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1.
Cell ; 181(2): 293-305.e11, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32142653

RESUMO

Pulmonary tuberculosis, a disease caused by Mycobacterium tuberculosis (Mtb), manifests with a persistent cough as both a primary symptom and mechanism of transmission. The cough reflex can be triggered by nociceptive neurons innervating the lungs, and some bacteria produce neuron-targeting molecules. However, how pulmonary Mtb infection causes cough remains undefined, and whether Mtb produces a neuron-activating, cough-inducing molecule is unknown. Here, we show that an Mtb organic extract activates nociceptive neurons in vitro and identify the Mtb glycolipid sulfolipid-1 (SL-1) as the nociceptive molecule. Mtb organic extracts from mutants lacking SL-1 synthesis cannot activate neurons in vitro or induce cough in a guinea pig model. Finally, Mtb-infected guinea pigs cough in a manner dependent on SL-1 synthesis. Thus, we demonstrate a heretofore unknown molecular mechanism for cough induction by a virulent human pathogen via its production of a complex lipid.


Assuntos
Tosse/fisiopatologia , Glicolipídeos/metabolismo , Nociceptores/fisiologia , Fatores de Virulência/metabolismo , Adulto , Animais , Linhagem Celular , Tosse/etiologia , Tosse/microbiologia , Feminino , Glicolipídeos/fisiologia , Cobaias , Interações Hospedeiro-Patógeno , Humanos , Lipídeos/fisiologia , Pulmão/microbiologia , Macrófagos/microbiologia , Masculino , Camundongos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Cultura Primária de Células , Tuberculose/microbiologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/fisiopatologia , Fatores de Virulência/fisiologia
2.
mSphere ; 4(3)2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31167949

RESUMO

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.IMPORTANCE Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/metabolismo , Fatores de Virulência/metabolismo , Animais , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Membrana Celular/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Feminino , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Tuberculose/microbiologia
3.
Cell Rep ; 16(5): 1253-1258, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27452467

RESUMO

The prevailing paradigm is that tuberculosis infection is initiated when patrolling alveolar macrophages and dendritic cells within the terminal alveolus ingest inhaled Mycobacterium tuberculosis (Mtb). However, definitive data for this model are lacking. Among the epithelial cells of the upper airway, a specialized epithelial cell known as a microfold cell (M cell) overlies various components of mucosa-associated lymphatic tissue. Here, using multiple mouse models, we show that Mtb invades via M cells to initiate infection. Intranasal Mtb infection in mice lacking M cells either genetically or by antibody depletion resulted in reduced invasion and dissemination to draining lymph nodes. M cell-depleted mice infected via aerosol also had delayed dissemination to lymph nodes and reduced mortality. Translocation of Mtb across two M cell transwell models was rapid and transcellular. Thus, M cell translocation is a vital entry mechanism that contributes to the pathogenesis of Mtb.


Assuntos
Células Epiteliais/virologia , Mycobacterium tuberculosis/patogenicidade , Tuberculose/virologia , Animais , Células CACO-2 , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/virologia , Feminino , Humanos , Linfonodos/virologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Alvéolos Pulmonares/virologia
4.
J Immunol ; 196(11): 4641-9, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27183573

RESUMO

Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for 1.5 million deaths annually. We previously showed that M. tuberculosis infection in mice induces expression of the CO-producing enzyme heme oxygenase (HO1) and that CO is sensed by M. tuberculosis to initiate a dormancy program. Further, mice deficient in HO1 succumb to M. tuberculosis infection more readily than do wild-type mice. Although mouse macrophages control intracellular M. tuberculosis infection through several mechanisms, such as NO synthase, the respiratory burst, acidification, and autophagy, how human macrophages control M. tuberculosis infection remains less well understood. In this article, we show that M. tuberculosis induces and colocalizes with HO1 in both mouse and human tuberculosis lesions in vivo, and that M. tuberculosis induces and colocalizes with HO1 during primary human macrophage infection in vitro. Surprisingly, we find that chemical inhibition of HO1 both reduces inflammatory cytokine production by human macrophages and restricts intracellular growth of mycobacteria. Thus, induction of HO1 by M. tuberculosis infection may be a mycobacterial virulence mechanism to enhance inflammation and bacterial growth.


Assuntos
Heme Oxigenase-1/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/metabolismo , Tuberculose/microbiologia , Animais , Linhagem Celular , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células U937
5.
Cell Host Microbe ; 17(6): 820-8, 2015 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-26048137

RESUMO

Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection.


Assuntos
DNA Bacteriano/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Mycobacterium tuberculosis/genética , Nucleotidiltransferases/metabolismo , Tuberculose/microbiologia , Animais , Autofagia , Proteínas de Ligação a DNA/metabolismo , Humanos , Imunidade Inata , Interferon beta/imunologia , Interferon beta/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Nucleotidiltransferases/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/metabolismo , Tuberculose/mortalidade
6.
Chem Res Toxicol ; 24(9): 1457-9, 2011 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-21812477

RESUMO

Previously, we determined that diethyl acetylenedicarboxylate (DAD), a protein cross-linker, was significantly more toxic than analogous monofunctional electrophiles. We hypothesized that other protein cross-linkers enhance toxicity similarly. In agreement with this hypothesis, the bifunctional electrophile divinyl sulfone (DVSF) was 6-fold more toxic than ethyl vinyl sulfone (EVSF) in colorectal carcinoma cells and greater than 10-fold more toxic in Saccharomyces cerevisiae. DVSF and DAD caused oligomerization of yeast thioredoxin 2 (Trx2p) in vitro and promoted Trx2p cross-linking to other proteins in yeast at cytotoxic doses. Our results suggest that protein cross-linking is considerably more detrimental to cellular homeostasis than simple alkylation.


Assuntos
Alcinos/toxicidade , Reagentes de Ligações Cruzadas/toxicidade , Citotoxinas/toxicidade , Proteínas/química , Sulfonas/toxicidade , Alquilação , Linhagem Celular Tumoral , Humanos , Saccharomyces cerevisiae/citologia
7.
Chem Res Toxicol ; 24(1): 81-8, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21105742

RESUMO

Many α,ß-unsaturated carbonyl compounds are used in biochemical and medical research. Their biological effects are due in large part to their electrophilic properties, whereby they undergo reaction with nucleophilic sites in proteins and nucleic acids. Here, we describe a structure-activity comparison of the cytotoxic properties of diethyl maleate (DEM) and closely related chemical analogs. All molecules that contained an α,ß-unsaturated carbonyl group were cytotoxic to human colorectal carcinoma cells, causing apoptotic cell death. However, related molecules lacking this chemical moiety were not cytotoxic. One of the molecules screened, diethyl acetylenedicarboxylate (DAD), was considerably more cytotoxic than DEM and other analogues. Induction of cell death by DAD was significantly decreased following preincubation of cells with N-acetylcysteine, suggesting that its reactivity with thiols in cells might account for its cytotoxicity. By use of a model thiol compound, it was found that DAD can undergo addition reactions with two equivalents of thiol. When the reactivity of DAD with proteins was explored, it was determined that DAD induces oligomerization of Gpx3p, a yeast glutathione peroxidase with highly reactive cysteine residues in its active site. Our results suggest that DAD functions as a protein-thiol cross-linker, providing a potential chemical explanation for its cytotoxic potency.


Assuntos
Alcinos/química , Reagentes de Ligações Cruzadas/química , Maleatos/química , Compostos de Sulfidrila/química , Acetilcisteína/química , Alcinos/toxicidade , Domínio Catalítico , Linhagem Celular Tumoral , Glutationa Peroxidase/química , Glutationa Peroxidase/metabolismo , Humanos , Maleatos/toxicidade , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade
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