Excess exogenous pyruvate inhibits lactate dehydrogenase activity in live cells in an MCT1-dependent manner

Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-¹³C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging. However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECARs) as a measure of lactate production, and hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various heterotetrameric isoforms comprising LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates in cellulo. Of importance, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and monocarboxylate transporter-1–dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-¹³C]pyruvate magnetic resonance spectroscopic imaging and stable isotope tracer experiments.     

Pyruvate Carboxylase Plays a Crucial Role in Carbon Metabolism of Extra- and Intracellularly Replicating Listeria monocytogenes

The human pathogen L. monocytogenes is a facultatively intracellular bacterium that survives and replicates in the cytosol of many mammalian cells. The listerial metabolism, especially under intracellular conditions, is still poorly understood. Recent studies analyzed the carbon metabolism of L. monocytogenes by the ¹³C isotopologue perturbation method in a defined minimal medium containing [U-¹³C₆]glucose. It was shown that these bacteria produce oxaloacetate mainly by carboxylation of pyruvate due to an incomplete tricarboxylic acid cycle. Here, we report that a pycA insertion mutant defective in pyruvate carboxylase (PYC) still grows, albeit at a reduced rate, in brain heart infusion (BHI) medium but is unable to multiply in a defined minimal medium with glucose or glycerol as a carbon source. Aspartate and glutamate of the pycA mutant, in contrast to the wild-type strain, remain unlabeled when [U-¹³C₆]glucose is added to BHI, indicating that the PYC-catalyzed carboxylation of pyruvate is the predominant reaction leading to oxaloacetate in L. monocytogenes. The pycA mutant is also unable to replicate in mammalian cells and exhibits high virulence attenuation in the mouse sepsis model.Listeria monocytogenes is a human pathogen that can cause systemic infections, especially in immunocompromised people, with symptoms such as septicemia, (encephalo)meningitis, placentitis, and stillbirth. These Gram-positive bacteria are able to enter the cytosol of many mammalian cells after being taken up via normal or induced phagocytosis by professional phagocytes, mainly macrophages and dendritic cells, and nonphagocytic cells, such as epithelial cells, fibroblasts, and endothelial cells (1, 8, 13). While the virulence genes and their regulation (4, 21), as well as the encoded virulence factors (20, 22), necessary for the various steps of the intracellular replication cycle of L. monocytogenes have been extensively studied in the past few decades, there is still little information concerning the metabolic capacities and the metabolic adaptation processes (10) that enable these bacteria to efficiently replicate in the cytosol of their host cells.The information on listerial metabolism obtained from the genome sequence (7) suggests that these heterotrophic bacteria are capable of utilizing a variety of carbohydrates as carbon sources, since a large number of genes encoding phosphoenolpyruvate (PEP)-phosphotransferase systems (PTS) were identified. Furthermore, all genes encoding the enzymes necessary for the catabolism of glycerol and dihydroxyacetone are present in the L. monocytogenes genome (7, 11). This genomic information is in accord with data from previous and more recent physiological studies (11, 17, 24).Most genes encoding the enzymes for the major catabolic pathways, namely, glycolysis, the citrate cycle, and the pentose phosphate cycle, are present in L. monocytogenes. The citrate cycle, however, seems to be interrupted, since the genes encoding 2-oxoglutarate dehydrogenase have not been identified in all L. monocytogenes strains sequenced so far, including EGD-e (7), or in Listeria innocua strain Clip 11262. This enzymatic gap in the citrate cycle was recently confirmed by ¹³C isotopologue perturbation studies using uniformly ¹³C-labeled glucose. The results showed that two C₄ amino acids, aspartate and threonine, are generated in L. monocytogenes, predominantly from building blocks comprising one or three ¹³C atoms, respectively (2). These data suggested that oxaloacetate, the direct or indirect precursor of both amino acids, is generated by an anaplerotic reaction assembling precursors composed of one and three carbon atoms, respectively. This can be afforded by the carboxylation of pyruvate catalyzed by the ATP-dependent pyruvate carboxylase (PYC) encoded by pycA.The genes encoding the enzymes for most anabolic pathways, but not those for the biosynthesis of thiamine (vitamin B₁), riboflavin (vitamin B₂), biotin, and thiotic acid (lipoate), were also identified in L. monocytogenes. However, these bacteria grow efficiently in a mineral salt medium containing a suitable carbon source (e.g., glucose) and these four cofactors only when the amino acids cysteine, methionine, glutamine, arginine, valine, isoleucine, and leucine are also added (17). According to Tsai and Hodgson, strain 10403S requires only methionine and cysteine (24). The missing sulfate reductase in L. monocytogenes readily explains the strict requirement for cysteine/methionine as a sulfur source, while the missing nitrate reductase may be the reason for the stimulatory growth effect of glutamine and arginine as reduced nitrogen sources. However, the need for the three branched-chain amino acids (BCAA) valine, isoleucine, and leucine for efficient growth of L. monocytogenes EGD-e (references 17 and 24 and our unpublished results) is less obvious, since L. monocytogenes has the complete genetic set for synthesis of the BCAA, indicating the role of metabolic intermediates in listerial growth.The central precursor for the biosynthesis of the BCAA is pyruvate, which is channeled into their biosynthetic pathways either directly, via oxidative decarboxylation of pyruvate to acetyl-coenzyme A (CoA), or more indirectly via oxaloacetate (generated by pyruvate carboxylation) to aspartate and further to threonine. Thus, biosynthesis of the BCAA may compete with the PYC-mediated generation of oxaloacetate for the common substrate pyruvate. These data suggest that PYC may play an important role in the carbon metabolism of L. monocytogenes.To more precisely determine the significance of this anaplerotic enzyme for listerial metabolism and pathogenesis, we generated a mutant of L. monocytogenes EGD-e defective in pycA, the gene encoding PYC, and studied the replication of this mutant under different extra- and intracellular growth conditions. The results show that PYC indeed plays a crucial role in the intracellular replication of L. monocytogenes and hence in the infection process.     

Src-dependent Tyrosine Phosphorylation of Non-muscle Myosin Heavy Chain-IIA Restricts Listeria monocytogenes Cellular Infection

Bacterial pathogens often interfere with host tyrosine phosphorylation cascades to control host responses and cause infection. Given the role of tyrosine phosphorylation events in different human infections and our previous results showing the activation of the tyrosine kinase Src upon incubation of cells with Listeria monocytogenes, we searched for novel host proteins undergoing tyrosine phosphorylation upon L. monocytogenes infection. We identify the heavy chain of the non-muscle myosin IIA (NMHC-IIA) as being phosphorylated in a specific tyrosine residue in response to L. monocytogenes infection. We characterize this novel post-translational modification event and show that, upon L. monocytogenes infection, Src phosphorylates NMHC-IIA in a previously uncharacterized tyrosine residue (Tyr-158) located in its motor domain near the ATP-binding site. In addition, we found that other intracellular and extracellular bacterial pathogens trigger NMHC-IIA tyrosine phosphorylation. We demonstrate that NMHC-IIA limits intracellular levels of L. monocytogenes, and this is dependent on the phosphorylation of Tyr-158. Our data suggest a novel mechanism of regulation of NMHC-IIA activity relying on the phosphorylation of Tyr-158 by Src.     

Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro

Background

Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection.

Methodology/Principal Findings

The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp^LAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp^LAP for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, Lbp^LAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp^LAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp^LAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h.

Conclusions/Significance

Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp^LAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations.     

A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication

Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocytogenes mutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified gene menD or pepP were defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus validating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of the menD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, the menD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both the menD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multiple bacterial systems.     

Identification and Characterization of Di- and Tripeptide Transporter DtpT of Listeria monocytogenes EGD-e

Listeria monocytogenes is a gram-positive intracellular pathogen responsible for opportunistic infections in humans and animals. Here we identified and characterized the dtpT gene (lmo0555) of L. monocytogenes EGD-e, encoding the di- and tripeptide transporter, and assessed its role in growth under various environmental conditions as well as in the virulence of L. monocytogenes. Uptake of the dipeptide Pro-[¹⁴C]Ala was mediated by the DtpT transporter and was abrogated in a ΔdtpT isogenic deletion mutant. The DtpT transporter was shown to be required for growth when the essential amino acids leucine and valine were supplied as peptides. The protective effect of glycine- and proline-containing peptides during growth in defined medium containing 3% NaCl was noted only in L. monocytogenes EGD-e, not in the ΔdtpT mutant strain, indicating that the DtpT transporter is involved in salt stress protection. Infection studies showed that DtpT contributes to pathogenesis in a mouse infection model but has no role in bacterial growth following infection of J774 macrophages. These studies reveal that DptT may contribute to the virulence of L. monocytogenes.     

Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-¹³C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained ¹³C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis (¹³C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.     

Carbon metabolism of Listeria monocytogenes growing inside macrophages

The intracellular metabolism of Listeria monocytogenes was studied by ¹³C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-¹³C₆]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-¹³C₆]glucose was provided during the infection period, ¹³C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C₃-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-¹³C₆]glucose into bacterial amino acids under the same experimental settings. The ¹³C pattern suggests that (i) significant fractions (50–100%) of bacterial amino acids were provided by the host cell, (ii) a C₃-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.     

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.     

Leishmania infantum Modulates Host Macrophage Mitochondrial Metabolism by Hijacking the SIRT1-AMPK Axis

Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis.     

Listeria monocytogenes cytosolic metabolism promotes replication,survival, and evasion of innate immunity

Listeria monocytogenes, the causative agent of listeriosis, is an intracellular pathogen that is exquisitely evolved to survive and replicate in the cytosol of eukaryotic cells. Eukaryotic cells typically restrict bacteria from colonising the cytosol, likely through a combination of cell autonomous defences, nutritional immunity, and innate immune responses including induction of programmed cell death. This suggests that L. monocytogenes and other professional cytosolic pathogens possess unique metabolic adaptations, not only to support replication but also to facilitate resistance to host‐derived stresses/defences and avoidance of innate immune activation. In this review, we outline our current understanding of L. monocytogenes metabolism in the host cytosol and highlight major metabolic processes which promote intracellular replication and survival.     

13C Nuclear Magnetic Resonance Studies of Citrate and Glucose Cometabolism by Lactococcus lactis

¹³C nuclear magnetic resonance (¹³C-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongrowing cells of Lactococcus lactis subsp. lactis 19B under anaerobic conditions. The metabolism of citrate plus glucose during growth was also monitored directly by in vivo NMR. Although pyruvate is a common intermediate metabolite in the metabolic pathways of both citrate and glucose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4-¹³C]citrate, [3-¹³C]pyruvate, and [2-¹³C]glucose. The presence of an additional substrate caused a considerable stimulation in the rates of substrate utilization, and the pattern of end products was changed. Acetate plus acetoin and butanediol represented more than 80% (molar basis) of the end products of the metabolism of citrate (or pyruvate) alone, but when glucose was also added, 80% of the citrate (or pyruvate) was converted to lactate. This result can be explained by the activation of lactate dehydrogenase by fructose 1,6-bisphosphate, an intermediate in glucose metabolism. The effect of different concentrations of glucose on the metabolism of citrate by dilute cell suspensions was also probed by using analytical methods other than NMR. Pyruvate dehydrogenase (but not pyruvate formate-lyase) was active in the conversion of pyruvate to acetyl coenzyme A. α-Acetolactate was detected as an intermediate metabolite of citrate or pyruvate metabolism, and the labeling pattern of the end products agrees with the α-acetolactate pathway. It was demonstrated that the contribution of the acetyl coenzyme A pathway for the synthesis of diacetyl, should it exist, is lower than 10%. Evidence for the presence of internal carbon reserves in L. lactis is presented.     

13C metabolic flux analysis clarifies distinct metabolic phenotypes of cancer cell spheroid mimicking tumor hypoxia

Cancer cells adapt their intracellular energy metabolism to the oxygen-deprived tumor microenvironment (TME) to ensure tumor progression. This adaptive mechanism has focused attention on the metabolic phenotypes of tumor cells under hypoxic TME for developing novel cancer therapies. Although widely used monolayer (2D) culture does not fully reflect in vivo hypoxic TME, spheroid (3D) culture can produce a milieu similar to the TME in vivo. However, how different metabolic phenotypes are expressed in 3D cultures mimicking tumor hypoxia compared with 2D cultures under hypoxia remains unclear. To address this issue, we investigated the metabolic phenotypes of 2D- and 3D-cultured cancer cells by ¹³C-metabolic flux analysis (¹³C-MFA). Principal component analysis of ¹³C mass isotopomer distributions clearly demonstrated distinct metabolic phenotypes of 3D-cultured cells. ¹³C-MFA clarified that 3D culture significantly upregulated pyruvate carboxylase flux in line with the pyruvate carboxylase protein expression level. On the other hand, 3D culture downregulated glutaminolytic flux. Consistent with our findings, 3D-cultured cells are more resistant to a glutaminase inhibitor than 2D-cultured cells. This study suggests the importance of considering the metabolic characteristics of the particular in vitro model used for research on cancer metabolism.     

Suppression of major histocompatibility complex class I and class II gene expression in Listeria monocytogenes-infected murine macrophages

Macrophage cells play a central role during infection with Listeria monocytogenes by both providing a major habitat for bacterial multiplication and presenting bacterial antigens to the immune system. In this study, we investigated the influence of L. monocytogenes infection on the expression of MHC class I and class II genes in two murine macrophage cell lines. Steady-state levels of I-Aβ chain mRNA were decreased in both resting J774A.1 and P388D₁ macrophages infected with L. monocytogenes whereas reduction of H-2K mRNA was only observed in P388D₁ cells. In addition, L. monocytogenes suppressed induction of MHC class I and class II mRNAs in response to γ-interferon as well as the maintenance of the induced state in activated P388D₁ macrophages. Exposure to the non-pathogenic species L. innocua or a deletion mutant of L. monocytogenes, which lacks the lecithinase operon, did not cause a reduction in H-2K and I-Aβ mRNA levels nor suppress expression of Ia antigens. Inhibition of MHC gene expression may represent an important part of the cross-talk between L. monocytogenes and the macrophage that probably influences the efficiency of a T cell-mediated immune response and thus the outcome of a listerial infection.     

Listeria monocytogenes Triggers the Cell Surface Expression of Gp96 Protein and Interacts with Its N Terminus to Support Cellular Infection

Listeria monocytogenes is an intracellular food-borne pathogen causing listeriosis in humans. This bacterium deploys an arsenal of virulence factors that act in concert to promote cellular infection. Bacterial surface proteins are of primary importance in the process of host cell invasion. They interact with host cellular receptors, inducing/modulating specific cellular responses. We previously identified Vip, a Listeria surface protein covalently attached to the bacterial cell wall acting as a key virulence factor. We have shown that Vip interacts with Gp96 localized at the surface of host cells during invasion and that this interaction is critical for a successful infection in vivo. To better understand the importance of Vip-Gp96 interaction during infection, we aimed to characterize this interaction at the molecular level. Here we demonstrate that, during infection, L. monocytogenes triggers the cellular redistribution of Gp96, inducing its exposure at the cell surface. Upon infection, Gp96 N-terminal domain is exposed to the extracellular milieu in L2071 fibroblasts and interacts with Vip expressed by Listeria. We identified Gp96 (Asp¹–Leu¹⁷⁰) as sufficient to interact with Vip; however, we also showed that the region Tyr¹⁷⁹–Leu³⁹⁰ of Gp96 is important for the interaction. Our findings unravel the Listeria-induced surface expression of Gp96 and the topology of its insertion on the plasma membrane and improve our knowledge on the Vip-Gp96 interaction during Listeria infection.     

Listeriolysin O Is Necessary and Sufficient to Induce Autophagy during Listeria monocytogenes Infection

Background

Recent studies have suggested that autophagy is utilized by cells as a protective mechanism against Listeria monocytogenes infection.

Methodology/Principal Findings

However we find autophagy has no measurable role in vacuolar escape and intracellular growth in primary cultured bone marrow derived macrophages (BMDMs) deficient for autophagy (atg5^−/−). Nevertheless, we provide evidence that the pore forming activity of the cholesterol-dependent cytolysin listeriolysin O (LLO) can induce autophagy subsequent to infection by L. monocytogenes. Infection of BMDMs with L. monocytogenes induced microtubule-associated protein light chain 3 (LC3) lipidation, consistent with autophagy activation, whereas a mutant lacking LLO did not. Infection of BMDMs that express LC3-GFP demonstrated that wild-type L. monocytogenes was encapsulated by LC3-GFP, consistent with autophagy activation, whereas a mutant lacking LLO was not. Bacillus subtilis expressing either LLO or a related cytolysin, perfringolysin O (PFO), induced LC3 colocalization and LC3 lipidation. Further, LLO-containing liposomes also recruited LC3-GFP, indicating that LLO was sufficient to induce targeted autophagy in the absence of infection. The role of autophagy had variable effects depending on the cell type assayed. In atg5^−/− mouse embryonic fibroblasts, L. monocytogenes had a primary vacuole escape defect. However, the bacteria escaped and grew normally in atg5^−/− BMDMs.

Conclusions/Significance

We propose that membrane damage, such as that caused by LLO, triggers bacterial-targeted autophagy, although autophagy does not affect the fate of wild-type intracellular L. monocytogenes in primary BMDMs.