RESUMEN
Wolbachia are obligate intracellular bacteria that occur in insects and filarial worms. Strains that infect insects have genomes that encode mobile genetic elements, including diverse lambda-like prophages called Phage WO. Phage WO packages an approximately 65 kb viral genome that includes a unique eukaryotic association module, or EAM, that encodes unusually large proteins thought to mediate interactions between the bacterium, its virus, and the eukaryotic host cell. The Wolbachia supergroup B strain, wStri from the planthopper Laodelphax striatellus, produces phage-like particles that can be recovered from persistently infected mosquito cells by ultracentrifugation. Illumina sequencing, assembly, and manual curation of DNA from two independent preparations converged on an identical 15,638 bp sequence that encoded packaging, assembly, and structural proteins. The absence of an EAM and regulatory genes defined for Phage WO from the wasp, Nasonia vitripennis, was consistent with the possibility that the 15,638 bp sequence represents an element related to a gene transfer agent (GTA), characterized by a signature head-tail region encoding structural proteins that package host chromosomal DNA. Future investigation of GTA function will be supported by the improved recovery of physical particles, electron microscopic examination of potential diversity among particles, and rigorous examination of DNA content by methods independent of sequence assembly.
RESUMEN
The springtail, Folsomia candida, is a soil arthropod commonly used to evaluate environmental toxins. Conflicting data on the toxicity of the herbicide paraquat prompted re-evaluation of its effects on F. candida survival and reproduction. Paraquat has an LC50 of about 80 µM when tested in the absence of charcoal; charcoal, often used in test arenas to facilitate visualization of the white Collembola, has a protective effect. Survivors of paraquat treatment fail to resume molting and oviposition, suggesting an irreversible effect on the Wolbachia symbiont that restores diploidy during parthenogenetic reproduction of this species.
Asunto(s)
Artrópodos , Wolbachia , Femenino , Animales , Paraquat , Suelo , Carbón Orgánico , ReproducciónRESUMEN
Wolbachia is an obligate intracellular bacterium that infects many species of insects, and has been of particular interest in recent efforts to reduce disease transmission by mosquitoes. Two aspects of Wolbachia biology underlie its applications for insect control: first, the bacterium behaves as a natural gene drive agent and, second, when introduced into mosquitoes that do not harbor Wolbachia in nature, infection reduces survival of pathogens. These properties support efforts to explore the basic biology of Wolbachia in insect cell lines, which can produce sufficient infectious material for microbiological studies and microinjection into novel hosts. When introduced into naïve C7-10 Aedes albopictus mosquito cells, the yield of Wolbachia strain wStri improves, roughly in proportion to the size of the inoculum, as exponential growth of the host cell ceases. Wolbachia yields also increase when persistently infected C/wStri1 cells or naive, newly infected cells are treated with 20-hydroxyecdysone (20E), which inhibits growth in the G1 phase of the cell cycle. These observations suggest that Wolbachia infection and replication are independent of exponential growth and mitosis of host cells. To explore yields of infectious bacteria in cells arrested prior to infection, I tested host cells pre-treated with mitomycin C, an agent that crosslinks DNA and prevents cell division that is used to produce "feeder layers" with mammalian cells. Yields of wStri per plate increased by about 50-fold relative to exponentially growing cells, and the multiplicity of infection necessary for a robust infection was reduced to a single bacterium per cell. These results suggest that Wolbachia infection and replication are supported by mitotically arrested cells and provide new insights into biological processes that influence maintenance of a widespread obligate intracellular bacterium.
Asunto(s)
Aedes , Wolbachia , Animales , Línea Celular , MamíferosRESUMEN
In anautogenous mosquitoes, synchronous development of terminal ovarian follicles after a blood meal provides an important model for studies on insect reproduction. Removal and implantation of ovaries, in vitro culture of dissected tissues and immunological assays for vitellogenin synthesis by the fat body showed that the Aedes aegypti (L.) (Diptera, Culicidae) mosquito ovary produces a factor essential for egg production. The discovery that the ovarian factor was the insect steroid hormone, ecdysone, provided a model for co-option of the larval hormones as reproductive hormones in adult insects. In later work on cultured mosquito cells, ecdysone was shown to arrest the cell cycle, resulting in an accumulation of diploid cells in G1, prior to initiation of DNA synthesis. Some mosquito species, such as Culex pipiens L. (Diptera, Culicidae), harbor the obligate intracellular bacterium, Wolbachia pipientis Hertig (Rickettsiales, Anaplasmataceae), in their reproductive tissues. When maintained in mosquito cell lines, Wolbachia abundance increases in ecdysone-arrested cells. This observation facilitated the recovery of high levels of Wolbachia from cultured cells for microinjection and genetic manipulation. In female Culex pipiens, it will be of interest to explore how hormonal cues that support initiation and progression of the vitellogenic cycle influence Wolbachia replication and transmission to subsequent generations via infected eggs.
RESUMEN
Genomes of the obligate intracellular alpha proteobacterium Wolbachia pipientis often encode prophage-like regions, and in a few cases, purified particles have been recovered. Because the structure of a conserved WO phage genome has been difficult to establish, we examined paired terminase and portal genes in Wolbachia phages and prophages, relative to those encoded by the gene transfer agent RcGTA from the free-living alpha proteobacterium Rhodobacter capsulatus. Terminase and portal proteins from Wolbachia have higher similarity to orthologs encoded by RcGTA than to orthologs encoded by bacteriophage lambda. In lambdoid phages, these proteins play key roles in assembly of mature phage particles, while in less well-studied gene transfer agents, terminase and portal proteins package random fragments of bacterial DNA, which could confound elucidation of WO phage genomes. In WO phages and prophages, terminase genes followed by a short gpW gene may be separated from the downstream portal gene by open-reading frames encoding a GH_25 hydrolase/muramidase, a PD-(D/E)XK nuclease, a hypothetical protein and/or a RelE/ParE toxin-antitoxin module. These aspects of gene organization, coupled with evidence for a low, non-inducible yield of WO phages, and the small size of WO phage particles described in the literature raise the possibility that Wolbachia prophage regions participate in processes that extend beyond conventional bacteriophage lysogeny and lytic replication. These intervening genes, and their possible relation to functions associated with GTAs, may contribute to variability among WO phage genomes recovered from physical particles and impact the ability of WO phages to act as transducing agents.
Asunto(s)
Bacteriófagos , Wolbachia , Bacteriófagos/genética , Empaquetamiento del ADN , Endodesoxirribonucleasas , Muramidasa/genética , Profagos/genética , Wolbachia/genéticaRESUMEN
The obligate intracellular microbe, Wolbachia pipientis (Rickettsiales; Anaplasmataceae), is a Gram-negative member of the alpha proteobacteria that infects arthropods and filarial worms. Although closely related to the genera Anaplasma and Ehrlichia, which include pathogens of humans, Wolbachia is uniquely associated with invertebrate hosts in the clade Ecdysozoa. Originally described in Culex pipiens mosquitoes, Wolbachia is currently represented by 17 supergroups and is believed to occur in half of all insect species. In mosquitoes, Wolbachia acts as a gene drive agent, with the potential to modify vector populations; in filarial worms, Wolbachia functions as a symbiont, and is a target for drug therapy. A small number of Wolbachia strains from supergroups A, B, and F have been maintained in insect cell lines, which are thought to provide a more permissive environment than the natural host. When transferred back to an insect host, Wolbachia produced in cultured cells are infectious and retain reproductive phenotypes. Here, I review applications of insect cell lines in Wolbachia research and describe conditions that facilitate Wolbachia infection and replication in naive host cells. Progress in manipulation of Wolbachia in vitro will enable genetic and biochemical advances that will facilitate eventual genetic engineering of this important biological control agent.
RESUMEN
Wolbachia is an obligate intracellular bacterium that has undergone extensive genomic streamlining in its arthropod and nematode hosts. Because the gene encoding the bacterial DNA recombination/repair protein RecA is not essential in Escherichia coli, abundant expression of this protein in a mosquito cell line persistently infected with Wolbachia strain wStri was unexpected. However, RecA's role in the lytic cycle of bacteriophage lambda provides an explanation for retention of recA in strains known to encode lambda-like WO prophages. To examine DNA recombination/repair capacities in Wolbachia, a systematic examination of RecA and related proteins in complete or nearly complete Wolbachia genomes from supergroups A, B, C, D, E, F, J and S was undertaken. Genes encoding proteins including RecA, RecF, RecO, RecR, RecG and Holliday junction resolvases RuvA, RuvB and RuvC are uniformly absent from Wolbachia in supergroup C and have reduced representation in supergroups D and J, suggesting that recombination and repair activities are compromised in nematode-associated Wolbachia, relative to strains that infect arthropods. An exception is filarial Wolbachia strain wMhie, assigned to supergroup F, which occurs in a nematode host from a poikilothermic lizard. Genes encoding LexA and error-prone polymerases are absent from all Wolbachia genomes, suggesting that the SOS functions induced by RecA-mediated activation of LexA do not occur, despite retention of genes encoding a few proteins that respond to LexA induction in E. coli. Three independent E. coli accessions converge on a single Wolbachia UvrD helicase, which interacts with mismatch repair proteins MutS and MutL, encoded in nearly all Wolbachia genomes. With the exception of MutL, which has been mapped to a eukaryotic association module in Phage WO, proteins involved in recombination/repair are uniformly represented by single protein annotations. Putative phage-encoded MutL proteins are restricted to Wolbachia supergroups A and B and show higher amino acid identity than chromosomally encoded MutL orthologs. This analysis underscores differences between nematode and arthropod-associated Wolbachia and describes aspects of DNA metabolism that potentially impact development of procedures for transformation and genetic manipulation of Wolbachia.
Asunto(s)
Artrópodos/microbiología , Reparación del ADN , Nematodos/microbiología , Rec A Recombinasas/genética , Wolbachia/genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Especificidad del Huésped , Familia de Multigenes , Rec A Recombinasas/metabolismo , Recombinación Genética , Serina Endopeptidasas/genética , Especificidad de la Especie , Wolbachia/clasificación , Wolbachia/metabolismoRESUMEN
Wolbachia is an obligate intracellular Gram-negative alpha-proteobacterium that has diverse effects on reproduction of arthropod hosts, including cytoplasmic incompatibility, male killing, feminization, and parthenogenesis. Some of these effects have important potential for control of insect pests, including mosquitoes that vector pathogens of humans. In mosquitoes, and in most other arthropods, elimination of Wolbachia by antibiotic treatment has no effect on host survival and reverses the Wolbachia-associated phenotype. Elimination of Wolbachia strain wFol, which enables parthenogenetic reproduction of the Collembolan, Folsomia candida, would result in population extinction. However, F. candida adults remain viable and resume reproduction when antibiotics are removed, suggesting that wFol survives antibiotic treatment in a quiescent persister state similar to that induced by chromosomally encoded toxin-antitoxin (TA) modules in free-living bacteria. Computational approaches were used to document the presence of antitoxin genes upstream of Wolbachia RelE/ParE, Fic, and AbiEii toxin genes. Moreover, this analysis revealed that Wolbachia RatA toxin is encoded by a single copy gene associated with an ssrS noncoding RNA gene. Documentation of potentially functional TA modules expands our understanding of the metabolic capabilities of Wolbachia, and provides an explanation for variable and sometimes contradictory results of antibiotic treatments. The presence of chromosomal TA modules in Wolbachia genomes suggests that wFol, and potentially other strains of Wolbachia, can enter a quiescent persister state.
Asunto(s)
Partenogénesis/genética , Reproducción/genética , Sistemas Toxina-Antitoxina/genética , Wolbachia/genética , Animales , Cromosomas Bacterianos/genética , Culicidae/microbiología , Topoisomerasa de ADN IV/genética , Genoma Bacteriano/genética , Humanos , Masculino , Control de Plagas , Simbiosis/genética , Wolbachia/patogenicidadRESUMEN
Factors that influence establishment of Wolbachia, an obligate intracellular bacterium, in novel insect hosts or uninfected insect cell lines are poorly understood. Infectivity of Wolbachia strain wStr was correlated with flow cytometric profiles to define optimal conditions for harvesting an infectious inoculum. Wolbachia recovered from the cell culture supernatant after gentle pipetting of infected cells represented about 1% of the total bacterial population and were more infectious than Wolbachia that remained associated with intact cells and/or membranes after low-speed centrifugation. Optimal establishment of a robust infection in naïve cells required 6 d, at a ratio of 80 to 160 bacteria per cell. Among Aedes albopictus mosquito cell lines, an aneuploid line with a 4n + 1 karyotype was more susceptible to infection than diploid lines. These findings contribute to the in vitro manipulation of Wolbachia, illustrate some of the many factors that influence infectivity, and identify areas for future investigation.
Asunto(s)
Aedes/microbiología , Infecciones Bacterianas/microbiología , Espacio Intracelular/microbiología , Wolbachia/fisiología , Aedes/citología , Aedes/efectos de los fármacos , Aneuploidia , Animales , Línea Celular , Metotrexato/farmacología , Wolbachia/efectos de los fármacos , Wolbachia/patogenicidadRESUMEN
Wolbachia pipientis (Rickettsiales; Anaplasmataceae) is an obligate intracellular alpha proteobacterium that occurs in arthropods and filarial worms. Some strains of Wolbachia can be maintained as persistent infections in insect cell lines. C/wStr1 cells from the mosquito Aedes albopictus maintain a robust infection with Wolbachia strain wStr, originally isolated from the planthopper, Laodelphax striatellus. To explore possible functions of penicillin-binding proteins expressed from the wStr genome, C/wStr1 cells were exposed to ampicillin. Absolute levels of Wolbachia increased 3.5-fold in ampicillin-treated cells and fivefold in naive cells newly infected with wStr. Because cell numbers were depressed by ampicillin treatment, Wolbachia yield on a per-cell basis increased by 15-fold. The absence of a similar effect on wAlbB in Aa23 host cells suggests that the Wolbachia strain, the presence/absence of genes encoding penicillin-binding proteins, or the interaction between wAlbB and its host cells may modulate the effects of ampicillin.
Asunto(s)
Aedes/microbiología , Ampicilina/farmacología , Wolbachia/fisiología , Aedes/efectos de los fármacos , Aedes/genética , Aedes/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Péptidos/química , Alineación de Secuencia , Especificidad de la Especie , Espectrometría de Masas en TándemRESUMEN
Neonicotinoid insecticides are the most widely used class of insecticides worldwide. Concern has grown over their widespread environmental presence and potential unintended adverse effects. The present study examined hydrolysis and photolysis reaction rates of neonicotinoids and assessed any residual toxicity of reaction products. Hydrolysis rates were tested between pH 4 and 10 and found to be base-catalyzed. Experiments revealed a nonelementary rate law for hydrolysis, with the hydroxide concentration raised to a power of 0.55 ± 0.09, which has implications for accurate prediction of environmental half-lives. Divalent metal ions (Cu2+ , Ni2+ , Zn2+ ) and minerals (kaolinite, goethite, TiO2 ) had no effect on hydrolysis rates. The hydrolysis rate in a natural water, however, was slower than that predicted by buffered experiments. Nitenpyram, imidacloprid, thiamethoxam, and clothianidin reacted via direct photolysis in both ultrapure and natural waters, with average quantum yields of 0.024 ± 0.001, 0.0105 ± 0.0002, 0.0140 ± 0.0002, and 0.0101 ± 0.0001, respectively. Acetamiprid primarily underwent indirect photolysis by reaction with OH· (1.7 ± [0.2] × 109 M-1 s-1 ). For all compounds, the urea derivative was the most commonly detected product in both hydrolysis and photolysis experiments. Using mosquito (Culex pipiens) larvae, no residual toxicity of reaction products was observed. Results indicate long environmental half-lives for the tested neonicotinoids, which may help to explain their ubiquitous presence in environmental matrices. Environ Toxicol Chem 2018;37:2797-2809. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.
Asunto(s)
Insecticidas/toxicidad , Neonicotinoides/toxicidad , Fotólisis , Pruebas de Toxicidad , Animales , Culex/efectos de los fármacos , Contaminación Ambiental/análisis , Hidrólisis , Insecticidas/química , Iones , Cinética , Larva , Metales/química , Neonicotinoides/química , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/toxicidadRESUMEN
The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales), is a widespread, vertically transmitted endosymbiont of filarial nematodes and arthropods. In insects, Wolbachia modifies reproduction, and in mosquitoes, infection interferes with replication of arboviruses, bacteria and plasmodia. Development of Wolbachia as a tool to control pest insects will be facilitated by an understanding of molecular events that underlie genetic exchange between Wolbachia strains. Here, we used nucleotide sequence, transcriptional and proteomic analyses to evaluate expression levels and establish the mosaic nature of genes flanking the T4SS virB8-D4 operon from wStr, a supergroup B-strain from a planthopper (Hemiptera) that maintains a robust, persistent infection in an Aedes albopictus mosquito cell line. Based on protein abundance, ribA, which contains promoter elements at the 5'-end of the operon, is weakly expressed. The 3'-end of the operon encodes an intact wspB, which encodes an outer membrane protein and is co-transcribed with the vir genes. WspB and vir proteins are expressed at similar, above average abundance levels. In wStr, both ribA and wspB are mosaics of conserved sequence motifs from Wolbachia supergroup A- and B-strains, and wspB is nearly identical to its homolog from wCobU4-2, an A-strain from weevils (Coleoptera). We describe conserved repeated sequence elements that map within or near pseudogene lesions and transitions between A- and B-strain motifs. These studies contribute to ongoing efforts to explore interactions between Wolbachia and its host cell in an in vitro system.
Asunto(s)
Genes Bacterianos/genética , Wolbachia/genética , Animales , Secuencia de Bases , Operón/genética , ProteómicaRESUMEN
Wolbachia pipientis (Rickettsiales), an obligate intracellular alphaproteobacterium in insects, manipulates host reproduction to maximize invasion of uninfected insect populations. Modification of host population structure has potential applications for control of pest species, particularly if Wolbachia can be maintained, manipulated, and genetically engineered in vitro. Although Wolbachia maintains an obligate mutualism with genome stability in nematodes, arthropods can be co-infected with distinct Wolbachia strains, and horizontal gene transfer between strains is potentially mediated by WO phages encoded within Wolbachia genomes. Proteomic analysis of a robust, persistent infection of a mosquito cell line with wStr from the planthopper, Laodelphax striatellus, revealed expression of a full array of WO phage genes, as well as nine of ten non-phage genes that occur between two distinct clusters of WOMelB genes in the genome of wMel, which infects Drosophila melanogaster. These non-phage genes encode potential host-adaptive proteins and are expressed in wStr at higher levels than phage structural proteins. A subset of seven of the non-phage genes is flanked by highly conserved non-coding sequences, including a putative promoter element, that are not present in a syntenically arranged array of homologs in plasmids from three tick-associated Rickettsia spp. These studies expand our understanding of wStr in a host cell line derived from the mosquito, Aedes albopictus, and provide a basis for investigating conditions that favor the lytic phase of the WO phage life cycle and recovery of infectious phage particles.
Asunto(s)
Bacteriófagos/genética , Drosophila melanogaster/genética , Control Biológico de Vectores , Proteoma/genética , Aedes/genética , Aedes/microbiología , Animales , Drosophila melanogaster/microbiología , Técnicas de Transferencia de Gen , Genoma Bacteriano , Hemípteros/genética , Filogenia , Wolbachia/genética , Wolbachia/patogenicidadRESUMEN
The plant allelochemical L-mimosine (ß-[N-(3-hydroxy-4-pyridone)]-α-aminopropionic acid; leucenol) resembles the nonessential amino acid, tyrosine. Because the obligate intracellular alphaproteobacterium, Wolbachia pipientis, metabolizes amino acids derived from host cells, the effects of mimosine on infected and uninfected mosquito cells were investigated. The EC50 for mimosine was 6-7 µM with Aedes albopictus C7-10 and C/wStr cell lines, and was not influenced by infection status. Mosquito cells responded to concentrations of mimosine substantially lower than those used to synchronize the mammalian cell cycle; at concentrations of 30-35 µM, mimosine reversibly arrested the mosquito cell cycle at the G1/S boundary and inhibited growth of Wolbachia strain wStr. Although lower concentrations of mimosine slightly increased wStr abundance, concentrations that suppressed the cell cycle reduced Wolbachia levels.
Asunto(s)
Aedes/citología , Aedes/efectos de los fármacos , Mimosina/farmacología , Wolbachia/efectos de los fármacos , Aedes/microbiología , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular/efectos de los fármacos , Línea Celular/microbiología , Relación Dosis-Respuesta a Droga , Feromonas/farmacología , Wolbachia/crecimiento & desarrollo , Wolbachia/patogenicidadRESUMEN
Conditions for flow cytometric evaluation of the intracellular bacterium, Wolbachia pipientis, in infected mosquito cells are described. This approach will streamline investigation of Wolbachia's interactions with host cells and facilitate identification of culture conditions that select for Wolbachia-infected cells.
Asunto(s)
Técnicas Bacteriológicas , Citoplasma/microbiología , Citometría de Flujo , Wolbachia , Animales , Antibacterianos/farmacología , Línea Celular , Culicidae/microbiología , Escherichia coli , Citometría de Flujo/métodos , Wolbachia/efectos de los fármacos , Wolbachia/fisiologíaRESUMEN
Wolbachia pipientis, a widespread vertically transmitted intracellular bacterium, provides a tool for insect control through manipulation of host-microbe interactions. We report proteomic characterization of wStr, a Wolbachia strain associated with a strong cytoplasmic incompatibility phenotype in its native host, Laodelphax striatellus. In the Aedes albopictusâ C/wStr1 mosquito cell line, wStr maintains a robust, persistent infection. MS/MS analyses of gel bands revealed a protein 'footprint' dominated by Wolbachia-encoded chaperones, stress response and cell membrane proteins, including the surface antigen WspA, a peptidoglycan-associated lipoprotein and a 73 kDa outer membrane protein. Functional classifications and estimated abundance levels of 790 identified proteins suggested that expression, stabilization and secretion of proteins predominate over bacterial genome replication and cell division. High relative abundances of cysteine desulphurase, serine/glycine hydroxymethyl transferase, and components of the α-ketoglutarate dehydrogenase complex in conjunction with above average abundances of glutamate dehydrogenase and proline utilization protein A support Wolbachia genome-based predictions for amino acid metabolism as a primary energy source. wStr expresses 15 Vir proteins of a Type IV secretion system and its transcriptional regulator. Proteomic characterization of a robust insect-associated Wolbachia strain provides baseline information that will inform further development of in vitro protocols for Wolbachia manipulation.
Asunto(s)
Proteínas Bacterianas/análisis , Proteoma/análisis , Wolbachia/crecimiento & desarrollo , Aedes , Animales , Línea Celular , Electroforesis en Gel Bidimensional , Espectrometría de Masas en TándemRESUMEN
Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7-10 mosquito cells decreases in riboflavin-depleted culture medium. A well-studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7-10 cells with an LC50 of approximately 12 µg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell.
Asunto(s)
Aedes/microbiología , Riboflavina/fisiología , Wolbachia , Aedes/química , Aedes/efectos de los fármacos , Animales , Células Cultivadas , Medios de Cultivo , Flavinas/farmacología , Proteómica , Riboflavina/análisis , Wolbachia/genética , Wolbachia/crecimiento & desarrolloRESUMEN
Cytoplasmic incompatibility (CI) is a conditional sterility induced by the bacterium Wolbachia pipientis that infects reproductive tissues in many arthropods. Although CI provides a potential tool to control insect vectors of arthropod-borne diseases, the molecular basis for CI induction is unknown. We hypothesized that a Wolbachia-encoded, CI-inducing factor would be enriched in sperm recovered from spermathecae of female mosquitoes. Using SDS-PAGE and mass spectrometry, we detected peptides from the 56 kDa hypothetical protein, encoded by wPip_0282, associated with sperm transferred to females by Wolbachia infected males. We also detected peptides from the same protein in Wolbachia infected ovaries. Homologs of wPip_0282 and the co-transcribed downstream gene, wPip_0283, occur as multiple divergent copies in genomes of CI-inducing strains of Wolbachia. The operon is located in a genomic context that includes mobile genetic elements. The absence of wPip_0282 and wPip_0283 homologs from genomes of Wolbachia in filarial nematodes, as well as other members of the Rickettsiales, suggests a role as a candidate CI effector.