RESUMO
Chronic coinfections of Staphylococcus aureus and Pseudomonas aeruginosa frequently fail to respond to antibiotic treatment, leading to significant patient morbidity and mortality. Currently, the impact of interspecies interaction on S. aureus antibiotic susceptibility remains poorly understood. In this study, we utilize a panel of P. aeruginosa burn wound and cystic fibrosis (CF) lung isolates to demonstrate that P. aeruginosa alters S. aureus susceptibility to bactericidal antibiotics in a variable, strain-dependent manner and further identify 3 independent interactions responsible for antagonizing or potentiating antibiotic activity against S. aureus. We find that P. aeruginosa LasA endopeptidase potentiates lysis of S. aureus by vancomycin, rhamnolipids facilitate proton-motive force-independent tobramycin uptake, and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) induces multidrug tolerance in S. aureus through respiratory inhibition and reduction of cellular ATP. We find that the production of each of these factors varies between clinical isolates and corresponds to the capacity of each isolate to alter S. aureus antibiotic susceptibility. Furthermore, we demonstrate that vancomycin treatment of a S. aureus mouse burn infection is potentiated by the presence of a LasA-producing P. aeruginosa population. These findings demonstrate that antibiotic susceptibility is complex and dependent not only upon the genotype of the pathogen being targeted, but also on interactions with other microorganisms in the infection environment. Consideration of these interactions will improve the treatment of polymicrobial infections.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Glicolipídeos/farmacologia , Interações Microbianas/fisiologia , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Animais , Queimaduras/microbiologia , Queimaduras/patologia , Coinfecção , Glicolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/crescimento & desenvolvimento , Vancomicina/farmacologia , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/patologiaRESUMO
Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.
Assuntos
Bacteroides/enzimologia , Endo-1,4-beta-Xilanases/genética , Glicosídeo Hidrolases/genética , Intestinos/microbiologia , Polissacarídeos/metabolismo , Simbiose/fisiologia , Bacteroides/genética , Endo-1,4-beta-Xilanases/metabolismo , Fermentação/fisiologia , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Glucuronatos/metabolismo , Glicosídeo Hidrolases/metabolismo , Humanos , Microbiota/fisiologia , Mutagênese Sítio-Dirigida , Oligossacarídeos/metabolismo , Filogenia , Transcriptoma , Xilanos/metabolismoRESUMO
The gut microbiota prevents harmful microbes from entering the body, a function known as colonization resistance. The enteric pathogen Salmonella enterica serovar (S.) Typhimurium uses its virulence factors to break colonization resistance through unknown mechanisms. Using metabolite profiling and genetic analysis, we show that the initial rise in luminal pathogen abundance was powered by a combination of aerobic respiration and mixed acid fermentation of simple sugars, such as glucose, which resulted in their depletion from the metabolome. The initial rise in the abundance of the pathogen in the feces coincided with a reduction in the cecal concentrations of acetate and butyrate and an increase in epithelial oxygenation. Notably, these changes in the host environment preceded changes in the microbiota composition. We conclude that changes in the host environment can weaken colonization resistance even in the absence of overt compositional changes in the gut microbiota.
RESUMO
The gut microbiota prevents infection by crowding out pathogens.
Assuntos
Microbioma Gastrointestinal , Interações Hospedeiro-Patógeno , Humanos , Animais , Camundongos , Salmonella entericaRESUMO
Listeria monocytogenes uses respiration to sustain a risky fermentative lifestyle during infection.
Assuntos
Listeria monocytogenes , Listeriose , Fermentação , HumanosRESUMO
Aminoglycosides are bactericidal drugs which require a proton motive force (PMF) for uptake into the bacterial cell. Low energy cells, such as persisters, maintain a PMF below the threshold for drug uptake and are tolerant to aminoglycosides. In this chapter, we discuss mechanisms to target the bacterial membrane and stimulate aminoglycoside uptake to kill Staphylococcus aureus persisters.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológicoRESUMO
Staphylococcus aureus is a major human pathogen that causes an array of infections ranging from minor skin infections to more serious infections, including osteomyelitis, endocarditis, necrotizing pneumonia and sepsis1. These more serious infections usually arise from an initial bloodstream infection and are frequently recalcitrant to antibiotic treatment1. Phagocytosis by macrophages and neutrophils is the primary mechanism through which S. aureus infection is controlled by the immune system2. Macrophages have been shown to be a major reservoir of S. aureus in vivo3, but the role of macrophages in the induction of antibiotic tolerance has not been explored. Here, we show that macrophages not only fail to efficiently kill phagocytosed S. aureus, but also induce tolerance to multiple antibiotics. Reactive oxygen species generated by respiratory burst attack iron-sulfur cluster-containing proteins, including TCA-cycle enzymes, result in decreased respiration, lower ATP and increased antibiotic tolerance. We further show that respiratory burst induces antibiotic tolerance in the spleen during a murine systemic infection. These results suggest that a major component of the innate immune response is antagonistic to the bactericidal activities of antibiotics.
Assuntos
Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/metabolismo , Animais , Linhagem Celular , Ciclo do Ácido Cítrico , Modelos Animais de Doenças , Farmacorresistência Bacteriana/imunologia , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunidade Inata , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/deficiência , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/imunologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Aminoglycoside antibiotics require proton motive force (PMF) for bacterial internalization. In non-respiring populations, PMF drops below the level required for drug influx, limiting the utility of aminoglycosides against strict and facultative anaerobes. We recently demonstrated that rhamnolipids (RLs), biosurfactant molecules produced by Pseudomonas aeruginosa, potentiate aminoglycoside activity against Staphylococcus aureus. Here, we demonstrate that RLs induce PMF-independent aminoglycoside uptake to restore sensitivity to otherwise tolerant persister, biofilm, small colony variant, and anaerobic populations of S. aureus. Furthermore, we show that this approach represses the rise of resistance, restores sensitivity to highly resistant clinical isolates, and is effective against other Gram-positive pathogens. Finally, while other membrane-acting agents can synergize with aminoglycosides, induction of PMF-independent uptake is uncommon, and distinct to RLs among several compounds tested. In all, small-molecule induction of PMF-independent aminoglycoside uptake circumvents phenotypic tolerance, overcomes genotypic resistance, and expands the utility of aminoglycosides against intrinsically recalcitrant bacterial populations.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Aminoglicosídeos/química , Animais , Antibacterianos/química , Linhagem Celular , Tolerância a Medicamentos , Camundongos , Testes de Sensibilidade MicrobianaRESUMO
Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels. S. aureus knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and ß-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. These findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation.IMPORTANCE Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Proteômica , Staphylococcus aureus/metabolismoRESUMO
Francisella tularensis is a Gram-negative, facultative, intracellular bacterial pathogen and one of the most virulent organisms known. A hallmark of F. tularensis pathogenesis is the bacterium's ability to replicate to high densities within the cytoplasm of infected cells in over 250 known host species, including humans. This demonstrates that F. tularensis is adept at modulating its metabolism to fluctuating concentrations of host-derived nutrients. The precise metabolic pathways and nutrients utilized by F. tularensis during intracellular growth, however, are poorly understood. Here, we use systematic mutational analysis to identify the carbon catabolic pathways and host-derived nutrients required for F. tularensis intracellular replication. We demonstrate that the glycolytic enzyme phosphofructokinase (PfkA), and thus glycolysis, is dispensable for F. tularensis SchuS4 virulence, and we highlight the importance of the gluconeogenic enzyme fructose 1,6-bisphosphatase (GlpX). We found that the specific gluconeogenic enzymes that function upstream of GlpX varied based on infection model, indicating that F. tularensis alters its metabolic flux according to the nutrients available within its replicative niche. Despite this flexibility, we found that glutamate dehydrogenase (GdhA) and glycerol 3-phosphate (G3P) dehydrogenase (GlpA) are essential for F. tularensis intracellular replication in all infection models tested. Finally, we demonstrate that host cell lipolysis is required for F. tularensis intracellular proliferation, suggesting that host triglyceride stores represent a primary source of glycerol during intracellular replication. Altogether, the data presented here reveal common nutritional requirements for a bacterium that exhibits characteristic metabolic flexibility during infection.IMPORTANCE The widespread onset of antibiotic resistance prioritizes the need for novel antimicrobial strategies to prevent the spread of disease. With its low infectious dose, broad host range, and high rate of mortality, F. tularensis poses a severe risk to public health and is considered a potential agent for bioterrorism. F. tularensis reaches extreme densities within the host cell cytosol, often replicating 1,000-fold in a single cell within 24 hours. This remarkable rate of growth demonstrates that F. tularensis is adept at harvesting and utilizing host cell nutrients. However, like most intracellular pathogens, the types of nutrients utilized by F. tularensis and how they are acquired is not fully understood. Identifying the essential pathways for F. tularensis replication may reveal new therapeutic strategies for targeting this highly infectious pathogen and may provide insight for improved targeting of intracellular pathogens in general.
Assuntos
Carbono/metabolismo , Citoplasma/microbiologia , Francisella tularensis/crescimento & desenvolvimento , Redes e Vias Metabólicas , Animais , Replicação do DNA , Feminino , Francisella tularensis/metabolismo , Frutose-Bifosfatase/metabolismo , Gluconeogênese , Glicólise , Macrófagos/microbiologia , Macrófagos/fisiologia , Análise do Fluxo Metabólico , Camundongos , Camundongos Endogâmicos C57BL , Fosfofrutoquinases/metabolismo , Tularemia/metabolismo , VirulênciaRESUMO
Macrophages are myeloid-derived phagocytic cells and one of the first immune cell types to respond to microbial infections. However, a number of bacterial pathogens are resistant to the antimicrobial activities of macrophages and can grow within these cells. Macrophages have other immune surveillance roles including the acquisition of cytosolic components from multiple types of cells. We hypothesized that intracellular pathogens that can replicate within macrophages could also exploit cytosolic transfer to facilitate bacterial spread. We found that viable Francisella tularensis, as well as Salmonella enterica bacteria transferred from infected cells to uninfected macrophages along with other cytosolic material through a transient, contact dependent mechanism. Bacterial transfer occurred when the host cells exchanged plasma membrane proteins and cytosol via a trogocytosis related process leaving both donor and recipient cells intact and viable. Trogocytosis was strongly associated with infection in mice, suggesting that direct bacterial transfer occurs by this process in vivo.