RESUMO
T. gondii is a eukaryotic parasite that has evolved a stage called tachyzoite which multiplies in host cells by producing two daughter cells internally. These nascent tachyzoites bud off their mother and repeat the division process until the expanding progenies escape to settle and multiply in other host cells. Over these intra- and extra-cellular phases, the tachyzoite maintains an essential apicobasal polarity that emerges through a unique bidirectional budding process of the elongating cells. This process requires the assembly of several molecular complexes that, at the nascent pole, encompass structural and myosin motor elements. To characterize a recently identified basal pole marker named BCC7 with respect to the posterior myosin J and myosin C motors, we used conventional biochemistry as well as advanced proteomic and in silico analysis in conjunction with live and super resolution microscopy of transgenic fluorescent tachyzoites. We document that BCC7 forms a ribbed ring below which myosin C motor entities distribute regularly. In addition, we identified-among 13 BCC7 putative partners-two novel and five known members of the inner membrane complex (IMC) family which ends at the apical side of the ring. Therefore, BCC7 could assist the stabilization of the IMC plaques and contribute to the parasite biomechanical properties.
Assuntos
Toxoplasma , Divisão Celular , Miosinas/metabolismo , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismoRESUMO
Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections.
Assuntos
Francisella tularensis/genética , Ferro/metabolismo , Porinas/genética , Tularemia/genética , Proteínas de Bactérias/genética , Vacinas Bacterianas , Transporte Biológico/genética , Transporte Biológico/imunologia , Armas Biológicas , Escherichia coli/genética , Fluoroquinolonas/efeitos adversos , Fluoroquinolonas/uso terapêutico , Francisella tularensis/metabolismo , Francisella tularensis/patogenicidade , Humanos , Porinas/metabolismo , Tularemia/tratamento farmacológico , Tularemia/microbiologiaRESUMO
Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae.
Assuntos
Produtos do Gene env/ultraestrutura , Glicoproteínas/ultraestrutura , Spumavirus/ultraestrutura , Western Blotting , Linhagem Celular , Microscopia Crioeletrônica , Humanos , Processamento de Imagem Assistida por Computador , Conformação Proteica , Spumavirus/química , TransfecçãoRESUMO
Fluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in Francisella novicida clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the in vitro FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were compared to the MICs of FQs against bacterial clones harboring the same mutations and were further validated through complementation experiments and structural modeling. Whole-genome sequencing of highly FQ-resistant lineages was also done. Supercoiling and DNA cleavage assays demonstrated that GyrA D87 is a hot spot FQ resistance target in F. novicida and pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition in F. novicida is that the first-step mutation occurs in GyrB, with direct or indirect consequences for FQ sensitivity. Insertion of P466 into GyrB leads to a 50% inhibitory concentration (IC50) comparable to that observed for a mutant gyrase carrying the GyrA D87Y substitution, while the D487E-ΔK488 mutation, while not active on its own, contributes to the high level of resistance that occurs following acquisition of the GyrA D87G substitution in double GyrA/GyrB mutants. The involvement of other putative targets is discussed, including that of a ParE mutation that was found to arise in the very late stage of antibiotic exposure. This study provides the first characterization of the molecular mechanisms responsible for FQ resistance in Francisella.
Assuntos
DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/genética , Francisella/genética , Genoma Bacteriano , Mutação , Motivos de Aminoácidos , Substituição de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Sítios de Ligação , Clonagem Molecular , DNA Girase/química , DNA Girase/metabolismo , DNA Topoisomerase IV/química , DNA Topoisomerase IV/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fluoroquinolonas/química , Fluoroquinolonas/farmacologia , Francisella/efeitos dos fármacos , Francisella/enzimologia , Francisella/crescimento & desenvolvimento , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Testes de Sensibilidade Microbiana , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Tetherin is an unusual surface glycoprotein that employs an N-terminal and a C-terminal region to anchor the protein into membranes. Structural analyses revealed an elongated structure for the ectodomain that is probably oriented parallel to cellular membranes. Expression of tetherin can be induced by interferon in selected cell types, which leads to the restriction of HIV-1 replication in the absence of the viral antagonist Vpu. This review focuses on recent progress on the understanding of the molecular mechanisms of tetherin function during HIV and other enveloped virus budding processes. We discuss the role of diverse viral antagonists in tetherin down regulation and place the structural information on the ectodomain into the context of tetherin's ability to physically link virions such as HIV-1 to the plasma membrane after completion of budding.
RESUMO
The Toxoplasma gondii tachyzoite is a singled-cell obligate intracellular parasite responsible for the acute phase of toxoplasmosis. This polarized cell exhibits an apical complex, a hallmark of the phylum Apicomplexa, essential for motility, invasion, and egress from the host cell. Located on the opposite end of the cell is the basal complex, an elaborated cytoskeletal structure that also plays critical roles in the lytic cycle of the parasite, being involved in motility, cell division, constriction and cytokinesis, as well as intravacuolar cell-cell communication. Nevertheless, only a few proteins of this structure have been described and functionally assessed. In this study, we used spatial proteomics to identify new basal complex components (BCC), and in situ imaging, including ultrastructure expansion microscopy, to position them. We thus confirmed the localization of nine BCCs out of the 12 selected candidates and assigned them to different sub-compartments of the basal complex, including two new domains located above the basal ring and below the posterior cup. Their functional investigation revealed that none of these BCCs are essential for parasite growth in vitro. However, one BCC is critical for constricting of the basal complex, likely through direct interaction with the class VI myosin heavy chain J (MyoJ), and for gliding motility. Four other BCCs, including a phosphatase and a guanylate-binding protein, are involved in the formation and/or maintenance of the intravacuolar parasite connection, which is required for the rosette organization and synchronicity of cell division.
Assuntos
Toxoplasma , Toxoplasmose , Humanos , Toxoplasma/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasmose/parasitologia , Citoesqueleto/metabolismo , Divisão CelularRESUMO
BACKGROUND: Rickettsia prowazekii is the etiological agent of epidemic typhus and is an obligate intracellular bacterium that grows as a parasite freely within the cytoplasm of a eukaryotic host cell. Previous studies have shown that rOmpA and rOmpB which belong to the family of rickettsial cell surface antigens are involved in vitro in the adhesion of Rickettsiae to epithelial cells. Recently, two putative rickettsial adhesins have been identified using high resolution 2D-PAGE coupled with mass spectrometry. In this study, we further characterize and describe the adhesin Adr2 from R. prowazekii. METHODOLOGY/PRINCIPAL FINDINGS: Using an overlay assay coupled with mass spectrometry two adhesins, Adr1 (RP827) and Adr2 (RP828), were identified from the R. prowazekii proteome Recombinant R. prowazekii Adr2 was expressed through fusion with Dsbc in Escherichia coli, purified and concentrated, thus allowing production of specific monoclonal antibodies, as confirmed by western blot assays. Finally, inhibition of rickettsiae-induced cytotoxicity with monoclonal anti-Adr2 antibody has showed a greatest impact on bacterial cell entry at 8 h post-infection (ca50%) and then decreased progressively to attempt 18% of inhibition at day 7. These, correlated to the inhibition of rickettsiae-induced cytotoxicity with monoclonal anti-rOmpB antibody. Thus, Adr2 is sufficient to mediate R. prowazekii entry into the cell at early stage of mammalian cell infection. CONCLUSIONS: Our results suggest that R. prowazekii Adr2 could be the main actor promoting the entry of rickettsiae into the host cells. The present study opens the framework for future investigations for better understanding of the Adr2 -mediated mechanisms involved in adhesion/invasion or intracellular survival of R. prowazekii.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Fibroblastos/microbiologia , Rickettsia prowazekii/genética , Rickettsia prowazekii/patogenicidade , Adesinas Bacterianas/isolamento & purificação , Animais , Anticorpos Antibacterianos/imunologia , Células Cultivadas , Clonagem Molecular , Eletroforese em Gel Bidimensional , Escherichia coli/genética , Expressão Gênica , Espectrometria de Massas , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.
Assuntos
Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Francisella tularensis/metabolismo , Estresse Oxidativo/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Reparo do DNA/fisiologia , Escherichia coli/metabolismo , Macrófagos/metabolismo , Camundongos , Proteômica/métodos , Alinhamento de Sequência , Tularemia/microbiologia , Virulência/fisiologiaRESUMO
"Nanobacteria" are nanometer-scale spherical and ovoid particles which have spurred one of the biggest controversies in modern microbiology. Their biological nature has been severely challenged by both geologists and microbiologists, with opinions ranging from considering them crystal structures to new life forms. Although the nature of these autonomously replicating particles is still under debate, their role in several calcification-related diseases has been reported. In order to gain better insights on this calciferous agent, we performed a large-scale project, including the analysis of "nanobacteria" susceptibility to physical and chemical compounds as well as the comprehensive nucleotide, biochemical, proteomic, and antigenic analysis of these particles. Our results definitively ruled out the existence of "nanobacteria" as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call "nanons." The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor.
Assuntos
Apatitas/metabolismo , Bactérias/metabolismo , Calcinose/metabolismo , alfa-Fetoproteínas/metabolismo , Acanthamoeba/microbiologia , Animais , Antibacterianos/farmacologia , Apatitas/química , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Sequência de Bases , Calcinose/microbiologia , Sobrevivência Celular , Feminino , Amplificação de Genes , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Monócitos/microbiologia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Trofozoítos/microbiologia , alfa-Fetoproteínas/químicaRESUMO
DNA microarrays are a powerful and promising approach to gain a detailed understanding of the bacterial response and the molecular cross-talk that can occur as a consequence of host-pathogen interactions. However, published studies mainly describe the host response to infection. Analysis of bacterial gene regulation in the course of infection has confronted many challenges. This review summarizes the different strategies used over the last few years to investigate, at the genomic scale, and using microarrays, the alterations in the bacterial transcriptome in response to interactions with host cells. Thirty-seven studies involving 19 different bacterial pathogens were compiled and analyzed. Our in silico comparison of the transcription profiles of bacteria grown in broth or in contact with eukaryotic cells revealed some features commonly observed when bacteria interact with host cells, including stringent response and cell surface remodeling.
Assuntos
Doenças dos Animais/genética , Bactérias/genética , Infecções Bacterianas/microbiologia , Infecções Bacterianas/veterinária , Interações Hospedeiro-Patógeno , Transcrição Gênica , Doenças dos Animais/microbiologia , Animais , Galinhas , Cricetinae , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/genética , Coelhos , RatosRESUMO
Biofilms are currently considered as a predominant lifestyle of many bacteria in nature. While they promote survival of microbes, biofilms also potentially increase the threats to animal and public health in case of pathogenic species. They not only facilitate bacteria transmission and persistence, but also promote spreading of antibiotic resistance leading to chronic infections. In the case of Francisella tularensis, the causative agent of tularemia, biofilms have remained largely enigmatic. Here, applying live and static confocal microscopy, we report growth and ultrastructural organization of the biofilms formed in vitro by these microorganisms over the early transition from coccobacillary into coccoid shape during biofilm assembly. Using selective dispersing agents, we provided evidence for extracellular DNA (eDNA) being a major and conserved structural component of mature biofilms formed by both F. subsp. novicida and a human clinical isolate of F. philomiragia. We also observed a higher physical robustness of F. novicida biofilm as compared to F. philomiragia one, a feature likely promoted by specific polysaccharides. Further, F. novicida biofilms resisted significantly better to ciprofloxacin than their planktonic counterparts. Importantly, when grown in biofilms, both Francisella species survived longer in cold water as compared to free-living bacteria, a trait possibly associated with a gain in fitness in the natural aquatic environment. Overall, this study provides information on survival of Francisella when embedded with biofilms that should improve both the future management of biofilm-related infections and the design of effective strategies to tackle down the problematic issue of bacteria persistence in aquatic ecosystems.
Assuntos
Biofilmes , Farmacorresistência Bacteriana , Francisella/fisiologia , Água Doce/microbiologia , Adaptação Fisiológica , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Sequência Conservada , DNA Bacteriano/química , Francisella/efeitos dos fármacos , Francisella/genética , Francisella/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , HumanosRESUMO
Among the eukaryotic cells that navigate through fully developed metazoan tissues, protozoans from the Apicomplexa phylum have evolved motile developmental stages that move much faster than the fastest crawling cells owing to a peculiar substrate-dependent type of motility, known as gliding. Best-studied models are the Plasmodium sporozoite and the Toxoplasma tachyzoite polarized cells for which motility is vital to achieve their developmental programs in the metazoan hosts. The gliding machinery is shared between the two parasites and is largely characterized. Localized beneath the cell surface, it includes actin filaments, unconventional myosin motors housed within a multimember glideosome unit, and apically secreted transmembrane adhesins. In contrast, less is known about the force mechanisms powering cell movement. Pioneered biophysical studies on the sporozoite and phenotypic analysis of tachyzoite actin-related mutants have added complexity to the general view that force production for parasite forward movement directly results from the myosin-driven rearward motion of the actin-coupled adhesion sites. Here, we have interrogated how forces and substrate adhesion-de-adhesion cycles operate and coordinate to allow the typical left-handed helical gliding mode of the tachyzoite. By combining quantitative traction force and reflection interference microscopy with micropatterning and expansion microscopy, we unveil at the millisecond and nanometer scales the integration of a critical apical anchoring adhesion with specific traction and spring-like forces. We propose that the acto-myoA motor directs the traction force which allows transient energy storage by the microtubule cytoskeleton and therefore sets the thrust force required for T. gondii tachyzoite vital helical gliding capacity.
Assuntos
Parasitos , Toxoplasma , Actinas , Animais , Proteínas de Protozoários , Torque , TraçãoRESUMO
BACKGROUND: The Rickettsia genus includes 25 validated species, 17 of which are proven human pathogens. Among these, the pathogenicity varies greatly, from the highly virulent R. prowazekii, which causes epidemic typhus and kills its arthropod host, to the mild pathogen R. africae, the agent of African tick-bite fever, which does not affect the fitness of its tick vector. RESULTS: We evaluated the clonality of R. africae in 70 patients and 155 ticks, and determined its genome sequence, which comprises a circular chromosome of 1,278,540 bp including a tra operon and an unstable 12,377-bp plasmid. To study the genetic characteristics associated with virulence, we compared this species to R. prowazekii, R. rickettsii and R. conorii. R. africae and R. prowazekii have, respectively, the less and most decayed genomes. Eighteen genes are present only in R. africae including one with a putative protease domain upregulated at 37 degrees C. CONCLUSION: Based on these data, we speculate that a loss of regulatory genes causes an increase of virulence of rickettsial species in ticks and mammals. We also speculate that in Rickettsia species virulence is mostly associated with gene loss.The genome sequence was deposited in GenBank under accession number [GenBank: NZ_AAUY01000001].
Assuntos
Genoma Bacteriano , Filogenia , Rickettsia/genética , Animais , DNA Bacteriano/genética , Feminino , Humanos , Masculino , Família Multigênica , Plasmídeos , Rickettsia/classificação , Rickettsia/patogenicidade , Análise de Sequência de DNA , Especificidade da Espécie , Carrapatos/microbiologia , Transcrição Gênica , VirulênciaRESUMO
The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair-long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic "melting pot" where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.
Assuntos
Amoeba/genética , Amoeba/microbiologia , Genoma Bacteriano , Modelos Genéticos , Rickettsia/genética , Rickettsia/patogenicidade , Actinas/metabolismo , Animais , Chlorocebus aethiops , Análise por Conglomerados , Família Multigênica , Fases de Leitura Aberta , Filogenia , Infecções por Rickettsia/genética , Células VeroRESUMO
Francisella tularensis is the causative agent in tularemia for which the high prevalence of treatment failure and relapse is a major concern. Directed-evolution experiments revealed that acquisition of fluoroquinolone (FQ) resistance was linked to factors in addition to mutations in DNA gyrase. Here, using F. tularensis live vaccine strain (LVS) as a model, we demonstrated that FupA/B (Fer-Utilization Protein) expression is linked to FQ susceptibility, and that the virulent strain F. tularensis subsp. tularensis SCHU S4 deleted for the homologous FupA protein exhibited even higher FQ resistance. In addition to an increased FQ minimal inhibitory concentration, LVSΔfupA/B displayed tolerance toward bactericidal compounds including ciprofloxacin and gentamicin. Interestingly, the FupA/B deletion was found to promote increased secretion of outer membrane vesicles (OMVs). Mass spectrometry-based quantitative proteomic characterization of vesicles from LVS and LVS∆fupA/B identified 801 proteins, including a subset of 23 proteins exhibiting differential abundance between both strains which may therefore contribute to the reduced antibiotic susceptibility of the FupA/B-deleted strain. We also demonstrated that OMVs are key structural elements of LVSΔfupA/B biofilms providing protection against FQ. These results provide a new basis for understanding and tackling antibiotic resistance and/or persistence of Francisella and other pathogenic members of the Thiotrichales class.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Biofilmes , Vesículas Extracelulares/metabolismo , Fluoroquinolonas/farmacologia , Francisella tularensis/efeitos dos fármacos , Francisella tularensis/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana , Vesículas Extracelulares/genética , Francisella tularensis/fisiologia , Deleção de Genes , Testes de Sensibilidade Microbiana , MutaçãoRESUMO
Spotted fever group rickettsiae are obligate intracellular pathogens able to manipulate the actin cytoskeleton, thus enabling cell-to-cell spreading during infection. While the RickA protein, which has similarity to the WASP family of Arp2/3-complex activators, was described as being responsible for actin-based motility, recent studies demonstrated that another factor, still unidentified, is also involved in this phenomenon. Here, using recombinant protein of Rickettsia conorii as an antigen, we produced a monoclonal antibody (mAb) directed against RickA. Its specificity was checked using two-dimensional polyacrylamide gel electrophoresis coupled with MS analysis. In Western-blot assays, our antibody recognized the RickA protein from all spotted fever group rickettsiae tested. This mAb would be useful to monitor the expression of RickA in spotted fever group rickettsiae grown under various culture conditions, associated or not with the motility phenotype, and thus to gain better knowledge about the molecular mechanisms involved in their cell-to-cell spreading.
Assuntos
Anticorpos Monoclonais , Proteínas de Bactérias/análise , Rickettsia/química , Animais , Anticorpos Monoclonais/isolamento & purificação , Western Blotting/métodos , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel Bidimensional , Feminino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de Sequência de DNARESUMO
We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular alpha-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp) and a long (62,829 bp) form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin-antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as beta-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria.
Assuntos
Genoma Bacteriano , Plasmídeos/genética , Rickettsia felis/genética , Aclimatação , Antígenos de Superfície/análise , Antígenos de Superfície/classificação , Mapeamento Cromossômico , DNA Bacteriano/genética , Genes Bacterianos , Genômica , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Fases de Leitura Aberta , Fenótipo , Plasmídeos/isolamento & purificação , Rickettsia felis/patogenicidade , Rickettsia felis/ultraestrutura , Homologia de Sequência do Ácido NucleicoRESUMO
BACKGROUND: The aim of this study was to analyze the genomic diversity of several Tropheryma whipplei strains by microarray-based comparative genomic hybridization. Fifteen clinical isolates originating from biopsy samples recovered from different countries were compared with the T. whipplei Twist strain. For each isolate, the genes were defined as either present or absent/divergent using the GACK analysis software. Genomic changes were then further characterized by PCR and sequencing. RESULTS: The results revealed a limited genetic variation among the T. whipplei isolates, with at most 2.24% of the probes exhibiting differential hybridization against the Twist strain. The main variation was found in genes encoding the WiSP membrane protein family. This work also demonstrated a 19.2 kb-pair deletion within the T. whipplei DIG15 strain. This deletion occurs in the same region as the previously described large genomic rearrangement between Twist and TW08/27. Thus, this can be considered as a major hot-spot for intra-specific T. whipplei differentiation. Analysis of this deleted region confirmed the role of WND domains in generating T. whipplei diversity. CONCLUSION: This work provides the first comprehensive genomic comparison of several T. whipplei isolates. It reveals that clinical isolates originating from various geographic and biological sources exhibit a high conservation rate, indicating that T. whipplei rarely interacts with exogenous DNA. Remarkably, frequent inter-strain variations were dicovered that affected members of the WiSP family.
Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Tropheryma/genética , DNA Bacteriano/genética , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Reação em Cadeia da Polimerase , Especificidade da Espécie , Tropheryma/classificação , Tropheryma/isolamento & purificaçãoRESUMO
The Rickettsia genus is composed of Gram-negative bacteria responsible for Typhus and spotted fevers. Because of the limitations imposed by their obligate intracellular location, the molecular mechanisms responsible for their pathogenicity remain poorly understood. Several rickettsial genomes are now available, thus providing the foundation for a new era of post-genomic research. Here, using Rickettsia conorii as model, we developed a suitable method for microarray-based transcriptome analysis of rickettsiae. Total RNA was extracted from infected Vero cells using a protocol preserving its integrity, as observed by Bioanalyzer (Agilent) profiles. By a subtractive hybridization method, the samples were subsequently depleted of eukaryotic RNA that represents up to 90% of the whole extract and that hampers fluorochrome labeling of rickettsial nucleic acids. To obtain the amount of material required for microarray hybridization, the bacterial RNA was then amplified using random primers. Hybridizations were carried out on microarrays specific for R. conorii but containing a limited number of selected targets. Our results show that this method yielded reproducible signals. Transcriptional changes observed following exposure of R. conorii to a nutrient stress were verified by real-time quantitative PCR and by quantitative reverse transcription PCR starting from amplified cDNA and total RNA as templates, respectively. We conclude that this approach has great potential for the study of mechanisms behind the virulence and intracellular survival of members of the genus Rickettsia.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , RNA Mensageiro/análise , Rickettsia conorii/metabolismo , Animais , Chlorocebus aethiops , Primers do DNA/química , RNA Mensageiro/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rickettsia conorii/genética , Rickettsia conorii/fisiologia , Células VeroRESUMO
Sequencing of the Rickettsia conorii genome and its comparison with its closest sequenced pathogenic relative, i.e., Rickettsia prowazekii, provided powerful insights into the evolution of these microbial pathogens. However, advances in our knowledge of rickettsial diseases are still hindered by the difficulty of working with strict intracellular bacteria and their hosts. Information gained from comparing the genomes of closely related organisms will shed new light on proteins susceptible to be targeted in specific diagnostic assays, by new antimicrobial drugs, and that could be employed in the generation of future rickettsial vaccines. In this review we present a detailed comparison of the metabolic pathways of these bacteria as well as the polymorphisms of their membrane proteins, transporters and putative virulence factors. Environmental adaptation of Rickettsia is also discussed.