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.     

Nucleolin, a shuttle protein promoting infection of human monocytes by Francisella tularensis

Background

Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells.

Methodology/Principal Findings

Here, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with an iglC mutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant.

Conclusions/Significance

We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages.     

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.     

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes.     

Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern,phagosome maturation,and escape to the cytosol

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication‐permissive compartment termed the Mycobacterium‐containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid‐borne genes. Fluorescence microscopy of M. marinum‐infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol‐3‐phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V‐ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.     

Deletion of the membrane protein Lmo0412 increases the virulence of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that causes gastroenteritis, meningitis, encephalitis and maternofetal infections. 20–30% of eubacterial ORFs are predicted to encode membrane proteins. The bacterial cytoplasmic membrane is a macromolecular structure, which plays a key role for the pathogenesis. Despite this, little knowledge exists regarding the function of cytoplasmic membrane proteins of Listeria during infection. Here, we investigated a predicted membrane protein of the pathogen L. monocytogenes, Lmo0412, of unknown function. Lmo0412 is only present in the Listeria genus and low conserved in the non-pathogenic species L. innocua. Bacterial fractionation and western blot analyses showed that Lmo0412 was only detectable in the membrane of L. monocytogenes EGDe during logarithmic growth phase. lmo0412 expression in L. monocytogenes was down-regulated during in vitro infection of JEG-3 epithelial cells. An L. monocytogenes mutant deficient in this membrane protein showed increased invasion of Caco-2 and NRK-49F host cells using in vitro infection models. Moreover, the lack of Lmo0412 in this deletion mutant increased the viable bacteria counts in the spleen and liver of mice compared to the wild type strain. Taken together, these data suggest a selective advantage conferred by the absence of Lmo0412 for the virulence of L. monocytogenes.     

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.     

Epigallocatechin gallate inhibits intracellular survival of Listeria monocytogenes in macrophages

Epigallocatechin gallate (EGCg), the major tea catechin, is known as a potent anti-microbial and anti-tumor compound. The effects of EGCg on host defense mechanisms against Listeria monocytogenes infection were examined in vitro using mouse peritoneal exudate cells. The study showed that EGCg inhibited the intracellular growth of L. monocytogenes in macrophages. The enhancement of in vitro anti-L. monocytogenes activity by EGCg is not due to the modulation of reactive oxygen intermediates or the production of reactive nitrogen intermediates but due to the inhibition of its escaping from the phagosome into cytosolic space. Anti-L. monocytogenes of EGCg is through the inhibition of hemolytic and cholesterol-binding activity of listeriolysin O, which usually disrupts the phagosomal membrane in the escaping phase of L. monocytogenes.     

Acquired resistance of Listeria monocytogenes in and escaped from liver parenchymal cells to gentamicin is caused by being coated with their plasma membrane

After systemic infection, a majority of Listeria monocytogenes invade liver parenchymal cells (LPC), replicate therein and spread to neighboring cells, suggesting that 3 different types of L. monocytogenes exist in the liver: L. monocytogenes being unable to invade LPC, residing in LPC, and escaped from infected LPC. Although listeriolysin O (LLO) participates in escape of L. monocytogenes from macrophages and L. monocytogenes is susceptible to gentamicin (Gm), it remains elusive whether LLO participates in invasion/escape of L. monocytogenes into/from LPC, and whether L. monocytogenes in/escaped from LPC are susceptible to Gm. In the present study, we examined whether LLO is involved in invasion/escape of L. monocytogenes into/from LPC and whether L. monocytogenes in/escaped from LPC are susceptible to Gm. Invasion/escape of L. monocytogenes were found in LPC lines regardless of LLO expression, and L. monocytogenes in/escaped from LPC lines showed resistance to Gm. L. monocytogenes escaped from LPC lines were coated with their plasma membrane and the acquired resistance to Gm was abrogated by saponin. Our results indicate that invasion/escape of L. monocytogenes into/from LPC occur independently of LLO, and suggest that the acquired resistance of L. monocytogenes in/escaped from LPC to Gm is caused by being coated with their plasma membrane.     

Strain-Specific Interactions of Listeria monocytogenes with the Autophagy System in Host Cells

Listeria monocytogenes is an intracellular bacterial pathogen that can replicate in the cytosol of host cells. These bacteria undergo actin-based motility in the cytosol via expression of ActA, which recruits host actin-regulatory proteins to the bacterial surface. L. monocytogenes is thought to evade killing by autophagy using ActA-dependent mechanisms. ActA-independent mechanisms of autophagy evasion have also been proposed, but remain poorly understood. Here we examined autophagy of non-motile (ΔactA) mutants of L. monocytogenes strains 10403S and EGD-e, two commonly studied strains of this pathogen. The ΔactA mutants displayed accumulation of ubiquitinated proteins and p62/SQSTM1 on their surface. However, only strain EGD-e ΔactA displayed colocalization with the autophagy marker LC3 at 8 hours post infection. A bacteriostatic agent (chloramphenicol) was required for LC3 recruitment to 10403S ΔactA, suggesting that these bacteria produce a factor for autophagy evasion. Internalin K was proposed to block autophagy of L. monocytogenes in the cytosol of host cells. However, deletion of inlK in either the wild-type or ΔactA background of strain 10403S had no impact on autophagy evasion by bacteria, indicating it does not play an essential role in evading autophagy. Replication of ΔactA mutants of strain EGD-e and 10403S was comparable to their parent wild-type strain in macrophages. Thus, ΔactA mutants of L. monocytogenes can block killing by autophagy at a step downstream of protein ubiquitination and, in the case of strain EGD-e, downstream of LC3 recruitment to bacteria. Our findings highlight the strain-specific differences in the mechanisms that L. monocytogenes uses to evade killing by autophagy in host cells.     

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.     

Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving—as recently confirmed—escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.     

Host Factors Required for Modulation of Phagosome Biogenesis and Proliferation of Francisella tularensis within the Cytosol

Francisella tularensis is a highly infectious facultative intracellular bacterium that can be transmitted between mammals by arthropod vectors. Similar to many other intracellular bacteria that replicate within the cytosol, such as Listeria, Shigella, Burkholderia, and Rickettsia, the virulence of F. tularensis depends on its ability to modulate biogenesis of its phagosome and to escape into the host cell cytosol where it proliferates. Recent studies have identified the F. tularensis genes required for modulation of phagosome biogenesis and escape into the host cell cytosol within human and arthropod-derived cells. However, the arthropod and mammalian host factors required for intracellular proliferation of F. tularensis are not known. We have utilized a forward genetic approach employing genome-wide RNAi screen in Drosophila melanogaster-derived cells. Screening a library of ∼21,300 RNAi, we have identified at least 186 host factors required for intracellular bacterial proliferation. We silenced twelve mammalian homologues by RNAi in HEK293T cells and identified three conserved factors, the PI4 kinase PI4KCA, the ubiquitin hydrolase USP22, and the ubiquitin ligase CDC27, which are also required for replication in human cells. The PI4KCA and USP22 mammalian factors are not required for modulation of phagosome biogenesis or phagosomal escape but are required for proliferation within the cytosol. In contrast, the CDC27 ubiquitin ligase is required for evading lysosomal fusion and for phagosomal escape into the cytosol. Although F. tularensis interacts with the autophagy pathway during late stages of proliferation in mouse macrophages, this does not occur in human cells. Our data suggest that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis. Our data will facilitate deciphering molecular ecology, patho-adaptation of F. tularensis to the arthropod vector and its role in bacterial ecology and patho-evolution to infect mammals.     

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.     

The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection

Phagocytosis is a key process in innate immunity and homeostasis. After particle uptake, newly formed phagosomes mature by acquisition of endolysosomal enzymes. Macrophage activation by interferon gamma (IFN‐γ) increases microbicidal activity, but delays phagosomal maturation by an unknown mechanism. Using quantitative proteomics, we show that phagosomal proteins harbour high levels of typical and atypical ubiquitin chain types. Moreover, phagosomal ubiquitylation of vesicle trafficking proteins is substantially enhanced upon IFN‐γ activation of macrophages, suggesting a role in regulating phagosomal functions. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN‐γ activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ligase activity enhanced phagosomal maturation and increased cytokine responses to bacterial infection, suggesting that both innate immune signalling from the phagosome and phagolysosomal trafficking are controlled through ubiquitylation. RNF115 knock‐out mice show less tissue damage in response to S. aureus infection, indicating a role of RNF115 in inflammatory responses in vivo. In conclusion, RNF115 and phagosomal ubiquitylation are important regulators of innate immune functions during bacterial infections.     

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.