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1.
mBio ; 12(1)2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33531394

RESUMEN

Zinc is an essential element in all domains of life. Nonetheless, how prokaryotes achieve selective acquisition of zinc from the extracellular environment remains poorly understood. Here, we elucidate a novel mechanism for zinc-binding in AdcA, a solute-binding protein of Streptococcus pneumoniae Crystal structure analyses reveal the two-domain organization of the protein and show that only the N-terminal domain (AdcAN) is necessary for zinc import. Zinc binding induces only minor changes in the global protein conformation of AdcA and stabilizes a highly mobile loop within the AdcAN domain. This loop region, which is conserved in zinc-specific solute-binding proteins, facilitates closure of the AdcAN binding site and is crucial for zinc acquisition. Collectively, these findings elucidate the structural and functional basis of selective zinc uptake in prokaryotes.IMPORTANCE Zinc is an essential nutrient for the virulence of bacterial pathogens such as Streptococcus pneumoniae Many Gram-positive bacteria use a two-domain lipoprotein for zinc acquisition, but how this class of metal-recruiting proteins acquire zinc and interact with the uptake machinery has remained poorly defined. We report the first structure of a two-domain lipoprotein, AdcA from S. pneumoniae, and use computational, spectroscopic, and microbiological approaches to provide new insights into the functional basis of zinc recruitment. Our findings reveal that AdcA employs a novel mechanism for zinc binding that we have termed the "trap-door" mechanism, and we show how the static metal-binding site of the protein, which confers its selectivity for zinc ions, is combined with a dynamic surface element to facilitate zinc recruitment and import into the bacterium. Together, these findings expand our understanding of how bacteria acquire zinc from the environment and provide a foundation for inhibiting this process, through antimicrobial targeting of the dynamic structural elements to block bacterial zinc scavenging.

2.
Int J Food Microbiol ; 340: 109042, 2021 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-33461002

RESUMEN

The development of antimicrobial resistance in foodborne pathogens is a growing public health concern. This study was undertaken to determine the antimicrobial susceptibility of Salmonella enterica subspecies enterica isolated from the Australian commercial egg layer industry. S. enterica subspecies enterica (n=307) isolated from Australian commercial layer flock environments (2015-2018) were obtained from reference, research and State Government laboratories from six Australian states. All Salmonella isolates were serotyped. Antimicrobial susceptibility testing (AST) for 16 antimicrobial agents was performed by broth microdilution. Antimicrobial resistance genes and sequence types (STs) were identified in significant isolates by whole genome sequencing (WGS). Three main serotypes were detected, S. Typhimurium (n=61, 19.9%), S. Senftenburg (n=45, 14.7%) and S. Agona (n=37, 12.1%). AST showed 293/307 (95.4%) isolates were susceptible to all tested antimicrobial agents and all isolates were susceptible to amoxicillin-clavulanate, azithromycin, ceftiofur, ceftriaxone, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin and trimethoprim-sulfamethoxazole. Low levels of non-susceptibility were observed to streptomycin (2.3%, n=7), sulfisoxazole (2.0%, n=6), chloramphenicol (1.3%, n=4) and tetracycline (1.0%, n=3). Very low levels of non-susceptibility were observed to ampicillin (2/307; 0.7%) and cefoxitin (2/307; 0.7%). Two isolates (S. Havana and S. Montevideo), exhibited multidrug-resistant phenotypes to streptomycin, sulfisoxazole and tetracycline and possessed corresponding antimicrobial resistance genes (aadA4, aac(6')-Iaa, sul1, tetB). One S. Typhimurium isolate was resistant to ampicillin and tetracycline, and possessed both tetA and blaTEM-1B. WGS also identified these isolates as belonging to ST4 (S. Montevideo), ST578 (S. Havana) and ST19 (S. Typhimurium). The absence of resistance to highest priority critically important antimicrobials as well as the extremely low level of AMR generally among Australian commercial egg layer Salmonella isolates likely reflect Australia's conservative antimicrobial registration policy in food-producing animals and low rates of antimicrobial use within the industry.

3.
Commun Biol ; 3(1): 694, 2020 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-33214631

RESUMEN

Metal ion homeostasis is essential for all forms of life. However, the breadth of intracellular impacts that arise upon dysregulation of metal ion homeostasis remain to be elucidated. Here, we used cadmium, a non-physiological metal ion, to investigate how the bacterial pathogen, Streptococcus pneumoniae, resists metal ion stress and dyshomeostasis. By combining transcriptomics, metabolomics and metalloproteomics, we reveal that cadmium stress dysregulates numerous essential cellular pathways including central carbon metabolism, lipid membrane biogenesis and homeostasis, and capsule production at the transcriptional and/or functional level. Despite the breadth of cellular pathways susceptible to metal intoxication, we show that S. pneumoniae is able to maintain viability by utilizing cellular pathways that are predominately metal-independent, such as the pentose phosphate pathway to maintain energy production. Collectively, this work provides insight into the cellular processes impacted by cadmium and how resistance to metal ion toxicity is achieved in S. pneumoniae.

4.
Sci Transl Med ; 12(570)2020 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-33208501

RESUMEN

The emergence of polymyxin resistance in carbapenem-resistant and extended-spectrum ß-lactamase (ESBL)-producing bacteria is a critical threat to human health, and alternative treatment strategies are urgently required. We investigated the ability of the hydroxyquinoline analog ionophore PBT2 to restore antibiotic sensitivity in polymyxin-resistant, ESBL-producing, carbapenem-resistant Gram-negative human pathogens. PBT2 resensitized Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa to last-resort polymyxin class antibiotics, including the less toxic next-generation polymyxin derivative FADDI-287, in vitro. We were unable to select for mutants resistant to PBT2 + FADDI-287 in polymyxin-resistant E. coli containing a plasmid-borne mcr-1 gene or K. pneumoniae carrying a chromosomal mgrB mutation. Using a highly invasive K. pneumoniae strain engineered for polymyxin resistance through mgrB mutation, we successfully demonstrated the efficacy of PBT2 + polymyxin (colistin or FADDI-287) for the treatment of Gram-negative sepsis in immunocompetent mice. In comparison to polymyxin alone, the combination of PBT2 + polymyxin improved survival and reduced bacterial dissemination to the lungs and spleen of infected mice. These data present a treatment modality to break antibiotic resistance in high-priority polymyxin-resistant Gram-negative pathogens.

5.
Metallomics ; 12(11): 1791-1801, 2020 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-33078811

RESUMEN

Nitric oxide (NO˙) is a radical molecule produced by mammalian phagocytic cells as part of the innate immune response to bacterial pathogens. It exerts its antimicrobial activity in part by impairing the function of metalloproteins, particularly those containing iron and zinc cofactors. The pathogenic Gram-negative bacterium Salmonella enterica serovar typhimurium undergoes dynamic changes in its cellular content of the four most common metal cofactors following exposure to NO˙ stress. Zinc, iron and magnesium all decrease in response to NO˙ while cellular manganese increases significantly. Manganese acquisition is driven primarily by increased expression of the mntH and sitABCD transporters following derepression of MntR and Fur. ZupT also contributes to manganese acquisition in response to nitrosative stress. S. Typhimurium mutants lacking manganese importers are more sensitive to NO˙, indicating that manganese is important for resistance to nitrosative stress.

6.
J Inorg Biochem ; 208: 111087, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32505855

RESUMEN

Zinc is a potent antimicrobial component of the innate immune response at the host-pathogen interface. Bacteria subvert or resist host zinc insults by metal efflux pathways that include cation diffusion facilitator (CDF) proteins. The structural and functional examination of this protein class has been limited, with only the structures of the zinc transporter YiiP proteins from E. coli and Shewanella oneidensis described to date. Here, we determine the metal binding properties, solution quaternary structures and three dimensional architectures of the C-terminal domains of the metal transporter CzcD proteins from Cupriavidus metallidurans, Pseudomonas aeruginosa and Thermotoga maritima. We reveal significant diversity in the metal-binding properties and structures of these proteins and discover a potential novel mechanism for metal-promoted dimerization for the Cupriavidus metallidurans and Pseudomonas aeruginosa proteins.

7.
mSystems ; 5(3)2020 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-32398278

RESUMEN

Experimental evolution is a powerful technique to understand how populations evolve from selective pressures imparted by the surrounding environment. With the advancement of whole-population genomic sequencing, it is possible to identify and track multiple contending genotypes associated with adaptations to specific selective pressures. This approach has been used repeatedly with model species in vitro, but only rarely in vivo Herein we report results of replicate experimentally evolved populations of Streptococcus pneumoniae propagated by repeated murine nasal colonization with the aim of identifying gene products under strong selection as well as the population genetic dynamics of infection cycles. Frameshift mutations in one gene, dltB, responsible for incorporation of d-alanine into teichoic acids on the bacterial surface, evolved repeatedly and swept to high frequency. Targeted deletions of dltB produced a fitness advantage during initial nasal colonization coupled with a corresponding fitness disadvantage in the lungs during pulmonary infection. The underlying mechanism behind the fitness trade-off between these two niches was found to be enhanced adherence to respiratory cells balanced by increased sensitivity to host-derived antimicrobial peptides, a finding recapitulated in the murine model. Additional mutations that are predicted to affect trace metal transport, central metabolism, and regulation of biofilm production and competence were also selected. These data indicate that experimental evolution can be applied to murine models of pathogenesis to gain insight into organism-specific tissue tropisms.IMPORTANCE Evolution is a powerful force that can be experimentally harnessed to gain insight into how populations evolve in response to selective pressures. Herein we tested the applicability of experimental evolutionary approaches to gain insight into how the major human pathogen Streptococcus pneumoniae responds to repeated colonization events using a murine model. These studies revealed the population dynamics of repeated colonization events and demonstrated that in vivo experimental evolution resulted in highly reproducible trajectories that reflect the environmental niche encountered during nasal colonization. Mutations impacting the surface charge of the bacteria were repeatedly selected during colonization and provided a fitness benefit in this niche that was counterbalanced by a corresponding fitness defect during lung infection. These data indicate that experimental evolution can be applied to models of pathogenesis to gain insight into organism-specific tissue tropisms.

8.
mSphere ; 5(2)2020 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-32188750

RESUMEN

Globally, more antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance (AMR). The development of novel ionophores, a class of antimicrobials used exclusively in animals, holds promise as a strategy to replace or reduce essential human antimicrobials in veterinary practice. PBT2 is a zinc ionophore with recently demonstrated antibacterial activity against several Gram-positive pathogens, although the underlying mechanism of action is unknown. Here, we investigated the bactericidal mechanism of PBT2 in the bovine mastitis-causing pathogen, Streptococcus uberis In this work, we show that PBT2 functions as a Zn2+/H+ ionophore, exchanging extracellular zinc for intracellular protons in an electroneutral process that leads to cellular zinc accumulation. Zinc accumulation occurs concomitantly with manganese depletion and the production of reactive oxygen species (ROS). PBT2 inhibits the activity of the manganese-dependent superoxide dismutase, SodA, thereby impairing oxidative stress protection. We propose that PBT2-mediated intracellular zinc toxicity in S. uberis leads to lethality through multiple bactericidal mechanisms: the production of toxic ROS and the impairment of manganese-dependent antioxidant functions. Collectively, these data show that PBT2 represents a new class of antibacterial ionophores capable of targeting bacterial metal ion homeostasis and cellular redox balance. We propose that this novel and multitarget mechanism of PBT2 makes the development of cross-resistance to medically important antimicrobials unlikely.IMPORTANCE More antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance. Therefore, the elimination of antimicrobial crossover between human and veterinary medicine is of great interest. Unfortunately, the development of new antimicrobials is an expensive high-risk process fraught with difficulties. The repurposing of chemical agents provides a solution to this problem, and while many have not been originally developed as antimicrobials, they have been proven safe in clinical trials. PBT2, a zinc ionophore, is an experimental therapeutic that met safety criteria but failed efficacy checkpoints against both Alzheimer's and Huntington's diseases. It was recently found that PBT2 possessed potent antimicrobial activity, although the mechanism of bacterial cell death is unresolved. In this body of work, we show that PBT2 has multiple mechanisms of antimicrobial action, making the development of PBT2 resistance unlikely.

9.
Infect Immun ; 88(6)2020 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-32205403

RESUMEN

To control infection, mammals actively withhold essential nutrients, including the transition metal manganese, by a process termed nutritional immunity. A critical component of this host response is the manganese-chelating protein calprotectin. While many bacterial mechanisms for overcoming nutritional immunity have been identified, the intersection between metal starvation and other essential inorganic nutrients has not been investigated. Here, we report that overexpression of an operon encoding a highly conserved inorganic phosphate importer, PstSCAB, increases the sensitivity of Staphylococcus aureus to calprotectin-mediated manganese sequestration. Further analysis revealed that overexpression of pstSCAB does not disrupt manganese acquisition or result in overaccumulation of phosphate by S. aureus However, it does reduce the ability of S. aureus to grow in phosphate-replete defined medium. Overexpression of pstSCAB does not aberrantly activate the phosphate-responsive two-component system PhoPR, nor was this two-component system required for sensitivity to manganese starvation. In a mouse model of systemic staphylococcal disease, a pstSCAB-overexpressing strain is significantly attenuated compared to wild-type S. aureus This defect is partially reversed in a calprotectin-deficient mouse, in which manganese is more readily available. Given that expression of pstSCAB is regulated by PhoPR, these findings suggest that overactivation of PhoPR would diminish the ability of S. aureus to resist nutritional immunity and cause infection. As PhoPR is also necessary for bacterial virulence, these findings imply that phosphate homeostasis represents a critical regulatory node whose activity must be precisely controlled in order for S. aureus and other pathogens to cause infection.

10.
J Bacteriol ; 202(9)2020 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-32071094

RESUMEN

The host restricts the availability of zinc to prevent infection. To overcome this defense, Staphylococcus aureus and Pseudomonas aeruginosa rely on zincophore-dependent zinc importers. Synthesis of the zincophore staphylopine by S. aureus and its import are both necessary for the bacterium to cause infection. In this study, we sought to elucidate how loss of zincophore efflux impacts bacterial resistance to host-imposed zinc starvation. In culture and during infection, mutants lacking CntE, the staphylopine efflux pump, were more sensitive to zinc starvation imposed by the metal-binding immune effector calprotectin than those lacking the ability to import staphylopine. However, disruption of staphylopine synthesis reversed the enhanced sensitivity phenotype of the ΔcntE mutant to calprotectin, indicating that intracellular toxicity of staphylopine is more detrimental than the impaired ability to acquire zinc. Unexpectedly, intracellular accumulation of staphylopine does not increase the expression of metal importers or alter cellular metal concentrations, suggesting that, contrary to prevailing models, the toxicity associated with staphylopine is not strictly due to intracellular chelation of metals. As P. aeruginosa and other pathogens produce zincophores with similar chemistry, our observations on the crucial importance of zincophore efflux are likely to be broadly relevant.IMPORTANCE Staphylococcus aureus and many other bacterial pathogens rely on metal-binding small molecules to obtain the essential metal zinc during infection. In this study, we reveal that export of these small molecules is critical for overcoming host-imposed metal starvation during infection and prevents toxicity due to accumulation of the metal-binding molecule within the cell. Surprisingly, we found that intracellular toxicity of the molecule is not due to chelation of cellular metals.

11.
J Chem Theory Comput ; 16(3): 1913-1923, 2020 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-32059108

RESUMEN

Divalent metal cations are essential for many biological processes; however, accurately modeling divalent metal ions has proved a significant challenge for molecular dynamics force fields. Here we show that the choice of ion model influences the observed dynamics in PsaA, a metal binding protein from Streptococcus pneumoniae. We conduct extensive unbiased simulations and free energy calculations of PsaA bound to its cognate ligand Mn2+ and inhibitory ligand Zn2+ using three nonbonded ion models: a 12-6 model, a 12-6-4 model, and a multisite model. The observed coordination geometries and metal binding dynamics are sensitive to the choice of ion model, with the most dramatic differences observed in free energy calculations of ion release. We show that the conformational ensemble of Mn-bound PsaA is more similar to the crystallographic metal bound open state. This work extends the current model of PsaA metal binding and provides a framework for the rationalization of experimentally determined metal binding behavior. Our findings support the use of the 12-6-4 ion model for further simulations of divalent cation binding proteins.


Asunto(s)
Proteínas Portadoras/metabolismo , Cationes Bivalentes/metabolismo , Metales/química , Streptococcus pneumoniae/química , Humanos , Conformación Molecular
12.
ACS Infect Dis ; 6(1): 150-158, 2020 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-31658418

RESUMEN

Acinetobacter baumannii is a ubiquitous Gram-negative bacterium, that is associated with significant disease in immunocompromised individuals. The success of A. baumannii is partly attributable to its high level of antibiotic resistance. Further, A. baumannii expresses a broad arsenal of putative zinc efflux systems that are likely to aid environmental persistence and host colonization, but detailed insights into how the bacterium deals with toxic concentrations of zinc are lacking. In this study we present the transcriptomic responses of A. baumannii to toxic zinc concentrations. Subsequent mutant analyses revealed a primary role for the resistance-nodulation-cell division heavy metal efflux system CzcCBA, and the cation diffusion facilitator transporter CzcD in zinc resistance. To examine the role of zinc at the host-pathogen interface we utilized a murine model of zinc deficiency and challenge with wild-type and czcA mutant strains, which identified highly site-specific roles for zinc during A. baumannii infection. Overall, we provide novel insight into the key zinc resistance mechanisms of A. baumannii and outline the role these systems play in enabling the bacterium to survive in diverse environments.

13.
J Bacteriol ; 202(1)2019 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-31611288

RESUMEN

The obligate intracellular pathogen Chlamydia trachomatis is a globally significant cause of sexually transmitted bacterial infections and the leading etiological agent of preventable blindness. The first-row transition metal iron (Fe) plays critical roles in chlamydial cell biology, and acquisition of this nutrient is essential for the survival and virulence of the pathogen. Nevertheless, how C. trachomatis acquires Fe from host cells is not well understood, since it lacks genes encoding known siderophore biosynthetic pathways, receptors for host Fe storage proteins, and the Fe acquisition machinery common to many bacteria. Recent studies have suggested that C. trachomatis directly acquires host Fe via the ATP-binding cassette permease YtgABCD. Here, we characterized YtgA, the periplasmic solute binding protein component of the transport pathway, which has been implicated in scavenging Fe(III) ions. The structure of Fe(III)-bound YtgA was determined at 2.0-Å resolution with the bound ion coordinated via a novel geometry (3 Ns, 2 Os [3N2O]). This unusual coordination suggested a highly plastic metal binding site in YtgA capable of interacting with other cations. Biochemical analyses showed that the metal binding site of YtgA was not restricted to interaction with only Fe(III) ions but could bind all transition metal ions examined. However, only Mn(II), Fe(II), and Ni(II) ions bound reversibly to YtgA, with Fe being the most abundant cellular transition metal in C. trachomatis Collectively, these findings show that YtgA is the metal-recruiting component of the YtgABCD permease and is most likely involved in the acquisition of Fe(II) and Mn(II) from host cells.IMPORTANCE Chlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. Infection by C. trachomatis is dependent on the ability to acquire essential nutrients, such as the transition metal iron, from host cells. In this study, we show that iron is the most abundant transition metal in C. trachomatis and report the structural and biochemical properties of the iron-recruiting protein YtgA. Knowledge of the high-resolution structure of YtgA will provide a platform for future structure-based antimicrobial design approaches.


Asunto(s)
Antígenos Bacterianos/química , Proteínas de Unión a Hierro/química , Hierro/metabolismo , Antígenos Bacterianos/metabolismo , Sitios de Unión , Proteínas de Unión a Hierro/metabolismo
14.
PLoS Pathog ; 15(8): e1007957, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31437249

RESUMEN

Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences.


Asunto(s)
Suplementos Dietéticos , Modelos Animales de Enfermedad , Enfermedades Pulmonares/inmunología , Infecciones Neumocócicas/inmunología , Streptococcus pneumoniae/inmunología , Virulencia/efectos de los fármacos , Zinc/administración & dosificación , Animales , Femenino , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/microbiología , Ratones , Infecciones Neumocócicas/tratamiento farmacológico , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/crecimiento & desarrollo
15.
Nat Commun ; 10(1): 3067, 2019 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-31296851

RESUMEN

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introducing the WalKH271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PASCYT domain reveal a metal-binding site, in which a zinc ion (Zn2+) is tetrahedrally-coordinated by four amino acids including H271. The WalKH271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.


Asunto(s)
Proteínas Bacterianas/metabolismo , Histidina Quinasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Staphylococcus aureus/metabolismo , Zinc/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cationes Bivalentes/metabolismo , Histidina/genética , Histidina Quinasa/química , Histidina Quinasa/genética , Simulación de Dinámica Molecular , Mutación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Regulón/genética , Staphylococcus aureus/genética , Tirosina/genética
16.
Elife ; 82019 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-30900991

RESUMEN

Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch from an open to a closed conformation upon substrate binding, providing specificity for transport. We investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs sample a range of conformations that activate transport. Certain non-transported ligands leave the structure largely unaltered or trigger a conformation distinct from that of transported substrates. Intriguingly, in some cases, similar SBP conformations are formed by both transported and non-transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP interactions, SBP conformational dynamics and substrate transport.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Cinética , Unión Proteica , Conformación Proteica , Imagen Individual de Molécula , Especificidad por Sustrato
17.
Int J Mol Sci ; 20(3)2019 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-30699983

RESUMEN

Acinetobacter baumannii has emerged as one of the leading causative agents of nosocomial infections. Due to its high level of intrinsic and adapted antibiotic resistance, treatment failure rates are high, which allows this opportunistic pathogen to thrive during infection in immune-compromised patients. A. baumannii can cause infections within a broad range of host niches, with pneumonia and bacteraemia being associated with the greatest levels of morbidity and mortality. Although its resistance to antibiotics is widely studied, our understanding of the mechanisms required for dealing with environmental stresses related to virulence and hospital persistence, such as copper toxicity, is limited. Here, we performed an in silico analysis of the A. baumannii copper resistome, examining its regulation under copper stress. Using comparative analyses of bacterial P-type ATPases, we propose that A. baumannii encodes a member of a novel subgroup of P1B-1 ATPases. Analyses of three putative inner membrane copper efflux systems identified the P1B-1 ATPase CopA as the primary mediator of cytoplasmic copper resistance in A. baumannii. Using a murine model of A. baumannii pneumonia, we reveal that CopA contributes to the virulence of A. baumannii. Collectively, this study advances our understanding of how A. baumannii deals with environmental copper toxicity, and it provides novel insights into how A. baumannii combats adversities encountered as part of the host immune defence.


Asunto(s)
Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/patogenicidad , ATPasas Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Proteínas de Escherichia coli/metabolismo , Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , ATPasas Transportadoras de Cobre/genética , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Proteínas de Escherichia coli/genética , Filogenia , Virulencia
18.
mBio ; 10(1)2019 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-30723122

RESUMEN

Free fatty acids hold important immune-modulatory roles during infection. However, the host's long-chain polyunsaturated fatty acids, not commonly found in the membranes of bacterial pathogens, also have significant broad-spectrum antibacterial potential. Of these, the omega-6 fatty acid arachidonic acid (AA) and the omega-3 fatty acid decosahexaenoic acid (DHA) are highly abundant; hence, we investigated their effects on the multidrug-resistant human pathogen Acinetobacter baumannii Our analyses reveal that AA and DHA incorporate into the A. baumannii bacterial membrane and impact bacterial fitness and membrane integrity, with DHA having a more pronounced effect. Through transcriptional profiling and mutant analyses, we show that the A. baumannii ß-oxidation pathway plays a protective role against AA and DHA, by limiting their incorporation into the phospholipids of the bacterial membrane. Furthermore, our study identified a second bacterial membrane protection system mediated by the AdeIJK efflux system, which modulates the lipid content of the membrane via direct efflux of lipids other than AA and DHA, thereby providing a novel function for this major efflux system in A. baumannii This is the first study to examine the antimicrobial effects of host fatty acids on A. baumannii and highlights the potential of AA and DHA to protect against A. baumannii infections.IMPORTANCE A shift in the Western diet since the industrial revolution has resulted in a dramatic increase in the consumption of omega-6 fatty acids, with a concurrent decrease in the consumption of omega-3 fatty acids. This decrease in omega-3 fatty acid consumption has been associated with significant disease burden, including increased susceptibility to infectious diseases. Here we provide evidence that DHA, an omega-3 fatty acid, has superior antimicrobial effects upon the highly drug-resistant pathogen Acinetobacter baumannii, thereby providing insights into one of the potential health benefits of omega-3 fatty acids. The identification and characterization of two novel bacterial membrane protective mechanisms against host fatty acids provide important insights into A. baumannii adaptation during disease. Furthermore, we describe a novel role for the major multidrug efflux system AdeIJK in A. baumannii membrane maintenance and lipid transport. This core function, beyond drug efflux, increases the appeal of AdeIJK as a therapeutic target.


Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/fisiología , Adaptación Fisiológica , Antibacterianos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Estrés Fisiológico , Transporte Biológico Activo , Membrana Celular/metabolismo , Perfilación de la Expresión Génica , Proteínas de Transporte de Membrana/metabolismo , Redes y Vías Metabólicas/genética , Oxidación-Reducción
19.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 1): 54-61, 2019 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-30605126

RESUMEN

The glutathione reductase (GR) from Streptococcus pneumoniae is a flavoenzyme that catalyzes the reduction of oxidized glutathione (GSSG) to its reduced form (GSH) in the cytoplasm of this bacterium. The maintenance of an intracellular pool of GSH is critical for the detoxification of reactive oxygen and nitrogen species and for intracellular metal tolerance to ions such as zinc. Here, S. pneumoniae GR (SpGR) was overexpressed and purified and its crystal structure determined at 2.56 Šresolution. SpGR shows overall structural similarity to other characterized GRs, with a dimeric structure that includes an antiparallel ß-sheet at the dimer interface. This observation, in conjunction with comparisons with the interface structures of other GR enzymes, allows the classification of these enzymes into three classes. Analyses of the kinetic properties of SpGR revealed a significantly higher value for Km(GSSG) (231.2 ± 24.7 µM) in comparison to other characterized GR enzymes.


Asunto(s)
Proteínas Bacterianas/química , Glutatión Reductasa/química , Glutatión/química , NADP/química , Streptococcus pneumoniae/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocatálisis , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glutatión/metabolismo , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Cinética , Modelos Moleculares , NADP/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/enzimología , Homología Estructural de Proteína , Especificidad por Sustrato
20.
Infect Immun ; 87(1)2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30348827

RESUMEN

During infection, the host utilizes a diverse array of processes to combat invaders, including the restriction of availability of essential nutrients such as manganese. Similarly to many other pathogens, Staphylococcus aureus possesses two manganese importers, MntH and MntABC. Several infection models have revealed a critical role for MntABC during staphylococcal infection. However, culture-based studies have suggested parity between the two transporters when cells are resisting manganese starvation imposed by the manganese binding immune effector calprotectin. In this investigation, initial elemental analysis revealed that MntABC is the primary transporter responsible for obtaining manganese in culture in the presence of calprotectin. MntABC was also necessary to maintain wild-type levels of manganese-dependent superoxide dismutase activity in the presence of calprotectin. Building on this framework, we investigated if MntABC enabled S. aureus to resist the synergistic actions of nutritional immunity and other host defenses. This analysis revealed that MntABC critically contributes to staphylococcal growth when S. aureus is subjected to manganese limitations and exposed to oxidative stress. This transporter was also important for growth in manganese-limited environments when S. aureus was forced to consume glucose as an energy source, which occurs when it encounters nitric oxide. MntABC also expanded the pH range conducive for S. aureus growth under conditions of manganese scarcity. Collectively, the data presented in this work provide a robust molecular basis for the crucial role of MntABC in staphylococcal virulence. Further, this work highlights the importance of synergy between host defenses and the necessity of evaluating the contribution of virulence factors to pathogenesis in the presence of multiple stressors.


Asunto(s)
Manganeso/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/metabolismo , Factores de Virulencia/metabolismo , Interacciones Huésped-Patógeno , Complejo de Antígeno L1 de Leucocito/metabolismo , Estrés Oxidativo , Virulencia
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