RESUMEN
Tuberculosis remains a significant global health pandemic. There is an urgent need for new anti-tubercular agents to combat the rising incidence of drug resistance and to offer effective and additive therapeutic options. High-throughput screening of a subset of the NatureBank marine fraction library (n = 2000) identified a sample derived from an Australian marine sponge belonging to the order Haplosclerida that displayed promising anti-mycobacterial activity. Bioassay-guided fractionation of the organic extract from this Haplosclerida sponge led to the purification of previously identified antimicrobial pyrrole alkaloids, axinellamines A (1) and B (2). The axinellamine compounds were found to have a 90% minimum inhibitory concentration (MIC90) of 18 µM and 15 µM, respectively. The removal of protein and complex carbon sources reduced the MIC90 of 1 and 2 to 0.6 and 0.8 µM, respectively. The axinellamines were not toxic to mammalian cells at 25 µM and significantly reduced the intracellular bacterial load by >5-fold. These data demonstrate that axinellamines A and B are effective anti-tubercular agents and promising targets for future medicinal chemistry efforts.
Asunto(s)
Antituberculosos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis , Poríferos , Antituberculosos/farmacología , Antituberculosos/química , Antituberculosos/aislamiento & purificación , Animales , Mycobacterium tuberculosis/efectos de los fármacos , Humanos , Alcaloides/farmacología , Alcaloides/química , Alcaloides/aislamiento & purificación , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Pirroles/farmacología , Pirroles/química , Pirroles/aislamiento & purificaciónRESUMEN
The ability of Mycobacterium tuberculosis (Mtb) to persist in the host complicates and prolongs tuberculosis (TB) patient chemotherapy. Here we demonstrate that a neglected two-component system (TCS) of Mtb, TcrXY, is an autoregulated acid-sensing TCS that controls a functionally diverse 70-gene regulon required for bacterial persistence. Characterisation of two representatives of this regulon, Rv3706c and Rv3705A, implicate these genes as key determinants for the survival of Mtb in vivo by serving as important effectors to mitigate redox stress at acidic pH. We show that genetic silencing of the response regulator tcrX using CRISPR interference attenuates the persistence of Mtb during chronic mouse infection and improves treatment with the two front-line anti-TB drugs, rifampicin and isoniazid. We propose that targeting TcrXY signal transduction blocks the ability of Mtb to sense and respond to acid stress, resulting in a disordered program of persistence to render the organism vulnerable to existing TB chemotherapy.
Asunto(s)
Genes Bacterianos , Mycobacterium tuberculosis , Animales , Humanos , Ratones , Antituberculosos/química , Genes Bacterianos/fisiología , Isoniazida , Mycobacterium tuberculosis/genética , Infección Persistente , RifampinRESUMEN
Oxidised derivatives of cholesterol have been shown to inhibit the growth of Mycobacterium tuberculosis (Mtb). The bacteriostatic activity of these compounds has been attributed to their inhibition of CYP125A1 and CYP142A1, two metabolically critical cytochromes P450 that initiate degradation of the sterol side chain. Here, we synthesise and characterise an extensive library of 28 cholesterol derivatives to develop a structure-activity relationship for this class of inhibitors. The candidate compounds were evaluated for MIC with virulent Mtb and in binding studies with CYP125A1 and CYP142A1 from Mtb.
Asunto(s)
Mycobacterium tuberculosis , Sistema Enzimático del Citocromo P-450/metabolismo , Colesterol/metabolismo , Relación Estructura-ActividadRESUMEN
Simple alkyl-sulfonylacetamides have potent antitubercular activity and significantly decrease mycolic acid levels in mycobacteria. Although these compounds were originally designed to inhibit the ketoacyl synthase domain of fatty acid synthase, structure-activity relationships and biochemical evidence do not fully support fatty acid synthase as the target. In 2004, an enzyme family involved in the activation and transfer of fatty acids as acyl-adenylates was identified in mycobacteria, separate from the universal acetyl-CoA carrier mechanism. These fatty acyl-AMP ligases (FAAL), encoded by the FadD family play important roles in the biosynthesis of mycolic acids along with fatty acid metabolism and are hypothesised here to be the molecular target of the sulfonylacetamides. Due to structural similarities with the ligase's natural substrate, it is believed these compounds are exerting action via competitive inhibition of these highly potent molecular targets. The primary aim of this investigation was to synthesize an extended library of sulfonylacetamide derivatives, building upon existing structural activity relations to validate the molecular mechanism with the aid of molecular modelling, while also attempting to explore novel structural isosteres for further drug design and development. Sulfonylacetamide derivatives were modified based on the putative molecular target resulting in derivatives with improved activities towards Mycobacteriumtuberculosis (H37Rv). The most active novel derivatives reported were 19, 22b, 22c and 46 displaying MIC90 levels of 1.4, 16.0, 13.0 and 5.9 µg/mL, respectively.
Asunto(s)
Mycobacterium tuberculosis , Acetamidas/farmacología , Antituberculosos/farmacología , Ácidos Micólicos/metabolismo , Ácidos Grasos/metabolismo , Ácido Graso SintasasRESUMEN
The unusual and sterically constrained amino acid, seco-1-azacubane-2-carboxylic acid, was incorporated into a range of bioactive chemical templates, including enalaprilat, perindoprilat, endomorphin-2 and isoniazid, and subjected to biological testing. The endomorphin-2 derivative displayed increased activity at the δ opioid receptor, but a loss in activity was observed in the other cases, although human normal cell line evaluation suggests limited cytotoxic effects.
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Ácidos Carboxílicos , Receptores Opioides mu , Humanos , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Aminoácidos , Línea CelularRESUMEN
To date, the clinical use of the anti-tubercular therapy bedaquiline has been somewhat limited due to safety concerns. Recent investigations determined that modification of the B- and C-ring units of bedaquiline delivered new diarylquinolines (for example TBAJ-587) with potent anti-tubercular activity yet an improved safety profile due to reduced affinity for the hERG channel. Building on our recent discovery that substitution of the quinoline motif (the A-ring subunit) for C5-aryl pyridine groups within bedaquiline analogues led to retention of anti-tubercular activity, we investigated the concurrent modification of A-, B- and C-ring units within bedaquiline variants. This led to the discovery that 4-trifluoromethoxyphenyl and 4-chlorophenyl pyridyl analogues of TBAJ-587 retained relatively potent anti-tubercular activity and for the 4-chlorophenyl derivative in particular, a significant reduction in hERG inhibition relative to bedaquiline was achieved, demonstrating that modifications of the A-, B- and C-ring units within the bedaquiline structure is a viable strategy for the design of effective, yet safer (and less lipophilic) anti-tubercular compounds.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Diarilquinolinas/farmacología , Diarilquinolinas/química , Antituberculosos/farmacología , Antituberculosos/químicaRESUMEN
Tuberculosis and parasitic infections continue to impose a significant threat to global public health and economic growth. There is an urgent need to develop new treatments to combat these diseases. Here, we report the in vitro and in vivo profiles of a new bicyclic nitroimidazole subclass, namely, nitroimidazopyrazinones, against mycobacteria and Trypanosoma cruzi. Derivatives with monocyclic side chains were selective against Mycobacterium tuberculosis and were able to reduce the bacterial load when dosed orally in mice. We demonstrated that deazaflavin-dependent nitroreductase (Ddn) could act effectively on nitroimidazopyrazinones, indicating the potential of Ddn as an activating enzyme for these new compounds in M. tuberculosis. Oral administration of compounds with extended biaryl side chains (73 and 74) was effective in suppressing infection in an acute T. cruzi-infected murine model. These findings demonstrate that active nitroimidazopyrazinones have potential to be developed as orally available clinical candidates against both tuberculosis and Chagas disease.
Asunto(s)
Enfermedad de Chagas , Mycobacterium tuberculosis , Nitroimidazoles , Trypanosoma cruzi , Tuberculosis , Animales , Enfermedad de Chagas/tratamiento farmacológico , Modelos Animales de Enfermedad , Ratones , Nitroimidazoles/farmacología , Nitroimidazoles/uso terapéutico , Nitrorreductasas , Tuberculosis/tratamiento farmacológicoRESUMEN
Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli Patógena Extraintestinal , Adhesinas Bacterianas/metabolismo , Adhesinas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones por Escherichia coli/metabolismo , Escherichia coli Patógena Extraintestinal/genética , Escherichia coli Patógena Extraintestinal/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Humanos , Enfermedades Intestinales , Polisacáridos/metabolismoRESUMEN
Intracellular bacterial pathogens such as Mycobacterium tuberculosis are remarkably adept at surviving within a host, employing a variety of mechanisms to counteract host defenses and establish a protected niche. Constant surveying of the environment is key for pathogenic mycobacteria to discern their immediate location and coordinate the expression of genes necessary for adaptation. Two-component systems efficiently perform this role, typically comprised of a transmembrane sensor kinase and a cytoplasmic response regulator. In this review, we describe the role of two-component systems in bacterial pathogenesis, focusing predominantly on the role of sensor kinases of M. tuberculosis. We highlight important features of sensor kinases in mycobacterial infection, discuss ways in which these signaling proteins sense and respond to environments, and how this is attuned to their intracellular lifestyle. Finally, we discuss recent studies which have identified and characterized inhibitors of two-component sensor kinases toward establishing a new strategy in anti-mycobacterial therapy.
Asunto(s)
Infecciones por Mycobacterium , Mycobacterium tuberculosis , Adaptación Fisiológica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Histidina/metabolismo , Histidina Quinasa/genética , Histidina Quinasa/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismoRESUMEN
Pretomanid and MCC7433, a novel nitroimidazopyrazinone analog, are promising antitubercular agents that belong to the bicyclic nitroimidazole family. Despite possessing high cell permeability, they suffer from poor aqueous solubility and require specialized formulations in order to be orally bioavailable. To address this limitation, we investigated the use of mesoporous silica nanoparticles (MCM-41) as drug carriers. MCM-41 nanoparticles were synthesized using a sol-gel method, and their surface was further modified with amine and phosphonate groups. A simple rotary evaporation method was used to incorporate the compounds of interest into the nanoparticles, leading to a high encapsulation efficiency of ≥86% with â¼10% loading (w/w). An overall significant improvement of solubility was also observed, and the pharmacological activity of pretomanid and MCC7433 was fully retained when tested in vitro against Mycobacterium tuberculosis using these nanocarriers. Amino-functionalized MCM-41 nanoparticles were found to enhance the systemic exposure of MCC7433 in mice (1.3-fold higher Cmax) compared to MCC7433 alone. The current work highlights the potential of using nanoparticles such as mesoporous silica as a carrier for oral delivery of poorly soluble antibacterial agents against tuberculosis.
Asunto(s)
Nanopartículas , Nitroimidazoles , Organofosfonatos , Aminas , Animales , Antituberculosos/farmacología , Portadores de Fármacos , Ratones , Nitroimidazoles/farmacología , Dióxido de SilicioRESUMEN
Despite promising efficacy, the clinical use of the anti-tubercular therapeutic bedaquiline has been restricted due to safety concerns. To date, limited SAR studies have focused on the quinoline ring (A-ring), and as such, we set out to explore modifications within this region in an attempt to discover new bedaquiline variants with an improved safety profile. We herein report the development of unique synthetic strategies that facilitated access to novel bedaquiline analogues leading to the discovery that anti-tubercular activity could be retained following replacement of the quinoline motif with pyridine heterocycles. This discovery is anticipated to open up multiple new avenues for exploration in the design of improved anti-tubercular therapeutics.
RESUMEN
New drugs to treat tuberculosis (TB) are urgently needed to combat the increase in resistance observed among the current first-line and second-line treatments. Here, we propose ketol-acid reductoisomerase (KARI) as a target for anti-TB drug discovery. Twenty-two analogues of IpOHA, an inhibitor of plant KARI, were evaluated as antimycobacterial agents. The strongest inhibitor of Mycobacterium tuberculosis (Mt) KARI has a Ki value of 19.7 nM, fivefold more potent than IpOHA (Ki = 97.7 nM). This and four other potent analogues are slow- and tight-binding inhibitors of MtKARI. Three compounds were cocrystallized with Staphylococcus aureus KARI and yielded crystals that diffracted to 1.6-2.0 Å resolution. Prodrugs of these compounds possess antimycobacterial activity against H37Rv, a virulent strain of human TB, with the most active compound having an MIC90 of 2.32 ± 0.04 µM. This compound demonstrates a very favorable selectivity window and represents a highly promising lead as an anti-TB agent.
Asunto(s)
Antituberculosos/farmacología , Herbicidas/farmacología , Cetoácido Reductoisomerasa/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Animales , Antituberculosos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Simulación del Acoplamiento Molecular , Profármacos , Staphylococcus aureus/enzimologíaRESUMEN
Antimicrobial resistance threatens the viability of modern medicine, which is largely dependent on the successful prevention and treatment of bacterial infections. Unfortunately, there are few new therapeutics in the clinical pipeline, particularly for Gram-negative bacteria. We now present a detailed evaluation of the antimicrobial activity of cannabidiol, the main non-psychoactive component of cannabis. We confirm previous reports of Gram-positive activity and expand the breadth of pathogens tested, including highly resistant Staphylococcus aureus, Streptococcus pneumoniae, and Clostridioides difficile. Our results demonstrate that cannabidiol has excellent activity against biofilms, little propensity to induce resistance, and topical in vivo efficacy. Multiple mode-of-action studies point to membrane disruption as cannabidiol's primary mechanism. More importantly, we now report for the first time that cannabidiol can selectively kill a subset of Gram-negative bacteria that includes the 'urgent threat' pathogen Neisseria gonorrhoeae. Structure-activity relationship studies demonstrate the potential to advance cannabidiol analogs as a much-needed new class of antibiotics.
Asunto(s)
Antibacterianos/farmacología , Cannabidiol/análogos & derivados , Cannabidiol/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Animales , Antibacterianos/química , Cannabidiol/química , Cannabidiol/toxicidad , Clostridioides difficile/efectos de los fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Femenino , Células HEK293 , Hemólisis/efectos de los fármacos , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones Endogámicos , Pruebas de Sensibilidad Microbiana , Neisseria gonorrhoeae/efectos de los fármacos , Enfermedades Cutáneas Bacterianas/tratamiento farmacológico , Enfermedades Cutáneas Bacterianas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Relación Estructura-ActividadRESUMEN
Following the approval of delamanid and pretomanid as new drugs to treat drug-resistant tuberculosis, there is now a renewed interest in bicyclic nitroimidazole scaffolds as a source of therapeutics against infectious diseases. We recently described a nitroimidazopyrazinone bicyclic subclass with promising antitubercular and antiparasitic activity, prompting additional efforts to generate analogs with improved solubility and enhanced potency. The key pendant aryl substituent was modified by (i) introducing polar functionality to the methylene linker, (ii) replacing the terminal phenyl group with less lipophilic heterocycles, or (iii) generating extended biaryl side chains. Improved antitubercular and antitrypanosomal activity was observed with the biaryl side chains, with most analogs achieved 2- to 175-fold higher activity than the monoaryl parent compounds, with encouraging improvements in solubility when pyridyl groups were incorporated. This study has contributed to understanding the existing structure-activity relationship (SAR) of the nitroimidazopyrazinone scaffold against a panel of disease-causing organisms to support future lead optimization.
Asunto(s)
Antituberculosos/química , Nitroimidazoles/química , Pirazinas/química , Animales , Antiprotozoarios/química , Antiprotozoarios/farmacología , Antituberculosos/metabolismo , Antituberculosos/farmacología , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Semivida , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Microsomas/metabolismo , Mycobacterium/efectos de los fármacos , Unión Proteica , Pirazinas/metabolismo , Pirazinas/farmacología , Solubilidad , Relación Estructura-Actividad , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
Bacterial nutrition is an essential aspect of host-pathogen interaction. For the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, fatty acids derived from lipid droplets are considered the major carbon source. However, many other soluble nutrients are available inside host cells and may be used as alternative carbon sources. Lactate and pyruvate are abundant in human cells and fluids, particularly during inflammation. In this work, we study Mtb metabolism of lactate and pyruvate combining classic microbial physiology with a 'multi-omics' approach consisting of transposon-directed insertion site sequencing (TraDIS), RNA-seq transcriptomics, proteomics and stable isotopic labelling coupled with mass spectrometry-based metabolomics. We discovered that Mtb is well adapted to use both lactate and pyruvate and that their metabolism requires gluconeogenesis, valine metabolism, the Krebs cycle, the GABA shunt, the glyoxylate shunt and the methylcitrate cycle. The last two pathways are traditionally associated with fatty acid metabolism and, unexpectedly, we found that in Mtb the methylcitrate cycle operates in reverse, to allow optimal metabolism of lactate and pyruvate. Our findings reveal a novel function for the methylcitrate cycle as a direct route for the biosynthesis of propionyl-CoA, the essential precursor for the biosynthesis of the odd-chain fatty acids.
Asunto(s)
Ácido Láctico/metabolismo , Mycobacterium tuberculosis/metabolismo , Ácido Pirúvico/metabolismo , Acilcoenzima A/metabolismo , Proteínas Bacterianas/metabolismo , Citrato (si)-Sintasa/metabolismo , Citratos/metabolismo , Ácidos Grasos/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Glioxilatos , Tuberculosis/microbiologíaRESUMEN
Autotransporters are the largest family of outer membrane and secreted proteins in Gram-negative bacteria. Most autotransporters are localised to the bacterial surface where they promote colonisation of host epithelial surfaces. Here we present the crystal structure of UpaB, an autotransporter that is known to contribute to uropathogenic E. coli (UPEC) colonisation of the urinary tract. We provide evidence that UpaB can interact with glycosaminoglycans and host fibronectin. Unique modifications to its core ß-helical structure create a groove on one side of the protein for interaction with glycosaminoglycans, while the opposite face can bind fibronectin. Our findings reveal far greater diversity in the autotransporter ß-helix than previously thought, and suggest that this domain can interact with host macromolecules. The relevance of these interactions during infection remains unclear.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glicosaminoglicanos/metabolismo , Escherichia coli Uropatógena/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Factores de Virulencia/química , Factores de Virulencia/metabolismoRESUMEN
Tuberculosis and parasitic diseases, such as giardiasis, amebiasis, leishmaniasis, and trypanosomiasis, all urgently require improved treatment options. Recently, it has been shown that antitubercular bicyclic nitroimidazoles such as pretomanid and delamanid have potential as repurposed therapeutics for the treatment of visceral leishmaniasis. Here, we show that pretomanid also possesses potent activity against Giardia lamblia and Entamoeba histolytica, thus expanding the therapeutic potential of nitroimidazooxazines. Synthetic analogues with a novel nitroimidazopyrazin-one/-e bicyclic nitroimidazole chemotype were designed and synthesized, and structure-activity relationships were generated. Selected derivatives had potent antiparasitic and antitubercular activity while maintaining drug-like properties such as low cytotoxicity, good metabolic stability in liver microsomes and high apparent permeability across Caco-2 cells. The kinetic solubility of the new bicyclic derivatives varied and was found to be a key parameter for future optimization. Taken together, these results suggest that promising subclasses of bicyclic nitroimidazoles containing different core architectures have potential for further development.
Asunto(s)
Antiparasitarios/química , Antiparasitarios/farmacología , Antituberculosos/química , Antituberculosos/farmacología , Animales , Antiparasitarios/síntesis química , Antituberculosos/síntesis química , Células CACO-2 , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Estabilidad de Medicamentos , Entamoeba histolytica/efectos de los fármacos , Giardia lamblia/efectos de los fármacos , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Microsomas Hepáticos/efectos de los fármacos , Nitroimidazoles/farmacología , Relación Estructura-ActividadRESUMEN
Homologous alignment cloning (HAC) is a rapid method of molecular cloning that facilitates low-cost, highly efficient cloning of polymerase chain reaction products into any plasmid vector in approximately 2 min. HAC facilitates insert integration due to a sequence alignment strategy, by way of short, vector-specific homology tails appended to insert during amplification. Simultaneous exposure of single-stranded fragment ends, utilising the 3'â5' exonuclease activity of T4 DNA polymerase, creates overlapping homologous DNA on each molecule. The exonuclease activity of T4 polymerase is quenched simply by the addition of EDTA and a simple annealing step ensures high yield and high fidelity vector formation. The resultant recombinant plasmids are transformed into standard E. coli cloning strains and screened via established methods as necessary. HAC exploits reagents commonly found in molecular research laboratories and achieves efficiencies that exceed conventional cloning methods, including another ligation-independent method we tested. HAC is also suitable for combining multiple fragments in a single reaction, thus extending its flexibility.
RESUMEN
Escherichia coli is a versatile pathogen capable of causing intestinal and extraintestinal infections that result in a huge burden of global human disease. The diversity of E. coli is reflected by its multiple different pathotypes and mosaic genome composition. E. coli strains are also a major driver of antibiotic resistance, emphasizing the urgent need for new treatment and prevention measures. Here, we used a large data set comprising 1,700 draft and complete genomes to define the core and accessory genome of E. coli and demonstrated the overlapping relationship between strains from different pathotypes. In combination with proteomic investigation, this analysis revealed core genes that encode surface-exposed or secreted proteins that represent potential broad-coverage vaccine antigens. One of these antigens, YncE, was characterized as a conserved immunogenic antigen able to protect against acute systemic infection in mice after vaccination. Overall, this work provides a genomic blueprint for future analyses of conserved and accessory E. coli genes. The work also identified YncE as a novel antigen that could be exploited in the development of a vaccine against all pathogenic E. coli strains-an important direction given the high global incidence of infections caused by multidrug-resistant strains for which there are few effective antibiotics. IMPORTANCEE. coli is a multifaceted pathogen of major significance to global human health and an important contributor to increasing antibiotic resistance. Given the paucity of therapies still effective against multidrug-resistant pathogenic E. coli strains, novel treatment and prevention strategies are urgently required. In this study, we defined the core and accessory components of the E. coli genome by examining a large collection of draft and completely sequenced strains available from public databases. This data set was mined by employing a reverse-vaccinology approach in combination with proteomics to identify putative broadly protective vaccine antigens. One such antigen was identified that was highly immunogenic and induced protection in a mouse model of bacteremia. Overall, our study provides a genomic and proteomic framework for the selection of novel vaccine antigens that could mediate broad protection against pathogenic E. coli.
RESUMEN
Extra-intestinal pathogenic Escherichia coli (ExPEC) are responsible for diverse infections including meningitis, sepsis and urinary tract infections. The alarming rise in anti-microbial resistance amongst ExPEC complicates treatment and has highlighted the need for alternative preventive measures. SslE is a lipoprotein secreted by a dedicated type II secretion system in E. coli that was first identified as a potential vaccine candidate using reverse genetics. Although the function and protective efficacy of SslE has been studied, the molecular mechanisms that regulate SslE expression remain to be fully elucidated. Here, we show that while the expression of SslE can be detected in E. coli culture supernatants, different strains express and secrete different amounts of SslE when grown under the same conditions. While the histone-like transcriptional regulator H-NS strongly represses sslE at ambient temperatures, the variation in SslE expression at human physiological temperature suggested a more complex mode of regulation. Using a genetic screen to identify novel regulators of sslE in the high SslE-expressing strain UTI89, we defined a new role for the nucleoid-associated regulator Fis and the ribosome-binding GTPase TypA as positive regulators of sslE transcription. We also showed that Fis-mediated enhancement of sslE transcription is dependent on a putative Fis-binding sequence located upstream of the -35 sequence in the core promoter element, and provide evidence to suggest that Fis may work in complex with H-NS to control SslE expression. Overall, this study has defined a new mechanism for sslE regulation and increases our understanding of this broadly conserved E. coli vaccine antigen.