PMID: Site Menu Home. Search Health Topics. Search the NIH Guide. NIH Research Matters. November 10, Coronaviruses hijack lysosomes to exit cells. The components of the normal biosynthetic secretory pathway top and the lysosome pathway bottom , which this study found coronaviruses use to exit cells. Lysosomes are involved with various cell processes. They break down excess or worn-out cell parts. They may be used to destroy invading viruses and bacteria.
If the cell is damaged beyond repair, lysosomes can help it to self-destruct in a process called programmed cell death, or apoptosis. Now, the lysosome is a specific type of organelle that's very acidic. Just as humans are susceptible to viruses, bacteria have their own viruses to contend with. To defend against a phage attack, bacteria have evolved a variety of immune systems. Once a match is found, the Cas proteins chop up the invading genetic material and destroy the phage.
Light microscopy in live cells combined with single-particle tracking shows that the clathrin coat is recruited to sites of VSV binding Cureton et al.
Because of the bullet shape and large size of VSV particles, the clathrin vesicles form slowly, they deviate from the round shape, and some carry only a partial clathrin coat Cureton et al.
Although VSV endocytosis is dependent on plasma membrane PI 4,5 P 2 , dynamin, and actin, it is unclear whether AP2 is the adaptor protein required for endocytosis and infection Cureton et al.
A general lesson from the observations with VSV and other viruses is that although the viruses differ in size, they have evolved to use CME very efficiently. They can induce the formation of clathrin-coated vesicles CCVs locally, and they can adapt the size and shape of the clathrin-coated pits CCPs for their needs.
It will be interesting to determine how VSV and other viruses guide and regulate the coat assembly process. The existence of a clathrin-, caveolin-, and dynamin-independent pathway for virus endocytosis was first described for members of the polyomaviruses: mouse polyoma virus, and SV40 Gilbert and Benjamin ; Damm et al.
These are small nonenveloped DNA viruses diameter 50 nm that replicate in the nucleus of host cells. The VP1 proteins that form the icosahedral viral coat bind to the sialic-acid-containing carbohydrate moiety of specific gangliosides that serve as cell-surface receptors Tsai et al.
Present as 72 homopentamers, VP1 constitutes the main building block in the capsid shell Stehle et al. In a process that in EM sections looks as if the virus particle would be budding into the cell, the PM wraps itself tightly around the virus particle Fig. The same can be seen when viruses are added to giant unilamellar liposomes containing GM1 with long acyl chains. When many viruses are present, long, narrow, tight-fitting, tubular invaginations with multiple viruses can form in the PM of cells and in liposomes.
Interestingly, pit and tube formation does not require the whole virus; isolated VP1 homopentamers are sufficient. Endocytosis of enveloped and nonenveloped viruses. These electron micrographs show viruses from different families during endocytic entry. A Bound to its ganglioside receptors GD1a and GT1b , an incoming mouse polyoma virus particle is seen in a tight-fitting indentation of the plasma membrane of a 3T6 cell.
Although viruses of the polyoma virus family can also use caveolae see arrow for entry, this particle is most likely making use of a clathrin- and caveolin-independent mechanism used by these viruses. B Human papilloma virus 16 enters HeLa cells via a macropinocytosis-like mechanism that involves binding to filopodia, surfing along filopodia to the cell body, and activation of an endocytic process independent of clathrin and caveolin Schelhaas et al. C Vesicular stomatitis virus is internalized by clathrin-coated vesicles Matlin et al.
D An influenza A virus particle is seen in an early endosome after endocytic internalization Matlin et al.
E In this cryoEM image, a vaccinia virus particle a poxvirus is seen in a macropinocytic vacuole after endocytosis in a HeLa cell. The particle is labeled with immunogold against the A5 core protein. F The contact between an SV40 particle and the plasma membrane is very tight during the clathrin- and caveolin-independent entry process.
The virus seems to bud into the cell. Scale bars, nm. For the fission reaction that detaches the virus-containing indentations, the virus alone is not sufficient. Energy in the form ATP is required, as well as cellular tyrosine kinases, cholesterol, and a change in actin dynamics.
Contrary to CCVs, vesicle fission is independent of dynamin Damm et al. The virus-containing vesicles formed are delivered to early endosomes, and the virus is eventually transported via late endosomes to the ER Fig. In all these cases, the receptors are glycosphingolipid molecules concentrated in lipid rafts. They differ from most other receptors in that they do not span the bilayer.
Caveolar endocytosis has been proposed for a variety of viruses. One of the problems in assigning viruses to this pathway is that the endocytic role of caveolae in cell life is still poorly defined. No single characteristic such as cholesterol dependence alone is sufficient as a criterion to define this pathway.
Although much less dynamic than CCVs, it is clear, however, that a fraction of the caveolae is mobile and can undergo endocytosis especially when activated by a ligand Kirkham and Parton ; Pelkmans and Zerial ; Tagawa et al. The best studied among the viral candidates for caveolar uptake is SV Morphological evidence from several laboratories using immune-electron and immunofluorescence microscopy shows colocalization of the viruses and caveolin-1 in plasma membrane spots and pits Anderson et al.
Addition of SV40 to cells causes a dramatic, tyrosine-phosphorylation-dependent elevation in caveolar vesicle formation and caveolar vesicle motility Tagawa et al. Recently, it was shown that depletion of EHD2 a peripheral caveolar ATPase leads to a dramatic increase in caveolar vesicle trafficking and increases the efficiency of SV40 infection Stoeber et al.
Thus, in summary, it seems that SV40 can make use of two parallel endocytic mechanisms. One corresponds to the caveolar pathway and relies on caveolin The other is caveolin independent, with the virus particle serving as the curvature-generating principle. To what extent viruses other than SV40 can use these pathways is not clear. There are reports suggesting that other polyomaviruses such as BK virus and mouse polyoma virus may also exploit caveolae Richterova et al.
Macropinocytosis offers incoming viruses an altogether different type of endocytic experience Fig. Transiently triggered by external ligands such as growth factors and PS-containing particles, this mechanism is known to induce internalization of fluid, membrane, and whatever happens to be associated with the membrane such as viruses Swanson and Watts The process is important for the clearing of cell remnants after apoptotic cell death.
The key events in macropinocytosis include the activation of receptor molecules such as receptor tyrosine kinases, integrins, and PS receptors Swanson and Watts This triggers downstream signaling cascades resulting in transient, global changes in actin dynamics that lead to cell-wide plasma membrane ruffling in the form of filopodia, lamellopodia, circular ruffles, or blebs.
Large, uncoated vacuoles macropinosomes are formed. After moving deeper into the cytoplasm, these usually end up fusing with late endosomes or lysosomes. Because there are many variations of the process, macropinocytosis must be viewed as a collective term for an assortment of related mechanisms.
Macropinocytosis plays a role in the infection of viruses of different families including large viruses such as pox-, filo-, paramyxo, and herpesviruses Lim and Gleeson ; Mercer and Helenius Some smaller enveloped and nonenveloped viruses such as influenza A virus seem to use pathways that share many properties with macropinocytosis Khan et al.
Epidermal growth factor receptor EGFR and other growth factor receptors are often activated and essential as accessory factors in infectivity Eierhoff et al. There are viruses for which endocytic entry does not fall into the categories listed above Table 1. However, the vesicles are small, Rho GTPases are not activated, and there is no elevation in fluid uptake. Some of these characteristics are shared by human rhinovirus 14 and one of the alternate pathways described for influenza A virus Matlin et al.
In addition, two Old World arenaviruses, LCMV and Lassa virus, enter by a clathrin-, caveolin-, and dynamin-independent pathway via multivesicular bodies bypassing early endosomes Quirin et al. In the case of Acanthamoeba polyphaga mimivirus, a giant enveloped virus nm in diameter that uses amoeba as a host, uptake into human macrophages seems to involve a phagocytosis-like mechanism Ghigo et al.
This uptake process resembles the phagocytosis of bacteria. However, because virus entry studies and the classification of endocytic mechanisms are not always straightforward, the situation may change. Viruses may, in fact, provide one of the most informative systems for further classification of endocytic processes.
The rate and efficiency of entry of bacteria into cells vary greatly with the cell type and other parameters such as the temperature or the growth phase at which bacteria have been harvested Fig.
Endocytosis of bacteria. These electron micrographs show various phases of bacterial endocytosis into mammalian cells.
A , left Binding of Listeria entering into cells. Two coated pits are detectable on this cross section. The tight apposition of the membrane illustrates what is meant by the zipper mechanism. A , middle micrograph A Shigell a entering into a cell and the huge membrane ruffles that engulf the bacterium. This micrograph illustrates the trigger mechanism. A , right Bartonella henselae enter as a group into an endothelial cell.
B Listeria is entering into the cell and the clathrin coat is visible as a thickening of the plasma membrane underneath the bacterium. C , left Listeria is present in a membrane-bound vacuole. C , right Chlamydia vacuole full of bacteria that have replicated.
The hallmarks of the zipper mechanism are a dedicated bacterial surface protein or component that interacts directly with a host cell receptor, thereby inducing a series of signaling events that culminates in endocytosis. The actin cytoskeleton and its dynamics as well as the membrane composition and its plasticity are critical elements Pizarro-Cerda and Cossart , ; Cossart and Roy The bacterial proteins involved in entry often mimic endogenous ligands and exploit the properties of their receptors maximally.
Alternatively, bacteria can like viruses described above interact with components that act as a bridge to a cell-surface receptor. It is clear that the affinity of the ligand and its density on the bacterial surface critically control the efficiency of the entry process. More recently, the CME machinery has been shown to be involved in early steps after initial contact between the incoming microbe and the receptor before the cytoskeleton rearrangements Boleti et al. Listeria monocytogenes is the prototype of a bacterium entering by the zipper mechanism Pizarro-Cerda et al.
It expresses two proteins involved in entry. The first, Internalin InlA , is a surface protein that interacts with E-cadherin, a cell—cell adhesion molecule expressed only in some epithelial cells Mengaud et al.
For entry into most cell types, Listeria uses a second invasin, the InlB protein, which activates the receptor tyrosine kinase Met, the hepatocyte growth factor receptor that is expressed on all cells of epithelial origin Shen et al. Integrity of the membrane and its lipid rafts is critical for the initial clustering of E-cadherin when bacteria enter by the InlA pathway.
In contrast, for the InlB pathway, the integrity of lipid rafts is critical for the correct localization of the phosphoinositides produced in the plasma membrane by PI3 kinase, which is recruited to the receptor activated upon bacterial entry Seveau et al. Investigations of Listeria endocytosis in cells that do not express E-cadherin have shown that InlB mimics HGF and first induces the autophosphorylation of Met Shen et al.
This, in turn, leads to the recruitment of Gab1, Cbl, and Shc Ireton et al. The ubiquitin ligase Cbl triggers the ubiquitination of Met as a prelude for the recruitment of the clathrin adaptor Dab2 and that of the clathrin heavy and light chains Veiga and Cossart Src-mediated phosphorylation of the clathrin heavy chain is critical for the whole process Veiga et al.
Strikingly, Hip1R, which interacts with the clathrin light chain and actin filaments, is then recruited followed by myosin VI, which interacts with Hip1R and actin. Because myosin VI has the capacity to move toward the minus end of actin filaments, it probably then pulls the bacteria toward the interior of the cell.
Dynamin is also recruited at the bacterial entry site, as well as cortactin. A second wave of actin rearrangements is then triggered more classically by the Gab1-mediated recruitment of PI3 kinase Ireton et al. Note that actin rearrangements are finally down-regulated by the recruitment of proteins such as cofilin Bierne et al.
Strikingly, although immunofluorescence data clearly show clathrin recruitment at the entry site Veiga and Cossart ; Veiga et al.
Clathrin-coated vesicles are not seen. That clathrin depletion prevents actin recruitment suggests that coated pits serve as a platform for cytoskeletal arrangements Lecuit et al. Interestingly like Met see above and many other receptors, E-cadherin undergoes several posttranslational modifications upon activation including phosphorylation and ubiquitination Bonazzi et al.
Ubiquitination is mediated by the E-cadherin-specific ubiquitin ligase Hakai and allows the recruitment of clathrin and other components of the clathrin-mediated endocytosis machinery upstream of actin rearrangements Bonazzi et al. Interestingly, colocalization of caveolin at the site of the InlA E-cadherin entry site and depletion experiments show that caveolin also participates in entry.
Listeria entry is thus a complex process implicating the clathrin-mediated endocytosis machinery, the actin cytoskeleton as in classical phagocytosis, and caveolae Pizarro-Cerda et al.
Entry of Yersinia pseudotuberculosis into cells resembles that of Listeria Wong and Isberg Unlike fibronectin, invasin does not possess an RGD motif, but has a domain structurally similar. Invasin has a higher affinity than fibronectin for integrins. It induces integrin clustering and efficient downstream signaling.
Thus, a lower affinity of the integrin for the cytoskeleton could allow higher mobility of the receptors in the membrane. The local concentration of PI 4,5 P 2 is critical for entry, and Arf6 may have a role in activation of PIP5 kinase, the control of cytoskeleton rearrangements and membrane traffic involved in the closure of the phagocytic cup Wong and Isberg The analysis of Yersinia entry has frequently involved the use of E. This strain has recently been used to show that as for Listeria the clathrin-mediated endocytosis machinery is critical for bacterial entry Veiga et al.
However, this has not been studied in detail yet. Several other bacteria such as some Streptococci and also Staphylococcus aureus use integrins for their uptake Ozeri et al. However, they express a variety of fibronectin-binding proteins and use a bridging mechanism. As for Listeria , uptake of these bacteria requires components of the clathrin-mediated endocytic pathway.
In addition to TARP, entry requires the concerted activation of growth factor receptors, cytoplasmic kinases, and small GTPases to remodel the actin cytoskeleton Lane et al. Additional host factors include clathrin and cholesterol-rich microdomains Boleti et al. Shigella and Salmonella are two bacteria that use the trigger mechanism to enter cells Tran Van Nhieu et al. The hallmark of this mechanism is the formation of huge, actin-rich membrane ruffles triggered by a signaling cascade that induces localized transient changes in actin dynamics.
It is triggered by the translocation of T3SS effectors into the host cytosol. In Salmonella , the secretion system involved in entry is encoded by a chromosomal pathogenicity island PAI. In Shigella , the PAI is carried by a plasmid. For many years, the nature of the initial contact between Shigella and cells remained elusive. More recently, it was shown that upon challenge with epithelial cells, Shigella establish contacts with filopodial-like extensions, which then retract to bring bacteria into contact with the cell body, where invasion occurs Romero et al.
Filopodia are cell-surface sensory organelles implicated in adhesive processes, including the formation of intercellular junctions. They are not induced by bacterial contact. Time-lapse video microscopy showed that bacterial capture by filopodia can be inhibited by antibodies against IpaB and IpaD, two proteins that are located at the tip of the T3SS, indicating that contact likely occurs between a cell-surface receptor present at the filopodial tip and the T3SS tip complex Carayol and Tran Van Nhieu Upon cell contact, IpaB and IpaC insert into the host cell plasma membrane to form a translocon complex that allows injection of T3SS effectors.
These induce localized membrane ruffling by polymerization of cortical actin. It is possible that these activities synergize for efficient actin polymerization. IpaA induces actin depolymerization at subsequent stages of the entry process. Filopodial capture might allow bacteria to target specific sites of the epithelium Romero et al. Indeed, Shigella invasion occurs mainly at multicellular junctions in the apical surface of polarized intestinal cells. These correspond to the intersection between several cells.
Interestingly, tricellulin a protein required for the integrity of multicellular junctions is essential for cell-to-cell spreading of Shigella , a process involving components of the clathrin-mediated endocytosis pathway Fukumatsu et al. In contrast to Shigella , which is nonadhesive and nonmotile and sampled by the finger-like extensions of the cell surface, Salmonella is a motile bacterium.
Indeed, a recent report shows that Salmonella can also enter cells by a zipper-like mechanism mediated by an outer membrane protein named Rck and a signaling pathway that ressembles that of Listeria Mijouin et al. Bartonella henselae is a Gram-negative bacterium that specifically colonizes the endothelium Dehio Attachment to nucleated cells is mediated by nonfimbrial outer membrane adhesion protein belonging to the type V secretion systems T5SS , for example, trimer autotransporters, including BadA Bartonella adhesin A and Vomps variably expressed outer membrane proteins.
The massive cytoskeletal rearrangements resulting in invasome-mediated uptake of bacterial aggregates are entirely dependent on the VirB type IV secretion system, but the specific effectors involved are unknown. Interestingly , Bartonella also invades erythrocytes, in this case, with an essential role for the invasion-associated locus proteins IalA and IalB. This entry probably uses a novel mechanism that deserves investigation.
After endocytic uptake, the incoming pathogens enter a complex network of heterogeneous but functionally interconnected endocytic vacuoles and vesicles with early endosomes and macropinosomes as commonly used gateways Fig. Viruses use the endocytic network of organelles for transit deeper into the cell and for penetration into the cytosol.
Unlike some bacteria, they do not modify the composition and functions of the organelles. In early endosomes, the viruses are generally localized in the vacuolar part, sharing this volume with intralumenal vesicles ILVs. If the particles have not penetrated already in early endosomes, they typically follow the pathway in the direction of late endosomes and endolysosomes Huotari and Helenius This pathway involves a complex maturation program that prepares the endosome for fusion with lysosomes see Klumperman and Raposa ; Wandinger-Ness and Zerial Macropinosomes can also deliver their cargo to lysosomes by fusing with late endosomes, endolysosomes, or lysosomes.
Before this can occur, they also undergo a maturation process similar to endosomes. Defects in endosome and macropinosome maturation inhibit the productive entry of many viruses Khor et al. The majority of viruses are acid-activated, that is, viral membrane fusion proteins and penetration mechanisms depend on exposure of the virus to low pH Helenius et al.
For viruses that penetrate from early endosomes, the pH threshold is 6 or above, and for late-penetrating viruses it is lower Lozach et al. Receptor interactions and proteolytic processing can also trigger escape with or without acid dependence Chandran et al.
Much is known about the structure and function of fusion factors that cover the surface of envelope of viruses Earp et al. They are oligomeric, type 1 membrane glycoproteins with large ectodomains. The prefusion conformation is metastable, which means that when triggered by low pH or other cues the proteins can undergo major conformational changes and alterations in oligomeric structure.
This occurs without the input of additional energy. The changes involve the exposure of hydrophobic or amphipathic peptide sequences fusion peptides that allow the protein to insert into the target membrane.
After generating a bridge between the two membranes, further conformational changes in clusters of these proteins allows the distance between the membrane to be reduced so that the hydration shell covering the lipid bilayer is disturbed in a focal site. This results in hemifusion the innermost leaflets of the two bilayers fuse followed by full fusion, when the outer leaflets also fuse. Of intracellular bacterial species, a minority, which includes Listeria , Shigella , Rickettsia, Francisella Celli and Zahrt , and also Salmonella Knodler et al.
In the case of M. Intracytosolic bacteria use several molecular tools to escape from the primary internalization vacuoles. The best characterized is the Listeria pore-forming toxin listeriolysin O Hamon et al. However, listeriolysin O is not always necessary for escape because listeriolysin O mutants can escape into the cytosol of some human cells, showing that escape can also be controlled by other unknown bacterial and cellular components.
In the cytosol, the bacteria subvert cellular defense mechanisms such as antimicrobial peptides, or autophagy. Note that recent data indicate that the induction of autophagy does not necessarily lead to bacterial killing.
There are even some bacteria that exploit the autophagic machinery for their own profit for a recent review, see Mostowy and Cossart
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