School of Medicine have found that some bacteria can use viruses to help them pass along the recipe for their favorite toxin, results published in the journal Science. Like humans, bacteria are also prone to infection by viruses. And most of these viruses—called bacteriophage, or phage for short—make their bacterial victims sick.
Or even dead. But in the laboratory, the scientists discovered that Staphylococcus aureus, the bug that causes toxic shock syndrome among other things, can actually co-opt phage, using them to shuttle the gene for toxic-shock toxin to another bacteria, in this case Listeria. But in this particular battle, ya gotta hope the viruses come out on top.
Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist. HCV has been recognized as a major cause of chronic liver disease and affects approximately million people worldwide at the present time. Persistent infection is associated with the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. A protective vaccine for HCV is not yet available and even the most recent combination of antiviral therapy is often poorly tolerated [1].
The HCV genome encodes one large open reading frame that is translated as a polyprotein which is proteolytically processed to yield the viral structural and nonstructural NS proteins.
Two virally encoded proteases participate in polyprotein processing, the NS autoprotease which cleaves in cis at the NS junction and the NSA serine protease which cleaves at four downstream NS protein junctions. The N-terminus of NS3, in complex with its endoplasmic reticulum ER membrane-anchored co-factor NS4A, primarily functions as a serine protease, which cleaves the viral polyprotein precursor downstream to NS3.
Zymogens are inactive enzyme precursors that are converted to their active form following a biochemical modification, such as proteolytic processing. Among the known and important groups of enzymes that are proteolytically activated are secreted digestive enzymes like pepsin and trypsin [6] , [7] , the cysteine aspartic acid proteases caspases which play an essential role at various stages of the apoptotic process [8] ; and blood coagulating factors [9].
Recently we described a proof of concept for potential new anti-viral agents that can specifically eradicate virally infected cells thus limiting virus production and spread. Upon binding and translocation into cells cytoplasm by virtue of the corresponding Pseudomonas exotoxin A domains, activation of these toxins is mediated by HCV-NS3 protease cleavage that separates between the toxic domains and a fused, rationally designed inhibitory peptide. These zymoxins showed a higher level of cytotoxicity when applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window.
Still, the zymoxins we described had two limitations: first, they required high levels of NS3 protease for efficient activation, and second, they were not totally inactive in the zymogenized state, and their basal cytotoxic activity without proteolytic activation resulted in quite narrow therapeutic windows [10]. Apparently, the zymoxins were only partially inhibited by the rationally designed inhibitory peptides.
Natural bacterial plasmids ensure their survival within the bacterial host by replicating inside their host cell and using mechanisms that function to segregate them prior to cell division [11]. The system consists of a pair of genes encoding for a stable toxin and an unstable antitoxin organized in a bicistronic operon that is transcriptionally autoregulated either by the toxin-antitoxin complex itself or by the antitoxin alone.
When coexpressed in plasmid harboring cells, the antitoxin component interferes with the lethal action of the toxin. If a cell loses the plasmid, the cellular concentration of the labile antitoxin that is degraded faster than the more stable toxin is rapidly diminished, enabling the toxin to exert its action which eventually results in cell death reviewed by [12] , [13] , [14] , [15] , [16].
Toxin-antitoxin systems have also been found integrated to the chromosome of various bacteria species where their function has been the subject of considerable speculation [14] , [17] , [18] , [19] , [20] , [21]. The system, which is activated by several stress conditions, was denoted MazEF after its two active protein components: the long lived MazF toxin and the labile MazE antitoxin. MazF induced toxicity is executed by blocking de-novo protein synthesis through its endoribonuclease activity that catalyze the cleavage of single-stranded mRNAs at ACA sequences [22].
The crystal structure of the MazE-MazF complex indicates that the interactions between the toxin and the antitoxin are primarily mediated by the acidic C terminus of MazE which wraps around the MazF homodimer crossing the edge of the dimer interface [24]. Interestingly, it was found that one inhibitory peptide, occupying a single active site on the MazF homodimer, affects the conformation of both sites that consequently become catalytically inactive.
The discovery of the MazF mechanism of action was soon followed by demonstrations that it is also toxic to eukaryotic cells, causing Bak-dependent programmed cell death in mammalian cells, suggesting it may be used as a tool for gene therapy against diseases such as cancer and AIDS [26] , [27]. Here we describe the design of a different zymoxin than those described in [10] , based on NS3-activable MazF ribonuclease that is delivered as a transgene by an adenoviral vector.
The delivered transgene encodes for a fusion between MazF, and a potent inhibitory peptide derived from its natural antidote MazE, through an NS3 cleavable linker. In contrast, in NS3 expressing or in HCV infected cells, NS3-mediated cleavage separates between the toxin and its inhibitor which results in inhibition of protein synthesis followed by death of the cells.
A short inhibitory peptide corresponding to MazE C-terminal 35 amino-acids which encompass the 23 amino-acids inhibitory peptide MazEp that has been described by Li et al. A flexible linker, followed by the C-terminal ER membrane anchor of the tyrosine phosphatase PTP1B [28] , was than fused to the C terminus of the inhibitory peptide and the whole construct was fused through its N terminus to the monomeric red fluorescence protein mCherry [29] see Figure 1.
In contrast, in HCV infected cells, the fusion protein is expected to colocalize with the ER membrane-bound viral NS3 protease in infected cells, NS3 is localized to the cytosolic side of the ER membrane and membranes of ER-like modified compartments [30] , [31] , [32] , [33] , [34]. As a result, the NS3-cleavable linker between the toxin and its inhibitory peptide is expected to be cleaved.
The toxic ribonuclease, no longer covalently tethered to its ER membrane-anchored inhibitor, is now free to diffuse into the cytoplasm which lacks the antidote and exert its cytotoxic activity. Finally, fusion to the fluorescent protein mCherry makes the whole construct trackable and facilitates the determination of its expression level and intracellular localization by fluorescence microscopy.
The amino-acid sequence of the MazF based zymoxins can be found in Text S1. The NS3-activated MazF zymoxin was constructed by fusing 5 elements in the following order from the N terminus : monomeric red fluorescence protein mCherry, E. The toxic ribonuclease, no longer covalently tethered to its ER-anchored inhibitor, is now free to diffuse to the cytoplasm which lacks the antidote and exert its destructive activity.
In this assay, the toxicity of an expressed transgene can be comparatively and qualitatively assessed by testing its effect on the competency of transfected cells to evolve into colonies under selection. After 10 days of selection, surviving colonies were stained. As expected, growth was severely inhibited when cells were transfected with the EGFP-fused active uninhibited toxin. A day before transfection, 7. Surviving colonies were fixed and stained with Giemsa upper panel.
Number of surviving Colonies from wells that were seeded with cells was determined by manual counting. As shown in Figure 3 A and B , both zymoxins have a similar cellular distribution, colocalizing with the ER marker calnexin at the juxtanuclear region of the ER.
These observations confirm that indeed both the protease and the modified ribonuclease are tethered to a common cellular compartment, presumably the cytoplasmic side of the ER membrane see scheme in Figure 1. After 24 h, the cells were fixed. Slides were then mounted and examined by confocal fluorescence microscope. Since expression of active MazF was found to inhibit de-novo protein synthesis in mammalian cells [27] , we hypothesized that such an effect may be observed also in cells in which MazF based zymoxin is proteolytically activated.
Levels of de-novo protein synthesis were than determined by [ 3 H]-leucine incorporation assay. As shown in Figure 4 upper panel , a complete shutoff in protein synthesis was observed as soon as 24 h post NS3 induction in cells that express the cleavable construct, indicating proteolytic activation of the zymoxin.
As expected, protein synthesis was not impaired following NS3 induction in cells that express the uncleavable toxin. Results are expressed as percent of the value obtained for cells which were not induced to express the NS3 protease No tet.
Numbers above each bar represent mean counts per minute CPM values for 7 micrograms total protein samples upper panel. Solid arrow: full length zymoxin. Hollow arrow: N' terminal portion of NS3-cleaved zymoxin lower panel.
In support of these findings, an immunoblot assay revealed a near complete cleavage of the zymoxin following tetracycline-induced expression of NS3 protease in NS3-activated MazF expressing cells Figure 4 , lower panel. Presumably, this proteolytic activation of the ribonuclease toxin resulted in a cessation of cellular protein synthesis soon after induction, what explains the detection of relatively low levels of NS3 protease in these cells.
As expected, no zymoxin cleavage could be detected in cells expressing the uncleavable form of the toxin. A faint band, corresponding to a cleaved zymoxin, can also be detected in uninduced cells that express the NS3 cleavable construct. Apparently, this very low, basal proteolytic activity is well tolerated by NS3 activated zymoxin expressing cells that shows no indication of de-novo protein synthesis inhibition in comparison to cells that express the uncleavable toxin see CPM values in the upper panel of Figure 4.
After 72 h, the relative fraction of viable cells was determined using an enzymatic MTT assay. These findings demonstrate the deleterious effect of the MazF based zymoxin specifically toward NS3 protease expressing cells, as well as its competence to be activated by very low cellular levels of protease.
The results show that both lower and higher induction levels of NS3 protease caused growth inhibition and rounding of cells that constitutively expresses the cleavable MazF. Furthermore, both green and red fluorescence were faint in these cells, probably as a result of the destructive ribonuclease activity of the cleaved toxin toward the NS3 protease and its own mRNA.
As expected, none of the above observations was evident when these cells were not supplemented with tetracycline or when NS3 expression was induced to high level in cells that constitutively express the uncleavable toxin Figure 6. Natural hosts for SV40 are Asian macaques, where it induces persistent infections in the kidneys. However, it was also shown that SV40 is significantly associated with human brain tumours and bone cancer [ 77 ], indicating its cell transforming properties.
The viral capsid is mainly composed of 72 VP1 protein pentamers in an icosahedral organisation [ 78 ]. Minor differences in the carbohydrate moiety of GM1, which is exposed into the extracellular space, strongly affect the binding affinity of the virus [ 80 ].
The multivalent binding of the VP1 pentamer to GM1 enables the tight association of the virus to the host cell despite the otherwise low affinity of individual binding sites of SV40 to GM1 [ 79 ].
In addition, a recent study on cellular and artificial membranes revealed that by virtue of this multivalent binding of GM1, SV40 induces the reorganization of membrane lipids and the segregation of specific lipids into membrane nanoscale domains, and thereby actively promotes its own uptake into the host cell [ 6 ].
This process critically depends on the lipid structure of GM1 and is essential for efficient infection by SV40 Figure 2. The precise physiological function of caveolae — uncoated, flask-like pits, enriched in cholesterol and glycosphingolipids e. GM1, GM3 — still remains debated. A recent study supports the notion that caveolae act as a membrane reservoir to counter mechanical stress [ 81 ]. The role of caveolae in clathrin-independent endocytosis is equally a matter of much debate [ 82 , 83 ].
FRAP fluorescence recovery after photobleaching -experiments on cells, which express GFP-tagged caveolin-1 the major protein component of caveolae in epithelial cells show that caveolae are rather immobile structures, a finding that argues against a major role of caveolae in constitutive endocytosis [ 84 ].
Though earlier studies suggest that the uptake of SV40 occurs via caveolae [ 85 - 88 ], recent work shows that the majority of SV40 does not partition into caveolae and that SV40 efficiently infects cells devoid of caveolin-1 [ 6 , 89 ], corroborating the idea that the caveolin-independent, lipid-induced pathway represents the major route for efficient SVinfection.
Influenza A Virus IAV is the causative agent of flu, which is an infectious disease, primary affecting the deep respiratory tract. Infectious particles of influenza viruses are pleomorphic, filamentous or spherically shaped particles with a mean diameter of nm [ 90 ]. Following receptor binding, virions undergo endocytosis and become uncoated in a pH-dependent manner [ 91 ].
The low pH of late endosomes induces a conformational change in the HA, resulting in the fusion of HA with the endosomal membrane [ 92 , 93 ] and the release of the RNA into the cytosol of the infected cell. Electron microscopy-based studies revealed that plasma membrane-derived vesicles containing IAV are surrounded by clathrin, indicating that clathrin is involved in the uptake of IAV [ 93 ]. Since IAV particles have also been observed in smooth, non-coated vesicles, it was speculated that IAV also enter host cells by clathrin- and caveolin-independent endocytosis.
This notion was supported by the observation that cells expressing dominant negative Eps15 a clathrin adaptor and caveolin-1 were still infected by IAV. Interestingly, subsequent studies showed that IAV actively induce the de novo formation of clathrin-coated pits by binding to the host cell surface [ 72 ]. The mechanism that triggers the recruitment of clathrin is unknown. It was speculated that IAV by binding to the host cell surface induces negative membrane curvatures that are sensed by BAR-domain containing proteins, which in turn recruit clathrin.
However, the membrane-bending properties of IAV have not yet been shown. Although experimental data are missing to this end, it is conceivable that IAV is able to bend membranes through clustering of sialic acid receptors for entry Figure 2.
The process of membrane bending and receptor clustering would likely be more complex than for Shiga toxin and SV40 VP1, considering that two different membranes i. Although more specific receptors than sialic acids are not yet identified for IAV, the virus activates specific cellular kinases for its efficient uptake, for example PI3K. PI3K is activated during the first 60 min of infection and was demonstrated to be required for efficient uptake [ 94 ]. In the context of bacterial invasion, PI3K activation is often associated with dramatic actin re-arrangements leading to macropinocytosis of the bacterium.
Interestingly, this pathway has been reported recently as an alternative entry pathway for IAV that is dependent on the kinases Rac1 and Src, but independent of dynamin [ 95 ]. It was speculated that the virus activates this pathway by interacting with receptor tyrosine kinases RTK in the plasma membrane of the host cell.
This study hints to RTK, such as the EGF receptor, as entry receptors that promote the efficient uptake of the virus in a sialic acid dependent manner. So far, PI3K activation has not been linked to the re-arrangements of the cytoskeleton by actin polymerisation, although it was shown for polarized epithelial cells; actin dynamics and the motor protein myosin IV are apparently indispensable for the internalisation of IAV [ 97 ].
Despite the vast number of reports analysing the entry processes of IAV at the plasma membrane, additional studies are required to understand the exact mechanistic role of sialic acids during the entry mechanism. In particular, less is known about the trans-bilayer signalling of sialic acids on the outer membrane leaflet towards the cytosolic machinery in the context of IAV entry. So far, we have addressed the endocytotic mechanisms of toxins and viruses.
In the following, we will review the internalization strategies of invasive bacteria. The most significant difference to toxin molecules and viruses regarding the initial entry steps, is that bacteria sense environmental changes e. By doing so, these pathogens can manipulate their local microenvironment to a certain level, which makes the invasion process more complex as compared to toxins and viruses.
A limited number of immune cells, such as macrophages, monocytes, dendritic cells, and neutrophils, are able to incorporate large particles in an actin-dependent process called phagocytosis and thus eliminate cellular debris, apoptotic bodies and pathogens [ 99 ].
During phagocytosis, the tight interaction between the particle and cell surface receptors of the host cell e. Fc or complement receptors induces a transient reorganization of the actin cytoskeleton and the generation of local membrane protrusions that engulf the particle.
Several pathogens, such as Mycobacteria including M. During the invasion by means of phagocytosis, the pathogen is passively taken up into the cell together with extracellular fluid. After internalization, pathogens alter the cellular machinery e. The uptake of M. Under normal conditions PI 4,5 P 2 and PI 3,4,5 P 3 are mainly localized and formed at the plasma membrane and recruit proteins important for phagocytosis. These include the early endosomal antigen 1 EEA1 , which is critical for the further maturation of pre-mature phagocytic vesicles into phagolysosomes [ ].
By secreting the phosphatidylinositol analogon lipoarabinomannan LAM , M. Phosphatidylinositol mannoside PIM , which is another mycobacterium-secreted phosphoinositide, stimulates early endosome fusion and consequently blocks phagosomal maturation [ ]. PI 3 P is also directly dephosphorylated by the mycobacterium-secreted PI phosphatase SapM, which additionally contributes to the arrest of phagosomal maturation.
These examples illustrate the powerful defence of M. Swanson and Baer were the first who proposed these mechanisms for particle phagocytosis in [ 99 ]. These cells respond with a re-arrangement of the cytoskeleton that promotes the entry of the bacterium. As a result of both strategies, these bacteria are tightly engulfed by the host cell plasma membrane [ ] Figure 3. Salmonella , as well as Shigella , described in the toxin section above , Listeria and Yersinia described below , are foodborne pathogens, which cause gastritic infections by ingestion of contaminated food or water.
Typical symptoms of an infection by Salmonella species, e. Salmonella enterica, are diarrhoea, abdominal cramps and fever. The T3SS spans the bacterial membrane and is then inserted into the host cell membrane. For an efficient invasion, a specific membrane microenvironment is critical.
The translocated bacterial virulence factors subvert various cellular activities of the host cell, which leads to a massive polymerization of actin and enables the internalization of the pathogen into the target cell [ , ]. The resulting membrane protrusions engulf the pathogen, which is then i. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen leading to acute infections of the respiratory tract, the urinary tract and the skin.
Like other Gram-negative bacteria mentioned above, P. Among the different strains presented so far, those that produce the T3SS effector proteins ExoT and ExoS are more efficiently internalized than those that do not produce these proteins [ ]. Both functions redundantly disrupt the actin cytoskeleton [ ].
Surprisingly, ExoS and ExoT act as anti-internalization factors [ ], probably by interfering with components of the Abl pathway [ ]: Rac1, Cdc42 and Crk were demonstrated to be activated by, and necessary for, the cellular uptake of P. Early studies demonstrate that P. Among these, asialo-GM1 seems to be important for the attachment of the bacteria to target cells in the respiratory tract by type IV pili [ 3 ].
Surprisingly, studies applying the small-molecule inhibitor PPMP, which inhibits the glucosylceramide synthase and consequently the biosynthesis of glycosphingolipids [ ], revealed that glycosphingolipids are rather important for the internalization instead of the adhesion to target cells [ ]. Rasmussen P. Bech F. Atlung T.
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