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1.
J Bacteriol ; 205(4): e0047522, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-37010281

RESUMO

Lytic transglycosylases cut peptidoglycan backbones, facilitating a variety of functions within bacteria, including cell division, pathogenesis, and insertion of macromolecular machinery into the cell envelope. Here, we identify a novel role of a secreted lytic transglycosylase associated with the predatory lifestyle of Bdellovibrio bacteriovorus strain HD100. During wild-type B. bacteriovorus prey invasion, the predator rounds up rod-shaped prey into spherical prey bdelloplasts, forming a spacious niche within which the predator grows. Deleting the MltA-like lytic transglycosylase Bd3285 still permitted predation but resulted in three different, invaded prey cell shapes: spheres, rods, and "dumbbells." Amino acid D321 within the catalytic C-terminal 3D domain of Bd3285 was essential for wild-type complementation. Microscopic analyses revealed that dumbbell-shaped bdelloplasts are derived from Escherichia coli prey undergoing cell division at the moment of Δbd3285 predator invasion. Prelabeling of E. coli prey peptidoglycan prior to predation with the fluorescent D-amino acid HADA showed that the dumbbell bdelloplasts invaded by B. bacteriovorus Δbd3285 contained a septum. Fluorescently tagged Bd3285, expressed in E. coli, localized to the septum of dividing cells. Our data indicate that B. bacteriovorus secretes the lytic transglycosylase Bd3285 into the E. coli periplasm during prey invasion to cleave the septum of dividing prey, facilitating prey cell occupation. IMPORTANCE Antimicrobial resistance is a serious and rapidly growing threat to global health. Bdellovibrio bacteriovorus can prey upon an extensive range of Gram-negative bacterial pathogens and thus has promising potential as a novel antibacterial therapeutic and is a source of antibacterial enzymes. Here, we elucidate the role of a unique secreted lytic transglycosylase from B. bacteriovorus which acts on the septal peptidoglycan of its prey. This improves our understanding of mechanisms that underpin bacterial predation.


Assuntos
Bdellovibrio bacteriovorus , Bdellovibrio , Animais , Bdellovibrio bacteriovorus/genética , Bdellovibrio/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Comportamento Predatório , Aminoácidos/metabolismo
2.
Sci Rep ; 9(1): 4293, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862785

RESUMO

In assessing the potential of predatory bacteria, such as Bdellovibrio bacteriovorus, to become live therapeutic agents against bacterial infections, it is crucial to understand and quantify Bdellovibrio host cell interactions at a molecular level. Here, we quantify the interactions of live B. bacteriovorus with human phagocytic cells, determining the uptake mechanisms, persistence, associated cytokine responses and intracellular trafficking of the non-growing B. bacteriovorus in PMA-differentiated U937 cells. B. bacteriovorus are engulfed by U937 cells and persist for 24 h without affecting host cell viability and can be observed microscopically and recovered and cultured post-uptake. The uptake of predators is passive and depends on the dynamics of the host cell cytoskeleton; the engulfed predators are eventually trafficked through the phagolysosomal pathway of degradation. We have also studied the prevalence of B. bacteriovorus specific antibodies in the general human population. Together, these results quantify a period of viable persistence and the ultimate fate of B. bacteriovorus inside phagocytic cells. They provide new knowledge on predator availability inside hosts, plus potential longevity and therefore potential efficacy as a treatment in humans and open up future fields of work testing if predators can prey on host-engulfed pathogenic bacteria.


Assuntos
Bdellovibrio/patogenicidade , Fagócitos/microbiologia , Actinas/metabolismo , Bdellovibrio bacteriovorus/patogenicidade , Sobrevivência Celular/fisiologia , Células Cultivadas , Humanos , Microtúbulos/metabolismo , Fagócitos/citologia , Fagossomos/microbiologia , Células U937
3.
Eur J Immunol ; 45(10): 2937-44, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26202849

RESUMO

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease involving recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNF receptor superfamily 1A gene localised to exons encoding the ectodomain of the p55 TNF receptor, TNF receptor-1 (TNFR1). The aim of this study was to investigate the role of cell surface TNFR1 in TRAPS, and the contribution of TNF-dependent and TNF-independent mechanisms to the production of cytokines. HEK-293 and SK-HEP-1 cell lines were stably transfected with WT or TRAPS-associated variants of human TNF receptor superfamily 1A gene. An anti-TNFR1 single domain antibody (dAb), and an anti-TNFR1 mAb, bound to cell surface WT and variant TNFR1s. In HEK-293 cells transfected with death domain-inactivated (R347A) TNFR1, and in SK-HEP-1 cells transfected with normal (full-length) TNFR1, cytokine production stimulated in the absence of exogenous TNF by the presence of certain TNFR1 variants was not inhibited by the anti-TNFR1 dAb. In SK-Hep-1 cells, specific TRAPS mutations increased the level of cytokine response to TNF, compared to WT, and this augmented cytokine production was suppressed by the anti-TNFR1 dAb. Thus, TRAPS-associated variants of TNFR1 enhance cytokine production by a TNF-independent mechanism and by sensitising cells to a TNF-dependent stimulation. The TNF-dependent mechanism requires cell surface expression of TNFR1, as this is blocked by TNFR1-specific dAb.


Assuntos
Doenças Autoimunes/imunologia , Doenças Genéticas Inatas/imunologia , Mutação de Sentido Incorreto , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/imunologia , Substituição de Aminoácidos , Anticorpos/imunologia , Anticorpos/farmacologia , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Linhagem Celular , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Células HEK293 , Humanos , Estrutura Terciária de Proteína , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Síndrome , Fator de Necrose Tumoral alfa/genética
4.
Arthritis Rheum ; 63(4): 1151-5, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21225679

RESUMO

In this report, we describe treatment outcomes in the first case of a patient with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) treated with the anti-interleukin-6 (anti-IL-6) receptor monoclonal antibody tocilizumab. Since IL-6 levels are elevated in TRAPS, we hypothesized that tocilizumab might be effective. The patient, a 52-year-old man with lifelong TRAPS in whom treatment with etanercept and anakinra had failed, was administered tocilizumab for 6 months, and the therapeutic response was assessed by measurement of monocyte CD16 expression and cytokine levels. Following treatment, the evolving acute attack was aborted and further attacks of TRAPS were prevented. The patient did not require corticosteroids and showed significant clinical improvement in scores for pain, stiffness, and well-being. Moreover, the acute-phase response diminished significantly with treatment. Monocyte CD16 expression was reduced and the numbers of circulating CD14+CD16+ and CD14++CD16- monocytes were transiently decreased. However, cytokine levels were not reduced. This case supports the notion of a prominent role for IL-6 in mediating the inflammatory attacks in TRAPS, but blockade of IL-6 did not affect the underlying pathogenesis. These preliminary findings require confirmation.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Febre Familiar do Mediterrâneo/tratamento farmacológico , Febre Familiar do Mediterrâneo/fisiopatologia , Interleucina-6/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Anticorpos Monoclonais Humanizados , Etanercepte , Humanos , Imunoglobulina G/uso terapêutico , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Masculino , Pessoa de Meia-Idade , Receptores de Interleucina-6/antagonistas & inibidores , Receptores do Fator de Necrose Tumoral/uso terapêutico , Falha de Tratamento , Resultado do Tratamento
6.
Arthritis Rheum ; 60(1): 269-80, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19116900

RESUMO

OBJECTIVE: To analyze the effects of tumor necrosis factor receptor-associated periodic syndrome (TRAPS)-associated mutant tumor necrosis factor receptor type I (TNFRI) expression in a cell type directly relevant to the inflammation in TRAPS, and to identify novel markers associated with mutant TNFRI expression. METHODS: Transcriptome analysis on 30,000 human genes was performed on SK-Hep-1 human endothelial cells transfected with either wild-type (WT) or TRAPS-associated mutant TNFRI. Quantitative reverse transcriptase-polymerase chain reaction and protein expression levels measured by enzyme-linked immunosorbent assay verified transcriptional changes for selected genes both in supernatants from cells expressing mutant TNFRI and in patient plasma. RESULTS: Cells expressing mutant TNFRI showed up-regulation of multiple proinflammatory genes relative to WT transfectants, including genes for pentraxin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, CCL2, and CCL5, which were also expressed as proteins. In addition, the expression of most of these markers was increased in the plasma and peripheral blood mononuclear cells from TRAPS patients relative to those from healthy controls. The cysteine mutations (C33Y and C52F), which are associated with a more severe clinical phenotype, induced more genes than the low-penetrance mutation R92Q, which is associated with a milder phenotype. The expression of most genes was induced by a death domain (DD)-dependent mechanism, since they were not induced by expression of TNFRI mutants with an inactivated DD. CONCLUSION: TRAPS-associated TNFRI mutants induce the expression of multiple genes encoding inflammatory molecules, cellular receptors, transcription factors, and regulators of apoptosis in endothelial cells that require the cytoplasmic signaling properties of the receptor. Different mutants have specific expression profiles, indicating mutation-specific effects. The expression of some of these markers was also elevated in samples from TRAPS patients.


Assuntos
Biomarcadores , Febre Familiar do Mediterrâneo/genética , Febre Familiar do Mediterrâneo/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Endotélio/citologia , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/normas , Compostos Organometálicos , Reprodutibilidade dos Testes , Transfecção
7.
Arthritis Rheum ; 56(12): 4182-8, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18050249

RESUMO

OBJECTIVE: Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an inherited autosomal-dominant autoinflammatory condition caused by mutations in the ectodomain of the 55-kd tumor necrosis factor (TNF) receptor superfamily 1A. Proinflammatory blood monocytes with the phenotype CD14+,CD16+,HLA-DR++ are a major source of TNF, and the number of such monocytes is increased during infection and inflammation. The aim of this study was to investigate whether the expression of circulating CD16+ monocytes is affected in patients with TRAPS. METHODS: Peripheral blood obtained from patients with TRAPS and healthy control subjects was stained with monoclonal antibodies to detect CD14++,CD16- monocytes and CD14+,CD16+ monocytes, using flow cytometry. Lipopolysaccharide-induced TNF production was measured by intracellular cytokine staining. Activation-induced shedding of CD16 was investigated by treating blood samples with phorbol myristate acetate. RESULTS: The level of CD16 expression by CD14+,CD16+ monocytes, but not their absolute number, was significantly elevated in patients with TRAPS, even though the patients were not experiencing clinically overt episodes of autoinflammation at the time of sampling. These findings are similar to those for the C-reactive protein levels and erythrocyte sedimentation rates in the same patients. The enhanced level of CD16 expression by monocytes from patients with TRAPS was not attributable to a defect in activation-induced shedding of CD16. The CD14+,CD16+ monocytes were the predominant source of TNF in both patients and healthy control subjects. CONCLUSION: The level of CD16 expression by monocytes was elevated in patients with TRAPS, as a feature of the underlying constitutive inflammation status.


Assuntos
Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Monócitos/metabolismo , Mutação/genética , Periodicidade , Receptores de IgG/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Adulto , Doenças Autoimunes/patologia , Sedimentação Sanguínea , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Receptores de IgG/genética , Síndrome
8.
Arthritis Rheum ; 56(8): 2765-73, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17665435

RESUMO

OBJECTIVE: To investigate the effect of mutations in tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) in TNFR-associated periodic syndrome (TRAPS) on the binding of anti-TNFRSF1A monoclonal antibodies (mAb), and to investigate the subcellular distribution of mutant versus wild-type (WT) TNFRSF1A in patients with TRAPS. METHODS: HEK 293 cells transfected with WT and/or mutant TNFRSF1A were used to investigate the interaction of anti-TNFRSF1A mAb with the WT and mutant proteins. Monoclonal antibodies that differentially bound to C33Y TNFRSF1A were used to investigate the distribution of WT and mutant TNFRSF1A in TRAPS patients with the C33Y mutation. RESULTS: We identified a mAb whose binding to TNFRSF1A was completely abolished by the C33Y or C52F TRAPS-associated mutations, whereas other mutations (T50M, C88Y, R92Q) had lesser effects on the binding of this mAb. A different mAb was found to bind efficiently to all of the mutant forms of TNFRSF1A examined as well as to the WT receptor. Exploitation of the differential binding properties of these mAb indicated that mutant (as distinct from WT) TNFRSF1A showed abnormal intracellular retention in the neutrophils of TRAPS patients with the C33Y mutation, with little if any expression of mutant TNFRSF1A on the cell surface or as soluble receptor in plasma. CONCLUSION: TRAPS-associated mutant TNFRSF1A has an antigenically altered structure and shows abnormal retention in the leukocytes of patients with TRAPS, which is consistent with previous findings from in vitro and transgenic model systems. This is consistent with a misfolded protein response contributing to the pathophysiology of TRAPS.


Assuntos
Diversidade de Anticorpos/genética , Febre Familiar do Mediterrâneo/genética , Mutação , Neutrófilos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Diversidade de Anticorpos/imunologia , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linhagem Celular , Febre Familiar do Mediterrâneo/imunologia , Humanos , Neutrófilos/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transfecção
9.
Arthritis Rheum ; 54(8): 2674-87, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16871532

RESUMO

OBJECTIVE: To investigate the effect of mutations in the tumor necrosis factor receptor superfamily 1A (TNFRSF1A) gene on the conformation and behavior of the TNFRSF1A protein. Mutations in TNFRSF1A cause the autosomal-dominant, autoinflammatory TNFR-associated periodic syndrome (TRAPS). METHODS: The expression of recombinant TNFRSF1A was compared in SK-HEp-1 endothelial cells and HEK 293 epithelial cells stably transfected with full-length R347A or Deltasig constructs of wild-type or TRAPS-associated mutant TNFRSF1A. TNF binding was assessed in HEK 293 cell lines expressing R347A wild-type or mutant TNFRSF1A. Homology modeling of the 3-dimensional structure of the ectodomains of wild-type and mutant TNFRSF1A was performed. RESULTS: TRAPS-associated mutant and wild-type TNFRSF1A behaved differently and had different localization properties within the cell, as a direct result of mutations in the ectodomains of TNFRSF1A. From a structural perspective, mutants with a predicted structure similar to that of the wild-type protein (e.g., R92Q) behaved similarly to wild-type TNFRSF1A, whereas forms of TNFRSF1A with mutations predicted to drastically destabilize the protein structure (e.g., cysteine mutations) showed defects in cell surface expression and TNF binding. CONCLUSION: The results obtained from the in vitro experiments, in combination with the modeled structures, indicate that the phenotype and clinical differences between different TRAPS-associated mutants of TNFRSF1A result from different conformations of the TNFRSF1A ectodomains.


Assuntos
Febre Familiar do Mediterrâneo/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Febre Familiar do Mediterrâneo/metabolismo , Febre Familiar do Mediterrâneo/patologia , Humanos , Rim/citologia , Rim/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Receptores Tipo I de Fatores de Necrose Tumoral/química , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
10.
Arthritis Rheum ; 50(8): 2651-9, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15334481

RESUMO

OBJECTIVE: To investigate the effect of mutations in tumor necrosis factor receptor superfamily 1A (TNFRSF1A) on the ability of the receptors to be cleaved from the cell surface upon stimulation. The mutations we studied are associated with clinically distinct forms of TNF receptor-associated periodic syndrome (TRAPS). We also investigated different cell types within the same form of TRAPS. METHODS: The shedding of TNFRSF1A in response to stimulation with phorbol myristate acetate was assessed in leukocytes and dermal fibroblasts from patients with C33Y TRAPS, and in HEK 293 cell lines stably transfected with constructs containing wild-type TNFRSF1A and/or TNFRSF1A mutants identified in TRAPS patients. RESULTS: The shedding of TNFRSF1A differed between cell types within the same form of TRAPS. In particular, dermal fibroblasts, but not leukocytes, from C33Y TRAPS patients demonstrated reduced shedding of TNFRSF1A. Shedding of both wild-type and mutant TNFRSF1A from the transfected HEK 293 cells showed minor differences, but was in all cases induced to a substantial extent. CONCLUSION: Differences in TNFRSF1A shedding are not purely a function of the TNFRSF1A structure, but are also influenced by other features of genetic makeup and/or cellular differentiation. It is unlikely that a defect in TNFRSF1A shedding per se can fully explain the clinical features that are common to TRAPS patients with different TNFRSF1A mutations.


Assuntos
Antígenos CD/genética , Febre Familiar do Mediterrâneo/genética , Receptores do Fator de Necrose Tumoral/genética , Antígenos CD/metabolismo , Fibroblastos/fisiologia , Humanos , Leucócitos/fisiologia , Mutação , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Acetato de Tetradecanoilforbol/farmacologia
11.
Immunology ; 113(1): 65-79, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15312137

RESUMO

Tumour necrosis factor (TNF)-receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder involving autosomal-dominant missense mutations in TNF receptor superfamily 1A (TNFRSF1A) ectodomains. To elucidate the molecular effects of TRAPS-related mutations, we transfected HEK-293 cells to produce lines stably expressing high levels of either wild-type (WT) or single mutant recombinant forms of TNFRSF1A. Mutants with single amino acid substitutions in the first cysteine-rich domain (CRD1) were produced both as full-length receptor proteins and as truncated forms lacking the cytoplasmic signalling domain (deltasig). High-level expression of either WT or mutant full-length TNFRSF1A spontaneously induced apoptosis and interleukin-8 production, indicating that the mutations in CRD1 did not abrogate signalling. Consistent with this, WT and mutant full-length TNFRSF1A formed cytoplasmic aggregates that co-localized with ubiquitin and chaperones, and with the signal transducer TRADD, but not with the inhibitor, silencer of death domain (SODD). Furthermore, as expected, WT and mutant deltasig forms of TNFRSF1A did not induce apoptosis or interleukin-8 production. However, whereas the WT full-length TNFRSF1A was expressed both in the cytoplasm and on the cell surface, the mutant receptors showed strong cytoplasmic expression but reduced cell-surface expression. The WT and mutant deltasig forms of TNFRSF1A were all expressed at the cell surface, but a proportion of the mutant receptors were also retained in the cytoplasm and co-localized with BiP. Furthermore, the mutant forms of surface-expressed deltasig TNFRSF1A were defective in binding TNF-alpha. We conclude that TRAPS-related CRD1 mutants of TNFRSF1A possess signalling properties associated with the cytoplasmic death domain, but other behavioural features of the mutant receptors are abnormal, including intracellular trafficking and TNF binding.


Assuntos
Antígenos CD/genética , Febre Familiar do Mediterrâneo/genética , Mutação de Sentido Incorreto , Receptores do Fator de Necrose Tumoral/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Apoptose/imunologia , Linhagem Celular , Membrana Celular/imunologia , Citocinas/biossíntese , Citoplasma/imunologia , Febre Familiar do Mediterrâneo/imunologia , Humanos , Microscopia Confocal , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais/genética , Transfecção , Fator de Necrose Tumoral alfa/metabolismo
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