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1.
Microbiol Res ; 270: 127338, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-36854232

RESUMEN

Autophagy is a crucial innate immune response that clears pathogens intracellularly. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. Here, we reported that dTDP-4-dehydro-ß-ւ-rhamnose reductase (RfbD) was able to enhance bacterial colonization in vivo and in vitro by regulating autophagy. We screened the transposon mutant library of Salmonella Enteritidis strain Z11 by High-Content Analysis System, found that rfbD gene has an effect on autophagy. The Z11ΔrfbD-infected group showed greater expression of LC3-II than the Z11-infected group in HeLa, RAW264.7, and J774A.1 cells. Overall, the survival of Z11ΔrfbD in RAW264.7 cells was reduced after 8 h of infection compared to that of the Z11 wild-type strain. In addition, we observed that inhibition of autophagic flux significantly increased the survival of Z11ΔrfbD in RAW264.7 cells. Mice infection experiments revealed that Z11ΔrfbD virulence was significantly reduced, and bacterial load was reduced in the liver and cecum in mice model, and LC3-II expression was significantly increased. These findings indicate an important role of Salmonella Enteritidis protein as a strategy to suppress autophagy and provides new ideas for manipulating autophagy as a novel strategy to treat infectious diseases.


Asunto(s)
Salmonelosis Animal , Salmonella enteritidis , Animales , Humanos , Ratones , Autofagia/genética , Células HeLa/microbiología , Inmunidad Innata , Células RAW 264.7/microbiología , Salmonella enteritidis/genética , Salmonelosis Animal/microbiología , Virulencia/genética
2.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-33805570

RESUMEN

Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.


Asunto(s)
Corynebacterium/patogenicidad , Células Epiteliales/microbiología , Animales , Línea Celular , Chlorocebus aethiops , Corynebacterium/genética , Corynebacterium/aislamiento & purificación , Infecciones por Corynebacterium/microbiología , Impedancia Eléctrica , Proteínas Fluorescentes Verdes/genética , Células HeLa/microbiología , Interacciones Huésped-Patógeno , Humanos , Larva/microbiología , Lepidópteros/microbiología , Receptor Toll-Like 2/metabolismo , Células Vero/microbiología , Virulencia
3.
Sci Rep ; 9(1): 7694, 2019 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-31118484

RESUMEN

Staphylococcus aureus causes serious medical problems in human and animals. Here we show that S. aureus can compromise host genomic integrity as indicated by bacteria-induced histone H2AX phosphorylation, a marker of DNA double strand breaks (DSBs), in human cervix cancer HeLa and osteoblast-like MG-63 cells. This DNA damage is mediated by alpha phenol-soluble modulins (PSMα1-4), while a specific class of lipoproteins (Lpls), encoded on a pathogenicity island in S. aureus, dampens the H2AX phosphorylation thus counteracting the DNA damage. This DNA damage is mediated by reactive oxygen species (ROS), which promotes oxidation of guanine forming 7,8-dihydro-8-oxoguanine (8-oxoG). DNA damage is followed by the induction of DNA repair that involves the ATM kinase-signaling pathway. An examination of S. aureus strains, isolated from the same patient during acute initial and recurrent bone and joint infections (BJI), showed that recurrent strains produce lower amounts of Lpls, induce stronger DNA-damage and prompt the G2/M transition delay to a greater extent that suggest an involvement of these mechanisms in adaptive processes of bacteria during chronicization. Our findings redefine our understanding of mechanisms of S. aureus-host interaction and suggest that the balance between the levels of PSMα and Lpls expression impacts the persistence of the infection.


Asunto(s)
Daño del ADN , Staphylococcus aureus/patogenicidad , Acetilcisteína/farmacología , Artritis Infecciosa/microbiología , Toxinas Bacterianas/farmacología , Línea Celular Tumoral , Reparación del ADN , Etopósido/farmacología , Puntos de Control de la Fase G2 del Ciclo Celular , Islas Genómicas , Guanina/análogos & derivados , Guanina/metabolismo , Células HeLa/microbiología , Histonas/análisis , Interacciones Huésped-Patógeno , Humanos , Lipoproteínas/farmacología , Osteítis/microbiología , Osteoblastos/microbiología , Estrés Oxidativo , Fosforilación , Procesamiento Proteico-Postraduccional , Especies Reactivas de Oxígeno , Infecciones Estafilocócicas/microbiología
4.
Int J Med Microbiol ; 309(2): 97-107, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30606692

RESUMEN

Autophagy is an evolutionary conserved self-balancing process that plays an important role in maintaining cellular homeostasis via the clearance of damaged organelles and misfolded proteins. Infection-triggered autophagy specifically inhibits the invasion of intracellular bacterial replication and hence protects the cells from microbial infections. It has been reported that Acinetobacter baumannii trigger cell autophagy. However, the role of its virulence protein OmpA remains unclear. Therefore, this study aimed to explore the effects of Acinetobacter baumannii OmpA on cell autophagy and its underlying molecular mechanisms. The results showed that OmpA induced autophagy in HeLa and RAW264.7 cells, increased LC3BII expression, and hindered p62 degradation. Moreover, OmpA triggered incomplete autophagy by interfering the fusion of autophagosomes with lysosomes. Besides, OmpA activated MAPK/JNK signaling pathway and enhanced the phosphorylation levels of JNK, p38, and ERK, c-Jun. Inhibition of JNK signaling pathway suppressed OmpA-induced autophagy in HeLa cells. Ab wild-type strains carrying OmpA triggered incomplete autophagy and resulted in a large number of IL-1ß production. Ab-△OmpA strain (OmpA gene mutation) restored autophagic flux and reduced the accumulation of p62 and the release of IL-1ß in HeLa cells. Rapamycin activated autophagy to inhibit OmpA-induced IL-1ß secretion and protect HeLa cells from inflammatory damage. Collectively, these results suggest that OmpA can induce autophagy in HeLa cells through MAPK/JNK signaling pathway. Pre-treatment with Rapamycin activates autophagy and protects against cell death.


Asunto(s)
Acinetobacter baumannii/inmunología , Autofagia , Proteínas de la Membrana Bacteriana Externa/metabolismo , Células HeLa/inmunología , Células HeLa/microbiología , Sistema de Señalización de MAP Quinasas , Animales , Supervivencia Celular/efectos de los fármacos , Humanos , Ratones , Células RAW 264.7 , Sirolimus/metabolismo
5.
Nat Commun ; 9(1): 3607, 2018 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-30190478

RESUMEN

Innate immunity is the first line of defense against infections. Pathways regulating innate responses can also modulate other processes, including stress resistance and longevity. Increasing evidence suggests a role for the nucleolus in regulating cellular processes implicated in health and disease. Here we show the highly conserved nucleolar protein, fibrillarin, is a vital factor regulating pathogen resistance. Fibrillarin knockdown enhances resistance in C. elegans against bacterial pathogens, higher levels of fibrillarin induce susceptibility to infection. Pathogenic infection reduces nucleolar size, ribsosomal RNA, and fibrillarin levels. Genetic epistasis reveals fibrillarin functions independently of the major innate immunity mediators, suggesting novel mechanisms of pathogen resistance. Bacterial infection also reduces nucleolar size and fibrillarin levels in mammalian cells. Fibrillarin knockdown prior to infection increases intracellular bacterial clearance, reduces inflammation, and enhances cell survival. Collectively, these findings reveal an evolutionarily conserved role of fibrillarin in infection resistance and suggest the nucleolus as a focal point in innate immune responses.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiología , Resistencia a la Enfermedad , Interacciones Huésped-Patógeno , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/microbiología , Nucléolo Celular/patología , Proteínas Cromosómicas no Histona/metabolismo , Enterococcus faecalis/patogenicidad , Evolución Molecular , Femenino , Técnicas de Silenciamiento del Gen , Células HeLa/microbiología , Humanos , Ratones Endogámicos C57BL , Mutación , Pseudomonas aeruginosa/patogenicidad , Staphylococcus aureus/patogenicidad
6.
J Microbiol Methods ; 152: 31-35, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30031738

RESUMEN

To establish a successful infection, microorganisms have developed strategies to invade host cells. One of the most important human pathogens and the greatest cause of urinary tract infections, Escherichia coli, still do not have its invasion mechanisms fully understood. This work aims to present a new approach for detecting bacterial invasion of lineage cells, based on an enzymatic-fluorogenic method. The focus of this technique is the detection of E. coli invasion of HeLa cells, exploring ß-glucuronidase, a specific constitutive enzyme of this bacterium. This enzyme hydrolyses the key substrate of this work, 4-methylumbelliferyl-ß-d-glucuronide (MUG), resulting in a fluorogenic molecule, 4-methylumbelliferone. The fluorescence curve created by this method, analyzed by Tukey statistical test, demonstrated that this detection can be efficiently performed after 5 h incubation with MUG. When testing uropathogenic E. coli and E. coli isolated from human gastrointestinal microbiota, the proposed method presented similar results to those exhibited by plate counting invasion detection. Data examination by Duncan statistical test allowed the creation of an intensity range of bacterial invasion, which is part of the process of results interpretation. Detection by this enzymatic-fluorogenic method, compared to other existing bacterial invasion detection techniques, is less burdensome, more sensitive and allows fast achievement of reliable results.


Asunto(s)
Técnicas Bacteriológicas/métodos , Escherichia coli/aislamiento & purificación , Células HeLa/microbiología , Infecciones Urinarias/diagnóstico , Escherichia coli Uropatógena/aislamiento & purificación , Técnicas de Cultivo de Célula , Recuento de Colonia Microbiana , Colorantes Fluorescentes , Microbioma Gastrointestinal , Glucuronidasa , Humanos , Himecromona/análogos & derivados , Reproducibilidad de los Resultados , Especificidad por Sustrato , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/patogenicidad
7.
Rev Inst Med Trop Sao Paulo ; 59: e84, 2017 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-29267592

RESUMEN

A study with transmission electron microscopy of mycoplasma-contaminated HeLa cells using five cell donors referred to as donors A, B, C, D and E, observations are herein presented. Experiments performed with cells from donors B, C and D, revealed the presence of Mycoplasma hyorhinis after PCR and sequencing experiments. Bacteria probably originated from a cytoplasm with compacted tiny granular particles replacing the normal cytosol territories, or from the contact with the cytoplasm through a clear semi-solid material. The compact granularity (CG) of the cytoplasm was crossed by stripes of smooth and rough endoplasmic reticulum cisternae. Among apparently normal mitochondria, it was noted, in variable proportions, mitochondria with crista-delimited lucent central regions that expand to and occupied the interior of a crista-less organelle, which can undergo fission. Other components of the scenarios of mycoplasma-induced cell demolition are villus-like structures with associated 80-200 nm vesicles and a clear, flexible semi-solid, process-sensitive substance that we named jam-like material. This material coated the cytoplasmic surface, its recesses, irregular protrusions and detached cytoplasmic fragments. It also cushioned forming bacteria. Cyst-like structures were often present in the cytoplasm. Cells, mainly apoptotic, exhibiting ample cytoplasmic sectors with characteristic net-like profile due to adjoined vacuoles, as well as ovoid or elongated profiles, consistently appeared in all cells from the last four cell donors. These cells were named "modified host cells" because bacteria arose in the vacuoles. The possibility that, in some samples, there was infection and/or coinfection of the host cell by another organism(s) cannot be ruled out.


Asunto(s)
Citosol/microbiología , Retículo Endoplásmico/microbiología , Células HeLa/microbiología , Mitocondrias/microbiología , Mycoplasma hyorhinis/aislamiento & purificación , Vacuolas/microbiología , Células Cultivadas , Citosol/patología , ADN Bacteriano , Retículo Endoplásmico/patología , Células HeLa/patología , Humanos , Microscopía Electrónica de Transmisión , Mitocondrias/patología , Reacción en Cadena de la Polimerasa , Estaurosporina/farmacología , Vacuolas/patología
8.
PLoS One ; 12(2): e0172630, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28212413

RESUMEN

Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus. Our new dual-functional plasmid pWRG730 allows for heat-inducible expression of the λ Red recombinase and tet-inducible production of I-SceI. Methyl-accepting chemotaxis proteins (MCP) are transmembrane chemoreceptors for a vast set of environmental signals including amino acids, sugars, ions and oxygen. Based on the sensory input of MCPs, chemotaxis is a key component for Salmonella virulence. To determine the contribution of individual MCPs we sequentially deleted seven MCP genes. The individual mutations were validated by PCR and genetic integrity of the final seven MCP mutant WRG279 was confirmed by whole genome sequencing. The successive MCP mutants were functionally tested in a HeLa cell infection model which revealed increased invasion rates for non-chemotactic mutants and strains lacking the MCP CheM (Tar). The phenotype of WRG279 was reversed with plasmid-based expression of CheM. The complemented WRG279 mutant showed also partially restored chemotaxis in swarming assays on semi-solid agar. Our optimized scarless deletion protocol enables efficient and precise manipulation of the Salmonella genome. As demonstrated with whole genome sequencing, multiple subsequent mutagenesis steps can be realized without the introduction of unwanted mutations. The sequential deletion of seven MCP genes revealed a significant role of CheM for the interaction of S. Typhimurium with host cells which might give new insights into mechanisms of Salmonella host cell sensing.


Asunto(s)
Quimiotaxis/genética , Mutagénesis Sitio-Dirigida/métodos , Salmonella typhimurium/genética , Proteínas Bacterianas/genética , Eliminación de Gen , Genes Bacterianos , Células HeLa/microbiología , Humanos , Polimorfismo de Nucleótido Simple , Infecciones por Salmonella/genética , Infecciones por Salmonella/microbiología , Salmonella typhimurium/patogenicidad
9.
PLoS One ; 11(9): e0161965, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27627128

RESUMEN

Salmonella Typhimurium (S. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and S. TmSipA, a sopBsopE2sopE mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that S. TmSipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the S. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of S. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between S. Tm and SPIRE proteins or to an indirect effect.


Asunto(s)
Proteínas Bacterianas/fisiología , Estudio de Asociación del Genoma Completo/métodos , Interacciones Huésped-Patógeno/fisiología , Proteínas de Microfilamentos/fisiología , Proteínas Nucleares/fisiología , Salmonella typhimurium/patogenicidad , Animales , Línea Celular , Técnica del Anticuerpo Fluorescente , Células HeLa/metabolismo , Células HeLa/microbiología , Humanos , Ratones , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Salmonella typhimurium/fisiología
10.
Cell Microbiol ; 18(7): 982-97, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27282465

RESUMEN

Invasion and multiplication of the facultative, cytosolic, enteropathogen Shigella flexneri within the colonic epithelial lining leads to an acute inflammatory response, fever and diarrhea. During the inflammatory process, infected cells are subjected to numerous stresses including heat, oxidative stress and genotoxic stress. The evolutionarily conserved pathway of cellular stress management is the formation of stress granules that store translationally inactive cellular mRNAs and interfere with cellular signalling pathways by sequestering signalling components. In this study, we investigated the ability of S. flexneri-infected cells to form stress granules in response to exogenous stresses. We found that S. flexneri infection inhibits movement of the stress granule markers eIF3 and eIF4B into stress granules and prevents the aggregation of G3BP1 and eIF4G-containing stress granules. This inhibition occurred only with invasive, but not with non-invasive bacteria and occurred in response to stresses that induce translational arrest through the phosphorylation of eIF2α and by treating cells with pateamine A, a drug that induces stress granules by inhibiting the eIF4A helicase. The S. flexneri-mediated stress granule inhibition could be largely phenocopied by the microtubule-destabilizing drug nocodazole and while S. flexneri infection did not lead to microtubule depolymerization, infection greatly enhanced acetylation of alpha-tubulin. Our data suggest that qualitative differences in the microtubule network or subversion of the microtubule-transport machinery by S. flexneri may be involved in preventing the full execution of this cellular stress response.


Asunto(s)
Interacciones Huésped-Patógeno/fisiología , Shigella flexneri/patogenicidad , Estrés Fisiológico/fisiología , Actinas/metabolismo , Proteínas Portadoras/metabolismo , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas , Disentería Bacilar/metabolismo , Disentería Bacilar/microbiología , Disentería Bacilar/patología , Compuestos Epoxi/farmacología , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/microbiología , Células HeLa/microbiología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Macrólidos/farmacología , Microtúbulos/metabolismo , Mutación , Fosforilación , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Shigella flexneri/efectos de los fármacos , Shigella flexneri/genética , Tiazoles/farmacología
11.
Protein Sci ; 25(5): 1037-48, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26947936

RESUMEN

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents.


Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Shigella flexneri/patogenicidad , Adenosina Trifosfatasas/genética , Dominio Catalítico , Cromatografía en Gel , Células HeLa/microbiología , Humanos , Mutación Puntual , Multimerización de Proteína , Shigella flexneri/genética , Shigella flexneri/metabolismo , Sistemas de Secreción Tipo III/química , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo
12.
Cell Microbiol ; 18(3): 305-18, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26381674

RESUMEN

Phosphatidylcholine is a constituent of Chlamydia trachomatis membranes that must be acquired from its mammalian host to support bacterial proliferation. The CLA1 (SR-B1) receptor is a bi-directional phosphatidylcholine/cholesterol transporter that is recruited to the inclusion of Chlamydia-infected cells along with ABCA1. C. trachomatis growth was inhibited in a dose-dependent manner by BLT-1, a selective inhibitor of CLA1 function. Expression of a BLT-1-insensitive CLA1(C384S) mutant ameliorated the effect of the drug on chlamydial growth. CLA1 knockdown using shRNAs corroborated an important role for CLA1 in the growth of C. trachomatis. Trafficking of a fluorescent phosphatidylcholine analogue to Chlamydia was blocked by the inhibition of CLA1 or ABCA1 function, indicating a critical role for these transporters in phosphatidylcholine acquisition by this organism. Our analyses using a dual-labelled fluorescent phosphatidylcholine analogue and mass spectrometry showed that the phosphatidylcholine associated with isolated Chlamydia was unmodified host phosphatidylcholine. These results indicate that C. trachomatis co-opts host phospholipid transporters normally used to assemble lipoproteins to acquire host phosphatidylcholine essential for growth.


Asunto(s)
Chlamydia trachomatis/crecimiento & desarrollo , Interacciones Huésped-Patógeno , Fosfatidilcolinas/metabolismo , Receptores Depuradores de Clase B/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Membrana Celular/metabolismo , Chlamydia trachomatis/efectos de los fármacos , Chlamydia trachomatis/patogenicidad , Ciclopentanos/farmacología , Células HeLa/efectos de los fármacos , Células HeLa/microbiología , Humanos , Receptores Depuradores de Clase B/genética , Esfingomielinas/metabolismo , Tiosemicarbazonas/farmacología
13.
Cell Microbiol ; 18(7): 949-69, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-26676327

RESUMEN

Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.


Asunto(s)
Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno/fisiología , Fosfatos de Fosfatidilinositol/metabolismo , Salmonella typhimurium/metabolismo , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Membrana Celular/microbiología , Células HeLa/microbiología , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/patogenicidad , Vacuolas/metabolismo , Factores de Virulencia/genética
14.
PLoS One ; 10(6): e0131220, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26098675

RESUMEN

Healthy vaginal microbiota is dominated by Lactobacillus spp., which form a critical line of defence against pathogens, including Candida spp. The present study aims to identify vaginal lactobacilli exerting in vitro activity against Candida spp. and to characterize their antifungal mechanisms of action. Lactobacillus strains were isolated from vaginal swabs of healthy premenopausal women. The isolates were taxonomically identified to species level (L. crispatus B1-BC8, L. gasseri BC9-BC14 and L. vaginalis BC15-BC17) by sequencing the 16S rRNA genes. All strains produced hydrogen peroxide and lactate. Fungistatic and fungicidal activities against C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis and C. lusitaniae were evaluated by broth micro-dilution method. The broadest spectrum of activity was observed for L. crispatus BC1, BC4, BC5 and L. vaginalis BC15, demonstrating fungicidal activity against all isolates of C. albicans and C. lusitaniae. Metabolic profiles of lactobacilli supernatants were studied by 1H-NMR analysis. Metabolome was found to be correlated with both taxonomy and activity score. Exclusion, competition and displacement experiments were carried out to investigate the interference exerted by lactobacilli toward the yeast adhesion to HeLa cells. Most Lactobacillus strains significantly reduced C. albicans adhesion through all mechanisms. In particular, L. crispatus BC2, L. gasseri BC10 and L. gasseri BC11 appeared to be the most active strains in reducing pathogen adhesion, as their effects were mediated by both cells and supernatants. Inhibition of histone deacetylases was hypothesised to support the antifungal activity of vaginal lactobacilli. Our results are prerequisites for the development of new therapeutic agents based on probiotics for prophylaxis and adjuvant therapy of Candida infection.


Asunto(s)
Candidiasis Vulvovaginal/microbiología , Lactobacillus/aislamiento & purificación , Vagina/microbiología , Adolescente , Adulto , Antifúngicos/metabolismo , Secuencia de Bases , Candida/fisiología , Femenino , Células HeLa/microbiología , Humanos , Peróxido de Hidrógeno/metabolismo , Lactobacillus/genética , Lactobacillus/fisiología , Espectroscopía de Resonancia Magnética , Metabolómica , Microbiota/fisiología , Persona de Mediana Edad , Datos de Secuencia Molecular , Adulto Joven
15.
Infect Genet Evol ; 32: 74-88, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25745888

RESUMEN

Lymphogranuloma venereum (LGV) is a human sexually transmitted disease caused by the obligate intracellular bacterium Chlamydia trachomatis (serovars L1-L3). LGV clinical manifestations range from severe ulcerative proctitis (anorectal syndrome), primarily caused by the epidemic L2b strains, to painful inguinal lymphadenopathy (the typical LGV bubonic form). Besides potential host-related factors, the differential disease severity and tissue tropism among LGV strains is likely a function of the genetic backbone of the strains. We aimed to characterize the genetic variability among LGV strains as strain- or serovar-specific mutations may underlie phenotypic signatures, and to investigate the mutational events that occurred throughout the pathoadaptation of the epidemic L2b lineage. By analyzing 20 previously published genomes from L1, L2, L2b and L3 strains and two new genomes from L2b strains, we detected 1497 variant sites and about 100 indels, affecting 453 genes and 144 intergenic regions, with 34 genes displaying a clear overrepresentation of nonsynonymous mutations. Effectors and/or type III secretion substrates (almost all of those described in the literature) and inclusion membrane proteins showed amino acid changes that were about fivefold more frequent than silent changes. More than 120 variant sites occurred in plasmid-regulated virulence genes, and 66% yielded amino acid changes. The identified serovar-specific variant sites revealed that the L2b-specific mutations are likely associated with higher fitness and pointed out potential targets for future highly discriminatory diagnostic/typing tests. By evaluating the evolutionary pathway beyond the L2b clonal radiation, we observed that 90.2% of the intra-L2b variant sites occurring in coding regions involve nonsynonymous mutations, where CT456/tarp has been the main target. Considering the progress on C. trachomatis genetic manipulation, this study may constitute an important contribution for prioritizing study targets for functional genomics aiming to dissect the impact of the identified intra-LGV polymorphisms on virulence or tropism dissimilarities among LGV strains.


Asunto(s)
Chlamydia trachomatis/genética , Genes Bacterianos/genética , Linfogranuloma Venéreo/microbiología , Secuencia de Bases , Chlamydia trachomatis/aislamiento & purificación , Chlamydia trachomatis/patogenicidad , Genes Bacterianos/fisiología , Variación Genética/genética , Genoma Bacteriano/genética , Células HeLa/microbiología , Humanos , Datos de Secuencia Molecular , Mutación/genética , Alineación de Secuencia , Virulencia/genética
18.
Nat Commun ; 5: 5201, 2014 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-25392082

RESUMEN

Chlamydia, a major human bacterial pathogen, assumes effective strategies to protect infected cells against death-inducing stimuli, thereby ensuring completion of its developmental cycle. Paired with its capacity to cause extensive host DNA damage, this poses a potential risk of malignant transformation, consistent with circumstantial epidemiological evidence. Here we reveal a dramatic depletion of p53, a tumor suppressor deregulated in many cancers, during Chlamydia infection. Using biochemical approaches and live imaging of individual cells, we demonstrate that p53 diminution requires phosphorylation of Murine Double Minute 2 (MDM2; a ubiquitin ligase) and subsequent interaction of phospho-MDM2 with p53 before induced proteasomal degradation. Strikingly, inhibition of the p53-MDM2 interaction is sufficient to disrupt intracellular development of Chlamydia and interferes with the pathogen's anti-apoptotic effect on host cells. This highlights the dependency of the pathogen on a functional MDM2-p53 axis and lends support to a potentially pro-carcinogenic effect of chlamydial infection.


Asunto(s)
Infecciones por Chlamydia/metabolismo , Chlamydia trachomatis/patogenicidad , Proteínas Proto-Oncogénicas c-mdm2/fisiología , Proteína p53 Supresora de Tumor/fisiología , Apoptosis/fisiología , Western Blotting , Transformación Celular Neoplásica/metabolismo , Chlamydia/patogenicidad , Chlamydia/fisiología , Infecciones por Chlamydia/fisiopatología , Chlamydia trachomatis/fisiología , Células HeLa/microbiología , Humanos , Fosforilación
19.
Biol Res ; 47: 38, 2014 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-25299837

RESUMEN

BACKGROUND: Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. RESULTS: The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. CONCLUSIONS: The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.


Asunto(s)
Apoptosis/fisiología , Infecciones por Ureaplasma/fisiopatología , Ureaplasma/patogenicidad , Citoesqueleto de Actina/ultraestructura , Adhesión Bacteriana , Caspasa 2/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Supervivencia Celular , Femenino , Citometría de Flujo , Expresión Génica , Gentamicinas/farmacología , Células HeLa/microbiología , Humanos , Microscopía Confocal , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Estadísticas no Paramétricas , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Ureaplasma/efectos de los fármacos
20.
Nat Commun ; 5: 4718, 2014 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-25146723

RESUMEN

Increasing evidence suggests an important role for miRNAs in the molecular interplay between bacterial pathogens and host cells. Here we perform a fluorescence microscopy-based screen using a library of miRNA mimics and demonstrate that miRNAs modulate Salmonella infection. Several members of the miR-15 miRNA family were among the 17 miRNAs that more efficiently inhibit Salmonella infection. We discovered that these miRNAs are downregulated during Salmonella infection, through the inhibition of the transcription factor E2F1. Analysis of miR-15 family targets revealed that derepression of cyclin D1 and the consequent promotion of G1/S transition are crucial for Salmonella intracellular proliferation. In addition, Salmonella induces G2/M cell cycle arrest in infected cells, further promoting its replication. Overall, these findings uncover a mechanism whereby Salmonella renders host cells more susceptible to infection by controlling cell cycle progression through the active modulation of host cell miRNAs.


Asunto(s)
Interacciones Huésped-Patógeno/genética , MicroARNs/genética , Infecciones por Salmonella/genética , Animales , Puntos de Control del Ciclo Celular , Ciclina D1/genética , Factor de Transcripción E2F1/genética , Regulación de la Expresión Génica , Células HeLa/efectos de los fármacos , Células HeLa/microbiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Lipopolisacáridos/farmacología , Ratones , Familia de Multigenes , Células RAW 264.7/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad
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