RESUMEN
Legionella pneumophila, the causative agent of Legionnaires' disease, replicates as an intracellular parasite of amoebae and persists in the environment as a free-living microbe. Here we have analyzed the complete genome sequences of L. pneumophila Paris (3,503,610 bp, 3,077 genes), an endemic strain that is predominant in France, and Lens (3,345,687 bp, 2,932 genes), an epidemic strain responsible for a major outbreak of disease in France. The L. pneumophila genomes show marked plasticity, with three different plasmids and with about 13% of the sequence differing between the two strains. Only strain Paris contains a type V secretion system, and its Lvh type IV secretion system is encoded by a 36-kb region that is either carried on a multicopy plasmid or integrated into the chromosome. Genetic mobility may enhance the versatility of L. pneumophila. Numerous genes encode eukaryotic-like proteins or motifs that are predicted to modulate host cell functions to the pathogen's advantage. The genome thus reflects the history and lifestyle of L. pneumophila, a human pathogen of macrophages that coevolved with fresh-water amoebae.
Asunto(s)
Fenómenos Fisiológicos Celulares , Genoma Bacteriano , Interacciones Huésped-Parásitos , Legionella pneumophila/genética , Adaptación Biológica , Secuencia de Aminoácidos , Amoeba/microbiología , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Humanos , Macrófagos Alveolares/microbiología , Datos de Secuencia Molecular , FilogeniaRESUMEN
PURPOSE: We recently demonstrated increased frequency and growth potential of late outgrowth endothelial progenitor cells (OECs) in patients with neovascular age-related macular degeneration (nvAMD). This study investigated the effects of short- and long-term in vitro inhibition of vascular endothelial growth factor (VEGF) Receptor-2 (VEGFR-2) signaling by SU5416 and other inhibitors of the VEGF signaling pathway in OECs. METHODS: OECs, from the peripheral blood of patients with nvAMD, and human umbilical vein endothelial cells were grown in the presence of SU5416, other VEGFR-2 tyrosine kinase inhibitors (TKIs), and inhibitors of phosphatidylinositol 3'-Kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) in complete angiogenic medium. Apotosis was assessed after 48 h using the fluorescein isothiocyanate Annexin V method. Cell counts were performed for 10 days, and features of senescence were analyzed using senescence-associated ß-galactosidase staining, the telomeric repeat amplification protocol for telomerase activity, Southern blot analysis for mean telomere length, flow cytometric analysis for cell-cycle arrest, and western blot for p53 and p21. Control OECs, cells treated for 7 days with inhibitors, as well as naturally senescent OECs were analyzed for expression of different endothelial antigens, including VEGFR-2 and the receptor for stromal cell-derived factor 1, chemokine receptor 4 (CXCR-4). Migration in vitro to VEGF and stromal cell-derived factor 1 of OECs was assessed. RESULTS: SU5416, other VEGFR-2 TKIs, and inhibitors of PI3K, Akt, and PKC induced apoptosis, inhibited long-term proliferation, reduced telomerase activity, and induced premature senescence and cell-cycle arrest in OECs as well as in human umbilical vein endothelial cells. Naturally senescent cells and cells rendered senescent by VEGFR-2 TKIs had reduced VEGFR-2 and CXCR-4 expression and demonstrated reduced migratory ability to VEGF. CONCLUSIONS: This study demonstrates apoptosis upon short-term inhibition and inhibition of long-term survival of OECs from patients with nvAMD by SU5416, presumably via PI3K/Akt and/or PKC-mediated reduction in telomerase activity and subsequent induction of premature senescence, which is accompanied by impaired endothelial activity. Therefore, induction of premature senescence in endothelial cells may represent a potential therapeutic target in nvAMD.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Indoles/farmacología , Degeneración Macular/tratamiento farmacológico , Pirroles/farmacología , Células Madre/efectos de los fármacos , Apoptosis , Células Endoteliales/citología , Humanos , Degeneración Macular/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Receptores CXCR4/biosíntesis , Transducción de Señal , Células Madre/citología , Resultado del Tratamiento , Venas Umbilicales/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Bacterial populations are subject to complex processes of diversification that involve mutation and horizontal DNA transfer mediated by transformation, transduction, or conjugation. Tracing the evolutionary events leading to genetic changes allows us to infer the history of a microbe. Here, we combine experimental and in silico approaches to explore the forces that drive the genome dynamics of Streptococcus agalactiae, the leading cause of neonatal infections. We demonstrate that large DNA segments of up to 334 kb of the chromosome of S. agalactiae can be transferred through conjugation from multiple initiation sites. Consistently, a genome-wide map analysis of nucleotide polymorphisms among eight human isolates demonstrated that each chromosome is a mosaic of large chromosomal fragments from different ancestors suggesting that large DNA exchanges have contributed to the genome dynamics in the natural population. The analysis of the resulting genetic flux led us to propose a model for the evolutionary history of this species in which clonal complexes of clinical importance derived from a single clone that evolved by exchanging large chromosomal regions with more distantly related strains. The emergence of this clone could be linked to selective sweeps associated with the reduction of genetic diversity in three regions within a large panel of human isolates. Up to now sex in bacteria has been assumed to involve mainly small regions; our results define S. agalactiae as an alternative paradigm in the study of bacterial evolution.
Asunto(s)
Evolución Biológica , Cromosomas Bacterianos/genética , Genoma Bacteriano , Streptococcus agalactiae/genética , Conjugación Genética , Variación Genética , Humanos , Modelos Genéticos , Polimorfismo GenéticoRESUMEN
Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.
Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Genoma Bacteriano , Photorhabdus/química , Photorhabdus/metabolismo , Proteoma/química , Proteoma/metabolismo , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Animales , Toxinas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , Datos de Secuencia Molecular , Photorhabdus/genética , Photorhabdus/patogenicidad , Rhabditoidea/microbiología , Alineación de Secuencia/métodos , Homología de Secuencia de Aminoácido , Simbiosis/genéticaRESUMEN
BACKGROUND: A chikungunya virus outbreak of unprecedented magnitude is currently ongoing in Indian Ocean territories. In Réunion Island, this alphavirus has already infected about one-third of the human population. The main clinical symptom of the disease is a painful and invalidating poly-arthralgia. Besides the arthralgic form, 123 patients with a confirmed chikungunya infection have developed severe clinical signs, i.e., neurological signs or fulminant hepatitis. METHODS AND FINDINGS: We report the nearly complete genome sequence of six selected viral isolates (isolated from five sera and one cerebrospinal fluid), along with partial sequences of glycoprotein E1 from a total of 127 patients from Réunion, Seychelles, Mauritius, Madagascar, and Mayotte islands. Our results indicate that the outbreak was initiated by a strain related to East-African isolates, from which viral variants have evolved following a traceable microevolution history. Unique molecular features of the outbreak isolates were identified. Notably, in the region coding for the non-structural proteins, ten amino acid changes were found, four of which were located in alphavirus-conserved positions of nsP2 (which contains helicase, protease, and RNA triphosphatase activities) and of the polymerase nsP4. The sole isolate obtained from the cerebrospinal fluid showed unique changes in nsP1 (T301I), nsP2 (Y642N), and nsP3 (E460 deletion), not obtained from isolates from sera. In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Homology 3D modelling allowed mapping of these two changes to regions that are important for membrane fusion and virion assembly. Change E1-A226V was absent in the initial strains but was observed in >90% of subsequent viral sequences from Réunion, denoting evolutionary success possibly due to adaptation to the mosquito vector. CONCLUSIONS: The unique molecular features of the analyzed Indian Ocean isolates of chikungunya virus demonstrate their high evolutionary potential and suggest possible clues for understanding the atypical magnitude and virulence of this outbreak.
Asunto(s)
Infecciones por Alphavirus/epidemiología , Infecciones por Alphavirus/genética , Virus Chikungunya/genética , Brotes de Enfermedades , Genoma Viral , Secuencia de Bases , Líquido Cefalorraquídeo/virología , Virus Chikungunya/aislamiento & purificación , Evolución Molecular , Variación Genética , Genoma Viral/genética , Glicosilación , Humanos , Inmunoensayo , Islas del Oceano Índico/epidemiología , Fenotipo , Filogenia , Análisis de Secuencia de ADN , Análisis de Secuencia de ARNRESUMEN
Streptococcus agalactiae is a leading cause of invasive infections in neonates, and responsible for bovine mastitis. It is also a commensal bacterium adapted to asymptomatic colonization of the mammalian gut and of the genitourinary tract. Here, we report the analysis of a collection of 75 strains of human and animal origin by using serotyping, multilocus sequence typing, whole genome DNA-array hybridizations and sequence comparison of putatively virulence-associated loci. Although the most variable parts of the genome are the previously predicted genomic islands, significant genetic variations were present in the genome backbone. Evolution within genes encoding surface and secreted proteins and those involved in the biosynthesis of different capsular types is mainly due to recombination events leading to the replacement of a locus of several genes or to the allelic exchange of the internal part of a gene. These two processes, which led to a broad diversity of surface protein patterns, are probably involved in the diversity of interactions with the host and its immune system. According to gene content comparisons and phylogeny, recent gene replacements by horizontal gene transfer may occur but are rare events. Although specific gene patterns, with respect to the origin of the strains and the epidemiological characteristics, were not identified, we show that the recently described hypervirulent ST-17 lineage is a homogeneous group. The study highlights for the first time that this lineage contains a specific and conserved set of surface proteins, probably accounting for its high capacity to cause infections in newborns.
Asunto(s)
Evolución Molecular , Variación Genética , Streptococcus agalactiae/clasificación , Streptococcus agalactiae/genética , Adulto , Animales , Proteínas Bacterianas/genética , Gatos , Bovinos , Preescolar , ADN Bacteriano/análisis , Perros , Femenino , Genoma Bacteriano , Cobayas , Humanos , Recién Nacido , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Conejos , Análisis de Secuencia de ADN , Serotipificación , Streptococcus agalactiae/patogenicidad , Virulencia/genéticaRESUMEN
Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. Multilocus sequence typing (MLST) revealed that the sequence type ST-17 defines a "highly virulent" serotype III clone strongly associated with neonatal invasive infections. Our aim was to identify a target sequence enabling rapid, simple, and specific detection of this clone by a real-time PCR assay. Conventional methods for DNA manipulation and gene analyses were used to characterize the gbs2018 gene variant specific for ST-17 clone and to design ST-17- and GBS-specific primers. Conventional and real-time PCR assays were developed to detect GBS and ST-17 clones in bacterial cultures and directly on clinical samples. One hundred and fifty-six French GBS strains from various geographical areas in France isolated between 1990 and 2005 were screened by PCR with ST-17-specific primers. Forty strains were positive, and all were validated by MLST as ST-17. A representative sampling of 49 ST-17-PCR-negative strains was confirmed by MLST as non-ST-17. Real-time PCR was further used to directly test 85 vaginal samples. Among these, 13 were GBS-positive, and one was identified as ST-17. The association between strain invasiveness and ST-17 lineage in neonates with late onset disease was highly significant: 78% (P<0.0001) of strains isolated were ST-17. In conclusion, an ST-17-specific gbs2018 allele was identified and used to develop a sensitive and specific rapid-screening molecular assay for identifying ST-17 "highly virulent" GBS. Using this technique, accurate identification of women and neonates colonized by ST-17 can be readily achieved within less than 2 h.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , Infecciones Estreptocócicas/diagnóstico , Streptococcus agalactiae/aislamiento & purificación , Adulto , Niño , Preescolar , Cartilla de ADN , Femenino , Genes Bacterianos , Variación Genética , Humanos , Recién Nacido , Embarazo , Sensibilidad y Especificidad , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/clasificación , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidadRESUMEN
Analysis of the Photorhabdus luminescens genome sequence revealed that the pts region is related to the tail synthesis gene core of the P2 phage. The pts locus encodes a DNA invertase homologue. PCR-RFLP analysis showed the two potential tail fiber regions of the pts locus present DNA inversions. Electron microscopy revealed a phage tail-like particle, related to the R-type family and named R-photorhabdicin, in the culture supernatant of P. luminescens. Mass spectrometry analysis of two sub-units of R-photorhabdicin revealed that they are encoded by the pts locus. The role of this P2-related prophage remnant in the Photorhabdus genome is discussed.
Asunto(s)
Bacteriófago P2/genética , Photorhabdus/genética , Photorhabdus/virología , beta-Fructofuranosidasa/genética , Bacteriófago P2/enzimología , Bacteriófago P2/ultraestructura , Microscopía Electrónica , Profagos/enzimología , Profagos/genética , Profagos/ultraestructura , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , beta-Fructofuranosidasa/metabolismoRESUMEN
Listeria monocytogenes, an invasive opportunistic, food-borne pathogen, remains one of the leading causes of mortality from food-borne infections. The recently determined complete genome sequences of L. monocytogenes strain EGDe and of that of the closely related non-pathogenic species Listeria innocua strain CLIP11262 enhance our knowledge of the genetic basis of the virulence of L. monocytogenes and advance our understanding of the evolution of these Listeria species. Both genomes encode a high number of surface, transport and regulatory proteins. Comparison of the genome organisation revealed a perfect synteny between the two Listeria genomes. Comparison with other closely related bacteria also showed a high conservation in genome organisation among the Listeria, Staphylococcus and Bacillus group of low G+C content bacteria. Distinct G+C content of a number of strain-specific genes suggests intensive lateral gene transfer. The identification of a 55-kb locus encoding proteins with high homology to Salmonella enterica serovar Typhimurium vitamin B(12) synthesis proteins as well as those necessary for degradation of ethanolamine and propanediol further indicates acquisition of a complete metabolic pathway by horizontal gene transfer and a probable role of this locus in anaerobic growth in the host.
Asunto(s)
Genoma Bacteriano , Listeria monocytogenes/genética , Listeria/genética , Anaerobiosis , Animales , Bacillus/genética , Proteínas Bacterianas/genética , Bacteriófagos/genética , Bacteriófagos/fisiología , Composición de Base , Evolución Molecular , Humanos , Listeria/metabolismo , Listeria/patogenicidad , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidad , Operón , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidad de la Especie , Staphylococcus/genética , Transducción Genética , Virulencia , Vitamina B 12/biosíntesisRESUMEN
PURPOSE: To evaluate endothelial progenitor cell [late outgrowth endothelial progenitor cells (OECs)], vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1α (SDF-1α) plasma levels as potential biomarkers before and during ranibizumab (Lucentis(®)) treatment for neovascular age-related macular degeneration (nvAMD). METHODS: Thirty-one patients with untreated nvAMD presenting for 3 consecutive intravitreal ranibizumab injections and a follow-up visit at 4 weeks intervals were enrolled. Peripheral blood was collected before each injection and at the follow-up visit and OEC clusters were cultured and evaluated according to previously published protocols. VEGF and SDF-1α plasma levels were measured by enzyme-linked immunosorbent assay and compared to values from healthy young and old control. RESULTS: Patients with a high OEC count before treatment presented significantly more often with a short symptom duration and a smaller choroidal neovascularization size. VEGF plasma levels were significantly higher in nvAMD (282.4±195.2 pg/mL) compared to young (45.5±6.8 pg/mL) and old control (46.1±8.5 pg/mL). OEC levels decreased nonsignificantly during ranibizumab treatment, returning to baseline levels after the third injection. VEGF and SDF-1α plasma levels decreased significantly during treatment toward control values. Patients needing retreatment after 3 ranibizumab injections had significantly higher VEGF plasma levels at pretreatment compared to patients not needing further treatment. CONCLUSIONS: The results presented here suggest that VEGF plasma levels may warrant further evaluation regarding biological, therapeutical, and predictive implications in nvAMD.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Quimiocina CXCL12/sangre , Neovascularización Coroidal/tratamiento farmacológico , Células Endoteliales/patología , Degeneración Macular/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/sangre , Anciano , Anciano de 80 o más Años , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/efectos adversos , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/efectos adversos , Biomarcadores/sangre , Recuento de Células , Neovascularización Coroidal/sangre , Neovascularización Coroidal/complicaciones , Esquema de Medicación , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Femenino , Humanos , Inyecciones Intravítreas , Degeneración Macular/sangre , Degeneración Macular/etiología , Masculino , Ranibizumab , Resultado del TratamientoRESUMEN
A continuous intraocular delivery of neurotrophic factors (NFs) is being explored as a strategy to rescue photoreceptor cells and visual functions in degenerative retinal disorders that are currently untreatable. To establish a cell-based intraocular delivery system for a sustained administration of NFs to the dystrophic mouse retina, we used a polycistronic lentiviral vector to genetically modify adherently cultivated murine neural stem (NS) cells. The vector concurrently encoded a gene of interest, a reporter gene, and a resistance gene and thus facilitated the selection, cloning, and in vivo tracking of the modified cells. To evaluate whether modified NS cells permit delivery of functionally relevant quantities of NFs to the dystrophic mouse retina, we expressed a secretable variant of ciliary neurotrophic factor (CNTF) in NS cells and grafted the cells into the vitreous space of Pde6b(rd1) and Pde6b(rd10) mice, two animal models of retinitis pigmentosa. In both mouse lines, grafted cells attached to the retina and lens, where they differentiated into astrocytes and some neurons. Adverse effects of the transplanted cells on the morphology of host retinas were not observed. Importantly, the CNTF-secreting NS cells significantly attenuated photoreceptor degeneration in both mutant mouse lines. The neuroprotective effect was significantly more pronounced when clonally derived NS cell lines selected for high expression levels of CNTF were grafted into Pde6b(rd1) mice. Intravitreal transplantations of modified NS cells may thus represent a useful method for preclinical studies aimed at evaluating the therapeutic potential of a cell-based intraocular delivery of NFs in mouse models of photoreceptor degeneration.
Asunto(s)
Factor Neurotrófico Ciliar/metabolismo , Terapia Genética , Células-Madre Neurales/metabolismo , Células-Madre Neurales/trasplante , Retina/metabolismo , Retinitis Pigmentosa/terapia , Animales , Células Cultivadas , Factor Neurotrófico Ciliar/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Modelos Animales de Enfermedad , Vectores Genéticos , Lentivirus/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación , Neurogénesis , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/patología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Esferoides Celulares , Transducción Genética , TransfecciónRESUMEN
Comparative genomics is the cornerstone of identification of gene functions. The immense number of living organisms precludes experimental identification of functions except in a handful of model organisms. The bacterial domain is split into large branches, among which the Firmicutes occupy a considerable space. Bacillus subtilis has been the model of Firmicutes for decades and its genome has been a reference for more than 10 years. Sequencing the genome involved more than 30 laboratories, with different expertises, in a attempt to make the most of the experimental information that could be associated with the sequence. This had the expected drawback that the sequencing expertise was quite varied among the groups involved, especially at a time when sequencing genomes was extremely hard work. The recent development of very efficient, fast and accurate sequencing techniques, in parallel with the development of high-level annotation platforms, motivated the present resequencing work. The updated sequence has been reannotated in agreement with the UniProt protein knowledge base, keeping in perspective the split between the paleome (genes necessary for sustaining and perpetuating life) and the cenome (genes required for occupation of a niche, suggesting here that B. subtilis is an epiphyte). This should permit investigators to make reliable inferences to prepare validation experiments in a variety of domains of bacterial growth and development as well as build up accurate phylogenies.
Asunto(s)
Bacillus subtilis/fisiología , Genoma Bacteriano , Compartimento Celular , Bases de Datos Genéticas , Ecosistema , Regulación Bacteriana de la Expresión Génica , Fenómenos Genéticos , Variación Genética , Metabolismo , Proteoma/análisis , Análisis de Secuencia de ADNRESUMEN
Genomics can provide the basis for understanding the evolution of emerging, lethal human pathogens such as Legionella pneumophila, the causative agent of Legionnaires' disease. This bacterium replicates within amoebae and persists in the environment as a free-living microbe. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease and within the 15 serogroups (Sg), L. pneumophila Sg1 causes over 84% of Legionnaires' disease worldwide. Why L. pneumophila Sg1 is so predominant is unknown. Here, we report the first comprehensive screen of the gene content of 217 L. pneumophila and 32 non-L. pneumophila strains isolated from humans and the environment using a Legionella DNA-array. Strikingly, we uncovered a high conservation of virulence- and eukaryotic-like genes, indicating strong environmental selection pressures for their preservation. No specific hybridization profile differentiated clinical and environmental strains or strains of different serogroups. Surprisingly, the gene cluster coding the determinants of the core and the O side-chain synthesis of the lipopolysaccaride (LPS cluster) determining Sg1 was present in diverse genomic backgrounds, strongly implicating the LPS of Sg1 itself as a principal cause of the high prevalence of Sg1 strains in human disease and suggesting that the LPS cluster can be transferred horizontally. Genomic analysis also revealed that L. pneumophila is a genetically diverse species, in part due to horizontal gene transfer of mobile genetic elements among L. pneumophila strains, but also between different Legionella species. However, the genomic background also plays a role in disease causation as demonstrated by the identification of a globally distributed epidemic strain exhibiting the genotype of the sequenced L. pneumophila strain Paris.
Asunto(s)
Variación Genética , Legionella pneumophila/genética , Brotes de Enfermedades , Transferencia de Gen Horizontal , Genes Bacterianos , Genómica , Humanos , Secuencias Repetitivas Esparcidas , Legionella/clasificación , Legionella/genética , Legionella pneumophila/clasificación , Legionella pneumophila/patogenicidad , Enfermedad de los Legionarios/epidemiología , Enfermedad de los Legionarios/microbiología , Lipopolisacáridos/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , FilogeniaRESUMEN
Listeria monocytogenes is a food-borne, opportunistic, bacterial pathogen causing a wide spectrum of diseases, including meningitis, septicemia, abortion, and gastroenteritis, in humans and animals. Among the 13 L. monocytogenes serovars described, human listeriosis is mostly associated with strains of serovars 4b, 1/2b, and 1/2a. Within the species L. monocytogenes, three phylogenetic lineages are described. Serovar 1/2a belongs to phylogenetic lineage I, while serovars 4b and 1/2b group in phylogenetic lineage II. To explore the role of gene expression in the adaptation of L. monocytogenes strains of these two major lineages to different environments, as well as in virulence, we performed whole-genome expression profiling of six L. monocytogenes isolates of serovars 4b, 1/2b, and 1/2a of distinct origins, using a newly constructed Listeria multigenome DNA array. Comparison of the global gene expression profiles revealed differences among strains. The expression profiles of two strains having distinct 50% lethal doses, as assessed in the mouse model, were further analyzed. Gene ontology term enrichment analysis of the differentially expressed genes identified differences in protein-, nucleic acid-, carbon metabolism-, and virulence-related gene expression. Comparison of the expression profiles of the core genomes of all strains revealed differences between the two lineages with respect to cell wall synthesis, the stress-related sigma B regulon and virulence-related genes. These findings suggest different patterns of interaction with host cells and the environment, key factors for host colonization and survival in the environment.
Asunto(s)
Proteínas Bacterianas/metabolismo , Genoma Bacteriano , Listeria monocytogenes/metabolismo , Factor sigma/metabolismo , Virulencia/genética , Proteínas Bacterianas/genética , Pared Celular/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Listeria monocytogenes/clasificación , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Redes y Vías Metabólicas , Factor sigma/genética , Transcripción GenéticaRESUMEN
Adaptation to the host environment and exploitation of host cell functions are critical to the success of intracellular pathogens. Here, insight to these virulence mechanisms was obtained for the first time from the transcriptional program of the human pathogen Legionella pneumophila during infection of its natural host, Acanthamoeba castellanii. The biphasic life cycle of L. pneumophila was reflected by a major shift in gene expression from replicative to transmissive phase, concerning nearly half of the genes predicted in the genome. However, three different L. pneumophila strains showed similar in vivo gene expression patterns, indicating that common regulatory mechanisms govern the Legionella life cycle, despite the plasticity of its genome. During the replicative phase, in addition to components of aerobic metabolism and amino acid catabolism, the Entner-Doudoroff pathway, a NADPH producing mechanism used for sugar and/or gluconate assimilation, was expressed, suggesting for the first time that intracellular L. pneumophila may also scavenge host carbohydrates as nutrients and not only proteins. Identification of genes only upregulated in vivo but not in vitro, may explain higher virulence of in vivo grown L. pneumophila. Late in the life cycle, L. pneumophila upregulates genes predicted to promote transmission and manipulation of a new host cell, therewith priming it for the next attack. These including substrates of the Dot/Icm secretion system, other factors associated previously with invasion and virulence, the motility and the type IV pilus machineries, and > 90 proteins not characterized so far. Analysis of a fliA (sigma28) deletion mutant identified genes coregulated with the flagellar regulon, including GGDEF/EAL regulators and factors that promote host cell entry and survival.
Asunto(s)
Acanthamoeba castellanii/microbiología , Legionella pneumophila/genética , Legionella pneumophila/patogenicidad , Fagocitos/microbiología , Aminoácidos/metabolismo , Animales , Proteínas Bacterianas/genética , Secuencia de Bases , Metabolismo de los Hidratos de Carbono , ADN Bacteriano/genética , Flagelos/genética , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Humanos , Legionella pneumophila/crecimiento & desarrollo , Legionella pneumophila/metabolismo , Modelos Biológicos , Regulón , Factor sigma/genética , Especificidad de la Especie , Transcripción Genética , VirulenciaRESUMEN
Photorhabdus is an entomopathogenic bacterium belonging to the Enterobacteriaceae. The genome of the TT01 strain of Photorhabdus luminescens was recently sequenced and a large number of toxin-encoding genes were found. Genomic analysis predicted the presence on the chromosome of genes encoding a type three secretion system (TTSS), the main role of which is the delivery of effector proteins directly into eukaryotic host cells. We report here the functional characterization of the TTSS. The locus identified encodes the secretion/translocation apparatus, gene expression regulators and an effector protein - LopT - homologous to the Yersinia cysteine protease cytotoxin YopT. Heterologous expression in Yersinia demonstrated that LopT was translocated into mammal cells in an active form, as shown by the appearance of a form of the RhoA GTPase with modified electrophoretic mobility. In vitro study showed that recombinant LopT was able to release RhoA and Rac from human and insect cell membrane. In vivo assays of infection of the cutworm Spodoptera littoralis and the locust Locusta migratoria with a TT01 strain carrying a translational fusion of the lopT gene with the gfp reporter gene revealed that the lopT gene was switched on only at sites of cellular defence reactions, such as nodulation, in insects. TTSS-mutant did not induce nodule formation and underwent phagocytosis by insect macrophage cells, suggesting that the LopT effector plays an essential role in preventing phagocytosis and indicating an unexpected link between TTSS expression and the nodule reaction in insects.
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Proteínas Bacterianas/fisiología , Locusta migratoria/microbiología , Fagocitosis/inmunología , Photorhabdus/metabolismo , Spodoptera/microbiología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Membrana Celular/metabolismo , Cisteína Endopeptidasas/genética , GTP Fosfohidrolasas/metabolismo , Genoma Bacteriano , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Locusta migratoria/inmunología , Locusta migratoria/ultraestructura , Datos de Secuencia Molecular , Photorhabdus/inmunología , Photorhabdus/ultraestructura , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/inmunología , Spodoptera/ultraestructura , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal pneumonia, sepsis, and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes 35 proteins containing an LPXTG motif which are thought to be covalently linked to the peptidoglycan by an enzyme called sortase. The role of these cell wall-anchored proteins in GBS pathogenesis was evaluated on a global level by inactivating the srtA gene. This gene encodes the major sortase SrtA that anchors most of the LPXTG-containing proteins. We chose the C5a peptidase (ScpB) and Alp2, an abundant immunogenic protein, as prototypical LPXTG-containing proteins. As expected, the SrtA knockout mutant was unable to anchor the C5a peptidase (ScpB) and Alp2 to the cell wall. Complementation with plasmid-borne srtA inserted into the chromosome restored the correct surface localization of both ScpB and Alp2. Interestingly, the SrtA mutant was impaired for binding to the major extracellular matrix components fibronectin and fibrinogen and displayed a significant reduction in adherence to human (A549, HeLa, and Caco-2) and murine (L2) epithelial cells compared to the parental wild-type strain. Surprisingly, the inactivation of srtA had no effect on the virulence of the type III strain of GBS in a neonatal rat model (measured by the 50% lethal dose and lung colonization) but strongly impaired the capacity of the strain to colonize the intestines of gnotobiotic mice in a competition assay. These results demonstrate that LPXTG-containing proteins are involved in cell adhesion and GBS persistence in vivo.
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Aminoaciltransferasas/fisiología , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Intestinos/microbiología , Streptococcus agalactiae/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoaciltransferasas/genética , Animales , Pared Celular/metabolismo , Células Cultivadas , Cisteína Endopeptidasas , Células Epiteliales/microbiología , Fibronectinas/fisiología , Humanos , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Ratas , Ratas Sprague-DawleyRESUMEN
In silico subtractive/differential genome analysis is a powerful approach for identifying genus- or species-specific genes, or groups of genes that are responsible for a unique phenotype. By this method, one searches for genes present in one group of bacteria and absent in another group. A software package has been developed, named FindTarget, that has a user-friendly web interface to facilitate differential genome analysis. The user chooses the genomes to compare, the similarity criteria and the thresholds to decide if a gene has a counterpart in another genome. The searches are based on BLASTP comparisons of proteomes. FindTarget also includes access to sequences, coloured multiple alignments, phylogenetic trees of conserved proteins and links to public annotated databases which provide a means for validation of the results. To illustrate this approach, a FindTarget search for genes putatively involved in the specificity of cell envelope synthesis of Gram-negative bacteria is presented. The results show that most of the identified genes are clearly involved in cell wall processes, underlining the power of such an approach in general and that of FindTarget in particular.
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Biología Computacional , Genoma Bacteriano , Programas Informáticos , Proteínas Bacterianas/genética , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/metabolismo , Proteoma , Interfaz Usuario-ComputadorRESUMEN
UNLABELLED: DiffTool is a resource to build and visualize protein clusters computed from a sequence database. The package provides a clustering tool to construct protein families according to sequence similarities and a web interface to query the corresponding clusters. A subtractive genome analysis tool selects protein families specific for a genome or a group of genomes. For each protein cluster, DiffTool includes access to sequences, coloured multiple alignments and phylogenetic trees. AVAILABILITY: A cluster database built from yeast and complete prokaryotic genomes is queryable at http://bioweb.pasteur.fr/seqanal/difftool. All the Perl sources are freely available to non-profit organizations upon request.
Asunto(s)
Análisis por Conglomerados , Sistemas de Administración de Bases de Datos , Bases de Datos de Proteínas , Almacenamiento y Recuperación de la Información/métodos , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Gráficos por Computador , Genoma , Internet , Alineación de Secuencia/métodos , Homología de SecuenciaRESUMEN
Photorhabdus luminescens is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ, a two-component system associated with virulence in intracellular bacterial pathogens. This organism was shown to respond to the availability of environmental magnesium. A mutant with a knockout mutation in the regulatory component of this system (phoP) had no obvious growth defect. It was, however, more motile and more sensitive to antimicrobial peptides than its wild-type parent. Remarkably, the mutation eliminated virulence in an insect model. No insect mortality was observed after injection of a large number of the phoP bacteria, while very small amounts of parental cells killed insect larvae in less than 48 h. At the molecular level, the PhoPQ system mediated Mg(2+)-dependent modifications in lipopolysaccharides and controlled a locus (pbgPE) required for incorporation of 4-aminoarabinose into lipid A. Mg(2+)-regulated gene expression of pbgP1 was absent in the mutant and was restored when phoPQ was complemented in trans. This finding highlights the essential role played by PhoPQ in the virulence of an entomopathogen.