RESUMO
Pathogenic Shigella bacteria are a paradigm to address key issues of cell and infection biology. Polar localisation of the Shigella autotransporter protein IcsA is essential for actin tail formation, which is necessary for the bacterium to travel from cell-to-cell; yet how proteins are targeted to the bacterial cell pole is poorly understood. The bacterial actin homologue MreB has been extensively studied in broth culture using model organisms including Escherichia coli, Bacillus subtilis and Caulobacter crescentus, but has never been visualised in rod-shaped pathogenic bacteria during infection of host cells. Here, using single-cell analysis of intracellular Shigella, we discover that MreB accumulates at the cell pole of bacteria forming actin tails, where it colocalises with IcsA. Pharmacological inhibition of host cell actin polymerisation and genetic deletion of IcsA is used to show, respectively, that localisation of MreB to the cell poles precedes actin tail formation and polar localisation of IcsA. Finally, by exploiting the MreB inhibitors A22 and MP265, we demonstrate that MreB polymerisation can support actin tail formation. We conclude that Shigella MreB promotes polar IcsA positioning for actin tail formation, and suggest that understanding the bacterial cytoskeleton during host-pathogen interactions can inspire development of new therapeutic regimes for infection control.This article has an associated First Person interview with the first author of the paper.
Assuntos
Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Shigella flexneri , Fatores de Transcrição/metabolismo , Citoesqueleto de Actina , Proteínas de Escherichia coli , Células HeLa , Interações entre Hospedeiro e Microrganismos , Humanos , Shigella flexneri/citologia , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidadeRESUMO
Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.
Assuntos
Neutrófilos/imunologia , Antígenos O/imunologia , Shigella sonnei/imunologia , Shigella sonnei/patogenicidade , Virulência/imunologia , Animais , Disenteria Bacilar , Humanos , Peixe-ZebraRESUMO
Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane-bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram-positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid-encoded and secreted virulence-associated protein A (VapA) participates in exclusion of the proton-pumping vacuolar-ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH-neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid-less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH-neutral and hence growth-promoting intracellular niche. VapA represents a new type of Gram-positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton-pumping ATPase, and consequently disarming host defences.
Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Fagossomos/microbiologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Rhodococcus equi/crescimento & desenvolvimento , Rhodococcus equi/metabolismo , Fatores de Virulência/metabolismo , Animais , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , VirulênciaRESUMO
Antimicrobial resistance is a major threat the world is currently facing. Development of new antibiotics and the assessment of their toxicity represent important challenges. Current methods for addressing antibiotic toxicity rely on measuring mitochondrial damage using ATP and/or membrane potential as a readout. In this study, we propose an alternative readout looking at changes in the lipidome on intact and unprocessed cells by matrix-assisted laser desorption ionization mass spectrometry. As a proof of principle, we evaluated the impact of known antibiotics (levofloxacin, ethambutol, and kanamycin) on the lipidome of HeLa cells and mouse bone marrow-derived macrophages. Our methodology revealed that clinically relevant concentrations of kanamycin alter the ratio of cardiolipins to phosphatidylinositols. Unexpectedly, only kanamycin had this effect even though all antibiotics used in this study led to a decrease in the maximal mitochondrial respiratory capacity. Altogether, we report that intact cell-targeted lipidomics can be used as a qualitative method to rapidly assess the toxicity of aminoglycosides in HeLa and primary cells. Moreover, these results demonstrate there is no direct correlation between the ratio of cardiolipins to phosphatidylinositols and the maximal mitochondrial respiratory capacity.
Assuntos
Antibacterianos/farmacologia , Cardiolipinas/metabolismo , Canamicina/farmacologia , Fosfatidilinositóis/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antibacterianos/administração & dosagem , Relação Dose-Resposta a Droga , Etambutol/farmacologia , Células HeLa , Humanos , Canamicina/administração & dosagem , Levofloxacino/farmacologia , Metabolismo dos Lipídeos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single-cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri-infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti-Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria.
Assuntos
Autofagia , Mitocôndrias/metabolismo , Septinas/metabolismo , Shigella/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Humanos , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Ligação ProteicaAssuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Encefalite por Herpes Simples/metabolismo , Fibroblastos/fisiologia , Herpesvirus Humano 1/fisiologia , Mutação/genética , Proteínas Serina-Treonina Quinases/genética , Receptor 3 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Autofagia/genética , Células Cultivadas , Criança , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferons/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Poli I-C/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
The major human bacterial pathogen Pseudomonas aeruginosa causes multidrug-resistant infections in people with underlying immunodeficiencies or structural lung diseases such as cystic fibrosis (CF). We show that a few environmental isolates, driven by horizontal gene acquisition, have become dominant epidemic clones that have sequentially emerged and spread through global transmission networks over the past 200 years. These clones demonstrate varying intrinsic propensities for infecting CF or non-CF individuals (linked to specific transcriptional changes enabling survival within macrophages); have undergone multiple rounds of convergent, host-specific adaptation; and have eventually lost their ability to transmit between different patient groups. Our findings thus explain the pathogenic evolution of P. aeruginosa and highlight the importance of global surveillance and cross-infection prevention in averting the emergence of future epidemic clones.
Assuntos
Fibrose Cística , Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Fibrose Cística/microbiologia , Evolução Molecular , Transferência Genética Horizontal , Adaptação ao Hospedeiro , Especificidade de Hospedeiro , Macrófagos/microbiologia , Macrófagos/imunologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Infecções por Pseudomonas/microbiologia , Interações Hospedeiro-PatógenoRESUMO
Septins are cytoskeletal proteins widely recognized for their role in eukaryotic cell division. Septins also assemble into cage-like structures that entrap cytosolic Shigella flexneri targeted to macroautophagy/autophagy. Although the Shigella septin cage was discovered ~10 y ago, how septins recognize Shigella was poorly understood. We found that septins are recruited to regions of micrometer-scale curvature presented by dividing bacterial cells, and cardiolipin (a curvature-specific phospholipid) promotes septin recruitment to these regions. Chemical manipulation of bacteria revealed that following recruitment, septins assemble into cages around growing bacterial cells. Once assembled, septin cages inhibit Shigella cell division by autophagy and fusion with lysosomes. Thus, recognition of dividing bacterial cells by the septin cytoskeleton targets intracellular pathogens to antibacterial autophagy.
Assuntos
Autofagia , Shigella , Divisão Celular , Citoesqueleto , SeptinasRESUMO
Shigella flexneri, a Gram-negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. During infection of epithelial cells, Shigella escape from the phagosome to the cytosol, where they reroute host cell glycolysis to obtain nutrients for proliferation. Septins, a poorly understood component of the cytoskeleton, can entrap cytosolic Shigella targeted to autophagy in cage-like structures to restrict bacterial proliferation. Although bacterial entrapment by septin caging has been the subject of intense investigation, the role of septins and the autophagy machinery in the proliferation of noncaged Shigella is mostly unknown. Here, we found that intracellular Shigella fail to efficiently proliferate in SEPT2-, SEPT7-, or p62/SQSTM1-depleted cells. Consistent with a failure to proliferate, single cell analysis of bacteria not entrapped in septin cages showed that the number of metabolically active Shigella in septin- or p62-depleted cells is reduced. Targeted metabolomic analysis revealed that host cell glycolysis is dysregulated in septin-depleted cells, suggesting a key role for septins in modulation of glycolysis. Together, these results suggest that septins and the autophagy machinery may regulate metabolic pathways that promote the proliferation of intracellular Shigella not entrapped in septin cages.
Assuntos
Autofagia/fisiologia , Proliferação de Células/fisiologia , Septinas/metabolismo , Shigella/patogenicidade , Autofagia/genética , Proliferação de Células/genética , Células HeLa , Humanos , Septinas/genéticaRESUMO
Septins are cytoskeletal proteins implicated in cytokinesis and host-pathogen interactions. During macroautophagy/autophagy of Shigella flexneri, septins assemble into cage-like structures to entrap actin-polymerizing bacteria and restrict their dissemination. How septins assemble to entrap bacteria is not fully known. We discovered that mitochondria support septin cage assembly to promote autophagy of Shigella. Consistent with roles for the cytoskeleton in mitochondrial dynamics, we showed that DNM1L/DRP1 (dynamin 1 like) can interact with septins to enhance mitochondrial fission. Remarkably, Shigella fragment mitochondria and escape from septin cage entrapment in order to avoid autophagy. These results uncover a close relationship between mitochondria and septin assembly, and identify a new role for mitochondria in bacterial autophagy.
Assuntos
Autofagia , Shigella , Mitocôndrias , Dinâmica Mitocondrial , SeptinasRESUMO
The cytoskeleton occupies a central role in cellular immunity by promoting bacterial sensing and antibacterial functions. Septins are cytoskeletal proteins implicated in various cellular processes, including cell division. Septins also assemble into cage-like structures that entrap cytosolic Shigella, yet how septins recognize bacteria is poorly understood. Here, we discover that septins are recruited to regions of micron-scale membrane curvature upon invasion and division by a variety of bacterial species. Cardiolipin, a curvature-specific phospholipid, promotes septin recruitment to highly curved membranes of Shigella, and bacterial mutants lacking cardiolipin exhibit less septin cage entrapment. Chemically inhibiting cell separation to prolong membrane curvature or reducing Shigella cell growth respectively increases and decreases septin cage formation. Once formed, septin cages inhibit Shigella cell division upon recruitment of autophagic and lysosomal machinery. Thus, recognition of dividing bacterial cells by the septin cytoskeleton is a powerful mechanism to restrict the proliferation of intracellular bacterial pathogens.
Assuntos
Lisossomos/metabolismo , Pseudomonas aeruginosa/fisiologia , Septinas/metabolismo , Shigella flexneri/fisiologia , Staphylococcus aureus/fisiologia , Autofagia , Cardiolipinas/genética , Cardiolipinas/metabolismo , Divisão Celular , Proliferação de Células , Citoesqueleto/metabolismo , Células HeLa , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Septinas/genética , Shigella flexneri/genética , Shigella flexneri/patogenicidade , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidadeRESUMO
Autophagy, an intracellular degradation process, is increasingly recognized as having important roles in host defense. Interactions between Shigella flexneri and the autophagy machinery were first discovered in 2005. Since then, work has shown that multiple autophagy pathways are triggered by S. flexneri, and autophagic responses can have different roles during Shigella infection. Here, we review the interactions between S. flexneri and the autophagy machinery, highlighting that studies using Shigella can reveal the breadth of autophagic responses available to the host.
Assuntos
Autofagia/imunologia , Disenteria Bacilar/patologia , Shigella flexneri/imunologia , Shigella flexneri/patogenicidade , Citoplasma/microbiologia , Disenteria Bacilar/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/imunologia , Inflamação/imunologia , Fagocitose/imunologia , Vacúolos/imunologiaRESUMO
Bdellovibrio bacteriovorus are predatory bacteria that invade and kill a range of Gram-negative bacterial pathogens in natural environments and in vitro [1, 2]. In this study, we investigated Bdellovibrio as an injected, antibacterial treatment in vivo, using zebrafish (Danio rerio) larvae infected with an antibiotic-resistant strain of the human pathogen Shigella flexneri. When injected alone, Bdellovibrio can persist for more than 24 hr in vivo yet exert no pathogenic effects on zebrafish larvae. Bdellovibrio injection of zebrafish containing a lethal dose of Shigella promotes pathogen killing, leading to increased zebrafish survival. Live-cell imaging of infected zebrafish reveals that Shigella undergo rounding induced by the invasive predation from Bdellovibrio in vivo. Furthermore, Shigella-dependent replication of Bdellovibrio was captured inside the zebrafish larvae, indicating active predation in vivo. Bdellovibrio can be engulfed and ultimately eliminated by host neutrophils and macrophages, yet have a sufficient dwell time to prey on pathogens. Experiments in immune-compromised zebrafish reveal that maximal therapeutic benefits of Bdellovibrio result from the synergy of both bacterial predation and host immunity, but that in vivo predation contributes significantly to the survival outcome. Our results demonstrate that successful antibacterial therapy can be achieved via the host immune system working together with bacterial predation by Bdellovibrio. Such cooperation may be important to consider in the fight against antibiotic-resistant infections in vivo.