Regulation of chaperone function by coupled folding and oligomerization

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

Tissue compartmentalization enables; Salmonella; persistence during chemotherapy

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

Disruption of Coronin 1 Signaling in T Cells Promotes Allograft Tolerance while Maintaining Anti-Pathogen Immunity

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

The Italian National Project of Astrobiology-Life in Space-Origin, Presence, Persistence of Life in Space, from Molecules to Extremophiles

The \u2018\u2018Life in Space\u2019\u2019 project was funded in the wake of the Italian Space Agency\u2019s proposal for the development of a network of institutions and laboratories conceived to implement Italian participation in space astrobiology experiments

Parallel Exploitation of Diverse Host Nutrients Enhances Salmonella Virulence

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

Structural basis for maintenance of bacterial outer membrane lipid asymmetry

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.The crystal structure of MlaA, coupled with simulations of its interaction with phospholipids, elucidates how this outer membrane lipoprotein acts as a phospholipid translocation channel to maintain the asymmetric composition of the outer membrane

Zebrafish Larvae as an; in vivo; Model for Antimicrobial Activity Tests against Intracellular; Salmonella;

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.; We established suitable; Salmonella; -infection conditions using genetically engineered zebrafish and; Salmonella; -expressing fluorescent proteins (; green fluorescent protein; (; GFP; ) and/or; mCherry; ).; 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.; 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

Outer membrane permeability: Antimicrobials and diverse nutrients bypass porins in Pseudomonas aeruginosa

International audienceGram-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

Immunity to Intracellular Salmonella Depends on Surface-associated Antigens

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