations for effective models. Thoughtful decisions on disease replication, parameter monitoring, and data collection are crucial for model reliability. By meticulously replicating human diseases and addressing limitations, researchers maximize the potential of animal infection models. This aids in targeted therapeutic development, bridges knowledge gaps, and helps combat MDR ESKAPE pathogens, safeguarding public health.
Assuntos
Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla , Animais , Humanos , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/fisiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Klebsiella pneumoniae/efeitos dos fármacos , Acinetobacter baumannii/efeitos dos fármacos , Infecção Hospitalar/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Enterobacter/efeitos dos fármacos , Infecções Bacterianas/microbiologiaRESUMO
Deoxynivalenol (DON) is the most abundant mycotoxin in cereal crops and derived foods and is of great concern in agriculture. Bioremediation strategies have long been sought to minimize the impact of mycotoxin contamination, but few direct and effective enzyme-catalyzed detoxification methods are currently available. In this study, we established a multi-enzymatic cascade reaction and successfully achieved detoxification at double sites: glutathionylation for the C-12,13 epoxide group and epimerization for the C-3 hydroxyl group. This yielded novel derivatives of DON, 3-epi-DON-13-glutathione (3-epi-DON-13-GSH) as well as its by-product, 3-keto-DON-13-GSH, for which precise structures were validated via liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Both cell viability and DNA synthesis assays demonstrated dramatically decreased cytotoxicity of the double-site modified product 3-epi-DON-13-GSH. These findings provide a promising and urgently needed novel method for addressing the problem of DON contamination in agricultural and industrial settings.
Assuntos
Tricotecenos , Tricotecenos/química , Tricotecenos/metabolismo , Contaminação de Alimentos/análise , Humanos , Fusarium/metabolismo , Fusarium/química , Inativação Metabólica , Micotoxinas/química , Micotoxinas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Glutationa/química , Glutationa/metabolismo , Biodegradação Ambiental , Espectrometria de Massas em TandemRESUMO
Zero-valent iron (ZVI) has been extensively studied for its capacity to remove various contaminants in the environments. However, whether ZVI affects bacterial resistance to antibiotics has not been fully explored. Herein, it was unexpected that, compared with microscale ZVI (mZVI), nanoscale ZVI (nZVI) facilitated the susceptibility of Pseudomonas aeruginosa (P. aeruginosa) to chloramphenicol (CAP), with a decrease in the minimal inhibitory concentration (MIC) of about 60 %, demonstrating a nanosize-specific effect. nZVI enhanced CAP accumulation in P. aeruginosa via inhibitory effect on efflux pumps activated by MexT, thus conferring the susceptibility of P. aeruginosa to CAP. Circular dichroism spectroscopy revealed that the structure of MexT was changed during the evolution. More importantly, molecular dynamic simulations uncovered that, once the structure of MexT changed, it would be more likely to interact with nZVI, resulting in more serious changes in its secondary structure, which was consistent with the increasing susceptibility of P. aeruginosa to CAP. Collectively, this study elucidated the size-specific effect and the underlying mechanism of ZVI on the bacterial evolution of susceptibility toward antibiotics, highlighting the potentials of nZVI-based technologies on the prevention of bacterial resistance to antibiotics, one of the most important issue for globally public health.
Assuntos
Antibacterianos , Cloranfenicol , Farmacorresistência Bacteriana , Ferro , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa , Pseudomonas aeruginosa/efeitos dos fármacos , Cloranfenicol/farmacologia , Cloranfenicol/química , Antibacterianos/farmacologia , Antibacterianos/química , Ferro/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Nanopartículas Metálicas/química , Simulação de Dinâmica Molecular , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genéticaRESUMO
The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host's ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA. The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting.
Assuntos
Carbapenêmicos , Infecções por Klebsiella , Klebsiella pneumoniae , Regiões Promotoras Genéticas , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Virulência/genética , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/tratamento farmacológico , Regiões Promotoras Genéticas/genética , Carbapenêmicos/farmacologia , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Camundongos , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/patogenicidade , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Polimorfismo de Nucleotídeo Único , Infecção Hospitalar/microbiologia , Infecção Hospitalar/tratamento farmacológicoRESUMO
Trichothecene (TCN) contamination in food and feed is a serious challenge due to the negative health and economic impacts. Here, we confirmed that the glutathione S-transferase (GST) Fhb7-GST could broadly catalyze type A, type B and type D TCNs into glutathione epoxide adducts (TCN-13-GSHs). To evaluate the toxicity of TCN-13-GSH adducts, we performed cell proliferation assays in vitro, which demonstrated decreased cytotoxicity of the adducts. Moreover, in vivo assays (repeated-dose treatment in mice) confirmed that TCN-13-GSH adducts were dramatically less toxic than the corresponding TCNs. To establish whether TCN-13-GSH was metabolized back to free toxin during digestion, single-dose metabolic tests were performed in rats; DON-13-GSH was not hydrolyzed in vivo, but rather was quickly metabolized to another low-toxicity compound, DON-13-N-acetylcysteine. These results demonstrate the promise of Fhb7-GST as a candidate of detoxification enzyme potentially applied in TCN-contaminated agricultural samples, minimizing the detrimental effects of the mycotoxin.
Assuntos
Glutationa Transferase , Tricotecenos , Ratos , Camundongos , Animais , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Tricotecenos/toxicidade , Tricotecenos/metabolismo , Glutationa/metabolismo , CatáliseRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that predominantly causes nosocomial and community-acquired lung infections. As a member of ESKAPE pathogens, carbapenem-resistant P. aeruginosa (CRPA) compromises the limited therapeutic options, raising an urgent demand for the development of lead compounds against previously-unrecognized drug targets. Biotin is an important cofactor, of which the de novo synthesis is an attractive antimicrobial target in certain recalcitrant infections. Here we report genetic and biochemical definition of P. aeruginosa BioH (PA0502) that functions as a gatekeeper enzyme allowing the product pimeloyl-ACP to exit from fatty acid synthesis cycle and to enter the late stage of biotin synthesis pathway. In relative to Escherichia coli, P. aeruginosa physiologically requires 3-fold higher level of cytosolic biotin, which can be attributed to the occurrence of multiple biotinylated enzymes. The BioH protein enables the in vitro reconstitution of biotin synthesis. The repertoire of biotin abundance is assigned to different mouse tissues and/or organ contents, and the plasma biotin level of mouse is around 6-fold higher than that of human. Removal of bioH renders P. aeruginosa biotin auxotrophic and impairs its intra-phagosome persistence. Based on a model of CD-1 mice mimicking the human environment, lung challenge combined with systemic infection suggested that BioH is necessary for the full virulence of P. aeruginosa. As expected, the biotin synthesis inhibitor MAC13772 is capable of dampening the viability of CRPA. Notably, MAC13772 interferes the production of pyocyanin, an important virulence factor of P. aeruginosa. Our data expands our understanding of P. aeruginosa biotin synthesis relevant to bacterial infectivity. In particular, this study represents the first example of an extracellular pathogen P. aeruginosa that exploits biotin cofactor as a fitness determinant, raising the possibility of biotin synthesis as an anti-CRPA target.
Assuntos
Biotina , Infecções por Pseudomonas , Animais , Humanos , Camundongos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Biotina/química , Biotina/metabolismo , Pseudomonas aeruginosa/metabolismoRESUMO
Staphylococcus and Streptococcus, two groups of major human pathogens, are equipped with a fatty acid kinase (Fak) machinery to scavenge host fatty acids. The Fak complex is contains an ATP-binding subunit FakA, which interacts with varied FakB isoforms, and synthesizes acyl-phosphate from extracellular fatty acids. However, how FakA recognizes its FakB partners and then activates different fatty acids is poorly understood. Here, we systematically describe the Fak system from the zoonotic pathogen, Streptococcus suis. The crystal structure of SsFakA complexed with SsFakB2 was determined at 2.6 Å resolution. An in vitro system of Fak-PlsX (phosphate: acyl-ACP transacylase) was developed to track acyl-phosphate intermediate and its final product acyl-ACP. Structure-guided mutagenesis enabled us to characterize a mechanism for streptococcal FakA working with FakB partners engaged in host fatty acid scavenging. These findings offer a comprehensive description of the Fak kinase machinery, thus advancing the discovery of attractive targets against deadly infections with Streptococcus.
RESUMO
Tuberculosis (TB) is one of the leading infectious diseases of global concern, and one quarter of the world's population are TB carriers. Biotin metabolism appears to be an attractive anti-TB drug target. However, the first-stage of mycobacterial biotin synthesis is fragmentarily understood. Here we report that three evolutionarily-distinct BioH isoenzymes (BioH1 to BioH3) are programmed in biotin synthesis of Mycobacterium smegmatis. Expression of an individual bioH isoform is sufficient to allow the growth of an Escherichia coli ΔbioH mutant on the non-permissive condition lacking biotin. The enzymatic activity in vitro combined with biotin bioassay in vivo reveals that BioH2 and BioH3 are capable of removing methyl moiety from pimeloyl-ACP methyl ester to give pimeloyl-ACP, a cognate precursor for biotin synthesis. In particular, we determine the crystal structure of dimeric BioH3 at 2.27Å, featuring a unique lid domain. Apart from its catalytic triad, we also dissect the substrate recognition of BioH3 by pimeloyl-ACP methyl ester. The removal of triple bioH isoforms (ΔbioH1/2/3) renders M. smegmatis biotin auxotrophic. Along with the newly-identified Tam/BioC, the discovery of three unusual BioH isoforms defines an atypical 'BioC-BioH(3)' paradigm for the first-stage of mycobacterial biotin synthesis. This study solves a long-standing puzzle in mycobacterial nutritional immunity, providing an alternative anti-TB drug target.
Assuntos
Antituberculosos , Biotina , Biotina/química , Biotina/metabolismo , Escherichia coli/metabolismo , Ésteres/metabolismo , Isoenzimas/metabolismoRESUMO
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important extracellular human pathogen. The initial adherence of EHEC to host cells is a major cue for transcriptional induction of the locus of enterocyte effacement (LEE) genes to promote colonization and pathogenesis, but the mechanism through which this adherence is sensed and the LEE is induced remains largely elusive. Here, we report a complete signal transduction pathway for this virulence activation process. In this pathway, the outer-membrane lipoprotein NlpE senses a mechanical cue generated from initial host adherence and activates the BaeSR two-component regulatory system; the response regulator BaeR then directly activates the expression of airA located on O-island-134 and encoding a LEE transcriptional activator. Disruption of this pathway severely attenuates EHEC O157:H7 virulence both in vitro and in vivo. This study provides further insights into the evolution of EHEC pathogenesis and the host-pathogen interaction.
Assuntos
Escherichia coli Êntero-Hemorrágica , Infecções por Escherichia coli , Escherichia coli O157 , Proteínas de Escherichia coli , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Fosfoproteínas/metabolismo , Virulência/genéticaRESUMO
The amino acid sequence of a protein contains all the necessary information to specify its shape, which dictates its biological activities. However, it is challenging and expensive to experimentally determine the three-dimensional structure of proteins. The backbone torsion angles play a critical role in protein structure prediction, and accurately predicting the angles can considerably advance the tertiary structure prediction by accelerating efficient sampling of the large conformational space for low energy structures. Here we first time propose evolutionary signatures computed from protein sequence profiles, and a novel recurrent architecture, termed ESIDEN, that adopts a straightforward architecture of recurrent neural networks with a small number of learnable parameters. The ESIDEN can capture efficient information from both the classic and new features benefiting from different recurrent architectures in processing information. On the other hand, compared to widely used classic features, the new features, especially the Ramachandran basin potential, provide statistical and evolutionary information to improve prediction accuracy. On four widely used benchmark datasets, the ESIDEN significantly improves the accuracy in predicting the torsion angles by comparison to the best-so-far methods. As demonstrated in the present study, the predicted angles can be used as structural constraints to accurately infer protein tertiary structures. Moreover, the proposed features would pave the way to improve machine learning-based methods in protein folding and structure prediction, as well as function prediction. The source code and data are available at the website https://kornmann.bioch.ox.ac.uk/leri/resources/download.html .
Assuntos
Bases de Dados de Proteínas , Evolução Molecular , Redes Neurais de Computação , Proteínas/química , Software , Valor Preditivo dos Testes , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/genéticaRESUMO
The transferability of bacterial resistance to tigecycline, the 'last-resort' antibiotic, is an emerging challenge of global health concern. The plasmid-borne tet(X) that encodes a flavin-dependent monooxygenase represents a new mechanism for tigecycline resistance. Natural source for an ongoing family of Tet(X) resistance determinants is poorly understood. Here, we report the discovery of 26 new variants [tet(X18) to tet(X44)] from the poultry pathogen Riemerella anatipestifer, which expands extensively the current Tet(X) family. R. anatipestifer appears as a natural reservoir for tet(X), of which the chromosome harbours varied copies of tet(X) progenitors. Despite that an inactive ancestor rarely occurs, the action and mechanism of Tet(X2/4)-P, a putative Tet(X) progenitor, was comprehensively characterized, giving an intermediate level of tigecycline resistance. The potential pattern of Tet(X) dissemination from ducks to other animals and humans was raised, in the viewpoint of ecological niches. Therefore, this finding defines a large pool of natural sources for Tet(X) tigecycline resistance, heightening the need of efficient approaches to manage the inter-species transmission of tet(X) resistance determinants.
Assuntos
Doenças das Aves Domésticas , Riemerella , Animais , Antibacterianos/farmacologia , Patos , Testes de Sensibilidade Microbiana , Aves Domésticas , Riemerella/genética , Tigeciclina/farmacologiaRESUMO
An evolving family of mobile colistin resistance (MCR) enzymes is threatening public health. However, the molecular mechanism by which the MCR enzyme as a rare member of lipid A-phosphoethanolamine (PEA) transferases gains the ability to confer phenotypic colistin resistance remains enigmatic. Here, we report an unusual example that genetic duplication and amplification produce a functional variant (Ah762) of MCR-3 in certain Aeromonas species. The lipid A-binding cavity of Ah762 is functionally defined. Intriguingly, we locate a hinge linker of Ah762 (termed Linker 59) that determines the MCR. Genetic and biochemical characterization reveals that Linker 59 behaves as a facilitator to render inactive MCR variants to regain the ability of colistin resistance. Along with molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC) suggests that this facilitator guarantees the formation of substrate phosphatidylethanolamine (PE)-accessible pocket within MCR-3-like enzymes. Therefore, our finding defines an MCR-3 inside facilitator for colistin resistance.
Assuntos
Colistina/química , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Biotin is an essential micro-nutrient across the three domains of life. The paradigm earlier step of biotin synthesis denotes "BioC-BioH" pathway in Escherichia coli. Here we report that BioZ bypasses the canonical route to begin biotin synthesis. In addition to its origin of Rhizobiales, protein phylogeny infers that BioZ is domesticated to gain an atypical role of ß-ketoacyl-ACP synthase III. Genetic and biochemical characterization demonstrates that BioZ catalyzes the condensation of glutaryl-CoA (or ACP) with malonyl-ACP to give 5'-keto-pimeloyl ACP. This intermediate proceeds via type II fatty acid synthesis (FAS II) pathway, to initiate the formation of pimeloyl-ACP, a precursor of biotin synthesis. To further explore molecular basis of BioZ activity, we determine the crystal structure of Agrobacterium tumefaciens BioZ at 1.99 Å, of which the catalytic triad and the substrate-loading tunnel are functionally defined. In particular, we localize that three residues (S84, R147, and S287) at the distant bottom of the tunnel might neutralize the charge of free C-carboxyl group of the primer glutaryl-CoA. Taken together, this study provides molecular insights into the BioZ biotin synthesis pathway.
Assuntos
Vias Biossintéticas , Biotina/biossíntese , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteína de Transporte de Acila/metabolismo , Acil Coenzima A/metabolismo , Agrobacterium/crescimento & desenvolvimento , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Filogenia , Multimerização Proteica , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Tigecycline and colistin are few of 'last-resort' antibiotic defences used in anti-infection therapies against carbapenem-resistant bacterial pathogens. The successive emergence of plasmid-borne tet(X) tigecycline resistance mechanism and mobile colistin resistance (mcr) determinant, renders them clinically useless. Here, we report that co-carriage of tet(X6) and mcr-1 gives co-resistance to both classes of antibiotics by a single plasmid in Escherichia coli. Tet(X6), the new tigecycline resistance enzyme is functionally defined. Both Tet(X6) and MCR-1 robustly interfere accumulation of antibiotic-induced reactive oxygen species (ROS). Unlike that mcr-1 exerts fitness cost in E. coli, tet(X6) does not. In the tet(X6)-positive strain that co-harbors mcr-1, tigecycline resistance is independently of colistin resistance caused by MCR-1-mediated lipid A remodelling, and vice versa. In general consistency with that of MCR-1, Tet(X6) leads to the failure of tigecycline treatment in the infection model of G. mellonella. Taken together, the co-production of Tet(X) and MCR-1 appears as a major clinic/public health concern.
Assuntos
Farmacorresistência Bacteriana , Proteínas de Escherichia coli , Escherichia coli , Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Tigeciclina/farmacologiaRESUMO
MCR-4 and MCR-8 are two recently identified members of an ongoing MCR family of colistin resistance. Although that aquatic reservoir for MCR-4 is proposed, the origin and mechanism of MCR-8 is poorly understood. Here we report a previously unrecognized non-mobile colistin resistance enzyme, termed NMCR-2, originating from the plant pathogen Kosakonia pseudosacchari. NMCR-2 (551aa) gives 67.3% identity to MCR-8 (565aa). NMCR-2 is placed as a progenitor/ancestor for MCR-8 in phylogeny of MCR members. Genetic study reveals that nmcr-2 is comparable to mcr-8 in the ability of producing phenotypic colistin resistance. Biochemical analyses determine that these two enzymes catalyse the transfer of PEA from the donor PE lipid substrate to the recipient lipid A molecule by a putative 'ping-pong' trade-off. Further experiment of protein engineering demonstrates that the two motifs (TM region and catalytic domain) of NMCR-2 are functionally exchangeable with that of MCR-8, rather than MCR-1. Physiological impacts of nmcr-2 and/or mcr-8 are detected in Escherichia coli, featuring with fitness cost. Evidently, the action and mechanism of NMCR-2 is analogous to that of MCR-8. Therefore, our finding underlines that NMCR-2 might be a possible progenitor of MCR-8.
Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Enterobacteriaceae/classificação , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Testes de Sensibilidade Microbiana , Família Multigênica , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismoRESUMO
A growing number of tet(X)-type tigecycline resistance determinants [tet(X1) to tet(X5)] constitutes an expanding family of tetracycline-inactivating enzymes, posing a potential risk to global public health. Here, we report the development of an efficient multiplex PCR method to detect the family of tet(X) variants. This method is successfully applied in the screen and validation of tet(X) genes in the field and clinic bacterial samples. In addition, we found that the formerly proposed tet(X1) is a premature truncated version by the inappropriate annotation, and fixed this error. Overall, it might be the first genetic tool for the detection of different Tet(X) members.
Assuntos
Genes Bacterianos/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Resistência a Tetraciclina/genética , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Sensibilidade e Especificidade , TigeciclinaRESUMO
Polymyxins are a group of detergent-like antimicrobial peptides that are the ultimate line of defense against carbapenem-resistant pathogens in clinical settings. Polymyxin resistance primarily originates from structural remodeling of lipid A anchored on bacterial surfaces. We integrate genetic, structural, and biochemical aspects of three major types of lipid A modifiers that have been shown to confer intrinsic colistin resistance. Namely, we highlight ArnT, a glycosyltransferase, EptA, a phosphoethanolamine transferase, and the AlmEFG tripartite system, which is restricted to EI Tor biotype of Vibrio cholerae O1. We also discuss the growing family of mobile colistin resistance (MCR) enzymes, each of which is analogous to EptA, and which pose great challenges to global public health.
Assuntos
Antibacterianos/química , Lipídeo A/metabolismo , Polimixinas/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Farmacorresistência Bacteriana , Etanolaminas/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicosiltransferases/metabolismo , Humanos , Modelos Moleculares , Fosfotransferases/metabolismo , Polimixinas/farmacologia , Ligação Proteica , Conformação ProteicaRESUMO
Colistin is the last-resort antibiotic against lethal infections with multidrug-resistant bacterial pathogens. A rainbow coalition of mobile colistin resistance (mcr) genes raises global health concerns. Here, we describe the action and mechanism of colistin resistance imparted by MCR-4, a recently-identified member from the broader MCR family. We found that MCR-4 originates from the silenced variant of Shewanella frigidimarina via progressive evolution and forms a phylogenetically-distinct group from the well-studied MCR-1/2 family. Domain-swapping experiments further confirmed that MCR-1 and MCR-4 transmembrane and catalytic domains are not functionally-interchangeable. However, structural and functional analyses demonstrated that MCR-4 possesses a similar PE lipid substrate-recognizable cavity and exploits an almost-identical ping-pong catalysis mechanism. MCR-4 also can alleviate colistin-triggered accumulation of reactive oxygen species (ROS). Taken together, this finding constitutes a functional proof that MCR-4 proceeds in a distinct evolutionary path to fulfill a consistent molecular mechanism, resulting in phenotypic colistin resistance.