A Novel Class of Lipoprotein Lipase-Sensitive Molecules Mediates Toll-Like Receptor 2 Activation by Porphyromonas gingivalis

ABSTRACTInfection by the chronic periodontitis-associated pathogenPorphyromonas gingivalisactivates a Toll-like receptor 2 (TLR2) response that triggers inflammation in the host but also promotes bacterial persistence. Our aim was to define ligands on the surfaces of intactP. gingivaliscells that determine its ability to activate TLR2. Molecules previously reported as TLR2 agonists include lipopolysaccharide (LPS), fimbriae, the lipoprotein PG1828, and phosphoceramides. We demonstrate that these molecules do not comprise the major factors responsible for stimulating TLR2 by whole bacterial cells. First,P. gingivalismutants devoid of the reported protein agonists, PG1828 and fimbriae, activate TLR2 as strongly as the wild type. Second, two-phase extraction of whole bacteria resulted in a preponderance of TLR2 agonist activity partitioning to the hydrophilic phase, demonstrating that phosphoceramides are not a major TLR2 ligand. Third, analysis of LPS revealed that TLR2 activation is independent of lipid A structural variants. Instead, activation of TLR2 and TLR2/TLR1 by LPS is in large part due to copurifying molecules that are sensitive to the action of the enzyme lipoprotein lipase. Strikingly, intactP. gingivalisbacterial cells treated with lipoprotein lipase were attenuated in their ability to activate TLR2. We propose that a novel class of molecules comprised by lipoproteins constitutes the major determinants that confer toP. gingivalisthe ability to stimulate TLR2 signaling.

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Identification of PGN_1123 as the Gene Encoding Lipid A Deacylase, an Enzyme Required for Toll-Like Receptor 4 Evasion, inPorphyromonas gingivalis

Journal of Bacteriology ◽

10.1128/jb.00683-18 ◽

2019 ◽

Vol 201 (11) ◽

Cited By ~ 3

Author(s):

Sumita Jain ◽

Ana M. Chang ◽

Manjot Singh ◽

Jeffrey S. McLean ◽

Stephen R. Coats ◽

...

Keyword(s):

Immune Response ◽

Porphyromonas Gingivalis ◽

Innate Immune Response ◽

Lipid A ◽

Innate Immune ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Gene Encoding ◽

Content Type ◽

Chronic Inflammatory Condition

ABSTRACTRemoval of one acyl chain from bacterial lipid A by deacylase activity is a mechanism used by many pathogenic bacteria to evade the host's Toll-like receptor 4 (TLR4)-mediated innate immune response. InPorphyromonas gingivalis, a periodontal pathogen, lipid A deacylase activity converts a majority of the initially synthesized penta-acylated lipid A, a TLR4 agonist, to tetra-acylated structures, which effectively evade TLR4 sensing by being either inert or antagonistic at TLR4. In this paper, we report successful identification of the gene that encodes theP. gingivalislipid A deacylase enzyme. This gene, PGN_1123 inP. gingivalis33277, is highly conserved withinP. gingivalis, and putative orthologs are phylogenetically restricted to theBacteroidetesphylum. Lipid A of ΔPGN_1123 mutants is penta-acylated and devoid of tetra-acylated structures, and the mutant strain provokes a strong TLR4-mediated proinflammatory response, in contrast to the negligible response elicited by wild-typeP. gingivalis. Heterologous expression of PGN_1123 inBacteroides thetaiotaomicronpromoted lipid A deacylation, confirming that PGN_1123 encodes the lipid A deacylase enzyme.IMPORTANCEPeriodontitis, commonly referred to as gum disease, is a chronic inflammatory condition that affects a large proportion of the population.Porphyromonas gingivalisis a bacterium closely associated with periodontitis, although how and if it is a cause for the disease are not known. It has a formidable capacity to dampen the host's innate immune response, enabling its persistence in diseased sites and triggering microbial dysbiosis in animal models of infection.P. gingivalisis particularly adept at evading the host's TLR4-mediated innate immune response by modifying the structure of lipid A, the TLR4 ligand. In this paper, we report identification of the gene encoding lipid A deacylase, a key enzyme that modifies lipid A to TLR4-evasive structures.

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Toll-Like Receptor 2 Ligand Pretreatment Attenuates Retinal Microglial Inflammatory Response but Enhances Phagocytic Activity toward Staphylococcus aureus

Infection and Immunity ◽

10.1128/iai.00149-12 ◽

2012 ◽

Vol 80 (6) ◽

pp. 2076-2088 ◽

Cited By ~ 41

Author(s):

Travis Kochan ◽

Anuj Singla ◽

Joaquin Tosi ◽

Ashok Kumar

Keyword(s):

Staphylococcus Aureus ◽

Inflammatory Response ◽

Phagocytic Activity ◽

Toll Like Receptor ◽

Innate Response ◽

Content Type ◽

Proinflammatory Mediators ◽

Retinal Microglia ◽

Tlr2 Ligand ◽

Toll Like Receptor 2

ABSTRACTStaphylococcus aureusis a leading cause of severe endophthalmitis, which often results in vision loss in some patients. Previously, we showed that Toll-like receptor 2 (TLR2) ligand pretreatment prevented the development of staphylococcal endophthalmitis in mice and suggested that microglia might be involved in this protective effect (Kumar A, Singh CN, Glybina IV, Mahmoud TH, Yu FS. J. Infect. Dis. 201:255–263, 2010). The aim of the present study was to understand how microglial innate response is modulated by TLR2 ligand pretreatment. Here, we demonstrate thatS. aureusinfection increased the CD11b+CD45+microglial/macrophage population in the C57BL/6 mouse retina. Using cultured primary retinal microglia and a murine microglial cell line (BV-2), we found that these cells express TLR2 and that its expression is increased upon stimulation with bacteria or an exclusive TLR2 ligand, Pam3Cys. Furthermore, challenge of primary retinal microglia withS. aureusand its cell wall components peptidoglycan (PGN) and lipoteichoic acid (LTA) induced the secretion of proinflammatory mediators (tumor necrosis factor alpha [TNF-α] and MIP-2). This innate response was attenuated by a function-blocking anti-TLR2 antibody or by small interfering RNA (siRNA) knockdown of TLR2. In order to assess the modulation of the innate response, microglia were pretreated with a low dose (0.1 or 1 μg/ml) of Pam3Cys and then challenged with liveS. aureus. Our data showed thatS. aureus-induced production of proinflammatory mediators is dramatically reduced in pretreated microglia. Importantly, microglia pretreated with the TLR2 agonist phagocytosed significantly more bacteria than unstimulated cells. Together, our data suggest that TLR2 plays an important role in retinal microglial innate response toS. aureus, and its sensitization inhibits inflammatory response while enhancing phagocytic activity.

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Impact of Toll-Like Receptor 2 Deficiency on Immune Responses to Mycobacterial Antigens

Infection and Immunity ◽

10.1128/iai.05724-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4649-4656 ◽

Cited By ~ 5

Author(s):

Muhammad J. Rahman ◽

Olga D. Chuquimia ◽

Dagbjort H. Petursdottir ◽

Natalia Periolo ◽

Mahavir Singh ◽

...

Keyword(s):

Immune Responses ◽

Cellular Responses ◽

Mouse Strains ◽

Mycobacterial Antigen ◽

Toll Like Receptor ◽

Partial Explanation ◽

Content Type ◽

Tlr2 Ligand ◽

Toll Like Receptor 2

ABSTRACTIn the present study, we addressed the question of whether Toll-like receptor 2 (TLR2)-mediated innate immunity can contribute to the development of acquired immune responses. We immunized TLR2−/−and wild-type (WT) mice three times subcutaneously with the mycobacterial antigen (Ag19kDa) (a TLR2 ligand) or Ag85A (not a TLR2 ligand). One week after the last immunization, sera and spleens were collected. To evaluate cellular responses, we measured gamma interferon (IFN-γ) afterin vitrorestimulation of spleen cells with antigen alone or antigen-pulsed bone marrow-derived macrophages (BMMAg) or pulmonary macrophages (PuMAg). Antibody responses were comparable in the two mouse strains, but we observed differences in the cellular responses. Recall responses to Ag85A were similar in the two strains, but responses to Ag19kDa given alone or presented by BMM or PuM were lower in TLR2−/−than in WT mice. The largest differences in cellular responses were observed when Ag19kDa was presented by PuM. To understand this, we analyzed phenotypic and functional differences between BMM and PuM upon stimulation with various ligands. Generally, PuM had a lower response to the TLR2 ligand Pam3Cys-Ser-(Lys)4trihydrochloride and to anti-CD40 than BMM, as measured by cytokine secretion and upregulation of costimulatory molecules. This might provide a partial explanation for the lower capacity of PuM when pulsed with Ag19kDa, also a TLR2 ligand. Altogether, our results revealed weaknesses in the T cell and antigen-presenting cell (APC) compartments of the Ag19kDa-immunized TLR2−/−mice but indicated that specific immune responses could be generated in the absence of TLR2 regardless of the characteristics of the antigen used.

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The Importance of Being an Antagonist as Well as Persistent

Journal of Bacteriology ◽

10.1128/jb.00146-19 ◽

2019 ◽

Vol 201 (11) ◽

Cited By ~ 1

Author(s):

Ann Progulske-Fox

Keyword(s):

Porphyromonas Gingivalis ◽

Lipid A ◽

Current Work ◽

Phenotypic Characterization ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Content Type ◽

Link Type ◽

Tlr4 Agonist

ABSTRACT The current work by Jain et al. (S. Jain, A. M. Chang, M. Singh, J. S. McLean, et al., J Bacteriol 201:e00683-18, 2019, https://doi.org/10.1128/JB.00683-18) reports the cloning of the lipid A deacylase gene of Porphyromonas gingivalis and the phenotypic characterization of the enzyme. Attempts to clone the gene and thus provide proof of the existence of this enzyme had gone on for 2 decades. The enzyme is central to the bacterium’s ability to modify and tailor the structure of its lipid A, changing a lipid A that is a moderate Toll-like receptor 4 (TLR4) agonist to an antagonist or silencer and thereby potentially changing the course of infection.

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Glycine Lipids of Porphyromonas gingivalis Are Agonists for Toll-Like Receptor 2

Infection and Immunity ◽

10.1128/iai.00877-19 ◽

2020 ◽

Vol 88 (4) ◽

Cited By ~ 2

Author(s):

Frank C. Nichols ◽

Robert B. Clark ◽

Yaling Liu ◽

Anthony A. Provatas ◽

Christopher J. Dietz ◽

...

Keyword(s):

Liquid Chromatography ◽

Porphyromonas Gingivalis ◽

Multiple Reaction Monitoring ◽

Lipid Classes ◽

Periodontal Pathogen ◽

Broth Culture ◽

Toll Like Receptor ◽

Content Type ◽

Periodontal Tissues ◽

Toll Like Receptor 2

ABSTRACT The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis. The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids.

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Copper-Induced Expression of a Transmissible Lipoprotein Intramolecular Transacylase Alters Lipoprotein Acylation and the Toll-Like Receptor 2 Response to Listeria monocytogenes

Journal of Bacteriology ◽

10.1128/jb.00195-19 ◽

2019 ◽

Vol 201 (13) ◽

Cited By ~ 2

Author(s):

Krista M. Armbruster ◽

Gloria Komazin ◽

Timothy C. Meredith

Keyword(s):

Listeria Monocytogenes ◽

Bacterial Species ◽

Acyl Chain ◽

Constitutive Expression ◽

Innate Immune ◽

Copper Resistance ◽

Toll Like Receptor ◽

General Role ◽

Content Type ◽

Toll Like Receptor 2

ABSTRACT Bacterial lipoproteins are globular proteins anchored to the extracytoplasmic surfaces of cell membranes through lipidation at a conserved N-terminal cysteine. Lipoproteins contribute to an array of important cellular functions for bacteria, as well as being a focal point for innate immune system recognition through binding to Toll-like receptor 2 (TLR2) heterodimer complexes. Although lipoproteins are conserved among nearly all classes of bacteria, the presence and type of α-amino-linked acyl chain are highly variable and even strain specific within a given bacterial species. The reason for lyso-lipoprotein formation and N-acylation variability in general is presently not fully understood. In Enterococcus faecalis, lipoproteins are anchored by an N-acyl-S-monoacyl-glyceryl cysteine (lyso form) moiety installed by a chromosomally encoded lipoprotein intramolecular transacylase (Lit). Here, we describe a mobile genetic element common to environmental isolates of Listeria monocytogenes and Enterococcus spp. encoding a functional Lit ortholog (Lit2) that is cotranscribed with several well-established copper resistance determinants. Expression of Lit2 is tightly regulated, and induction by copper converts lipoproteins from the diacylglycerol-modified form characteristic of L. monocytogenes type strains to the α-amino-modified lyso form observed in E. faecalis. Conversion to the lyso form through either copper addition to media or constitutive expression of lit2 decreases TLR2 recognition when using an activated NF-κB secreted embryonic alkaline phosphatase reporter assay. While lyso formation significantly diminishes TLR2 recognition, lyso-modified lipoprotein is still predominantly recognized by the TLR2/TLR6 heterodimer. IMPORTANCE The induction of lipoprotein N-terminal remodeling in response to environmental copper in Gram-positive bacteria suggests a more general role in bacterial cell envelope physiology. N-terminal modification by lyso formation, in particular, simultaneously modulates the TLR2 response in direct comparison to their diacylglycerol-modified precursors. Thus, use of copper as a frontline antimicrobial control agent and ensuing selection raises the potential of diminished innate immune sensing and enhanced bacterial virulence.

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Mycobacterium tuberculosisLipoprotein and Lipoglycan Binding to Toll-Like Receptor 2 Correlates with Agonist Activity and Functional Outcomes

Infection and Immunity ◽

10.1128/iai.00450-18 ◽

2018 ◽

Vol 86 (10) ◽

Cited By ~ 10

Author(s):

Supriya Shukla ◽

Edward T. Richardson ◽

Michael G. Drage ◽

W. Henry Boom ◽

Clifford V. Harding

Keyword(s):

Immune Responses ◽

Agonist Activity ◽

Toll Like Receptor ◽

Necrosis Factor Alpha ◽

Tlr2 Agonist ◽

Content Type ◽

Host Immune Responses ◽

Extracellular Signal ◽

Toll Like Receptor 2 ◽

Tlr2 Signaling

ABSTRACTMycobacterium tuberculosiscauses persistent infection due to its ability to evade host immune responses.M. tuberculosisinduces Toll-like receptor 2 (TLR2) signaling, which influences immune responses toM. tuberculosis. TLR2 agonists expressed byM. tuberculosisinclude lipoproteins (e.g., LprG), the glycolipid phosphatidylinositol mannoside 6 (PIM6), and the lipoglycan lipomannan (LM). AnotherM. tuberculosislipoglycan, mannose-capped lipoarabinomannan (ManLAM), lacks TLR2 agonist activity. In contrast, PILAM, fromMycobacterum smegmatis, does have TLR2 agonist activity. Our understanding of howM. tuberculosislipoproteins and lipoglycans interact with TLR2 is limited, and binding of these molecules to TLR2 has not been measured directly. Here, we directly measuredM. tuberculosislipoprotein and lipoglycan binding to TLR2 and its partner receptor, TLR1. LprG, LAM, and LM were all found to bind to TLR2 in the absence of TLR1, but not to TLR1 in the absence of TLR2. Trimolecular interactions were revealed by binding of TLR2-LprG or TLR2-PIM6 complexes to TLR1, whereas binding of TLR2 to TLR1 was not detected in the absence of the lipoprotein or glycolipid. ManLAM exhibited low affinity for TLR2 in comparison to PILAM, LM, and LprG, which correlated with reduced ability of ManLAM to induce TLR2-mediated extracellular-signal-regulated kinase (ERK) activation and tumor necrosis factor alpha (TNF-α) secretion in macrophages. We provide the first direct affinity measurement and kinetic analysis ofM. tuberculosislipoprotein and lipoglycan binding to TLR2. Our results demonstrate that binding affinity correlates with the functional ability of agonists to induce TLR2 signaling.

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Porphyromonas gingivalis lipopolysaccharide promotes T- helper 17 cell differentiation from human CD4+ naïve T cells via toll-like receptor-2 in vitro

Archives of Oral Biology ◽

10.1016/j.archoralbio.2019.104483 ◽

2019 ◽

Vol 107 ◽

pp. 104483 ◽

Cited By ~ 3

Author(s):

Liping Zhang ◽

Li Gao ◽

Chenrong Xu ◽

Xiting Li ◽

Panpan Wang ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

Porphyromonas Gingivalis ◽

Toll Like Receptor ◽

T Helper ◽

T Helper 17 ◽

T Helper 17 Cell ◽

Porphyromonas Gingivalis Lipopolysaccharide ◽

Toll Like Receptor 2

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Staphylococcus aureus Releases Proinflammatory Membrane Vesicles To Resist Antimicrobial Fatty Acids

mSphere ◽

10.1128/msphere.00804-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Arnaud Kengmo Tchoupa ◽

Andreas Peschel

Keyword(s):

Fatty Acids ◽

Staphylococcus Aureus ◽

Immune Responses ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Innate Immune ◽

Toll Like Receptor ◽

Content Type ◽

Healthy Humans ◽

Toll Like Receptor 2

ABSTRACT Staphylococcus aureus is a major pathogen, which colonizes one in three otherwise healthy humans. This significant spread of S. aureus is largely due to its ability to circumvent innate immune responses, including antimicrobial fatty acids (AFAs) on the skin and in nasal secretions. In response to AFAs, S. aureus swiftly induces resistance mechanisms, which have yet to be completely elucidated. Here, we identify membrane vesicle (MV) release as a resistance strategy used by S. aureus to sequester host-specific AFAs. MVs protect S. aureus against a wide array of AFAs. Strikingly, beside MV production, S. aureus modulates MV composition upon exposure to AFAs. MVs purified from bacteria grown in the presence of linoleic acid display a distinct protein content and are enriched in lipoproteins, which strongly activate Toll-like receptor 2 (TLR2). Cumulatively, our findings reveal the protective capacities of MVs against AFAs, which are counteracted by an increased TLR2-mediated innate immune response. IMPORTANCE The nares of one in three humans are colonized by Staphylococcus aureus. In these environments, and arguably on all mucosal surfaces, bacteria encounter fatty acids with antimicrobial properties. Our study uncovers that S. aureus releases membrane vesicles (MVs) that act as decoys to protect the bacterium against antimicrobial fatty acids (AFAs). The AFA-neutralizing effects of MVs were neither strain specific nor restricted to one particular AFA. Hence, MVs may represent “public goods” playing an overlooked role in shaping bacterial communities in AFA-rich environments such as the skin and nose. Intriguingly, in addition to MV biogenesis, S. aureus modulates MV composition in response to exposure to AFAs, including an increased release of lipoproteins. These MVs strongly stimulate the innate immunity via Toll-like receptor 2 (TLR2). TLR2-mediated inflammation, which helps to fight infections, may exacerbate inflammatory disorders like atopic dermatitis. Our study highlights intricate immune responses preventing infections from colonizing bacteria.

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