RESUMO
Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.
Assuntos
Autofagia/genética , Metabolismo dos Lipídeos/genética , Lisossomos/fisiologia , Macrófagos/fisiologia , MicroRNAs/metabolismo , Mycobacterium tuberculosis/fisiologia , Tuberculose/genética , Animais , Células Cultivadas , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Lisossomos/microbiologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
To define cellular immunity to the intracellular pathogen Toxoplasma gondii, we performed a genome-wide CRISPR loss-of-function screen to identify genes important for (interferon gamma) IFN-γ-dependent growth restriction. We revealed a role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as necessary for IFN-γ-mediated control of T. gondii in multiple human cell types. RNF213 was also important for control of bacterial (Mycobacterium tuberculosis) and viral (Vesicular Stomatitis Virus) pathogens in human cells. RNF213-mediated ubiquitination of the parasitophorous vacuole membrane (PVM) led to growth restriction of T. gondii in response to IFN-γ. Moreover, overexpression of RNF213 in naive cells also impaired growth of T. gondii. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a previously described noncanonical autophagy pathway. Mutational analysis revealed that the ATPase domain of RNF213 was required for its recruitment to the PVM, while loss of a critical histidine in the RZ finger domain resulted in partial reduction of recruitment to the PVM and complete loss of ubiquitination. Both RNF213 mutants lost the ability to restrict growth of T. gondii, indicating that both recruitment and ubiquitination are required. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity that is both necessary and sufficient for control of intracellular pathogens in human cells.
Assuntos
Toxoplasma , Toxoplasmose , Humanos , Interferon gama/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Toxoplasma/metabolismo , Fatores de Transcrição , Adenosina Trifosfatases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Interleukin-1 (IL-1) signaling is essential for controlling virulent Mycobacterium tuberculosis (Mtb) infection since antagonism of this pathway leads to exacerbated pathology and increased susceptibility. In contrast, the triggering of type I interferon (IFN) signaling is associated with the progression of tuberculosis (TB) disease and linked with negative regulation of IL-1 signaling. However, mice lacking IL-1 signaling can control Mtb infection if infected with an Mtb strain carrying the rifampin-resistance conferring mutation H445Y in its RNA polymerase ß subunit (rpoB-H445Y Mtb). The mechanisms that govern protection in the absence of IL-1 signaling during rpoB-H445Y Mtb infection are unknown. In this study, we show that in the absence of IL-1 signaling, type I IFN signaling controls rpoB-H445Y Mtb replication, lung pathology, and excessive myeloid cell infiltration. Additionally, type I IFN is produced predominantly by monocytes and recruited macrophages and acts on LysM-expressing cells to drive protection through nitric oxide (NO) production to restrict intracellular rpoB-H445Y Mtb. These findings reveal an unexpected protective role for type I IFN signaling in compensating for deficiencies in IL-1 pathways during rpoB-H445Y Mtb infection.
Assuntos
Proteínas de Bactérias , RNA Polimerases Dirigidas por DNA , Interferon Tipo I , Mycobacterium tuberculosis , Rifampina , Transdução de Sinais , Interferon Tipo I/metabolismo , Animais , Camundongos , Rifampina/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mutação , Camundongos Endogâmicos C57BL , Farmacorresistência Bacteriana/genética , Tuberculose/microbiologia , Tuberculose/imunologia , Tuberculose/genética , Camundongos KnockoutRESUMO
Drug tolerance in bacteria is widely believed to be due to metabolic changes that accompany growth arrest. A study in this issue of Cell reveals a drug tolerance mechanism in replicating mycobacteria that is induced by residence in macrophages and depends on drug efflux.
RESUMO
Rapid and accurate quantification of low-abundance protein biomarkers in biofluids can transform the diagnosis of a range of pathologies, including infectious diseases. Here, we harness ultrabright plasmonic fluors as "digital nanolabels" and demonstrate the detection and quantification of subfemtomolar concentrations of human IL-6 and SARS-CoV-2 alpha and variant proteins in clinical nasopharyngeal swab and saliva samples from COVID-19 patients. The resulting digital plasmonic fluor-linked immunosorbent assay (digital p-FLISA) enables detection of SARS-CoV-2 nucleocapsid protein, both in solution and in live virions. Digital p-FLISA outperforms the "gold standard" enzyme-linked immunosorbent assay (ELISA), having a nearly 7000-fold lower limit-of-detection, and outperforms a commercial antigen test, having over 5000-fold improvement in analytical sensitivity. Detection and quantification of very low concentrations of target proteins holds potential for early detection of pathological conditions, treatment monitoring, and personalized medicine.
Assuntos
COVID-19 , Humanos , Ensaio de Imunoadsorção Enzimática , COVID-19/diagnóstico , Fluorimunoensaio , SARS-CoV-2 , Biomarcadores , Sensibilidade e EspecificidadeRESUMO
Streptococcus pneumoniae is a leading cause of pneumonia and invasive disease, particularly, in the elderly. S. pneumoniae lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against S. pneumoniae or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing S. pneumoniae triggered Atg5- and Atg7-dependent recruitment of LC3 to S. pneumoniae-containing vesicles. The association of LC3 with S. pneumoniae-containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, S. pneumoniae was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with S. pneumoniae were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced S. pneumoniae survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control S. pneumoniae infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.
Assuntos
Envelhecimento/imunologia , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fagocitose , Streptococcus pneumoniae/imunologia , Animais , Autofagia , Proteínas de Bactérias/metabolismo , Lipídeos/química , Macrófagos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pneumoniae/ultraestrutura , Estreptolisinas/metabolismoRESUMO
Autophagy can degrade intracellular bacteria, but how this pathway contributes to phagocytosis is unclear. In this issue of Immunity, Bonilla et al. (2013) demonstrate an additional role for autophagy in Mycobacterium tuberculosis internalization by macrophages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia/imunologia , Proteínas de Choque Térmico/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fagocitose/imunologia , Receptores Imunológicos/biossíntese , Receptores Depuradores Classe A/biossíntese , Animais , Proteína 7 Relacionada à Autofagia , Proteína Sequestossoma-1RESUMO
Placenta accreta spectrum includes the full range of abnormal placental attachment to the uterus or other structures, encompassing placenta accreta, placenta increta, placenta percreta, morbidly adherent placenta, and invasive placentation. The incidence of placenta accreta spectrum has increased in recent years, largely driven by increasing rates of cesarean delivery. Prenatal detection of placenta accreta spectrum is primarily made by ultrasound and is important to reduce maternal morbidity associated with the condition. Despite a large body of research on various placenta accreta spectrum ultrasound markers and their screening performance, inconsistencies in the literature persist. In response to the need for standardizing the definitions of placenta accreta spectrum markers and the approach to the ultrasound examination, the Society for Maternal-Fetal Medicine convened a task force with representatives from the American Institute of Ultrasound in Medicine, the American College of Obstetricians and Gynecologists, the American College of Radiology, the International Society of Ultrasound in Obstetrics and Gynecology, the Society for Radiologists in Ultrasound, the American Registry for Diagnostic Medical Sonography, and the Gottesfeld-Hohler Memorial Ultrasound Foundation. The goals of the task force were to assess placenta accreta spectrum sonographic markers on the basis of available data and expert consensus, provide a standardized approach to the prenatal ultrasound evaluation of the uterus and placenta in pregnancies at risk of placenta accreta spectrum, and identify research gaps in the field. This manuscript provides information on the Placenta Accreta Spectrum Task Force process and findings.
Assuntos
Placenta Acreta/diagnóstico por imagem , Ultrassonografia Pré-Natal/métodos , Ultrassonografia Pré-Natal/normas , Cesárea/efeitos adversos , Cicatriz/diagnóstico por imagem , Feminino , Idade Gestacional , Ginecologia , Humanos , Obstetrícia , Placenta/diagnóstico por imagem , Placenta Acreta/epidemiologia , Gravidez , Sensibilidade e Especificidade , Sociedades Médicas , Estados Unidos , Útero/diagnóstico por imagemRESUMO
OBJECTIVE: This study aims to compare completion rates and reproducibility of myocardial performance index (MPI) using conventional spectral Doppler versus tissue Doppler in an unselected high-risk third trimester population. STUDY DESIGN: This was a prospective cross-sectional study of high-risk pregnancies at ≥28 + 0 weeks' gestation. Conventional spectral and tissue Doppler MPI of the left ventricle (LV) and right ventricle (RV) was attempted on all patients. RESULTS: Seventy-nine pregnancies were evaluated. LV tissue Doppler MPI was completed more frequently than LV conventional spectral Doppler MPI (63/79, 79.7% vs. 45/79, 55.7%), p-value <0.01. RV tissue Doppler MPI was completed more frequently than RV conventional spectral Doppler MPI (68/79, 86% vs. 42/79, 53.2%), p-value <0.01. In obese subjects (n = 50) LV tissue Doppler MPI was completed more frequently than LV conventional spectral Doppler MPI (37/50, 74% vs. 26/50, 52%), p-value <0.01. RV tissue Doppler MPI was completed more frequently than RV conventional spectral Doppler MPI (40/50, 80% vs. 25/50, 50%), p-value <0.01. intraclass correlation coefficient for all modalities ranged between 0.73 and 0.93, except for LV conventional spectral Doppler intraobserver variability which was 0.22. CONCLUSION: Tissue Doppler had statistically higher completion rates than conventional spectral Doppler, including the obese subgroup, with evidence of strong reproducibility in the third trimester.
Assuntos
Coração Fetal/diagnóstico por imagem , Coração Fetal/fisiologia , Contração Miocárdica/fisiologia , Terceiro Trimestre da Gravidez , Função Ventricular/fisiologia , Adulto , Estudos Transversais , Ecocardiografia Doppler/métodos , Estudos de Viabilidade , Feminino , Idade Gestacional , Humanos , Gravidez , Estudos Prospectivos , Reprodutibilidade dos Testes , Ultrassonografia Pré-Natal , Adulto JovemRESUMO
Mycobacterium tuberculosis' success as a pathogen comes from its ability to evade degradation by macrophages. Normally macrophages clear microorganisms that activate pathogen-recognition receptors (PRRs) through a lysosomal-trafficking pathway called "LC3-associated phagocytosis" (LAP). Although Mtuberculosis activates numerous PRRs, for reasons that are poorly understood LAP does not substantially contribute to Mtuberculosis control. LAP depends upon reactive oxygen species (ROS) generated by NADPH oxidase, but Mtuberculosis fails to generate a robust oxidative response. Here, we show that CpsA, a LytR-CpsA-Psr (LCP) domain-containing protein, is required for Mtuberculosis to evade killing by NADPH oxidase and LAP. Unlike phagosomes containing wild-type bacilli, phagosomes containing the ΔcpsA mutant recruited NADPH oxidase, produced ROS, associated with LC3, and matured into antibacterial lysosomes. Moreover, CpsA was sufficient to impair NADPH oxidase recruitment to fungal particles that are normally cleared by LAP. Intracellular survival of the ΔcpsA mutant was largely restored in macrophages missing LAP components (Nox2, Rubicon, Beclin, Atg5, Atg7, or Atg16L1) but not in macrophages defective in a related, canonical autophagy pathway (Atg14, Ulk1, or cGAS). The ΔcpsA mutant was highly impaired in vivo, and its growth was partially restored in mice deficient in NADPH oxidase, Atg5, or Atg7, demonstrating that CpsA makes a significant contribution to the resistance of Mtuberculosis to NADPH oxidase and LC3 trafficking in vivo. Overall, our findings reveal an essential role of CpsA in innate immune evasion and suggest that LCP proteins have functions beyond their previously known role in cell-wall metabolism.
Assuntos
Proteínas de Bactérias/metabolismo , Macrófagos/imunologia , Proteínas Associadas aos Microtúbulos/fisiologia , NADPH Oxidase 2/fisiologia , Fagocitose/fisiologia , Tuberculose/prevenção & controle , Animais , Autofagia , Proteínas de Bactérias/genética , Feminino , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Mycobacterium tuberculosis/patogenicidade , Óxido Nítrico Sintase Tipo II/fisiologia , Fagossomos , Espécies Reativas de Oxigênio/metabolismo , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
Mycobacterium tuberculosis (Mtb) encodes five type VII secretion systems (T7SS), designated ESX-1-ESX-5, that are critical for growth and pathogenesis. The best characterized is ESX-1, which profoundly impacts host cell interactions. In contrast, the ESX-3 T7SS is implicated in metal homeostasis, but efforts to define its function have been limited by an inability to recover deletion mutants. We overcame this impediment using medium supplemented with various iron complexes to recover mutants with deletions encompassing select genes within esx-3 or the entire operon. The esx-3 mutants were defective in uptake of siderophore-bound iron and dramatically accumulated cell-associated mycobactin siderophores. Proteomic analyses of culture filtrate revealed that secretion of EsxG and EsxH was codependent and that EsxG-EsxH also facilitated secretion of several members of the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) protein families (named for conserved PE and PPE N-terminal motifs). Substrates that depended on EsxG-EsxH for secretion included PE5, encoded within the esx-3 locus, and the evolutionarily related PE15-PPE20 encoded outside the esx-3 locus. In vivo characterization of the mutants unexpectedly showed that the ESX-3 secretion system plays both iron-dependent and -independent roles in Mtb pathogenesis. PE5-PPE4 was found to be critical for the siderophore-mediated iron-acquisition functions of ESX-3. The importance of this iron-acquisition function was dependent upon host genotype, suggesting a role for ESX-3 secretion in counteracting host defense mechanisms that restrict iron availability. Further, we demonstrate that the ESX-3 T7SS secretes certain effectors that are important for iron uptake while additional secreted effectors modulate virulence in an iron-independent fashion.
Assuntos
Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Aerossóis , Animais , Polaridade Celular/efeitos dos fármacos , Genótipo , Hemina/farmacologia , Proteínas de Homeodomínio/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Ferro/farmacologia , Macrófagos/citologia , Macrófagos/microbiologia , Espectrometria de Massas , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Mutação/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Oxazóis/metabolismo , Fenótipo , Proteômica , Sideróforos/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Virulência/efeitos dos fármacosRESUMO
OBJECTIVE: This study was aimed to evaluate the prevalence of sonographic markers for placenta accreta spectrum (PAS) in pregnancies at low-risk for PAS. STUDY DESIGN: Pregnant women at low-risk for PAS presenting for routine second trimester ultrasound who enrolled in the study were evaluated prospectively for sonographic markers of PAS during two ultrasounds at 18 to 24 and 28 to 34 weeks. Frequencies of PAS markers were compared between the second and third trimester and between those with and without prior cesarean deliveries (CD). RESULTS: Overall, 174 women were included. Several markers were seen frequently in the second trimester: vascular cervical invasion (57%), lacunae (46%), subplacental hypervascularity (37%), and irregularity of the posterior bladder wall (37%). Other markers were seen infrequently or not at all: loss of the retroplacental clear zone, uterovesical interface < 1 mm, bridging vessels, placental bulge or focal exophytic mass. Frequencies of markers did not differ between women with and without prior CD. Lacunae were larger and more numerous in the third trimester. Two or more PAS markers were observed in 98% of second trimester ultrasounds. CONCLUSION: Several PAS sonographic markers occur commonly in low-risk pregnancies. In the absence of risk factors, the independent predictive value of these markers is questionable.
Assuntos
Placenta Acreta/diagnóstico por imagem , Placenta/diagnóstico por imagem , Ultrassonografia Pré-Natal , Adulto , Biomarcadores , Reações Falso-Positivas , Feminino , Humanos , Miométrio/anatomia & histologia , Miométrio/diagnóstico por imagem , Placenta/anatomia & histologia , Placenta/irrigação sanguínea , Gravidez , Segundo Trimestre da Gravidez , Terceiro Trimestre da Gravidez , Prevalência , Estudos Prospectivos , Fatores de Risco , Ultrassonografia Doppler em CoresRESUMO
The success of Mycobacterium tuberculosis (Mtb) as a pathogen rests upon its ability to grow intracellularly in macrophages. Interferon-gamma (IFN-γ) is critical in host defense against Mtb and stimulates macrophage clearance of Mtb through an autophagy pathway. Here we show that the host protein ubiquilin 1 (UBQLN1) promotes IFN-γ-mediated autophagic clearance of Mtb. Ubiquilin family members have previously been shown to recognize proteins that aggregate in neurodegenerative disorders. We find that UBQLN1 can interact with Mtb surface proteins and associates with the bacilli in vitro. In IFN-γ activated macrophages, UBQLN1 co-localizes with Mtb and promotes the anti-mycobacterial activity of IFN-γ. The association of UBQLN1 with Mtb depends upon the secreted bacterial protein, EsxA, which is involved in permeabilizing host phagosomes. In autophagy-deficient macrophages, UBQLN1 accumulates around Mtb, consistent with the idea that it marks bacilli that traffic through the autophagy pathway. Moreover, UBQLN1 promotes ubiquitin, p62, and LC3 accumulation around Mtb, acting independently of the E3 ligase parkin. In summary, we propose a model in which UBQLN1 recognizes Mtb and in turn recruits the autophagy machinery thereby promoting intracellular control of Mtb. Thus, polymorphisms in ubiquilins, which are known to influence susceptibility to neurodegenerative illnesses, might also play a role in host defense against Mtb.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Interferon gama/metabolismo , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia , Camundongos , Fagossomos/metabolismoRESUMO
Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodeficiency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune system such as macrophages and dendritic cells (DCs) and may provide Ca(2+) signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca(2+) signaling strongly impairs many effector functions of bone marrow-derived macrophages and bone marrow-derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/imunologia , Células Dendríticas/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/metabolismo , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Cálcio/metabolismo , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Transporte/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Retículo Endoplasmático/metabolismo , Humanos , Imunidade Inata/imunologia , Inflamassomos/imunologia , Ativação Linfocitária/imunologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína ORAI1 , Fagocitose/imunologia , Imunodeficiência Combinada Severa/genética , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Linfócitos T/imunologiaRESUMO
OBJECTIVES: The 2012 Eunice Kennedy Shriver National Institute of Child Health fetal imaging consensus suggested that fetal anatomy ultrasound in obese pregnancies be performed at 20 to 22 weeks, with follow-up in 2 to 4 weeks if anatomy is incomplete. It was postulated that imaging in early gestation may improve visualization, although no prospective trials had been done to date. METHODS: We performed a prospective longitudinal blinded trial comparing an early gestation ultrasound (13 + 0 to 15 + 6 weeks) with the traditional second-trimester ultrasound for completion of the fetal anatomy survey in obese patients. Inclusion criteria included singleton gestation, body mass index (BMI) more than 30, less than 16 + 0 weeks' gestation, and no karyotype abnormality; exclusion criteria included age younger than 18 years, more than 16 weeks' gestation at time of consent, and BMI less than 30. Participants received a transvaginal and/or transabdominal sonogram for fetal anatomic survey at 13 + 0 to 15 + 6 weeks' gestation (US1). Images from US1 were blinded to physicians and sonographers performing subsequent examinations. All participants underwent the traditional transabdominal sonogram at 18 to 24 weeks (US2). If US2 failed to complete the anatomic survey, a repeat transabdominal sonogram (2-US2) was performed 2 to 4 weeks later. RESULTS: A total of 152 pregnancies met the criteria. Anatomy completion rate was 57.2% for US1 and 62.5% for US2, which was not statistically significant, even when stratified by BMI. Excluding the philtrum, the US1 performed better than US2 for class III obesity (65.5% versus 45.5% [P = .035]). Combination of US1 + US2 yielded a higher completion rate than US2 + 2-US2 (94.1% versus 83.6% [P = .0023]). CONCLUSIONS: In the setting of maternal obesity, the addition of an ultrasound in early gestation may be of highest benefit for patients with class III obesity (BMI > 40 kg/m2 ).
Assuntos
Diagnóstico Precoce , Feto/anatomia & histologia , Feto/diagnóstico por imagem , Idade Gestacional , Aumento da Imagem/métodos , Obesidade/diagnóstico por imagem , Ultrassonografia Pré-Natal/métodos , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gravidez , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Método Simples-Cego , Adulto JovemRESUMO
More people die every year from Mycobacterium tuberculosis infection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogen Mycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In M. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. With M. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated the esx-3 expression in both M. tuberculosis and M. smegmatis, there is a significant difference in the dynamics of this regulation. In M. smegmatis, the esx-3 locus behaved like other iron-regulated genes such as mbtB In M. tuberculosis, both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of M. tuberculosis with macrophages, leading to impaired intracellular M. tuberculosis survival. Our findings detail the regulatory differences of esx-3 in M. tuberculosis and M. smegmatis and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in M. tuberculosis.
Assuntos
Proteínas de Bactérias/metabolismo , Metais/metabolismo , Mycobacterium tuberculosis/fisiologia , Tuberculose/microbiologia , Sistemas de Secreção Tipo II , Animais , Regulação Bacteriana da Expressão Gênica , Loci Gênicos , Ferro/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana , Proteínas Recombinantes , Tuberculose/imunologia , Zinco/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Mycobacterium tuberculosis/patogenicidade , Fosfoproteínas/metabolismo , Tuberculose/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Parede Celular/genética , Parede Celular/imunologia , Parede Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/imunologia , Endossomos/genética , Endossomos/imunologia , Endossomos/metabolismo , Células HEK293 , Humanos , Membranas Intracelulares/imunologia , Membranas Intracelulares/metabolismo , Lisossomos/genética , Lisossomos/imunologia , Lisossomos/metabolismo , Lisossomos/microbiologia , Fusão de Membrana/genética , Fusão de Membrana/imunologia , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Tuberculose/genética , Tuberculose/imunologiaRESUMO
Mycobacterium tuberculosis causes tuberculosis (TB), one of the world's most deadly infections. Lipids play an important role in M. tuberculosis pathogenesis. M. tuberculosis grows intracellularly within lipid-laden macrophages and extracellularly within the cholesterol-rich caseum of necrotic granulomas and pulmonary cavities. Evolved from soil saprophytes that are able to metabolize cholesterol from organic matter in the environment, M. tuberculosis inherited an extensive and highly conserved machinery to metabolize cholesterol. M. tuberculosis uses this machinery to degrade host cholesterol; the products of cholesterol degradation are incorporated into central carbon metabolism and used to generate cell envelope lipids, which play important roles in virulence. The host also modifies cholesterol by enzymatically oxidizing it to a variety of derivatives, collectively called oxysterols, which modulate cholesterol homeostasis and the immune response. Recently, we found that M. tuberculosis converts host cholesterol to an oxidized metabolite, cholestenone, that accumulates in the lungs of individuals with TB. M. tuberculosis encodes cholesterol-modifying enzymes, including a hydroxysteroid dehydrogenase, a putative cholesterol oxidase, and numerous cytochrome P450 monooxygenases. Here, we review what is known about cholesterol and its oxidation products in the pathogenesis of TB. We consider the possibility that the biological function of cholesterol metabolism by M. tuberculosis extends beyond a nutritional role.
RESUMO
Mycobacterium tuberculosis (Mtb) possesses an arsenal of virulence factors to evade host immunity. Previously, we showed that the Mtb protein CpsA, which protects Mtb against the host NADPH oxidase, is required in mice during the first 3 weeks of infection but is thereafter dispensable for full virulence. Using flow cytometry, we find that ΔcpsA Mtb is retained in alveolar macrophages, impaired in recruiting and disseminating into monocyte-derived cells, and more likely to be localized in airway cells than wild-type Mtb. The lungs of ΔcpsA-infected mice also have markedly fewer antigen-specific T cells, indicating a delay in adaptive immunity. Thus, we conclude that CpsA promotes dissemination of Mtb from alveolar macrophages and the airways and generation of an adaptive immune response. Our studies of ΔcpsA Mtb show that a more effective innate immune response against Mtb can be undermined by a corresponding delay in the adaptive immune response.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Camundongos , Animais , Pulmão , Macrófagos Alveolares , Imunidade InataRESUMO
Mycobacterium tuberculosis (Mtb)-infected neutrophils are often found in the airways of patients with active tuberculosis (TB), and excessive recruitment of neutrophils to the lung is linked to increased bacterial burden and aggravated pathology in TB. The basis for the permissiveness of neutrophils for Mtb and the ability to be pathogenic in TB has been elusive. Here, we identified metabolic and functional features of neutrophils that contribute to their permissiveness in Mtb infection. Using single-cell metabolic and transcriptional analyses, we found that neutrophils in the Mtb-infected lung displayed elevated mitochondrial metabolism, which was largely attributed to the induction of activated neutrophils with enhanced metabolic activities. The activated neutrophil subpopulation was also identified in the lung granulomas from Mtb-infected non-human primates. Functionally, activated neutrophils harbored more viable bacteria and displayed enhanced lipid uptake and accumulation. Surprisingly, we found that interferon-γ promoted the activation of lung neutrophils during Mtb infection. Lastly, perturbation of lipid uptake pathways selectively compromised Mtb survival in activated neutrophils. These findings suggest that neutrophil heterogeneity and metabolic diversity are key to their permissiveness for Mtb and that metabolic pathways in neutrophils represent potential host-directed therapeutics in TB.