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1.
Nature ; 584(7821): 479-483, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32788728

RESUMO

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Assuntos
Membrana Celular/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/patogenicidade , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Estabilidade Enzimática , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Genes Essenciais , Hidrolases/química , Hidrolases/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/biossíntese , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , Virulência
2.
Proc Natl Acad Sci U S A ; 119(38): e2123117119, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36099298

RESUMO

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.


Assuntos
Acinetobacter baumannii , Antibacterianos , Ácido Aspártico Endopeptidases , Proteínas de Bactérias , Farmacorresistência Bacteriana , Furanos , Deleção de Genes , Lipoproteínas , Inibidores de Proteases , Piridinas , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Ácido Aspártico Endopeptidases/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Furanos/farmacologia , Lipoproteínas/biossíntese , Lipoproteínas/genética , Peptídeos/farmacologia , Inibidores de Proteases/farmacologia , Sinais Direcionadores de Proteínas/genética , Piridinas/farmacologia
3.
J Bacteriol ; 206(10): e0010224, 2024 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-39235234

RESUMO

Inosine 5'-monophosphate dehydrogenase (IMPDH), known as GuaB in bacteria, catalyzes the rate-limiting step in de novo guanine biosynthesis and is conserved from humans to bacteria. We developed a series of potent inhibitors that selectively target GuaB over its human homolog. Here, we show that these GuaB inhibitors are bactericidal, generate phenotypic signatures that are distinct from other antibiotics, and elicit different time-kill kinetics and regulatory responses in two important Gram-negative pathogens: Acinetobacter baumannii and Escherichia coli. Specifically, the GuaB inhibitor G6 rapidly kills A. baumannii but only kills E. coli after 24 h. After exposure to G6, the expression of genes involved in purine biosynthesis and stress responses change in opposite directions while siderophore biosynthesis is downregulated in both species. Our results suggest that different species respond to GuaB inhibition using distinct regulatory programs and possibly explain the different bactericidal kinetics upon GuaB inhibition. The comparison highlights opportunities for developing GuaB inhibitors as novel antibiotics.IMPORTANCEA. baumannii is a priority bacterial pathogen for which development of new antibiotics is urgently needed due to the emergence of multidrug resistance. We recently developed a series of specific inhibitors against GuaB, a bacterial inosine 5'-monophosphate dehydrogenase, and achieved sub-micromolar minimum inhibitory concentrations against A. baumannii. GuaB catalyzes the rate-limiting step of de novo guanine biosynthesis and is highly conserved across bacterial pathogens. This study shows that inhibition of GuaB induced a bacterial morphological profile distinct from that of other classes of antibiotics, highlighting a novel mechanism of action. Moreover, our transcriptomic analysis showed that regulation of de novo purine biosynthesis and stress responses of A. baumannii upon GuaB inhibition differed significantly from that of E. coli.


Assuntos
Acinetobacter baumannii , Antibacterianos , Inibidores Enzimáticos , Escherichia coli , IMP Desidrogenase , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/enzimologia , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/metabolismo , IMP Desidrogenase/genética , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
4.
Nature ; 561(7722): 189-194, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30209367

RESUMO

Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.


Assuntos
Antibacterianos/classificação , Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Biocatálise/efeitos dos fármacos , Produtos Biológicos/classificação , Produtos Biológicos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/enzimologia , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/patogenicidade , Lisina/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos/química , Porinas , Ligação Proteica , Domínios Proteicos , Serina Endopeptidases , Especificidade por Substrato
5.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443214

RESUMO

Tandem gene amplification is a frequent and dynamic source of antibiotic resistance in bacteria. Ongoing expansions and contractions of repeat arrays during population growth are expected to manifest as cell-to-cell differences in copy number (CN). As a result, a clonal bacterial culture could comprise subpopulations of cells with different levels of antibiotic sensitivity that result from variable gene dosage. Despite the high potential for misclassification of heterogenous cell populations as either antibiotic-susceptible or fully resistant in clinical settings, and the concomitant risk of inappropriate treatment, CN distribution among cells has defied analysis. Here, we use the MinION single-molecule nanopore sequencer to uncover CN heterogeneity in clonal populations of Escherichia coli and Acinetobacter baumannii grown from single cells isolated while selecting for resistance to an optimized arylomycin, a member of a recently discovered class of Gram-negative antibiotic. We found that gene amplification of the arylomycin target, bacterial type I signal peptidase LepB, is a mechanism of unstable arylomycin resistance and demonstrate in E. coli that amplification instability is independent of RecA. This instability drives the emergence of a nonuniform distribution of lepB CN among cells with a range of 1 to at least 50 copies of lepB identified in a single clonal population. In sum, this remarkable heterogeneity, and the evolutionary plasticity it fuels, illustrates how gene amplification can enable bacterial populations to respond rapidly to novel antibiotics. This study establishes a rationale for further nanopore-sequencing studies of heterogeneous cell populations to uncover CN variability at single-molecule resolution.


Assuntos
Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Amplificação de Genes/efeitos dos fármacos , Proteínas de Membrana/genética , Sequenciamento por Nanoporos/métodos , Peptídeos Cíclicos/genética , Serina Endopeptidases/genética , Variações do Número de Cópias de DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Testes de Sensibilidade Microbiana , Mutação , Sequenciamento por Nanoporos/instrumentação , Recombinases Rec A/metabolismo
6.
Proc Natl Acad Sci U S A ; 116(3): 934-943, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30598433

RESUMO

Plastid genomes (plastomes) vary enormously in size and gene content among the many lineages of nonphotosynthetic plants, but key lineages remain unexplored. We therefore investigated plastome sequence and expression in the holoparasitic and morphologically bizarre Balanophoraceae. The two Balanophora plastomes examined are remarkable, exhibiting features rarely if ever seen before in plastomes or in any other genomes. At 15.5 kb in size and with only 19 genes, they are among the most reduced plastomes known. They have no tRNA genes for protein synthesis, a trait found in only three other plastid lineages, and thus Balanophora plastids must import all tRNAs needed for translation. Balanophora plastomes are exceptionally compact, with numerous overlapping genes, highly reduced spacers, loss of all cis-spliced introns, and shrunken protein genes. With A+T contents of 87.8% and 88.4%, the Balanophora genomes are the most AT-rich genomes known save for a single mitochondrial genome that is merely bloated with AT-rich spacer DNA. Most plastid protein genes in Balanophora consist of ≥90% AT, with several between 95% and 98% AT, resulting in the most biased codon usage in any genome described to date. A potential consequence of its radical compositional evolution is the novel genetic code used by Balanophora plastids, in which TAG has been reassigned from stop to tryptophan. Despite its many exceptional properties, the Balanophora plastome must be functional because all examined genes are transcribed, its only intron is correctly trans-spliced, and its protein genes, although highly divergent, are evolving under various degrees of selective constraint.


Assuntos
Balanophoraceae/genética , Evolução Molecular , Código Genético , Genomas de Plastídeos , Proteínas de Plantas/genética
7.
Proc Natl Acad Sci U S A ; 115(14): 3692-3697, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29555747

RESUMO

The folding and insertion of integral ß-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ß-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli ß-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ß-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ß-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.


Assuntos
Antibacterianos/farmacologia , Anticorpos Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Fluidez de Membrana/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Escherichia coli/imunologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína
8.
Artigo em Inglês | MEDLINE | ID: mdl-29339384

RESUMO

The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral ß-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of Escherichia coli growth followed by selection of compounds that induced the extracytoplasmic σE stress response. Mutations in lolC, lolD, and lolE conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of E. coli cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolCQ258K), which confers high-level resistance to G0507 in vivo but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.


Assuntos
Bactérias Gram-Negativas/metabolismo , Lipoproteínas/metabolismo , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Lipoproteínas/genética , Mutação/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética
9.
Proc Natl Acad Sci U S A ; 112(27): E3515-24, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26100885

RESUMO

Despite the enormous diversity among parasitic angiosperms in form and structure, life-history strategies, and plastid genomes, little is known about the diversity of their mitogenomes. We report the sequence of the wonderfully bizarre mitogenome of the hemiparasitic aerial mistletoe Viscum scurruloideum. This genome is only 66 kb in size, making it the smallest known angiosperm mitogenome by a factor of more than three and the smallest land plant mitogenome. Accompanying this size reduction is exceptional reduction of gene content. Much of this reduction arises from the unexpected loss of respiratory complex I (NADH dehydrogenase), universally present in all 300+ other angiosperms examined, where it is encoded by nine mitochondrial and many nuclear nad genes. Loss of complex I in a multicellular organism is unprecedented. We explore the potential relationship between this loss in Viscum and its parasitic lifestyle. Despite its small size, the Viscum mitogenome is unusually rich in recombinationally active repeats, possessing unparalleled levels of predicted sublimons resulting from recombination across short repeats. Many mitochondrial gene products exhibit extraordinary levels of divergence in Viscum, indicative of highly relaxed if not positive selection. In addition, all Viscum mitochondrial protein genes have experienced a dramatic acceleration in synonymous substitution rates, consistent with the hypothesis of genomic streamlining in response to a high mutation rate but completely opposite to the pattern seen for the high-rate but enormous mitogenomes of Silene. In sum, the Viscum mitogenome possesses a unique constellation of extremely unusual features, a subset of which may be related to its parasitic lifestyle.


Assuntos
DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/genética , Genoma Mitocondrial/genética , Proteínas de Plantas/genética , Viscum/genética , Sequência de Bases , DNA Mitocondrial/classificação , Genes Mitocondriais/genética , Variação Genética , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Filogenia , RNA de Plantas/genética , RNA Ribossômico/genética , Homologia de Sequência do Ácido Nucleico
10.
BMC Plant Biol ; 17(1): 49, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28222679

RESUMO

BACKGROUND: Aerobically respiring eukaryotes usually contain four respiratory-chain complexes (complexes I-IV) and an ATP synthase (complex V). In several lineages of aerobic microbial eukaryotes, complex I has been lost, with an alternative, nuclear-encoded NADH dehydrogenase shown in certain cases to bypass complex I and oxidize NADH without proton translocation. The first loss of complex I in any multicellular eukaryote was recently reported in two studies; one sequenced the complete mitogenome of the hemiparasitic aerial mistletoe, Viscum scurruloideum, and the other sequenced the V. album mitogenome. The V. scurruloideum study reported no significant additional loss of mitochondrial genes or genetic function, but the V. album study postulated that mitochondrial genes encoding all ribosomal RNAs and proteins of all respiratory complexes are either absent or pseudogenes, thus raising questions as to whether the mitogenome and oxidative respiration are functional in this plant. RESULTS: To determine whether these opposing conclusions about the two Viscum mitogenomes reflect a greater degree of reductive/degenerative evolution in V. album or instead result from interpretative and analytical differences, we reannotated and reanalyzed the V. album mitogenome and compared it with the V. scurruloideum mitogenome. We find that the two genomes share a complete complement of mitochondrial rRNA genes and a typical complement of genes encoding respiratory complexes II-V. Most Viscum mitochondrial protein genes exhibit very high levels of divergence yet are evolving under purifying, albeit relaxed selection. We discover two cases of horizontal gene transfer in V. album and show that the two Viscum mitogenomes differ by 8.6-fold in size (66 kb in V. scurruloideum; 565 kb in V. album). CONCLUSIONS: Viscum mitogenomes are extraordinary compared to other plant mitogenomes in terms of their wide size range, high rates of synonymous substitutions, degree of relaxed selection, and unprecedented loss of respiratory complex I. However, contrary to the initial conclusions regarding V. album, both Viscum mitogenomes possess conventional sets of rRNA and, excepting complex I, respiratory genes. Both plants should therefore be able to carry out aerobic respiration. Moreover, with respect to size, the V. scurruloideum mitogenome has experienced a greater level of reductive evolution.


Assuntos
Complexo I de Transporte de Elétrons/genética , Evolução Molecular , Transferência Genética Horizontal , Variação Genética , Genoma de Planta , Viscum/genética , DNA de Plantas , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Deleção de Genes , Genes de Plantas , Genoma Mitocondrial , Anotação de Sequência Molecular , Proteínas de Plantas/genética , RNA de Plantas , RNA Ribossômico , Análise de Sequência de DNA , Especificidade da Espécie , Viscum/metabolismo , Viscum album/genética , Viscum album/metabolismo
11.
Proc Natl Acad Sci U S A ; 111(15): 5694-9, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24706808

RESUMO

Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum ß-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000-2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL-resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Genoma Bacteriano/genética , Filogenia , Sequência de Bases , Biologia Computacional , Fluoroquinolonas , Funções Verossimilhança , Modelos Genéticos , Dados de Sequência Molecular , Filogeografia , Polimorfismo de Nucleotídeo Único/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , beta-Lactamases/metabolismo
12.
Infect Immun ; 84(3): 775-81, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26729762

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) is an important cause of diarrhea and hemolytic-uremic syndrome (HUS) worldwide. Australia's worst outbreak of HUS occurred in Adelaide in 1995 and was one of the first major HUS outbreaks attributed to a non-O157 Shiga-toxigenic E. coli (STEC) strain. Molecular analyses conducted at the time suggested that the outbreak was caused by an O111:H(-) clone, with strains from later in the outbreak harboring an extra copy of the genes encoding the potent Shiga toxin 2 (Stx2). Two decades later, we have used next-generation sequencing to compare two isolates from early and late in this important outbreak. We analyzed genetic content, single-nucleotide polymorphisms (SNPs), and prophage insertion sites; for the latter, we demonstrate how paired-end sequence data can be leveraged to identify such insertion sites. The two strains are genetically identical except for six SNP differences and the presence of not one but two additional Stx2-converting prophages in the later isolate. Isolates from later in the outbreak were associated with higher levels of morbidity, suggesting that the presence of the additional Stx2-converting prophages is significant in terms of the virulence of this clone.


Assuntos
Escherichia coli Êntero-Hemorrágica/genética , Infecções por Escherichia coli/microbiologia , Síndrome Hemolítico-Urêmica/microbiologia , Austrália/epidemiologia , Surtos de Doenças , Escherichia coli Êntero-Hemorrágica/classificação , Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Genômica , Humanos , Filogenia
13.
J Crohns Colitis ; 18(7): 985-1001, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-38267224

RESUMO

BACKGROUND AND AIMS: This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.


Assuntos
Toxinas Bacterianas , Clostridium perfringens , Fezes , Doenças Inflamatórias Intestinais , Mucosa Intestinal , Humanos , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/genética , Clostridium perfringens/patogenicidade , Criança , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Doenças Inflamatórias Intestinais/microbiologia , Toxinas Bacterianas/genética , Fezes/microbiologia , Feminino , Masculino , Adolescente , Biópsia , Infecções por Clostridium/microbiologia , Proteínas Hemolisinas
14.
mBio ; 15(10): e0089724, 2024 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-39207111

RESUMO

Guanine nucleotides are required for growth and viability of cells due to their structural role in DNA and RNA, and their regulatory roles in translation, signal transduction, and cell division. The natural antibiotic mycophenolic acid (MPA) targets the rate-limiting step in de novo guanine nucleotide biosynthesis executed by inosine-5´-monophosphate dehydrogenase (IMPDH). MPA is used clinically as an immunosuppressant, but whether in vivo inhibition of bacterial IMPDH (GuaB) is a valid antibacterial strategy is controversial. Here, we describe the discovery of extremely potent small molecule GuaB inhibitors (GuaBi) specific to pathogenic bacteria with a low frequency of on-target spontaneous resistance and bactericidal efficacy in vivo against Acinetobacter baumannii mouse models of infection. The spectrum of GuaBi activity includes multidrug-resistant pathogens that are a critical priority of new antibiotic development. Co-crystal structures of A. baumannii, Staphylococcus aureus, and Escherichia coli GuaB proteins bound to inhibitors show comparable binding modes of GuaBi across species and identifies key binding site residues that are predictive of whole-cell activity across both Gram-positive and Gram-negative clades of Bacteria. The clear in vivo efficacy of these small molecule GuaB inhibitors in a model of A. baumannii infection validates GuaB as an essential antibiotic target. IMPORTANCE: The emergence of multidrug-resistant bacteria worldwide has renewed interest in discovering antibiotics with novel mechanism of action. For the first time ever, we demonstrate that pharmacological inhibition of de novo guanine biosynthesis is bactericidal in a mouse model of Acinetobacter baumannii infection. Structural analyses of novel inhibitors explain differences in biochemical and whole-cell activity across bacterial clades and underscore why this discovery may have broad translational impact on treatment of the most recalcitrant bacterial infections.


Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , IMP Desidrogenase , Acinetobacter baumannii/efeitos dos fármacos , Animais , Camundongos , Antibacterianos/farmacologia , Antibacterianos/química , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/metabolismo , Modelos Animais de Doenças , Testes de Sensibilidade Microbiana , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Descoberta de Drogas , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Feminino , Farmacorresistência Bacteriana Múltipla
15.
Microbiome ; 11(1): 47, 2023 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-36894983

RESUMO

BACKGROUND: IL-22 is induced by aryl hydrocarbon receptor (AhR) signaling and plays a critical role in gastrointestinal barrier function through effects on antimicrobial protein production, mucus secretion, and epithelial cell differentiation and proliferation, giving it the potential to modulate the microbiome through these direct and indirect effects. Furthermore, the microbiome can in turn influence IL-22 production through the synthesis of L-tryptophan (L-Trp)-derived AhR ligands, creating the prospect of a host-microbiome feedback loop. We evaluated the impact IL-22 may have on the gut microbiome and its ability to activate host AhR signaling by observing changes in gut microbiome composition, function, and AhR ligand production following exogenous IL-22 treatment in both mice and humans. RESULTS: Microbiome alterations were observed across the gastrointestinal tract of IL-22-treated mice, accompanied by an increased microbial functional capacity for L-Trp metabolism. Bacterially derived indole derivatives were increased in stool from IL-22-treated mice and correlated with increased fecal AhR activity. In humans, reduced fecal concentrations of indole derivatives in ulcerative colitis (UC) patients compared to healthy volunteers were accompanied by a trend towards reduced fecal AhR activity. Following exogenous IL-22 treatment in UC patients, both fecal AhR activity and concentrations of indole derivatives increased over time compared to placebo-treated UC patients. CONCLUSIONS: Overall, our findings indicate IL-22 shapes gut microbiome composition and function, which leads to increased AhR signaling and suggests exogenous IL-22 modulation of the microbiome may have functional significance in a disease setting. Video Abstract.


Assuntos
Microbioma Gastrointestinal , Humanos , Animais , Camundongos , Receptores de Hidrocarboneto Arílico/metabolismo , Interleucinas , Indóis , Interleucina 22
16.
BMC Genomics ; 12: 532, 2011 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-22035052

RESUMO

BACKGROUND: Changes in transcriptional regulation underlie many of the phenotypic differences observed within and between species of bacteria. Lateral genetic transfer (LGT) can significantly impact the transcription factor (TF) genes which drive these transcriptional changes. Although much emphasis has been placed on LGT of intact genes, the units of transfer and recombination do not necessarily correspond to regions delineated by exact gene boundaries. Here we apply phylogenetic and network-based methods to investigate the relationship between units of lateral transfer and recombination within the Escherichia coli - Shigella clade and the topological properties of genes in the E. coli transcriptional regulatory network (TRN). RESULTS: We carried out a systematic phylogenetic study of genetic transfer among 5282 sets of putatively orthologous genes from 27 strains belonging to the E. coli - Shigella clade. We then used these results to examine the evolutionary histories of TF genes, as well as the transcriptional regulation of lateral genes. We found evidence of LGT in 2655 (50.3%) gene sets: 678 (12.8%) show evidence of recombination breakpoints within the gene boundaries. Thus, within- and whole- gene lateral transfer is widespread among strains of E. coli and Shigella. We found that unlike global regulators, which have mostly evolved vertically, neighbour regulators (genes which regulate adjacent genes on the chromosome) have frequently been subject to transfer within the E. coli - Shigella clade. At least 56 (62%) of the 90 neighbour regulator gene sets examined show evidence of LGT, 19 (34%) of which have internal recombination breakpoints. Neighbour regulators show no evidence of co-transfer with their nearby target genes. Rather, the frequency of recombination breakpoints, and conflicting evolutionary histories among neighbour regulators and their target genes, suggest that the genomic regions encoding these genes have been constructed through successive layering of LGT events within the clade. We find no difference in the relative complexity of regulation (i.e. the number of regulators) of lateral versus vertical genes. CONCLUSIONS: Neighbour regulators show higher frequencies of transfer than other types of regulatory genes. This implicates the topological properties of regulatory genes in the TRN, and their physical proximity to targets on the chromosome, as contributing to successful LGT. The prevalence of recombination breakpoints within regulatory and target gene sets indicates that within-gene transfer has had a significant cumulative effect on the evolution of regulatory interactions in E. coli and Shigella.


Assuntos
Evolução Biológica , Escherichia coli/genética , Transferência Genética Horizontal , Shigella/genética , Escherichia coli/classificação , Redes Reguladoras de Genes , Filogenia , Shigella/classificação , Fatores de Transcrição/genética
17.
Cell Host Microbe ; 29(10): 1521-1530.e10, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34492225

RESUMO

The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability.


Assuntos
Proteínas de Bactérias/metabolismo , Disenteria Bacilar/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Shigella flexneri/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Bactérias/genética , Disenteria Bacilar/genética , Disenteria Bacilar/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Proteólise , Shigella flexneri/genética , Shigella flexneri/fisiologia , Ubiquitina-Proteína Ligases/genética
18.
mBio ; 11(5)2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900806

RESUMO

Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use.IMPORTANCE Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.


Assuntos
Antibacterianos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Farmacorresistência Bacteriana/genética , Lipoproteínas/metabolismo , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/enzimologia , Animais , Ácido Aspártico Endopeptidases/genética , Proteínas de Bactérias/genética , Enterobacteriaceae/classificação , Enterobacteriaceae/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Peptídeos/farmacologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade
19.
Microb Cell ; 4(4): 108-111, 2017 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-28435837

RESUMO

Secretion of proteins into the extracellular environment is crucial for the normal physiology and virulence of pathogenic bacteria. Type I signal peptidase (SPase I) mediates the final step of bacterial secretion, by cleaving proteins at their signal peptide once they are translocated by the Sec or twin-arginine (Tat) translocon. SPase I has long been thought to be essential for viability in multiple bacterial pathogens. Challenging this view, we and others have recently created Staphylococcus aureus bacteria lacking the SPase I SpsB that are viable and able to grow in vitro when over-expressing a native gene cassette encoding for a putative ABC transporter. This transporter apparently compensates for SpsB's essential function by mediating alternative cleavage of a subset of proteins at a site distinct from the SpsB-cleavage site, leading to SpsB-independent secretion. This alternative secretion system also drives the main mechanism of resistance to an arylomycin-derived SpsB inhibitor, by means of mutations in a putative transcriptional repressor (cro/cI) causing over-expression of the ABC transporter. These findings raise multiple interesting biological questions. Unraveling the mechanism of SpsB-independent secretion may provide an interesting twist to the paradigm of bacterial secretion.

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