RESUMO
Previously, we showed that Legionella pneumophila secretes rhizoferrin, a polycarboxylate siderophore that promotes bacterial growth in iron-deplete media and the murine lung. Yet, past studies failed to identify a role for the rhizoferrin biosynthetic gene (lbtA) in L. pneumophila infection of host cells, suggesting the siderophore's importance was solely linked to extracellular survival. To test the possibility that rhizoferrin's relevance to intracellular infection was missed due to functional redundancy with the ferrous iron transport (FeoB) pathway, we characterized a new mutant lacking both lbtA and feoB. This mutant was highly impaired for growth on bacteriological media that were only modestly depleted of iron, confirming that rhizoferrin-mediated ferric iron uptake and FeoB-mediated ferrous iron uptake are critical for iron acquisition. The lbtA feoB mutant, but not its lbtA-containing complement, was also highly defective for biofilm formation on plastic surfaces, demonstrating a new role for the L. pneumophila siderophore in extracellular survival. Finally, the lbtA feoB mutant, but not its complement containing lbtA, proved to be greatly impaired for growth in Acanthamoeba castellanii, Vermamoeba vermiformis, and human U937 cell macrophages, revealing that rhizoferrin does promote intracellular infection by L. pneumophila. Moreover, the application of purified rhizoferrin triggered cytokine production from the U937 cells. Rhizoferrin-associated genes were fully conserved across the many sequenced strains of L. pneumophila examined but were variably present among strains from the other species of Legionella. Outside of Legionella, the closest match to the L. pneumophila rhizoferrin genes was in Aquicella siphonis, another facultative intracellular parasite of amoebae.
Assuntos
Amoeba , Legionella pneumophila , Animais , Camundongos , Humanos , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Sideróforos/metabolismo , Amoeba/metabolismo , Células U937 , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Macrófagos/microbiologia , BiofilmesRESUMO
Cytokines made by macrophages play a critical role in determining the course of Legionella pneumophila infection. Prior murine-based modeling indicated that this cytokine response is initiated upon recognition of L. pneumophila by a subset of Toll-like receptors, namely TLR2, TLR5, and TLR9. Through the use of shRNA/siRNA knockdowns and subsequently CRISPR/Cas9 knockouts (KO), we determined that TRIF, an adaptor downstream of endosomal TLR3 and TLR4, is required for full cytokine secretion by human primary and cell-line macrophages. By characterizing a further set of TLR KO's in human U937 cells, we discerned that, contrary to the viewpoint garnered from murine-based studies, TLR3 and TLR4 (along with TLR2 and TLR5) are in fact vital to the macrophage response in the early stages of L. pneumophila infection. This conclusion was bolstered by showing that i) chemical inhibitors of TLR3 and TLR4 dampen the cytokine output of primary human macrophages and ii) transfection of TLR3 and TLR4 into HEK cells conferred an ability to sense L. pneumophila. TLR3- and TLR4-dependent cytokines promoted migration of human HL-60 neutrophils across an epithelial layer, pointing to the biological importance for the newfound signaling pathway. The response of U937 cells to L. pneumophila LPS was dependent upon TLR4, a further contradiction to murine-based studies, which had concluded that TLR2 is the receptor for Legionella LPS. Given the role of TLR3 in sensing nucleic acid (i.e., dsRNA), we utilized newly-made KO U937 cells to document that DNA-sensing by cGAS-STING and DNA-PK are also needed for the response of human macrophages to L. pneumophila. Given the lack of attention given them in the bacterial field, C-type lectin receptors were similarly examined; but, they were not required. Overall, this study arguably represents the most extensive, single-characterization of Legionella-recognition receptors within human macrophages.
Assuntos
Doença dos Legionários/imunologia , Macrófagos/imunologia , Moléculas com Motivos Associados a Patógenos/imunologia , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Proteínas de Bactérias/imunologia , Humanos , Legionella pneumophila/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Camundongos , Moléculas com Motivos Associados a Patógenos/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires' disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface, but this is not dependent on ChiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. We also show that the ChiA C-terminal chitinase domain has novel Zn2+-dependent peptidase activity against mammalian mucin-like proteins, namely MUC5AC and the C1-esterase inhibitor, and that ChiA promotes bacterial penetration of mucin gels. Our findings suggest that ChiA can facilitate passage of L. pneumophila through the alveolar mucosa, can modulate the host complement system and that ChiA may be a promising target for vaccine development.
Assuntos
Quitinases/metabolismo , Legionella pneumophila/metabolismo , Acetilglucosamina/metabolismo , Proteínas de Bactérias/metabolismo , Quitina/metabolismo , Quitinases/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Doença dos Legionários/metabolismo , Metais , Mucina-1/metabolismo , Mucinas/metabolismo , Proteólise , Relação Estrutura-Atividade , Fatores de Virulência/metabolismoRESUMO
The epithelium that lines the epididymal duct establishes the optimal milieu in which spermatozoa mature, acquire motility, and are stored. This finely tuned environment also protects antigenic sperm against pathogens and autoimmunity, which are potential causes of transient or permanent infertility. The epididymal epithelium is pseudostratified and contains basal cells (BCs) that are located beneath other epithelial cells. Previous studies showed that in the mouse epididymis, BCs possess macrophage-like characteristics. However, we previously identified a dense population of cells belonging to the mononuclear phagocyte (MP) system (comprised of macrophages and dendritic cells) in the basal compartment of the mouse epididymis and showed that a subset of MPs express the macrophage marker F4/80. In the present study, we evaluate the distribution of BCs and MPs in the epididymis of transgenic CD11c-EYFP mice, in which EYFP is expressed exclusively in MPs, using antibodies against the BC marker keratin 5 (KRT5) and the macrophage marker F4/80. Immunofluorescence labeling for laminin, a basement membrane marker, showed that BCs and most MPs are located in the basal region of the epithelium. Confocal microscopy showed that in the initial segment, both BCs and MPs project intraepithelial extensions and establish a very intricate network. Flow cytometry experiments demonstrated that epididymal MPs and BCs are phenotypically distinct. BCs do not express F4/80, and MPs do not express KRT5. Therefore, despite their proximity and some morphological similarities with peritubular macrophages and dendritic cells, BCs do not belong to the MP system.
Assuntos
Células Dendríticas/imunologia , Epididimo/imunologia , Epitélio/imunologia , Macrófagos/imunologia , Animais , Antígenos de Diferenciação/imunologia , Antígenos CD11/imunologia , Epididimo/citologia , Células Epiteliais/imunologia , Citometria de Fluxo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis after MI. Here, we report that defective efferocytosis by macrophages after experimental MI led to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling, and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA-Seq and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophage-produced VEGFC was directly effective at suppressing proinflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines.
Assuntos
Traumatismos Cardíacos , Infarto do Miocárdio , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Traumatismos Cardíacos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Fagocitose , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
Legionella pneumophila, a Gram-negative bacterium and the causative agent of Legionnaires' disease, exports over 300 effector proteins/virulence factors, through its type II (T2SS) and type IV secretion systems (T4SS). One such T2SS virulence factor, ChiA, not only functions as a chitinase, but also as a novel mucinase, which we believe aids ChiA-dependent virulence during lung infection. Previously published protocols manipulated wild-type L. pneumophila strain 130b and its chiA mutant to express plasmid-encoded GFP. Similarly, earlier studies demonstrated that wheat germ agglutinin (WGA) can be fluorescently labeled and can bind to mucins. In the current protocol, GFP-labeled bacteria were incubated with type II and type III porcine stomach mucins, which were then labeled with TexasRed-tagged WGA and analyzed by flow-cytometry to measure the binding of bacteria to mucins in the presence or absence of endogenous ChiA. In addition, we analysed binding of purified ChiA to type II and type III porcine stomach mucins. This protocol couples both bacterial and direct protein binding to mucins and is the first to measure Gram-negative bacterial binding to mucins using WGA and flow-cytometric analysis. Graphic abstract: Strategy for assessing bacterial and protein binding to mucins.
RESUMO
Lung adenocarcinomas (LAC) of smokers and never-smokers differ from one another in epidemiology, and clinical and molecular characteristics. The pathogenetic differences between these tumors are potential biomarkers and therapeutic targets. Mouse carcinogenesis models of human LAC are proven tools applicable for the identification of these molecular changes. Allelic loss frequency on human chromosome 6q is higher in LAC of smokers compared with never smokers. We analyzed the orthologous region on mouse chromosome 10 and found this region similarly was a more frequent site of allelic loss in carcinogen-induced LAC compared with non-induced or spontaneous LAC. We then conducted high resolution quantitative PCR-based deletion mapping of this region and identified the FoxO3a gene as the focus of bi-allelic or homozygous deletion (HD). HDs were detected in 5 out of 15 (33.3%) LAC cell lines and in 6 out of 75 (8%) carcinogen-induced primary LAC. FoxO3a was exclusively affected by HD in 7 of the samples examined, as loss of both alleles did extend to the nearest flanking genes of FoxO3a. Deletion of FoxO3a, either by HD or subclonal loss was detected in 38 out of 75 (50.7%) of carcinogen-induced LAC in contrast to only 1 out of 10 (10%) of LAC of untreated mice. Several of the samples also were subjected to direct sequence analysis; however, no intragenic mutations were detected. These results implicate FoxO3a as a selective target of deletion in mouse LAC. Significant association with carcinogenic induction suggests that deletion of FoxO3a contributes to the development of carcinogen-initiated tumors.
Assuntos
Adenocarcinoma/genética , Fatores de Transcrição Forkhead/genética , Neoplasias Pulmonares/genética , Aflatoxina B1/toxicidade , Animais , Carcinógenos/toxicidade , Modelos Animais de Doenças , Proteína Forkhead Box O3 , Deleção de Genes , Perda de Heterozigosidade , Camundongos , Nitrosaminas/toxicidade , Reação em Cadeia da Polimerase , Fumar/efeitos adversos , Uretana/análogos & derivados , Uretana/toxicidadeRESUMO
Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)(+) macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE(-/-) mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.
Assuntos
Hematopoese Extramedular/imunologia , Células-Tronco Hematopoéticas/imunologia , Macrófagos/imunologia , Mielopoese/imunologia , Baço/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/imunologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Hematopoese Extramedular/genética , Células-Tronco Hematopoéticas/patologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Mielopoese/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Nanopartículas , Placa Aterosclerótica/genética , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Interferência de RNA , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Baço/patologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Heterozygous mutations in the UBIAD1 gene cause Schnyder corneal dystrophy characterized by abnormal cholesterol and phospholipid deposits in the cornea. Ubiad1 protein was recently identified as Golgi prenyltransferase responsible for biosynthesis of vitamin K2 and CoQ10, a key protein in the mitochondrial electron transport chain. Our study shows that silencing UBIAD1 in cultured human hepatocellular carcinoma cells causes dramatic morphological changes and cholesterol storage in the mitochondria, emphasizing an important role of UBIAD1 in mitochondrial function.