RESUMEN
While a third of the world carries the burden of tuberculosis, disease control has been hindered by a lack of tools, including a rapid, point-of-care diagnostic and a protective vaccine. In many infectious diseases, antibodies (Abs) are powerful biomarkers and important immune mediators. However, in Mycobacterium tuberculosis (Mtb) infection, a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach, we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses, such that Ltb infection is associated with unique Ab Fc functional profiles, selective binding to FcγRIII, and distinct Ab glycosylation patterns. Moreover, compared to Abs from Atb, Abs from Ltb drove enhanced phagolysosomal maturation, inflammasome activation, and, most importantly, macrophage killing of intracellular Mtb. Combined, these data point to a potential role for Fc-mediated Ab effector functions, tuned via differential glycosylation, in Mtb control.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Humoral , Tuberculosis Latente/inmunología , Mycobacterium tuberculosis/inmunología , Adulto , Femenino , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Activación de Macrófagos , Masculino , Persona de Mediana Edad , Polisacáridos/inmunología , Análisis por Matrices de Proteínas , Receptores de IgG/inmunología , Adulto JovenRESUMEN
The activation of an immune response in tomato (Solanum lycopersicum) against Pseudomonas syringae relies on the recognition of E3 ligase-deficient forms of AvrPtoB by the host protein kinase, Fen. To investigate the mechanisms by which Fen-mediated immunity is regulated, we characterize in this study a Fen-interacting protein, Fni3, and its cofactor, S. lycoperiscum Uev (Suv). Fni3 encodes a homolog of the Ubc13-type ubiquitin-conjugating enzyme that catalyzes exclusively Lys-63-linked ubiquitination, whereas Suv is a ubiquitin-conjugating enzyme variant. The C-terminal region of Fen was necessary for interaction with Fni3, and this interaction was required for cell death triggered by overexpression of Fen in Nicotiana benthamiana leaves. Fni3 was shown to be an active E2 enzyme, but Suv displayed no ubiquitin-conjugating activity; Fni3 and Suv together directed Lys-63-linked ubiquitination. Decreased expression of Fni3, another tomato Ubc13 homolog, Sl-Ubc13-2, or Suv in N. benthamiana leaves diminished cell death associated with Fen-mediated immunity and cell death elicited by several other resistance (R) proteins and their cognate effectors. We also discovered that coexpression of Fen and other R proteins/effectors with a Fni3 mutant that is compromised for ubiquitin-conjugating activity diminished the cell death. These results suggest that Fni3/Sl-Ubc13-2 and Suv regulate the immune response mediated by Fen and other R proteins through Lys-63-linked ubiquitination.
Asunto(s)
Nicotiana/enzimología , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta , Proteínas de Plantas/metabolismo , Pseudomonas syringae/fisiología , Solanum lycopersicum/enzimología , Secuencia de Bases , Silenciador del Gen , Solanum lycopersicum/genética , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Análisis de Secuencia de ADN , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/microbiología , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
Many bacterial pathogens of plants and animals use a type III secretion system to deliver diverse virulence-associated 'effector' proteins into the host cell. The mechanisms by which these effectors act are mostly unknown; however, they often promote disease by suppressing host immunity. One type III effector, AvrPtoB, expressed by the plant pathogen Pseudomonas syringae pv. tomato, has a carboxy-terminal domain that is an E3 ubiquitin ligase. Deletion of this domain allows an amino-terminal region of AvrPtoB (AvrPtoB(1-387)) to be detected by certain tomato varieties leading to immunity-associated programmed cell death. Here we show that a host kinase, Fen, physically interacts with AvrPtoB(1-387 )and is responsible for activating the plant immune response. The AvrPtoB E3 ligase specifically ubiquitinates Fen and promotes its degradation in a proteasome-dependent manner. This degradation leads to disease susceptibility in Fen-expressing tomato lines. Various wild species of tomato were found to exhibit immunity in response to AvrPtoB(1-387 )and not to full-length AvrPtoB. Thus, by acquiring an E3 ligase domain, AvrPtoB has thwarted a highly conserved host resistance mechanism.
Asunto(s)
Enfermedades de las Plantas/inmunología , Proteínas Quinasas/metabolismo , Pseudomonas syringae/enzimología , Solanum lycopersicum/inmunología , Solanum lycopersicum/microbiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Solanum lycopersicum/clasificación , Solanum lycopersicum/enzimología , Fenotipo , Enfermedades de las Plantas/microbiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Pseudomonas syringae/inmunología , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Variability in bacterial sterilization is a key feature of Mycobacterium tuberculosis (Mtb) disease. In a population of human macrophages, there are macrophages that restrict Mtb growth and those that do not. However, the sources of heterogeneity in macrophage state during Mtb infection are poorly understood. Here, we perform RNAseq on restrictive and permissive macrophages and reveal that the expression of genes involved in GM-CSF signaling discriminates between the two subpopulations. We demonstrate that blocking GM-CSF makes macrophages more permissive of Mtb growth while addition of GM-CSF increases bacterial control. In parallel, we find that the loss of bacterial control that occurs in HIV-Mtb coinfected macrophages correlates with reduced GM-CSF secretion. Treatment of coinfected cells with GM-CSF restores bacterial control. Thus, we leverage the natural variation in macrophage control of Mtb to identify a critical cytokine response for regulating Mtb survival and identify components of the antimicrobial response induced by GM-CSF.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Transducción de Señal/inmunología , Tuberculosis/inmunología , Capa Leucocitaria de la Sangre/citología , Células Cultivadas , Perfilación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , VIH/inmunología , VIH/patogenicidad , Infecciones por VIH/inmunología , Infecciones por VIH/microbiología , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Macrófagos/microbiología , Mycobacterium tuberculosis/patogenicidad , Cultivo Primario de Células , Análisis de Secuencia de ARN , Tuberculosis/microbiología , Vitamina D/inmunología , Vitamina D/metabolismoRESUMEN
There is increasing evidence that phenotypically drug-resistant bacteria may be important determinants of antibiotic treatment failure. Using high-throughput imaging, we defined distinct subpopulations of mycobacterial cells that exhibit heritable but semi-stable drug resistance. These subpopulations have distinct transcriptional signatures and growth characteristics at both bulk and single-cell levels, which are also heritable and semi-stable. We find that the mycobacterial histone-like protein HupB is required for the formation of these subpopulations. Using proteomic approaches, we further demonstrate that HupB is posttranslationally modified by lysine acetylation and lysine methylation. Mutation of a single posttranslational modification site specifically abolishes the formation of one of the drug-resistant subpopulations of cells, providing the first evidence in prokaryotes that posttranslational modification of a bacterial nucleoid-associated protein may epigenetically regulate cell state.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Farmacorresistencia Bacteriana , Isoniazida/farmacología , Mycobacterium/efectos de los fármacos , Mycobacterium/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/química , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Histonas/química , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Mutación , Mycobacterium/genética , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Mycobacterium tuberculosis persists within macrophages in an arrested phagosome and depends upon necrosis to elude immunity and disseminate. Although apoptosis of M. tuberculosis-infected macrophages is associated with reduced bacterial growth, the bacteria are relatively resistant to other forms of death, leaving the mechanism underlying this observation unresolved. We find that after apoptosis, M. tuberculosis-infected macrophages are rapidly taken up by uninfected macrophages through efferocytosis, a dedicated apoptotic cell engulfment process. Efferocytosis of M. tuberculosis sequestered within an apoptotic macrophage further compartmentalizes the bacterium and delivers it along with the apoptotic cell debris to the lysosomal compartment. M. tuberculosis is killed only after efferocytosis, indicating that apoptosis itself is not intrinsically bactericidal but requires subsequent phagocytic uptake and lysosomal fusion of the apoptotic body harboring the bacterium. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, these data indicate that it can also function as an antimicrobial effector mechanism.