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1.
mBio ; : e0131724, 2024 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-39240091

RESUMEN

Biofilm is a dynamic structure from which individual bacteria and micro-aggregates are released to subsequently colonize new niches by either detachment or dispersal. Screening of a transposon mutant library identified genes associated with the alteration of Klebsiella pneumoniae biofilm including fabR, which encodes a transcriptional regulator involved in membrane lipid homeostasis. An isogenic ∆fabR mutant formed more biofilm than the wild-type (WT) strain and its trans-complemented strain. The thick and round aggregates observed with ∆fabR were resistant to extensive washes, unlike those of the WT strain. Confocal microscopy and BioFlux microfluidic observations showed that fabR deletion was associated with biofilm robustness and impaired erosion over time. The genes fabB and yqfA associated with fatty acid metabolism were significantly overexpressed in the ∆fabR strain, in both planktonic and biofilm conditions. Two monounsaturated fatty acids, palmitoleic acid (C16:1) and oleic acid (C18:1), were found in higher proportion in biofilm cells than in planktonic forms, whereas heptadecenoic acid (C17:1) and octadecanoic acid, 11-methoxy (C18:0-OCH3) were found in higher proportion in the planktonic lifestyle. The fabR mutation induced variations in the fatty acid composition, with no clear differences in the amounts of saturated fatty acids (SFA) and unsaturated fatty acids for the planktonic lifestyle but lower SFA in the biofilm form. Atomic force microscopy showed that deletion of fabR is associated with decreased K. pneumoniae cell rigidity in the biofilm lifestyle, as well as a softer, more elastic biofilm with increased cell cohesion compared to the wild-type strain.IMPORTANCEKlebsiella pneumoniae is an opportunistic pathogen responsible for a wide range of nosocomial infections. The success of this pathogen is due to its high resistance to antibiotics and its ability to form biofilms. The molecular mechanisms involved in biofilm formation have been largely described but the dispersal process that releases individual and aggregate cells from mature biofilm is less well documented while it is associated with the colonization of new environments and thus new threats. Using a multidisciplinary approach, we show that modifications of bacterial membrane fatty acid composition lead to variations in the biofilm robustness, and subsequent bacterial detachment and biofilm erosion over time. These results enhance our understanding of the genetic requirements for biofilm formation in K. pneumoniae that affect the time course of biofilm development and the embrittlement step preceding its dispersal that will make it possible to control K. pneumoniae infections.

2.
Microorganisms ; 9(6)2021 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-34204632

RESUMEN

Klebsiella pneumoniae is a Gram-negative pathogen responsible for community-acquired and nosocomial infections. The strains of this species belong to the opportunistic group, which is comprised of the multidrug-resistant strains, or the hypervirulent group, depending on their accessory genome, which determines bacterial pathogenicity and the host immune response. The aim of this survey is to present an overview of the murine models mimicking K. pneumoniae infectious processes (i.e., gastrointestinal colonization, urinary, pulmonary, and systemic infections), and the bacterial functions deployed to colonize and disseminate into the host. These in vivo approaches are pivotal to develop new therapeutics to limit K. pneumoniae infections via a modulation of the immune responses and/or microbiota.

3.
mBio ; 10(6)2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31848284

RESUMEN

Listeria monocytogenes is a pathogenic bacterium causing potentially fatal foodborne infections in humans and animals. While the mechanisms used by Listeria to manipulate its host have been thoroughly characterized, how the host controls bacterial virulence factors remains to be extensively deciphered. Here, we found that the secreted Listeria virulence protein InlC is monoubiquitinated by the host cell machinery on K224, restricting infection. We show that the ubiquitinated form of InlC interacts with the intracellular alarmin S100A9, resulting in its stabilization and in increased reactive oxygen species production by neutrophils in infected mice. Collectively, our results suggest that posttranslational modification of InlC exacerbates the host response upon Listeria infection.IMPORTANCE The pathogenic potential of Listeria monocytogenes relies on the production of an arsenal of virulence determinants that have been extensively characterized, including surface and secreted proteins of the internalin family. We have previously shown that the Listeria secreted internalin InlC interacts with IκB kinase α to interfere with the host immune response (E. Gouin, M. Adib-Conquy, D. Balestrino, M.-A. Nahori, et al., Proc Natl Acad Sci USA, 107:17333-17338, 2010, https://doi.org/10.1073/pnas.1007765107). In the present work, we report that InlC is monoubiquitinated on K224 upon infection of cells and provide evidence that ubiquitinated InlC interacts with and stabilizes the alarmin S100A9, which is a critical regulator of the immune response and inflammatory processes. Additionally, we show that ubiquitination of InlC causes an increase in reactive oxygen species production by neutrophils in mice and restricts Listeria infection. These findings are the first to identify a posttranscriptional modification of an internalin contributing to host defense.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Listeria/fisiología , Listeriosis/metabolismo , Listeriosis/microbiología , Calgranulina B/metabolismo , Susceptibilidad a Enfermedades , Células Epiteliales , Humanos , Ubiquitinación
4.
NPJ Biofilms Microbiomes ; 5(1): 25, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31583108

RESUMEN

Biofilm-dispersal is a key determinant for further dissemination of biofilm-embedded bacteria. Recent evidence indicates that biofilm-dispersed bacteria have transcriptional features different from those of both biofilm and planktonic bacteria. In this study, the in vitro and in vivo phenotypic properties of Klebsiella pneumoniae cells spontaneously dispersed from biofilm were compared with those of planktonic and sessile cells. Biofilm-dispersed cells, whose growth rate was the same as that of exponential planktonic bacteria but significantly higher than those of sessile and stationary planktonic forms, colonized both abiotic and biotic surfaces more efficiently than their planktonic counterparts regardless of their initial adhesion capabilities. Microscopy studies suggested that dispersed bacteria initiate formation of microcolonies more rapidly than planktonic bacteria. In addition, dispersed cells have both a higher engulfment rate and better survival/multiplication inside macrophages than planktonic cells and sessile cells. In an in vivo murine pneumonia model, the bacterial load in mice lungs infected with biofilm-dispersed bacteria was similar at 6, 24 and 48 h after infection to that of mice lungs infected with planktonic or sessile bacteria. However, biofilm-dispersed and sessile bacteria trend to elicit innate immune response in lungs to a lesser extent than planktonic bacteria. Collectively, the findings from this study suggest that the greater ability of K. pneumoniae biofilm-dispersed cells to efficiently achieve surface colonization and to subvert the host immune response confers them substantial advantages in the first steps of the infection process over planktonic bacteria.


Asunto(s)
Adhesión Bacteriana , Biopelículas/crecimiento & desarrollo , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/crecimiento & desarrollo , Klebsiella pneumoniae/inmunología , Fenotipo , Neumonía Bacteriana/microbiología , Animales , Carga Bacteriana , Modelos Animales de Enfermedad , Evasión Inmune , Inmunidad Innata , Infecciones por Klebsiella/inmunología , Pulmón/microbiología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Viabilidad Microbiana , Fagocitosis , Neumonía Bacteriana/inmunología , Factores de Tiempo
5.
Infect Immun ; 87(11)2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31481408

RESUMEN

Some respiratory infections have been associated with dysbiosis of the intestinal microbiota. The underlying mechanism is incompletely understood, but cross talk between the intestinal microbiota and local immune cells could influence the immune response at distal mucosal sites. This has led to the concept of enhancing respiratory defenses by modulating the intestinal microbiota with exogenous supplementation of beneficial strains. In this study, we examined the effect of Lactobacillus plantarum CIRM653 on the inflammatory response induced by the pathogen Klebsiella pneumoniae Oral administration of L. plantarum CIRM653 to mice subsequently infected by K. pneumoniae via the nasal route (i) reduced the pulmonary inflammation response, with decreased numbers of lung innate immune cells (macrophages and neutrophils) and cytokines (mouse keratinocyte-derived chemokine [KC], interleukin-6 [IL-6], and tumor necrosis factor alpha [TNF-α]) in the bronchoalveolar fluid, and (ii) induced an immunosuppressive Treg response in lungs. In vitro coincubation of L. plantarum CIRM653 and K. pneumoniae with human dendritic cells and peripheral blood mononuclear cells resulted in decreased Th1 (IL-12p70 and interferon gamma [IFN-γ]) and Th17 (IL-23 and IL-17) and increased Treg (IL-10) cytokine levels compared to those observed for K. pneumoniae-infected cells. Neither K. pneumoniae nor L. plantarum CIRM653 had any effect on cytokine production by intestinal epithelial cells in vitro, but the induction of the NF-κB pathway and IL-8 and IL-6 production by K. pneumoniae in airway epithelial cells was significantly reduced when the pathogen was coincubated with L. plantarum CIRM653. The remote IL-10-mediated modulation of the K. pneumoniae inflammatory response by L. plantarum CIRM653 supports the concept of immunomodulation by beneficial bacteria through the gut-lung axis.


Asunto(s)
Inflamación/microbiología , Infecciones por Klebsiella/inmunología , Lactobacillus plantarum/fisiología , Neumonía Bacteriana/microbiología , Animales , Infecciones por Klebsiella/metabolismo , Klebsiella pneumoniae , Ratones , Ratones Endogámicos C57BL , Neumonía Bacteriana/patología , Probióticos
6.
Artículo en Inglés | MEDLINE | ID: mdl-29560270

RESUMEN

Persisters form sub-populations of stress-tolerant cells that play a major role in the capacity of biofilms to survive and recover from disturbances such as antibiotic treatments. The mechanisms of persistence are diverse and influenced by environmental conditions, and persister populations are more heterogeneous than formerly suspected. We used computational modeling to assess the impact of three switching strategies between susceptible and persister cells on the capacity of bacterial biofilms to grow, survive and recover from antibiotic treatments. The strategies tested were: (1) constant switches, (2) substrate-dependent switches and (3) antibiotic-dependent switches. We implemented these strategies in an individual-based biofilm model and simulated antibiotic shocks on virtual biofilms. Because of limited available data on switching rates in the literature, nine parameter sets were assessed for each strategy. Substrate and antibiotic-dependent switches allowed high switching rates without affecting the growth of the biofilms. Compared to substrate-dependent switches, constant and antibiotic-dependent switches were associated with higher proportions of persisters in the top of the biofilms, close to the substrate source, which probably confers a competitive advantage within multi-species biofilms. The constant and substrate-dependent strategies need a compromise between limiting the wake-up and death of persisters during treatments and leaving the persister state fast enough to recover quickly after antibiotic-removal. Overall, the simulations gave new insights into the relationships between the dynamics of persister populations in biofilms and their dynamics of growth, survival and recovery when faced with disturbances.

7.
Microb Biotechnol ; 10(6): 1616-1627, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28730700

RESUMEN

We developed and compared two mathematical models of variable phenotypic switching rates between normal and persister cells that depend on substrate concentration and antibiotic presence. They could be used to simulate the formation of persisters in environments with concentration gradients such as biofilms. Our models are extensions of a previous model of the dynamics of normal and persistent cell populations developed by Balaban et al. (2004, Science 305: 1622). We calibrated the models' parameters with experimental killing curves obtained after ciprofloxacin treatment of samples regularly harvested from planktonic batch cultures of Klebsiella pneumoniae. Our switching models accurately reproduced the dynamics of normal and persistent populations in planktonic batch cultures and under antibiotic treatment. Results showed that the models are valid for a large range of substrate concentrations and for zero or high doses of antibiotics.


Asunto(s)
Antibacterianos/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Antibacterianos/análisis , Biopelículas/efectos de los fármacos , Cinética , Klebsiella pneumoniae/química , Klebsiella pneumoniae/crecimiento & desarrollo , Modelos Teóricos
8.
Mol Microbiol ; 105(2): 188-210, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28422332

RESUMEN

In most environments, microorganisms evolve in a sessile mode of growth, designated as biofilm, which is characterized by cells embedded in a self-produced extracellular matrix. Although a biofilm is commonly described as a "cozy house" where resident bacteria are protected from aggression, bacteria are able to break their biofilm bonds and escape to colonize new environments. This regulated process is observed in a wide variety of species; it is referred to as biofilm dispersal, and is triggered in response to various environmental and biological signals. The first part of this review reports the main regulatory mechanisms and effectors involved in biofilm dispersal. There is some evidence that dispersal is a necessary step between the persistence of bacteria inside biofilm and their dissemination. In the second part, an overview of the main methods used so far to study the dispersal process and to harvest dispersed bacteria was provided. Then focus was on the properties of the biofilm-dispersed bacteria and the fundamental role of the dispersal process in pathogen dissemination within a host organism. In light of the current body of knowledge, it was suggested that dispersal acts as a potent means of disseminating bacteria with enhanced colonization properties in the surrounding environment.


Asunto(s)
Bacterias/metabolismo , Biopelículas/crecimiento & desarrollo , Bacterias/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica/genética
9.
Methods Mol Biol ; 1535: 173-195, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27914079

RESUMEN

Listeria monocytogenes is a bacterial pathogen which invades and multiplies within non-professional phagocytes. Signaling cascades involved in cellular entry have been extensively analyzed, but the events leading to vacuolar escape remain less clear. In this chapter, we detail a microscopy FRET-based assay which allows quantitatively measuring L. monocytogenes infection and escape from its internalization vacuole, as well as a correlative light/electron microscopy method to investigate the morphological features of the vacuolar compartments containing L. monocytogenes.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Listeria monocytogenes/metabolismo , Listeria monocytogenes/ultraestructura , Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Vacuolas/metabolismo , Transporte Biológico , Vacuolas/ultraestructura
10.
PLoS One ; 11(7): e0159475, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27441664

RESUMEN

BACKGROUND AND OBJECTIVES: Interdialytic lock solutions should maintain catheter patency and prevent catheter infections. We aimed to determine in which conditions injectable anticoagulant agents (IAAs) combined with ethanol are compatible and to assess the antibiofilm activity of the selected combination and its effects on dialysis catheters (DC). METHODS: The solubility and compatibility of unfractionated heparin (UFH), low molecular weight heparins (LMWHs), heparinoids and fondaparinux (50 to 2,500 U/mL) in 30 to 70% ethanol were determined by visual observation. The stability of enoxaparin in ethanol and the ethanol content were assessed by high performance liquid chromatography (HPLC) and titrimetric control, respectively. The bactericidal effect was determined on 24h-old biofilms embedded in silicone-DC. The integrity of polyurethane-DC immersed in anticoagulant-ethanol was assessed by gas chromatography-mass spectrometry (GC-MS) and compared with previously published results. RESULTS: The compatibility of IAAs and ethanol varied according to IAA type and concentration, and ethanol content. UFH in 40% ethanol was not compatible, whatever the UFH concentration used. Established limits of compatibility of enoxaparin, nadroparin, dalteparin and tinzaparin in 40% ethanol were 1350, 575, 307 and 207 U/ml, respectively, and up to 300 U/ml for danaparoid and 1 mg/mL for fondaparinux. Enoxaparin 400 U/mL in 40% ethanol (Enox/Eth) eradicated biofilm after 4 hours of exposure for Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans and after 24 hours for Klebsiella pneumoniae and S. aureus. Aliphatic carbonate and alcohol compounds were released by polyurethane-DC after Enox/Eth exposure, as after 40% ethanol or saline exposure. There was no significant difference between the amounts released after 30 minutes of exposure to Enox/Eth and 15 days to saline. CONCLUSIONS: A 40% ethanol solution can be combined with all IAAs but UFH. Enox/Eth was effective as an anti-biofilm agent with minor impacts on DC integrity and could be a useful interdialytic lock solution.


Asunto(s)
Anticoagulantes/farmacología , Biopelículas/efectos de los fármacos , Catéteres , Enoxaparina/farmacología , Etanol/química , Inyecciones , Poliuretanos/farmacología , Albúminas/metabolismo , Fraccionamiento Químico , Precipitación Química , Cromatografía Líquida de Alta Presión , Cromatografía de Gases y Espectrometría de Masas , Hemólisis/efectos de los fármacos , Heparina de Bajo-Peso-Molecular , Humanos , Solubilidad , Soluciones
11.
BMC Genomics ; 17: 237, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26979871

RESUMEN

BACKGROUND: Surface-associated communities of bacteria, known as biofilms, play a critical role in the persistence and dissemination of bacteria in various environments. Biofilm development is a sequential dynamic process from an initial bacterial adhesion to a three-dimensional structure formation, and a subsequent bacterial dispersion. Transitions between these different modes of growth are governed by complex and partially known molecular pathways. RESULTS: Using RNA-seq technology, our work provided an exhaustive overview of the transcriptomic behavior of the opportunistic pathogen Klebsiella pneumoniae derived from free-living, biofilm and biofilm-dispersed states. For each of these conditions, the combined use of Z-scores and principal component analysis provided a clear illustration of distinct expression profiles. In particular, biofilm-dispersed cells appeared as a unique stage in the bacteria lifecycle, different from both planktonic and sessile states. The K-means cluster analysis showed clusters of Coding DNA Sequences (CDS) and non-coding RNA (ncRNA) genes differentially transcribed between conditions. Most of them included dominant functional classes, emphasizing the transcriptional changes occurring in the course of K. pneumoniae lifestyle transitions. Furthermore, analysis of the whole transcriptome allowed the selection of an overall of 40 transcriptional signature genes for the five bacterial physiological states. CONCLUSIONS: This transcriptional study provides additional clues to understand the key molecular mechanisms involved in the transition between biofilm and the free-living lifestyles, which represents an important challenge to control both beneficial and harmful biofilm. Moreover, this exhaustive study identified physiological state specific transcriptomic reference dataset useful for the research community.


Asunto(s)
Adhesión Bacteriana/genética , Biopelículas , Klebsiella pneumoniae/genética , Transcriptoma , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Klebsiella pneumoniae/fisiología , ARN Bacteriano/genética , Análisis de Secuencia de ARN
12.
Appl Environ Microbiol ; 82(1): 211-7, 2016 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-26497455

RESUMEN

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a ß-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface ß-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes.


Asunto(s)
Citoplasma/microbiología , Interacciones Huésped-Patógeno , Listeria monocytogenes/fisiología , Vacuolas/microbiología , Proteínas Bacterianas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Humanos , Listeria/enzimología , Listeria/metabolismo , Listeria monocytogenes/enzimología , Microscopía Fluorescente , beta-Lactamasas/metabolismo
13.
Genome Announc ; 3(6)2015 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-26564039

RESUMEN

We report here the genome sequence of Klebsiella pneumoniae CH1034, a sequence type 6 (ST6) strain isolated in 2012 from a central venous catheter of a hospitalized patient.

14.
PLoS One ; 10(3): e0116215, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25751658

RESUMEN

Chaperone/usher (CU) assembly pathway is used by a wide range of Enterobacteriaceae to assemble adhesive surface structures called pili or fimbriae that play a role in bacteria-host cell interactions. In silico analysis revealed that the genome of Klebsiella pneumoniae LM21 harbors eight chromosomal CU loci belonging to γκп and Ï­ clusters. Of these, only two correspond to previously described operons, namely type 1 and type 3-encoding operons. Isogenic usher deletion mutants of K. pneumoniae LM21 were constructed for each locus and their role in adhesion to animal (Intestine 407) and plant (Arabidopsis thaliana) cells, biofilm formation and murine intestinal colonization was investigated. Type 3 pili usher deleted mutant was impaired in all assays, whereas type 1 pili usher deleted mutant only showed attenuation in adhesion to plant cells and in intestinal colonization. The LM21ΔkpjC mutant was impaired in its capacity to adhere to Arabidopsis cells and to colonize the murine intestine, either alone or in co-inoculation experiments. Deletion of LM21kpgC induced a significant decrease in biofilm formation, in adhesion to animal cells and in colonization of the mice intestine. The LM21∆kpaC and LM21∆kpeC mutants were only attenuated in biofilm formation and the adhesion abilities to Arabidopsis cells, respectively. No clear in vitro or in vivo effect was observed for LM21∆kpbC and LM21∆kpdC mutants. The multiplicity of CU loci in K. pneumoniae genome and their specific adhesion pattern probably reflect the ability of the bacteria to adhere to different substrates in its diverse ecological niches.


Asunto(s)
Proteínas Bacterianas/genética , Klebsiella pneumoniae/genética , Chaperonas Moleculares/genética , Animales , Arabidopsis/microbiología , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Biopelículas , Línea Celular , Femenino , Expresión Génica , Genes Bacterianos , Sitios Genéticos , Humanos , Infecciones por Klebsiella/microbiología , Ratones , Chaperonas Moleculares/metabolismo , Filogenia
15.
PLoS One ; 9(6): e99995, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24932475

RESUMEN

Competition and cooperation phenomena occur within highly interactive biofilm communities and several non-biocides molecules produced by microorganisms have been described as impairing biofilm formation. In this study, we investigated the anti-biofilm capacities of an ubiquitous and biofilm producing bacterium, Klebsiella pneumoniae. Cell-free supernatant from K. pneumoniae planktonic cultures showed anti-biofilm effects on most Gram positive bacteria tested but also encompassed some Gram negative bacilli. The anti-biofilm non-bactericidal activity was further investigated on Staphylococcus epidermidis, by determining the biofilm biomass, microscopic observations and agglutination measurement through a magnetic bead-mediated agglutination test. Cell-free extracts from K. pneumoniae biofilm (supernatant and acellular matrix) also showed an influence, although to a lesser extend. Chemical analyses indicated that the active molecule was a high molecular weight polysaccharide composed of five monosaccharides: galactose, glucose, rhamnose, glucuronic acid and glucosamine and the main following sugar linkage residues [→ 2)-α-L-Rhap-(1 →]; [→ 4)-α-L-Rhap-(1 →]; [α-D-Galp-(1 →]; [→ 2,3)-α-D-Galp-(1 →]; [→ 3)-ß-D-Galp-(1 →] and, [→ 4)-ß-D-GlcAp-(1 →]. Characterization of this molecule indicated that this component was more likely capsular polysaccharide (CPS) and precoating of abiotic surfaces with CPS extracts from different serotypes impaired the bacteria-surface interactions. Thus the CPS of Klebsiella would exhibit a pleiotropic activity during biofilm formation, both stimulating the initial adhesion and maturation steps as previously described, but also repelling potential competitors.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Klebsiella pneumoniae/fisiología , Polisacáridos Bacterianos/farmacología , Biopelículas/efectos de los fármacos , Biomasa , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Sistema Libre de Células , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Klebsiella pneumoniae/efectos de los fármacos , Plancton/efectos de los fármacos , Espectroscopía de Protones por Resonancia Magnética , Staphylococcus epidermidis/efectos de los fármacos , Staphylococcus epidermidis/fisiología
16.
Cell Microbiol ; 16(3): 311-23, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24128258

RESUMEN

Recognition of bacteria by metazoans is mediated by receptors that recognize different types of microorganisms and elicit specific cellular responses. The soil amoebae Dictyostelium discoideum feeds upon a variable mixture of environmental bacteria, and it is expected to recognize and adapt to various food sources. To date, however, no bacteria-sensing mechanisms have been described. In this study, we isolated a Dictyostelium mutant (fspA KO) unable to grow in the presence of non-capsulated Klebsiella pneumoniae bacteria, but growing as efficiently as wild-type cells in the presence of other bacteria, such as Bacillus subtilis. fspA KO cells were also unable to respond to K. pneumoniae and more specifically to bacterially secreted folate in a chemokinetic assay, while they responded readily to B. subtilis. Remarkably, both WT and fspA KO cells were able to grow in the presence of capsulated LM21 K. pneumoniae, and responded to purified capsule, indicating that capsule recognition may represent an alternative, FspA-independent mechanism for K. pneumoniae sensing. When LM21 capsule synthesis genes were deleted, growth and chemokinetic response were lost for fspA KO cells, but not for WT cells. Altogether, these results indicate that Dictyostelium amoebae use specific recognition mechanisms to respond to different K. pneumoniae elements.


Asunto(s)
Cápsulas Bacterianas , Dictyostelium/microbiología , Conducta Alimentaria/fisiología , Fagocitosis/genética , Bacillus subtilis , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Klebsiella pneumoniae
17.
Microbiologyopen ; 2(3): 402-14, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23520129

RESUMEN

Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47-48%) were rare sequences (<5 copies). Prokaryotic phyla represented the species richness, and eukaryotic phyla accounted for a small part. Among the prokaryotic phyla, Proteobacteria (beta and alpha) predominated, followed by Bacteroidetes together with a large number of nonaffiliated bacterial sequences. Bacillariophyta plastids were abundant. The remaining bacterial phyla, Verrucomicrobia and Cyanobacteria, made up the rest of the bulk biodiversity. The most abundant eukaryotic phyla were annelid worms, followed by Diatoms, and Chlorophytes. These latter phyla attest to the abundance of plastids and the importance of photosynthetic activity for the biofilm. These findings highlight the existence and plasticity of multiple trophic levels within these complex biological systems.


Asunto(s)
Archaea/clasificación , Bacterias/clasificación , Biopelículas/crecimiento & desarrollo , Biota , Eucariontes/clasificación , Ríos/microbiología , Ríos/parasitología , Archaea/genética , Bacterias/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Eucariontes/genética , Francia , Genes de ARNr , Fenómenos Fisiológicos , Análisis de Secuencia de ADN
18.
Proc Natl Acad Sci U S A ; 107(40): 17333-8, 2010 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-20855622

RESUMEN

Listeria monocytogenes is an intracellular pathogen responsible for severe foodborne infections. It can replicate in both phagocytic and nonphagocytic mammalian cells. The infectious process at the cellular level has been studied extensively, but how the bacterium overcomes early host innate immune responses remains largely unknown. Here we show that InlC, a member of the internalin family, is secreted intracellularly and directly interacts with IKKα, a subunit of the IκB kinase complex critical for the phosphorylation of IκB and activation of NF-κB, the major regulator of innate immune responses. Infection experiments with WT Listeria or the inlC-deletion mutant and transfection of cells with InlC reveal that InlC expression impairs phosphorylation and consequently delays IκB degradation normally induced by TNF-α, a classical NF-κB stimulator. Moreover, infection of RAW 264.7 macrophages by the inlC mutant leads to increased production of proinflammatory cytokines compared with that obtained with the WT. Finally, in a peritonitis mouse model, we show that infection with the inlC mutant induces increased production of chemokines and increased recruitment of neutrophils in the peritoneal cavity compared with infection with WT. Together, these results demonstrate that InlC, by interacting with IKKα, dampens the host innate response induced by Listeria during the infection process.


Asunto(s)
Proteínas Bacterianas/inmunología , Quinasa I-kappa B/metabolismo , Inmunidad Innata , Subunidades de Proteína/metabolismo , Animales , Línea Celular , Humanos , Quinasa I-kappa B/genética , Listeria monocytogenes/inmunología , Listeria monocytogenes/patogenicidad , Ratones , Regiones Promotoras Genéticas , Subunidades de Proteína/genética , Factor de Necrosis Tumoral alfa/metabolismo , Técnicas del Sistema de Dos Híbridos
19.
Res Microbiol ; 161(7): 595-603, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20600864

RESUMEN

Biofilm formation by Klebsiella pneumoniae is modulated by quorum sensing through the synthesis of interspecies AI-2 autoinducers. We characterized in K. pneumoniae the genes homologous to those described in Escherichia coli involved in AI-2 transport, and created two isogenic mutants deleted of lsrCD and tqsA. The levels of extracellular AI-2 with lsrCD and tqsA knockout mutants showed increased and lowered concentrations of AI-2, respectively. The level of transcripts of luxS, the gene responsible for AI-2 synthesis, was increased in sessile cells of the tqsA mutant. In contrast, the expression of the AI-2 import regulator genes lsrR and lsrK was decreased. In addition, the two mutants lsrCD and tqsA formed biofilms with greater biomass but impaired architecture. Since exopolysaccharides play a main role in K. pneumoniae biofilm formation, we investigated their relationship with AI-2 synthesis. None of the mutations in luxS and the AI-2 transport systems affected the expression of three capsule polysaccharide-related genes (wzi, wza and wzx), but all induced an increase in the expression of two lipopolysaccharide (LPS)-synthesis-related genes, wbbM and wzm. AI-2 therefore seems to act as a regulator of biofilm formation and LPS synthesis in sessile K. pneumoniae cells.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Homoserina/análogos & derivados , Klebsiella pneumoniae/fisiología , Lactonas/metabolismo , Percepción de Quorum , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , Genes Bacterianos , Homoserina/genética , Homoserina/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Lipopolisacáridos/biosíntesis , Mutación , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia , Transducción de Señal/genética
20.
Appl Environ Microbiol ; 76(11): 3625-36, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20363781

RESUMEN

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen which invades different cell types, including nonphagocytic cells, where it is able to replicate and survive. The different steps of the cellular infectious process have been well described and consist of bacterial entry, lysis of the endocytic vacuole, intracellular replication, and spreading to neighboring cells. To study the listerial infectious process, gentamicin survival assays, plaque formation, and direct microscopy observations are typically used; however, there are some caveats with each of these techniques. In this study we describe new single-cell techniques based on use of an array of integrative fluorescent plasmids (green, cyan, and yellow fluorescent proteins) to easily, rapidly, and quantitatively detect L. monocytogenes in vitro and in vivo. We describe construction of 13 integrative and multicopy plasmids which can be used for detecting intracellular bacteria, for measuring invasion, cell-to-cell spreading, and intracellular replication, for monitoring in vivo infections, and for generating transcriptional or translational reporters. Furthermore, we tested these plasmids in a variety of epifluorescence- and flow cytometry-based assays. We showed that we could (i) determine the expression of a particular promoter during the cell cycle, (ii) establish in one rapid experiment at which step in the cell cycle a particular mutant is defective, and (iii) easily measure the number of infected cells in vitro and in mouse organs. The plasmids that are described and the methods to detect them are new powerful tools to study host-Listeria interactions in a fast, robust, and high-throughput manner.


Asunto(s)
Técnicas Bacteriológicas/métodos , Listeria monocytogenes/patogenicidad , Listeriosis/microbiología , Biología Molecular/métodos , Coloración y Etiquetado/métodos , Animales , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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