Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death

Staphylococcus aureus is a common bacterial etiology of serious infectious diseases. S. aureus can invade various types of non-professional phagocytes to produce host cell death. We show here that shortly after invasion of HeLa cells S. aureus transit to autophagosomes was characterized by double membranes and co-localization with LC3. S. aureus were not able to replicate and produce cell death in autophagy-deficient atg5-/- mouse embryonic fibroblasts. S. aureus-containing autophagosomes do not acidify nor do they acquire lysosome-associated membrane protein-2, indicating that S. aureus inhibits autophagosome maturation and fusion with lysosomes. Eventually, S. aureus escape from autophagosomes into the cytoplasm, which results in caspase-independent host cell death. S. aureus strains deficient for agr, a global regulator of S. aureus virulence, were not targeted by autophagy and did not produce host-cell death. Autophagy induction by rapamycin restored both replication and cytotoxicity of agr-deficient S. aureus strains, indicating that an agr-regulated factor(s) is required for autophagy-mediated cytotoxicity. The results of this study suggest that rapid induction of autophagy is essential for S. aureus replication, escape into the cytoplasm, and host cell killing.     

Specific Behavior of Intracellular Streptococcus pyogenes That Has Undergone Autophagic Degradation Is Associated with Bacterial Streptolysin O and Host Small G Proteins Rab5 and Rab7

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLO-deficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells.     

Expression of listeriolysin O by intracellular Listeria monocytogenes following infection of lipopolysaccharide-treated or untreated J774.1 macrophage-like cells

Abstract Listeria monocytogenes replicates in a phagocytic cell following escape into the host cytoplasm. Listeriolysin O, secreted by L. monocytogenes , which belongs to the thiol-activated hemolysin family, is known to play an important role in the escape of the bacterium into the host cytoplasm. In this study, we demonstrated that expression of listeriolysin O by infecting L. monocytogenes was lightly induced in J774.1 macrophage-like cells pretreated with lipopolysaccharide, although the growth of the bacteria was suppressed. The number of viable L. monocytogenes decreased until 4 h post-infection and then increased between 4 and 8 h post-infection in untreated J774.1 host cells, but it decreased until 8 h post-infection in lipopolysaccharide-treated host cells. However, expression of listeriolysin O by L. monocytogenes was not induced in the untreated host cells, while it increased between 0 and 4 h post-infection in the lipopolysaccharide-treated host cells. Expression of listeriolysin O mRNA in the lipopolysaccharide-treated host cells was also induced at 2 h post-infection, suggesting that listeriolysin O was newly synthesized in the macrophage-like cells. These results suggest that macrophage activation induced with lipopolysaccharide could lead to the expression of the listeriolysin O gene and the synthesis of listeriolysin O protein under suppression of the intracellular growth of L. monocytogenes .     

The expression of alpha-haemolysin is required for Staphylococcus aureus phagosomal escape after internalization in CFT-1 cells

Staphylococcus aureus colonizes the lungs of cystic fibrosis patients and treatment with antibiotics usually results in recurrent and relapsing infections. We have shown that S. aureus can invade and replicate within a cystic fibrosis epithelial cell line (CFT-1), and that these internalized bacteria subsequently escape from the endocytic vesicle. The a ccessory g ene r egulator, agr , in S. aureus has been shown to control the expression of a large number of secreted toxins involved in virulence. Here we show that an agr mutant of S. aureus strain RN6390 was unable to escape from the endocytic vesicle after invasion of the CFT-1 cells using markers of vesicular trafficking (LAMP-1 and 2, LysoTracker and Vacuolar-ATPase). Trafficking analysis of live S. aureus which did not express alpha-haemolysin, a specific agr regulated toxin, revealed a defect in vesicular escape that was undistinguishable from the trafficking defect exhibited by the agr mutant. Furthermore, overexpression of alpha-haemolysin under an inducible promoter in an agr mutant of S. aureus partially restored the phagosome-escaping phenotype of an agr mutant. These results demonstrate that the expression of agr is required for vesicular escape, and that biologically active alpha-haemolysin is required for S. aureus escape from the endocytic vesicle into the cytosol of CFT-1 cells.     

QseC-mediated dephosphorylation of QseB is required for expression of genes associated with virulence in uropathogenic Escherichia coli

Bacteria sense environmental cues and regulate gene expression accordingly so as to persist in diverse niches. QseC is a membrane sensor kinase shown in enterohemorrhagic Escherichia coli to respond to host and bacterial signals by phosphorylating the QseB response regulator at residue D51, resulting in QseB activation and presumably upregulation of virulence genes. We studied QseBC in uropathogenic E. coli (UPEC). UPEC establish infection by colonizing and invading bladder cells. After invasion, UPEC can escape into the cytoplasm where they can form intracellular bacterial communities. Deletion of qseC significantly attenuated intracellular bacterial community formation and virulence, whereas paradoxically qseB deletion did not impact pathogenesis. We found that QseB upregulates its own expression in the qseC mutant, arguing that it is activated even in the absence of QseC. However, expression of QseB, but not a QseB_D51A mutant, in the absence of QseC resulted in downregulation of type 1 pili, curli and flagella. We observed similar phenotypes with enterohemorrhagic E. coli , showing that this is not a UPEC-specific phenomenon. Target gene expression is restored when QseC is present. We discovered that QseC has phosphatase activity required for QseB dephosphorylation. Thus, the QseC phosphatase capacity is critical for modulating QseB activity and subsequent gene expression.     

The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells

Yersinia virulence is dependent on the expression of plasmid-encoded secreted proteins called Yops. After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm. Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways. In contrast to this active inhibition of phagocytosis by Yersinia , other pathogens such as Salmonella , Shigella , Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways. We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens. We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen. We then measured the level of bacterial invasion. Preinfection with Yersinia inhibited invasion of Edwardsiella , Shigella and Listeria , but not Salmonella . Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion. We correlated the inhibitory effect of the Yops with the inhibitory action of the cell-signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways. We also speculate that YopE affects the rho pathway.     

Spontaneous formation of IpaB ion channels in host cell membranes reveals how Shigella induces pyroptosis in macrophages

The Gram-negative bacterium Shigella flexneri invades the colonic epithelium and causes bacillary dysentery. S. flexneri requires the virulence factor invasion plasmid antigen B (IpaB) to invade host cells, escape from the phagosome and induce macrophage cell death. The mechanism by which IpaB functions remains unclear. Here, we show that purified IpaB spontaneously oligomerizes and inserts into the plasma membrane of target cells forming cation selective ion channels. After internalization, IpaB channels permit potassium influx within endolysosomal compartments inducing vacuolar destabilization. Endolysosomal leakage is followed by an ICE protease-activating factor-dependent activation of Caspase-1 in macrophages and cell death. Our results provide a mechanism for how the effector protein IpaB with its ion channel activity causes phagosomal destabilization and induces macrophage death. These data may explain how S. flexneri uses secreted IpaB to escape phagosome and kill the host cells during infection and, may be extended to homologs from other medically important enteropathogenic bacteria.     

A role for type I signal peptidase in Staphylococcus aureus quorum sensing

The Staphylococcus aureus Agr quorum-sensing system modulates the expression of extracellular virulence factors. The Agr system is controlled by an autoinducing peptide (AIP) molecule that is secreted during growth. In the AIP biosynthetic pathway, two proteolytic events are required to remove the leader and tail segments of AgrD, the peptide precursor of AIP. The only protein known to be involved in this pathway is AgrB, a membrane endopeptidase that removes the AgrD carboxy-tail. We designed a synthetic peptide substrate and developed an assay to detect peptidases that can remove the N-terminal leader of AIP. Several peptidase activities were detected in S. aureus extracts and these activities were present in both wild-type and agr mutant strains. Only one of these peptidases cleaved in the correct position and all properties of this enzyme were consistent with type I signal peptidase. Subsequent cloning and purification of the two known S. aureus signal peptidases, SpsA and SpsB, demonstrated that only SpsB catalysed this activity in vitro. To investigate the role of SpsB in AIP biosynthesis, SpsB peptide inhibitors were designed and characterized. The most effective inhibitor blocked SpsB activity in vitro and showed antibacterial activity against S. aureus. Importantly, the inhibitor reduced expression of an Agr-dependent reporter and inhibited AIP production in S. aureus, indicating a role for SpsB in quorum sensing.     

Influence of Sae‐regulated and Agr‐regulated factors on the escape of Staphylococcus aureus from human macrophages

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild‐type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol‐soluble modulins (PSMs) for phagosomal escape. Thus, Sae‐regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis‐related caspase 3, 7 or 8 nor to NLRP3‐dependent inflammasome activation.     

Host cell egress and invasion induce marked relocations of glycolytic enzymes in Toxoplasma gondii tachyzoites

Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K(+)] experienced during egress and invasion, a signal that requires changes of [Ca(2+)](c) in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites.     

An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals that is endemic in subtropical areas. B. pseudomallei is a facultative intracellular pathogen that may invade and survive within eukaryotic cells for prolonged periods. After internalization, the bacteria escape from endocytic vacuoles into the cytoplasm of infected cells and form membrane protrusions by inducing actin polymerization at one pole. It is believed that survival within phagocytic cells and cell-to-cell spread via actin protrusions is required for full virulence. We have studied the role of a putative type III protein secretion apparatus (Bsa) in the interaction between B. pseudomallei and host cells. The Bsa system is very similar to the Inv/Mxi-Spa type III secretion systems of Salmonella and Shigella. Moreover, B. pseudomallei encodes proteins that are very similar to Salmonella and Shigella Inv/Mxi-Spa secreted proteins required for invasion, escape from endocytic vacuoles, intercellular spread and pathogenesis. Antibodies to putative Bsa-secreted proteins were detected in convalescent serum from a melioidosis patient, suggesting that the system is functionally expressed in vivo. B. pseudomallei mutant strains lacking components of the Bsa secretion and translocation apparatus were constructed. The mutant strains exhibited reduced replication in J774.2 murine macrophage-like cells, an inability to escape from endocytic vacuoles and a complete absence of formation of membrane protrusions and actin tails. These findings indicate that the Bsa type III secretion system plays an essential role in modulating the intracellular behaviour of B. pseudomallei.     

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.     

A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.     

Staphylococcus aureus produces membrane-derived vesicles that induce host cell death

Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs) produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.     

Modeling the step of endosomal escape during cell infection by a nonenveloped virus

Widely disparate viruses enter the host cell through an endocytic pathway and travel the cytoplasm inside an endosome. For the viral genetic material to be delivered into the cytoplasm, these viruses have to escape the endosomal compartment, an event triggered by the conformational changes of viral endosomolytic proteins. We focus here on small nonenveloped viruses such as adeno-associated viruses, which contain few penetration proteins. The first time a penetration protein changes conformation defines the slowest timescale responsible for the escape. To evaluate this time, we construct what to our knowledge is a novel biophysical model based on a stochastic approach that accounts for the small number of proteins, the endosomal maturation, and the protease activation dynamics. We show that the escape time increases with the endosomal size, whereas decreasing with the number of viral particles inside the endosome. We predict that the optimal escape probability is achieved when the number of proteases in the endosome is in the range of 250-350, achieved for three viral particles.     

Shigella and autophagy

Bacterial invasion of eukaryotic cells, and host recognition and elimination of the invading bacteria, determines the fate of bacterial infection. Once inside mammalian cells, many pathogenic bacteria enter the host cytosol to escape from the lytic compartment and gain a replicative niche. Recent studies indicate that autophagy also recognizes intracellular bacteria. Although autophagy is a conserved membrane trafficking pathway in eukaryotic cells that sequesters undesirable or recyclable cytoplasmic components or organelles and delivers them to lysosomes, autophagy has recently been described as playing a pivotal role as an intracellular surveillance system for recognition and eradication of the pathogens that have invaded the cytoplasm. Indeed, unless they are able to circumvent entrapping by autophagosomes, bacteria ultimately undergo degradation by delivery into autolysosomes. In this review we discuss recent discoveries regarding Shigella strategies for infecting mammalian cells, and then focus on recent studies of an elegant bacterial survival strategy against autophagic degradation.