RESUMEN
The Mycobacterium abscessus drug development pipeline is poorly populated, with particularly few validated target-lead couples to initiate de novo drug discovery. Trimethoprim, an inhibitor of dihydrofolate reductase (DHFR) used for the treatment of a range of bacterial infections, is not active against M. abscessus. Thus, evidence that M. abscessus DHFR is vulnerable to pharmacological intervention with a small molecule inhibitor is lacking. Here, we show that the pyrrolo-quinazoline PQD-1, previously identified as a DHFR inhibitor active against Mycobacterium tuberculosis, exerts whole cell activity against M. abscessus. Enzyme inhibition studies showed that PQD-1, in contrast to trimethoprim, is a potent inhibitor of M. abscessus DHFR and over-expression of DHFR causes resistance to PQD-1, providing biochemical and genetic evidence that DHFR is a vulnerable target and mediates PQD-1's growth inhibitory activity in M. abscessus. As observed in M. tuberculosis, PQD-1 resistant mutations mapped to the folate pathway enzyme thymidylate synthase (TYMS) ThyA. Like trimethoprim in other bacteria, PQD-1 synergizes with the dihydropteroate synthase (DHPS) inhibitor sulfamethoxazole (SMX), offering an opportunity to exploit the successful dual inhibition of the folate pathway and develop similarly potent combinations against M. abscessus. PQD-1 is active against subspecies of M. abscessus and a panel of clinical isolates, providing epidemiological validation of the target-lead couple. Leveraging a series of PQD-1 analogs, we have demonstrated a dynamic structure-activity relationship (SAR). Collectively, the results identify M. abscessus DHFR as an attractive target and PQD-1 as a chemical starting point for the discovery of novel drugs and drug combinations that target the folate pathway in M. abscessus.
Asunto(s)
Antagonistas del Ácido Fólico , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Mycobacterium tuberculosis , Humanos , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Antagonistas del Ácido Fólico/farmacología , Trimetoprim/farmacología , Mycobacterium tuberculosis/metabolismo , Inhibidores Enzimáticos/farmacología , Ácido Fólico , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológicoRESUMEN
Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.
Asunto(s)
Mycobacterium tuberculosis , Myxococcales , Antibacterianos/química , Ribosomas/metabolismo , Biosíntesis de ProteínasRESUMEN
OBJECTIVES: To determine how resistance to macrolides is conferred in field isolates of Pasteurella multocida and Mannheimia haemolytica that lack previously identified resistance determinants for rRNA methylation, efflux and macrolide-modifying enzymes. METHODS: Isolates of P. multocida and M. haemolytica identified as being highly resistant (MICs >64 mg/L) to the macrolides erythromycin, gamithromycin, tilmicosin, tildipirosin and tulathromycin were screened by multiplex PCR for the previously identified resistance genes erm(42), msr(E) and mph(E). Strains lacking these determinants were analysed by genome sequencing and primer extension on the rRNAs. RESULTS: Macrolide resistance in one M. haemolytica isolate was conferred by the 23S rRNA mutation A2058G; resistance in three P. multocida isolates were caused by mutations at the neighbouring nucleotide A2059G. In each strain, all six copies of the rrn operons encoded the respective mutations. There were no mutations in the ribosomal protein genes rplD or rplV, and no other macrolide resistance mechanism was evident. CONCLUSIONS: High-level macrolide resistance can arise from 23S rRNA mutations in P. multocida and M. haemolytica despite their multiple copies of rrn. Selective pressures from exposure to different macrolide or lincosamide drugs presumably resulted in consolidation of either the A2058G or the A2059G mutation.
Asunto(s)
Farmacorresistencia Bacteriana , Genes de ARNr , Macrólidos/farmacología , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/genética , Mutación , Pasteurella multocida/efectos de los fármacos , Pasteurella multocida/genética , Antibacterianos/farmacología , Genoma Bacteriano , Humanos , Mannheimia haemolytica/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Operón , Pasteurella multocida/aislamiento & purificación , ARN Ribosómico 23S/genéticaRESUMEN
Mannheimia haemolytica and Pasteurella multocida are aetiological agents commonly associated with respiratory tract infections in cattle. Recent isolates of these pathogens have been shown to be resistant to macrolides and other ribosome-targeting antibiotics. Direct analysis of the 23S rRNAs by mass spectrometry revealed that nucleotide A2058 is monomethylated, consistent with a Type I erm phenotype conferring macrolide-lincosamide resistance. The erm resistance determinant was identified by full genome sequencing of isolates. The sequence of this resistance determinant, now termed erm(42), has diverged greatly from all previously characterized erm genes, explaining why it has remained undetected in PCR screening surveys. The sequence of erm(42) is, however, completely conserved in six independent M. haemolytica and P. multocida isolates, suggesting relatively recent gene transfer between these species. Furthermore, the composition of neighbouring chromosomal sequences indicates that erm(42) was acquired from other members of the Pasteurellaceae. Expression of recombinant erm(42) in Escherichia coli demonstrated that the enzyme retains its properties as a monomethyltransferase without any dimethyltransferase activity. Erm(42) is a novel addition to the Erm family: it is phylogenetically distant from the other Erm family members and it is unique in being a bona fide monomethyltransferase that is disseminated between bacterial pathogens.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/enzimología , Metiltransferasas/metabolismo , Pasteurella multocida/efectos de los fármacos , Pasteurella multocida/enzimología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Farmacorresistencia Bacteriana/fisiología , Mannheimia haemolytica/genética , Metiltransferasas/genética , Pruebas de Sensibilidad Microbiana , Pasteurella multocida/química , Pasteurella multocida/genética , Reacción en Cadena de la Polimerasa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC(50)], 0.23 ± 0.01 µM) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site.
Asunto(s)
Antibacterianos/química , Mannheimia haemolytica/efectos de los fármacos , Pasteurella multocida/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/efectos de los fármacos , Tilosina/análogos & derivados , Drogas Veterinarias/química , Antibacterianos/farmacología , Sitios de Unión , Disacáridos/química , Disacáridos/farmacología , Escherichia coli/química , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Mannheimia haemolytica/química , Mannheimia haemolytica/genética , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mutación , Nucleótidos/genética , Pasteurella multocida/química , Pasteurella multocida/genética , ARN Ribosómico/química , ARN Ribosómico/genética , Ribosomas/química , Fracciones Subcelulares/química , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Transcripción Genética/efectos de los fármacos , Tilosina/química , Tilosina/farmacología , Drogas Veterinarias/farmacologíaRESUMEN
The bacterial pathogens Mannheimia haemolytica and Pasteurella multocida are major etiological agents in respiratory tract infections of cattle. Although these infections can generally be successfully treated with veterinary macrolide antibiotics, a few recent isolates have shown resistance to these drugs. Macrolide resistance in members of the family Pasteurellaceae is conferred by combinations of at least three genes: erm(42), which encodes a monomethyltransferase and confers a type I MLS(B) (macrolide, lincosamide, and streptogramin B) phenotype; msr(E), which encodes a macrolide efflux pump; and mph(E), which encodes a macrolide-inactivating phosphotransferase. Here, we describe a multiplex PCR assay that detects the presence of erm(42), msr(E), and mph(E) and differentiates between these genes. In addition, the assay distinguishes P. multocida from M. haemolytica by amplifying distinctive fragments of the 23S rRNA (rrl) genes. One rrl fragment acts as a general indicator of gammaproteobacterial species and confirms whether the PCR assay has functioned as intended on strains that are negative for erm(42), msr(E), and mph(E). The multiplex system has been tested on more than 40 selected isolates of P. multocida and M. haemolytica and correlated with MICs for the veterinary macrolides tulathromycin and tilmicosin, and the newer compounds gamithromycin and tildipirosin. The multiplex PCR system gives a rapid and robustly accurate determination of macrolide resistance genotypes and bacterial genus, matching results from microbiological methods and whole-genome sequencing.
Asunto(s)
Antibacterianos/farmacología , Macrólidos/farmacología , Mannheimia haemolytica/efectos de los fármacos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Pasteurella multocida/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Lincosamidas/farmacología , Mannheimia haemolytica/genética , Pasteurella multocida/genética , Estreptogramina B/farmacologíaRESUMEN
Respiratory tract infections in cattle are commonly associated with the bacterial pathogens Mannheimia haemolytica and Pasteurella multocida. These infections can generally be successfully treated in the field with one of several groups of antibiotics, including macrolides. A few recent isolates of these species exhibit resistance to veterinary macrolides with phenotypes that fall into three distinct classes. The first class has type I macrolide, lincosamide, and streptogramin B antibiotic resistance and, consistent with this, the 23S rRNA nucleotide A2058 is monomethylated by the enzyme product of the erm(42) gene. The second class shows no lincosamide resistance and lacks erm(42) and concomitant 23S rRNA methylation. Sequencing of the genome of a representative strain from this class, P. multocida 3361, revealed macrolide efflux and phosphotransferase genes [respectively termed msr(E) and mph(E)] that are arranged in tandem and presumably expressed from the same promoter. The third class exhibits the most marked drug phenotype, with high resistance to all of the macrolides tested, and possesses all three resistance determinants. The combinations of erm(42), msr(E), and mph(E) are chromosomally encoded and intermingled with other exogenous genes, many of which appear to have been transferred from other members of the Pasteurellaceae. The presence of some of the exogenous genes explains recent reports of resistance to additional drug classes. We have expressed recombinant versions of the erm(42), msr(E), and mph(E) genes within an isogenic Escherichia coli background to assess their individually contributions to resistance. Our findings indicate what types of compounds might have driven the selection for these resistance determinants.
Asunto(s)
Lincosamidas/farmacología , Macrólidos/farmacología , Mannheimia haemolytica/efectos de los fármacos , Pasteurella multocida/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Mannheimia haemolytica/genética , Pruebas de Sensibilidad Microbiana , Pasteurella multocida/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , ARN Ribosómico 23S/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estreptogramina B/farmacologíaRESUMEN
The veterinary antibiotic tildipirosin (20,23-dipiperidinyl-mycaminosyl-tylonolide, Zuprevo) was developed recently to treat bovine and swine respiratory tract infections caused by bacterial pathogens such as Pasteurella multocida. Tildipirosin is a derivative of the naturally occurring compound tylosin. Here, we define drug-target interactions by combining chemical footprinting with structure modeling and show that tildipirosin, tylosin, and an earlier tylosin derivative, tilmicosin (20-dimethylpiperidinyl-mycaminosyl-tylonolide, Micotil), bind to the same macrolide site within the large subunit of P. multocida and Escherichia coli ribosomes. The drugs nevertheless differ in how they occupy this site. Interactions of the two piperidine components, which are unique to tildipirosin, distinguish this drug from tylosin and tilmicosin. The 23-piperidine of tildipirosin contacts ribosomal residues on the tunnel wall while its 20-piperidine is oriented into the tunnel lumen and is positioned to interfere with the growing nascent peptide.
Asunto(s)
Macrólidos/química , Ribosomas/química , Tilosina/análogos & derivados , Antibacterianos/química , Sitios de Unión , Escherichia coli/metabolismo , Modelos Químicos , Modelos Moleculares , Pasteurella multocida/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , ARN Ribosómico/metabolismo , Tilosina/químicaRESUMEN
(3'-5')-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a "one-pot" three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1ß, tumor necrosis factor α, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.