RESUMO
Many pathogenic bacteria translocate virulence factors into their eukaryotic hosts by means of type IV secretion systems (T4SS) spanning the inner and outer membranes. Genes encoding components of these systems have been identified within the order Rickettsiales based upon their sequence similarities to other prototypical systems. Anaplasma phagocytophilum strains are obligate intracellular, tick-borne bacteria that are members of this order. The organization of these components at the genomic level was determined in several Anaplasma phagocytophilum strains, showing overall conservation, with the exceptions of the virB2 and virB6 genes. The virB6 loci are characterized by the presence of four virB6 copies (virB6-1 through virB6-4) arranged in tandem within a gene cluster known as the sodB-virB operon. Interestingly, the virB6-4 gene varies significantly in length among different strains due to extensive tandem repeats at the 3' end. To gain an understanding of how these enigmatic virB6 genes function in A. phagocytophilum, we investigated their expression in infected human and tick cells. Our results show that these genes are expressed by A. phagocytophilum replicating in both cell types and that VirB6-3 and VirB6-4 proteins are surface exposed. Analysis of an A. phagocytophilum mutant carrying the Himar1 transposon within the virB6-4 gene demonstrated that the insertion not only disrupted its expression but also exerted a polar effect on the sodB-virB operon. Moreover, the altered expression of genes within this operon was associated with the attenuated in vitro growth of A. phagocytophilum in human and tick cells, indicating the importance of these genes in the physiology of this obligate intracellular bacterium in such different environments.IMPORTANCE Knowledge of the T4SS is derived from model systems, such as Agrobacterium tumefaciens The structure of the T4SS in Rickettsiales differs from the classical arrangement. These differences include missing and duplicated components with structural alterations. Particularly, two sequenced virB6-4 genes encode unusual C-terminal structural extensions resulting in proteins of 4,322 (GenBank accession number AGR79286.1) and 9,935 (GenBank accession number ANC34101.1) amino acids. To understand how the T4SS is used in A. phagocytophilum, we describe the expression of the virB6 paralogs and explore their role as the bacteria replicate within its host cell. Conclusions about the importance of these paralogs for colonization of human and tick cells are supported by the deficient phenotype of an A. phagocytophilum mutant isolated from a sequence-defined transposon insertion library.
Assuntos
Anaplasma phagocytophilum/crescimento & desenvolvimento , Anaplasma phagocytophilum/genética , Proteínas de Bactérias/genética , Anaplasma phagocytophilum/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Linhagem Celular , Ehrlichiose/microbiologia , Humanos , Mutagênese Insercional , Óperon , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismoRESUMO
Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.
Assuntos
Anaplasma phagocytophilum/enzimologia , Ehrlichiose/microbiologia , Ixodes/microbiologia , Metiltransferases/metabolismo , Carrapatos/microbiologia , Animais , Ehrlichiose/genética , Ixodes/imunologia , Metiltransferases/genética , Ativação Transcricional , Regulação para CimaRESUMO
Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.
Assuntos
Anaplasma phagocytophilum/enzimologia , Di-Hidrolipoamida Desidrogenase/imunologia , Di-Hidrolipoamida Desidrogenase/metabolismo , Ehrlichiose/imunologia , Ehrlichiose/patologia , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo , Adulto , Anaplasma phagocytophilum/imunologia , Anaplasma phagocytophilum/patogenicidade , Animais , Ehrlichiose/microbiologia , Feminino , Deleção de Genes , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Neutrófilos/imunologia , Neutrófilos/microbiologia , Baço/microbiologia , Baço/patologiaRESUMO
Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions. As the density of mutants was non-saturating, genes without insertions were either essential for Ap, or spared randomly. To resolve this question, we applied a biostatistical method for prediction of essential genes. Since the chances that a transposon was inserted into genomic TA dinucleotide sites should be the same for all loci, we used a Markov chain Monte Carlo model to estimate the probability that a non-mutated gene was essential for Ap. Predicted essential genes included those coding for structural ribosomal proteins, enzymes involved in metabolism, components of the type IV secretion system, antioxidant defense molecules and hypothetical proteins. We have used an in silico post-genomic approach to predict genes with high probability of being essential for replication of Ap in HL-60 cells. These results will help target genes to investigate their role in the pathogenesis of human anaplasmosis.
Assuntos
Anaplasma phagocytophilum/genética , DNA Bacteriano/genética , Ehrlichiose , Genes Essenciais/genética , Células Precursoras de Granulócitos , Linhagem Celular , Elementos de DNA Transponíveis/genética , Ehrlichiose/genética , Ehrlichiose/microbiologia , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cadeias de MarkovRESUMO
The incidence of human diseases caused by tick-borne pathogens is increasing but little is known about the molecular interactions between the agents and their vectors and hosts. Anaplasma phagocytophilum (Ap) is an obligate intracellular, tick-borne bacterium that causes granulocytic anaplasmosis in humans, dogs, sheep, and horses. In mammals, neutrophil granulocytes are a primary target of infection, and in ticks, Ap has been found in gut and salivary gland cells. To identify bacterial genes that enable Ap to invade and proliferate in human and tick cells, labeled mRNA from Ap bound to or replicating within human and tick cells (lines HL-60 and ISE6), and replicating in primary human granulocytes ex vivo, was hybridized to a custom tiling microarray containing probes representing the entire Ap genome. Probe signal values plotted over a map of the Ap genome revealed antisense transcripts and unannotated genes. Comparisons of transcript levels from each annotated gene between test conditions (e.g., Ap replicating in HL-60 vs. ISE6) identified those that were differentially transcribed, thereby highlighting genes associated with each condition. Bacteria replicating in HL-60 cells upregulated 122 genes compared to those in ISE6, including numerous p44 paralogs, five HGE-14 paralogs, and 32 hypothetical protein genes, of which 47% were predicted to be secreted or localized to the membrane. By comparison, 60% of genes upregulated in ISE6 encoded hypothetical proteins, 60% of which were predicted to be secreted or membrane associated. In granulocytes, Ap upregulated 120 genes compared to HL-60, 33% of them hypothetical and 43% of those predicted to encode secreted or membrane associated proteins. HL-60-grown bacteria binding to HL-60 cells barely responded transcriptionally, while ISE6-grown bacteria binding to ISE6 cells upregulated 48 genes. HL-60-grown bacteria, when incubated with ISE6 cells, upregulated the same genes that were upregulated by ISE6-grown bacteria exposed to uninfected ISE6. Hypothetical genes (constituting about 29% of Ap genes) played a disproportionate role in most infection scenarios, and particular sets of them were consistently upregulated in bacteria binding/entering both ISE6 and HL-60 cells. This suggested that the encoded proteins played central roles in establishing infection in ticks and humans.
RESUMO
Mutational analysis is an efficient approach to identifying microbial gene function. Until recently, lack of an effective tool for Anaplasmataceae yielding reproducible results has created an obstacle to functional genomics, because surrogate systems, e.g., ectopic gene expression and analysis in E. coli, may not provide accurate answers. We chose to focus on a method for high-throughput generation of mutants via random mutagenesis as opposed to targeted gene inactivation. In our search for a suitable mutagenesis tool, we considered attributes of the Himar1 transposase system, i.e., random insertion into AT dinucleotide sites, which are abundant in Anaplasmataceae, and lack of requirement for specific host factors. We chose the Anaplasma marginale tr promoter, and the clinically irrelevant antibiotic spectinomycin for selection, and in addition successfully implemented non-antibiotic selection using an herbicide resistance gene. These constructs function reasonably well in Anaplasma phagocytophilum harvested from human promyelocyte HL-60 cells or Ixodes scapularis tick cells. We describe protocols developed in our laboratory, and discuss what likely makes them successful. What makes Anaplasmataceae electroporation competent is unknown and manipulating electroporation conditions has not improved mutational efficiency. A concerted effort is needed to resolve remaining problems that are inherent to the obligate intracellular bacteria. Finally, using this approach, we describe the discovery and characterization of a putative secreted effector necessary for Ap survival in HL-60 cells.
Assuntos
Anaplasmataceae/genética , Genes Bacterianos , Mutagênese , Anaplasma marginale/genética , Anaplasma phagocytophilum/genética , Animais , Análise Mutacional de DNA , Elementos de DNA Transponíveis , Genômica , Células HL-60 , Humanos , Ixodes/citologia , Transformação BacterianaRESUMO
BACKGROUND: Anaplasma phagocytophilum (Ap) is an obligate intracellular bacterium and the agent of human granulocytic anaplasmosis, an emerging tick-borne disease. Ap alternately infects ticks and mammals and a variety of cell types within each. Understanding the biology behind such versatile cellular parasitism may be derived through the use of tiling microarrays to establish high resolution, genome-wide transcription profiles of the organism as it infects cell lines representative of its life cycle (tick; ISE6) and pathogenesis (human; HL-60 and HMEC-1). RESULTS: Detailed, host cell specific transcriptional behavior was revealed. There was extensive differential Ap gene transcription between the tick (ISE6) and the human (HL-60 and HMEC-1) cell lines, with far fewer differentially transcribed genes between the human cell lines, and all disproportionately represented by membrane or surface proteins. There were Ap genes exclusively transcribed in each cell line, apparent human- and tick-specific operons and paralogs, and anti-sense transcripts that suggest novel expression regulation processes. Seven virB2 paralogs (of the bacterial type IV secretion system) showed human or tick cell dependent transcription. Previously unrecognized genes and coding sequences were identified, as were the expressed p44/msp2 (major surface proteins) paralogs (of 114 total), through elevated signal produced to the unique hypervariable region of each - 2/114 in HL-60, 3/114 in HMEC-1, and none in ISE6. CONCLUSION: Using these methods, whole genome transcription profiles can likely be generated for Ap, as well as other obligate intracellular organisms, in any host cells and for all stages of the cell infection process. Visual representation of comprehensive transcription data alongside an annotated map of the genome renders complex transcription into discernable patterns.
Assuntos
Anaplasma phagocytophilum/genética , Biologia Computacional , Perfilação da Expressão Gênica/métodos , Genoma Bacteriano , Carrapatos/microbiologia , Animais , Linhagem Celular , DNA Complementar/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Bacteriano/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Transcrição GênicaRESUMO
The genome project of the black legged tick, Ixodes scapularis, provides sequence data for testing gene function and regulation in this important pathogen vector. We tested Sleeping Beauty (SB), a Tc1/mariner group transposable element, and cationic lipid-based transfection reagents for delivery and genomic integration of transgenes into I. scapularis cell line ISE6. Plasmid DNA and dsRNA were effectively transfected into ISE6 cells and they were successfully transformed to express a red fluorescent protein (DsRed2) and a selectable marker, neomycin phosphotransferase (NEO). Frequency of transformation was estimated as 1 transformant per 5000-10,000 cells and cultures were incubated for 2-3 months in medium containing the neomycin analog G418 in order to isolate transformants. Genomic integration of the DsRed2 transgene was confirmed by inverse PCR and sequencing that demonstrated a TA nucleotide pair inserted between SB inverted/direct repeat sequences and tick genomic sequences, indicating that insertion of the DsRed2 gene into the tick cell genome occurred through the activity of SB transposase. RNAi using dsRNA transcribed from the DsRed2 gene silenced expression of red fluorescent protein in transformed ISE6 cells. SB transposition in cell line ISE6 provides an effective means to explore the functional genomics of I. scapularis.
Assuntos
Ixodes/genética , Transfecção/métodos , Animais , Linhagem Celular , Elementos de DNA Transponíveis , Inativação Gênica , GenômicaRESUMO
BACKGROUND: Tick-borne pathogens cause emerging zoonoses, and include fastidious organisms such as Anaplasma phagocytophilum. Because of their obligate intracellular nature, methods for mutagenesis and transformation have not been available. RESULTS: To facilitate genetic manipulation, we transformed A. phagocytophilum (Ap) to express a green fluorescent protein (GFP) with the Himar1 transposase system and selection with the clinically irrelevant antibiotic spectinomycin. CONCLUSION: These transformed bacteria (GFP/Ap) grow at normal rates and are brightly fluorescent in human, monkey, and tick cell culture. Molecular characterization of the GFP/Ap genomic DNA confirmed transposition and the flanking genomic insertion locations were sequenced. Three mice inoculated with GFP/Ap by intraperitoneal injection became infected as demonstrated by the appearance of morulae in a peripheral blood neutrophil and re-isolation of the bacteria in culture.
Assuntos
Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Transformação Bacteriana/fisiologia , Animais , Proteínas de Fluorescência Verde/genética , Haplorrinos , Humanos , CarrapatosRESUMO
Anaplasma phagocytophilum, the causative agent of human granulocytotropic anaplasmosis, can infect white-tailed deer (WTD; Odocoileus virginianus), and this species is a crucial host for adult Ixodes scapularis, the primary vector of A. phagocytophilum. The goal of this study was to determine the geographic distribution of A. phagocytophilum among WTD across a 19 state region and to evaluate the utility of WTD as natural sentinels. Serologic testing using the indirect fluorescent antibody (IFA) assay was conducted on WTD serum samples and molecular and xenodiagnostic tests were performed to confirm serologic results. The surveillance system was assessed through examination of vital attributes including WTD age and gender associations with serologic status, sample size adequacy for accurate infection status classification, and presence of the vector, I. scapularis. Six hundred thirty-three of 2,666 (24%) WTD in 17 states tested positive for antibodies (>or=128) when tested by IFA assay. Testing for p44 and/or 16S rRNA gene targets identified 73 (16%) PCR positive WTD among 458 animals tested, all of which originated from seropositive populations. Attempts to culture A. phagocytophilum from WTD were unsuccessful; however, xenodiagnostic mice inoculated with blood from 3 WTD became infected. Seroprevalence did not differ by deer age or gender; however, WTDAssuntos
Anaplasma phagocytophilum/imunologia
, Anticorpos Antibacterianos/sangue
, Cervos/microbiologia
, Reservatórios de Doenças/veterinária
, Ehrlichiose/veterinária
, Distribuição por Idade
, Anaplasma phagocytophilum/isolamento & purificação
, Animais
, Animais Selvagens/microbiologia
, Vetores Aracnídeos/microbiologia
, Sequência de Bases
, DNA Bacteriano/análise
, Reservatórios de Doenças/microbiologia
, Ehrlichiose/epidemiologia
, Ehrlichiose/transmissão
, Feminino
, Técnica Indireta de Fluorescência para Anticorpo/métodos
, Técnica Indireta de Fluorescência para Anticorpo/veterinária
, Ixodes/microbiologia
, Masculino
, Dados de Sequência Molecular
, Reação em Cadeia da Polimerase/métodos
, Reação em Cadeia da Polimerase/veterinária
, Vigilância de Evento Sentinela/veterinária
, Estudos Soroepidemiológicos
, Distribuição por Sexo
, Estados Unidos/epidemiologia
RESUMO
To determine the mechanisms by which adoptive immunotherapy could reduce lethality to acute myelogenous leukemia (AML), a novel technique was developed to track both leukemic blasts and adoptively transferred cytotoxic T cells (CTLs) independently and simultaneously in mice. To follow the fate of ex vivo generated anti-AML-reactive CTLs, splenocytes obtained from enhanced green fluorescent protein transgenic mice were cocultured with AML lysate-pulsed dendritic cells, which subsequently were expanded by exposure to anti-CD3/CD28 monoclonal antibody-coated magnetic microspheres. To track AML cells, stable transfectants of C1498 expressing DsRed2, a red fluorescent protein, were generated. Three factors related to CTLs correlated with disease-free survival: (a). CTL L-selectin expression. L-Selectin high fractions resulted in 70% disease-free survival, whereas L-selectin low-expressing CTLs resulted in only 30% disease-free survival. (b). Duration of ex vivo expansion (9 versus 16 days). Short-term expanded CTLs could be found at high frequency in lymphoid organs for longer than 4 weeks after transfer, whereas long-term expanded CTLs were cleared from the system after 2 weeks. Duration of expansion correlated inversely with L-selectin expression. (c). CTL dose. A higher dose (40 versus 5 x 10(6)) resulted in superior disease-free survival. This survival advantage was achieved with short-term expanded CTLs only. The site of treatment failure was mainly the central nervous system where no CTLs could be identified at AML sites.
Assuntos
Imunoterapia Adotiva/métodos , Leucemia Mieloide/imunologia , Leucemia Mieloide/terapia , Linfócitos T Citotóxicos/imunologia , Animais , Movimento Celular/imunologia , Humanos , Selectina L/biossíntese , Selectina L/imunologia , Tecido Linfoide/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T Citotóxicos/metabolismoRESUMO
Functional and anatomical relationships among primary afferent fibers, blood vessels, and cancers are poorly understood. However, recent evidence suggests that physical and biochemical interactions between these peripheral components are important to both tumor biology and cancer-associated pain. To determine the role of these peripheral components in a mouse model of cancer pain, we quantified the change in nerve and blood vessel density within a fibrosarcoma tumor mass using stereological analysis of serial confocal optical sections of immunostained hind paw. To this end we introduced the Discoma coral-derived red fluorescent protein (DsRed2) into the NCTC 2472 fibrosarcoma line using the Sleeping Beauty transposon methodology, thus providing a unique opportunity to visualize tumor-nerve-vessel associations in context with behavioral assessment of tumor-associated hyperalgesia. Tumors from hyperalgesic mice are more densely innervated with calcitonin gene related peptide (CGRP)-immunoreactive nerve fibers and less densely vascularized than tumors from non-hyperalgesic mice. As hyperalgesia increased from Day 5 to 12 post-implantation, the density of protein gene product 9.5 (PGP9.5)-immunoreactive nerves and CD31-immunoreactive blood vessels in tumors decreased, whereas CGRP-immunoreactive nerve density remained unchanged. Importantly, intra-tumor injection of a CGRP1 receptor antagonist (CGRP 8-37) partially blocked the tumor-associated mechanical hyperalgesia, indicating that local production of CGRP may contribute to tumor-induced nociception through a receptor-mediated process. The results describe for the first time the interaction among sensory nerves, blood vessels and tumor cells in otherwise healthy tissue, and our assessment supports the hypothesis that direct tumor cell-axon communication may underlie, at least in part, the occurrence of cancer pain.
Assuntos
Fibrossarcoma/irrigação sanguínea , Fibrossarcoma/metabolismo , Membro Posterior/irrigação sanguínea , Membro Posterior/inervação , Hiperalgesia/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Animais , Linhagem Celular Tumoral , Fibrossarcoma/complicações , Membro Posterior/metabolismo , Hiperalgesia/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Proteínas de Plantas , Proteínas Inativadoras de Ribossomos Tipo 1RESUMO
Ixodes scapularis ticks transmit Anaplasma phagocytophilum (Ap), agent of human granulocytic anaplasmosis (HGA). Invasion of neutrophil granulocytes (PMN) by Ap is the hallmark of the disease, but these short-lived phagocytes are not likely the sole cell type required for productive infection. We analyzed infection of microvascular endothelial cells during pathogenesis of anaplasmosis in vivo and in vitro. Organs from Ap-infected mice were processed for confocal microscopy 41 days p.i. Fluorescent labeling of heart and liver sections using anti-factor VIII and anti-MSP2 antibodies allowed colocalization of Ap and vascular endothelium, indicating infection. Ap rapidly invaded and grew within HMEC-1 human microvascular endothelial cells and readily transferred to PMN. Over 50% of PMN became infected within two hours of coincubation with HMEC-1. PMN adhered to, polarized, and migrated upon infected endothelial monolayers. The Ap receptor on human PMN is PSGL-1, and infected endothelial cells upregulate ICAM-1 (CD54), but the mechanisms of transfer of Ap remain unknown. To elucidate the cellular determinants involved, we tested relevant antibodies and lectins. Anti-PSGL-1 reduced infection of PMN, but did not inhibit adherence of PMN to Ap infected HMEC-1 cells while anti-CD18 did. Sialidase pretreatment increased, and EDTA and fucoidan decreased binding of Ap to HMEC-1, whereas several other lectins had no effect. An endothelial reservoir of Ap offers opportunities for ongoing, direct cell-to-cell infection of PMN, avoidance of host immune effectors, and completion of the Ap life cycle by infection of circulating leukocytes available for transfer to blood-feeding ticks.
Assuntos
Anaplasma phagocytophilum/fisiologia , Células Endoteliais/microbiologia , Neutrófilos/microbiologia , Anaplasma phagocytophilum/patogenicidade , Animais , Comunicação Celular/imunologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Endotélio Vascular/imunologia , Endotélio Vascular/microbiologia , Endotélio Vascular/patologia , Feminino , Células HL-60 , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos SCID , Neutrófilos/imunologia , Neutrófilos/patologiaRESUMO
Anaplasma marginale and A. phagocytophilum are obligate intracellular, tick-borne pathogens that target erythrocytes and neutrophil granulocytes, respectively. Because ticks do not directly tap blood vessels, an intermediate tissue may mediate infection of blood cells. We considered that vascular endothelium interacts with circulating blood cells in vivo, and could be involved in pathogenesis and dissemination of the organisms. We used light and electron microscopy and immune labeling to show that A. phagocytophilum invaded rhesus (RF/6A), human (HMEC-1, MVEC), as well as bovine (BCE C/D-1b) endothelial cell lines, whereas A. marginale infected rhesus and bovine endothelial cells. A. marginale formed large intracellular inclusions that appeared smooth and solid at first, and subsequently coalesced into discrete granules. A. phagocytophilum formed numerous smaller inclusions in each cell. Within 1-3 weeks, the monolayers were destroyed, and lysed cultures were diluted onto fresh monolayers. Electron microscopy demonstrated uneven distribution of A. marginale inside large inclusions, with reticulated forms grouped more tightly than denser cells, whereas in A. phagocytophilum individual organisms appeared more evenly spaced. Specific polyclonal and monoclonal antibodies both labeled A. marginale and A. phagocytophilum in endothelial cells, and oligonucleotide primers complimentary to either A. marginale or A. phagocytophilum amplified their expected target from these cultures. In conclusion, we demonstrate that relevant microvascular endothelium is susceptible to anaplasmas in vitro and may present a link that could explain development of the immune response and persistent infection.
Assuntos
Anaplasma marginale/patogenicidade , Anaplasma phagocytophilum/crescimento & desenvolvimento , Anaplasmose/microbiologia , Ehrlichiose/microbiologia , Células Endoteliais/microbiologia , Anaplasma marginale/genética , Anaplasma marginale/crescimento & desenvolvimento , Anaplasma marginale/imunologia , Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/imunologia , Anaplasmose/imunologia , Animais , Bovinos , DNA Bacteriano/química , DNA Bacteriano/genética , Ehrlichiose/imunologia , Eletroforese em Gel de Ágar , Células Endoteliais/imunologia , Células Endoteliais/ultraestrutura , Humanos , Macaca mulatta , Microscopia Eletrônica , Microscopia de Contraste de Fase , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genéticaRESUMO
We developed and applied transposon-based transformation vectors for molecular manipulation and analysis of spotted fever group rickettsiae, which are obligate intracellular bacteria that infect ticks and, in some cases, mammals. Using the Epicentre EZ::TN transposon system, we designed transposons for simultaneous expression of a reporter gene and a chloramphenicol acetyltransferase (CAT) resistance marker. Transposomes (transposon-transposase complexes) were electroporated into Rickettsia monacensis, a rickettsial symbiont isolated from the tick Ixodes ricinus. Each transposon contained an expression cassette consisting of the rickettsial ompA promoter and a green fluorescent protein (GFP) reporter gene (GFPuv) or the ompB promoter and a red fluorescent protein reporter gene (DsRed2), followed by the ompA transcription terminator and a second ompA promoter CAT gene cassette. Selection with chloramphenicol gave rise to rickettsial populations with chromosomally integrated single-copy transposons as determined by PCR, Southern blotting, and sequence analysis. Reverse transcription-PCR and Northern blots demonstrated transcription of all three genes. GFPuv transformant rickettsiae exhibited strong fluorescence in individual cells, but DsRed2 transformants did not. Western blots confirmed expression of GFPuv in R. monacensis and in Escherichia coli, but DsRed2 was expressed only in E. coli. The DsRed2 gene, but not the GFPuv gene, contains many GC-rich amino acid codons that are rare in the preferred codon suite of rickettsiae, possibly explaining the failure to express DsRed2 protein in R. monacensis. We demonstrated that our vectors provide a means to study rickettsia-host cell interactions by visualizing GFPuv-fluorescent R. monacensis associated with actin tails in tick host cells.