Critical role of the N-terminal residues of listeriolysin O in phagosomal escape and virulence of Listeria monocytogenes

A putative PEST sequence was recently identified close to the N-terminus of listeriolysin O (LLO), a major virulence factor secreted by the pathogenic Listeria monocytogenes. The deletion of this motif did not affect the secretion and haemolytic activity of LLO, but abolished bacterial virulence. Here, we first tested whether the replacement of the PEST motif of LLO by two different sequences, with either a very high or no PEST score, would affect phagosomal escape, protein stability and, ultimately, the virulence of L. monocytogenes. Then, we constructed LLO mutants with an intact PEST sequence but carrying mutations on either side, or on both sides, of the PEST motif. The properties of these mutants prompted us to construct three LLO mutants carrying single amino acid substitutions in the distal portion of the PEST region (P49A, K50A and P52A; preprotein numbering). Our data demonstrate that the susceptibility of LLO to intracellular proteolytic degradation is not related to the presence of a high PEST score sequence and that the insertion of two residues immediately downstream of the intact PEST sequence is sufficient to impair phagosomal escape and abolish bacterial virulence. Furthermore, we show that single amino acid substitutions in the distal portion of the PEST motif are sufficient to attenuate bacterial -virulence significantly, unravelling the critical role of this region of LLO in the pathogenesis of L. -monocytogenes.     

单增李斯特菌溶血素O的PEST序列激活ERK1/2磷酸化的作用

[目的]本研究旨在构建单核细胞增多性李斯特菌(Listeria monocytogenes,简称单增李斯特菌)溶血素O(Listeriolysin O,LLO)的关键结构域PEST序列(包含S44、S48和T51关键磷酸化位点)突变体,并针对其生物学功能展开研究。[方法]以李斯特菌参考菌株EGD-e为模板扩增编码LLO的hly基因,克隆至pET30a(+)原核表达载体,在此基础上利用氨基酸突变技术获得表达PEST突变体(LLO△PEST、LLOS44A、LLOS48A和LLOT51A)的重组质粒,转入E.coli Rosetta感受态细胞中,诱导表达重组蛋白经镍离子亲和层析纯化后进行SDS-PAGE分析。利用红细胞裂解试验检测重组蛋白的溶血活性,并通过Western blotting检测重组突变蛋白刺激Caco-2细胞后对MAPK关键信号分子ERK1/2磷酸化水平变化的影响。[结果]结果显示,本研究成功获得重组LLO及其突变体蛋白LLO△PEST、LLOS44A、LLOS48A和LLOT51A。在pH5.5和7.4条件下,LLO△PEST、LLOS44A、LLOS48A和LLOT51A均具有和LLO相当的溶血活性,说明PEST序列缺失或突变并不影响LLO的膜裂解活性。研究进一步发现,重组LLO及其突变蛋白刺激Caco-2细胞后均能激活ERK1/2的磷酸化。[结论]研究表明LLO的关键结构域PEST序列对于维持该蛋白的膜裂解能力及穿孔活性并非必需,且该结构域的缺失不影响李斯特菌在感染宿主时依赖LLO介导ERK1/2磷酸化的生物学过程。本研究将为进一步探索细菌感染过程中PEST序列对于LLO发挥生物学功能的潜在作用及分子机制奠定基础。     

Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes

The hly-encoded listeriolysin O (LLO) is a major virulence factor secreted by the intracellular pathogen Listeria monocytogenes, which plays a crucial role in the escape of bacteria from the phagosomal compartment. Here, we identify a putative PEST sequence close to the N-terminus of LLO and focus on the role of this motif in the biological activities of LLO. Two LLO variants were constructed: a deletion mutant protein, lacking the 19 residues comprising this sequence (residues 32-50), and a recombinant protein of wild-type size, in which all the P, E, S or T residues within this motif have been substituted. The two mutant proteins were fully haemolytic and were secreted in culture supernatants of L. monocytogenes in quantities comparable with that of the wild-type protein. Strikingly, both mutants failed to restore virulence to a hly-negative strain in vivo. In vitro assays showed that L. monocytogenes expressing the LLO deletion mutant was strongly impaired in its ability to escape from the phagosomal vacuole and, subsequently, to divide in the cytosol of infected cells. This work reveals for the first time that the N-terminal portion of LLO plays an important role in the development of the infectious process of L. monocytogenes.     

Listeriolysin O: a phagosome-specific lysin

Listeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin family and a primary virulence factor of the gram-positive, facultative intracellular pathogen Listeria monocytogenes. During the intracellular life cycle of L. monocytogenes, LLO is largely responsible for mediating rupture of the phagosomal membrane, thereby allowing the bacterium access to the host cytosol, its replicative niche. In the host cytosol, LLO activity is controlled at numerous levels to prevent perforation of the plasma membrane and loss of the intracellular environment. In this review, we focus primarily on the role of LLO in phagosomal escape and the multiple regulatory mechanisms that control LLO activity in the host cytosol.     

Perturbation of vacuolar maturation promotes listeriolysin O-independent vacuolar escape during Listeria monocytogenes infection of human cells

Listeria monocytogenes is a bacterial pathogen that replicates within the cytosol of infected host cells. The ability to rapidly escape the phagocytic vacuole is essential for efficient intracellular replication. In the murine model of infection, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for vacuolar dissolution, as LLO-deficient (ΔLLO) mutants remain trapped within vacuoles. In contrast, in many human cell types ΔLLO L. monocytogenes are capable of vacuolar escape at moderate to high frequencies. To better characterize the mechanism of LLO-independent vacuolar escape in human cells, we conducted an RNA interference screen to identify vesicular trafficking factors that play a role in altering vacuolar escape efficiency of ΔLLO L. monocytogenes . RNA interference knockdown of 18 vesicular trafficking factors resulted in increased LLO-independent vacuolar escape. Our results suggest that knockdown of one factor, RABEP1 (rabaptin-5), decreased the maturation of vacuoles containing ΔLLO L. monocytogenes . Thus, we provide evidence that increased vacuolar escape of ΔLLO L. monocytogenes in human cells correlates with slower vacuolar maturation. We also determined that increased LLO-independent dissolution of vacuoles during RABEP1 knockdown required the bacterial broad-range phospholipase C (PC-PLC). We hypothesize that slowing the kinetics of vacuolar maturation generates an environment conducive for vacuolar escape mediated by the bacterial phospholipases.     

Sec16p potentiates the action of COPII proteins to bud transport vesicles

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is approximately 10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.     

Listeriolysin O suppresses phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection

The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L.?monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O,?a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allow L.?monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst.     

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 (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.     

The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization

The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors.     

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.     

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.     

Antitopes define preferential proteasomal cleavage site usage

Protein degradation by proteasomes is a major source of peptides presented by major histocompatibility v complex class I proteins. Importantly, interferon gamma-induced immunoproteasomes in many cases strongly enhance the generation of antigenic peptides both in vitro and in vivo. Whether this is due to enhanced substrate turnover or to a change in proteasomal cleavage specificity is, however, largely unresolved. To overcome the problems of peptide quantification inherent to mass spectrometry, we introduced the "antitope" as substrate-specific internal standard. The antitope is a non-functional peptide that is generated by proteasomal cleavage within the epitope, resulting in partial overlaps with the functional epitope. Using antitopes as internal standards we demonstrate that the observed enhanced immunoproteasome-dependent presentation of the bacterial listeriolysin O T-cell epitope LLO(296-304) is indeed due to altered cleavage preferences. This method is also applicable to other major histocompatibility class I epitopes as is shown for two potential epitopes derived from Coxsackievirus.     

Ubiquitination of intracellular bacteria: a new bacteria-sensing system?

Ubiquitination is a protein modification generally used by cells to tag proteins that are destined for proteasomal degradation. In a recent article, Perrin et al. reported that the ubiquitination system has a role in the recognition of bacterial pathogens in the cytosol of mammalian cells. They showed that polyubiquitinated proteins accumulate on the surface of cytosolic Salmonella typhimurium. In macrophages, but not epithelial cells, proteasomes become associated with the surface of cytosolic bacteria. The authors proposed that the ubiquitin-proteasome machinery might be implicated indirectly in bacterial clearance.     

Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops

'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors.     

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.     

Listeria monocytogenes listeriolysin O and phosphatidylinositol-specific phospholipase C affect adherence to epithelial cells

Listeria monocytogenes, a foodborn intracellular animal and human pathogen, produces several exotoxins contributing to virulence. Among these are listeriolysin O (LLO), a pore-forming cholesterol-dependent hemolysin, and a phosphatidylinositol-specific phospholipase C (PI-PLC). LLO is known to play an important role in the escape of bacteria from the primary phagocytic vacuole of macrophages, and PI-PLC supports this process. Evidence is accumulating that LLO and PI-PLC are multifunctional virulence factors with many important roles in the host-parasite interaction other than phagosomal membrane disruption. LLO and PI-PLC may induce a number of host cell responses by modulating signal transduction of infected cells via intracellular Ca2+ levels and the metabolism of phospholipids. This would result in the activation of host phospholipase C and protein kinase C. In the present study, using Bacillus sub tilis strains expressing LLO, PI-PLC, and simultaneously LLO and PI-PLC, we show that LLO and PI-PLC enhance bacterial binding to epithelial cells Int407, with LLO being necessary and PI-PLC playing an accessory role. The results of this work suggest that these two listerial proteins act on epithelial cells prior to internalization.     

Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.

Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.     

The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.