scholarly journals A New ESX-1 Substrate inMycobacterium marinumThat Is Required for Hemolysis but Not Host Cell Lysis

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Rachel E. Bosserman ◽  
Kathleen R. Nicholson ◽  
Matthew M. Champion ◽  
Patricia A. Champion
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Response ◽  
Secretion System ◽  
Mycobacterial Species ◽  
Macrophage Infection ◽  
Content Type ◽  
Family Protein ◽  
Species Specific ◽  
System 1 ◽  
Insight Into

ABSTRACTThe ESX-1 (ESAT-6 system 1) secretion system plays a conserved role in the virulence of diverse mycobacterial pathogens, including the human pathogenMycobacterium tuberculosisandM. marinum, an environmental mycobacterial species. The ESX-1 system promotes the secretion of protein virulence factors to the extracytoplasmic environment. The secretion of these proteins triggers the host response by lysing the phagosome during macrophage infection. Using proteomic analyses of theM. marinumsecretome in the presence and absence of a functional ESX-1 system, we and others have hypothesized that MMAR_2894, a PE family protein, is a potential ESX-1 substrate inM. marinum. We used genetic and quantitative proteomic approaches to determine if MMAR_2894 is secreted by the ESX-1 system, and we defined the requirement ofMMAR_2894for ESX-1-mediated secretion and virulence. We show that MMAR_2894 is secreted by the ESX-1 system inM. marinumand is itself required for the optimal secretion of the known ESX-1 substrates inM. marinum. Moreover, we found that MMAR_2894 was differentially required for hemolysis and cytolysis of macrophages, two lytic activities ascribed to theM. marinumESX-1 system.IMPORTANCEBothMycobacterium tuberculosis, the cause of human tuberculosis (TB), andMycobacterium marinum, a pathogen of ectotherms, use the ESX-1 secretion system to cause disease. There are many established similarities between the ESX-1 systems inM. tuberculosisand inM. marinum. Yet the two bacteria infect different hosts, hinting at species-specific functions of the ESX-1 system. Our findings demonstrate that MMAR_2894 is a PE protein secreted by the ESX-1 system ofM. marinum. We show that MMAR_2894 is required for the optimal secretion of mycobacterial proteins required for disease. Because theMMAR_2894gene is not conserved inM. tuberculosis, our findings demonstrate that MMAR_2894 may contribute to a species-specific function of the ESX-1 system inM. marinum, providing new insight into how theM. marinumandM. tuberculosissystems differ.

scholarly journals Mycobacterium tuberculosisType VII Secretion System Effectors Differentially Impact the ESCRT Endomembrane Damage Response

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Ekansh Mittal ◽  
Michael L. Skowyra ◽  
Grace Uwase ◽  
Emir Tinaztepe ◽  
Alka Mehra ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Recent Work ◽  
Host Response ◽  
Membrane Damage ◽  
Secretion System ◽  
Intracellular Pathogens ◽  
Dependent Manner ◽  
Membrane Repair ◽  
Content Type ◽  
Endosomal Sorting

ABSTRACTIntracellular pathogens have varied strategies to breach the endolysosomal barrier so that they can deliver effectors to the host cytosol, access nutrients, replicate in the cytoplasm, and avoid degradation in the lysosome. In the case ofMycobacterium tuberculosis, the bacterium perforates the phagosomal membrane shortly after being taken up by macrophages. Phagosomal damage depends upon the mycobacterial ESX-1 type VII secretion system (T7SS). Sterile insults, such as silica crystals or membranolytic peptides, can also disrupt phagosomal and endolysosomal membranes. Recent work revealed that the host endosomal sorting complex required for transport (ESCRT) machinery rapidly responds to sterile endolysosomal damage and promotes membrane repair. We hypothesized that ESCRTs might also respond to pathogen-induced phagosomal damage and thatM. tuberculosiscould impair this host response. Indeed, we found that ESCRT-III proteins were recruited toM. tuberculosisphagosomes in anESX-1-dependent manner. We previously demonstrated that the mycobacterial effectors EsxG/TB9.8 and EsxH/TB10.4, both secreted by the ESX-3 T7SS, can inhibit ESCRT-dependent trafficking of receptors to the lysosome. Here, we additionally show that ESCRT-III recruitment to sites of endolysosomal damage is antagonized by EsxG and EsxH, both within the context ofM. tuberculosisinfection and sterile injury. Moreover, EsxG and EsxH themselves respond within minutes to membrane damage in a manner that is independent of calcium and ESCRT-III recruitment. Thus, our study reveals that T7SS effectors and ESCRT participate in a series of measures and countermeasures for control of phagosome integrity.IMPORTANCEMycobacterium tuberculosiscauses tuberculosis, which kills more people than any other infection.M. tuberculosisgrows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease,M. tuberculosisdamages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer ofM. tuberculosiscan inhibit ESCRT, raising the possibility thatM. tuberculosisimpairs this host response. Here, we show that ESCRT is recruited to damagedM. tuberculosisphagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.

scholarly journals EspM Is a Conserved Transcription Factor That Regulates Gene Expression in Response to the ESX-1 System

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kevin G. Sanchez ◽  
Micah J. Ferrell ◽  
Alexandra E. Chirakos ◽  
Kathleen R. Nicholson ◽  
Robert B. Abramovitch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Protein Transport ◽  
Transcriptional Response ◽  
Secretion System ◽  
Pathogenic Species ◽  
Macrophage Infection ◽  
Content Type ◽  
Link Type ◽  
Phagosomal Membrane

ABSTRACT Pathogenic mycobacteria encounter multiple environments during macrophage infection. Temporally, the bacteria are engulfed into the phagosome, lyse the phagosomal membrane, and interact with the cytosol before spreading to another cell. Virulence factors secreted by the mycobacterial ESX-1 (ESAT-6-system-1) secretion system mediate the essential transition from the phagosome to the cytosol. It was recently discovered that the ESX-1 system also regulates mycobacterial gene expression in Mycobacterium marinum (R. E. Bosserman, T. T. Nguyen, K. G. Sanchez, A. E. Chirakos, et al., Proc Natl Acad Sci U S A 114:E10772–E10781, 2017, https://doi.org/10.1073/pnas.1710167114), a nontuberculous mycobacterial pathogen, and in the human-pathogenic species M. tuberculosis (A. M. Abdallah, E. M. Weerdenburg, Q. Guan, R. Ummels, et al., PLoS One 14:e0211003, 2019, https://doi.org/10.1371/journal.pone.0211003). It is not known how the ESX-1 system regulates gene expression. Here, we identify the first transcription factor required for the ESX-1-dependent transcriptional response in pathogenic mycobacteria. We demonstrate that the gene divergently transcribed from the whiB6 gene and adjacent to the ESX-1 locus in mycobacterial pathogens encodes a conserved transcription factor (MMAR_5438, Rv3863, now espM). We prove that EspM from both M. marinum and M. tuberculosis directly and specifically binds the whiB6-espM intergenic region. We show that EspM is required for ESX-1-dependent repression of whiB6 expression and for the regulation of ESX-1-associated gene expression. Finally, we demonstrate that EspM functions to fine-tune ESX-1 activity in M. marinum. Taking the data together, this report extends the esx-1 locus, defines a conserved regulator of the ESX-1 virulence pathway, and begins to elucidate how the ESX-1 system regulates gene expression. IMPORTANCE Mycobacterial pathogens use the ESX-1 system to transport protein substrates that mediate essential interactions with the host during infection. We previously demonstrated that in addition to transporting proteins, the ESX-1 secretion system regulates gene expression. Here, we identify a conserved transcription factor that regulates gene expression in response to the ESX-1 system. We demonstrate that this transcription factor is functionally conserved in M. marinum, a pathogen of ectothermic animals; M. tuberculosis, the human-pathogenic species that causes tuberculosis; and M. smegmatis, a nonpathogenic mycobacterial species. These findings provide the first mechanistic insight into how the ESX-1 system elicits a transcriptional response, a function of this protein transport system that was previously unknown.

scholarly journals A Genetic Screen for Mycobacterium tuberculosis Mutants Defective for Phagosome Maturation Arrest Identifies Components of the ESX-1 Secretion System

2007 ◽  
Vol 75 (6) ◽  
pp. 2668-2678 ◽  
Author(s):  
Jason A. MacGurn ◽  
Jeffery S. Cox
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Mechanisms ◽  
Lipid Synthesis ◽  
Secretion System ◽  
Growth Defect ◽  
Phagosome Maturation ◽  
Macrophage Infection ◽  
Maturation Arrest ◽  
System A ◽  
Early Endosomes

ABSTRACT After phagocytosis, the intracellular pathogen Mycobacterium tuberculosis arrests the progression of the nascent phagosome into a phagolysosome, allowing for replication in a compartment that resembles early endosomes. To better understand the molecular mechanisms that govern phagosome maturation arrest, we performed a visual screen on a set of M. tuberculosis mutants specifically attenuated for growth in mice to identify strains that failed to arrest phagosome maturation and trafficked to late phagosomal compartments. We identified 10 such mutants that could be partitioned into two classes based on the kinetics of trafficking. Importantly, four of these mutants harbor mutations in genes that encode components of the ESX-1 secretion system, a pathway critical for M. tuberculosis virulence. Although ESX-1 is required, the known ESX-1 secreted proteins are dispensable for phagosome maturation arrest, suggesting that a novel effector required for phagosome maturation arrest is secreted by ESX-1. Other mutants identified in this screen had mutations in genes involved in lipid synthesis and secretion and in molybdopterin biosynthesis, as well as in genes with unknown functions. Most of these trafficking mutants exhibited a corresponding growth defect during macrophage infection, but two mutants grew like wild-type M. tuberculosis during macrophage infection. Our results support the emerging consensus that multiple factors from M. tuberculosis, including the ESX-1 secretion system, are involved in modulating trafficking within the host.

scholarly journals Identification and Characterization of Mycobacterium tuberculosis Beijing Genotype Strain SBH163, Isolated in Sabah, Malaysia

2020 ◽  
Vol 9 (2) ◽  
Author(s):  
Jaeyres Jani ◽  
Siti Fatimah Abu Bakar ◽  
Zainal Arifin Mustapha ◽  
Chin Kai Ling ◽  
Roddy Teo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequence ◽  
Beijing Genotype ◽  
Molecular Characteristics ◽  
Whole Genome ◽  
Content Type ◽  
Identification And Characterization ◽  
Insight Into ◽  
Malaysian Borneo

This is a report on the whole-genome sequence of Mycobacterium tuberculosis strain SBH163, which was isolated from a patient in the Malaysian Borneo state of Sabah. This report provides insight into the molecular characteristics of an M. tuberculosis Beijing genotype strain related to strains from Russia and South Africa.

scholarly journals Transcriptional Profiling of Mycobacterium tuberculosis Exposed toIn VitroLysosomal Stress

2016 ◽  
Vol 84 (9) ◽  
pp. 2505-2523 ◽  
Author(s):  
Wenwei Lin ◽  
Paola Florez de Sessions ◽  
Garrett Hor Keong Teoh ◽  
Ahmad Naim Nazri Mohamed ◽  
Yuan O. Zhu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Metabolic Reprogramming ◽  
Human Macrophage ◽  
Host Immune System ◽  
Activated Macrophages ◽  
Intrinsic Resistance ◽  
Macrophage Infection ◽  
Content Type

Increasing experimental evidence supports the idea thatMycobacterium tuberculosishas evolved strategies to survive within lysosomes of activated macrophages. To further our knowledge ofM. tuberculosisresponse to the hostile lysosomal environment, we profiled the global transcriptional activity ofM. tuberculosiswhen exposed to the lysosomal soluble fraction (SF) prepared from activated macrophages. Transcriptome sequencing (RNA-seq) analysis was performed using various incubation conditions, ranging from noninhibitory to cidal based on the mycobacterial replication or killing profile. Under inhibitory conditions that led to the absence of apparent mycobacterial replication,M. tuberculosisexpressed a unique transcriptome with modulation of genes involved in general stress response, metabolic reprogramming, respiration, oxidative stress, dormancy response, and virulence. The transcription pattern also indicates characteristic cell wall remodeling with the possible outcomes of increased infectivity, intrinsic resistance to antibiotics, and subversion of the host immune system. Among the lysosome-specific responses, we identified theglgE-mediated 1,4 α-glucan synthesis pathway and a defined group of VapBC toxin/anti-toxin systems, both of which represent toxicity mechanisms that potentially can be exploited for killing intracellular mycobacteria. A meta-analysis including previously reported transcriptomic studies in macrophage infection andin vitrostress models was conducted to identify overlapping and nonoverlapping pathways. Finally, the Tap efflux pump-encoding geneRv1258cwas selected for validation. AnM. tuberculosis ΔRv1258cmutant was constructed and displayed increased susceptibility to killing by lysosomal SF and the antimicrobial peptide LL-37, as well as attenuated survival in primary murine macrophages and human macrophage cell line THP-1.

scholarly journals SseK1 and SseK3 Type III Secretion System Effectors Inhibit NF-κB Signaling and Necroptotic Cell Death in Salmonella-Infected Macrophages

2017 ◽  
Vol 85 (3) ◽  
Author(s):  
Regina A. Günster ◽  
Sophie A. Matthews ◽  
David W. Holden ◽  
Teresa L. M. Thurston
Keyword(s):  
Cell Death ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Macrophage Infection ◽  
Content Type ◽  
Type Iii ◽  
Tnf Α ◽  
Arginine Residues

ABSTRACT Within host cells such as macrophages, Salmonella enterica translocates virulence (effector) proteins across its vacuolar membrane via the SPI-2 type III secretion system. Previously, it was shown that when expressed ectopically, the effectors SseK1 and SseK3 inhibit tumor necrosis factor alpha (TNF-α)-induced NF-κB activation. In this study, we show that ectopically expressed SseK1, SseK2, and SseK3 suppress TNF-α-induced, but not Toll-like receptor 4- or interleukin-induced, NF-κB activation. Inhibition required a DXD motif in SseK1 and SseK3, which is essential for the transfer of N-acetylglucosamine to arginine residues (arginine-GlcNAcylation). During macrophage infection, SseK1 and SseK3 inhibited NF-κB activity in an additive manner. SseK3-mediated inhibition of NF-κB activation did not require the only known host-binding partner of this effector, the E3-ubiquitin ligase TRIM32. SseK proteins also inhibited TNF-α-induced cell death during macrophage infection. Despite SseK1 and SseK3 inhibiting TNF-α-induced apoptosis upon ectopic expression in HeLa cells, the percentage of infected macrophages undergoing apoptosis was SseK independent. Instead, SseK proteins inhibited necroptotic cell death during macrophage infection. SseK1 and SseK3 caused GlcNAcylation of different proteins in infected macrophages, suggesting that these effectors have distinct substrate specificities. Indeed, SseK1 caused the GlcNAcylation of the death domain-containing proteins FADD and TRADD, whereas SseK3 expression resulted in weak GlcNAcylation of TRADD but not FADD. Additional, as-yet-unidentified substrates are likely to explain the additive phenotype of a Salmonella strain lacking both SseK1 and SseK3.

scholarly journals Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria

2016 ◽  
Vol 198 (23) ◽  
pp. 3220-3232 ◽  
Author(s):  
Oren Mayer ◽  
Paras Jain ◽  
Torin R. Weisbrod ◽  
Daniel Biro ◽  
Libby Ho ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nontuberculous Mycobacteria ◽  
Mycobacterium Abscessus ◽  
Striking Difference ◽  
Mycobacterial Species ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Specific Phage ◽  
Phage Production ◽  
Specific Restriction

ABSTRACTMycobacteriophage DS6A is unique among the more than 8,000 isolated mycobacteriophages due to its ability to form plaques exclusively on mycobacteria belonging to theMycobacterium tuberculosiscomplex (MTBC). Speculation surrounding this specificity has led to unsupported assertions in published studies and patents that nontuberculous mycobacteria (NTM) are wholly resistant to DS6A infection. In this study, we identified two independent nonessential regions in the DS6A genome and replaced them with an mVenus-expressing plasmid to generate fluorescent reporter phages Φ2GFP12 and Φ2GFP13. We show that even though DS6A is able to form plaques only on MTBC bacteria, infection of various NTM results in mVenus expression in transduced cells. The efficiency of DS6A in delivering DNA varied between NTM species. Additionally, we saw a striking difference in the efficiency of DNA delivery between the closely related members of theMycobacterium abscessuscomplex,M. abscessusandMycobacterium massiliense. We also demonstrated that TM4 and DS6A, two phages that do not form plaques onM. massiliense, differ in their ability to deliver DNA, suggesting that there is a phage-specific restriction between mycobacterial species. Phylogenetic analysis reveals that the DS6A genome has a characteristically mosaic structure but provided few insights into the basis for the specificity for MTBC hosts. This study demonstrates that the inability of the MTBC-specific phage DS6A to form plaques on NTM is more complex than previously thought. Moreover, the DS6A-derived fluorophages provide important new tools for the study of mycobacterial biology.IMPORTANCEThe coevolution of bacteria and their infecting phages involves a constant arms race for bacteria to prevent phage infection and phage to overcome these preventions. Although a diverse array of phage defense systems is well characterized in bacteria, very few phage restriction systems are known in mycobacteria. The DS6A mycobacteriophage is unique in the mycobacterial world in that it forms plaques only on members of theMycobacterium tuberculosiscomplex. However, the novel DS6A reporter phages developed in this work demonstrate that DS6A can infect nontuberculous mycobacteria at various efficiencies. By comparing the abilities of DS6A and another phage, TM4, to infect and form plaques on various mycobacterial species, we can begin to discern new phage restriction systems employed within the genus.

scholarly journals Biphasic Dynamics of Macrophage Immunometabolism during Mycobacterium tuberculosis Infection

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Lanbo Shi ◽  
Qingkui Jiang ◽  
Yuri Bushkin ◽  
Selvakumar Subbian ◽  
Sanjay Tyagi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Immune Cells ◽  
Aerobic Glycolysis ◽  
Defense Responses ◽  
Bioactive Lipids ◽  
Macrophage Infection ◽  
Mycobacterium Tuberculosis Infection ◽  
Metabolic Switch ◽  
Content Type ◽  
M1 Polarization

ABSTRACT Macrophages are the primary targets of Mycobacterium tuberculosis infection; the early events of macrophage interaction with M. tuberculosis define subsequent progression and outcome of infection. M. tuberculosis can alter the innate immunity of macrophages, resulting in suboptimal Th1 immunity, which contributes to the survival, persistence, and eventual dissemination of the pathogen. Recent advances in immunometabolism illuminate the intimate link between the metabolic states of immune cells and their specific functions. In this review, we describe the little-studied biphasic metabolic dynamics of the macrophage response during progression of infection by M. tuberculosis and discuss their relevance to macrophage immunity and M. tuberculosis pathogenicity. The early phase of macrophage infection, which is marked by M1 polarization, is accompanied by a metabolic switch from mitochondrial oxidative phosphorylation to hypoxia-inducible factor 1 alpha (HIF-1α)-mediated aerobic glycolysis (also known as the Warburg effect in cancer cells), as well as by an upregulation of pathways involving oxidative and antioxidative defense responses, arginine metabolism, and synthesis of bioactive lipids. These early metabolic changes are followed by a late adaptation/resolution phase in which macrophages transition from glycolysis to mitochondrial oxidative metabolism, with a consequent dampening of macrophage proinflammatory and antimicrobial responses. Importantly, the identification of upregulated metabolic pathways and/or metabolic regulatory mechanisms with immunomodulatory functions during M1 polarization has revealed novel mechanisms of M. tuberculosis pathogenicity. These advances can lead to the development of novel host-directed therapies to facilitate bacterial clearance in tuberculosis by targeting the metabolic state of immune cells.

scholarly journals A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism

2016 ◽  
Vol 85 (2) ◽  
Author(s):  
Emily A. Williams ◽  
Felix Mba Medie ◽  
Rachel E. Bosserman ◽  
Benjamin K. Johnson ◽  
Cristal Reyna ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Nonsense Mutation ◽  
Gene Disruption ◽  
Cell Envelope ◽  
Mycobacterium Marinum ◽  
Nonsense Suppression ◽  
Whole Genome ◽  
Content Type ◽  
System 1 ◽  
Insight Into

ABSTRACT Mycobacterial pathogens use the ESAT-6 system 1 (Esx-1) exporter to promote virulence. Previously, we used gene disruption and complementation to conclude that the MMAR_0039 gene in Mycobacterium marinum is required to promote Esx-1 export. Here we applied molecular genetics, proteomics, and whole-genome sequencing to demonstrate that the MMAR_0039 gene is not required for Esx-1 secretion or virulence. These findings suggest that we initially observed an indirect mechanism of genetic complementation. We identified a spontaneous nonsense mutation in a known Esx-1-associated gene which causes a loss of Esx-1 activity. We show that the Esx-1 function was restored by nonsense suppression. Moreover, we identified a polar mutation in the ppsC gene which reduced cellular impermeability but did not impact cytotoxicity in macrophages. Our studies reveal insight into Esx-1 export, nonsense suppression, and cell envelope lipid biogenesis.

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