RESUMO
The mechanism by which only some individuals infected with Mycobacterium tuberculosis develop necrotic granulomas with progressive disease while others form controlled granulomas that contain the infection remains poorly defined. Mice carrying the sst1-suscepible (sst1S) genotype develop necrotic inflammatory lung lesions, similar to human tuberculosis (TB) granulomas, which are linked to macrophage dysfunction, while their congenic counterpart (B6) mice do not. In this study we report that (a) sst1S macrophages developed aberrant, biphasic responses to TNF characterized by superinduction of stress and type I interferon pathways after prolonged TNF stimulation; (b) the late-stage TNF response was driven via a JNK/IFN-ß/protein kinase R (PKR) circuit; and (c) induced the integrated stress response (ISR) via PKR-mediated eIF2α phosphorylation and the subsequent hyperinduction of ATF3 and ISR-target genes Chac1, Trib3, and Ddit4. The administration of ISRIB, a small-molecule inhibitor of the ISR, blocked the development of necrosis in lung granulomas of M. tuberculosis-infected sst1S mice and concomitantly reduced the bacterial burden. Hence, induction of the ISR and the locked-in state of escalating stress driven by the type I IFN pathway in sst1S macrophages play a causal role in the development of necrosis in TB granulomas. Interruption of the aberrant stress response with inhibitors such as ISRIB may offer novel host-directed therapy strategies.
Assuntos
Granuloma do Sistema Respiratório/imunologia , Pulmão/imunologia , Mycobacterium tuberculosis/imunologia , Estresse Fisiológico/imunologia , Tuberculose Pulmonar/imunologia , Animais , Modelos Animais de Doenças , Granuloma do Sistema Respiratório/microbiologia , Granuloma do Sistema Respiratório/patologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos SCID , Necrose , Tuberculose Pulmonar/patologiaRESUMO
The strains serotyped as M71 from group A Streptococcus are common causes of pharyngeal and skin diseases worldwide. Here we characterize the genome of a unique non-invasive M71 human isolate, NS53. The genome does not contain structural rearrangements or large-scale gene gains/losses, but encodes a full set of non-truncated known virulence factors, thus providing an ideal reference for comparative studies. However, the NS53 genome showed incongruent phenotypic implications from distinct genotypic markers. NS53 is characterized as an emm pattern D and FCT (fibronectin-collagen-T antigen) type-3 strain, typical of skin tropic strains, but is phylogenetically close to emm pattern E strains with preference for both skin and pharyngeal infections. We propose that this incongruence could result from recombination within the emm gene locus, or, alternatively, selection has been against those genetic alterations. Combined with the inability to select for CovS switching, a process is indicated whereby NS53 has been pre-adapted to specific host niches selecting against variations in CovS and many other genes. This may allow the strain to attain successful colonization and long-term survival. A balance between genetic variations and fitness may exist for this bacterium to form a stabilized genome optimized for survival in specific host environments.
Assuntos
Genoma Bacteriano , Genótipo , Fenótipo , Streptococcus pyogenes/genética , Streptococcus pyogenes/fisiologia , Fatores de Virulência/genética , Adaptação Biológica , Variação Genética , Humanos , Filogenia , Sorogrupo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/patogenicidade , VirulênciaRESUMO
Evasion of complement-mediated opsonophagocytosis enables group A Streptococcus pyogenes (GAS) to establish infection. Different strain-dependent mechanisms are employed by the host to accomplish this goal. In general, GAS inhibits the amplification of the complement cascade on its cell surface by facilitating the degradation of C3b, an opsonin, to an inactive product, inactivated C3b (iC3b), in a step catalyzed by factor I (FI) and its cofactor, factor H (FH), with or without the participation of human host plasmin (hPm). GAS recruits FH to its cell surface via FH receptors, which are transcriptionally controlled by the two-component cluster of virulence responder-sensor system. The manner in which FI-FH and hPm function together on GAS cells is unknown. Using GAS strain AP53, which strongly binds host human plasminogen/plasmin (hPg/hPm) directly via an hPg/hPm surface receptor (PAM), we show that both FI-FH and hPm sequentially cleave C3b. Whereas FI-FH proteolytically cleaves C3b into iC3b, PAM-bound hPm catalyzes cleavage of iC3b into multiple smaller peptides. Unlike AP53, GAS strain M23ND weakly binds FH and recruits hPg/hPm to its cell surface indirectly via fibrinogen bound to M-protein, M23. In this case, FH-FI cleaves C3b into iC3b, with negligible degradation of iC3b by hPm that is bound to fibrinogen on the cells. AP53 and M23ND display similar resistance to human neutrophil-mediated phagocytosis, which results in a corresponding high lethality in mice after injection of these cells. These results suggest that GAS utilizes diverse mechanisms to degrade C3b and thus to protect bacterial cells from the complement response of the host.
Assuntos
Complemento C3b/imunologia , Neutrófilos/imunologia , Fagocitose , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Complemento C3b/genética , Humanos , Camundongos , Camundongos Transgênicos , Neutrófilos/patologia , Especificidade da Espécie , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/genéticaRESUMO
The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates â¼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection.
Assuntos
Regulação Bacteriana da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Desoxirribonucleases/metabolismo , Engenharia Genética , Histidina Quinase , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/patogenicidade , Análise de SobrevidaRESUMO
Group A Streptococcus pyogenes (GAS) strain AP53 is a primary isolate from a patient with necrotizing fasciitis. These AP53 cells contain an inactivating mutation in the sensor component of the cluster of virulence (cov) responder (R)/sensor (S) two-component gene regulatory system (covRS), which enhances the virulence of the primary strain, AP53/covR(+)S(-). However, specific mechanisms by which the covRS system regulates the survival of GAS in humans are incomplete. Here, we show a key role for covRS in the regulation of opsonophagocytosis of AP53 by human neutrophils. AP53/covR(+)S(-) cells displayed potent binding of host complement inhibitors of C3 convertase, viz. Factor H (FH) and C4-binding protein (C4BP), which concomitantly led to minimal C3b deposition on AP53 cells, further showing that these plasma protein inhibitors are active on GAS cells. This resulted in weak killing of the bacteria by human neutrophils and a corresponding high death rate of mice after injection of these cells. After targeted allelic alteration of covS(-) to wild-type covS (covS(+)), a dramatic loss of FH and C4BP binding to the AP53/covR(+)S(+) cells was observed. This resulted in elevated C3b deposition on AP53/covR(+)S(+) cells, a high level of opsonophagocytosis by human neutrophils, and a very low death rate of mice infected with AP53/covR(+)S(+). We show that covRS is a critical transcriptional regulator of genes directing AP53 killing by neutrophils and regulates the levels of the receptors for FH and C4BP, which we identify as the products of the fba and enn genes, respectively.
Assuntos
Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neutrófilos/metabolismo , Fagocitose , Proteínas Repressoras/metabolismo , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Complemento C3b/genética , Complemento C3b/metabolismo , Proteína de Ligação ao Complemento C4b/genética , Proteína de Ligação ao Complemento C4b/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Feminino , Frutose-Bifosfato Aldolase , Histidina Quinase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Neutrófilos/microbiologia , Neutrófilos/patologia , Ligação Proteica , Proteínas Repressoras/genética , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Fatores de Virulência/genéticaRESUMO
A skin-tropic invasive group A Streptococcus pyogenes (GAS) strain, AP53, contains a natural inactivating mutation in the covS gene (covS(M)) of the two-component responder (CovR)/sensor (CovS) gene regulatory system. The effects of this mutation on specific GAS virulence determinants have been assessed, with emphasis on expression of the extracellular protease, streptococcal pyrogenic exotoxin B (SpeB), capsular hyaluronic acid, and proteins that allow host plasmin assembly on the bacterial surface, viz. a high affinity plasminogen (Pg)/plasmin receptor, Pg-binding group A streptococcal M protein (PAM), and the human Pg activator streptokinase. To further illuminate mechanisms of the functioning of CovRS in the virulence of AP53, two AP53 isogenic strains were generated, one in which the natural covS(M) gene was mutated to WT-covS (AP53/covS(WT)) and a strain that contained an inactivated covR gene (AP53/ΔcovR). Two additional strains that do not contain PAM, viz. WT-NS931 and NS931/covS(M), were also employed. SpeB was not measurably expressed in strains containing covR(WT)/covS(M), whereas in strains with natural or engineered covR(WT)/covS(WT), SpeB expression was highly up-regulated. Alternatively, capsule synthesis via the hasABC operon was enhanced in strain AP53/covS(M), whereas streptokinase expression was only slightly affected by the covS inactivation. PAM expression was not substantially influenced by the covS mutation, suggesting that covRS had minimal effects on the mga regulon that controls PAM expression. These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase.