Metabolic Responses of Primary and Transformed Cells to Intracellular Listeria monocytogenes

The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon Listeria monocytogenes infection using ¹³C-labelled glucose or glutamine as carbon tracers. The ¹³C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.     

Avoiding death by autophagy: Interactions of Listeria monocytogenes with the macrophage autophagy system

Autophagy restricts the growth of a variety of intracellular pathogens. However, cytosol-adapted pathogens have evolved ways to evade restriction by this innate immune mechanism. Listeria monocytogenes is a Gram-positive bacterial pathogen that utilizes a cholesterol-dependent pore-forming toxin, listeriolysin O (LLO), to escape from the phagosome. Autophagy targets L. monocytogenes in LLO-damaged phagosomes and also in the cytosol under some experimental conditions. However, this bacterium has evolved multiple mechanisms to evade restriction by autophagy, including actin-based motility in the cytosol and an as yet undefined mechanism mediated by bacterial phospholipases C’s (PLCs). A population of L. monocytogenes with inefficient LLO activity forms Spacious Listeria-containing Phagosomes (SLAPs), which are autophagosome-like compartments that do not mature, allowing slow bacterial growth within enlarged vesicles. SLAPs may represent a stalemate between bacterial LLO action and the host autophagy system, resulting in persistent infection.

Addendum to: Birmingham CL, Canadien V, Gouin E, Troy EB, Yoshimori T, Cossart P, Higgins DE, Brumell JH. Listeria monocytogenes evades killing by autophagy during colonization of host cells. Autophagy 2007; 3:442-51.andBirmingham CL, Canadien V, Kaniuk NA, Steinberg BE, Higgins DE, Brumell JH. Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles. Nature 2008; 451:350-4.     

Biotechnological applications of Listeria's sophisticated infection strategies

Listeria monocytogenes is a Gram‐positive bacterium that is able to survive both in the environment and to invade and multiply within eukaryotic cells. Currently L. monocytogenes represents one of the most well‐studied and characterized microorganisms in bacterial pathogenesis. A hallmark of L. monocytogenes virulence is its ability to breach bodily barriers such as the intestinal epithelium, the blood–brain barrier as well as the placental barrier to cause severe systemic disease. Curiously, this theme is repeated at the level of the interaction between the individual cell and the bacterium where its virulence factors contribute to the ability of the bacteria to breach cellular barriers. L. monocytogenes is a model to study metabolic requirements of bacteria growing in an intracellular environment, modulation of signalling pathways in the infected cell and interactions with cellular defences involving innate and adaptive immunity. Technical advances such as the creation of LISTERIA‐susceptible mouse strains, had added interest in the study of the natural pathogenesis of the disease via oral infection. The use of attenuated strains of L. monocytogenes as vaccines has gained considerable interest because they can be used to express heterologous antigens as well as to somatically deliver recombinant DNA to eukaryotic cells. A novel vaccine concept, the use of non‐viable but metabolically active bacteria to induced immunoprotective responses, has been developed with L. monocytogenes. In this mini‐review, we review the strategies used by L. monocytogenes to subvert the cellular functions at different stages of the infection cycle in the host and examine how these properties are being exploited in biotechnological and clinical applications.     

Suppression of Cell-Mediated Immunity following Recognition of Phagosome-Confined Bacteria

Listeria monocytogenes is a facultative intracellular pathogen capable of inducing a robust cell-mediated immune response to sub-lethal infection. The capacity of L. monocytogenes to escape from the phagosome and enter the host cell cytosol is paramount for the induction of long-lived CD8 T cell–mediated protective immunity. Here, we show that the impaired T cell response to L. monocytogenes confined within a phagosome is not merely a consequence of inefficient antigen presentation, but is the result of direct suppression of the adaptive response. This suppression limited not only the adaptive response to vacuole-confined L. monocytogenes, but negated the response to bacteria within the cytosol. Co-infection with phagosome-confined and cytosolic L. monocytogenes prevented the generation of acquired immunity and limited expansion of antigen-specific T cells relative to the cytosolic L. monocytogenes strain alone. Bacteria confined to a phagosome suppressed the production of pro-inflammatory cytokines and led to the rapid MyD88-dependent production of IL-10. Blockade of the IL-10 receptor or the absence of MyD88 during primary infection restored protective immunity. Our studies demonstrate that the presence of microbes within a phagosome can directly impact the innate and adaptive immune response by antagonizing the signaling pathways necessary for inflammation and the generation of protective CD8 T cells.     

Autophagy Limits Listeria monocytogenes Intracellular Growth in the Early Phase of Primary Infection

Autophagy has been recently proposed to be a component of the innate cellular immune response against several types of intracellular microorganisms. However, other intracellular bacteria including Listeria monocytogenes have been thought to evade the autophagic cellular surveillance. Here, we show that cellular infection by L. monocytogenes induces an autophagic response, which inhibits the growth of both the wild-type and a delta actA mutant strain, the latter being impaired in cell-to-cell spreading. The onset of early intracellular growth is accelerated in autophagy-deficient cells, but the growth rate once bacteria begin to multiply in the cytosol does not change. Moreover, a significant fraction of the intracellular bacteria co-localize with autophagosomes at the early time-points after infection. Thus, autophagy targets L. monocytogenes during primary infection by limiting the onset of early bacterial growth. The bacterial expression of listeriolysin O but not phospholipases is necessary for the induction of autophagy, suggesting a possible role for permeabilization of the vacuole in the induction of autophagy. Interestingly, the growth of a delta plcA/B L. monocytogenes strain deficient for bacterial phospholipases is impaired in wild-type cells, but restored in the absence of autophagy, suggesting that bacterial phospholipases may facilitate the escape of bacteria from autophagic degradation. We conclude that L. monocytogenes are targeted for degradation by autophagy during the primary infection, in the early phase of the intracellular cycle, following listeriolysin O-dependent vacuole perforation but preceding active multiplication in the cytosol, and that expression of bacterial phospholipases is necessary for the evasion of autophagy.     

Conversion of an extracellular cytolysin into a phagosome-specific lysin which supports the growth of an intracellular pathogen

Listeria monocytogenes is a facultative intracellular pathogen which secretes a pore-forming cytolysin, listeriolysin O (LLO), necessary for intracellular growth. Clostridium perfringens is an extracellular pathogen which secretes a related cytolysin, perfrlngolysln O (PFO). When PFO is secreted by intracellular L. monocytogenes, it is toxic to the infected host cell. PFO-mediated toxicity renders the infected host cell permeable to gentamicin and leads to the death of the intracellular bacteria. In this study, we selected for L. monocytogenes mutants in which PFO supported the intracellular growth of L. monocytogenes. Six independent mutants were isolated, each containing a single amino acid change within the PFO protein. Three classes of PFO mutations were identified, all capable of mediating lysis of the vacuole but without a toxic effect upon the infected host cell. The first class had a severe defect in haemolytic activity. The second class had a change in the pH optimum of PFO. The third class had nearly wild-type levels of haemolytic activity, but had a decrease in protein half-life in the host-cell cytosol. Acquisition of single amino acid changes in PFO were sufficient to convert an extracellular cytolysin into a vacuole-specific lysin which mediated growth of L. monocytogenes in cultured cells.     

Listeria monocytogenes Evades Killing by Autophagy During Colonization of Host Cells

Listeria monocytogenes is an intracellular pathogen that is able to colonize the cytosol of macrophages. Here we examined the interaction of this pathogen with autophagy, a host cytosolicdegradative pathway that constitutes an important component of innate immunity towards microbial invaders. L. monocytogenes infection induced activation of the autophagy system in macrophages. At 1 h post infection (p.i.), a population of intracellular bacteria (~37%) colocalized with the autophagy marker LC3. These bacteria were within vacuoles and were targeted by autophagy in an LLO-dependent manner. At later stages in infection (by 4 h p.i.), the majority of L. monocytogenes escaped into the cytosol and rapidly replicated. At these times, less than 10% of intracellular bacteria colocalized with LC3. We found that ActA expression was sufficient to prevent autophagy of bacteria in the cytosol of macrophages. Surprisingly, ActA expression was not strictly necessary, indicating that other virulence factors were involved. Accordingly, we also found a role for the bacterial phospholipases, PI-PLC and PC-PLC, in autophagy evasion, as bacteria lacking phospholipase expression were targeted by autophagy at later times in infection. Together, our results demonstratethat L. monocytogenes utilizes multiple mechanisms to avoid destruction by the autophagy system during colonization of macrophages.     

Why is Listeria monocytogenes such a potent inducer of CD8+ T‐cells?

Listeria monocytogenes is a rapidly growing, Gram‐positive, facultative intracellular pathogen that has been used for over 5 decades as a model to study basic aspects of infection and immunity. In a murine intravenous infection model, immunisation with a sublethal infection of L. monocytogenes initially leads to rapid intracellular multiplication followed by clearance of the bacteria and ultimately culminates in the development of long‐lived cell‐mediated immunity (CMI) mediated by antigen‐specific CD8+ cytotoxic T‐cells. Importantly, effective immunisation requires live, replicating bacteria. In this review, we summarise the cell and immunobiology of L. monocytogenes infection and discuss aspects of its pathogenesis that we suspect lead to robust CMI. We suggest five specific features of L. monocytogenes infection that positively impact the development of CMI: (a) the bacteria have a predilection for professional antigen‐presenting cells; (b) the bacteria escape from phagosomes, grow, and secrete antigens into the host cell cytosol; (c) bacterial‐secreted proteins enter the major histocompatibility complex (MHC) class I pathway of antigen processing and presentation; (d) the bacteria do not induce rapid host cell death; and (e) cytosolic bacteria induce a cytokine response that favours CMI. Collectively, these features make L. monocytogenes an attractive vaccine vector for both infectious disease applications and cancer immunotherapy.     

Manipulation of the host cell death pathway by Shigella

Host cells deploy multiple defences against microbial infection. One prominent host defence mechanism, the death of infected cells, plays a pivotal role in clearing damaged cells, eliminating pathogens, removing replicative niches, exposing intracellular bacterial pathogens to extracellular immune surveillance and presenting bacteria‐derived antigens to the adaptive immune system. Although cell death can occur under either physiological or pathophysiological conditions, it acts as an innate defence mechanism against bacterial pathogens by limiting their persistent colonization. However, many bacterial pathogens, including Shigella, have evolved mechanisms that manipulate host cell death for their own benefit.     

STING-Dependent Type I IFN Production Inhibits Cell-Mediated Immunity to Listeria monocytogenes

Infection with Listeria monocytogenes strains that enter the host cell cytosol leads to a robust cytotoxic T cell response resulting in long-lived cell-mediated immunity (CMI). Upon entry into the cytosol, L. monocytogenes secretes cyclic diadenosine monophosphate (c-di-AMP) which activates the innate immune sensor STING leading to the expression of IFN-β and co-regulated genes. In this study, we examined the role of STING in the development of protective CMI to L. monocytogenes. Mice deficient for STING or its downstream effector IRF3 restricted a secondary lethal challenge with L. monocytogenes and exhibited enhanced immunity that was MyD88-independent. Conversely, enhancing STING activation during immunization by co-administration of c-di-AMP or by infection with a L. monocytogenes mutant that secretes elevated levels of c-di-AMP resulted in decreased protective immunity that was largely dependent on the type I interferon receptor. These data suggest that L. monocytogenes activation of STING downregulates CMI by induction of type I interferon.     

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.     

Listeriolysin O: A phagosome‐specific cytolysin revisited

Listeriolysin O (LLO) is an essential determinant of Listeria monocytogenes pathogenesis that mediates the escape of L. monocytogenes from host cell vacuoles, thereby allowing replication in the cytosol without causing appreciable cell death. As a member of the cholesterol‐dependent cytolysin (CDC) family of pore‐forming toxins, LLO is unique in that it is secreted by a facultative intracellular pathogen, whereas all other CDCs are produced by pathogens that are largely extracellular. Replacement of LLO with other CDCs results in strains that are extremely cytotoxic and 10,000‐fold less virulent in mice. LLO has structural and regulatory features that allow it to function intracellularly without causing cell death, most of which map to a unique N‐terminal region of LLO referred to as the proline, glutamic acid, serine, threonine (PEST)‐like sequence. Yet, while LLO has unique properties required for its intracellular site of action, extracellular LLO, like other CDCs, affects cells in a myriad of ways. Because all CDCs form pores in cholesterol‐containing membranes that lead to rapid Ca²⁺ influx and K⁺ efflux, they consequently trigger a wide range of host cell responses, including mitogen‐activated protein kinase activation, histone modification, and caspase‐1 activation. There is no debate that extracellular LLO, like all other CDCs, can stimulate multiple cellular activities, but the primary question we wish to address in this perspective is whether these activities contribute to L. monocytogenes pathogenesis.     

Erratum to: Florez-McClure ML,Hohsfield LA,Fonte G,Bealor MT,Link CD. Decreased insulin-receptor signaling promotes the autophagic degradation of β-amyloid peptide in C. elegans. Autophagy 2007; 3:569-80

Autophagy is a cell-autonomous mechanism of innate immunity that protects the cytosol against bacterial infection. Invasive bacteria, including Listeria monocytogenes, have thus evolved strategies to counteract a process that limits their intracellular growth. ActA is a surface protein produced by L. monocytogenes to polymerize actin and mediate intra- and intercellular movements, which plays a critical role in autophagy escape. We have recently investigated the role of another L. monocytogenes surface protein, the internalin InlK, in the infection process. We showed that in the cytosol of infected cells, InlK interacts with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoprotein particles named vaults. Although MVP has been implicated in a variety of key cellular process, its role remains elusive. We demonstrated that L. monocytogenes is able, via InlK, to decorate its surface with MVP in order to escape autophagic recognition. Strikingly, this new strategy used by L. monocytogenes to avoid autophagy is independent of ActA, suggesting that InlK-MVP interactions and actin polymerization are two processes that favor in the same manner the infection process. Understanding the role of MVP may provide new insights into bacterial infection and autophagy.     

Listeria-derived ActA is an effective adjuvant for primary and metastatic tumor immunotherapy

Tumor immunotherapy is currently at the cusp of becoming an important aspect of comprehensive cancer treatment in the clinic. However, the need for improved adjuvants to augment immune responses against tumor antigens is always present. In this paper, we characterize the Listeria monocytogenes-derived actin-nucleating protein, ActA, as a novel adjuvant for use in tumor immunotherapy. ActA is a virulence factor that is expressed on the cell surface of L. monocytogenes and facilitates the production of actin tails that propel Listeria throughout the cytosol of an infected host cell. It is believed that this ActA-dependent cytosolic motility allows Listeria to evade adaptive host cell defenses and facilitates its invasion into a proximal uninfected host cell. However, there is evidence that ActA fused to a tumor antigen and delivered by L. monocytogenes can perform a beneficial function in tumor immunotherapy as an adjuvant. Our investigation of this adjuvant activity demonstrates that ActA, either fused to or administered as a mixture with a tumor antigen, can augment anti-tumor immune responses, break immune tolerance and facilitate tumor eradication, which suggests that ActA is not only an effective adjuvant in tumor immunotherapy but can also be applied in a number of therapeutic settings.     

The innate immune response in human tuberculosis

Mycobacterium tuberculosis (Mtb) infection can be cleared by the innate immune system before the initiation of an adaptive immune response. This innate protection requires a variety of robust cell autonomous responses from many different host immune cell types. However, Mtb has evolved strategies to circumvent some of these defences. In this mini‐review, we discuss these host–pathogen interactions with a focus on studies performed in human cells and/or supported by human genetics studies (such as genome‐wide association studies).     

Actin‐based motility allows Listeria monocytogenes to avoid autophagy in the macrophage cytosol

Listeria monocytogenes grows in the host cytosol and uses the surface protein ActA to promote actin polymerisation and mediate actin‐based motility. ActA, along with two secreted bacterial phospholipases C, also mediates avoidance from autophagy, a degradative process that targets intracellular microbes. Although it is known that ActA prevents autophagic recognition of L. monocytogenes in epithelial cells by masking the bacterial surface with host factors, the relative roles of actin polymerisation and actin‐based motility in autophagy avoidance are unclear in macrophages. Using pharmacological inhibition of actin polymerisation and a collection of actA mutants, we found that actin polymerisation prevented the colocalisation of L. monocytogenes with polyubiquitin, the autophagy receptor p62, and the autophagy protein LC3 during macrophage infection. In addition, the ability of L. monocytogenes to stimulate actin polymerisation promoted autophagy avoidance and growth in macrophages in the absence of phospholipases C. Time‐lapse microscopy using green fluorescent protein‐LC3 macrophages and a probe for filamentous actin showed that bacteria undergoing actin‐based motility moved away from LC3‐positive membranes. Collectively, these results suggested that although actin polymerisation protects the bacterial surface from autophagic recognition, actin‐based motility allows escape of L. monocytogenes from autophagic membranes in the macrophage cytosol.     

Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster

The facultative intracellular bacterial pathogen Listeria monocytogenes is capable of replicating within a broad range of host cell types and host species. We report here the establishment of the fruit fly Drosophila melanogaster as a new model host for the exploration of L. monocytogenes pathogenesis and host response to infection. Listeria monocytogenes was capable of establishing lethal infections in adult fruit flies and larvae with extensive bacterial replication occurring before host death. Bacteria were found in the cytosol of insect phagocytic cells, and were capable of directing host cell actin polymerization. Bacterial gene products necessary for intracellular replication and cell-to-cell spread within mammalian cells were similarly found to be required within insect cells, and although previous work has suggested that L. monocytogenes virulence gene expression requires temperatures above 30 degrees C, bacteria within insect cells were found to express virulence determinants at 25 degrees C. Mutant strains of Drosophila that were compromised for innate immune responses demonstrated increased susceptibility to L. monocytogenes infection. These data indicate L. monocytogenes infection of fruit flies shares numerous features of mammalian infection, and thus that Drosophila has the potential to serve as a genetically tractable host system that will facilitate the analysis of host cellular responses to L. monocytogenes infection.     

Innate immunity to mycobacteria: vitamin D and autophagy

Autophagy is an ancient mechanism of protein degradation and a novel antimicrobial strategy. With respect to host defences against mycobacteria, autophagy plays a crucial role in antimycobacterial resistance, and contributes to immune surveillance of intracellular pathogens and vaccine efficacy. Vitamin D3 contributes to host immune responses against Mycobacterium tuberculosis through LL‐37/hCAP‐18, which is the only cathelicidin identified to date in humans. In this review, we discuss recent advances in our understanding of host immune strategies against mycobacteria, including vitamin D‐mediated innate immunity and autophagy activation. This review also addresses our current understanding regarding the autophagy connection to principal innate machinery, such as ubiquitin‐ or inflammasome‐involved pathways. Integrated dialog between autophagy and innate immunity may contribute to adequate host immune defences against mycobacterial infection.