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1.
J Infect Dis ; 222(3): 372-380, 2020 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-31605125

RESUMEN

Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model.


Asunto(s)
Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/inmunología , Serogrupo , Streptococcus/clasificación , Vacunas Conjugadas/inmunología , Administración Oral , Animales , Cápsulas Bacterianas/inmunología , Femenino , Ratones , Ratones Endogámicos BALB C , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/microbiología , Vacunas Neumococicas/administración & dosificación , Polisacáridos Bacterianos/inmunología , Streptococcus/inmunología , Virulencia
2.
J Infect Dis ; 222(11): 1882-1893, 2020 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-32492702

RESUMEN

BACKGROUND: Streptococcus pneumoniae infection can result in bacteremia with devastating consequences including heart damage. Necroptosis is a proinflammatory form of cell death instigated by pore-forming toxins such as S. pneumoniae pneumolysin. Necroptosis-inhibiting drugs may lessen organ damage during invasive pneumococcal disease (IPD). METHODS: In vitro experiments were carried out with human and mouse cardiomyocytes. Long-term cardiac damage was assessed using high-resolution echocardiography in ampicillin-rescued mice 3 months after challenge with S. pneumoniae. Ponatinib, a necroptosis-inhibiting and Food and Drug Administration-approved drug for lymphocytic leukemia treatment, was administered intraperitoneally alongside ampicillin to test its therapeutic efficacy. Histology of heart sections included hematoxylin-eosin staining for overt damage, immunofluorescence for necroptosis, and Sirius red/fast green staining for collagen deposition. RESULTS: Cardiomyocyte death and heart damage was due to pneumolysin-mediated necroptosis. IPD leads to long-term cardiac damage, as evidenced by de novo collagen deposition in mouse hearts and a decrease in fractional shortening. Adjunct necroptosis inhibition reduced the number of S. pneumoniae foci observed in hearts of acutely infected mice and serum levels of troponin I. Ponatinib reduced collagen deposition and protected heart function in convalescence. CONCLUSIONS: Acute and long-term cardiac damage incurred during IPD is due in part to cardiomyocyte necroptosis. Necroptosis inhibitors may be a viable adjunct therapy.


Asunto(s)
Corazón , Necroptosis , Neumonía Neumocócica/complicaciones , Animales , Bacteriemia , Muerte Celular , Modelos Animales de Enfermedad , Femenino , Imidazoles , Leucemia/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Infecciones Neumocócicas , Proteínas Quinasas , Piridazinas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Streptococcus pneumoniae
3.
J Bacteriol ; 201(21)2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31405914

RESUMEN

Streptococcus pneumoniae rapidly kills Staphylococcus aureus by producing membrane-permeable hydrogen peroxide (H2O2). The mechanism by which S. pneumoniae-produced H2O2 mediates S. aureus killing was investigated. An in vitro model that mimicked S. pneumoniae-S. aureus contact during colonization of the nasopharynx demonstrated that S. aureus killing required outcompeting densities of S. pneumoniae Compared to the wild-type strain, isogenic S. pneumoniae ΔlctO and S. pneumoniae ΔspxB, both deficient in production of H2O2, required increased density to kill S. aureus While residual H2O2 activity produced by single mutants was sufficient to eradicate S. aureus, an S. pneumoniae ΔspxB ΔlctO double mutant was unable to kill S. aureus A collection of 20 diverse methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains showed linear sensitivity (R2 = 0.95) for S. pneumoniae killing, but the same strains had different susceptibilities when challenged with pure H2O2 (5 mM). There was no association between the S. aureus clonal complex and sensitivity to either S. pneumoniae or H2O2 To kill S. aureus, S. pneumoniae produced ∼180 µM H2O2 within 4 h of incubation, while the killing-defective S. pneumoniae ΔspxB and S. pneumoniae ΔspxB ΔlctO mutants produced undetectable levels. Remarkably, a sublethal dose (1 mM) of pure H2O2 incubated with S. pneumoniae ΔspxB eradicated diverse S. aureus strains, suggesting that S. pneumoniae bacteria may facilitate conversion of H2O2 to a hydroxyl radical (·OH). Accordingly, S. aureus killing was completely blocked by incubation with scavengers of ·OH radicals, dimethyl sulfoxide (Me2SO), thiourea, or sodium salicylate. The ·OH was detected in S. pneumoniae cells by spin trapping and electron paramagnetic resonance. Therefore, S. pneumoniae produces H2O2, which is rapidly converted to a more potent oxidant, hydroxyl radicals, to rapidly intoxicate S. aureus strains.IMPORTANCEStreptococcus pneumoniae strains produce hydrogen peroxide (H2O2) to kill bacteria in the upper airways, including pathogenic Staphylococcus aureus strains. The targets of S. pneumoniae-produced H2O2 have not been discovered, in part because of a lack of knowledge about the underlying molecular mechanism. We demonstrated that an increased density of S. pneumoniae kills S. aureus by means of H2O2 produced by two enzymes, SpxB and LctO. We discovered that SpxB/LctO-produced H2O2 is converted into a hydroxyl radical (·OH) that rapidly intoxicates and kills S. aureus We successfully inhibited the toxicity of ·OH with three different scavengers and detected ·OH in the supernatant. The target(s) of the hydroxyl radicals represents a new alternative for the development of antimicrobials against S. aureus infections.


Asunto(s)
Peróxido de Hidrógeno/metabolismo , Radical Hidroxilo/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Streptococcus pneumoniae/metabolismo , Nasofaringe/metabolismo , Infecciones Estafilocócicas/microbiología
4.
PLoS Pathog ; 13(7): e1006495, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28704569

RESUMEN

Neisseria meningitidis is a commensal of human nasopharynx. In some circumstances, this bacteria can invade the bloodstream and, after crossing the blood brain barrier, the meninges. A filamentous phage, designated MDAΦ for Meningococcal Disease Associated, has been associated with invasive disease. In this work we show that the prophage is not associated with a higher virulence during the bloodstream phase of the disease. However, looking at the interaction of N. meningitidis with epithelial cells, a step essential for colonization of the nasopharynx, we demonstrate that the presence of the prophage, via the production of viruses, increases colonization of encapsulated meningococci onto monolayers of epithelial cells. The analysis of the biomass covering the epithelial cells revealed that meningococci are bound to the apical surface of host cells by few layers of heavily piliated bacteria, whereas, in the upper layers, bacteria are non-piliated but surrounded by phage particles which (i) form bundles of filaments, and/or (ii) are in some places associated with bacteria. The latter are likely to correspond to growing bacteriophages during their extrusion through the outer membrane. These data suggest that, as the biomass increases, the loss of piliation in the upper layers of the biomass does not allow type IV pilus bacterial aggregation, but is compensated by a large production of phage particles that promote bacterial aggregation via the formation of bundles of phage filaments linked to the bacterial cell walls. We propose that MDAΦ by increasing bacterial colonization in the mucosa at the site-of-entry, increase the occurrence of diseases.


Asunto(s)
Inovirus/fisiología , Infecciones Meningocócicas/microbiología , Neisseria meningitidis/patogenicidad , Neisseria meningitidis/virología , Animales , Adhesión Bacteriana , Células Epiteliales/microbiología , Femenino , Fimbrias Bacterianas/fisiología , Humanos , Ratones , Ratones SCID , Nasofaringe/microbiología , Neisseria meningitidis/crecimiento & desarrollo , Neisseria meningitidis/fisiología , Profagos/fisiología , Virulencia
5.
PLoS Pathog ; 13(8): e1006582, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28841717

RESUMEN

For over 130 years, invasive pneumococcal disease has been associated with the presence of extracellular planktonic pneumococci, i.e. diplococci or short chains in affected tissues. Herein, we show that Streptococcus pneumoniae that invade the myocardium instead replicate within cellular vesicles and transition into non-purulent biofilms. Pneumococci within mature cardiac microlesions exhibited salient biofilm features including intrinsic resistance to antibiotic killing and the presence of an extracellular matrix. Dual RNA-seq and subsequent principal component analyses of heart- and blood-isolated pneumococci confirmed the biofilm phenotype in vivo and revealed stark anatomical site-specific differences in virulence gene expression; the latter having major implications on future vaccine antigen selection. Our RNA-seq approach also identified three genomic islands as exclusively expressed in vivo. Deletion of one such island, Region of Diversity 12, resulted in a biofilm-deficient and highly inflammogenic phenotype within the heart; indicating a possible link between the biofilm phenotype and a dampened host-response. We subsequently determined that biofilm pneumococci released greater amounts of the toxin pneumolysin than did planktonic or RD12 deficient pneumococci. This allowed heart-invaded wildtype pneumococci to kill resident cardiac macrophages and subsequently subvert cytokine/chemokine production and neutrophil infiltration into the myocardium. This is the first report for pneumococcal biofilm formation in an invasive disease setting. We show that biofilm pneumococci actively suppress the host response through pneumolysin-mediated immune cell killing. As such, our findings contradict the emerging notion that biofilm pneumococci are passively immunoquiescent.


Asunto(s)
Biopelículas , Macrófagos/inmunología , Miocarditis/inmunología , Miocarditis/microbiología , Infecciones Neumocócicas/inmunología , Transcriptoma , Animales , Western Blotting , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Infecciones Neumocócicas/genética , Análisis de Componente Principal , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/inmunología , Virulencia/genética , Virulencia/inmunología
6.
Infect Immun ; 86(1)2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29061707

RESUMEN

Streptococcus pneumoniae (the pneumococcus) is the leading cause of community-acquired pneumonia and is now recognized to be a direct contributor to adverse acute cardiac events. During invasive pneumococcal disease, S. pneumoniae can gain access to the myocardium, kill cardiomyocytes, and form bacterium-filled "microlesions" causing considerable acute and long-lasting cardiac damage. While the molecular mechanisms responsible for bacterial translocation into the heart have been elucidated, the initial interactions of heart-invaded S. pneumoniae with cardiomyocytes remain unclear. In this study, we used a model of low multiplicity of S. pneumoniae infection with HL-1 mouse cardiomyocytes to investigate these early events. Using adhesion/invasion assays and immunofluorescent and transmission electron microscopy, we showed that S. pneumoniae rapidly adhered to and invaded cardiomyocytes. What is more, pneumococci existed as intravacuolar bacteria or escaped into the cytoplasm. Pulse-chase assays with BrdU confirmed intracellular replication of pneumococci within HL-1 cells. Using endocytosis inhibitors, bacterial isogenic mutants, and neutralizing antibodies against host proteins recognized by S. pneumoniae adhesins, we showed that S. pneumoniae uptake by cardiomyocytes is not through the well-studied canonical interactions identified for vascular endothelial cells. Indeed, S. pneumoniae invasion of HL-1 cells occurred through clathrin-mediated endocytosis (CME) and independently of choline binding protein A (CbpA)/laminin receptor, CbpA/polymeric immunoglobulin receptor, or cell wall phosphorylcholine/platelet-activating factor receptor. Subsequently, we determined that pneumolysin and streptococcal pyruvate oxidase-derived H2O2 production were required for cardiomyocyte killing. Finally, we showed that this cytotoxicity could be abrogated using CME inhibitors or antioxidants, attesting to intracellular replication of S. pneumoniae as a key first step in pneumococcal pathogenesis within the heart.


Asunto(s)
Peróxido de Hidrógeno , Miocitos Cardíacos/microbiología , Infecciones Neumocócicas/microbiología , Piruvato Oxidasa/metabolismo , Streptococcus pneumoniae , Animales , Proteínas Bacterianas/metabolismo , Supervivencia Celular/efectos de los fármacos , Femenino , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Infecciones Neumocócicas/inmunología , Estreptolisinas/metabolismo
7.
Mol Microbiol ; 98(3): 518-34, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26192619

RESUMEN

Intracellular multiplication and dissemination of the infectious bacterial pathogen Francisella tularensis implies the utilization of multiple host-derived nutrients. Here, we demonstrate that gluconeogenesis constitutes an essential metabolic pathway in Francisella pathogenesis. Indeed, inactivation of gene glpX, encoding the unique fructose 1,6-bisphosphatase of Francisella, severely impaired bacterial intracellular multiplication when cells were supplemented by gluconeogenic substrates such as glycerol or pyruvate. The ΔglpX mutant also showed a severe virulence defect in the mouse model, confirming the importance of this pathway during the in vivo life cycle of the pathogen. Isotopic profiling revealed the major role of the Embden-Meyerhof (glycolysis) pathway in glucose catabolism in Francisella and confirmed the importance of glpX in gluconeogenesis. Altogether, the data presented suggest that gluconeogenesis allows Francisella to cope with the limiting glucose availability it encounters during its infectious cycle by relying on host amino acids. Hence, targeting the gluconeogenic pathway might constitute an interesting therapeutic approach against this pathogen.


Asunto(s)
Francisella tularensis/metabolismo , Animales , Femenino , Francisella tularensis/genética , Francisella tularensis/patogenicidad , Genes Bacterianos , Gluconeogénesis , Células Hep G2 , Humanos , Espectrometría de Masas , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos BALB C , Tularemia/microbiología , Virulencia
8.
Microbiology (Reading) ; 162(2): 268-282, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26602366

RESUMEN

The mechanism by which Neisseria meningitidis becomes invasive is not well understood. Comparative genomics identified the presence of an 8 kb island in strains belonging to invasive clonal complexes. This island was designated MDA for meningococcal disease associated. MDA is highly conserved among meningococcal isolates and its analysis revealed a genomic organization similar to that of a filamentous prophage such as CTXΦ of Vibrio cholerae. Subsequent molecular investigations showed that the MDA island has indeed the characteristics of a filamentous prophage, which can enter into a productive cycle and is secreted using the type IV pilus (tfp) secretin PilQ. At least three genes of the prophage are necessary for the formation of the replicative cytoplasmic form (orf1, orf2 and orf9). Immunolabelling of the phage with antibodies against the major capsid protein, ORF4, confirmed that filamentous particles, about 1200 nm long, covered with ORF4 are present at the bacterial surface forming bundles in some places and interacting with pili. The MDA bacteriophage is able to infect different N. meningitidis strains, using the type IV pili as a receptor via an interaction with the adsorption protein ORF6. Altogether, these data demonstrate that the MDA island encodes a functional prophage able to produce infectious filamentous phage particles.


Asunto(s)
Sitios de Ligazón Microbiológica/genética , Inovirus/genética , Neisseria meningitidis/genética , Neisseria meningitidis/virología , Profagos/genética , Receptores Virales/genética , Secuencia de Bases , ADN Viral/genética , Fimbrias Bacterianas/virología , Infecciones Meningocócicas/microbiología , Neisseria meningitidis/patogenicidad , Análisis de Secuencia de ADN
9.
Microlife ; 5: uqae014, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38993744

RESUMEN

Streptococcus agalactiae is among the few pathogens that have not developed resistance to ß-lactam antibiotics despite decades of clinical use. The molecular basis of this long-lasting susceptibility has not been investigated, and it is not known whether specific mechanisms constrain the emergence of resistance. In this study, we first report ß-lactam tolerance due to the inactivation of the c-di-AMP phosphodiesterase GdpP. Mechanistically, tolerance depends on antagonistic regulation by the repressor BusR, which is activated by c-di-AMP and negatively regulates ß-lactam susceptibility through the BusAB osmolyte transporter and the AmaP/Asp23/GlsB cell envelope stress complex. The BusR transcriptional response is synergistic with the simultaneous allosteric inhibition of potassium and osmolyte transporters by c-di-AMP, which individually contribute to low-level ß-lactam tolerance. Genome-wide transposon mutagenesis confirms the role of GdpP and highlights functional interactions between a lysozyme-like hydrolase, the KhpAB RNA chaperone and the protein S immunomodulator in the response of GBS to ß-lactam. Overall, we demonstrate that c-di-AMP acts as a turgor pressure rheostat, coordinating an integrated response at the transcriptional and post-translational levels to cell wall weakening caused by ß-lactam activity, and reveal additional mechanisms that could foster resistance.

10.
FEBS J ; 290(11): 2968-2992, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36629470

RESUMEN

Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.


Asunto(s)
Proteínas Portadoras , Streptococcus agalactiae , Streptococcus agalactiae/genética , Streptococcus agalactiae/metabolismo , Guanosina Pentafosfato , Guanosina Tetrafosfato , Proteínas Bacterianas/metabolismo , AMP Cíclico , Fosfatos de Dinucleósidos/metabolismo , Potasio/metabolismo
11.
Infect Immun ; 80(9): 3297-306, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22778100

RESUMEN

Neisseria meningitidis crosses the blood-brain barrier (BBB) following the activation of the ß2-adrenergic receptor by the type IV pili (TFP). Two components of the type IV pili recruit the ß2-adrenergic receptor, the major pilin PilE and the minor pilin PilV. Here, we report that a strain deleted of PilX, one of the three minor pilins, is defective in endothelial cell signaling. The signaling role of PilX was abolished when pili were not retractable. Purified PilX was unable to recruit the ß2-adrenergic receptor, thus suggesting that PilX was playing an indirect role in endothelial cell signaling. Considering the recent finding that type IV pili can transition into a new conformation (N. Biais, D. L. Higashi, J. Brujic, M. So, and M. P. Sheetz, Proc. Natl. Acad. Sci. U. S. A. 107:11358-11363, 2010), we hypothesized that PilX was responsible for a structural modification of the fiber and allowed hidden epitopes to be exposed. To confirm this hypothesis, we showed that a monoclonal antibody which recognizes a linear epitope of PilE bound fibers only when bacteria adhered to endothelial cells. On the other hand, this effect was not observed in PilX-deleted pili. A deletion of a region of PilX exposed on the surface of the fiber had phenotypical consequences identical to those of a PilX deletion. These data support a model in which surface-exposed motifs of PilX use forces generated by pilus retraction to promote conformational changes required for TFP-mediated signaling.


Asunto(s)
Células Endoteliales/microbiología , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Sustancias Macromoleculares/metabolismo , Neisseria meningitidis/fisiología , Transducción de Señal , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Epítopos/inmunología , Proteínas Fimbrias/genética , Fimbrias Bacterianas/química , Fimbrias Bacterianas/ultraestructura , Eliminación de Gen , Humanos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/ultraestructura , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Neisseria meningitidis/patogenicidad , Receptores Adrenérgicos beta 2/metabolismo , Coloración y Etiquetado/métodos
12.
mBio ; 12(5): e0251621, 2021 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-34634940

RESUMEN

The polysaccharide capsule that surrounds Streptococcus pneumoniae (Spn) is one of its most important virulence determinants, serving to protect against phagocytosis. To date, 100 biochemical and antigenically distinct capsule types, i.e., serotypes, of Spn have been identified. Yet how capsule influences pneumococcal translocation across vascular endothelial cells (VEC), a key step in the progression of invasive disease, was unknown. Here, we show that despite capsule being inhibitory of Spn uptake by VEC, capsule enhances the escape rate of internalized pneumococci and thereby promotes translocation. Upon investigation, we determined that capsule protected Spn against intracellular killing by VEC and H2O2-mediated killing in vitro. Using a nitroblue tetrazolium reduction assay and nuclear magnetic resonance (NMR) analyses, purified capsule was confirmed as having antioxidant properties which varied according to serotype. Using an 11-member panel of isogenic capsule-switch mutants, we determined that serotype affected levels of Spn resistance to H2O2-mediated killing in vitro, with killing resistance correlated positively with survival duration within VEC, rate of transcytosis to the basolateral surface, and human attack rates. Experiments with mice supported our in vitro findings, with Spn producing oxidative-stress-resistant type 4 capsule being more organ-invasive than that producing oxidative-stress-sensitive type 2 capsule during bacteremia. Capsule-mediated protection against intracellular killing was also observed for Streptococcus pyogenes and Staphylococcus aureus. We conclude that capsular polysaccharide plays an important role within VEC, serving as an intracellular antioxidant, and that serotype-dependent differences in antioxidant capabilities impact the efficiency of VEC translocation and a serotype's potential for invasive disease. IMPORTANCE Streptococcus pneumoniae (Spn) is the leading cause of invasive disease. Importantly, only a subset of the 100 capsule types carried by Spn cause the majority of serious infections, suggesting that the biochemical properties of capsular polysaccharide are directly tied to virulence. Here, we describe a new function for Spn's capsule-conferring resistance to oxidative stress. Moreover, we demonstrate that capsule promotes intracellular survival of pneumococci within vascular endothelial cells and thereby enhances bacterial translocation across the vasculature and into organs. Using isogenic capsule-switch mutants, we show that different capsule types, i.e., serotypes, vary in their resistance to oxidative stress-mediated killing and that resistance is positively correlated with intracellular survival in an in vitro model, organ invasion during bacteremia in vivo, and epidemiologically established pneumococcal attack rates in humans. Our findings define a new role of capsule and provide an explanation for why certain serotypes of Spn more frequently cause invasive pneumococcal disease.


Asunto(s)
Cápsulas Bacterianas/fisiología , Traslocación Bacteriana , Células Endoteliales/microbiología , Streptococcus pneumoniae/fisiología , Streptococcus pneumoniae/patogenicidad , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Viabilidad Microbiana , Estrés Oxidativo , Fagocitosis , Infecciones Neumocócicas/microbiología , Virulencia , Factores de Virulencia
13.
Environ Microbiol ; 12(8): 2371-83, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21966926

RESUMEN

Archaea may be involved in global energy cycles, and are known for their ability to interact with eukaryotic species (sponges, corals and ascidians) or as archaeal-bacterial consortia. The recently proposed phylum Thaumarchaeota may represent the deepest branching lineage in the archaeal phylogeny emerging before the divergence between Euryarchaeota and Crenarchaeota. Here we report the first characterization of two marine thaumarchaeal species from shallow waters that consist of multiple giant cells. One species is coated with sulfur-oxidizing γ-Proteobacteria. These new uncultured thaumarchaeal species are able to live in the sulfide-rich environments of a tropical mangrove swamp, either on living tissues such as roots or on various kinds of materials such as stones, sunken woods, etc. These archaea and archaea/bacteria associations have been studied using light microscopy, transmission electron microscopy and scanning electron microscopy. Species identification of archaeons and the putative bacterial symbiont have been assessed by 16S small subunit ribosomal RNA analysis. The sulfur-oxidizing ability of the bacteria has been assessed by genetic investigation on alpha-subunit of the adenosine-5'-phosphosulfate reductase/oxidase's (AprA). Species identifications have been confirmed by fluorescence in situ hybridization using specific probes designed in this study. In this article, we describe two new giant archaeal species that form the biggest archaeal filaments ever observed. One of these species is covered by a specific biofilm of sulfur-oxidizing γ-Proteobacteria. This study highlights an unexpected morphological and genetic diversity of the phylum Thaumarchaeota.


Asunto(s)
Archaea/clasificación , Bacterias Reductoras del Azufre/genética , Simbiosis , Microbiología del Agua , Archaea/genética , Archaea/crecimiento & desarrollo , Archaea/ultraestructura , ADN de Archaea/genética , ADN Bacteriano/genética , Ecosistema , Gammaproteobacteria/genética , Gammaproteobacteria/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Hibridación Fluorescente in Situ , Filogenia , ARN Ribosómico 16S/genética , Agua de Mar/microbiología , Análisis de Secuencia de ADN , Sulfuros/análisis , Bacterias Reductoras del Azufre/crecimiento & desarrollo
14.
Genetics ; 178(4): 2145-60, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18430940

RESUMEN

Wolbachia-induced cytoplasmic incompatibility (CI) is expressed when infected males are crossed with either uninfected females or females infected with Wolbachia of different CI specificity. In diploid insects, CI results in embryonic mortality, apparently due to the the loss of the paternal set of chromosomes, usually during the first mitotic division. The molecular basis of CI has not been determined yet; however, several lines of evidence suggest that Wolbachia exhibits two distinct sex-dependent functions: in males, Wolbachia somehow "imprints" the paternal chromosomes during spermatogenesis (mod function), whereas in females, the presence of the same Wolbachia strain(s) is able to restore embryonic viability (resc function). On the basis of the ability of Wolbachia to induce the modification and/or rescue functions in a given host, each bacterial strain can be classified as belonging in one of the four following categories: mod(+) resc(+), mod(-) resc(+), mod(-) resc(-), and mod(+) resc(-). A so-called "suicide" mod(+) resc(-) strain has not been found in nature yet. Here, a combination of embryonic cytoplasmic injections and introgression experiments was used to transfer nine evolutionary, distantly related Wolbachia strains (wYak, wTei, wSan, wRi, wMel, wHa, wAu, wNo, and wMa) into the same host background, that of Drosophila simulans (STCP strain), a highly permissive host for CI expression. We initially characterized the modification and rescue properties of the Wolbachia strains wYak, wTei, and wSan, naturally present in the yakuba complex, upon their transfer into D. simulans. Confocal microscopy and multilocus sequencing typing (MLST) analysis were also employed for the evaluation of the CI properties. We also tested the compatibility relationships of wYak, wTei, and wSan with all other Wolbachia infections. So far, the cytoplasmic incompatibility properties of different Wolbachia variants are explained assuming a single pair of modification and rescue factors specific to each variant. This study shows that a given Wolbachia variant can possess multiple rescue determinants corresponding to different CI systems. In addition, our results: (a) suggest that wTei appears to behave in D. simulans as a suicide mod(+) resc(-) strain, (b) unravel unique CI properties, and (c) provide a framework to understand the diversity and the evolution of new CI-compatibility types.


Asunto(s)
Genes Bacterianos , Wolbachia/genética , Animales , Técnicas de Tipificación Bacteriana , Citoplasma/microbiología , Drosophila/clasificación , Drosophila/microbiología , Embrión no Mamífero/citología , Embrión no Mamífero/microbiología , Femenino , Técnica del Anticuerpo Fluorescente , Modelos Lineales , Masculino , Filogenia , Infecciones por Rickettsiaceae/microbiología , Wolbachia/clasificación , Wolbachia/citología
15.
Methods Mol Biol ; 1968: 137-146, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30929212

RESUMEN

Physical interactions of bacteria with host cells are often a principal aspect of bacterial pathogenesis. In the case of Streptococcus pneumoniae (Spn), which does not produce a secreted toxin, adhesion to and/or invasion of host cells is necessary for colonization of the nasopharynx and subsequently to cause opportunistic disease in its human host. Knowledge of how pneumococci interact with host cells thereby helps to explain its biology and may identify potential targets for intervention. One of the simplest, yet powerful, assays that can be leveraged to dissect the molecular basis of this vital host-pathogen interaction is the in vitro adhesion and invasion assay. Among many key results, this assay has been used to discover the bacterial and host determinants involved in bacterial attachment, identify host signaling networks required for uptake of the bacteria into an endosome, and the characterization of the intracellular trafficking machinery that is subverted by Spn during development of bacteremia and meningitis. These assays have also been used to characterize the epithelial, endothelial, and/or immune cell response to these bacteria, and to learn how pneumococci disperse from an established biofilm to a planktonic phenotype to colonize another niche and/or transmit. Herein, we will review this protocol, highlighting how simple changes in the bacterial strain or host cell line can elucidate the underlying molecular mechanisms for Spn virulence.


Asunto(s)
Adhesión Bacteriana/fisiología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/patogenicidad , Proteínas Bacterianas/metabolismo , Gentamicinas/farmacología , Interacciones Huésped-Patógeno , Humanos , Streptococcus pneumoniae/metabolismo
16.
Front Immunol ; 10: 615, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31019504

RESUMEN

Pore-forming toxin (PFT) induced necroptosis exacerbates pulmonary injury during bacterial pneumonia. However, its role during asymptomatic nasopharyngeal colonization and toward the development of protective immunity was unknown. Using a mouse model of Streptococcus pneumoniae (Spn) asymptomatic colonization, we determined that nasopharyngeal epithelial cells (nEC) died of pneumolysin (Ply)-dependent necroptosis. Mice deficient in MLKL, the necroptosis effector, or challenged with Ply-deficient Spn showed less nEC sloughing, increased neutrophil infiltration, and altered IL-1α, IL-33, CXCL2, IL-17, and IL-6 levels in nasal lavage fluid (NALF). Activated MLKL correlated with increased presence of CD11c+ antigen presenting cells in Spn-associated submucosa. Colonized MLKL KO mice and wildtype mice colonized with Ply-deficient Spn produced less antibody against the bacterial surface protein PspA, were delayed in bacterial clearance, and were more susceptible to a lethal secondary Spn challenge. We conclude that PFT-induced necroptosis is instrumental in the natural development of protective immunity against opportunistic PFT-producing bacterial pathogens.


Asunto(s)
Inmunidad Adaptativa , Necroptosis/inmunología , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/inmunología , Animales , Formación de Anticuerpos/inmunología , Apoptosis/inmunología , Proteínas Bacterianas/inmunología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Femenino , Humanos , Inmunidad Innata , Masculino , Ratones , Necroptosis/genética , Infecciones Neumocócicas/genética , Infecciones Neumocócicas/metabolismo , Estreptolisinas/inmunología
17.
PLoS One ; 13(9): e0204032, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30216364

RESUMEN

Streptococcus pneumoniae is an opportunistic Gram-positive pathogen that can cause invasive disease. Recent studies have shown that S. pneumoniae is able to invade the myocardium and kill cardiomyocytes, with one-in-five adults hospitalized for pneumococcal pneumonia having a pneumonia-associated adverse cardiac event. Furthermore, clinical reports have shown up to a 10-year increased risk of adverse cardiac events in patients formerly hospitalized for pneumococcal bacteremia. In this study, we investigated the ability of nine S. pneumoniae clinical isolates, representing eight unique serotypes, to cause cardiac damage in a mouse model of invasive disease. Following intraperitoneal challenge of C57BL/6 mice, four of these strains (D39, WU2, TIGR4, and 6A-10) caused high-grade bacteremia, while CDC7F:2617-97 and AMQ16 caused mid- and low-grade bacteremia, respectively. Three strains did not cause any discernible disease. Of note, only the strains capable of high-grade bacteremia caused cardiac damage, as inferred by serum levels of cardiac troponin-I. This link between bacteremia and heart damage was further corroborated by Hematoxylin & Eosin and Trichrome staining which showed cardiac cytotoxicity only in D39, WU2, TIGR4, and 6A-10 infected mice. Finally, hearts infected with these strains showed varying histopathological characteristics, such as differential lesion formation and myocytolysis, suggesting that the mechanism of heart damage varied between strains.


Asunto(s)
Cardiomiopatías/etiología , Infecciones Neumocócicas/complicaciones , Streptococcus pneumoniae/patogenicidad , Animales , Bacteriemia/complicaciones , Bacteriemia/microbiología , Bacteriemia/patología , Carga Bacteriana , Cardiomiopatías/microbiología , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/microbiología , Miocitos Cardíacos/patología , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/patología , Serogrupo , Especificidad de la Especie , Streptococcus pneumoniae/clasificación , Troponina T/sangre , Virulencia
18.
Sci Rep ; 8(1): 5846, 2018 04 11.
Artículo en Inglés | MEDLINE | ID: mdl-29643440

RESUMEN

Pore-forming toxins are the most common virulence factor in pathogenic bacteria. They lead to membrane permeabilization and cell death. Herein, we show that respiratory epithelial cells (REC) undergoing bacterial pore-forming toxin (PFT)-induced necroptosis simultaneously experienced caspase activation independently of RIPK3. MLKL deficient REC treated with a pan-caspase inhibitor were protected in an additive manner against PFT-induced death. Subsequently, cleaved versions of caspases-2, -4 and -10 were detected within REC undergoing necroptosis by immunoblots and monoclonal antibody staining. Caspase activation was observed in lung samples from mice and non-human primates experiencing Gram-negative and Gram-positive bacterial pneumonia, respectively. During apoptosis, caspase activation normally leads to cell shrinkage, nuclear condensation, and immunoquiescent death. In contrast, caspase activity during PFT-induced necroptosis increased the release of alarmins to the extracellular milieu. Caspase-mediated alarmin release was found sufficient to activate resting macrophages, leading to Interleukin-6 production. In a mouse model of Gram-negative pneumonia, deletion of caspases -2 and -11, the mouse orthologue of caspase-4, reduced pulmonary inflammation, immune cell infiltration and lung damage. Thus, our study describes a previously unrecognized role for caspase activation in parallel to necroptosis, and indicates that their activity plays a critical pro-inflammatory role during bacterial pneumonia.


Asunto(s)
Alarminas/metabolismo , Toxinas Bacterianas/metabolismo , Caspasas/metabolismo , Neumonía Bacteriana/inmunología , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Células A549 , Alarminas/inmunología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Toxinas Bacterianas/inmunología , Inhibidores de Caspasas/farmacología , Caspasas/genética , Caspasas/inmunología , Membrana Celular/ultraestructura , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/patología , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Necrosis/inmunología , Papio , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/patología , Proteínas Citotóxicas Formadoras de Poros/inmunología
19.
mBio ; 5(1): e01024-13, 2014 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-24520062

RESUMEN

UNLABELLED: Type IV pili (Tfp) are expressed by many Gram-negative bacteria to promote aggregation, adhesion, internalization, twitching motility, or natural transformation. Tfp of Neisseria meningitidis, the causative agent of cerebrospinal meningitis, are involved in the colonization of human nasopharynx. After invasion of the bloodstream, Tfp allow adhesion of N. meningitidis to human endothelial cells, which leads to the opening of the blood-brain barrier and meningitis. To achieve firm adhesion, N. meningitidis induces a host cell response that results in elongation of microvilli surrounding the meningococcal colony. Here we study the role of the major pilin subunit PilE during host cell response using human dermal microvascular endothelial cells and the pharynx carcinoma-derived FaDu epithelial cell line. We first show that some PilE variants are unable to induce a host cell response. By engineering PilE mutants, we observed that the PilE C-terminus domain, which contains a disulfide bonded region (D-region), is critical for the host cell response and that hypervariable regions confer different host cell specificities. Moreover, the study of point mutants of the pilin D-region combined with structural modeling of PilE revealed that the D-region contains two independent regions involved in signaling to human dermal microvascular endothelial cells (HDMECs) or FaDu cells. Our results indicate that the diversity of the PilE D-region sequence allows the induction of the host cell response via several receptors. This suggests that Neisseria meningitidis has evolved a powerful tool to adapt easily to many niches by modifying its ability to interact with host cells. IMPORTANCE: Type IV pili (Tfp) are long appendages expressed by many Gram-negative bacteria, including Neisseria meningitidis, the causative agent of cerebrospinal meningitis. These pili are involved in many aspects of pathogenesis: natural competence, aggregation, adhesion, and twitching motility. More specifically, Neisseria meningitidis, which is devoid of a secretion system to manipulate its host, has evolved its Tfp to signal to brain endothelial cells and open the blood-brain barrier. In this report, we investigate, at the molecular level, the involvement of the major pilin subunit PilE in host cell response. Our results indicate that the PilE C-terminal domain, which contains a disulfide bonded region (D-region), is critical for the host cell response and contains two independent regions involved in host cell signaling.


Asunto(s)
Variación Antigénica , Adhesión Celular , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Neisseria meningitidis/fisiología , Células Cultivadas , Células Endoteliales/microbiología , Células Epiteliales/microbiología , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Neisseria meningitidis/genética
20.
FEMS Microbiol Ecol ; 75(1): 63-76, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21091519

RESUMEN

The first studies of the 16S rRNA gene diversity of the bacterial symbionts found in lucinid clams did not clarify how symbiotic associations had evolved in this group. Indeed, although species-specific associations deriving from a putative ancestral symbiotic association have been described (coevolution scenario), associations between the same bacterial species and various host species (opportunistic scenario) have also been described. Here, we carried out a comparative molecular analysis of hosts, based on 18S and 28S rRNA gene sequences, and of symbionts, based on 16S rRNA gene sequences, to determine as to which evolutionary scenario led to modern lucinid/symbiont associations. For all sequences analyzed, we found only three bacterial symbiont species, two of which are harbored by lucinids colonizing mangrove swamps. The last symbiont is the most common and was found to be independent of biotope or depth. Another interesting feature is the similarity of ctenidial organization of lucinids from the Philippines to those described previously, with the exception that two bacterial morphotypes were observed in two different species (Gloverina rectangularis and Myrtea flabelliformis). Thus, there is apparently no specific association between Lucinidae and their symbionts, the association taking place according to which bacterial species is present in the environment.


Asunto(s)
Bivalvos/genética , Bivalvos/microbiología , Filogenia , Bacterias Reductoras del Azufre/genética , Simbiosis , Animales , Evolución Biológica , Bivalvos/ultraestructura , Filipinas , ARN Ribosómico 16S/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 28S/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Bacterias Reductoras del Azufre/clasificación , Bacterias Reductoras del Azufre/ultraestructura
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