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1.
Annu Rev Microbiol ; 73: 621-638, 2019 09 08.
Article in English | MEDLINE | ID: mdl-31226022

ABSTRACT

Bacteria need to deliver large molecules out of the cytosol to the extracellular space or even across membranes of neighboring cells to influence their environment, prevent predation, defeat competitors, or communicate. A variety of protein-secretion systems have evolved to make this process highly regulated and efficient. The type VI secretion system (T6SS) is one of the largest dynamic assemblies in gram-negative bacteria and allows for delivery of toxins into both bacterial and eukaryotic cells. The recent progress in structural biology and live-cell imaging shows the T6SS as a long contractile sheath assembled around a rigid tube with associated toxins anchored to a cell envelope by a baseplate and membrane complex. Rapid sheath contraction releases a large amount of energy used to push the tube and toxins through the membranes of neighboring target cells. Because reach of the T6SS is limited, some bacteria dynamically regulate its subcellular localization to precisely aim at their targets and thus increase efficiency of toxin translocation.


Subject(s)
Gram-Negative Bacteria/metabolism , Type VI Secretion Systems , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Secretion Systems , Cell Membrane/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Lipoproteins/chemistry , Lipoproteins/metabolism , Signal Transduction , Type VI Secretion Systems/biosynthesis , Type VI Secretion Systems/chemistry , Type VI Secretion Systems/metabolism , Type VI Secretion Systems/ultrastructure
2.
Infect Immun ; 89(7): e0057920, 2021 06 16.
Article in English | MEDLINE | ID: mdl-33875476

ABSTRACT

Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a noncanonical type VI secretion system (T6SS), in an arthropod in vivo infection model is missing. Here, we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. tularensis subsp. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of the T6SS effectors PdpC, PdpD, or OpiA is sufficient to kill G. mellonella larvae, while sheath recycling by ClpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK, OpiB1, OpiB2, and OpiB3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into the function of T6SS and pathogenesis of Francisella.


Subject(s)
Bacterial Proteins/genetics , Francisella tularensis/physiology , Larva/microbiology , Moths/microbiology , Type VI Secretion Systems/physiology , Animals , Bacterial Proteins/metabolism , Disease Models, Animal , Francisella tularensis/drug effects , Polyethylene Glycols/pharmacology , Tularemia , Type VI Secretion Systems/drug effects , Virulence/genetics , Virulence Factors/genetics , Virulence Factors/metabolism
3.
Proc Natl Acad Sci U S A ; 111(27): 9929-34, 2014 Jul 08.
Article in English | MEDLINE | ID: mdl-24958876

ABSTRACT

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.


Subject(s)
Cell Division , Shigella/physiology , Acetates/metabolism , Carbon/metabolism , Cytosol/metabolism , Genome, Bacterial , HeLa Cells , Humans , Metabolomics , Nuclear Magnetic Resonance, Biomolecular , Oxygen/metabolism , Pyruvic Acid/metabolism , Shigella/genetics , Shigella/metabolism
4.
Antimicrob Agents Chemother ; 60(8): 4991-5000, 2016 08.
Article in English | MEDLINE | ID: mdl-27297477

ABSTRACT

The clinical development of antibiotics with a new mode of action combined with efficient pulmonary drug delivery is a priority against untreatable Pseudomonas aeruginosa lung infections. POL7001 is a macrocycle antibiotic belonging to the novel class of protein epitope mimetic (PEM) molecules with selective and potent activity against P. aeruginosa We investigated ventilator-associated pneumonia (VAP) and cystic fibrosis (CF) as indications of the clinical potential of POL7001 to treat P. aeruginosa pulmonary infections. MICs of POL7001 and comparators were measured for reference and clinical P. aeruginosa strains. The therapeutic efficacy of POL7001 given by pulmonary administration was evaluated in murine models of P. aeruginosa acute and chronic pneumonia. POL7001 showed potent in vitro activity against a large panel of P. aeruginosa isolates from CF patients, including multidrug-resistant (MDR) isolates with adaptive phenotypes such as mucoid or hypermutable phenotypes. The efficacy of POL7001 was demonstrated in both wild-type and CF mice. In addition to a reduced bacterial burden in the lung, POL7001-treated mice showed progressive body weight recovery and reduced levels of inflammatory markers, indicating an improvement in general condition. Pharmacokinetic studies indicated that POL7001 reached significant concentrations in the lung after pulmonary administration, with low systemic exposure. These results support the further evaluation of POL7001 as a novel therapeutic agent for the treatment of P. aeruginosa pulmonary infections.


Subject(s)
Anti-Bacterial Agents/pharmacology , Pseudomonas aeruginosa/drug effects , Animals , Anti-Bacterial Agents/pharmacokinetics , Cystic Fibrosis/microbiology , Lung/drug effects , Lung/microbiology , Male , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Pneumonia, Ventilator-Associated/microbiology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/pathogenicity , Respiratory Tract Infections/microbiology
5.
Nat Commun ; 11(1): 5395, 2020 10 26.
Article in English | MEDLINE | ID: mdl-33106492

ABSTRACT

Tit-for-tat is a familiar principle from animal behavior: individuals respond in kind to being helped or harmed by others. Remarkably some bacteria appear to display tit-for-tat behavior, but how this evolved is not understood. Here we combine evolutionary game theory with agent-based modelling of bacterial tit-for-tat, whereby cells stab rivals with poisoned needles (the type VI secretion system) after being stabbed themselves. Our modelling shows tit-for-tat retaliation is a surprisingly poor evolutionary strategy, because tit-for-tat cells lack the first-strike advantage of preemptive attackers. However, if cells retaliate strongly and fire back multiple times, we find that reciprocation is highly effective. We test our predictions by competing Pseudomonas aeruginosa (a tit-for-tat species) with Vibrio cholerae (random-firing), revealing that P. aeruginosa does indeed fire multiple times per incoming attack. Our work suggests bacterial competition has led to a particular form of reciprocation, where the principle is that of strong retaliation, or 'tits-for-tat'.


Subject(s)
Bacterial Proteins/metabolism , Biological Evolution , Pseudomonas aeruginosa/physiology , Type VI Secretion Systems/metabolism , Vibrio cholerae/physiology , Bacterial Proteins/genetics , Pseudomonas aeruginosa/genetics , Type VI Secretion Systems/genetics , Vibrio cholerae/genetics
6.
Article in English | MEDLINE | ID: mdl-30234022

ABSTRACT

Francisella tularensis is the causative agent of the life-threatening disease tularemia. However, the molecular tools to study Francisella are limited. Especially, expression plasmids are sparse and difficult to use, as they are unstable and prone to spontaneous loss. Most Francisella expression plasmids lack inducible promoters making it difficult to control gene expression levels. In addition, available expression plasmids are mainly designed for F. tularensis, however, genetic differences including restriction-modification systems impede the use of these plasmids in F. novicida, which is often used as a model organism to study Francisella pathogenesis. Here we report construction and characterization of two mobilizable plasmids (pFNMB1 and pFNMB2) designed for regulated gene expression in F. novicida. pFNMB plasmids contain a tetracycline inducible promoter to control gene expression levels and oriT for RP4 mediated mobilization. We show that both plasmids are stably maintained in bacteria for more than 40 generations over 4 days of culturing in the absence of selection against plasmid loss. Expression levels are dependent on anhydrotetracycline concentration and homogeneous in a bacterial population. pFNMB1 and pFNMB2 plasmids differ in the sequence between promoter and translation start site and thus allow to reach different maximum levels of protein expression. We used pFNMB1 and pFNMB2 for complementation of Francisella Pathogenicity Island mutants ΔiglF, ΔiglI, and ΔiglC in-vitro and pFNMB1 to complement ΔiglI mutant in bone marrow derived macrophages.


Subject(s)
Francisella/genetics , Gene Expression Regulation, Bacterial , Genetic Engineering/methods , Genetic Vectors , Genetics, Microbial/methods , Plasmids , Gene Deletion , Genes, Bacterial , Genetic Complementation Test , Genomic Instability , Genomic Islands , Interspersed Repetitive Sequences , Promoter Regions, Genetic , Transcriptional Activation/drug effects
7.
Nat Commun ; 8: 15853, 2017 06 16.
Article in English | MEDLINE | ID: mdl-28621333

ABSTRACT

Francisella tularensis is an intracellular pathogen that causes the fatal zoonotic disease tularaemia. Critical for its pathogenesis is the ability of the phagocytosed bacteria to escape into the cell cytosol. For this, the bacteria use a non-canonical type VI secretion system (T6SS) encoded on the Francisella pathogenicity island (FPI). Here we show that in F. novicida T6SS assembly initiates at the bacterial poles both in vitro and within infected macrophages. T6SS dynamics and function depends on the general purpose ClpB unfoldase, which specifically colocalizes with contracted sheaths and is required for their disassembly. T6SS assembly depends on iglF, iglG, iglI and iglJ, whereas pdpC, pdpD, pdpE and anmK are dispensable. Importantly, strains lacking pdpC and pdpD are unable to escape from phagosome, activate AIM2 inflammasome or cause disease in mice. This suggests that PdpC and PdpD are T6SS effectors involved in phagosome rupture.


Subject(s)
Bacterial Proteins/metabolism , Endopeptidase Clp/metabolism , Francisella tularensis/metabolism , Phagosomes/immunology , Tularemia/microbiology , Type VI Secretion Systems/metabolism , Animals , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Endopeptidase Clp/genetics , Endopeptidase Clp/immunology , Female , Francisella tularensis/genetics , Francisella tularensis/immunology , Gene Expression Regulation, Bacterial , Humans , Mice , Mice, Inbred C57BL , Phagosomes/microbiology , Tularemia/immunology , Type VI Secretion Systems/genetics , Type VI Secretion Systems/immunology
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