RESUMEN
The germ theory states that pathogenic microorganisms are responsible for causing infectious diseases. The theory is inherently microbe-centric and does not account for variability in disease severity among individuals and asymptomatic carriership-two phenomena indicating an important role for host variability in infection outcome. The basic tenet of the germ theory was recently challenged, and a radically host-centric paradigm referred to as the "full-blown host theory" was proposed. According to this view, the pathogen is reduced to a passive environmental trigger, and the development of disease is instead due to pre-existing immunodeficiencies of the host. Here, we consider the factors that determine disease severity using established knowledge concerning evolutionary biology, microbial pathogenesis, and host-pathogen interactions. We note that the available data support a noncentric view that recognizes key roles for both the causative microbe and the host in dictating infection outcome.
Asunto(s)
Teoría del Gérmen de la Enfermedad , Interacciones Huésped-Patógeno , HumanosRESUMEN
Following mycobacterial entry into macrophages the ESX-1 type VII secretion system promotes phagosomal permeabilization and type I IFN production, key features of tuberculosis pathogenesis. The current model states that the secreted substrate ESAT-6 is required for membrane permeabilization and that a subsequent passive leakage of extracellular bacterial DNA into the host cell cytosol is sensed by the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) pathway to induce type I IFN production. We employed a collection of Mycobacterium marinum ESX-1 transposon mutants in a macrophage infection model and show that permeabilization of the phagosomal membrane does not require ESAT-6 secretion. Moreover, loss of membrane integrity is insufficient to induce type I IFN production. Instead, type I IFN production requires intact ESX-1 function and correlates with release of mitochondrial and nuclear host DNA into the cytosol, indicating that ESX-1 affects host membrane integrity and DNA release via genetically separable mechanisms. These results suggest a revised model for major aspects of ESX-1-mediated host interactions and put focus on elucidating the mechanisms by which ESX-1 permeabilizes host membranes and induces the type I IFN response, questions of importance for our basic understanding of mycobacterial pathogenesis and innate immune sensing.
Asunto(s)
Antígenos Bacterianos/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Interferón Tipo I/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Mycobacterium marinum/patogenicidad , Fagosomas/metabolismo , Antígenos Bacterianos/genética , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Mitocondrias/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/genética , Mycobacterium marinum/inmunología , Mycobacterium marinum/metabolismo , Tuberculosis/inmunología , Sistemas de Secreción Tipo VIIRESUMEN
From an evolutionary point of view a pathogen might benefit from regulating the inflammatory response, both in order to facilitate establishment of colonization and to avoid life-threatening host manifestations, such as septic shock. In agreement with this notion Streptococcus pyogenes exploits type I IFN-signaling to limit detrimental inflammation in infected mice, but the host-pathogen interactions and mechanisms responsible for induction of the type I IFN response have remained unknown. Here we used a macrophage infection model and report that S. pyogenes induces anti-inflammatory IL-10 in an M protein-dependent manner, a function that was mapped to the B- and C-repeat regions of the M5 protein. Intriguingly, IL-10 was produced downstream of type I IFN-signaling, and production of type I IFN occurred via M protein-dependent activation of the STING signaling pathway. Activation of STING was independent of the cytosolic double stranded DNA sensor cGAS, and infection did not induce detectable release into the cytosol of either mitochondrial, nuclear or bacterial DNA-indicating DNA-independent activation of the STING pathway in S. pyogenes infected macrophages. These findings provide mechanistic insight concerning how S. pyogenes induces the type I IFN response and identify a previously unrecognized macrophage-modulating role for the streptococcal M protein that may contribute to curb the inflammatory response to infection.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Interacciones Huésped-Patógeno , Interleucina-10/metabolismo , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Infecciones Estreptocócicas/metabolismo , Streptococcus pyogenes/fisiología , Animales , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Portadoras/genética , Células Cultivadas , Inmunidad Innata , Interferón Tipo I/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Nucleotidiltransferasas/genética , Transducción de Señal , Infecciones Estreptocócicas/genética , Infecciones Estreptocócicas/microbiologíaRESUMEN
Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the bactericidal enzyme hGIIA to exert its bactericidal function.
Asunto(s)
Antibacterianos/farmacología , Pared Celular/metabolismo , Fosfolipasas A2 Grupo II/inmunología , Inmunidad Innata/efectos de los fármacos , Polisacáridos Bacterianos/farmacología , Infecciones Estreptocócicas/microbiología , Streptococcus/inmunología , Actividad Bactericida de la Sangre , Fosfolipasas A2 Grupo II/sangre , Fosfolipasas A2 Grupo II/genética , Interacciones Huésped-Patógeno , Humanos , Infecciones Estreptocócicas/sangre , Infecciones Estreptocócicas/enzimología , Streptococcus/patogenicidadRESUMEN
The ability of macrophages to eradicate intracellular pathogens is normally greatly enhanced by IFNγ, a cytokine produced mainly after onset of adaptive immunity. However, adaptive immunity is unable to provide sterilizing immunity against mycobacteria, suggesting that mycobacteria have evolved virulence strategies to inhibit the bactericidal effect of IFNγ-signalling in macrophages. Still, the host-pathogen interactions and cellular mechanisms responsible for this feature have remained elusive. We demonstrate that the ESX-1 type VII secretion systems of Mycobacterium tuberculosis and Mycobacterium marinum exploit type I IFN-signalling to promote an IL-12(low) /IL-10(high) regulatory macrophage phenotype characterized by secretion of IL-10, IL-27 and IL-6. This mechanism had no impact on intracellular growth in the absence of IFNγ but suppressed IFNγ-mediated autophagy and growth restriction, indicating that the regulatory phenotype extends to function. The IFNγ-refractory phenotype was partly mediated by IL-27-signalling, establishing functional relevance for this downstream cytokine. These findings identify a novel macrophage-modulating function for the ESX-1 secretion system that may contribute to suppress the efficacy of adaptive immunity and provide mechanistic insight into the antagonistic cross talk between type I IFNs and IFNγ in mycobacterial infection.
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Antígenos Bacterianos/fisiología , Autofagia/inmunología , Proteínas Bacterianas/fisiología , Macrófagos/metabolismo , Mycobacterium tuberculosis/fisiología , Tuberculosis/microbiología , Animales , Sistemas de Secreción Bacterianos , Células Cultivadas , Interacciones Huésped-Patógeno , Inmunidad Innata , Interferón Tipo I/fisiología , Interferón gamma/fisiología , Interleucinas/metabolismo , Interleucinas/normas , Macrófagos/microbiología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Transducción de Señal , Tuberculosis/inmunologíaRESUMEN
Anti-neutrophil cytoplasmic antibody associated vasculitides (AAV) are conditions defined by an autoimmune small vessel inflammation. Dying neutrophils are found around the inflamed vessels and the balance between infiltrating neutrophils and macrophages is important to prevent autoimmunity. Here we investigate how sera from AAV patients may regulate macrophage polarization and function. Macrophages from healthy individuals were differentiated into M0, M1, M2a, M2b or M2c macrophages using a standardized protocol, and phenotyped according to their expression surface markers and cytokine production. These phenotypes were compared with those of macrophages stimulated with serum from AAV patients or healthy controls. While the healthy control sera induced a M0 macrophage, AAV serum promoted polarization towards the M2c subtype. No sera induced M1, M2a or M2b macrophages. The M2c subtype showed increased phagocytosis capacity compared with the other subtypes. The M2c polarization found in AAV is consistent with previous reports of increased levels of M2c-associated cytokines.
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Polaridad Celular/inmunología , Macrófagos/inmunología , Vasculitis Sistémica/sangre , Vasculitis Sistémica/inmunología , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Autoinmunidad/inmunología , Diferenciación Celular/inmunología , Línea Celular , Polaridad Celular/efectos de los fármacos , Citocinas/inmunología , Glucocorticoides/farmacología , Humanos , Células Jurkat , Activación de Macrófagos/inmunología , Macrófagos/clasificación , Neutrófilos/inmunología , Fagocitosis/inmunología , Fenotipo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Tirosina Quinasa c-MerRESUMEN
Serine protease inhibitors (serpins) constitute the largest family of protease inhibitors expressed in humans, but their role in infection remains largely unexplored. In infected macrophages, the mycobacterial ESX-1 type VII secretion system permeabilizes internal host membranes and causes leakage into the cytosol of host DNA, which induces type I interferon (IFN) production via the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) surveillance pathway, and promotes infection in vivo. Using the Mycobacterium marinum infection model, we show that ESX-1-mediated type I IFN signaling in macrophages selectively induces the expression of serpina3f and serpina3g, two cytosolic serpins of the clade A3. The membranolytic activity of ESX-1 also caused leakage of cathepsin B into the cytosol where it promoted cell death, suggesting that the induction of type I IFN comes at the cost of lysosomal rupture and toxicity. However, the production of cytosolic serpins suppressed the protease activity of cathepsin B in this compartment and thus limited cell death, a function that was associated with increased bacterial growth in infected mice. These results suggest that cytosolic serpins act in a type I IFN-dependent cytoprotective feedback loop to counteract the inevitable toxic effect of ESX-1-mediated host membrane rupture. IMPORTANCE: The ESX-1 type VII secretion system is a key virulence determinant of pathogenic mycobacteria. The ability to permeabilize host cell membranes is critical for several ESX-1-dependent virulence traits, including phagosomal escape and induction of the type I interferon (IFN) response. We find that it comes at the cost of lysosomal leakage and subsequent host cell death. However, our results suggest that ESX-1-mediated type I IFN signaling selectively upregulates serpina3f and serpina3g and that these cytosolic serpins limit cell death caused by cathepsin B that has leaked into the cytosol, a function that is associated with increased bacterial growth in vivo. The ability to rupture host membranes is widespread among bacterial pathogens, and it will be of interest to evaluate the role of cytosolic serpins and this type I IFN-dependent cytoprotective feedback loop in the context of human infection.
RESUMEN
Mycobacterial infections, including tuberculosis, are a major health problem globally. Prevention and treatments of tuberculosis are challenging due to the poor efficacy of the current vaccine and the emergence of drug-resistant strains. Therefore, it is critical to increase our basic understanding of mycobacterial virulence strategies as well as the host immune response during infection in the complex in vivo setting. While existing infection models provide valuable tools for investigating mycobacterial pathogenesis, they also exhibit limitations that can be addressed by the development of complementary models. Here we describe recent advances to the murine Mycobacterium marinum infection model, in which the bacteria produce a local infection restricted to the tail tissue. The M. marinum model has the advantage of mimicking some of the key hallmarks of human tuberculosis not replicated in the conventional murine Mycobacterium tuberculosis model, such as the formation of granulomas with central caseating necrosis and the spontaneous development of a latency-like stage. Moreover, the model is non-lethal and enables longitudinal analysis of disease development in live animals. In this chapter, we report protocols to prepare infected tissue samples for detailed and quantitative analysis of the immune response by flow cytometry, immunofluorescence microscopy, RT-qPCR, ELISA, and Western blot, as well as for the analysis of bacterial load and localization.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas , Mycobacterium marinum , Mycobacterium tuberculosis , Tuberculosis , Animales , Ratones , Humanos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Tuberculosis/microbiología , Virulencia , Pez Cebra/microbiologíaRESUMEN
BACKGROUND: Selection pressure exerted by pathogens can influence patterns of genetic diversity in the host. In the immune system especially, numerous genes encode proteins involved in antagonistic interactions with pathogens, paving the way for coevolution that results in increased genetic diversity as a consequence of balancing selection. The complement system is a key component of innate immunity. Many complement proteins interact directly with pathogens, either by recognising pathogen molecules for complement activation, or by serving as targets of pathogen immune evasion mechanisms. Complement genes can therefore be expected to be important targets of pathogen-mediated balancing selection, but analyses of such selection on this part of the immune system have been limited. RESULTS: Using a population sample of whole-genome resequencing data from wild bank voles (n = 31), we estimated the extent of genetic diversity and tested for signatures of balancing selection in multiple complement genes (n = 44). Complement genes showed higher values of standardised ß (a statistic expected to be high under balancing selection) than the genome-wide average of protein coding genes. One complement gene, FCNA, a pattern recognition molecule that interacts directly with pathogens, was found to have a signature of balancing selection, as indicated by the Hudson-Kreitman-Aguadé test (HKA) test. Scans for localised signatures of balancing selection in this gene indicated that the target of balancing selection was found in exonic regions involved in ligand binding. CONCLUSION: The present study adds to the growing evidence that balancing selection may be an important evolutionary force on components of the innate immune system. The identified target in the complement system typifies the expectation that balancing selection acts on genes encoding proteins involved in direct interactions with pathogens.
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Proteínas del Sistema Complemento , Selección Genética , Análisis de Secuencia de ADN , Proteínas del Sistema Complemento/genéticaRESUMEN
The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum. ESX-1 is known to interact with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. Using a murine M. marinum infection model, we identify neutrophils and Ly6C+MHCII+ monocytes as the main cellular reservoirs for the bacteria. We show that ESX-1 promotes intragranuloma accumulation of neutrophils and that neutrophils have a previously unrecognized required role in executing ESX-1-mediated pathology. To explore if ESX-1 also regulates the function of recruited neutrophils, we performed a single-cell RNA-sequencing analysis that indicated that ESX-1 drives newly recruited uninfected neutrophils into an inflammatory phenotype via an extrinsic mechanism. In contrast, monocytes restricted the accumulation of neutrophils and immunopathology, demonstrating a major host-protective function for monocytes specifically by suppressing ESX-1-dependent neutrophilic inflammation. Inducible nitric oxide synthase (iNOS) activity was required for the suppressive mechanism, and we identified Ly6C+MHCII+ monocytes as the main iNOS-expressing cell type in the infected tissue. These results suggest that ESX-1 mediates immunopathology by promoting neutrophil accumulation and phenotypic differentiation in the infected tissue, and they demonstrate an antagonistic interplay between monocytes and neutrophils by which monocytes suppress host-detrimental neutrophilic inflammation. IMPORTANCE The ESX-1 type VII secretion system is required for virulence of pathogenic mycobacteria, including Mycobacterium tuberculosis. ESX-1 interacts with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. We demonstrate that ESX-1 promotes immunopathology by driving intragranuloma accumulation of neutrophils, which upon arrival adopt an inflammatory phenotype in an ESX-1-dependent manner. In contrast, monocytes limited the accumulation of neutrophils and neutrophil-mediated pathology via an iNOS-dependent mechanism, suggesting a major host-protective function for monocytes specifically by restricting ESX-1-dependent neutrophilic inflammation. These findings provide insight into how ESX-1 promotes disease, and they reveal an antagonistic functional relationship between monocytes and neutrophils that might regulate immunopathology not only in mycobacterial infection but also in other infections as well as in inflammatory conditions and cancer.
Asunto(s)
Mycobacterium marinum , Mycobacterium tuberculosis , Sistemas de Secreción Tipo VII , Animales , Ratones , Neutrófilos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Tipo VII/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium marinum/genética , Inflamación/microbiología , Diferenciación CelularRESUMEN
Variability in disease severity caused by a microbial pathogen is impacted by each infection representing a unique combination of host and pathogen genomes. Here, we show that the outcome of invasive Streptococcus pyogenes infection is regulated by an interplay between human STING genotype and bacterial NADase activity. S. pyogenes-derived c-di-AMP diffuses via streptolysin O pores into macrophages where it activates STING and the ensuing type I IFN response. However, the enzymatic activity of the NADase variants expressed by invasive strains suppresses STING-mediated type I IFN production. Analysis of patients with necrotizing S. pyogenes soft tissue infection indicates that a STING genotype associated with reduced c-di-AMP-binding capacity combined with high bacterial NADase activity promotes a 'perfect storm' manifested in poor outcome, whereas proficient and uninhibited STING-mediated type I IFN production correlates with protection against host-detrimental inflammation. These results reveal an immune-regulating function for bacterial NADase and provide insight regarding the host-pathogen genotype interplay underlying invasive infection and interindividual disease variability.
Asunto(s)
NAD+ Nucleosidasa , Streptococcus pyogenes , Humanos , Proteínas Bacterianas/genética , Genotipo , Macrófagos/microbiología , NAD+ Nucleosidasa/genética , Streptococcus pyogenes/genéticaRESUMEN
The Esx-1 (type VII) secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium marinum. However, the molecular events and host-pathogen interactions underlying Esx-1-mediated virulence in vivo remain unclear. Here we address this problem in a non-lethal mouse model of M. marinum infection that allows detailed quantitative analysis of disease progression. M. marinum established local infection in mouse tails, with Esx-1-dependent formation of caseating granulomas similar to those formed in human tuberculosis, and bone deterioration reminiscent of skeletal tuberculosis. Analysis of tails infected with wild type or Esx-1-deficient bacteria showed that Esx-1 enhanced generation of proinflammatory cytokines, including the secreted form of IL-1beta, suggesting that Esx-1 promotes inflammasome activation in vivo. In vitro experiments indicated that Esx-1-dependent inflammasome activation required the host NLRP3 and ASC proteins. Infection of wild type and ASC-deficient mice demonstrated that Esx-1-dependent inflammasome activation exacerbated disease without restricting bacterial growth, indicating a host-detrimental role of this inflammatory pathway in mycobacterial infection. These findings define an immunoregulatory role for Esx-1 in a specific host-pathogen interaction in vivo, and indicate that the Esx-1 secretion system promotes disease and inflammation through its ability to activate the inflammasome.
Asunto(s)
Proteínas Bacterianas/inmunología , Interacciones Huésped-Patógeno/inmunología , Macrófagos/microbiología , Infecciones por Mycobacterium no Tuberculosas/inmunología , Mycobacterium marinum/crecimiento & desarrollo , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Femenino , Inflamación/inmunología , Inflamación/microbiología , Interleucina-1beta/metabolismo , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/inmunología , Mycobacterium marinum/patogenicidad , Proteína con Dominio Pirina 3 de la Familia NLR , Fagosomas/inmunología , Cola (estructura animal)/microbiología , Tuberculoma/inmunología , Tuberculoma/microbiología , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
All living cells require specific mechanisms that target proteins to the cell surface. In eukaryotes, the first part of this process involves recognition in the endoplasmic reticulum of amino-terminal signal sequences and translocation through Sec translocons, whereas subsequent targeting to different surface locations is promoted by internal sorting signals. In bacteria, N-terminal signal sequences promote translocation across the cytoplasmic membrane, which surrounds the entire cell, but some proteins are nevertheless secreted in one part of the cell by poorly understood mechanisms. Here we analyse localized secretion in the Gram-positive pathogen Streptococcus pyogenes, and show that the signal sequences of two surface proteins, M protein and protein F (PrtF), direct secretion to different subcellular regions. The signal sequence of M protein promotes secretion at the division septum, whereas that of PrtF preferentially promotes secretion at the old pole. Our work therefore shows that a signal sequence may contain information that directs the secretion of a protein to one subcellular region, in addition to its classical role in promoting secretion. This finding identifies a new level of complexity in protein translocation and emphasizes the potential of bacterial systems for the analysis of fundamental cell-biological problems.
Asunto(s)
Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Antígenos Bacterianos/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Señales de Clasificación de Proteína/fisiología , Streptococcus pyogenes/citología , Streptococcus pyogenes/metabolismo , Adhesinas Bacterianas/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Portadoras/genética , Señales de Clasificación de Proteína/genética , Estructura Terciaria de Proteína , Streptococcus pyogenes/clasificación , Streptococcus pyogenes/genéticaRESUMEN
The Esx-1 (type VII) secretion system is critical for virulence of both Mycobacterium tuberculosis and Mycobacterium marinum, and is highly conserved between the two species. Despite its importance, there has been no direct visualization of Esx-1 secretion until now. In M. marinum, we show that secretion of Mh3864, a novel Esx-1 substrate that remains partially cell wall-associated after translocation, occurred in polar regions, indicating that Esx-1 secretion takes place in these regions. Analysis of Esx-1 secretion in infected host cells suggested that Esx-1 activity is similarly localized in vivo. A core component of the Esx-1 apparatus, Mh3870, also localized to bacterial poles, showing a preference for new poles with active cell wall peptidoglycan (PGN) synthesis. This work demonstrates that the Esx-1 secretion machine localizes to, and is active at, the bacterial poles. Thus, virulence-related protein secretion is localized in mycobacteria, suggesting new potential therapeutic targets, which are urgently needed.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium marinum/metabolismo , Factores de Virulencia/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Actinas , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Línea Celular , Polaridad Celular , Pared Celular/metabolismo , Ratones , Mutagénesis , Mycobacterium marinum/genética , Peptidoglicano/metabolismo , Factores de Virulencia/genéticaRESUMEN
Like several other intracellular pathogens, Mycobacterium marinum (Mm) escapes from phagosomes into the host cytosol where it can polymerize actin, leading to motility that promotes spread to neighboring cells. However, only approximately 25% of internalized Mm form actin tails, and the fate of the remaining bacteria has been unknown. Here we show that cytosolic access results in a new and intricate host pathogen interaction: host macrophages ubiquitinate Mm, while Mm shed their ubiquitinated cell walls. Phagosomal escape and ubiquitination of Mm occurred rapidly, prior to 3.5 hours post infection; at the same time, ubiquitinated Mm cell wall material mixed with host-derived dense membrane networks appeared in close proximity to cytosolic bacteria, suggesting cell wall shedding and association with remnants of the lysed phagosome. At 24 hours post-infection, Mm that polymerized actin were not ubiquitinated, whereas ubiquitinated Mm were found within LAMP-1-positive vacuoles resembling lysosomes. Though double membranes were observed which sequestered Mm away from the cytosol, targeting of Mm to the LAMP-1-positive vacuoles was independent of classical autophagy, as demonstrated by absence of LC3 association and by Atg5-independence of their formation. Further, ubiquitination and LAMP-1 association did not occur with mutant avirulent Mm lacking ESX-1 (type VII) secretion, which fail to escape the primary phagosome; apart from its function in phagosome escape, ESX-1 was not directly required for Mm ubiquitination in macrophages or in vitro. These data suggest that virulent Mm follow two distinct paths in the cytosol of infected host cells: bacterial ubiquitination is followed by sequestration into lysosome-like organelles via an autophagy-independent pathway, while cell wall shedding may allow escape from this fate to permit continued residence in the cytosol and formation of actin tails.
Asunto(s)
Citosol/microbiología , Lisosomas/microbiología , Proteínas Asociadas a Microtúbulos/metabolismo , Mycobacterium marinum/metabolismo , Fagosomas/microbiología , Actinas/metabolismo , Proteína 5 Relacionada con la Autofagia , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/ultraestructura , Microscopía Fluorescente , Mycobacterium marinum/ultraestructura , Fagosomas/metabolismo , UbiquitinaciónRESUMEN
IgA nephropathy (IgAN) and Henoch-Schönlein purpura (HSP) are diseases characterized by IgA deposits in the kidney and/or skin. Both may arise after upper respiratory tract infections, but the pathogenic mechanisms governing these diseases remain unclear. Patients with IgAN (n = 16) and HSP (n = 17) were included in this study aimed at examining whether IgA-binding M proteins of group A streptococci could be involved. As M proteins vary in sequence, the study focused on the IgA-binding-region (IgA-BR) of three different M proteins: M4, M22, and M60. Renal tissue from IgAN and HSP patients and skin from HSP patients were examined for deposits of streptococcal IgA-BR by immunohistochemistry and electron microscopy using specific antibodies, and a skin sample from a HSP patient was examined by mass spectrometry. IgA-BR deposits were detected in 10/16 IgAN kidneys and 7/13 HSP kidneys. Electron microscopy demonstrated deposits of IgA-BRs in the mesangial matrix and glomerular basement membrane, which colocalized with IgA. Skin samples exhibited IgA-BR deposits in 4/5 biopsies, a result confirmed by mass spectrometry in one patient. IgA-BR deposits were not detected in normal kidney and skin samples. Taken together, these results demonstrate IgA-BR from streptococcal M proteins in patient tissues. IgA-BR, would on gaining access to the circulation, encounter circulatory IgA and form a complex with IgA-Fc that could deposit in tissues and contribute to the pathogenesis of IgAN and HSP.
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Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Glomerulonefritis por IGA/metabolismo , Vasculitis por IgA/metabolismo , Inmunoglobulina A/metabolismo , Precipitinas/metabolismo , Adolescente , Adulto , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Biopsia , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Niño , Preescolar , Femenino , Glomerulonefritis por IGA/patología , Humanos , Vasculitis por IgA/patología , Inmunohistoquímica , Riñón/metabolismo , Riñón/patología , Masculino , Microscopía Electrónica , Datos de Secuencia Molecular , Precipitinas/ultraestructura , Unión Proteica , Piel/metabolismo , Piel/patología , Piel/ultraestructura , Adulto JovenRESUMEN
The M protein of Streptococcus pyogenes is a major bacterial virulence factor that confers resistance to phagocytosis. To analyze how M protein allows evasion of phagocytosis, we used the M22 protein, which has features typical of many M proteins and has two well-characterized regions binding human plasma proteins: the hypervariable NH2-terminal region binds C4b-binding protein (C4BP), which inhibits the classical pathway of complement activation; and an adjacent semivariable region binds IgA-Fc. Characterization of chromosomal S. pyogenes mutants demonstrated that each of the ligand-binding regions contributed to phagocytosis resistance, which could be fully explained as cooperation between the two regions. Deposition of complement on S. pyogenes occurred almost exclusively via the classical pathway, even under nonimmune conditions, but was down-regulated by bacteria-bound C4BP, providing an explanation for the ability of bound C4BP to inhibit phagocytosis. Different opsonizing antisera shared the ability to block binding of both C4BP and IgA, suggesting that the two regions in M22 play important roles also under immune conditions, as targets for protective antibodies. These data indicate that M22 and similar M proteins confer resistance to phagocytosis through ability to bind two components of the human immune system.
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Antígenos Bacterianos , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Portadoras/fisiología , Proteínas Inactivadoras de Complemento/metabolismo , Glicoproteínas , Inmunoglobulina A/metabolismo , Fagocitosis , Streptococcus pyogenes/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Sitios de Unión , Proteínas Portadoras/química , Vía Clásica del Complemento , Humanos , Datos de Secuencia Molecular , ConejosRESUMEN
Specialized secretion systems of pathogenic bacteria commonly transport multiple effectors that act in concert to control and exploit the host cell as a replication-permissive niche. Both the Mycobacterium marinum and the Mycobacterium tuberculosis genomes contain an extended region of difference 1 (extRD1) locus that encodes one such pathway, the early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1) secretion apparatus. ESX-1 is required for virulence and for secretion of the proteins ESAT-6, culture filtrate protein 10 (CFP-10), and EspA. Here, we show that both Rv3881c and its M. marinum homolog, Mh3881c, are secreted proteins, and disruption of RD1 in either organism blocks secretion. We have renamed the Rv3881c/Mh3881c gene espB for ESX-1 substrate protein B. Secretion of M. marinum EspB (EspBM) requires both the Mh3879c and Mh3871 genes within RD1, while CFP-10 secretion is not affected by disruption of Mh3879c. In contrast, disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM. Mutants that fail to secrete only EspBM or only CFP-10 are less attenuated in macrophages than mutants failing to secrete both substrates. EspBM physically interacts with Mh3879c; the M. tuberculosis homolog, EspBT, physically interacts with Rv3879c; and mutants of EspBM that fail to bind Mh3879c fail to be secreted. We also found interaction between Rv3879c and Rv3871, a component of the ESX-1 machine, suggesting a mechanism for the secretion of EspB. The results establish EspB as a substrate of ESX-1 that is required for virulence and growth in macrophages and suggests that the contribution of ESX-1 to virulence may arise from the secretion of multiple independent substrates.
Asunto(s)
Mycobacterium marinum/genética , Mycobacterium marinum/patogenicidad , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Factores de Virulencia/genética , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mutación , Mycobacterium marinum/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMEN
BACKGROUND: Diagnosis of tuberculosis (TB) in human immunodeficiency virus (HIV)-coinfected individuals is challenging. We hypothesized that combinations of inflammatory markers could facilitate identification of active TB in HIV-positive individuals. METHODS: Participants were HIV-positive, treatment-naive adults systematically investigated for TB at Ethiopian health centers. Plasma samples from 130 subjects with TB (HIV+/TB+) and 130 subjects without TB (HIV+/TB-) were tested for concentration of the following markers: CCL5, C-reactive protein (CRP), interleukin (IL)-6, IL12-p70, IL-18, IL-27, interferon-γ-induced protein-10 (IP-10), procalcitonin (PCT), and soluble urokinase-type plasminogen activator receptor (suPAR). Analyzed markers were then assessed, either individually or in combination, with regard to infection status, CD4 cell count, and HIV ribonucleic acid (RNA) levels. RESULTS: The HIV+/TB+ subjects had higher levels of all markers, except IL12p70, compared with HIV+/TB- subjects. The CRP showed the best performance for TB identification (median 27.9 vs 1.8 mg/L for HIV+/TB+ and HIV+/TB-, respectively; area under the curve [AUC]: 0.80). Performance was increased when CRP was combined with suPAR analysis (AUC, 0.83 [0.93 for subjects with CD4 cell count <200 cells/mm3]). Irrespective of TB status, IP-10 concentrations correlated with HIV RNA levels, and both IP-10 and IL-18 were inversely correlated to CD4 cell counts. CONCLUSIONS: Although CRP showed the best single marker discriminatory potential, combining CRP and suPAR analyses increased performance for TB identification.
RESUMEN
Glycosylation defects occur in several human diseases. In IgA nephropathy, IgA1 contains O-glycans that are galactose-deficient and consist mostly of core 1 alpha2,6 sialylated N-acetylgalactosamine, a configuration suspected to prevent beta1,3 galactosylation. We confirmed the same aberrancy in IgA1 secreted by the human DAKIKI B cell line. Biochemical assays indicated CMP-NeuAc:GalNAc-IgA1 alpha2,6-sialyltransferase activity in this cell line. However, a candidate enzyme, ST6-GalNAcI, was not transcribed in DAKIKI cells, B cells isolated from blood, or Epstein-Barr virus (EBV)-immortalized IgA1-producing cells from the blood of IgAN patients and healthy controls. Instead, ST6-GalNAcII transcription was detected at a high level. Expression of the ST6-GalNAcII gene and activity of the CMP-NeuAc:GalNAc-IgA1 alpha2,6-sialyltransferase were higher in IgA1-producing cell lines from IgAN patients than in such cells from healthy controls. These data are the first evidence that human cells that lack ST6-GalNAcI can sialylate core 1 GalNAc-Ser/Thr.