RESUMEN
INTRODUCTION: Blood infections from multi-drug-resistant Salmonella pose a major health burden. This is especially true because Salmonella can survive and replicate intracellularly, and the development of new treatment strategies is dependent on expensive and time-consuming in vivo trials. The aim of this study was to develop a Salmonella-infection model that makes it possible to directly observe Salmonella infections of macrophages in vivo and to use this model to test the effect of antimicrobials against intra- and extracellular Salmonella in order to close the gap between in vitro and rodent-infection models. METHODS: We established suitable Salmonella-infection conditions using genetically engineered zebrafish and Salmonella-expressing fluorescent proteins (green fluorescent protein (GFP) and/or mCherry). RESULTS: We detected Salmonella inside and outside zebrafish larvae macrophages. Administration of the cell-impermeable antibiotic tobramycin removed Salmonella residing outside macrophages but did not affect Salmonella in macrophages, whereas ceftriaxone successfully cleared both types of Salmonella. Salmonella inside and outside macrophages experienced substantial DNA damage after administration of fluoroquinolones consistent with the excellent cell penetration of these antibiotics. CONCLUSIONS: The zebrafish-larvae model enables testing of antimicrobials for efficacy against extra- and intracellular Salmonella in a complex in vivo environment. This model thus might serve for antimicrobial lead optimization prior to using rodent models.
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Antibacterianos , Pez Cebra , Animales , Larva , Antibacterianos/farmacología , Antibacterianos/metabolismo , Macrófagos/metabolismo , Salmonella/genéticaRESUMEN
Virulence and persistence of the obligate intracellular parasite Toxoplasma gondii involve the secretion of effector proteins belonging to the family of dense granule proteins (GRAs) that act notably as modulators of the host defense mechanisms and participate in cyst wall formation. The subset of GRAs residing in the parasitophorous vacuole (PV) or exported into the host cell, undergo proteolytic cleavage in the Golgi upon the action of the aspartyl protease 5 (ASP5). In tachyzoites, ASP5 substrates play central roles in the morphology of the PV and the export of effectors across the translocon complex MYR1/2/3. Here, we used N-terminal amine isotopic labeling of substrates to identify novel ASP5 cleavage products by comparing the N-terminome of wild-type and Δasp5 lines in tachyzoites and bradyzoites. Validated substrates reside within the PV or PVM in an ASP5-dependent manner. Remarkably, Δasp5 bradyzoites are impaired in the formation of the cyst wall in vitro and exhibit a considerably reduced cyst burden in chronically infected animals. More specifically two-photon serial tomography of infected mouse brains revealed a comparatively reduced number and size of the cysts throughout the establishment of persistence in the absence of ASP5.
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Proteasas de Ácido Aspártico , Toxoplasma , Animales , Ratones , Toxoplasma/metabolismo , Proteasas de Ácido Aspártico/metabolismo , Proteínas Protozoarias/metabolismo , Infección Persistente , Vacuolas/metabolismo , Ácido Aspártico Endopeptidasas/metabolismoRESUMEN
Antimicrobial chemotherapy can fail to eradicate the pathogen, even in the absence of antimicrobial resistance. Persisting pathogens can subsequently cause relapsing diseases. In vitro studies suggest various mechanisms of antibiotic persistence, but their in vivo relevance remains unclear because of the difficulty of studying scarce pathogen survivors in complex host tissues. Here, we localized and characterized rare surviving Salmonella in mouse spleen using high-resolution whole-organ tomography. Chemotherapy cleared >99.5% of the Salmonella but was inefficient against a small Salmonella subset in the white pulp. Previous models could not explain these findings: drug exposure was adequate, Salmonella continued to replicate, and host stresses induced only limited Salmonella drug tolerance. Instead, antimicrobial clearance required support of Salmonella-killing neutrophils and monocytes, and the density of such cells was lower in the white pulp than in other spleen compartments containing higher Salmonella loads. Neutrophil densities declined further during treatment in response to receding Salmonella loads, resulting in insufficient support for Salmonella clearance from the white pulp and eradication failure. However, adjunctive therapies sustaining inflammatory support enabled effective clearance. These results identify uneven Salmonella tissue colonization and spatiotemporal inflammation dynamics as main causes of Salmonella persistence and establish a powerful approach to investigate scarce but impactful pathogen subsets in complex host environments.
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Antibacterianos/uso terapéutico , Enrofloxacina/uso terapéutico , Salmonelosis Animal/microbiología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/fisiología , Animales , Ratones , Ratones Endogámicos BALB C , Salmonelosis Animal/tratamiento farmacológicoRESUMEN
Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.
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Antibacterianos/metabolismo , Membrana Celular/fisiología , Nutrientes/metabolismo , Porinas/metabolismo , Pseudomonas aeruginosa/fisiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico/fisiología , Permeabilidad de la Membrana CelularRESUMEN
The homotrimeric molecular chaperone Skp of Gram-negative bacteria facilitates the transport of outer membrane proteins across the periplasm. It has been unclear how its activity is modulated during its functional cycle. Here, we report an atomic-resolution characterization of the Escherichia coli Skp monomer-trimer transition. We find that the monomeric state of Skp is intrinsically disordered and that formation of the oligomerization interface initiates folding of the α-helical coiled-coil arms via a unique "stapling" mechanism, resulting in the formation of active trimeric Skp. Native client proteins contact all three Skp subunits simultaneously, and accordingly, their binding shifts the Skp population toward the active trimer. This activation mechanism is shown to be essential for Salmonella fitness in a mouse infection model. The coupled mechanism is a unique example of how an ATP-independent chaperone can modulate its activity as a function of the presence of client proteins.
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The ability of the immune system to discriminate self from non-self is essential for eradicating microbial pathogens but is also responsible for allograft rejection. Whether it is possible to selectively suppress alloresponses while maintaining anti-pathogen immunity remains unknown. We found that mice deficient in coronin 1, a regulator of naive T cell homeostasis, fully retained allografts while maintaining T cell-specific responses against microbial pathogens. Mechanistically, coronin 1-deficiency increased cyclic adenosine monophosphate (cAMP) concentrations to suppress allo-specific T cell responses. Costimulation induced on microbe-infected antigen presenting cells was able to overcome cAMP-mediated immunosuppression to maintain anti-pathogen immunity. In vivo pharmacological modulation of this pathway or a prior transfer of coronin 1-deficient T cells actively suppressed allograft rejection. These results define a coronin 1-dependent regulatory axis in T cells important for allograft rejection and suggest that modulation of this pathway may be a promising approach to achieve long-term acceptance of mismatched allografts.
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Rechazo de Injerto/inmunología , Trasplante de Corazón , Infecciones/inmunología , Proteínas de Microfilamentos/metabolismo , Trasplante de Piel , Linfocitos T/inmunología , Aloinjertos/inmunología , Animales , Antígenos Bacterianos/inmunología , Antígenos Fúngicos/inmunología , Antígenos Virales/inmunología , Células Cultivadas , AMP Cíclico/inmunología , Supervivencia de Injerto , Homeostasis/genética , Humanos , Inmunidad , Terapia de Inmunosupresión , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Tolerancia al TrasplanteRESUMEN
The Gram-negative bacterial outer membrane (OM) is a unique bilayer that forms an efficient permeation barrier to protect the cell from noxious compounds 1,2 . The defining characteristic of the OM is lipid asymmetry, with phospholipids comprising the inner leaflet and lipopolysaccharides comprising the outer leaflet 1-3 . This asymmetry is maintained by the Mla pathway, a six-component system that is widespread in Gram-negative bacteria and is thought to mediate retrograde transport of misplaced phospholipids from the outer leaflet of the OM to the cytoplasmic membrane 4 . The OM lipoprotein MlaA performs the first step in this process via an unknown mechanism that does not require external energy input. Here we show, using X-ray crystallography, molecular dynamics simulations and in vitro and in vivo functional assays, that MlaA is a monomeric α-helical OM protein that functions as a phospholipid translocation channel, forming a ~20-Å-thick doughnut embedded in the inner leaflet of the OM with a central, amphipathic pore. This architecture prevents access of inner leaflet phospholipids to the pore, but allows outer leaflet phospholipids to bind to a pronounced ridge surrounding the channel, followed by diffusion towards the periplasmic space. Enterobacterial MlaA proteins form stable complexes with OmpF/C 5,6 , but the porins do not appear to play an active role in phospholipid transport. MlaA represents a lipid transport protein that selectively removes outer leaflet phospholipids to help maintain the essential barrier function of the bacterial OM.
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Bacterias/química , Proteínas de la Membrana Bacteriana Externa/química , Membrana Celular/química , Lípidos de la Membrana/química , Bacterias/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transporte Biológico , Cristalografía por Rayos X , ADN Bacteriano/genética , Difusión , Vectores Genéticos , Bacterias Gramnegativas/química , Bacterias Gramnegativas/metabolismo , Membrana Dobles de Lípidos , Lipopolisacáridos/química , Simulación de Dinámica Molecular , Mutación , Fosfolípidos/química , Fosfolípidos/metabolismo , Porinas/química , Conformación ProteicaRESUMEN
The host endolysosomal compartment is often manipulated by intracellular bacterial pathogens. Salmonella (Salmonella enterica serovar Typhimurium) secrete numerous effector proteins, including SifA, through a specialized type III secretion system to hijack the host endosomal system and generate the Salmonella-containing vacuole (SCV). To form this replicative niche, Salmonella targets the Rab7 GTPase to recruit host membranes through largely unknown mechanisms. We show that Pleckstrin homology domain-containing protein family member 1 (PLEKHM1), a lysosomal adaptor, is targeted by Salmonella through direct interaction with SifA. By binding the PLEKHM1 PH2 domain, Salmonella utilize a complex containing PLEKHM1, Rab7, and the HOPS tethering complex to mobilize phagolysosomal membranes to the SCV. Depletion of PLEKHM1 causes a profound defect in SCV morphology with multiple bacteria accumulating in enlarged structures and significantly dampens Salmonella proliferation in multiple cell types and mice. Thus, PLEKHM1 provides a critical interface between pathogenic infection and the host endolysosomal system.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Bacterianas/metabolismo , Glicoproteínas/metabolismo , Interacciones Huésped-Patógeno , Glicoproteínas de Membrana/metabolismo , Salmonella typhimurium/crecimiento & desarrollo , Vacuolas/microbiología , Animales , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Antibiotic therapy often fails to eliminate a fraction of transiently refractory bacteria, causing relapses and chronic infections. Multiple mechanisms can induce such persisters with high antimicrobial tolerance in vitro, but their in vivo relevance remains unclear. Using a fluorescent growth rate reporter, we detected extensive phenotypic variation of Salmonella in host tissues. This included slow-growing subsets as well as well-nourished fast-growing subsets driving disease progression. Monitoring of Salmonella growth and survival during chemotherapy revealed that antibiotic killing correlated with single-cell division rates. Nondividing Salmonella survived best but were rare, limiting their impact. Instead, most survivors originated from abundant moderately growing, partially tolerant Salmonella. These data demonstrate that host tissues diversify pathogen physiology, with major consequences for disease progression and control.
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Antibacterianos/administración & dosificación , Fluoroquinolonas/administración & dosificación , Imagen Óptica/métodos , Salmonella typhimurium/efectos de los fármacos , Fiebre Tifoidea/tratamiento farmacológico , Fiebre Tifoidea/microbiología , Animales , Proteínas Bacterianas/análisis , Enrofloxacina , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Proteoma/análisis , Salmonella typhimurium/citología , Salmonella typhimurium/crecimiento & desarrollo , Bazo/microbiología , Bazo/patologíaRESUMEN
Reactive oxygen and nitrogen species function in host defense via mechanisms that remain controversial. Pathogens might encounter varying levels of these species, but bulk measurements cannot resolve such heterogeneity. We used single-cell approaches to determine the impact of oxidative and nitrosative stresses on individual Salmonella during early infection in mouse spleen. Salmonella encounter and respond to both stresses, but the levels and impact vary widely. Neutrophils and inflammatory monocytes kill Salmonella by generating overwhelming oxidative stress through NADPH oxidase and myeloperoxidase. This controls Salmonella within inflammatory lesions but does not prevent their spread to more permissive resident red pulp macrophages, which generate only sublethal oxidative bursts. Regional host expression of inducible nitric oxide synthase exposes some Salmonella to nitrosative stress, triggering effective local Salmonella detoxification through nitric oxide denitrosylase. Thus, reactive oxygen and nitrogen species influence dramatically different outcomes of disparate Salmonella-host cell encounters, which together determine overall disease progression.
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Monocitos/inmunología , Neutrófilos/inmunología , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/fisiología , Bazo/inmunología , Animales , Femenino , Expresión Génica , Interacciones Huésped-Patógeno , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Monocitos/metabolismo , Monocitos/microbiología , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Neutrófilos/microbiología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Oxigenasas/genética , Oxigenasas/metabolismo , Peroxidasa/genética , Peroxidasa/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio/inmunología , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/patología , Salmonella typhimurium/patogenicidad , Análisis de la Célula Individual , Bazo/microbiología , Bazo/patologíaRESUMEN
Pathogen access to host nutrients in infected tissues is fundamental for pathogen growth and virulence, disease progression, and infection control. However, our understanding of this crucial process is still rather limited because of experimental and conceptual challenges. Here, we used proteomics, microbial genetics, competitive infections, and computational approaches to obtain a comprehensive overview of Salmonella nutrition and growth in a mouse typhoid fever model. The data revealed that Salmonella accessed an unexpectedly diverse set of at least 31 different host nutrients in infected tissues but the individual nutrients were available in only scarce amounts. Salmonella adapted to this situation by expressing versatile catabolic pathways to simultaneously exploit multiple host nutrients. A genome-scale computational model of Salmonella in vivo metabolism based on these data was fully consistent with independent large-scale experimental data on Salmonella enzyme quantities, and correctly predicted 92% of 738 reported experimental mutant virulence phenotypes, suggesting that our analysis provided a comprehensive overview of host nutrient supply, Salmonella metabolism, and Salmonella growth during infection. Comparison of metabolic networks of other pathogens suggested that complex host/pathogen nutritional interfaces are a common feature underlying many infectious diseases.
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Salmonella enterica/patogenicidad , Fiebre Tifoidea/microbiología , Animales , Línea Celular , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos BALB C , Proteómica , Salmonella enterica/genética , Salmonella enterica/metabolismo , Fiebre Tifoidea/metabolismoRESUMEN
Invasive Salmonella infection is an important health problem that is worsening because of rising antimicrobial resistance and changing Salmonella serovar spectrum. Novel vaccines with broad serovar coverage are needed, but suitable protective antigens remain largely unknown. Here, we tested 37 broadly conserved Salmonella antigens in a mouse typhoid fever model, and identified antigen candidates that conferred partial protection against lethal disease. Antigen properties such as high in vivo abundance or immunodominance in convalescent individuals were not required for protectivity, but all promising antigen candidates were associated with the Salmonella surface. Surprisingly, this was not due to superior immunogenicity of surface antigens compared to internal antigens as had been suggested by previous studies and novel findings for CD4 T cell responses to model antigens. Confocal microscopy of infected tissues revealed that many live Salmonella resided alone in infected host macrophages with no damaged Salmonella releasing internal antigens in their vicinity. In the absence of accessible internal antigens, detection of these infected cells might require CD4 T cell recognition of Salmonella surface-associated antigens that could be processed and presented even from intact Salmonella. In conclusion, our findings might pave the way for development of an efficacious Salmonella vaccine with broad serovar coverage, and suggest a similar crucial role of surface antigens for immunity to both extracellular and intracellular pathogens.