SNARE motif: A common motif used by pathogens to manipulate membrane fusion

Abstract

To penetrate host cells through their membranes, pathogens use a variety of molecular components in which the presence of heptad repeat motifs seems to be a prevailing element. Heptad repeats are characterized by a pattern of seven, generally hydrophobic, residues. In order to initiate membrane fusion, viruses use glycoproteins-containing heptad repeats. These proteins are structurally and functionally similar to the SNARE proteins known to be involved in eukaryotic membrane fusion. SNAREs also display a heptad repeat motif called the "SNARE motif". As bacterial genomes are being sequenced, microorganisms also appear to be carrying membrane proteins resembling eukaryotic SNAREs. This category of SNARE-like proteins might share similar functions and could be used by microorganisms to either promote or block membrane fusion. Such a recurrence across pathogenic organisms suggests that this architectural motif was evolutionarily selected because it most effectively ensures the survival of pathogens within the eukaryotic environment.

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Membrane Fusion in Eukaryotes is Mediated by SNARE Proteins

In eukaryotes, membrane fusion is a fundamental biological process for organelle formation, nutrient uptake, and the secretion of molecules. It is central to all aspects of immune function, including the secretion of immune mediators and the ingestion and destruction of pathogens.1 Intracellular transport is accomplished by vesicles, which deliver cargo proteins and membranes from one compartment to another. All of these intracellular pathways are controlled by a series of fusion events in which specialized proteins intervene, the so-called SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) family.2,3 The SNARE family shares an alpha-helical motif of 70 amino acids (also called SNARE motif) evolutionarily conserved across all eukaryotes.4,5

The essential role for SNARE proteins in membrane fusion was first outlined by in vivo experiments which demonstrated that fusion cannot occur when SNAREs are genetically removed or destroyed by neurotoxins.6,7 These toxins are known to disrupt brain transmission and provoke the paralytic stage of botulism or tetanus depending upon the infectious microorganism (Botulism is caused by Clostridium botulinum's toxins BoNT/A, B, C, D, E, F, G while Tetanus is caused by Clostridium tetanus' toxin TeNT). In the early 90s, the neuronal SNAREs were identified as the targets of these toxins.7,8 Later, SNARE gene overexpression and knock-down confirmed their implication in intra-cellular trafficking.9–15 The direct implication of SNARE proteins in membrane fusion itself has been demonstrated using different in vitro fusion assays.16,17 Remarkably, complementary SNAREs reconstituted into separate populations of liposomes, and more precisely their SNARE motif, are entirely sufficient to drive membrane fusion.18,19

Functional SNARE complexes are composed of four sub-units: three constituting the t-SNARE (target-SNARE), and one constituting the v-SNARE (vesicular-SNARE).20 These proteins, present on the surface of most intra-cellular compartments, interact to form a stable complex, bringing two membranes into close apposition and triggering their fusion (Fig. 1A). Structural data shows that this complex forms a very characteristic rod-shaped coiled-coil of four α-helices aligned in parallel [one v-SNARE and three t-SNAREs21–23] and highlights the importance of heptad repeat motifs to manipulate biological membranes.

Coiled-Coil Proteins are Involved in Both Eukaryotic and Viral Membrane Fusion

Viruses are the ultimate cellular hitchhikers, carrying little more than genetic information, which is then replicated, packaged and preserved by the host cells. Enveloped viruses are covered by a lipid bilayer derived from the host-cell membrane during virus budding. Fusion of the viral envelope membrane with a host cell membrane initiates infection.

In class I enveloped viruses, membrane fusion is mediated by virus surface glycoproteins that form α-helical rods (Fig. 1B).24 A well-studied example is the Influenza virus protein hemagglutinin (HA), for which both the biology and the mechanism of fusion are well understood. HA, present on the surface of the influenza virus, is constituted by two subunits linked by a disulfide bond, a surface subunit HA1 responsible for binding sialic acid on the surface of the host cell (surface receptor of the virus) and a transmembrane subunit HA2 containing the fusion peptide. Additionally, HA encodes heptad repeat motifs. By directly expressing HA, Judith White and colleagues developed a cell-cell fusion assay, which demonstrated the sufficiency of HA for membrane fusion.25 Supporting its role in fusion, specific point mutations in the predicted coiled-coil domain of HA strongly impact the glycoprotein-mediated membrane fusion.26–29

Later, X-ray crystal structures depicting enveloped viral fusion proteins were published, demonstrating the presence of a common heptad repeat motif in all fusogenic proteins. Among class I viral families, retrovirus [HIV-1,30–32 and SIV33,34], paramyxovirus [simian parainfluenza virus 5,35,36 Nipah and Hendra viruses37], coronavirus [Hepatitis virus;38 SARS39] and filovirus [Ebola40] generated the most extensive structural information.

Most of the new structures show a common coiled-coil helical bundle already described for influenza, and common to the vesicle SNARE membrane fusion machinery.41,42 Structural and functional parallels between intracellular and viral membrane fusion proteins were established.

Bacteria Also Use Coiled-Coil Motifs to Manipulate Membrane Fusion

Intracellular pathogens enter cells via phagocytosis. In their host cells, a significant number of intracellular microorganisms such as Mycobacterium, Salmonella, Chlamydia or Legionella, have the ability to create favorable intracellular compartments within which to multiply.43 This strategy relies on the capacity of bacteria to block undesirable fusion events with the cell's degradative compartments and their capacity to create new advantageous fusion pathways with host vesicles.44,45 To do so, bacteria remodel the surface of their infectious vacuoles by expressing their own proteins on the surface, using specialized syringe-like secretion systems that allow bacterial proteins to be delivered directly inside the host cell.46 In virtually all cases where responses to killed organisms have been examined, dead bacteria were rapidly trafficked to lysosomes. Viable organisms avoided this fate.

Genetic analysis of Legionella has identified a series of Dot/Icm genes for which mutants are rapidly trafficked to, and degraded within lysosomal compartments.47 In Chlamydia, a rapidly expanding class of bacterial proteins that modify the vacuolar membrane are called the chlamydial inclusion membrane proteins (Inc.). Chlamydial inclusions are not fusogenic with endosomal or lysosomal compartments.48,49 However, Chlamydia is fusogenic with exocytic compartments that contain sphingomyelin and on their way from the Golgi to the plasma membrane, demonstrating a masterful reorganization of host vesicular trafficking.50 In addition to these modifications, Chlamydia psittaci is able to associate its vacuole with mitochondria. Similarly, Legionella pneumophila vacuoles escape lysosomal fusion but associate with smooth vesicles, mitochondria and ultimately with rough endoplasmic reticulum, as does Brucella. In fact, Brucella abortus vacuoles segregate from the degradative endocytic pathway after initial interactions with its early compartments. Brucella vacuoles can then interact with the endoplasmic reticulum in a sustained manner leading to fusion between both organelles. This generates an ER-derived vacuole suitable for bacterial replication.51

Some SNARE-like proteins have been identified in intracellular bacteria.52–54 Among them, the vacuolar protein IncA expressed by Chlamydia trachomatis (called CtrIncA), displays two SNARE-like motifs.53,54 The modeling of a tetramer of SNARE-like motifs from CtrIncA gives a rod-like structure, very similar to the SNARE complex structure.53 Furthermore, it has been shown that CtrIncA interacts with mammalian SNAREs.54

Recently, we established that CtrIncA and CcaIncA (CcaIncA: IncA expressed by Chlamydia caviae) are able to manipulate membrane fusion.55 We showed that CtrIncA specifically inhibits endocytic fusion with little impact on exocytic fusion, suggesting that this protein displays some level of specificity and does not inhibit SNAREs indiscriminately. This result is consistent with the observation that Chlamydiae primarily protect their infectious vacuoles against fusion with the endocytic compartments.56 It is not yet clear if this discrimination is based on the SNARE sequence or if it is dependent on the structure of the SNARE complex (the endocytic t-SNARE complex is composed of three separate trans-membrane SNARE proteins versus two in the case of the exocytic complexes). Next, we used a series of CtrIncA mutants containing either the entire N-terminal SNARE-like motif [CtrIncA_1–141] or different truncated forms of the SNARE-like motif to demonstrate that the N-terminal SNARE-like motif was sufficient for sustaining the inhibition of the endocytic SNARE fusion.55 Interestingly, more SNARE-like proteins have been described in Chlamydia, although their function needs further investigation.54 Altogether, this demonstrates the implication of coiled-coil proteins in manipulating membrane fusion in yet another biological system. The similarity of structure and function between eukaryotic SNAREs, viral fusion proteins, and the Chlamydia SNARE-like protein IncA is striking. It suggests that heptad repeat motifs are used by a wide variety of organisms to manipulate membrane fusion.

Supporting this hypothesis, other intracellular bacteria express similar heptad repeat-containing proteins, capable of disrupting vesicular trafficking (See Fig. 2 describing our working model). Legionella pneumophila encodes a variety of effector proteins, which not only interfere with host cellular trafficking machinery, but also promote membrane fusion with non-endo-lysosomal compartments to allow for the development of a permissive ER-like replication vacuole. A subset of these proteins—LegC7, LegC2 and LegC3—are members of the Legionella eukaryotic-like gene (Leg) family, which are translocated via the Dot/Icm type-4 secretion system.57 The Dot/Icm secretion system is a syringe-like apparatus used to transport proteins from the Legionella cytosol, across both bacterial membranes to deliver proteins into the host cytosol.46 These three translocated proteins possess multiple coiled-coil domains linked to a hydrophobic α-helical transmembrane domain, analogous to the SNARE protein structure. When expressed in yeast, LegC2, LegC3 and LegC7 block endosomal maturation. Additionally, deletions and mutations in the N-terminus containing the coiled-coil domains indicate that these heptad repeat motifs are functional and required for endo-lysosomal interference.57,58 These observations are consistent with a model in which LegC2, LegC3 and LegC7 are expressed on the cytoplasmic face of the Legionella-containing vacuole where they utilize their SNARE-like coiled-coil motifs to interact with host trafficking machinery and disrupt normal endosomal maturation.

Although these coiled-coil proteins cause trafficking defects when expressed in yeast, mutant strains of Legionella harboring deletions of these three proteins do not show defects in replication.57,58 This highlights the strong redundancy of the protective system in place. To protect its infectious vacuole from host interference and to promote successful replication, it is likely that Legionella blocks fusion with the endocytic pathway at multiple levels. For example, it has been shown that Legionella manipulates small Rab GTPases, which are also involved in regulating the host membrane fusion.59 Legionella secretes soluble factors that activate Rab1, an ER-golgi Rab protein essential for the development of the ER-like replication vacuole.60,61 Manipulating Rab protein function is a common survival mechanism among intracellular bacteria such as Mycobacterium tuberculosis, Salmonella and Chlamydia.62 Blocking host SNAREs, in addition to corrupting Rab proteins, indicates the importance of protecting the vacuole and supports the existence of functional redundancy. The complexity of the bacterial protective system also highlights the necessity of using a variety of biochemical approaches to study bacterial effectors: (1) Occasionally it is impossible to genetically manipulate intracellular bacteria such as Chlamydia. Directly working with their proteins will allow for the characterization of host-pathogen interactions. (2) Since microorganisms likely express proteins with redundant functions on the surface of their infectious vacuole, working with their isolated proteins will lead to the identification of their specific role. (3) Obtaining conclusive results in vivo may be precluded by the low level of bacterial proteins expressed during infection. A biochemical approach allows for protein expression levels that will lead to definitive data.

Conclusion and Future Directions

Evidence has established the implication of bacterial coiled-coil proteins in manipulating membrane trafficking, but we have only just begun to study their detailed function. Investigating whether intracellular bacteria use SNARE-like proteins as a general mechanism to corrupt host membrane vesicular trafficking is the next important step in understanding host-bacteria interactions. Deciphering the crystal structure of such proteins with and without their SNARE binding partners will be crucial to further understand the host-pathogen biology.

The discovery of SNARE-like proteins in different pathogenic microorganisms will have a major impact. It would suggest that the SNARE motif has been selected during evolution because it is highly capable of manipulating membrane fusion, and thus will contribute to pathogen survival. Such a recurrence would allow for the development of a common strategy that targets a wide array of bacterial SNARE-like proteins. These bacterial proteins would likely be viable therapeutic targets since disruption of their protective function would render intracellular bacteria more susceptible to phagosomal clearance. Thorough understanding of the bacterial SNARE-like protein system will give us the necessary tools to design such therapeutics.

Figures and Tables

Figure 1 Eukaryotic SNAREs and viral coiled-coil proteins trigger membrane fusion. (A) During a fusion event in eukaryotic cells, the t-SNAREs present on the target membrane interact with the v-SNAREs present on the vesicle. This interaction brings both membranes into a close apposition and leads to membrane fusion. (B) During a viral infection, enveloped viruses enter their host cells through fusion with the host membrane (plasma membrane or endosome membrane). For example, HIV envelope protein gp41 utilizes three α-helical domains, which collapse into a trimer of hairpins to facilitate fusion between the host plasma membrane and the viral envelope thereby providing its viral contents access to the host cytosol.

Figure 2 Bacterial coiled-coil proteins inhibit membrane fusion (working model). Left: Non-pathogenic bacteria (blue) are internalized. Normal phagosome maturation is initiated and results in lysosomal fusion (formation of phagolysosomes) and destruction of the phagosomal content. Right: Intracellular bacteria (red) are internalized and express their own proteins on the surface of the phagosome (red coiled-coil proteins), blocking its maturation.

Acknowledgements

We thank the members of the Paumet laboratory, especially Benjamin Scheinfeld, for technical assistance in the project. We are grateful to Dr. James McNew (Rice University, TX) for critical reading of the manuscript and helpful discussion. This research is supported by the National Institutes of Health grant # RO1 AI073486 (to F.P.).

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