scholarly journals Sphingomyelin Biosynthesis Is Essential for Phagocytic Signaling during Mycobacterium tuberculosis Host Cell Entry

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Patrick Niekamp ◽  
Gaelen Guzman ◽  
Hans C. Leier ◽  
Ali Rashidfarrokhi ◽  
Veronica Richina ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Alveolar Macrophages ◽  
Biosynthetic Pathway ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Small Gtpases ◽  
Actin Dynamics ◽  
Content Type ◽  
Contact Sites ◽  
Phosphoinositide Turnover

ABSTRACT Phagocytosis by alveolar macrophages is the obligate first step in Mycobacterium tuberculosis (Mtb) infection, yet the mechanism underlying this process is incompletely understood. Here, we show that Mtb invasion relies on an intact sphingolipid biosynthetic pathway. Inhibition or knockout of early sphingolipid biosynthetic enzymes greatly reduces Mtb uptake across multiple phagocytic cell types without affecting other forms of endocytosis. While the phagocytic receptor dectin-1 undergoes normal clustering at the pathogen contact sites, sphingolipid biosynthetic mutant cells fail to segregate the regulatory phosphatase CD45 from the clustered receptors. Blocking sphingolipid production also impairs downstream activation of Rho GTPases, actin dynamics, and phosphoinositide turnover at the nascent phagocytic cup. Moreover, we found that production of sphingomyelin, not glycosphingolipids, is essential for Mtb uptake. Collectively, our data support a critical role of sphingomyelin biosynthesis in an early stage of Mtb infection and provide novel insights into the mechanism underlying phagocytic entry of this pathogen. IMPORTANCE Mycobacterium tuberculosis (Mtb) invades alveolar macrophages through phagocytosis to establish infection and cause disease. The molecular mechanisms underlying Mtb entry are still poorly understood. Here, we report that an intact sphingolipid biosynthetic pathway is essential for the uptake of Mtb by phagocytes. Disrupting sphingolipid production affects the segregation of the regulatory phosphatase CD45 from the nascent phagosome, a critical step in the progression of phagocytosis. We also show that blocking sphingolipid biosynthesis impairs activation of small GTPases and phosphoinositide turnover at the host-pathogen contact sites. Moreover, production of sphingomyelin, not glycosphingolipids, is critical for the phagocytic uptake of Mtb. These data demonstrate a vital role for sphingomyelin biosynthesis in an early step of Mtb infection, defining a potential target for antimycobacterial therapeutics.

scholarly journals The AF-6 Homolog Canoe Acts as a Rap1 Effector During Dorsal Closure of the Drosophila Embryo

Genetics ◽  
2003 ◽  
Vol 165 (1) ◽  
pp. 159-169
Author(s):  
Benjamin Boettner ◽  
Phoebe Harjes ◽  
Satoshi Ishimaru ◽  
Michael Heke ◽  
Hong Qing Fan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Interaction ◽  
Critical Role ◽  
Adherens Junction ◽  
Small Gtpases ◽  
Drosophila Embryo ◽  
Dorsal Closure ◽  
Cell Sheets ◽  
Jnk Pathway

Abstract Rap1 belongs to the highly conserved Ras subfamily of small GTPases. In Drosophila, Rap1 plays a critical role in many different morphogenetic processes, but the molecular mechanisms executing its function are unknown. Here, we demonstrate that Canoe (Cno), the Drosophila homolog of mammalian junctional protein AF-6, acts as an effector of Rap1 in vivo. Cno binds to the activated form of Rap1 in a yeast two-hybrid assay, the two molecules colocalize to the adherens junction, and they display very similar phenotypes in embryonic dorsal closure (DC), a process that relies on the elongation and migration of epithelial cell sheets. Genetic interaction experiments show that Rap1 and Cno act in the same molecular pathway during DC and that the function of both molecules in DC depends on their ability to interact. We further show that Rap1 acts upstream of Cno, but that Rap1, unlike Cno, is not involved in the stimulation of JNK pathway activity, indicating that Cno has both a Rap1-dependent and a Rap1-independent function in the DC process.

scholarly journals Mycobacterium tuberculosis Infection Up-Regulates Sialyl Lewis X Expression in the Lung Epithelium

2021 ◽  
Vol 9 (1) ◽  
pp. 99
Author(s):  
Rita Matos ◽  
Kaori L. Fonseca ◽  
Stefan Mereiter ◽  
Ana Raquel Maceiras ◽  
Joana Gomes ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Biosynthetic Pathway ◽  
Molecular Mechanisms ◽  
Tuberculosis Infection ◽  
Host Susceptibility ◽  
Sialyl Lewis X ◽  
Lung Pathology ◽  
Lung Epithelium ◽  
Lewis X ◽  
Sialyl Lewis

Glycans display increasingly recognized roles in pathological contexts, however, their impact in the host-pathogen interplay in many infectious diseases remains largely unknown. This is the case for tuberculosis (TB), one of the ten most fatal diseases worldwide, caused by infection of the bacteria Mycobacterium tuberculosis. We have recently reported that perturbing the core-2 O-glycans biosynthetic pathway increases the host susceptibility to M. tuberculosis infection, by disrupting the neutrophil homeostasis and enhancing lung pathology. In the present study, we show an increased expression of the sialylated glycan structure Sialyl-Lewis X (SLeX) in the lung epithelium upon M. tuberculosis infection. This increase in SLeX glycan epitope is accompanied by an altered lung tissue transcriptomic signature, with up-regulation of genes codifying enzymes that are involved in the SLeX core-2 O-glycans biosynthetic pathway. This study provides novel insights into previously unappreciated molecular mechanisms involving glycosylation, which modulate the host response to M. tuberculosis infection, possibly contributing to shape TB disease outcome.

scholarly journals ScreeningMycobacterium tuberculosisSecreted Proteins Identifies Mpt64 as a Eukaryotic Membrane-Binding Bacterial Effector

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Chelsea E. Stamm ◽  
Breanna L. Pasko ◽  
Sujittra Chaisavaneeyakorn ◽  
Luis H. Franco ◽  
Vidhya R. Nair ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mycobacterium Tuberculosis ◽  
Unfolded Protein Response ◽  
Molecular Mechanisms ◽  
Membrane Binding ◽  
Host Cells ◽  
Secreted Proteins ◽  
Content Type ◽  
Unfolded Protein ◽  
Protein Response

ABSTRACTMycobacterium tuberculosis(Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.IMPORTANCEAdvances have been made to identify secreted proteins ofMycobacterium tuberculosisduring animal infections. These data, combined with transposon screens identifying genes important forM. tuberculosisvirulence, have generated a vast resource of potentialM. tuberculosisvirulence proteins. However, the function of many of these proteins inM. tuberculosispathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200M. tuberculosissecreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) duringM. tuberculosisinfection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.

scholarly journals Metabolic Switching of Mycobacterium tuberculosis during Hypoxia Is Controlled by the Virulence Regulator PhoP

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Prabhat Ranjan Singh ◽  
Anil Kumar Vijjamarri ◽  
Dibyendu Sarkar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrogen Metabolism ◽  
Electron Acceptor ◽  
Latent Infection ◽  
Critical Role ◽  
Hypoxic Stress ◽  
Content Type ◽  
Metabolic Switching ◽  
Virulence Regulator

ABSTRACT Mycobacterium tuberculosis retains the ability to establish an asymptomatic latent infection. A fundamental question in mycobacterial physiology is to understand the mechanisms involved in hypoxic stress, a critical player in persistence. Here, we show that the virulence regulator PhoP responds to hypoxia, the dormancy signal, and effectively integrates hypoxia with nitrogen metabolism. We also provide evidence to demonstrate that both under nitrogen limiting conditions and during hypoxia, phoP locus controls key genes involved in nitrogen metabolism. Consistently, under hypoxia a ΔphoP strain shows growth attenuation even with surplus nitrogen, the alternate electron acceptor, and complementation of the mutant restores bacterial growth. Together, our observations provide new biological insights into the role of PhoP in integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR. The results have significant implications on the mechanism of intracellular survival and growth of the tubercle bacilli under a hypoxic environment within the phagosome. IMPORTANCE M. tuberculosis retains the unique ability to establish an asymptomatic latent infection. To understand the mechanisms involved in hypoxic stress which play a critical role in persistence, we show that the virulence regulator PhoP is linked to hypoxia, the dormancy signal. In keeping with this, phoP was shown to play a major role in M. tuberculosis growth under hypoxia even in the presence of surplus nitrogen, the alternate electron acceptor. Our results showing regulation of hypoxia-responsive genes provide new biological insights into role of the virulence regulator in metabolic switching by sensing hypoxia and integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR.

scholarly journals Vitamin D Rescues Impaired Mycobacterium tuberculosis-Mediated Tumor Necrosis Factor Release in Macrophages of HIV-Seropositive Individuals through an Enhanced Toll-Like Receptor Signaling PathwayIn Vitro

2012 ◽  
Vol 81 (1) ◽  
pp. 2-10 ◽  
Author(s):  
Asha Anandaiah ◽  
Sanjeev Sinha ◽  
Medhavi Bole ◽  
Surendra K. Sharma ◽  
Narendra Kumar ◽  
...  
Keyword(s):  
Vitamin D ◽  
Mycobacterium Tuberculosis ◽  
Tumor Necrosis Factor ◽  
Alveolar Macrophages ◽  
Tumor Necrosis ◽  
Nuclear Translocation ◽  
Content Type ◽  
Human Macrophages ◽  
Hiv Seropositive ◽  
Necrosis Factor

Mycobacterium tuberculosisdisease represents an enormous global health problem, with exceptionally high morbidity and mortality in HIV-seropositive (HIV+) persons. Alveolar macrophages from HIV+persons demonstrate specific and targeted impairment of critical host cell responses, including impairedM. tuberculosis-mediated tumor necrosis factor (TNF) release and macrophage apoptosis. Vitamin D may promote anti-M. tuberculosisresponses through upregulation of macrophage NO, NADPH oxidase, cathelicidin, and autophagy mechanisms, but whether vitamin D promotes anti-M. tuberculosismechanisms in HIV+macrophages is not known. In the current study, human macrophages exposed toM. tuberculosisdemonstrated robust release of TNF, IκB degradation, and NF-κB nuclear translocation, and these responses were independent of vitamin D pretreatment. In marked contrast, HIV+U1 human macrophages exposed toM. tuberculosisdemonstrated very low TNF release and no significant IκB degradation or NF-κB nuclear translocation, whereas vitamin D pretreatment restored these critical responses. The vitamin D-mediated restored responses were dependent in part on macrophage CD14 expression. Importantly, similar response patterns were observed with clinically relevant human alveolar macrophages from healthy individuals and asymptomatic HIV+persons at high clinical risk ofM. tuberculosisinfection. Taken together with the observation that local bronchoalveolar lavage fluid (BALF) levels of vitamin D are severely deficient in HIV+persons, the data from this study demonstrate that exogenous vitamin D can selectively rescue impaired critical innate immune responsesin vitroin alveolar macrophages from HIV+persons at risk forM. tuberculosisdisease, supporting a potential role for exogenous vitamin D as a therapeutic adjuvant inM. tuberculosisinfection in HIV+persons.

ADF/n-cofilin–dependent actin turnover determines platelet formation and sizing

Blood ◽  
2010 ◽  
Vol 116 (10) ◽  
pp. 1767-1775 ◽  
Author(s):  
Markus Bender ◽  
Anita Eckly ◽  
John H. Hartwig ◽  
Margitta Elvers ◽  
Irina Pleines ◽  
...  
Keyword(s):  
Actin Filament ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Actin Dynamics ◽  
Complex Process ◽  
First Time ◽  
Late Stages ◽  
Platelet Formation

Abstract The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin–mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.

Impact of the FcγII-receptor on quartz uptake and inflammatory response by alveolar macrophages

2008 ◽  
Vol 294 (6) ◽  
pp. L1137-L1148 ◽  
Author(s):  
Petra Haberzettl ◽  
Roel P. F. Schins ◽  
Doris Höhr ◽  
Verena Wilhelmi ◽  
Paul J. A. Borm ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Alveolar Macrophages ◽  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Inhibitory Effect ◽  
Small Gtpases ◽  
Downstream Signaling ◽  
Particle Uptake

The inflammatory response following particle inhalation is described as a key event in the development of lung diseases, e.g., fibrosis and cancer. The essential role of alveolar macrophages (AM) in the pathogenicity of particles through their functions in lung clearance and mediation of inflammation is well known. However, the molecular mechanisms and direct consequences of particle uptake are still unclear. Inhibition of different classic phagocytosis receptors by flow cytometry shows a reduction of the dose-dependent quartz particle (DQ12) uptake in the rat AM cell line NR8383. Thereby the strongest inhibitory effect was observed by blocking the FcγII-receptor (FcγII-R). Fluorescence immunocytochemistry, demonstrating FcγII-R clustering at particle binding sites as well as transmission electron microscopy, visualizing zippering mechanism-like morphological changes, confirmed the role of the FcγII-R in DQ12 phagocytosis. FcγII-R participation in DQ12 uptake was further strengthened by the quartz-induced activation of the Src-kinase Lyn, the phospho-tyrosine kinases Syk (spleen tyrosine kinase) and PI3K (phosphatidylinositol 3-kinase), as shown by Western blotting. Activation of the small GTPases Rac1 and Cdc42, shown by immunoprecipitation, as well as inhibition of tyrosine kinases, GTPases, or Rac1 provided further support for the role of the FcγII-R. Consistent with the uptake results, FcγII-R activation with its specific ligand caused a similar generation of reactive oxygen species and TNF-α release as observed after treatment with DQ12. In conclusion, our results indicate a major role of FcγII-R and its downstream signaling cascade in the phagocytosis of quartz particles in AM as well as in the associated generation and release of inflammatory mediators.

scholarly journals Critical Roles for Lipomannan and Lipoarabinomannan in Cell Wall Integrity of Mycobacteria and Pathogenesis of Tuberculosis

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Takeshi Fukuda ◽  
Takayuki Matsumura ◽  
Manabu Ato ◽  
Maho Hamasaki ◽  
Yukiko Nishiuchi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Critical Role ◽  
Protective Role ◽  
Cell Wall Integrity ◽  
Structural Defects ◽  
Wall Structure ◽  
Content Type ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

ABSTRACTLipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. InMycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to β-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalentMycobacterium tuberculosismutants became more sensitive to β-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis.IMPORTANCETuberculosis (TB) is a global burden, affecting millions of people worldwide.Mycobacterium tuberculosisis a causative agent of TB, and understanding the biology ofM. tuberculosisis essential for tackling this devastating disease. The cell wall ofM. tuberculosisis highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in allMycobacteriumspecies, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a chemotherapeutic target.

scholarly journals MicroRNA-325-3p Facilitates Immune Escape of Mycobacterium tuberculosis through Targeting LNX1 via NEK6 Accumulation to Promote Anti-Apoptotic STAT3 Signaling

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Beibei Fu ◽  
Weiwei Xue ◽  
Haiwei Zhang ◽  
Rui Zhang ◽  
Kelly Feldman ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Mechanisms ◽  
Immune Escape ◽  
Immune Surveillance ◽  
Intracellular Survival ◽  
Drug Resistant ◽  
Therapeutic Approaches ◽  
Content Type ◽  
Bacterial Proliferation ◽  
Stat3 Signaling

ABSTRACT Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that poses threats to the public. M. tuberculosis survives in macrophages by escaping from immune surveillance and clearance, which exacerbates the bacterial proliferation. However, the molecular mechanisms of this immune escape have not yet been fully understood. Using multiple cell and mouse models, we found that microRNA-325-3p (miR-325-3p) is upregulated after M. tuberculosis infection and Mir325-deficient mice show resistance to M. tuberculosis. We demonstrated that miR-325-3p directly targets LNX1, an E3 ubiquitin ligase of NEK6, and that this hampers the proteasomal degradation of NEK6 in macrophages. The abnormal accumulation of NEK6 leads to the activation of STAT3 signaling, thus inhibiting the process of apoptosis and promoting the intracellular survival of M. tuberculosis. Our findings not only reveal a new immune escape pathway of M. tuberculosis but also may provide new insights into the development of therapeutic approaches for drug-resistant TB. IMPORTANCE Intracellular survival of Mycobacterium tuberculosis results in bacterial proliferation and the spread of infection in lungs, consequently deteriorating the conditions of tuberculosis (TB) patients. This research discovers a new immune escape pathway of M. tuberculosis by modulating host miR-325-3p expression, thus leading to the intracellular survival of M. tuberculosis. These findings make a contribution to the understanding of the immune escape of M. tuberculosis, and they provide a theoretical basis for the development of therapeutic approaches for drug-resistant TB.

scholarly journals Modeling Tubercular ESX-1 Secretion Using Mycobacterium marinum

2020 ◽  
Vol 84 (4) ◽  
Author(s):  
Alexandra E. Chirakos ◽  
Ariane Balaram ◽  
William Conrad ◽  
Patricia A. Champion
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Chronic Disease ◽  
Protein Secretion ◽  
Molecular Mechanisms ◽  
Salt Water ◽  
Secretion System ◽  
Mycobacterium Marinum ◽  
Content Type

SUMMARY Pathogenic mycobacteria cause chronic and acute diseases ranging from human tuberculosis (TB) to nontubercular infections. Mycobacterium tuberculosis causes both acute and chronic human tuberculosis. Environmentally acquired nontubercular mycobacteria (NTM) cause chronic disease in humans and animals. Not surprisingly, NTM and M. tuberculosis often use shared molecular mechanisms to survive within the host. The ESX-1 system is a specialized secretion system that is essential for virulence and is functionally conserved between M. tuberculosis and Mycobacterium marinum. M. marinum is an NTM found in both salt water and freshwater that is often used to study mycobacterial virulence. Since the discovery of the secretion system in 2003, the use of both M. tuberculosis and M. marinum has defined the conserved molecular mechanisms underlying protein secretion and the lytic and regulatory activities of the ESX-1 system. Here, we review the trajectory of the field, including key discoveries regarding the ESX-1 system. We highlight the contributions of M. marinum studies and the conserved and unique aspects of the ESX-1 secretion system.

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