RESUMO
BACKGROUND: Anopheles cell lines are used in a variety of ways to better understand the major vectors of malaria in sub-Saharan Africa. Despite this, commonly used cell lines are not well characterized, and no tools are available for cell line identification and authentication. METHODS: Utilizing whole genome sequencing, genomes of 4a-3A and 4a-3B 'hemocyte-like' cell lines were characterized for insertions and deletions (indels) and SNP variation. Genomic locations of distinguishing sequence variation and species origin of the cell lines were also examined. Unique indels were targeted to develop a PCR-based cell line authentication assay. Mitotic chromosomes were examined to survey the cytogenetic landscape for chromosome structure and copy number in the cell lines. RESULTS: The 4a-3A and 4a-3B cell lines are female in origin and primarily of Anopheles coluzzii ancestry. Cytogenetic analysis indicates that the two cell lines are essentially diploid, with some relatively minor chromosome structural rearrangements. Whole-genome sequence was generated, and analysis indicated that SNPs and indels which differentiate the cell lines are clustered on the 2R chromosome in the regions of the 2Rb, 2Rc and 2Ru chromosomal inversions. A PCR-based authentication assay was developed to fingerprint three indels unique to each cell line. The assay distinguishes between 4a-3A and 4a-3B cells and also uniquely identifies two additional An. coluzzii cell lines tested, Ag55 and Sua4.0. The assay has the specificity to distinguish four cell lines and also has the sensitivity to detect cellular contamination within a sample of cultured cells. CONCLUSIONS: Genomic characterization of the 4a-3A and 4a-3B Anopheles cell lines was used to develop a simple diagnostic assay that can distinguish these cell lines within and across research laboratories. A cytogenetic survey indicated that the 4a-3A and Sua4.0 cell lines carry essentially normal diploid chromosomes, which makes them amenable to CRISPR/Cas9 genome editing. The presented simple authentication assay, coupled with screening for mycoplasma, will allow validation of the integrity of experimental resources and will promote greater experimental reproducibility of results.
Assuntos
Anopheles , Animais , Feminino , Masculino , Anopheles/genética , Hemócitos , Reprodutibilidade dos Testes , Mosquitos Vetores/genética , Linhagem CelularRESUMO
Streptococcus pyogenes (S. pyogenes) can thrive in its host during an infection, and, as a result, it must be able to respond to external stimuli and available carbon sources. The preclinical use of engineered pathogens capable of constitutive light production may provide real-time information on microbial-specific metabolic processes. In this study, we mapped the central metabolism of a luxABCDE-modified S. pyogenes Xen20 (Strep. Xen20) to its de novo synthesis of luciferase substrates as assessed by the rate of light production in response to different environmental triggers. Previous characterization predicted that the lux operon was under the myo-inositol iolE promotor. In this study, we revealed that supplementation with myo-inositol generated increased Strep. Xen20 luminescence. Surprisingly, when supplemented with infection-relevant carbon sources, such as glucose or glycine, light production was diminished. This was presumably due to the scavenging of pyruvate by L-lactate dehydrogenase (LDH). Inhibition of LDH by its inhibitor, oxamate, partially restored luminescent signal in the presence of glucose, presumably by allowing the resulting pyruvate to proceed to acetyl-coenzyme A (CoA). This phenomenon appeared specific to the lactic acid bacterial metabolism as glucose or glycine did not reduce signal in an analogous luxABCDE-modified Gram-positive pathogen, Staph. Xen29. The Strep. Xen20 cells produced light in a concentration-dependent manner, inversely related to the amount of glucose present. Taken together, our measures of microbial response could provide new information regarding the responsiveness of S. pyogenes metabolism to acute changes in its local environments and cellular health.
RESUMO
As a key vector for major arthropod-borne viruses (arboviruses) such as dengue, Zika and chikungunya, control of Aedes aegypti represents a major challenge in public health. Bloodmeal acquisition is necessary for the reproduction of vector mosquitoes and pathogen transmission. Blood contains potentially toxic amounts of iron while it provides nutrients for mosquito offspring; disruption of iron homeostasis in the mosquito may therefore lead to novel control strategies. We previously described a potential iron exporter in Ae. aegypti after a targeted functional screen of ZIP (zinc-regulated transporter/Iron-regulated transporter-like) and ZnT (zinc transporter) family genes. In this study, we performed an RNAseq-based screen in an Ae. aegypti cell line cultured under iron-deficient and iron-excess conditions. A subset of differentially expressed genes were analyzed via a cytosolic iron-sensitive dual-luciferase reporter assay with several gene candidates potentially involved in iron transport. In vivo gene silencing resulted in significant reduction of fecundity (egg number) and fertility (hatch rate) for one gene, termed dyspepsia. Silencing of dyspepsia reduced the induction of ferritin expression in the midgut and also resulted in delayed/impaired excretion and digestion. Further characterization of this gene, including a more direct confirmation of its substrate (iron or otherwise), could inform vector control strategies as well as to contribute to the field of metal biology.
Assuntos
Aedes/genética , Dispepsia/genética , Proteínas de Insetos/genética , Proteínas de Membrana Transportadoras/genética , Aedes/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Dispepsia/metabolismo , Inativação Gênica , Aptidão Genética , Proteínas de Insetos/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Análise de Sequência de RNA , Zinco/metabolismoRESUMO
The portion of the mosquito genome that does not code for proteins contains regulatory elements that likely underlie variation for important phenotypes including resistance and susceptibility to infection with arboviruses and Apicomplexan parasites. Filtering the non-coding genome to uncover these functional elements is an expanding area of research, though identification of non-coding regulatory elements is challenging due to the lack of an amino acid-like code for the non-coding genome and a lack of sequence conservation across species. This review focuses on three types of non-coding regulatory elements: (1) microRNAs (miRNAs), (2) long non-coding RNAs (lncRNAs), and (3) enhancers, and summarizes current advances in technical and analytical approaches for measurement of each of these elements on a genome-wide scale. The review also summarizes and highlights novel findings following application of these techniques in mosquito-borne disease research. Looking beyond the protein-coding genome is essential for understanding the complexities that underlie differential gene expression in response to arboviral or parasite infection in mosquito disease vectors. A comprehensive understanding of the regulation of gene and protein expression will inform transgenic and other vector control methods rooted in naturally segregating genetic variation.
RESUMO
BACKGROUND: Aedes aegypti is the principle vector of many arboviruses, including dengue virus and Zika virus, which are transmitted when an infected female mosquito takes a blood meal in order to initiate vitellogenesis. During blood digestion, ~ 10 mM heme-iron is ingested into the midgut lumen. While heme acts as both a nutrient and signaling molecule during blood digestion, it can also be highly toxic if left unchaperoned. Both signaling by, and degradation of, heme are intracellular processes, occurring in the nucleus and cytoplasm, respectively. However, the precise mechanism of heme uptake into the midgut epithelium is not currently known. RESULTS: We used next generation RNA sequencing with the goal to identify genes that code for membrane bound heme import protein(s) responsible for heme uptake into the midgut epithelium. Heme deprivation increased uptake of a heme fluorescent analog in cultured cells, while treatment of midguts with an excess of heme decreased uptake, confirming physiological changes were occurring in these heme-sensitive cells/tissues prior to sequencing. A list of candidate genes was assembled for each of the experimental sample sets, which included Aag2 and A20 cultured cells as well as midgut tissue, based on the results of a differential expression analysis, soft cluster analysis and number of predicted transmembrane domains. Lastly, the functions related to heme transport were examined through RNAi knockdown. CONCLUSIONS: Despite a large number of transmembrane domain containing genes differentially expressed in response to heme, very few were highly differentially expressed in any of the datasets examined. RNAi knockdown of a subset of candidates resulted in subtle changes in heme uptake, but minimal overall disruption to blood digestion/egg production. These results could indicate that heme import in Ae. aegypti may be controlled by a redundant system of multiple distinct transport proteins. Alternatively, heme membrane bound transport in Ae. aegypti could be regulated post-translationally.
Assuntos
Aedes/genética , Heme/metabolismo , Proteínas de Insetos/genética , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras/genética , Transcriptoma , Aedes/metabolismo , Animais , Células Cultivadas , Heme/deficiência , Proteínas de Insetos/metabolismo , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Flagelos/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Mutação , Polissacarídeos Bacterianos/biossíntese , Pseudomonas aeruginosa/patogenicidade , Seleção GenéticaRESUMO
Campylobacter jejuni is among the most common causes of diarrheal disease worldwide and efforts to develop protective measures against the pathogen are ongoing. One of the few defined virulence factors targeted for vaccine development is the capsule polysaccharide (CPS). We have developed a capsule conjugate vaccine against C. jejuni strain 81-176 (CPS-CRM) that is immunogenic in mice and nonhuman primates (NHPs) but only moderately immunogenic in humans when delivered alone or with aluminum hydroxide. To enhance immunogenicity, two novel liposome-based adjuvant systems, the Army Liposome Formulation (ALF), containing synthetic monophosphoryl lipid A, and ALF plus QS-21 (ALFQ), were evaluated with CPS-CRM in this study. In mice, ALF and ALFQ induced similar amounts of CPS-specific IgG that was significantly higher than levels induced by CPS-CRM alone. Qualitative differences in antibody responses were observed where CPS-CRM alone induced Th2-biased IgG1, whereas ALF and ALFQ enhanced Th1-mediated anti-CPS IgG2b and IgG2c and generated functional bactericidal antibody titers. CPS-CRM + ALFQ was superior to vaccine alone or CPS-CRM + ALF in augmenting antigen-specific Th1, Th2, and Th17 cytokine responses and a significantly higher proportion of CD4+ IFN-γ+ IL-2+ TNF-α+ and CD4+ IL-4+ IL-10+ T cells. ALFQ also significantly enhanced anti-CPS responses in NHPs when delivered with CPS-CRM compared to alum- or ALF-adjuvanted groups and showed the highest protective efficacy against diarrhea following orogastric challenge with C. jejuni This study provides evidence that the ALF adjuvants may provide enhanced immunogenicity of this and other novel C. jejuni capsule conjugate vaccines in humans.IMPORTANCECampylobacter jejuni is a leading cause of diarrheal disease worldwide, and currently no preventative interventions are available. C. jejuni is an invasive mucosal pathogen that has a variety of polysaccharide structures on its surface, including a capsule. In phase 1 studies, a C. jejuni capsule conjugate vaccine was safe but poorly immunogenic when delivered alone or with aluminum hydroxide. Here, we report enhanced immunogenicity of the conjugate vaccine delivered with liposome adjuvants containing monophosphoryl lipid A without or with QS-21, known as ALF and ALFQ, respectively, in preclinical studies. Both liposome adjuvants significantly enhanced immunity in mice and nonhuman primates and improved protective efficacy of the vaccine compared to alum in a nonhuman primate C. jejuni diarrhea model, providing promising evidence that these potent adjuvant formulations may enhance immunogenicity in upcoming human studies with this C. jejuni conjugate and other malaria and HIV vaccine platforms.
Assuntos
Vacinas Bacterianas/imunologia , Infecções por Campylobacter/prevenção & controle , Imunogenicidade da Vacina , Lipídeo A/análogos & derivados , Saponinas/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Infecções por Campylobacter/imunologia , Campylobacter jejuni/imunologia , Citocinas/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Lipídeo A/administração & dosagem , Lipossomos/administração & dosagem , Lipossomos/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Primatas , Células Th1/imunologia , Células Th2/imunologia , Vacinas Conjugadas/administração & dosagemRESUMO
Transcriptomic studies of Tribolium castaneum have led to significant advances in our understanding of co-regulation and differential expression of genes in development. However, previously used microarray approaches have covered only a subset of known genes. The aim of this study was to investigate gene expression patterns of beetle embryo, germ-line and somatic tissues. We identified 12,302 expressed genes and determined differentially expressed up and down-regulated genes among all samples. For example, 1624 and 3639 genes were differentially increased in expression greater than or equal to twofold change (FDR < 0.01) in testis vs. ovary (virgin female) and ovary vs. embryo (0-5 hr), respectively. Of these, many developmental, somatic and germ-line differentially expressed genes were identified. Furthermore, many maternally deposited transcripts were identified, whose expression either decreased rapidly or persisted during embryogenesis. Genes with the largest change in expression were predominantly decreased during early embryogenesis as compared to ovary or were increased in testis compared to embryo. We also identify zygotic genes induced after fertilization. The genome wide variation in transcript regulation in maternal and zygotic genes could provide additional information on how the anterior posterior axis formation is established in Tribolium embryos as compared to Drosophila Together, our data will facilitate studies of comparative developmental biology as well as help identify candidate genes for identifying cis-elements to drive transgenic constructs.
Assuntos
Embrião não Mamífero , Regulação da Expressão Gênica , Genes de Insetos , Células Germinativas/metabolismo , Tribolium/genética , Animais , Biologia Computacional/métodos , Desenvolvimento Embrionário/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Masculino , Anotação de Sequência Molecular , TranscriptomaRESUMO
Pseudomonas aeruginosa causes devastating infections in immunocompromised individuals. Once established, P. aeruginosa infections become incredibly difficult to treat due to the development of antibiotic tolerant, aggregated communities known as biofilms. A hyper-biofilm forming clinical variant of P. aeruginosa, known as a rugose small-colony variant (RSCV), is frequently isolated from chronic infections and is correlated with poor clinical outcome. The development of these mutants during infection suggests a selective advantage for this phenotype, but it remains unclear how this phenotype promotes persistence. While prior studies suggest RSCVs could survive by evading the host immune response, our study reveals infection with the RSCV, PAO1ΔwspF, stimulated an extensive inflammatory response that caused significant damage to the surrounding host tissue. In both a chronic wound model and acute pulmonary model of infection, we observed increased bacterial burden, host tissue damage, and a robust neutrophil response during RSCV infection. Given the essential role of neutrophils in P. aeruginosa-mediated disease, we investigated the impact of the RSCV phenotype on neutrophil function. The RSCV phenotype promoted phagocytic evasion and stimulated neutrophil reactive oxygen species (ROS) production. We also demonstrate that bacterial aggregation and TLR-mediated pro-inflammatory cytokine production contribute to the immune response to RSCVs. Additionally, RSCVs exhibited enhanced tolerance to neutrophil-produced antimicrobials including H2O2 and the antimicrobial peptide LL-37. Collectively, these data indicate RSCVs elicit a robust but ineffective neutrophil response that causes significant host tissue damage. This study provides new insight on RSCV persistence, and indicates this variant may have a critical role in the recurring tissue damage often associated with chronic infections.
Assuntos
Interações Hospedeiro-Patógeno , Neutrófilos/imunologia , Pneumonia Bacteriana/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Citocinas/metabolismo , Feminino , Variação Genética , Humanos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Microscopia Confocal , Mutação , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Neutrófilos/patologia , Fagocitose , Pneumonia Bacteriana/metabolismo , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/patologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Sus scrofa , CicatrizaçãoRESUMO
Heme and iron are essential molecules for many physiological processes and yet have the ability to cause oxidative damage such as lipid peroxidation, protein degradation, and ultimately cell death if not controlled. Blood-sucking arthropods have evolved diverse methods to protect themselves against iron/heme-related damage, as the act of bloodfeeding itself is high risk, high reward process. Protective mechanisms in medically important arthropods include the midgut peritrophic matrix in mosquitoes, heme aggregation into the crystalline structure hemozoin in kissing bugs and hemosomes in ticks. Once heme and iron pass these protective mechanisms they are presumed to enter the midgut epithelial cells via membrane-bound transporters, though relatively few iron or heme transporters have been identified in bloodsucking arthropods. Upon iron entry into midgut epithelial cells, ferritin serves as the universal storage protein and transport for dietary iron in many organisms including arthropods. In addition to its role as a nutrient, heme is also an important signaling molecule in the midgut epithelial cells for many physiological processes including vitellogenesis. This review article will summarize recent advancements in heme/iron uptake, detoxification and exportation in bloodfeeding arthropods. While initial strides have been made at ironing out the role of dietary iron and heme in arthropods, much still remains to be discovered as these molecules may serve as novel targets for the control of many arthropod pests.
RESUMO
PURPOSE: Generation of plasmin in vivo by Streptococcus pyogenes is thought to localize the active protease complexes to the pathogen surface to aid in tissue dissemination. Here, we chose to follow cutaneous streptococcal infections by the use of non-invasive bioluminescence imaging to determine if this pathogen can be followed by this approach and the extent of bacterial spread in the absence of canonical plasminogen activation by streptokinase. PROCEDURES: Mice were injected subcutaneously with either bioluminescent strains of streptococci, namely Xen20 and Xen10 or S. pyogenes ALAB49. Bioluminescence imaging was performed daily and results were correlated with microbiological and histological analyses. RESULTS: Comparative analysis of chronologic non-invasive datasets indicated that Xen20 did not disseminate from the initial infection site. Contrary to this, microbiological and histological analyses of Xen20 mice for total bacterial burden indicated sepsis and widespread pathogen involvement. CONCLUSIONS: The use of bioluminescence in microbe-based studies requires genomic and pathologic characterization to correlate imaging results with underlying pathology.
Assuntos
Modelos Animais de Doenças , Pele/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/patogenicidade , Animais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Aedes aegypti mosquitoes vector several arboviruses of global health significance, including dengue viruses and chikungunya virus. RNA interference (RNAi) plays an important role in antiviral immunity, gene regulation and protection from transposable elements. Double-stranded RNA binding proteins (dsRBPs) are important for efficient RNAi; in Drosophila functional specialization of the miRNA, endo-siRNA and exo-siRNA pathway is aided by the dsRBPs Loquacious (Loqs-PB, Loqs-PD) and R2D2, respectively. However, this functional specialization has not been investigated in other dipterans. We were unable to detect Loqs-PD in Ae. aegypti; analysis of other dipteran genomes demonstrated that this isoform is not conserved outside of Drosophila. Overexpression experiments and small RNA sequencing following depletion of each dsRBP revealed that R2D2 and Loqs-PA cooperate non-redundantly in siRNA production, and that these proteins exhibit an inhibitory effect on miRNA levels. Conversely, Loqs-PB alone interacted with mosquito dicer-1 and was essential for full miRNA production. Mosquito Loqs interacted with both argonaute 1 and 2 in a manner independent of its interactions with dicer. We conclude that the functional specialization of Loqs-PD in Drosophila is a recently derived trait, and that in other dipterans, including the medically important mosquitoes, Loqs-PA participates in both the miRNA and endo-siRNA based pathways.
Assuntos
Culicidae/genética , MicroRNAs/genética , Proteínas/genética , RNA Interferente Pequeno/genética , Animais , Linhagem Celular , Frações Subcelulares/metabolismoRESUMO
Molecular imaging by definition is the visualization of molecular and cellular processes within a given system. The modalities and reagents described here represent a diverse array spanning both pre-clinical and clinical applications. Innovations in probe design and technologies would greatly benefit therapeutic outcomes by enhancing diagnostic accuracy and assessment of acute therapy. Opportunistic pathogens continue to pose a worldwide threat, despite advancements in treatment strategies, which highlights the continued need for improved diagnostics. In this review, we present a summary of the current clinical protocol for the imaging of a suspected infection, methods currently in development to optimize this imaging process, and finally, insight into endocarditis as a model of infectious disease in immediate need of improved diagnostic methods.