NLRP11 is a pattern recognition receptor for bacterial lipopolysaccharide in the cytosol of human macrophages.

Endotoxin-bacterial lipopolysaccharide (LPS)-is a driver of lethal infection sepsis through excessive activation of innate immune responses. When delivered to the cytosol of macrophages, cytosolic LPS (cLPS) induces the assembly of an inflammasome that contains caspases-4/5 in humans or caspase-11 in mice. Whereas activation of all other inflammasomes is triggered by sensing of pathogen products by a specific host cytosolic pattern recognition receptor protein, whether pattern recognition receptors for cLPS exist has remained unclear, because caspase-4, caspase-5, and caspase-11 bind and activate LPS directly in vitro. Here, we show that the primate-specific protein NLRP11 is a pattern recognition receptor for cLPS that is required for efficient activation of the caspase-4 inflammasome in human macrophages. In human macrophages, NLRP11 is required for efficient activation of caspase-4 during infection with intracellular Gram-negative bacteria or upon electroporation of LPS. NLRP11 could bind LPS and separately caspase-4, forming a high-molecular weight complex with caspase-4 in HEK293T cells. NLRP11 is present in humans and other primates but absent in mice, likely explaining why it has been overlooked in screens looking for innate immune signaling molecules, most of which have been carried out in mice. Our results demonstrate that NLRP11 is a component of the caspase-4 inflammasome activation pathway in human macrophages.

Comparative study of GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and Shigella flexneri highlights differences in GBP repertoire and in GBP1 motif requirements.

Guanylate-Binding Proteins are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key GBP feature is the formation of supramolecular GBP complexes on the bacterial surface. Such complexes are observed when GBP1 binds lipopolysaccharide (LPS) from Shigella and Salmonella and further recruits GBP2-4. Here, we compared GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and S. flexneri. Francisella novicida was coated by GBP1 and GBP2 and to a lower extent by GBP4 in human macrophages. Contrary to S. flexneri, F. novicida was not targeted by GBP3, a feature independent of T6SS effectors. Multiple GBP1 features were required to promote targeting to F. novicida while GBP1 targeting to S. flexneri was much more permissive to GBP1 mutagenesis suggesting that GBP1 has multiple domains that cooperate to recognize F. novicida atypical LPS. Altogether our results indicate that the repertoire of GBPs recruited onto specific bacteria is dictated by GBP-specific features and by specific bacterial factors that remain to be identified.

A patatin-like phospholipase mediates Rickettsia parkeri escape from host membranes.

Rickettsia species of the spotted fever group are arthropod-borne obligate intracellular bacteria that can cause mild to severe human disease. These bacteria invade host cells, replicate in the cell cytosol, and spread from cell to cell. To access the host cytosol and avoid immune detection, they escape membrane-bound vacuoles by expressing factors that disrupt host membranes. Here, we show that a patatin-like phospholipase A2 enzyme (Pat1) facilitates Rickettsia parkeri infection by promoting escape from host membranes and cell-cell spread. Pat1 is important for infection in a mouse model and, at the cellular level, is crucial for efficiently escaping from single and double membrane-bound vacuoles into the host cytosol, and for avoiding host galectins that mark damaged membranes. Pat1 is also important for avoiding host polyubiquitin, preventing recruitment of autophagy receptor p62, and promoting actin-based motility and cell-cell spread.

Adaptations of intracellular bacteria to vacuolar or cytosolic niches.

Invasive bacteria colonise their host tissues by establishing niches inside eukaryotic cells, where they grow either in the cytosol or inside a specialised vacuole. These two distinct intracellular lifestyles both present benefits but also impose various constraints on pathogenic microorganisms, in terms of nutrient acquisition, space requirements, exposure to immune responses, and ability to disseminate. Here we review the major characteristics of cytosolic and vacuolar lifestyles and the strategies used by bacteria to overcome challenges specific to each compartment. Recent research providing evidence that these scenarios are not mutually exclusive is presented, with the dual lifestyles of two foodborne pathogens, Listeria monocytogenes and Salmonella Typhimurium, discussed in detail. Finally, we elaborate on the conceptual implications of polyvalence from the perspective of host-pathogen interactions.

Cytosolic detection of phagosomal bacteria-Mechanisms underlying PAMP exodus from the phagosome into the cytosol.

The metazoan innate immune system senses bacterial infections by detecting highly conserved bacterial molecules, termed pathogen-associated molecular patterns (PAMPs). PAMPs are detected by a variety of host pattern recognition receptors (PRRs), whose function is to coordinate downstream immune responses. PRR activities are, in part, regulated by their subcellular localizations. Accordingly, professional phagocytes can detect extracellular bacteria and their PAMPs via plasma membrane-oriented PRRs. Conversely, phagocytosed bacteria and their PAMPs are detected by transmembrane PRRs oriented toward the phagosomal lumen. Even though PAMPs are unable to passively diffuse across membranes, phagocytosed bacteria are also detected by PRRs localized within the host cell cytosol. This phenomenon is explained by phagocytosis of bacteria that specialize in phagosomal escape and cytosolic residence. Contrary to this cytosolic lifestyle, most bacteria studied to date spend their entire intracellular lifestyle contained within phagosomes, yet they also stimulate cytosolic PRRs. Herein, we will review our current understanding of how phagosomal PAMPs become accessible to cytosolic PRRs, as well as highlight knowledge gaps that should inspire future investigations.

Salmonella Infantis Delays the Death of Infected Epithelial Cells to Aggravate Bacterial Load by Intermittent Phosphorylation of Akt With SopB.

Salmonella Infantis has emerged as a major clinical pathogen causing gastroenteritis worldwide in recent years. As an intracellular pathogen, Salmonella has evolved to manipulate and benefit from the cell death signaling pathway. In this study, we discovered that S. Infantis inhibited apoptosis of infected Caco-2 cells by phosphorylating Akt. Notably, Akt phosphorylation was observed in a discontinuous manner: immediately 0.5 h after the invasion, then before peak cytosolic replication. Single-cell analysis revealed that the second phase was only induced by cytosolic hyper-replicating bacteria at 3-4 hpi. Next, Akt-mediated apoptosis inhibition was found to be initiated by Salmonella SopB. Furthermore, Akt phosphorylation increased mitochondrial localization of Bcl-2 to prevent Bax oligomerization on the mitochondrial membrane, maintaining the mitochondrial network homeostasis to resist apoptosis. In addition, S. Infantis induced pyroptosis, as evidenced by increased caspase-1 (p10) and GSDMS-N levels. In contrast, cells infected with the ΔSopB strain displayed faster but less severe pyroptosis and had less bacterial load. The results indicated that S. Infantis SopB-mediated Akt phosphorylation delayed pyroptosis, but aggravated its severity. The wild-type strain also caused more severe diarrhea and intestinal inflammatory damage than the ΔSopB strain in mice. These findings revealed that S. Infantis delayed the cells' death by intermittent activation of Akt, allowing sufficient time for replication, thereby causing more severe inflammation.

Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the epithelial cytosol, and the bacterial genes required for cytosolic colonization, remain largely unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes with cytosol-induced or vacuole-induced expression signatures. Using these genes as environmental biosensors, we defined that Salmonella is exposed to oxidative stress and iron and manganese deprivation in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS, mntH and sitA. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, showed increased expression in the cytosol compared to vacuole. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. This archetypical vacuole-adapted pathogen therefore requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.

A human apolipoprotein L with detergent-like activity kills intracellular pathogens.

Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.

Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.

SAC1 regulates autophagosomal phosphatidylinositol-4-phosphate for xenophagy-directed bacterial clearance.

Phosphoinositides are important molecules in lipid signaling, membrane identity, and trafficking that are spatiotemporally controlled by factors from both mammalian cells and intracellular pathogens. Here, using small interfering RNA (siRNA) directed against phosphoinositide kinases and phosphatases, we screen for regulators of the host innate defense response to intracellular bacterial replication. We identify SAC1, a transmembrane phosphoinositide phosphatase, as an essential regulator of xenophagy. Depletion or inactivation of SAC1 compromises fusion between Salmonella-containing autophagosomes and lysosomes, leading to increased bacterial replication. Mechanistically, the loss of SAC1 results in aberrant accumulation of phosphatidylinositol-4-phosphate [PI(4)P] on Salmonella-containing autophagosomes, thus facilitating recruitment of SteA, a PI(4)P-binding Salmonella effector protein, which impedes lysosomal fusion. Replication of Salmonella lacking SteA is suppressed by SAC-1-deficient cells, however, demonstrating bacterial adaptation to xenophagy. Our findings uncover a paradigm in which a host protein regulates the level of its substrate and impairs the function of a bacterial effector during xenophagy.

Intracellular localisation of Mycobacterium tuberculosis affects efficacy of the antibiotic pyrazinamide.

To be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of the pathogen, Mycobacterium tuberculosis. However, how host cell microenvironments affect antibiotic accumulation and efficacy remains unclear. Here, we use correlative light, electron, and ion microscopy to investigate how various microenvironments within human macrophages affect the activity of pyrazinamide (PZA), a key antibiotic against TB. We show that PZA accumulates heterogeneously among individual bacteria in multiple host cell environments. Crucially, PZA accumulation and efficacy is maximal within acidified phagosomes. Bedaquiline, another antibiotic commonly used in combined TB therapy, enhances PZA accumulation via a host cell-mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy. Our results may explain the potent in vivo efficacy of PZA, compared to its modest in vitro activity, and its critical contribution to TB combination chemotherapy.

Dot/Icm-Dependent Restriction of Legionella pneumophila within Neutrophils.

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is essential for lysosomal evasion and permissiveness of macrophages for intracellular proliferation of the pathogen. In contrast, we show that polymorphonuclear cells (PMNs) respond to a functional Dot/Icm system through rapid restriction of L. pneumophila. Specifically, we show that the L. pneumophila T4SS-injected amylase (LamA) effector catalyzes rapid glycogen degradation in the PMNs cytosol, leading to cytosolic hyperglucose. Neutrophils respond through immunometabolic reprogramming that includes upregulated aerobic glycolysis. The PMNs become activated with spatial generation of intracellular reactive oxygen species within the Legionella-containing phagosome (LCP) and fusion of specific and azurophilic granules to the LCP, leading to rapid restriction of L. pneumophila. We conclude that in contrast to macrophages, PMNs respond to a functional Dot/Icm system, and specifically to the effect of the injected amylase effector, through rapid engagement of major microbicidal processes and rapid restriction of the pathogen. IMPORTANCE Legionella pneumophila is commonly found in aquatic environments and resides within a wide variety of amoebal hosts. Upon aerosol transmission to humans, L. pneumophila invades and replicates with alveolar macrophages, causing pneumonia designated Legionnaires' disease. In addition to alveolar macrophages, neutrophils infiltrate into the lungs of infected patients. Unlike alveolar macrophages, neutrophils restrict and kill L. pneumophila, but the mechanisms were previously unclear. Here, we show that the pathogen secretes an amylase (LamA) enzyme that rapidly breakdowns glycogen stores within neutrophils, and this triggers increased glycolysis. Subsequently, the two major killing mechanisms of neutrophils, granule fusion and production of reactive oxygen species, are activated, resulting in rapid killing of L. pneumophila.

Cytosolic replication in epithelial cells fuels intestinal expansion and chronic fecal shedding of Salmonella Typhimurium.

Persistent and intermittent fecal shedding, hallmarks of Salmonella infections, are important for fecal-oral transmission. In the intestine, Salmonella enterica serovar Typhimurium (STm) actively invades intestinal epithelial cells (IECs) and survives in the Salmonella-containing vacuole (SCV) and the cell cytosol. Cytosolic STm replicate rapidly, express invasion factors, and induce extrusion of infected epithelial cells into the intestinal lumen. Here, we engineered STm that self-destruct in the cytosol (STmCytoKill), but replicates normally in the SCV, to examine the role of cytosolic STm in infection. Intestinal expansion and fecal shedding of STmCytoKill are impaired in mouse models of infection. We propose a model whereby repeated rounds of invasion, cytosolic replication, and release of invasive STm from extruded IECs fuels the high luminal density required for fecal shedding.

IL-1R1-Dependent Signals Improve Control of Cytosolic Virulent Mycobacteria In Vivo.

Mycobacterium tuberculosis infections claim more than a million lives each year, and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from phagolysosomes to the cytosol upon phagocytosis by macrophages. Translocation from the phagolysosome to the cytosol is an ESX-1-dependent process, as previously shown in vitro Here, we show that in vivo, mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillos, zebrafish, and patient material infected with M. tuberculosis, M. marinum, or M. leprae In contrast, when innate or adaptive immunity was compromised, as in severe combined immunodeficiency (SCID) or interleukin-1 receptor 1 (IL-1R1)-deficient mice, significant numbers of cytosolic M. tuberculosis bacilli were detected in the lungs of infected mice. Taken together, in vivo, translocation to the cytosol of M. tuberculosis is controlled by adaptive immune responses as well as IL-1R1-mediated signals.IMPORTANCE For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is subcellular localization, as M. tuberculosis can translocate from phagolysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study, we establish that high-level cytosolic escape of mycobacteria can indeed occur in vivo but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in the clearance of cells containing cytosolic mycobacteria connects two important fields, cell biology and immunology, which is vital for the understanding of the pathology of M. tuberculosis.

Type I IFNs facilitate innate immune control of the opportunistic bacteria Burkholderia cenocepacia in the macrophage cytosol.

The mammalian immune system is constantly challenged by signals from both pathogenic and non-pathogenic microbes. Many of these non-pathogenic microbes have pathogenic potential if the immune system is compromised. The importance of type I interferons (IFNs) in orchestrating innate immune responses to pathogenic microbes has become clear in recent years. However, the control of opportunistic pathogens-and especially intracellular bacteria-by type I IFNs remains less appreciated. In this study, we use the opportunistic, Gram-negative bacterial pathogen Burkholderia cenocepacia (Bc) to show that type I IFNs are capable of limiting bacterial replication in macrophages, preventing illness in immunocompetent mice. Sustained type I IFN signaling through cytosolic receptors allows for increased expression of autophagy and linear ubiquitination mediators, which slows bacterial replication. Transcriptomic analyses and in vivo studies also show that LPS stimulation does not replicate the conditions of intracellular Gram-negative bacterial infection as it pertains to type I IFN stimulation or signaling. This study highlights the importance of type I IFNs in protection against opportunistic pathogens through innate immunity, without the need for damaging inflammatory responses.

Cytoplasmic Incompatibility Variations in Relation with Wolbachia cid Genes Divergence in Culex pipiens.

In arthropods, Wolbachia endosymbionts induce conditional sterility, called cytoplasmic incompatibility (CI), resulting from embryonic lethality. CI penetrance (i.e., embryonic death rate) varies depending on host species and Wolbachia strains involved. All Culex pipiens mosquitoes are infected by the endosymbiotic alphaproteobacteria Wolbachia wPip. CI in Culex, characterized as a binary "compatible/incompatible" phenomenon, revealed an unparalleled diversity of patterns linked to the amplification-diversification of cidA and cidB genes. Here, we accurately studied CI penetrance variations in the light of cid genes divergence by generating a C. pipiens compatibility matrix between 11 lines hosting different phylogenetic wPip groups and exhibiting distinct cid gene repertoires. We showed, as expected, that crosses involving wPip from the same group were mostly compatible. In contrast, only 22% of the crosses involving different wPip groups were compatible, while 54% were fully incompatible. For the remaining 24% of the crosses, "intermediate" compatibilities were reported, and a cytological observation of the first zygotic division confirmed the occurrence of "canonical" CI phenotypes in a fraction of the eggs. Backcross experiments demonstrated that intermediate compatibilities were not linked to host genetic background but to the Wolbachia strains involved. This previously unstudied intermediate penetrance CI was more severe and frequent in crosses involving wPip-IV strains exhibiting cid variants markedly divergent from other wPip groups. Our data demonstrate that CI is not always a binary compatible/incompatible phenomenon in C. pipiens but that intermediate compatibilities putatively resulting from partial mismatch due to Cid proteins divergence exist in this species complex.IMPORTANCECulex pipiens mosquitoes are infected with wPip. These endosymbionts induce a conditional sterility called CI resulting from embryonic deaths, which constitutes a cornerstone for Wolbachia antivectorial methods. Recent studies revealed that (i) two genes, cidA and cidB, are central in Wolbachia-CI mechanisms, and (ii) compatibility versus incompatibility between mosquito lines depends on the wPip phylogenetic groups at play. Here, we studied CI variations in relation to wPip groups and cid genes divergence. We showed, as expected, that the crosses involving wPip from the same group were compatible. In contrast, 78% of the crosses involving different wPip groups were partially or fully incompatible. In such crosses, we reported defects during the first zygotic division, a hallmark of CI. We showed that CI was more severe and frequent in crosses involving wPip-IV strains exhibiting cid variants, which markedly diverge from those of other wPip groups.

The Enemy of My Enemy: New Insights Regarding Bacteriophage-Mammalian Cell Interactions.

Bacteriophages (phages) are the most abundant biological entity in the human body, but until recently the role that phages play in human health was not well characterized. Although phages do not cause infections in human cells, phages can alter the severity of bacterial infections by the dissemination of virulence factors amongst bacterial hosts. Recent studies, made possible with advances in genome engineering and microscopy, have uncovered a novel role for phages in the human body - the ability to modulate the physiology of the mammalian cells that can harbor intracellular bacteria. In this review, we synthesize key results on how phages traverse through mammalian cells - including uptake, distribution, and interaction with intracellular receptors - highlighting how these steps in turn influence host cell killing of bacteria. We discuss the implications of the growing field of phage-mammalian cell interactions for phage therapy.

Cytosolic Sensors for Pathogenic Viral and Bacterial Nucleic Acids in Fish.

Recognition of the non-self signature of invading pathogens is a crucial step for the initiation of the innate immune mechanisms of the host. The host response to viral and bacterial infection involves sets of pattern recognition receptors (PRRs), which bind evolutionarily conserved pathogen structures, known as pathogen-associated molecular patterns (PAMPs). Recent advances in the identification of different types of PRRs in teleost fish revealed a number of cytosolic sensors for recognition of viral and bacterial nucleic acids. These are DExD/H-box RNA helicases including a group of well-characterized retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and non-RLR DExD/H-box RNA helicases (e.g., DDX1, DDX3, DHX9, DDX21, DHX36 and DDX41) both involved in recognition of viral RNAs. Another group of PRRs includes cytosolic DNA sensors (CDSs), such as cGAS and LSm14A involved in recognition of viral and intracellular bacterial dsDNAs. Moreover, dsRNA-sensing protein kinase R (PKR), which has a role in antiviral immune responses in higher vertebrates, has been identified in fish. Additionally, fish possess a novel PKR-like protein kinase containing Z-DNA binding domain, known as PKZ. Here, we review the current knowledge concerning cytosolic sensors for recognition of viral and bacterial nucleic acids in teleosts.

Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles.

Pathogenic bacteria enter the cytosol of host cells through uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the vacuolar membrane [1]. Bacterial invaders are sensed either directly, through cytosolic pattern-recognition receptors specific for bacterial ligands, or indirectly, through danger receptors that bind host molecules displayed in an abnormal context, for example, glycans on damaged BCVs [2-4]. In contrast to damage caused by Listeria monocytogenes, a Gram-positive bacterium, BCV rupture by Gram-negative pathogens such as Shigella flexneri or Salmonella Typhimurium remains incompletely understood [5, 6]. The latter may cause membrane damage directly, when inserting their Type Three Secretion needles into host membranes, or indirectly through translocated bacterial effector proteins [7-9]. Here, we report that sphingomyelin, an abundant lipid of the luminal leaflet of BCV membranes, and normally absent from the cytosol, becomes exposed to the cytosol as an early predictive marker of BCV rupture by Gram-negative bacteria. To monitor subcellular sphingomyelin distribution, we generated a live sphingomyelin reporter from Lysenin, a sphingomyelin-specific toxin from the earthworm Eisenia fetida [10, 11]. Using super resolution live imaging and correlative light and electron microscopy (CLEM), we discovered that BCV rupture proceeds through two distinct successive stages: first, sphingomyelin is gradually translocated into the cytosolic leaflet of the BCV, invariably followed by cytosolic exposure of glycans, which recruit galectin-8, indicating bacterial entry into the cytosol. Exposure of sphingomyelin on BCVs may therefore act as an early danger signal alerting the cell to imminent bacterial invasion.