RESUMO
The pertussis agent Bordetella pertussis produces a number of virulence factors, of which the filamentous hemagglutinin (FhaB) plays a role in B. pertussis adhesion to epithelial and phagocytic cells. Moreover, FhaB was recently found to play a crucial role in nasal cavity infection and B. pertussis transmission to new hosts. The 367 kDa FhaB protein translocates through an FhaC pore to the outer bacterial surface and is eventually processed to a ~220 kDa N-terminal FHA fragment by the SphB1 protease. A fraction of the mature FHA then remains associated with bacterial cell surface, while most of FHA is shed into the bacterial environment. Previously reported indirect evidence suggested that FHA, or its precursor FhaB, may bind the ß2 integrin CD11b/CD18 of human macrophages. Therefore, we assessed FHA binding to various cells producing or lacking the integrin and show that purified mature FHA does not bind CD11b/CD18. Further results then revealed that the adhesion of B. pertussis to cells does not involve an interaction between the bacterial surface-associated FhaB and/or mature FHA and the ß2 integrin CD11b/CD18. In contrast, FHA binding was strongly inhibited at micromolar concentrations of heparin, corroborating that the cell binding of FHA is ruled by the interaction of its heparin-binding domain with sulfated glycosaminoglycans on the cell surface.
Assuntos
Bordetella pertussis , Coqueluche , Humanos , Bordetella pertussis/metabolismo , Fatores de Virulência de Bordetella , Hemaglutininas/metabolismo , Antígenos CD18 , Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Antígeno de Macrófago 1 , Integrinas , Heparina , Peptídeo Hidrolases , GlicosaminoglicanosRESUMO
Repeats-in-toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function-associated antigen 1 (LFA-1, integrin αL /ß2 )-dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA-1-expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA-1 with a cyan fluorescent protein-tagged cytosolic αL domain and a yellow fluorescent protein-tagged ß2 domain. Phorbol 12-myristate 13-acetate activation of LFA-1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both αL and ß2 were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the αL and ß2 subunits (Kd = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin αM , αX , and ß3 subunits (Kd = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of αL and ß2 show that LtxA binds membrane-proximal domain of αL and intermediate domain of ß2 .
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Aggregatibacter actinomycetemcomitans/imunologia , Exotoxinas/metabolismo , Interações Hospedeiro-Patógeno , Imunossupressores/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Células Jurkat , Microscopia de Fluorescência , Ligação ProteicaRESUMO
The Gram-negative coccobacillus Kingella kingae is increasingly recognized as an important invasive pediatric pathogen that causes mostly bacteremia and skeletal system infections. K. kingae secretes an RtxA toxin that belongs to a broad family of the RTX (Repeats in ToXin) cytotoxins produced by bacterial pathogens. Recently, we demonstrated that membrane cholesterol facilitates interaction of RtxA with target cells, but other cell surface structures potentially involved in toxin binding to cells remain unknown. We show that deglycosylation of cell surface structures by glycosidase treatment, or inhibition of protein N- and O-glycosylation by chemical inhibitors substantially reduces RtxA binding to target cells. Consequently, the deglycosylated cells were more resistant to cytotoxic activity of RtxA. Moreover, experiments on cells expressing or lacking cell surface integrins of the ß2 family revealed that, unlike some other cytotoxins of the RTX family, K. kingae RtxA does not bind target cells via the ß2 integrins. Our results, hence, show that RtxA binds cell surface oligosaccharides present on all mammalian cells but not the leukocyte-restricted ß2 integrins. This explains the previously observed interaction of the toxin with a broad range of cell types of various mammalian species and reveals that RtxA belongs to the group of broadly cytolytic RTX hemolysins.
Assuntos
Toxinas Bacterianas/metabolismo , Antígenos CD18/metabolismo , Membrana Celular/metabolismo , Kingella kingae/metabolismo , Oligossacarídeos/metabolismo , Animais , Morte Celular , Linhagem Celular , Feminino , Glicosídeo Hidrolases/metabolismo , Glicosilação , Humanos , Macrófagos/metabolismo , Camundongos , Oligossacarídeos/química , Ligação ProteicaRESUMO
Pediatric septic arthritis in patients under age of four is frequently caused by the oral Gram-negative bacterium Kingella kingae. This organism may be responsible for a severe form of infective endocarditis in otherwise healthy children and adults. A major virulence factor of K. kingae is RtxA, a toxin that belongs to the RTX (Repeats-in-ToXin) group of secreted pore forming toxins. To understand the RtxA effects on host cell membranes, the toxin activity was studied using planar lipid bilayers. K. kingae strain PYKK081 and its isogenic RtxA-deficient strain, KKNB100, were tested for their ability to form pores in artificial membranes of asolectin/n-decane. RtxA, purified from PYKK081, was able to rapidly form pores with an apparent diameter of 1.9nm as measured by the partition of nonelectrolytes in the pores. The RtxA channels are cation-selective and showed strong voltage-dependent gating. In contrast to supernatants of PYKK081, those of KKNB100 did not show any pore forming activity. We concluded that RtxA toxin is the only secreted protein from K. kingae forming large channels in host cell membranes where it induces cation flux leading to programmed cell death. Furthermore, our findings suggested that the planar lipid bilayer technique can effectively be used to test possible inhibitors of RTX toxin activity and to investigate the mechanism of the toxin binding to the membrane.
Assuntos
Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Kingella kingae/metabolismo , Bicamadas Lipídicas/metabolismo , Artrite Infecciosa/microbiologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/microbiologia , Citotoxinas/metabolismo , Citotoxinas/toxicidade , Eletroforese em Gel de Poliacrilamida , Interações Hospedeiro-Patógeno , Humanos , Lactente , Ativação do Canal Iônico/efeitos dos fármacos , Kingella kingae/genética , Kingella kingae/fisiologia , Masculino , Mutação , Ligação ProteicaRESUMO
Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its ß2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC(336) ((333)LEEYSKR(339)) is highly conserved among RTX toxins, whereas CRAC(503) ((501)VDYLK(505)) is unique to LtxA. A peptide corresponding to CRAC(336) inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC(336) and CRAC(503) bind cholesterol, only CRAC(336) competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC(336) site was essential for LtxA cytotoxicity. The conservation of CRAC(336) among RTX toxins suggests that this mechanism may be conserved among RTX toxins.
Assuntos
Toxinas Bacterianas/química , Colesterol/química , Exotoxinas/química , Microdomínios da Membrana/química , Pasteurellaceae/química , Motivos de Aminoácidos , Toxinas Bacterianas/metabolismo , Colesterol/metabolismo , Exotoxinas/metabolismo , Humanos , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Microdomínios da Membrana/metabolismo , Pasteurellaceae/metabolismo , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Kingella kingae is a human oral bacterium that can cause diseases of the skeletal system in children and infective endocarditis in children and adults. K. kingae produces a toxin of the RTX group, RtxA. To investigate the role of RtxA in disease pathogenesis in vivo, K. kingae strain PYKK081 and its isogenic RtxA-deficient strain KKNB100 were tested for their virulence and pathological consequences upon intraperitoneal injections in 7-day-postnatal (PN 7) rats. At the doses above 8.0 × 10(6) cells/animal, PYKK081 was able to cause a fatal illness, resulting in rapid weight loss, bacteremia, and abdominal necrotic lesion formation. Significant histopathology was observed in thymus, spleen, and bone marrow. Strain KKNB100 was less toxic to animals. Neither weight loss, bacteremia, nor histopathological changes were evident. Animals injected with KKNB100 exhibited a significantly elevated circulating white blood cell (WBC) count, whereas animals injected with PYKK081 had a WBC count that resembled that of the uninfected control. This observation parallels the subtleties associated with clinical presentation of K. kingae disease in humans and suggests that the toxin contributes to WBC depletion. Thus, our results demonstrate that RtxA is a key K. kingae virulence factor. Furthermore, our findings suggest that the PN 7 rat can serve as a useful model for understanding disease caused by K. kingae and for elucidating diagnostic parameters in human patients.
Assuntos
Toxinas Bacterianas , Kingella kingae/patogenicidade , Infecções por Neisseriaceae/microbiologia , Virulência/fisiologia , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Modelos Animais de Doenças , Contagem de Leucócitos , Infecções por Neisseriaceae/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Although Kingella kingae is the most common etiology of osteoarticular infections in young children, is a frequent cause of bacteremia in those younger than 4 years, and has been involved in clusters of invasive infections among daycare center attendees, the population structure of the species has not been systematically studied. Using multilocus sequence typing, we investigated the genetic diversity of the largest intercontinental collection of K. kingae strains to date. To facilitate typing of bacterial isolates, we developed a novel genotyping tool that targets the DNA uptake sequence (DUS). Among 324 strains isolated from asymptomatic carriers and patients from Israel, Europe, North America, and Australia with various invasive forms of the disease from 1960 to 2013, we identified 64 sequence types (STs) and 12 ST complexes (STcs). Five predominant STcs, comprising 72.2% of all strains, were distributed intercontinentally. ST-6 was the most frequent, showing a worldwide distribution, and appeared genotypically isolated by exhibiting few neighboring STs, suggesting an optimal fitness. ST-14 and ST-23 appeared to be the oldest groups of bacteria, while ST-25 probably emerged more recently from the highly evolutive ST-23. Using the DUS typing method, randomly chosen isolates were correctly classified to one of the major STcs. The comprehensive description of K. kingae evolution would help to detect new emerging clones and decipher virulence and fitness mechanisms. The rapid and reproducible DUS typing method may serve in the initial investigation of K. kingae outbreaks.
Assuntos
Variação Genética , Kingella kingae/classificação , Kingella kingae/isolamento & purificação , Tipagem de Sequências Multilocus/métodos , Infecções por Neisseriaceae/epidemiologia , Infecções por Neisseriaceae/microbiologia , Austrália/epidemiologia , Pré-Escolar , Análise por Conglomerados , Europa (Continente)/epidemiologia , Genótipo , Humanos , Lactente , Israel/epidemiologia , Kingella kingae/genética , Epidemiologia Molecular/métodos , América do Norte/epidemiologia , Osteoartrite/epidemiologia , Osteoartrite/microbiologia , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Kingella kingae is a human pathogen that causes pediatric osteoarticular infections and infective endocarditis in children and adults. The bacterium is usually susceptible to ß-lactam antibiotics, although ß-lactam resistance has been reported in rare isolates. This study was conducted to identify ß-lactam-resistant strains and to characterize the resistance mechanism. Screening of a set of 90 K. kingae clinical isolates obtained from different geographic locations revealed high-level resistance to penicillins among 25% of the strains isolated from Minnesota and Iceland. These strains produced TEM-1 ß-lactamase and were shown to contain additional ≥50-kb plasmids. Ion Torrent sequencing of extrachromosomal DNA from a ß-lactamase-producing strain confirmed the plasmid location of the blaTEM gene. An identical plasmid pattern was demonstrated by multiplex PCR in all ß-lactamase producers. The porin gene's fragments were analyzed to investigate the relatedness of bacterial strains. Phylogenetic analysis revealed 27 single-nucleotide polymorphisms (SNPs) in the por gene fragment, resulting in two major clusters with 11 allele types forming bacterial-strain subclusters. ß-Lactamase producers were grouped together based on por genotyping. Our results suggest that the ß-lactamase-producing strains likely originate from a single plasmid-bearing K. kingae isolate that traveled from Europe to the United States, or vice versa. This study highlights the prevalence of penicillin resistance among K. kingae strains in some regions and emphasizes the importance of surveillance for antibiotic resistance of the pathogen.
RESUMO
Diabetes Mellitus is a silent epidemic affecting >500 million, which claimed 6.7 million lives in 2021, a projected increase of >670% in <20 years old in the next two decades but insulin is unaffordable for the large majority of the globe. Therefore, we engineered proinsulin in plant cells to facilitate oral delivery. Stability of the proinsulin gene and expression in subsequent generations, after removal of the antibiotic-resistance gene, was confirmed using PCR, Southern and western blots. Proinsulin expression was high (up to 12 mg/g DW or 47.5% of total leaf protein), stable up to one year after storage of freeze-dried plant cells at ambient temperature and met FDA regulatory requirements of uniformity, moisture content and bioburden. GM1 receptor binding, required for uptake via gut epithelial cells was confirmed by pentameric assembly of CTB-Proinsulin. IP insulin injections (without C peptide) in STZ mice rapidly decreased blood glucose level leading to transient hypoglycemia, followed by hepatic glucose compensation. On the other hand, other than the 15-min lag period of oral proinsulin (transit time required to reach the gut), the kinetics of blood sugar regulation of oral CTB-Proinsulin in STZ mice was very similar to naturally secreted insulin in healthy mice (both contain C-peptide), without rapid decrease or hypoglycemia. Elimination of expensive fermentation, purification and cold storage/transportation should reduce cost and increase other health benefits of plant fibers. The recent approval of plant cell delivery of therapeutic proteins by FDA and approval of CTB-ACE2 for phase I/II human clinical studies augur well for advancing oral proinsulin to the clinic.
Assuntos
Hipoglicemia , Insulina , Humanos , Animais , Camundongos , Adulto Jovem , Adulto , Insulina/metabolismo , Proinsulina , Glicemia/análise , Células Vegetais/química , Células Vegetais/metabolismo , Peptídeo CRESUMO
Kingella kingae is a human oral bacterium that can cause infections of the skeletal system in children. The bacterium is also a cardiovascular pathogen causing infective endocarditis in children and adults. We report herein the draft genome sequence of septic arthritis K. kingae strain PYKK081.
Assuntos
Artrite Infecciosa/microbiologia , Genoma Bacteriano , Kingella kingae/genética , Infecções por Neisseriaceae/microbiologia , Sequência de Bases , Humanos , Lactente , Kingella kingae/classificação , Kingella kingae/isolamento & purificação , Masculino , Dados de Sequência MolecularRESUMO
Cell-free extracts prepared from Kingella kingae colony biofilms were found to inhibit biofilm formation by Aggregatibacter actinomycetemcomitans, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, and K. kingae. The extracts evidently inhibited biofilm formation by modifying the physicochemical properties of the cell surface, the biofilm matrix, and the substrate. Chemical and biochemical analyses indicated that the biofilm inhibition activity in the K. kingae extract was due to polysaccharide. Structural analyses showed that the extract contained two major polysaccharides. One was a linear polysaccharide with the structure â6)-α-d-GlcNAcp-(1â5)-ß-d-OclAp-(2â, which was identical to a capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 5. The second was a novel linear polysaccharide, designated PAM galactan, with the structure â3)-ß-d-Galf-(1â6)-ß-d-Galf-(1â. Purified PAM galactan exhibited broad-spectrum biofilm inhibition activity. A cluster of three K. kingae genes encoding UDP-galactopyranose mutase (ugm) and two putative galactofuranosyl transferases was sufficient for the synthesis of PAM galactan in Escherichia coli. PAM galactan is one of a growing number of bacterial polysaccharides that exhibit antibiofilm activity. The biological roles and potential technological applications of these molecules remain unknown.
Assuntos
Biofilmes/efeitos dos fármacos , Regulação para Baixo , Kingella kingae/metabolismo , Polissacarídeos Bacterianos/farmacologia , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fungos/efeitos dos fármacos , Fungos/fisiologia , Kingella kingae/química , Kingella kingae/genética , Dados de Sequência Molecular , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/químicaRESUMO
Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.
Assuntos
Drosophila , Genômica/educação , Universidades , Animais , Células Sanguíneas , Drosophila/genética , Humanos , EstudantesRESUMO
On February 11, 2019, we lost a colleague and friend Dr. Edward "Ned" Lally. [...].
RESUMO
The oral pathogen, Aggregatibacter actinomycetemcomitans, produces a number of virulence factors, including a leukotoxin (LtxA), which specifically kills human white blood cells, to provide a colonization advantage to the bacterium. Strains of A. actinomycetemcomitans that produce more LtxA have been more closely linked to disease, indicating that this toxin plays a key role in pathogenesis of the bacterium. Disruption of the activity of LtxA thus represents a promising approach to reducing the pathogenicity of the bacterium. Catechins are polyphenolic molecules derived from plants, which have shown potent antibacterial and antitoxin activities. We have previously shown that galloylated catechins are able to prevent LtxA delivery to host cells by altering the toxin's secondary structure and preventing binding to cholesterol on the host cell membrane. Here, we have investigated how one particular galloylated catechin, epigallocatechin gallate (EGCg), affects A. actinomycetemcomitans growth and toxin secretion. Our results demonstrate that EGCg, at micromolar concentrations, inhibits A. actinomycetemcomitans growth, as has been reported for other bacterial species. At subinhibitory concentrations, EGCg promotes LtxA production, but the toxicity of the bacterial supernatant against human immune cells is reduced. The results of our biophysical studies indicate that this seemingly contradictory result is caused by an EGCg-mediated enhancement of LtxA affinity for the bacterial cell surface. Together, these results demonstrate the potential of EGCg in the treatment of virulent A. actinomycetemcomitans infections.
Assuntos
Membrana Externa Bacteriana , Aggregatibacter actinomycetemcomitans , Animais , Bactérias , Catequina/análogos & derivados , Catequina/farmacologia , Exotoxinas , Humanos , CamundongosRESUMO
Leukotoxin (LtxA), from oral pathogen Aggregatibacter actinomycetemcomitans, is a secreted membrane-damaging protein. LtxA is internalized by ß2 integrin LFA-1 (CD11a/CD18)-expressing leukocytes and ultimately causes cell death; however, toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5a knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pHs. Our results demonstrate that the LtxA/LFA-1 complex gains access to the cytosol of Jurkat cells without evidence of plasma membrane damage, utilizing dynamin-dependent and presumably clathrin-independent mechanisms. Upon internalization, LtxA follows the LFA-1 endocytic trafficking pathways, as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp1) and CD11a. Knockdown of Rab5a resulted in the loss of susceptibility of Jurkat cells to LtxA cytotoxicity, suggesting that late events of LtxA endocytic trafficking are required for toxicity. Toxin trafficking via the degradative endocytic pathway may culminate in the delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes.
RESUMO
Screens for compounds and proteins with anti-cancer activity employ viability assays using relevant cancer cell lines. For leukaemia studies, the human leukaemia cell line, HL-60, is often used as a model system. To facilitate the discovery and investigation of anti-leukaemia therapeutics under physiological conditions, we have engineered HL-60 cells that stably express firefly luciferase and produce light that can be detected using an in vivo imaging system (IVIS). Bioluminescent HL-60luc cells could be rapidly detected in whole blood with a sensitivity of approximately 1000 viable cells/200 microl blood. Treatment of HL-60luc cells with the drug chlorambucil revealed that the bioluminescent viability assay is able to detect cell death earlier than the Trypan blue dye exclusion assay. HL-60luc cells administered intraperitoneally (i.p.) or intravenously (i.v.) were visualized in living mice. The rapidity and ease of detecting HL-60luc cells in biological fluid indicates that this cell line could be used in high-throughput screens for the identification of drugs with anti-leukaemia activity under physiological conditions.
Assuntos
Antineoplásicos/química , Luciferina de Vaga-Lumes/química , Leucemia/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Luciferina de Vaga-Lumes/administração & dosagem , Regulação Enzimológica da Expressão Gênica , Células HL-60 , Humanos , Cinética , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Luminescência , Medições Luminescentes , Camundongos , Camundongos SCID , Transplante de Neoplasias , Sensibilidade e EspecificidadeRESUMO
The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.
Assuntos
Exotoxinas/metabolismo , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Exotoxinas/toxicidade , Vesículas Extracelulares/metabolismo , Humanos , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Células THP-1RESUMO
A leukotoxin (LtxA) that is produced by Aggregatibacter actinomycetemcomitans (Aa) is an important virulence determinant in an aggressive form of periodontitis in adolescents. Understanding the function of this protein at the molecular level is critical to elucidating its role in the disease process. To accomplish genetic analysis of the protein structure and relating these observations to toxin function, we have developed an E. coli expression system for the generation and rapid purification of LtxA. Cloning the structural toxin gene, ltxA, from Aa strain JP2 under control of T7 promoter-1 of pCDFDuet-1 vector resulted in expression of a 114 KDa protein which could be easily purified by the presence of a carboxy-terminal engineered double hexahistidine (double-His6) tag and was immunologically reactive with an anti-LtxA monoclonal antibody, but was not cytotoxic. Cloning a second gene, ltxC, an acyltransferase gene, into the vector under control of T7 promoter-2, resulted in expression of the biologically active LtxA. The toxin was extracted from E. coli inclusion bodies, purified by immobilized metal affinity chromatography, and refolded by dialysis. When compared by circular dichroism (CD) spectroscopy analysis, acylated recombinant LtxA has a secondary structure consistent with wt LtxA, while variations in α-helical structure of nonacylated LtxA were observed. No modifications in α-helix were found upon the toxin's binding with liposome-incorporated cholesterol. Our results suggest that pure, biologically active recombinant LtxA can be isolated by a one-step affinity chromatography from E. coli. The toxic and structural properties of the recombinant LtxA are similar to its wt counterpart.
Assuntos
Aggregatibacter actinomycetemcomitans/genética , Toxinas Bacterianas/genética , Exotoxinas/genética , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/química , Toxinas Bacterianas/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Exotoxinas/biossíntese , Exotoxinas/química , Exotoxinas/isolamento & purificação , Humanos , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Células THP-1RESUMO
The oral bacterium, Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis, as well as systemic infections including endocarditis, produces numerous virulence factors, including a repeats-in-toxin (RTX) protein called leukotoxin (LtxA), which kills human immune cells. The strains of A. actinomycetemcomitans most closely associated with disease have been shown to produce the most LtxA, suggesting that LtxA plays a significant role in the virulence of this organism. LtxA, like many of the RTX toxins, can be divided into four functional domains: an N-terminal hydrophobic domain, which contains a significant fraction of hydrophobic residues and has been proposed to play a role in the membrane interaction of the toxin; the central domain, which contains two lysine residues that are the sites of post-translational acylation; the repeat domain that is characteristic of the RTX toxins, and a C-terminal domain thought to be involved in secretion. In its initial interaction with the host cell, LtxA must bind to both cholesterol and an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1). While both interactions are essential for toxicity, the domains of LtxA involved remain unknown. We therefore undertook a series of experiments, including tryptophan quenching and trypsin digestion, to characterize the structure of LtxA upon interaction with membranes of various lipid compositions. Our results demonstrate that LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane.