Salmonella-containing Vacuole Research Articles

Sorting nexin 3 (SNX3) is a component of a tubular endosomal network induced by Salmonella and involved in maturation of the Salmonella-containing vacuole

Salmonella enterica serovar Typhimurium is an intracellular pathogen that grows within a modified endomembrane compartment, the Salmonella-containing vacuole (SCV). Maturation of nascent SCVs involves the recruitment of early endosome markers and the remodelling of phosphoinositides at the membrane of the vacuole, in particular the production of phosphatidylinositol 3-phosphate [PI(3)P]. Sorting nexins (SNXs) are a family of proteins characterized by the presence of a phox homology (PX) domain that binds to phosphoinositides and are involved in intracellular trafficking in eukaryotic cells. We therefore studied whether sorting nexins, particularly sorting nexin 3 (SNX3), play a role in Salmonella infection. We found that SNX3 transiently localized to SCVs at early times post invasion (10 min) and presented a striking tubulation phenotype in the vicinity of SCVs at later times (30-60 min). The bacterial effector SopB, which is known to promote PI(3)P production on SCVs, was required for the formation of SNX3 tubules. In addition, RAB5 was also required for the formation of SNX3 tubules. Depletion of SNX3 by siRNA impaired RAB7 and LAMP1 recruitment to the SCV. Moreover, the formation of Salmonella-induced filaments (Sifs) was altered by SNX3 knock-down. Therefore, SNX3 plays a significant role in regulating the maturation of SCVs.

The Virulence Protein SopD2 Regulates Membrane Dynamics of Salmonella-Containing Vacuoles

Salmonella enterica serovar Typhimurium is a Gram-negative bacterial pathogen causing gastroenteritis in humans and a systemic typhoid-like illness in mice. The capacity of Salmonella to cause diseases relies on the establishment of its intracellular replication niche, a membrane-bound compartment named the Salmonella-containing vacuole (SCV). This requires the translocation of bacterial effector proteins into the host cell by type three secretion systems. Among these effectors, SifA is required for the SCV stability, the formation of Salmonella-induced filaments (SIFs) and plays an important role in the virulence of Salmonella. Here we show that the effector SopD2 is responsible for the SCV instability that triggers the cytoplasmic release of a sifA − mutant. Deletion of sopD2 also rescued intra-macrophagic replication and increased virulence of sifA− mutants in mice. Membrane tubular structures that extend from the SCV are the hallmark of Salmonella-infected cells. Until now, these unique structures have not been observed in the absence of SifA. The deletion of sopD2 in a sifA− mutant strain re-established membrane trafficking from the SCV and led to the formation of new membrane tubular structures, the formation of which is dependent on other Salmonella effector(s). Taken together, our data demonstrate that SopD2 inhibits the vesicular transport and the formation of tubules that extend outward from the SCV and thereby contributes to the sifA− associated phenotypes. These results also highlight the antagonistic roles played by SopD2 and SifA in the membrane dynamics of the vacuole, and the complex actions of SopD2, SifA, PipB2 and other unidentified effector(s) in the biogenesis and maintenance of the Salmonella replicative niche.

The Phosphoinositide Phosphatase SopB Manipulates Membrane Surface Charge and Trafficking of the Salmonella-Containing Vacuole

Shifts in electrostatic surface charge of membranes have recently been highlighted as a significant factor contributing to protein targeting to the plasma membrane and nascent phagosomes. Intracellular, vacuole-adapted pathogens may also regulate surface charge of their vacuoles to establish a replicative niche. Since Salmonella enterica serovar Typhimurium controls trafficking of the Salmonella-containing vacuole (SCV) and inhibits its fusion with lysosomes, we investigated the contribution of surface charge to this process. Using recently developed fluorescent biosensors, we show that the bacterial phosphoinositide phosphatase SopB controls membrane surface charge of nascent SCVs by reducing levels of negatively charged lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine. This SopB activity results in dissociation of a number of host-cell endocytic trafficking proteins from this compartment and inhibits SCV-lysosome fusion. Moreover, inducible reduction of negative charge rescues DeltasopB bacteria-containing SCVs from fusion with lysosomes. These results reveal a membrane-charge-based mechanism used by S. Typhimurium to control SCV maturation.

Carbon Metabolism of Enterobacterial Human Pathogens Growing in Epithelial Colorectal Adenocarcinoma (Caco-2) Cells

Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58) and Salmonella enterica Serovar Typhimurium (Stm 14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U-13C6]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The 13C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C3-compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C3-substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of 13C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens.

Salmonella vacuole maturation: PIKfyve leads the way

In this issue of The EMBO Journal, a study from Rohan Teasdale’s laboratory provides evidence that PIKfyve, a PtdIns D5-kinase, is required for maturation of Salmonellacontaining vacuole (SCV) and Salmonella replication. This report sheds light on a novel mechanism that mediates fusion of both macropinosomes and SCV with lysosomal/ late endosomal organelles.

Inhibition of the PtdIns(5) kinase PIKfyve disrupts intracellular replication of Salmonella

3-phosphorylated phosphoinositides (3-PtdIns) orchestrate endocytic trafficking pathways exploited by intracellular pathogens such as Salmonella to gain entry into the cell. To infect the host, Salmonellae subvert its normal macropinocytic activity, manipulating the process to generate an intracellular replicative niche. Disruption of the PtdIns(5) kinase, PIKfyve, be it by interfering mutant, siRNA-mediated knockdown or pharmacological means, inhibits the intracellular replication of Salmonella enterica serovar typhimurium in epithelial cells. Monitoring the dynamics of macropinocytosis by time-lapse 3D (4D) videomicroscopy revealed a new and essential role for PI(3,5)P(2) in macropinosome-late endosome/lysosome fusion, which is distinct from that of the small GTPase Rab7. This PI(3,5)P(2)-dependent step is required for the proper maturation of the Salmonella-containing vacuole (SCV) through the formation of Salmonella-induced filaments (SIFs) and for the engagement of the Salmonella pathogenicity island 2-encoded type 3 secretion system (SPI2-T3SS). Finally, although inhibition of PIKfyve in macrophages did inhibit Salmonella replication, it also appears to disrupt the macrophage's bactericidal response.

Imaging type-III secretion reveals dynamics and spatial segregation of Salmonella effectors

The type-III secretion system (T3SS) enables gram-negative bacteria to inject effector proteins into eukaryotic host cells. Upon entry, T3SS effectors work cooperatively to reprogram host cells, enabling bacterial survival. Progress in understanding when and where effectors localize within host cells has been hindered by a dearth of tools to study these proteins in the native cellular environment. We report a method to label and track T3SS effectors during infection using a split-GFP system. The breadth of this technique is demonstrated by labeling three effectors from Salmonella (PipB2, SteA, and SteC) and characterizing their localizations within host cells. PipB2 displays highly dynamic behavior on tubules emanating from the Salmonella containing vacuole labeled with both endo- and exocytic markers. SteA is preferentially enriched on tubules localizing with Golgi markers. This segregation suggests effector targeting and localization may play a functional role during infection.

The Role of SsrB And H-NS in Transcription Activation and Silencing/Anti-Silencing During Salmonella Pathogenesis

In bacterial pathogenesis, virulence gene regulation is controlled by two-component regulatory systems. In Escherichia coli, the EnvZ/OmpR two-component system regulates expression of outer membrane proteins, but in Salmonella enterica, OmpR activates transcription of the SsrA/B two-component system located on pathogenicity island 2 (SPI-2). SsrB controls expression of a type three secretory system whose effectors modify the vacuolar membrane and prevent its degradation via the endocytic pathway. Vacuolar modification enables Salmonella to survive and replicate in the macrophage phagosome and disseminate to the liver and spleen to cause systemic infection. The signals that activate EnvZ and SsrA are unknown, but are related to the acidic pH of the vacuole. SsrB binds to regions of DNA that are AT rich, with poor sequence conservation (Liao et al., J Biol Chem 284: 12008-12019, 2009). Although SsrB is a major virulence regulator in Salmonella, very little is known regarding how it binds DNA and activates transcription. Pathogenicity island genes are silenced by the heat-stable protein H-NS (Lucchini et al., PLoS Pathog 2: e81, 2006; Navarre et al., Science 313: 236-238, 2006). How transcription factors counter or relieve H-NS silencing is presently a major focus of study. The sifA gene is located outside of SPI-2 and encodes a product required for maintenance of the Salmonella-containing vacuole, involving formation of Salmonella-induced filaments or Sifs (Stein et al., Mol Microbiol 20: 151-164, 1996). SsrB directly activates expression of the sifA gene and relieves H-NS silencing to activate transcription. We used atomic force microscopy and single molecule experiments to examine H-NS/DNA interactions and the effect of SsrB on H-NS/DNA binding. Our recent findings will be discussed. Supported by NIH GM-058746, VA BX000372-01 to LJK and the RCE in Mechanobiology, National University of Singapore, Singapore.

Functional dissection of translocon proteins of the Salmonella Pathogenicity Island 2-encoded type III secretion system

BackgroundType III secretion systems (T3SS) are essential virulence factors of most Gram-negative bacterial pathogens. T3SS deliver effector proteins directly into the cytoplasm of eukaryotic target cells and for this function, the insertion of a subset of T3SS proteins into the target cell membrane is important. These proteins form hetero-oligomeric pores acting as translocon for the delivery of effector proteins. Salmonella enterica is a facultative intracellular pathogen that uses the Salmonella Pathogenicity Island 2 (SPI2)-encoded T3SS to manipulate host cells in order to survive and proliferate within the Salmonella-containing vacuole of host cells. Previous work showed that SPI2-encoded SseB, SseC and SseD act to form the translocon of the SPI2-T3SS.ResultsHere we investigated the structural requirements of SseB and SseD to form a functional translocon. Based on bioinformatic predictions, deletional analyses of SseB and SseD were performed and the effect on secretion by the T3SS, formation of a translocon, translocation of effector proteins and intracellular replication was investigated. Our data showed that both SseB and SseD are very sensitive towards alterations of the primary structure of the proteins. Although proteins encoded by mutant alleles were still secreted, we observed that all mutations resulted in a loss of function of the SPI2-T3SS.ConclusionThese observations indicate that translocon proteins of the SPI2-T3SS are highly evolved towards the formation of multi-subunit complex in the host cell membrane. Structural alterations are not tolerated and abrogate translocon function.

Interaction between the SifA Virulence Factor and Its Host Target SKIP Is Essential for Salmonella Pathogenesis

SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a DeltasifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.

Division of theSalmonella-Containing Vacuole and Depletion of Acidic Lysosomes inSalmonella-Infected Host Cells Are Novel Strategies ofSalmonella entericaTo Avoid Lysosomes

Salmonella has evolved several strategies to counteract intracellular microbicidal agents like reactive oxygen and nitrogen species. However, it is not yet clear how Salmonella escapes lysosomal degradation. Some studies have demonstrated that Salmonella can inhibit phagolysosomal fusion, whereas other reports have shown that the Salmonella-containing vacuole (SCV) fuses/interacts with lysosomes. Here, we have addressed this issue from a different perspective by investigating if the infected host cell has a sufficient quantity of lysosomes to target Salmonella. Our results suggest that SCVs divide along with Salmonella, resulting in a single bacterium per SCV. As a consequence, the SCV load per cell increases with the division of Salmonella inside the host cell. This demands more investment from the host cell to counteract Salmonella. Interestingly, we observed that Salmonella infection decreases the number of acidic lysosomes inside the host cell both in vitro and in vivo. These events potentially result in a condition in which an infected cell is left with insufficient acidic lysosomes to target the increasing number of SCVs, which favors the survival and proliferation of Salmonella inside the host cell.

Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane

The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K6R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K6R mutant. At later times, fewer SCV were decorated with SopB-K6R compared with SopB. Instead SopB-K6R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.

SCAMP3 is a component of theSalmonella-induced tubular network and reveals an interaction between bacterial effectors and post-Golgi trafficking

Salmonella enterica are facultative intracellular bacterial pathogens that proliferate within host cells in a membrane-bounded compartment, the Salmonella-containing vacuole (SCV). Intracellular replication of Salmonella is mediated by bacterial effectors translocated on to the cytoplasmic face of the SCV membrane by a type III secretion system. Some of these effectors manipulate the host endocytic pathway, resulting in the formation in epithelial cells of tubules enriched in late endosomal markers, known as Salmonella-induced filaments (SIFs). However, much less is known about possible interference of Salmonella with the secretory pathway. Here, a small-interference RNA screen revealed that secretory carrier membrane proteins (SCAMPs) 2 and 3 contribute to the maintenance of SCVs in the Golgi region of HeLa cells. This is likely to reflect a function of SCAMPs in vacuolar membrane dynamics. Moreover, SCAMP3, which accumulates on the trans-Golgi network in uninfected cells, marked tubules induced by Salmonella effectors that overlapped with SIFs but which also comprised distinct tubules lacking late endosomal proteins. We propose that SCAMP3 tubules reflect a manipulation of specific post-Golgi trafficking that might allow Salmonella to acquire nutrients and membrane, or to control host immune responses.

Glucose and Glycolysis Are Required for the Successful Infection of Macrophages and Mice by Salmonella enterica Serovar Typhimurium

Salmonella is a widespread zoonotic enteropathogen that causes gastroenteritis and fatal typhoidal disease in mammals. During systemic infection of mice, Salmonella enterica serovar Typhimurium resides and replicates in macrophages within the "Salmonella-containing vacuole" (SCV). It is surprising that the substrates and metabolic pathways necessary for growth of S. Typhimurium within the SCV of macrophages have not been identified yet. To determine whether S. Typhimurium utilized sugars within the SCV, we constructed a series of S. Typhimurium mutants that lacked genes involved in sugar transport and catabolism and tested them for replication in mice and macrophages. These mutants included a mutant with a mutation in the pfkAB-encoded phosphofructokinase, which catalyzes a key committing step in glycolysis. We discovered that a pfkAB mutant is severely attenuated for replication and survival within RAW 264.7 macrophages. We also show that disruption of the phosphoenolpyruvate:carbohydrate phosphotransferase system by deletion of the ptsHI and crr genes reduces S. Typhimurium replication within RAW 264.7 macrophages. We discovered that mutants unable to catabolize glucose due to deletion of ptsHI, crr, and glk or deletion of ptsG, manXYZ, and glk showed reduced replication within RAW 264.7 macrophages. This study proves that S. Typhimurium requires glycolysis for infection of mice and macrophages and that transport of glucose is required for replication within macrophages.

Ubiquitination—A Bacterial Effector's Ticket to Ride

Ubiquitination has many important cellular functions, including regulation of plasma membrane endocytosis. In their recent article in Cell, Patel et al. (2009) show that the activity of the multifunctional Salmonella virulence protein SopB is controlled by ubiquitin-dependent delivery from the plasma membrane to the Salmonella-containing vacuole.

The Salmonella Effector SptP Dephosphorylates Host AAA+ ATPase VCP to Promote Development of its Intracellular Replicative Niche

SummaryVirulence effectors delivered into intestinal epithelial cells by Salmonella trigger actin remodeling to direct pathogen internalization and intracellular replication in Salmonella-containing vacuoles (SCVs). One such effector, SptP, functions early during pathogen entry to deactivate Rho GTPases and reverse pathogen-induced cytoskeletal changes following uptake. SptP also harbors a C-terminal protein tyrosine phosphatase (PTPase) domain with no clear host substrates. Investigating SptP's longevity in infected cells, we uncover a late function of SptP, showing that it associates with SCVs, and its PTPase activity increases pathogen replication. Direct SptP binding and specific dephosphorylation of the AAA+ ATPase valosin-containing protein (VCP/p97), a facilitator of cellular membrane fusion and protein degradation, enhanced pathogen replication in SCVs. VCP and its adaptors p47 and Ufd1 were necessary for generating Salmonella-induced filaments on SCVs, a membrane fusion event characteristic of the pathogen replicative phase. Thus, Salmonella regulates the biogenesis of an intracellular niche through SptP-mediated dephosphorylation of VCP.

The DNA Bridging Protein H-NS and the SsrB Transcription Factor Counteract One Another to Silence and Activate Pathogenicity Island Genes in Salmonella

The transcription factor SsrB activates transcription of genes located on Pathogenicity Island 2 (SPI-2) in Salmonella enterica. These gene products are responsible for forming a type-three secretion system that secretes effectors that modify the host macrophage vacuole, enabling Salmonella to replicate and then disseminate to the liver and spleen. Recent studies have reported that pathogenicity island genes are silenced by the nucleoid-like protein H-NS1,2. How transcription factors counter or relieve H-NS silencing is a major focus of study. The sifA gene is located outside of SPI-2 and encodes a product required for maintenance of the Salmonella-containing vacuole, providing an intracellular niche conducive to Salmonella replication and survival. Part of this process involves formation of Salmonella-induced filaments (Sifs). We set out to determine whether SsrB directly activates expression of the sifA gene, and whether H-NS could counter or prevent this interaction. In vitro transcription assays indicate that SsrB directly activates sifA transcription and this stimulation is prevented in the presence of H-NS. SsrB activation requires super-coiled templates; in the presence of linear DNA, no sifA transcripts are observed. Using atomic force microscopy, we show that H-NS forms multiple bridging complexes on super-coiled DNA. Additional experiments are underway to examine the effect of SsrB on these complexes. Supported by NIH GM-58746 and NSF MCB-0613014 to LJK and NUS R144000171712 to Y.J.1. Navarre, W.W. et al., (2006) Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella. Science 313:236-238.2. Lucchini, S. et al., (2006) H-NS mediates the silencing of laterally acquired genes in bacteria. PLOS Pathog 2:e81-89.

Differentially Evolved Genes of Salmonella Pathogenicity Islands: Insights into the Mechanism of Host Specificity in Salmonella

BackgroundThe species Salmonella enterica (S. enterica) includes many serovars that cause disease in avian and mammalian hosts. These serovars differ greatly in their host range and their degree of host adaptation. The host specificity of S. enterica serovars appears to be a complex phenomenon governed by multiple factors acting at different stages of the infection process, which makes identification of the cause/s of host specificity solely by experimental methods difficult.Methodology/Principal FindingsIn this study, we have employed a molecular evolution and phylogenetics based approach to identify genes that might play important roles in conferring host specificity to different serovars of S. enterica. These genes are ‘differentially evolved’ in different S. enterica serovars. This list of ‘differentially evolved’ genes includes genes that encode translocon proteins (SipD, SseC and SseD) of both Salmonella pathogenicity islands 1 and 2 encoded type three secretion systems, sptP, which encodes an effector protein that inhibits the mitogen-activated protein kinase pathway of the host cell, and genes which encode effector proteins (SseF and SifA) that are important in placing the Salmonella-containing vacuole in a juxtanuclear position.Conclusions/SignificanceAnalysis of known functions of these ‘differentially evolved genes’ indicates that the products of these genes directly interact with the host cell and manipulate its functions and thereby confer host specificity, at least in part, to different serovars of S. enterica that are considered in this study.

Salmonella‐Containing Vacuoles: Directing Traffic and Nesting to Grow

Salmonella enterica serovar Typhimurium (S. typhimurium) is a gram-negative facultative intracellular pathogen that can infect a broad range of mammalian hosts. Following invasion of host cells, the majority of S. typhimurium are known to reside in a membrane-bound compartment known as the Salmonella-containing vacuole (SCV). S. typhimurium actively remodels this compartment using bacterial virulence proteins, called effectors, to establish a protected niche where it can replicate. S. typhimurium delivers more than 30 effectors into the host cell cytosol by bacterial type three secretion systems, encoded by Salmonella pathogenicity island 1 or 2 (SPI-1 or SPI-2). Recent studies have revealed a critical role for the SPI-1 effector SopB in 'directing traffic' at early stages of infection, allowing the bacteria to control SCV maturation by modulating its interaction with the endocytic system. At later stages of infection, the SCV establishes a 'nest' near the Golgi where optimal bacterial growth takes place. In this study, we highlight these recent developments in our understanding of SCV trafficking.

Effects of Lysosomal Membrane Protein Depletion on the Salmonella-Containing Vacuole

Salmonella is an intracellular bacterial pathogen that replicates within a membrane-bound vacuole in host cells. The major lysosomal membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are recruited to the Salmonella-containing vacuole as well as Salmonella- associated filaments (Sifs) that emerge from the vacuole. LAMP-1 is a dominant membrane marker for the vacuole and Sifs. Its colocalization with both is dependent on a major secreted bacterial virulence protein, SifA. Here, we show that SifA is required for the recruitment of LAMP-2 and can be used as a second independent marker for both the bacterial vacuolar membrane and Sifs. Further, RNAi studies revealed that in LAMP-1 depleted cells, the bacteria remain membrane bound as measured by their association with LAMP-2 protein. In contrast, LAMP-2 depletion increased the amount of LAMP-1 free bacteria. Together, the data suggests that despite its abundance, LAMP-1 is not essential, but LAMP-2 may be partially important for the Salmonella-containing vacuolar membrane.