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1.
PLoS Pathog ; 18(1): e1010166, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-35007292

RESUMEN

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.


Asunto(s)
Listeria monocytogenes/patogenicidad , Listeriosis/microbiología , Macrófagos/microbiología , Vacuolas/microbiología , Virulencia/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL
2.
PLoS Pathog ; 15(7): e1007959, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31339948

RESUMEN

The enteric bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), utilizes two type III secretion systems (T3SSs) to invade host cells, survive and replicate intracellularly. T3SS1 and its dedicated effector proteins are required for bacterial entry into non-phagocytic cells and establishment and trafficking of the nascent Salmonella-containing vacuole (SCV). Here we identify the first T3SS1 effector required to maintain the integrity of the nascent SCV as SopF. SopF associates with host cell membranes, either when translocated by bacteria or ectopically expressed. Recombinant SopF binds to multiple phosphoinositides in protein-lipid overlays, suggesting that it targets eukaryotic cell membranes via phospholipid interactions. In yeast, the subcellular localization of SopF is dependent on the activity of Mss4, a phosphatidylinositol 4-phosphate 5-kinase that generates PI(4,5)P2 from PI(4)P, indicating that membrane recruitment of SopF requires specific phospholipids. Ectopically expressed SopF partially colocalizes with specific phosphoinositide pools present on the plasma membrane in mammalian cells and with cytoskeletal-associated markers at the leading edge of cells. Translocated SopF concentrates on plasma membrane ruffles and around intracellular bacteria, presumably on the SCV. SopF is not required for bacterial invasion of non-phagocytic cells but is required for maintenance of the internalization vacuole membrane as infection with a S. Typhimurium ΔsopF mutant led to increased lysis of the SCV compared to wild type bacteria. Our structure-function analysis shows that the carboxy-terminal seven amino acids of SopF are essential for its membrane association in host cells and to promote SCV membrane stability. We also describe that SopF and another T3SS1 effector, SopB, act antagonistically to modulate nascent SCV membrane dynamics. In summary, our study highlights that a delicate balance of type III effector activities regulates the stability of the Salmonella internalization vacuole.


Asunto(s)
Salmonella typhimurium/fisiología , Sistemas de Secreción Tipo III/fisiología , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/microbiología , Células HeLa , Interacciones Microbiota-Huesped/fisiología , Humanos , Ratones , Fosfatidilinositoles/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Sistemas de Secreción Tipo III/química , Sistemas de Secreción Tipo III/genética , Vacuolas/metabolismo , Vacuolas/microbiología
3.
PLoS Pathog ; 12(12): e1006101, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28002452

RESUMEN

Coxiella burnetii is an intracellular bacterial pathogen that infects alveolar macrophages and replicates within a unique lysosome-derived vacuole. When Coxiella is trafficked to a host cell lysosome the essential Dot/Icm type IV secretion system is activated allowing over 130 bacterial effector proteins to be translocated into the host cytosol. This cohort of effectors is believed to manipulate host cell functions to facilitate Coxiella-containing vacuole (CCV) biogenesis and bacterial replication. Transposon mutagenesis has demonstrated that the Dot/Icm effector Cig57 is required for CCV development and intracellular replication of Coxiella. Here, we demonstrate a role for Cig57 in subverting clathrin-mediated traffic through its interaction with FCHO2, an accessory protein of clathrin coated pits. A yeast two-hybrid screen identified FCHO2 as a binding partner of Cig57 and this interaction was confirmed during infection using immunoprecipitation experiments. The interaction between Cig57 and FCHO2 is dependent on one of three endocytic sorting motif encoded by Cig57. Importantly, complementation analysis demonstrated that this endocytic sorting motif is required for full function of Cig57. Consistent with the intracellular growth defect in cig57-disrupted Coxiella, siRNA gene silencing of FCHO2 or clathrin (CLTC) inhibits Coxiella growth and CCV biogenesis. Clathrin is recruited to the replicative CCV in a manner that is dependent on the interaction between Cig57 and FCHO2. Creation of an FCHO2 knockout cell line confirmed the importance of this protein for CCV expansion, intracellular replication of Coxiella and clathrin recruitment to the CCV. Collectively, these results reveal Cig57 to be a significant virulence factor that co-opts clathrin-mediated trafficking, via interaction with FCHO2, to facilitate the biogenesis of the fusogenic Coxiella replicative vacuole and enable intracellular success of this human pathogen.


Asunto(s)
Sistemas de Secreción Bacterianos/metabolismo , Coxiella burnetii/metabolismo , Interacciones Huésped-Patógeno/fisiología , Fiebre Q/metabolismo , Factores de Virulencia/metabolismo , Proteínas Bacterianas/metabolismo , Western Blotting , Coxiella burnetii/crecimiento & desarrollo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Microscopía Fluorescente , Transporte de Proteínas , Técnicas del Sistema de Dos Híbridos , Vacuolas/metabolismo
4.
Infect Immun ; 83(10): 4081-92, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26216429

RESUMEN

Legionella organisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed the Legionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis of Legionella pneumophila. Here, we have characterized the Legionella longbeachae replicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV of L. pneumophila (pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deleting dotB and demonstrated the inability of this mutant to replicate inside THP-1 cells. L. longbeachae does not enter THP-1 cells as efficiently as L. pneumophila, and this is reflected in the observation that translocation of BlaM-RalFLLO (where RalFLLO is the L. longbeachae homologue of RalF) into THP-1 cells by the L. longbeachae Dot/Icm system is less efficient than that by L. pneumophila. This difference is negated in A549 cells where L. longbeachae and L. pneumophila infect with similar entry dynamics. A ß-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, the Rab-like effector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on the longbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to the longbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of the L. longbeachae replicative vacuole, highlighting phenotypic similarities to the vacuole of L. pneumophila as well as unique aspects of LCV biology.


Asunto(s)
Proteínas Bacterianas/metabolismo , Legionella longbeachae/metabolismo , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/microbiología , Sistemas de Secreción Tipo IV/metabolismo , Vacuolas/microbiología , Proteínas Bacterianas/genética , Línea Celular , Humanos , Legionella longbeachae/genética , Legionella pneumophila/genética , Transporte de Proteínas , Sistemas de Secreción Tipo IV/genética
5.
Microbiol Mol Biol Rev ; 83(2)2019 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-30814130

RESUMEN

The entry of pathogens into nonphagocytic host cells has received much attention in the past three decades, revealing a vast array of strategies employed by bacteria and viruses. A method of internalization that has been extensively studied in the context of viral infections is the use of the clathrin-mediated pathway. More recently, a role for clathrin in the entry of some intracellular bacterial pathogens was discovered. Classically, clathrin-mediated endocytosis was thought to accommodate internalization only of particles smaller than 150 nm; however, this was challenged upon the discovery that Listeria monocytogenes requires clathrin to enter eukaryotic cells. Now, with discoveries that clathrin is required during other stages of some bacterial infections, another paradigm shift is occurring. There is a more diverse impact of clathrin during infection than previously thought. Much of the recent data describing clathrin utilization in processes such as bacterial attachment, cell-to-cell spread and intracellular growth may be due to newly discovered divergent roles of clathrin in the cell. Not only does clathrin act to facilitate endocytosis from the plasma membrane, but it also participates in budding from endosomes and the Golgi apparatus and in mitosis. Here, the manipulation of clathrin processes by bacterial pathogens, including its traditional role during invasion and alternative ways in which clathrin supports bacterial infection, is discussed. Researching clathrin in the context of bacterial infections will reveal new insights that inform our understanding of host-pathogen interactions and allow researchers to fully appreciate the diverse roles of clathrin in the eukaryotic cell.


Asunto(s)
Clatrina/fisiología , Endocitosis , Células Eucariotas/microbiología , Interacciones Huésped-Patógeno , Animales , Proteínas Bacterianas/genética , Membrana Celular/microbiología , Coxiella burnetii/patogenicidad , Humanos , Listeria monocytogenes/patogenicidad , Shigella flexneri/patogenicidad
6.
Autophagy ; 14(10): 1710-1725, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29973118

RESUMEN

Coxiella burnetii is an intracellular bacterial pathogen which causes Q fever, a human infection with the ability to cause chronic disease with potentially life-threatening outcomes. In humans, Coxiella infects alveolar macrophages where it replicates to high numbers in a unique, pathogen-directed lysosome-derived vacuole. This compartment, termed the Coxiella-containing vacuole (CCV), has a low internal pH and contains markers both of lysosomes and autophagosomes. The CCV membrane is also enriched with CLTC (clathrin heavy chain) and this contributes to the success of the CCV. Here, we describe a role for CLTC, a scaffolding protein of clathrin-coated vesicles, in facilitating the fusion of autophagosomes with the CCV. During gene silencing of CLTC, CCVs are unable to fuse with each other, a phenotype also seen when silencing genes involved in macroautophagy/autophagy. MAP1LC3B/LC3B, which is normally observed inside the CCV, is excluded from CCVs in the absence of CLTC. Additionally, this study demonstrates that autophagosome fusion contributes to CCV size as cell starvation and subsequent autophagy induction leads to further CCV expansion. This is CLTC dependent, as the absence of CLTC renders autophagosomes no longer able to contribute to the expansion of the CCV. This investigation provides a functional link between CLTC and autophagy in the context of Coxiella infection and highlights the CCV as an important tool to explore the interactions between these vesicular trafficking pathways.


Asunto(s)
Autofagia , Cadenas Pesadas de Clatrina/metabolismo , Coxiella/metabolismo , Vacuolas/metabolismo , Autofagosomas/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Fusión de Membrana , Proteínas Asociadas a Microtúbulos/metabolismo , Fenotipo
7.
J Vis Exp ; (113)2016 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-27501079

RESUMEN

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on ß-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.


Asunto(s)
Coxiella burnetii , ARN Interferente Pequeño , Proteínas Bacterianas , Transferencia Resonante de Energía de Fluorescencia , Interacciones Huésped-Patógeno
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