37 results found
Thurston TLM, Holden DW, 2023, The Salmonella Typhi SPI-2 injectisome enigma., Microbiology (Reading), Vol: 169
The Salmonella pathogenicity island 2 (SPI-2)-encoded type III secretion system (injectisome) is assembled following uptake of bacteria into vacuoles in mammalian cells. The injectisome translocates virulence proteins (effectors) into infected cells. Numerous studies have established the requirement for a functional SPI-2 injectisome for growth of Salmonella Typhimurium in mouse macrophages, but the results of similar studies involving Salmonella Typhi and human-derived macrophages are not consistent. It is important to clarify the functions of the S. Typhi SPI-2 injectisome, not least because an inactivated SPI-2 injectisome forms the basis for live attenuated S. Typhi vaccines that have undergone extensive trials in humans. Intracellular expression of injectisome genes and effector delivery take longer in the S. Typhi/human macrophage model than for S. Typhimurium and we propose that this could explain the conflicting results. Furthermore, strains of both S. Typhimurium and S. Typhi contain intact genes for several 'core' effectors. In S. Typhimurium these cooperate to regulate the vacuole membrane and contribute to intracellular bacterial replication; similar functions are therefore likely in S. Typhi.
Baig MS, Thurston TLM, Sharma R, et al., 2023, Editorial: Targeting signalling pathways in inflammatory diseases, FRONTIERS IN IMMUNOLOGY, Vol: 14, ISSN: 1664-3224
Thurston TLM, Helaine S, 2023, Editorial overview: Two to tango: The intricate communications between host and bacteria, CURRENT OPINION IN MICROBIOLOGY, Vol: 74, ISSN: 1369-5274
Pillay TD, Hettiarachchi SU, Gan J, et al., 2023, Speaking the host language: how Salmonella effector proteins manipulate the host, Microbiology, Vol: 169, Pages: 1-30, ISSN: 1350-0872
Salmonella injects over 40 virulence factors, termed effectors, into host cells to subvert diverse host cellular processes. Of these 40 Salmonella effectors, at least 25 have been described as mediating eukaryotic-like, biochemical post-translational modifications (PTMs) of host proteins, altering the outcome of infection. The downstream changes mediated by an effector's enzymatic activity range from highly specific to multifunctional, and altogether their combined action impacts the function of an impressive array of host cellular processes, including signal transduction, membrane trafficking, and both innate and adaptive immune responses. Salmonella and related Gram-negative pathogens have been a rich resource for the discovery of unique enzymatic activities, expanding our understanding of host signalling networks, bacterial pathogenesis as well as basic biochemistry. In this review, we provide an up-to-date assessment of host manipulation mediated by the Salmonella type III secretion system injectosome, exploring the cellular effects of diverse effector activities with a particular focus on PTMs and the implications for infection outcomes. We also highlight activities and functions of numerous effectors that remain poorly characterized.
Panagi I, Thurston TLM, 2023, Ready, STAT3, Go! Bacteria in the race for M2 macrophage polarisation, CURRENT OPINION IN MICROBIOLOGY, Vol: 73, ISSN: 1369-5274
Rajpoot S, Kumar A, Gaponenko V, et al., 2023, Dorzolamide suppresses PKCd -TIRAP-p38 MAPK signaling axis to dampen the inflammatory response, FUTURE MEDICINAL CHEMISTRY, ISSN: 1756-8919
Aleksandrowicz A, Kolenda R, Baraniewicz K, et al., 2023, Membrane properties modulation by SanA: implications for xenobiotic resistance in Salmonella Typhimurium, Frontiers in Microbiology, Vol: 14
Introduction: Multidrug resistance in bacteria is a pressing concern, particularly among clinical isolates. Gram-negative bacteria like Salmonella employ various strategies, such as altering membrane properties, to resist treatment. Their two-membrane structure affects susceptibility to antibiotics, whereas specific proteins and the peptidoglycan layer maintain envelope integrity. Disruptions can compromise stability and resistance profile toward xenobiotics. In this study, we investigated the unexplored protein SanA’s role in modifying bacterial membranes, impacting antibiotic resistance, and intracellular replication within host cells. Methods: We generated a sanA deletion mutant and complemented it in trans to assess its biological function. High-throughput phenotypic profiling with Biolog Phenotype microarrays was conducted using 240 xenobiotics. Membrane properties and permeability were analyzed via cytochrome c binding, hexadecane adhesion, nile red, and ethidium bromide uptake assays, respectively. For intracellular replication analysis, primary bone marrow macrophages served as a host cells model. Results: Our findings demonstrated that the absence of sanA increased membrane permeability, hydrophilicity, and positive charge, resulting in enhanced resistance to certain antibiotics that target peptidoglycan synthesis. Furthermore, the sanA deletion mutant demonstrated enhanced replication rates within primary macrophages, highlighting its ability to evade the bactericidal effects of the immune system. Taking together, we provide valuable insights into a poorly known SanA protein, highlighting the complex interplay among bacterial genetics, membrane physiology, and antibiotic resistance, underscoring its significance in understanding Salmonella pathogenicity.
Periselneris J, Turner CT, Ercoli G, et al., 2022, Pneumolysin suppresses the initial macrophage pro-inflammatory response to <i>Streptococcus pneumoniae</i>, IMMUNOLOGY, Vol: 167, Pages: 413-427, ISSN: 0019-2805
Switzer A, Burchell L, Mitsidis P, et al., 2022, A role for the RNA polymerase gene specificity factor sigma(54) in the uniform colony growth of uropathogenic escherichia coli, Journal of Bacteriology, Vol: 204, Pages: 1-16, ISSN: 0021-9193
The canonical function of a bacterial sigma (σ) factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the σ54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the σ54-RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. Different activator ATPases couple diverse environmental cues to the σ54-RNAP to mediate adaptive changes in gene expression. Hence, the genes that rely upon σ54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by σ54, is not yet exhaustive. By comparing the growth patterns of prototypical enteropathogenic, uropathogenic, and nonpathogenic Escherichia coli strains devoid of σ54, we uncovered that the absence of σ54 results in two differently sized colonies that appear at different times specifically in the uropathogenic E. coli (UPEC) strain. Notably, UPEC bacteria devoid of individual activator ATPases of the σ54-RNAP do not phenocopy the σ54 mutant strain. Thus, it seems that σ54’s role as a determinant of uniform colony appearance in UPEC bacteria represents a putative non-canonical function of σ54 in regulating genetic information flow.
Schroeder GN, Pearson JS, Thurston TLM, 2021, Editorial: bacterial effectors as drivers of human disease: models, methods, mechanisms., Frontiers in Cellular and Infection Microbiology, Vol: 11, Pages: 1-4, ISSN: 2235-2988
The Toll-interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP) represents a key intracellular signalling molecule regulating diverse immune responses. Its capacity to function as an adaptor molecule has been widely investigated in relation to Toll-like Receptor (TLR)-mediated innate immune signalling. Since the discovery of TIRAP in 2001, initial studies were mainly focused on its role as an adaptor protein that couples Myeloid differentiation factor 88 (MyD88) with TLRs, to activate MyD88-dependent TLRs signalling. Subsequent studies delineated TIRAP’s role as a transducer of signalling events through its interaction with non-TLR signalling mediators. Indeed, the ability of TIRAP to interact with an array of intracellular signalling mediators suggests its central role in various immune responses. Therefore, continued studies that elucidate the molecular basis of various TIRAP-protein interactions and how they affect the signalling magnitude, should provide key information on the inflammatory disease mechanisms. This review summarizes the TIRAP recruitment to activated receptors and discusses the mechanism of interactions in relation to the signalling that precede acute and chronic inflammatory diseases. Furthermore, we highlighted the significance of TIRAP-TIR domain containing binding sites for several intracellular inflammatory signalling molecules. Collectively, we discuss the importance of the TIR domain in TIRAP as a key interface involved in protein interactions which could hence serve as a therapeutic target to dampen the extent of acute and chronic inflammatory conditions.
Breyer F, Hartlova A, Thurston T, et al., 2021, TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria, The EMBO Journal, Vol: 40, Pages: 1-19, ISSN: 0261-4189
Tumour progression locus 2 (TPL-2) kinase mediates Toll-like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a regulator of V-ATPases, to induce V-ATPase assembly and phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL-2 therefore controls innate immune responses of macrophages to bacteria via V-ATPase induction of phagosome maturation.
Mak H, Thurston T, 2021, Interesting biochemistries in the structure and function of bacterial effectors, Frontiers in Cellular and Infection Microbiology, Vol: 11, ISSN: 2235-2988
Bacterial effector proteins, delivered into host cells by specialized multiprotein secretion systems, are a key mediator of bacterial pathogenesis. Following delivery, they modulate a range of host cellular processes and functions. Strong selective pressures have resulted in bacterial effectors evolving unique structures that can mimic host protein biochemical activity or enable novel and distinct biochemistries. Despite the protein structure-function paradigm, effectors from different bacterial species that share biochemical activities, such as the conjugation of ubiquitin to a substrate, do not necessarily share structural or sequence homology to each other or the eukaryotic proteins that carry out the same function. Furthermore, some bacterial effectors have evolved structural variations to known protein folds which enable different or additional biochemical and physiological functions. Despite the overall low occurrence of intrinsically disordered proteins or regions in prokaryotic proteomes compared to eukaryotes proteomes, bacterial effectors appear to have adopted intrinsically disordered regions that mimic the disordered regions of eukaryotic signaling proteins. In this review, we explore examples of the diverse biochemical properties found in bacterial effectors that enable effector-mediated interference of eukaryotic signaling pathways and ultimately support pathogenesis. Despite challenges in the structural and functional characterisation of effectors, recent progress has been made in understanding the often unusual and fascinating ways in which these virulence factors promote pathogenesis. Nevertheless, continued work is essential to reveal the array of remarkable activities displayed by effectors.
Pham THM, Brewer SM, Thurston T, et al., 2020, Salmonella-driven polarization of granuloma macrophages antagonizes TNF-mediated pathogen restriction during persistent infection, Cell Host and Microbe, Vol: 27, Pages: 54-67.E5, ISSN: 1931-3128
Many intracellular bacteria can establish chronic infection and persist in tissues within granulomas composed of macrophages. Granuloma macrophages exhibit heterogeneous polarization states, or phenotypes, that may be functionally distinct. Here, we elucidate a host-pathogen interaction that controls granuloma macrophage polarization and long-term pathogen persistence during Salmonella Typhimurium ( STm) infection. We show that STm persists within splenic granulomas that are densely populated by CD11b +CD11c +Ly6C + macrophages. STm preferentially persists in granuloma macrophages reprogrammed to an M2 state, in part through the activity of the effector SteE, which contributes to the establishment of persistent infection. We demonstrate that tumor necrosis factor (TNF) signaling limits M2 granuloma macrophage polarization, thereby restricting STm persistence. TNF neutralization shifts granuloma macrophages toward an M2 state and increases bacterial persistence, and these effects are partially dependent on SteE activity. Thus, manipulating granuloma macrophage polarization represents a strategy for intracellular bacteria to overcome host restriction during persistent infection.
Panagi I, Jennings E, Zeng J, et al., 2020, Salmonella effector SteE converts the mammalian serine/threonine kinase GSK3 into a tyrosine kinase to direct macrophage polarization., Cell Host and Microbe, Vol: 27, Pages: 41-53.e6, ISSN: 1931-3128
Many Gram-negative bacterial pathogens antagonize anti-bacterial immunity through translocated effector proteins that inhibit pro-inflammatory signaling. In addition, the intracellular pathogen Salmonella enterica serovar Typhimurium initiates an anti-inflammatory transcriptional response in macrophages through its effector protein SteE. However, the target(s) and molecular mechanism of SteE remain unknown. Here, we demonstrate that SteE converts both the amino acid and substrate specificity of the host pleiotropic serine/threonine kinase GSK3. SteE itself is a substrate of GSK3, and phosphorylation of SteE is required for its activity. Remarkably, phosphorylated SteE then forces GSK3 to phosphorylate the non-canonical substrate signal transducer and activator of transcription 3 (STAT3) on tyrosine-705. This results in STAT3 activation, which along with GSK3 is required for SteE-mediated upregulation of the anti-inflammatory M2 macrophage marker interleukin-4Rα (IL-4Rα). Overall, the conversion of GSK3 to a tyrosine-directed kinase represents a tightly regulated event that enables a bacterial virulence protein to reprogram innate immune signaling and establish an anti-inflammatory environment.
Panagi I, Jennings E, Zeng J, et al., 2019, The Salmonella Effector SteE Converts the Mammalian Serine/Threonine Kinase GSK3 into a Tyrosine Kinase, Publisher: Elsevier BV
Stapels DAC, Hill PWS, Westermann AJ, et al., 2018, Salmonella persisters undermine host immune defenses during antibiotic treatment, Science, Vol: 362, Pages: 1156-1160, ISSN: 0036-8075
Many bacterial infections are hard to treat and tend to relapse, possibly due to the presence of antibiotic-tolerant persisters. In vitro, persister cells appear to be dormant. After uptake of Salmonella species by macrophages, nongrowing persisters also occur, but their physiological state is poorly understood. In this work, we show that Salmonella persisters arising during macrophage infection maintain a metabolically active state. Persisters reprogram macrophages by means of effectors secreted by the Salmonella pathogenicity island 2 type 3 secretion system. These effectors dampened proinflammatory innate immune responses and induced anti-inflammatory macrophage polarization. Such reprogramming allowed nongrowing Salmonella cells to survive for extended periods in their host. Persisters undermining host immune defenses might confer an advantage to the pathogen during relapse once antibiotic pressure is relieved.
Jennings E, Esposito D, Rittinger K, et al., 2018, Structure-function analyses of the bacterial zinc metalloprotease effector protein GtgA uncover key residues required for deactivating NF-B, Journal of Biological Chemistry, Vol: 293, Pages: 15316-15329, ISSN: 0021-9258
The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1′ residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1′ residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence.
Bourgeois JS, Zhou D, Thurston TLM, et al., 2018, Methylthioadenosine suppresses Salmonella virulence, Infection and Immunity, Vol: 86, ISSN: 0019-9567
In order to deploy virulence factors at appropriate times and locations, microbes must rapidly sense and respond to various metabolite signals. Previously we showed transient elevation of the methionine-derived metabolite methylthioadenosine (MTA) in serum during systemic Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Here we explored the functional consequences of increased MTA concentrations on S. Typhimurium virulence. We found that MTA-but not other related metabolites involved in polyamine synthesis and methionine salvage-reduced motility, host cell pyroptosis, and cellular invasion. Further, we developed a genetic model of increased bacterial endogenous MTA production by knocking out the master repressor of the methionine regulon, metJ Like MTA-treated S. Typhimurium, the ΔmetJ mutant displayed reduced motility, host cell pyroptosis, and invasion. These phenotypic effects of MTA correlated with suppression of flagellar and Salmonella pathogenicity island-1 (SPI-1) networks. ΔmetJ S. Typhimurium had reduced virulence in oral and intraperitoneal infection of C57BL/6J mice, independently of the effects of MTA on SPI-1. Finally, ΔmetJ bacteria induced a less severe inflammatory cytokine response in a mouse sepsis model. Together, these data indicate that exposure of S. Typhimurium to MTA or disruption of the bacterial methionine metabolism pathway suppresses S. Typhimurium virulence.
Esposito D, Günster RA, Martino L, et al., 2018, Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3, Journal of Biological Chemistry, Vol: 293, Pages: 5064-5078, ISSN: 0021-9258
The Salmonella secreted effector SseK3 translocates into host cells, targeting innate immune responses including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolysed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DXD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine-GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural re-arrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N-acetyl α-D-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N-glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1, reveal differences in the surface electrostatic charge distribution possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N-glycosyltransferase provides an important step towards a better understanding of this enzyme class and their roles as bacterial effectors.
Jennings E, Thurston TLM, Holden DW, 2017, <i>Salmonella</i> SPI-2 Type III Secretion System Effectors: Molecular Mechanisms And Physiological Consequences, CELL HOST & MICROBE, Vol: 22, Pages: 217-231, ISSN: 1931-3128
Gunster RA, Matthews SA, Holden DW, et al., 2017, SseK1 and SseK3 Type III Secretion System Effectors Inhibit NF-kappa B Signaling and Necroptotic Cell Death in Salmonella-Infected Macrophages (vol 85, e00010-17, 2017), Infection and Immunity, Vol: 85, ISSN: 0019-9567
Gunster R, Matthews SA, Holden DW, et al., 2017, SseK1 and SseK3 T3SS effectors inhibit NF-kB signalling and necroptotic cell death in Salmonella-infected macrophages, Infection and Immunity, Vol: 85, ISSN: 1098-5522
Within host cells such as macrophages, Salmonella enterica translocates virulence (effector) proteins across its vacuolar membrane using the SPI-2 type III secretion system. Previously it has been shown that when expressed ectopically the effectors SseK1 and SseK3 inhibit TNFα-induced NF-κB activation. In this study we show that ectopically expressed SseK1, SseK2 and SseK3 suppressed TNFα-, but not TLR4-, or interleukin-induced NF-κB activation. Inhibition required a DXD motif, which in SseK1 and SseK3 is essential for protein Arginine-N-acetylglucosamine (GlcNAc)-ylation. 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 TNFα-induced cell death during macrophage infection. Despite SseK1 and SseK3 inhibiting TNFα-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.
Boyle KB, Thurston TLM, Randow F, 2016, TBK1 directs WIPI2 against Salmonella, Autophagy, Vol: 12, Pages: 2508-2509, ISSN: 1554-8627
Defense of the mammalian cell cytosol against Salmonella invasion is reliant upon capture of the infiltrating bacteria by macroautophagy (hereafter autophagy), a process controlled by the kinase TBK1. In our recent study we showed that recruitment of TBK1 activity to Salmonella stabilizes the key autophagy regulator WIPI2 on those bacteria, a novel and essential function for TBK1 in the control of the early steps of antibacterial autophagy. Substantial redundancy exists in the precise recruitment mechanism for TBK1 because engagement with any of several Salmonella-associated ‘eat-me’ signals, including host-derived glycans, and K48- and K63-linked ubiquitin chains, suffices to recruit TBK1 functionality. We therefore propose that buffering TBK1 recruitment against potential bacterial interference might be of evolutionary advantage to the host.
Thurston T, Matthews S, Jennings E, et al., 2016, Growth inhibition of cytosolic Salmonella by caspase-1 and caspase-11 precedes host cell death, Nature Communications, Vol: 7, ISSN: 2041-1723
Sensing bacterial products in the cytosol of mammalian cells by NOD-like receptors leads to the activation of caspase-1 inflammasomes, and the production of the pro-inflammatory cytokines interleukin (IL)-18 and IL-1β. In addition, mouse caspase-11 (represented in humans by its orthologs, caspase-4 and caspase-5) detects cytosolic bacterial LPS directly. Activation of caspase-1 and caspase-11 initiates pyroptotic host cell death that releases potentially harmful bacteria from the nutrient-rich host cell cytosol into the extracellular environment. Here we use single cell analysis and time-lapse microscopy to identify a subpopulation of host cells, in which growth of cytosolic Salmonella Typhimurium is inhibited independently or prior to the onset of cell death. The enzymatic activities of caspase-1 and caspase-11 are required for growth inhibition in different cell types. Our results reveal that these proteases have important functions beyond the direct induction of pyroptosis and proinflammatory cytokine secretion in the control of growth and elimination of cytosolic bacteria.
Thurston TL, Boyle KB, Allen M, et al., 2016, Recruitment of TBK1 to cytosol‐invading Salmonella induces WIPI2‐dependent antibacterial autophagy, EMBO Journal, Vol: 35, Pages: 1779-1792, ISSN: 0261-4189
Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti-bacterial autophagy relies on the core autophagy machinery, cargo receptors, and "eat-me" signals such as galectin-8 and ubiquitin that label bacteria as autophagy cargo. Anti-bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti-bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella-associated "eat-me" signals, including host-derived glycans and K48- and K63-linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host.
O'Neill AM, Thurston TL, Holden DW, 2016, Erratum for O'Neill et al., Cytosolic Replication of Group A Streptococcus in Human Macrophages., mBio, Vol: 7, ISSN: 2161-2129
O'Neill A, Thurston T, Holden D, 2016, Cytosolic Replication of Group A Streptococcus in Human Macrophages, mBio, Vol: 7, ISSN: 2161-2129
As key components of innate immune defense, macrophages are essential in controlling bacterial pathogens, includinggroup A Streptococcus (GAS). Despite this, only a limited number of studies have analyzed the recovery of GAS from withinhuman neutrophils and macrophages. Here, we determined the intracellular fate of GAS in human macrophages by using severalquantitative approaches. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealedthat despite GAS-mediated cytotoxicity, replication occurred in viable, propidium iodide-negative macrophages. Whereas themajor virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (SLO) was impairedfor intracellular replication. SLO promoted bacterial escape from the GAS-containing vacuole (GCV) into the macrophagecytosol. Up to half of the cytosolic GAS colocalized with ubiquitin and p62, suggesting that the bacteria were targeted bythe autophagy machinery. Despite this, live imaging of U937 macrophages revealed proficient replication of GAS after GCV rupture,indicating that escape from the GCV is important for growth of GAS in macrophages. Our results reveal that GAS can replicatewithin viable human macrophages, with SLO promoting GCV escape and cytosolic growth, despite the recruitment of autophagyreceptors to bacteria.
Klionsky DJ, Abdelmohsen K, Abe A, et al., 2016, Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)., Autophagy, Vol: 12, Pages: 1-215, ISSN: 1554-8635
, 2016, Erratum., Autophagy, Vol: 12
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