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Patricia Renesto

Publications and source records attributed to Patricia Renesto.

18 recordsLinked to original sources
PubMed2006

Mimivirus giant particles incorporate a large fraction of anonymous and unique gene products.

Acanthamoeba polyphaga mimivirus is the largest known virus in both particle size and genome complexity. Its 1.2-Mb genome encodes 911 proteins, among which only 298 have predicted functions. The composition of purified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approach allowing the identification of 114 proteins. Besides the expected major structural components, the viral particle packages 12 proteins unambiguously associated with transcriptional machinery, 3 proteins associated with DNA repair, and 2 topoisomerases. Other main functional categories represented in the virion include oxidative pathways and protein modification. More than half of the identified virion-associated proteins correspond to anonymous genes of unknown function, including 45 "ORFans." As demonstrated by both Western blotting and immunogold staining, some of these "ORFans," which lack any convincing similarity in the sequence databases, are endowed with antigenic properties. Thus, anonymous and unique genes constituting the majority of the mimivirus gene complement encode bona fide proteins that are likely to participate in well-integrated processes.

Acanthamoeba↗
PubMed2006

Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity.

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPEgmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.

Amino Acid Sequence↗
PubMed2006

Genome sequence of Rickettsia bellii illuminates the role of amoebae in gene exchanges between intracellular pathogens.

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.

Actins↗
PubMed2006

Identification of two putative rickettsial adhesins by proteomic analysis.

The rickettsial membrane proteins that promote their uptake by eukaryotic host cells are unknown. To identify rickettsial ligand(s) that bind host cell surface proteins, biotinylated epithelial cells were used to probe a nitrocellulose membrane containing rickettsial extracts separated by SDS-PAGE. This overlay assay revealed that two close rickettsial ligands of approximately 32-30 kDa were recognized by host cells. Both proteins were identified using high resolution 2D-PAGE coupled with mass spectrometry analysis. One protein was identified as the C-terminal extremity of rOmpB called the beta-peptide. The second interacting protein was identified as a protein of unknown function encoded by RC1281 and RP828 in Rickettsia conorii and in Rickettsia prowazekii, respectively, that shares strong similarities with other bacterial adhesins. Both proteins are highly conserved within the Rickettsia genus and might play a critical role in their pathogenicity. These data may have important implications for the development of future vaccines against rickettsial infections.

Adhesins, Bacterial↗
PubMed2006

Global transcriptome analysis of Tropheryma whipplei in response to temperature stresses.

Tropheryma whipplei, the agent responsible for Whipple disease, is a poorly known pathogen suspected to have an environmental origin. The availability of the sequence of the 0.92-Mb genome of this organism made a global gene expression analysis in response to thermal stresses feasible, which resulted in unique transcription profiles. A few genes were differentially transcribed after 15 min of exposure at 43 degrees C. The effects observed included up-regulation of the dnaK regulon, which is composed of six genes and is likely to be under control of two HspR-associated inverted repeats (HAIR motifs) found in the 5' region. Putative virulence factors, like the RibC and IspDF proteins, were also overexpressed. While it was not affected much by heat shock, the T. whipplei transcriptome was strongly modified following cold shock at 4 degrees C. For the 149 genes that were differentially transcribed, eight regulons were identified, and one of them was composed of five genes exhibiting similarity with genes encoding ABC transporters. Up-regulation of these genes suggested that there was an increase in nutrient uptake when the bacterium was exposed to cold stress. As observed for other bacterial species, the major classes of differentially transcribed genes encode membrane proteins and enzymes involved in fatty acid biosynthesis, indicating that membrane modifications are critical. Paradoxically, the heat shock proteins GroEL2 and ClpP1 were up-regulated. Altogether, the data show that despite the lack of classical regulation pathways, T. whipplei exhibits an adaptive response to thermal stresses which is consistent with its specific environmental origin and could allow survival under cold conditions.

Actinomycetales↗
PubMed2005

Cryo-electron microscopy of the giant Mimivirus.

Mimivirus is the largest known virus. Using cryo-electron microscopy, the virus was shown to be icosahedral, covered by long fibers, and appears to have at least two lipid membranes within its protein capsid. A unique vertex, presumably for attachment and infection of the host, can be seen for particles that have a suitable orientation on the micrographs.

Capsid↗
PubMed2005

The genome sequence of Rickettsia felis identifies the first putative conjugative plasmid in an obligate intracellular parasite.

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.

Acclimatization↗
PubMed2005

Proteome analysis of Rickettsia conorii by two-dimensional gel electrophoresis coupled with mass spectrometry.

The availability of genome sequence offers the opportunity to further expand our knowledge about proteins expressed by Rickettsia conorii, strictly intracellular bacterium responsible for Mediterranean spotted fever. Using two-dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF mass spectrometry, we established the first reference map of R. conorii proteome. This approach also allowed identification of GroEL as the major antigen recognized by rabbit serum and sera of infected patients. Altogether, this work opens the way to characterize the proteome of R. conorii, to compare protein profiles of different isolates or of bacteria maintained under different experimental conditions and to identify immunogenic proteins as potential vaccine targets.

Animals↗
PubMed2005

Some lessons from Rickettsia genomics.

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.

Amino Acid Sequence↗
PubMed2005

Intervening sequence acquired by lateral gene transfer in Tropheryma whipplei results in 23S rRNA fragmentation.

Completion of Tropheryma whipplei genome sequencing may provide insights into the evolution of the molecular mechanisms underlying the pathogenicity of this microorganism. The first postgenomic application was the successful design of a comprehensive culture medium that allows axenic growth of this bacterium, which is particularly recalcitrant to cultivation. This achievement in turn permitted analysis of T. whipplei RNA without contaminating eukaryotic nucleic acids. To obtain high-quality RNA, several extraction methods were compared, but under all conditions tested an atypical profile was observed. By using a Northern blot assay we demonstrated that an insertion sequence previously described in T. whipplei 23S rRNA is in fact an intervening sequence excised during maturation. This cleavage could involve an RNase III identified in the genome of this microorganism. Among the bacteria with a 23S rRNA insertion sequence, T. whipplei is the only gram-positive microorganism. We present phylogenetic evidence that this mobile genetic element was acquired by lateral gene transfer from another enteric bacterium.

Actinomycetales↗
PubMed2005

Phylogenic analysis of rickettsial patatin-like protein with conserved phospholipase A2 active sites.

Genome analysis of Rickettsia felis highlighted the presence of three patatin-like protein (PLP) genes (pat1, pat2A, and pat2B), whereas only one PLP gene (pat1) is found in the other sequenced rickettsial genomes. Here, we aligned the rickettsial PLPs with characterized patatins from plants and found that they possess all the conserved amino acid residues identified as important for phospholipase A(2) activity. We also carried out a phylogenic analysis of the rickettsial PLPs together with bacterial and eukaryotic homologs. The phylogeny of the rickettsial Pat1 proteins is in conflict with the currently recognized Rickettsia phylogeny. Possible scenarios that might explain this incongruence are discussed and involve either gene conversion or gene duplication events.

Amino Acid Sequence↗
PubMed2004

Transcriptional response of Rickettsia conorii exposed to temperature variation and stress starvation.

Rickettsia conorii is an obligate intracellular bacterium transmitted to humans by Rhipicephalus sanguineus ticks. The success of this microorganism at surviving in nature implicates the ability to efficiently adapt to different environments, including the arthropod vector and the mammalian host. Numerous bacterial species possess a highly evolved system for stress adaptation. This so-called stringent response is mediated by guanosine 3',5'-bispyrophosphate and guanosine 3'-diphosphate 5'-triphosphate which are under spoT control in some Gram-negative bacteria. Interestingly, annotation of the R. conorii genome evidenced 5 spoT paralogs. We hypothesized that these spoT genes play a role in adaptation to environmental changes specifically encountered by rickettsiae during their different life cycles. Transcription of the spoT paralogs was examined by RT-PCR from infected Vero cells maintained in rich or deficient culture media, from infected C6/36 insect cells cultured at various temperatures and from infected ticks. Our results demonstrated that the 5 spoT genes can be transcribed. SpoT1 (RC0374) is only transcribed upon stringent response. Transcription of spoT3 (RC0888) was never observed in arthropod cells or ticks, but was specific to R. conorii RNA extracted from infected Vero cells. These results indicate that rickettsial spoT paralogs are independently transcribed, depending on the different infected hosts and the adaptive capacity of the pathogen. Bioinformatics analysis of these possibly encoded proteins is also reported.

Animals↗
PubMed2004

The 1.2-megabase genome sequence of Mimivirus.

We recently reported the discovery and preliminary characterization of Mimivirus, the largest known virus, with a 400-nanometer particle size comparable to mycoplasma. Mimivirus is a double-stranded DNA virus growing in amoebae. We now present its 1,181,404-base pair genome sequence, consisting of 1262 putative open reading frames, 10% of which exhibit a similarity to proteins of known functions. In addition to exceptional genome size, Mimivirus exhibits many features that distinguish it from other nucleocytoplasmic large DNA viruses. The most unexpected is the presence of numerous genes encoding central protein-translation components, including four amino-acyl transfer RNA synthetases, peptide release factor 1, translation elongation factor EF-TU, and translation initiation factor 1. The genome also exhibits six tRNAs. Other notable features include the presence of both type I and type II topoisomerases, components of all DNA repair pathways, many polysaccharide synthesis enzymes, and one intein-containing gene. The size and complexity of the Mimivirus genome challenge the established frontier between viruses and parasitic cellular organisms. This new sequence data might help shed a new light on the origin of DNA viruses and their role in the early evolution of eukaryotes.

Acanthamoeba↗
PubMed2003

Identification and characterization of a phospholipase D-superfamily gene in rickettsiae.

The completion of the sequencing of the genomes of both Rickettsia conorii and R. prowazekii provides the opportunity to identify putative virulence factors within these strictly intracellular pathogens. A role for a phospholipase A(2) (PLA(2)) in rickettsial pathogenicity was hypothesized, but the corresponding gene has not been identified. We have identified a gene that encodes a putative phospholipase D (PLD) and that has been detected by Southern blotting in 11 analyzed strains of rickettsiae. The recombinant protein is dimeric and has PLD activity, as demonstrated by its capacity to release [(3)H]-choline from phosphatidyl [(3)H]-choline. This PLD is present in whole rickettsial lysates and likely is a virulence factor, because incubation of rickettsiae with an anti-PLD antibody reduced their cytotoxic activity against Vero cells. This enzyme might account for the activity previously attributed to PLA(2) and might be critical for the intracellular life of these bacteria.

Amino Acid Sequence↗
PubMed2003

Genome-based design of a cell-free culture medium for Tropheryma whipplei.

Empirical approaches have guided the development of bacterial cultures. The availability of sequenced genomes now provides opportunities to define culture media for growth of fastidious pathogens with computer modelling of metabolic networks. A key issue is the possibility of growing host-dependent bacteria in cell-free conditions. The sequenced Tropheryma whipplei genome was analysed to identify specific metabolic deficiencies. We used this information to design a comprehensive medium that allowed three established T whipplei strains from culture with human cells and one new strain from a clinical sample to grow axenically. Genomic information can, therefore, provide sufficient clues for designing axenic media for fastidious and uncultured pathogens.

Actinomycetales↗
PubMed2002

Expression of green fluorescent protein in Rickettsia conorii.

Rickettsiae are obligate intracellular class III pathogens for which genetic manipulation has only recently been shown to be feasible. Such experiments were restricted to the typhus group rickettsiae, namely R. typhi and R. prowazekii. Here we report the first genetic manipulation of Rickettsia conorii, the bacterial agent responsible for the Mediterranean spotted fever. A gene encoding a variant of the green fluorescent protein under the control of the sterically repressed promoter (srp) from E. coli was integrated into the genome of this bacteria and detected by FACS analysis.

Animals↗