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1.
Toxins (Basel) ; 13(4)2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807365

RESUMO

The Hbl toxin is a three-component haemolytic complex produced by Bacillus cereus sensu lato strains and implicated as a cause of diarrhoea in B. cereus food poisoning. While the structure of the HblB component of this toxin is known, the structures of the other components are unresolved. Here, we describe the expression of the recombinant HblL1 component and the elucidation of its structure to 1.36 Å. Like HblB, it is a member of the alpha-helical pore-forming toxin family. In comparison to other members of this group, it has an extended hydrophobic beta tongue region that may be involved in pore formation. Molecular docking was used to predict possible interactions between HblL1 and HblB, and suggests a head to tail dimer might form, burying the HblL1 beta tongue region.


Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Hemolisinas/metabolismo , Bacillus cereus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
2.
Cell Host Microbe ; 28(3): 402-410.e5, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32544461

RESUMO

Bacteria and their toxins are associated with significant human morbidity and mortality. While a few bacterial toxins are well characterized, the mechanism of action for most toxins has not been elucidated, thereby limiting therapeutic advances. One such example is the highly potent pore-forming toxin, hemolysin BL (HBL), produced by the gram-positive pathogen Bacillus cereus. However, how HBL exerts its effects and whether it requires any host factors is unknown. Here, we describe an unbiased genome-wide CRISPR-Cas9 knockout screen that identified LPS-induced TNF-α factor (LITAF) as the HBL receptor. Using LITAF-deficient cells, a second, subsequent whole-genome CRISPR-Cas9 screen identified the LITAF-like protein CDIP1 as a second, alternative receptor. We generated LITAF-deficient mice, which exhibit marked resistance to lethal HBL challenges. This work outlines and validates an approach to use iterative genome-wide CRISPR-Cas9 screens to identify the complement of host factors exploited by bacterial toxins to exert their myriad biological effects.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Bacillus cereus/patogenicidade , Proteínas de Bactérias/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Hemolisinas/fisiologia , Receptores de Enterotoxina/fisiologia , Fatores de Transcrição/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Células CHO , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cricetulus , Proteínas de Ligação a DNA/genética , Células Endoteliais , Feminino , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , Humanos , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Enterotoxina/genética , Fatores de Transcrição/genética , Fatores de Virulência
3.
PLoS One ; 13(9): e0198862, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30208113

RESUMO

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.


Assuntos
Amidas/farmacologia , Vesícula/metabolismo , Técnicas de Cultura de Células/métodos , Epiderme/metabolismo , Queratinócitos/metabolismo , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Vesícula/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Epiderme/patologia , Humanos , Queratinócitos/patologia
4.
J Clin Invest ; 128(8): 3595-3604, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30035749

RESUMO

Autosomal dominant hyper IgE syndrome (AD-HIES), or Job's syndrome, is a primary immune deficiency caused by dominant-negative mutations in STAT3. Recurrent Staphylococcus aureus skin abscesses are a defining feature of this syndrome. A widely held hypothesis that defects in peripheral Th17 differentiation confer this susceptibility has never been directly evaluated. To assess the cutaneous immune response in AD-HIES, we induced suction blisters in healthy volunteers (HVs) and patients with AD-HIES and then challenged the wound with lethally irradiated bacteria. We show that cutaneous production of IL-17A and IL-17F was normal in patients with AD-HIES. Overproduction of TNF-α differentiated the responses in AD-HIES from HVs. This was associated with reduced IL-10 family signaling in blister-infiltrating cells and defective epithelial cell function. Mouse models of AD-HIES recapitulated these aberrant epithelial responses to S. aureus and involved defective epithelial-to-mesenchymal transition (EMT) rather than a failure of bacterial killing. Defective responses in mouse models of AD-HIES and primary keratinocyte cultures from patients with AD-HIES could be reversed by TNF-α blockade and by drugs with reported modulatory effects on EMT. Our results identify these as potential therapeutic approaches in patients with AD-HIES suffering S. aureus infections.


Assuntos
Células Epiteliais/imunologia , Furunculose/imunologia , Síndrome de Job/imunologia , Queratinócitos/imunologia , Staphylococcus aureus/imunologia , Fator de Necrose Tumoral alfa/imunologia , Adulto , Animais , Modelos Animais de Doenças , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/imunologia , Feminino , Furunculose/genética , Furunculose/patologia , Humanos , Interleucina-17/genética , Interleucina-17/imunologia , Síndrome de Job/genética , Síndrome de Job/patologia , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/genética
5.
Pathogens ; 6(2)2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28587312

RESUMO

Autosomal dominant hyper IgE syndrome (AD-HIES) is a primary immunodeficiency caused by a loss-of-function mutation in the Signal Transducer and Activator of Transcription 3 (STAT3). This immune disorder is clinically characterized by increased susceptibility to cutaneous and sinopulmonary infections, in particular with Candida and Staphylococcus aureus. It has recently been recognized that the skin microbiome of patients with AD-HIES is altered with an overrepresentation of certain Gram-negative bacteria and Gram-positive staphylococci. However, these alterations have not been characterized at the species- and strain-level. Since S. aureus infections are influenced by strain-specific expression of virulence factors, information on colonizing strain characteristics may provide insights into host-pathogen interactions and help guide management strategies for treatment and prophylaxis. The aim of this study was to determine whether the immunodeficiency of AD-HIES selects for unique strains of colonizing S. aureus. Using multi-locus sequence typing (MLST), protein A (spa) typing, and PCR-based detection of toxin genes, we performed a detailed analysis of the S. aureus isolates (n = 13) found on the skin of twenty-one patients with AD-HIES. We found a low diversity of sequence types, and an abundance of strains that expressed methicillin resistance, Panton-Valentine leukocidin (PVL), and staphylococcal enterotoxins K and Q (SEK, SEQ). Our results indicate that patients with AD-HIES may often carry antibiotic-resistant strains that harbor key virulence factors.

6.
JCI Insight ; 1(10)2016 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-27478874

RESUMO

Atopic dermatitis (AD) is characterized by reduced barrier function, reduced innate immune activation, and susceptibility to Staphylococcus aureus. Host susceptibility factors are suggested by monogenic disorders associated with AD-like phenotypes and can be medically modulated. S. aureus contributes to AD pathogenesis and can be mitigated by antibiotics and bleach baths. Recent work has revealed that the skin microbiome differs significantly between healthy controls and patients with AD, including decreased Gram-negative bacteria in AD. However, little is known about the potential therapeutic benefit of microbiome modulation. To evaluate whether parameters of AD pathogenesis are altered after exposure to different culturable Gram-negative bacteria (CGN) collected from human skin, CGN were collected from healthy controls and patients with AD. Then, effects on cellular and culture-based models of immune, epithelial, and bacterial function were evaluated. Representative strains were evaluated in the MC903 mouse model of AD. We found that CGN taken from healthy volunteers but not from patients with AD were associated with enhanced barrier function, innate immunity activation, and control of S. aureus. Treatment with CGN from healthy controls improved outcomes in a mouse model of AD. These findings suggest that a live-biotherapeutic approach may hold promise for treatment of patients with AD.

8.
BMC Microbiol ; 16: 60, 2016 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-27052736

RESUMO

BACKGROUND: Commensal Gram-negative (CGN) microbiota have been identified on human skin by DNA sequencing; however, methods to reliably culture viable Gram-negative skin organisms have not been previously described. RESULTS: Through the use of selective antibiotics and minimal media we developed methods to culture CGN from skin swabs. We identified several previously uncharacterized CGN at the species level by optimizing growth conditions and limiting the inhibitory effects of nutrient shock, temperature, and bacterial competition, factors that may have previously limited CGN isolation from skin cultures. CONCLUSIONS: Our protocol will permit future functional studies on the influences of CGN on skin homeostasis and disease.


Assuntos
Técnicas Bacteriológicas/métodos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Pele/microbiologia , Meios de Cultura , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Microbiota
9.
Cell Host Microbe ; 18(5): 520-2, 2015 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-26567505

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) are resistant to ß-lactam antibiotics, which inhibit bacterial cell wall synthesis. In this issue of Cell Host & Microbe, Müller et al. (2015) show that ß-lactam treatment of MRSA leads to synthesis of an altered cell wall that increases inflammasome activation and immunopathology during skin infection.


Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina , Parede Celular , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos
10.
BMC Genomics ; 15: 229, 2014 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-24661624

RESUMO

BACKGROUND: Upon infection of a mammalian host, Bacillus anthracis responds to host cues, and particularly to elevated temperature (37°C) and bicarbonate/CO2 concentrations, with increased expression of virulence factors that include the anthrax toxins and extracellular capsular layer. This response requires the presence of the pXO1 virulence plasmid-encoded pleiotropic regulator AtxA. To better understand the genetic basis of this response, we utilized a controlled in vitro system and Next Generation sequencing to determine and compare RNA expression profiles of the parental strain and an isogenic AtxA-deficient strain in a 2 × 2 factorial design with growth environments containing or lacking carbon dioxide. RESULTS: We found 15 pXO1-encoded genes and 3 chromosomal genes that were strongly regulated by the separate or synergistic actions of AtxA and carbon dioxide. The majority of the regulated genes responded to both AtxA and carbon dioxide rather than to just one of these factors. Interestingly, we identified two previously unrecognized small RNAs that are highly expressed under physiological carbon dioxide concentrations in an AtxA-dependent manner. Expression levels of the two small RNAs were found to be higher than that of any other gene differentially expressed in response to these conditions. Secondary structure and small RNA-mRNA binding predictions for the two small RNAs suggest that they may perform important functions in regulating B. anthracis virulence. CONCLUSIONS: A majority of genes on the virulence plasmid pXO1 that are regulated by the presence of either CO2 or AtxA separately are also regulated synergistically in the presence of both. These results also elucidate novel pXO1-encoded small RNAs that are associated with virulence conditions.


Assuntos
Bacillus anthracis/genética , Proteínas de Bactérias/genética , Dióxido de Carbono/metabolismo , Transativadores/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Análise de Sequência de RNA , Transativadores/metabolismo , Regulação para Cima , Fatores de Virulência/genética
11.
PLoS One ; 8(10): e76955, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24204713

RESUMO

Bacillus cereus is a spore-forming, Gram-positive bacterium commonly associated with outbreaks of food poisoning. It is also known as an opportunistic pathogen causing clinical infections such as bacteremia, meningitis, pneumonia, and gas gangrene-like cutaneous infections, mostly in immunocompromised patients. B. cereus secretes a plethora of toxins of which four are associated with the symptoms of food poisoning. Two of these, the non-hemolytic enterotoxin Nhe and the hemolysin BL (Hbl) toxin, are predicted to be structurally similar and are unique in that they require the combined action of three toxin proteins to induce cell lysis. Despite their dominant role in disease, the molecular mechanism of their toxic function is still poorly understood. We report here that B. cereus strain ATCC 10876 harbors not only genes encoding Nhe, but also two copies of the hbl genes. We identified Hbl as the major secreted toxin responsible for inducing rapid cell lysis both in cultured cells and in an intraperitoneal mouse toxicity model. Antibody neutralization and deletion of Hbl-encoding genes resulted in significant reductions of cytotoxic activity. Microscopy studies with Chinese Hamster Ovary cells furthermore showed that pore formation by both Hbl and Nhe occurs through a stepwise, sequential binding of toxin components to the cell surface and to each other. This begins with binding of Hbl-B or NheC to the eukaryotic membrane, and is followed by the recruitment of Hbl-L1 or NheB, respectively, followed by the corresponding third protein. Lastly, toxin component complementation studies indicate that although Hbl and Nhe can be expressed simultaneously and are predicted to be structurally similar, they are incompatible and cannot complement each other.


Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Enterotoxinas/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/farmacologia , Animais , Bacillus cereus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , Meios de Cultivo Condicionados/farmacologia , Enterotoxinas/genética , Enterotoxinas/farmacologia , Dosagem de Genes , Ordem dos Genes , Teste de Complementação Genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Microscopia Confocal , Mutação , Ligação Proteica
12.
BMC Genomics ; 14: 188, 2013 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-23506131

RESUMO

BACKGROUND: Signals of danger and damage in the cytosol of cells are sensed by NOD-like receptors (NLRs), which are components of multiprotein complexes called inflammasomes. Inflammasomes activate caspase-1, resulting in IL-1-beta and IL-18 secretion and an inflammatory response. To date, the only known activator of rodent Nlrp1 is anthrax lethal toxin (LT), a protease secreted by the bacterial pathogen Bacillus anthracis. Although susceptibility of mouse macrophages to LT has been genetically linked to Nlrp1b, mice harbor two additional Nlrp1 paralogs in their genomes (Nlrp1a and Nlrp1c). However, little is known about their expression profile and sequence in different mouse strains. Furthermore, simultaneous expression of these paralogs may lead to competitional binding of Nlrp1b interaction partners needed for inflammasome activation, thus influencing macrophages susceptibility to LT. To more completely understand the role(s) of Nlrp1 paralogs in mice, we surveyed for their expression in a large set of LT-resistant and sensitive mouse macrophages. In addition, we provide sequence comparisons for Nlrp1a and report on previously unrecognized splice variants of Nlrp1b. RESULTS: Our results show that macrophages from some inbred mouse strains simultaneously express different splice variants of Nlrp1b. In contrast to the highly polymorphic Nlrp1b splice variants, sequencing of expressed Nlrp1a showed the protein to be highly conserved across all mouse strains. We found that Nlrp1a was expressed only in toxin-resistant macrophages, with the sole exception of expression in LT-sensitive CAST/EiJ macrophages. CONCLUSIONS: Our data present a complex picture of Nlrp1 protein variations and provide a basis for elucidating their roles in murine macrophage function. Furthermore, the high conservation of Nlrp1a implies that it might be an important inflammasome sensor in mice.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Camundongos Endogâmicos/genética , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Processamento Alternativo , Animais , Antígenos de Bactérias/farmacologia , Proteínas Reguladoras de Apoptose/biossíntese , Toxinas Bacterianas/farmacologia , Inflamassomos/metabolismo , Camundongos
13.
PLoS One ; 7(11): e49741, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23152930

RESUMO

Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Toxinas Bacterianas/metabolismo , Macrófagos/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/toxicidade , Proteínas Reguladoras de Apoptose/química , Toxinas Bacterianas/toxicidade , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Caspase 1/metabolismo , Ativação Enzimática/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência
14.
PLoS Pathog ; 8(3): e1002638, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22479187

RESUMO

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1ß and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 ß release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.


Assuntos
Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Inflamassomos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Inflamassomos/biossíntese , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Ratos , Ratos Endogâmicos Lew
15.
Microbes Infect ; 14(5): 387-91, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22166343

RESUMO

Bacillus anthracis is a spore-forming, soil-dwelling bacterium. This review describes the occurrence of spontaneous mutations leading to loss of sporulation and the selective pressures that can lead to their enrichment. We also discuss recognition of the associated phenotypes on solid medium, thereby allowing researchers to employ measures that either prevent or favor selection of sporulation-deficient mutants.


Assuntos
Bacillus anthracis/citologia , Bacillus anthracis/crescimento & desenvolvimento , Técnicas Bacteriológicas/métodos , Esporos Bacterianos/citologia , Esporos Bacterianos/crescimento & desenvolvimento , Bacillus anthracis/genética , Bacillus anthracis/isolamento & purificação , Meios de Cultura/química , Seleção Genética , Esporos Bacterianos/genética
16.
Microbes Infect ; 14(5): 392-400, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22207185

RESUMO

Anthrax lethal toxin (LT), a major virulence determinant of anthrax disease, induces vascular collapse in mice and rats. LT activates the Nlrp1 inflammasome in macrophages and dendritic cells, resulting in caspase-1 activation, IL-1ß and IL-18 maturation and a rapid cell death (pyroptosis). This review presents the current understanding of LT-induced activation of Nlrp1 in cells and its consequences for toxin-mediated effects in rodent toxin and spore challenge models.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antraz/patologia , Antígenos de Bactérias/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Toxinas Bacterianas/metabolismo , Inflamassomos/metabolismo , Animais , Bacillus anthracis/patogenicidade , Morte Celular , Células Dendríticas/microbiologia , Células Dendríticas/fisiologia , Macrófagos/microbiologia , Macrófagos/fisiologia , Camundongos , Proteínas NLR , Ratos
17.
Mol Microbiol ; 83(1): 96-109, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22103323

RESUMO

Bacterial lipoproteins play a crucial role in virulence in some gram-positive bacteria. However, the role of lipoprotein biosynthesis in Bacillus anthracis is unknown. We created a B. anthracis mutant strain altered in lipoproteins by deleting the lgt gene encoding the enzyme prolipoprotein diacylglyceryl transferase, which attaches the lipid anchor to prolipoproteins. (14)C-palmitate labelling confirmed that the mutant strain lacked lipoproteins, and hydrocarbon partitioning showed it to have decreased surface hydrophobicity. The anthrax toxin proteins were secreted from the mutant strain at nearly the same levels as from the wild-type strain. The TLR2-dependent TNF-α response of macrophages to heat-killed lgt mutant bacteria was reduced. Spores of the lgt mutant germinated inefficiently in vitro and in mouse skin. As a result, in a murine subcutaneous infection model, lgt mutant spores had markedly attenuated virulence. In contrast, vegetative cells of the lgt mutant were as virulent as those of the wild-type strain. Thus, lipoprotein biosynthesis in B. anthracis is required for full virulence in a murine infection model.


Assuntos
Antraz/microbiologia , Bacillus anthracis/enzimologia , Bacillus anthracis/patogenicidade , Proteínas de Bactérias/metabolismo , Lipoproteínas/biossíntese , Precursores de Proteínas/biossíntese , Esporos Bacterianos/crescimento & desenvolvimento , Transferases/metabolismo , Animais , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Proteínas de Bactérias/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Esporos Bacterianos/enzimologia , Esporos Bacterianos/metabolismo , Transferases/genética , Virulência
18.
Infect Immun ; 80(2): 529-38, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22104108

RESUMO

The anthrax edema toxin (ET) of Bacillus anthracis is composed of the receptor-binding component protective antigen (PA) and of the adenylyl cyclase catalytic moiety, edema factor (EF). Uptake of ET into cells raises intracellular concentrations of the secondary messenger cyclic AMP, thereby impairing or activating host cell functions. We report here on a new consequence of ET action in vivo. We show that in mouse models of toxemia and infection, serum PA concentrations were significantly higher in the presence of enzymatically active EF. These higher concentrations were not caused by ET-induced inhibition of PA endocytosis; on the contrary, ET induced increased PA binding and uptake of the PA oligomer in vitro and in vivo through upregulation of the PA receptors TEM8 and CMG2 in both myeloid and nonmyeloid cells. ET effects on protein clearance from circulation appeared to be global and were not limited to PA. ET also impaired the clearance of ovalbumin, green fluorescent protein, and EF itself, as well as the small molecule biotin when these molecules were coinjected with the toxin. Effects on injected protein levels were not a result of general increase in protein concentrations due to fluid loss. Functional markers for liver and kidney were altered in response to ET. Concomitantly, ET caused phosphorylation and activation of the aquaporin-2 water channel present in the principal cells of the collecting ducts of the kidneys that are responsible for fluid homeostasis. Our data suggest that in vivo, ET alters circulatory protein and small molecule pharmacokinetics by an as-yet-undefined mechanism, thereby potentially allowing a prolonged circulation of anthrax virulence factors such as EF during infection.


Assuntos
Antraz/metabolismo , Antígenos de Bactérias/toxicidade , Bacillus anthracis/metabolismo , Toxinas Bacterianas/toxicidade , Animais , Antraz/imunologia , Antígenos de Bactérias/sangue , Bacillus anthracis/imunologia , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Células CHO , Cricetinae , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos , Receptores de Superfície Celular , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo
19.
PLoS Pathog ; 6(12): e1001222, 2010 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-21170303

RESUMO

Bacillus anthracis infects hosts as a spore, germinates, and disseminates in its vegetative form. Production of anthrax lethal and edema toxins following bacterial outgrowth results in host death. Macrophages of inbred mouse strains are either sensitive or resistant to lethal toxin depending on whether they express the lethal toxin responsive or non-responsive alleles of the inflammasome sensor Nlrp1b (Nlrp1b(S/S) or Nlrp1b(R/R), respectively). In this study, Nlrp1b was shown to affect mouse susceptibility to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1b(S/S) alleles (which allow activation of caspase-1 and IL-1ß release in response to anthrax lethal toxin challenge) effectively controlled bacterial growth and dissemination when compared to mice having Nlrp1b(R/R) alleles (which cannot activate caspase-1 in response to toxin). Nlrp1b(S)-mediated resistance to infection was not dependent on the route of infection and was observed when bacteria were introduced by either subcutaneous or intravenous routes. Resistance did not occur through alterations in spore germination, as vegetative bacteria were also killed in Nlrp1b(S/S) mice. Resistance to infection required the actions of both caspase-1 and IL-1ß as Nlrp1b(S/S) mice deleted of caspase-1 or the IL-1 receptor, or treated with the Il-1 receptor antagonist anakinra, were sensitized to infection. Comparison of circulating neutrophil levels and IL-1ß responses in Nlrp1b(S/S),Nlrp1b(R/) (R) and IL-1 receptor knockout mice implicated Nlrp1b and IL-1 signaling in control of neutrophil responses to anthrax infection. Neutrophil depletion experiments verified the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection, and establish roles for Nlrp1b(S), caspase-1, and IL-1ß in countering anthrax infection.


Assuntos
Antraz/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Caspase 1/imunologia , Interleucina-1/imunologia , Infiltração de Neutrófilos/imunologia , Transdução de Sinais/imunologia , Animais , Bacillus anthracis/imunologia , Bacillus anthracis/patogenicidade , Suscetibilidade a Doenças/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos
20.
Cell Host Microbe ; 8(5): 455-62, 2010 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-21075356

RESUMO

Bacillus anthracis kills through a combination of bacterial infection and toxemia. Anthrax toxin working via the CMG2 receptor mediates lethality late in infection, but its roles early in infection remain unclear. We generated myeloid-lineage specific CMG2-deficient mice to examine the roles of macrophages, neutrophils, and other myeloid cells in anthrax pathogenesis. Macrophages and neutrophils isolated from these mice were resistant to anthrax toxin. However, the myeloid-specific CMG2-deficient mice remained fully sensitive to both anthrax lethal and edema toxins, demonstrating that targeting of myeloid cells is not responsible for anthrax toxin-induced lethality. Surprisingly, the myeloid-specific CMG2-deficient mice were completely resistant to B. anthracis infection. Neutrophil depletion experiments suggest that B. anthracis relies on anthrax toxin secretion to evade the scavenging functions of neutrophils to successfully establish infection. This work demonstrates that anthrax toxin uptake through CMG2 and the resulting impairment of myeloid cells are essential to anthrax infection.


Assuntos
Antígenos de Bactérias/metabolismo , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/metabolismo , Interações Hospedeiro-Patógeno , Células Mieloides/microbiologia , Receptores de Peptídeos/metabolismo , Animais , Antraz/imunologia , Antraz/microbiologia , Bacillus anthracis/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Receptores de Peptídeos/genética
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