Cytosol Of Eukaryotic Cells Research Articles

Gene expression profiling of Yersinia pestis with deletion of lcrG, a known negative regulator for Yop secretion of type III secretion system

Yersinia pestis injects a set of virulent proteins into the cytosol of eukaryotic cells by a type III secretion system (T3SS). LcrG is a known negative regulator for secretion of Yersinia outer-membrane proteins (Yops) by blocking the secretion apparatus (Ysc) from the inner membrane. To further understand the effect of lcrG deletion on Y. pestis T3SS regulation, transcriptional profiles from the ΔlcrG mutant and wild-type Y. pestis strains were compared. The results showed that although the ΔlcrG mutant was markedly attenuated (600-fold increase of LD 50 in s.c. challenged BALB/c mice), transcriptions of almost all the type III genes were upregulated significantly in the ΔlcrG mutant. The immunoblotting analysis of YopM and LcrV demonstrated that their expressions were also increased in the ΔlcrG mutant in comparison to the wild-type strain. We speculate that, in addition to the negative regulation of the Yop secretion, LcrG could possibly play a negative regulatory role in the transcription of T3SS genes through indirect mechanisms. Furthermore, this report also revealed significant transcriptional changes in the genes encoding cell-envelope-related proteins and a virulence-related transcription factor RovA in the ΔlcrG mutant.

ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death

The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.

YscP and YscU Switch the Substrate Specificity of the Yersinia Type III Secretion System by Regulating Export of the Inner Rod Protein YscI

Pathogenic yersiniae utilize a type III secretion system to inject antihost factors, called Yops, directly into the cytosol of eukaryotic cells. The Yops are injected via a needle-like structure, comprising the YscF protein, on the bacterial surface. While the needle is being assembled, Yops cannot be secreted. YscP and YscU switch the substrate specificity of the secretion system to enable Yop export once the needle attains its proper length. Here, we demonstrate that the inner rod protein YscI plays a critical role in substrate specificity switching. We show that YscI is secreted by the type III secretion system and that YscI secretion by a yscP mutant is abnormally elevated. Furthermore, we show that mutations in the cytoplasmic domain of YscU reduce YscI secretion by the yscP null strain. We also demonstrate that mutants expressing one of three forms of YscI (those with mutations Q84A, L87A, and L96A) secrete substantial amounts of Yops yet exhibit severe defects in needle formation. In the absence of YscP, mutants with the same changes in YscI assemble needles but are unable to secrete Yops. Together, these results suggest that the formation of the inner rod, not the needle, is critical for substrate specificity switching and that YscP and YscU exert their effects on substrate export by controlling the secretion of YscI.

The Listeria monocytogenes Virulence Factor InlJ Is Specifically Expressed In Vivo and Behaves as an Adhesin

The food-borne pathogen Listeria monocytogenes is adapted to a diversity of environments, such as soil, food, body fluids, and the cytosol of eukaryotic cells. The transition between saprophytic and pathogenic life is mediated through complex regulatory pathways that modulate the expression of virulence factors. Here we examined the expression of inlJ, a recently identified gene encoding a protein of the LPXTG-internalin family and involved in pathogenesis. We show that inlJ expression is controlled neither by the major listerial regulator of virulence genes, PrfA, nor by AxyR, a putative AraC regulator encoded by a gene adjacent to inlJ and divergently transcribed. The InlJ protein is not produced by bacteria grown in vitro in brain heart infusion medium or replicating in the cytosol of tissue-cultured cells. In contrast, it is efficiently produced and localized at the surface of bacteria present in the liver and blood of infected animals. Strikingly, the expression of inlJ by a heterologous promoter in L. monocytogenes or L. innocua promotes bacterial adherence to human cells in vitro. Taken together, these results strongly suggest that InlJ acts as a novel L. monocytogenes sortase-anchored adhesin specifically expressed during infection in vivo.

Towards the control of intracellular protein turnover: Mitochondrial Lon protease inhibitors versus proteasome inhibitors

Cellular protein homeostasis results from the combination of protein biogenesis processes and protein quality control mechanisms, which contribute to the functional state of cells under normal and stress conditions. Proteolysis constitutes the final step by which short-lived, misfolded and damaged intracellular proteins are eliminated. Protein turnover and oxidatively modified protein degradation are mainly achieved by the proteasome in the cytosol and nucleus of eukaryotic cells while several ATP-dependent proteases including the matrix protease Lon take part in the mitochondrial protein degradation. Moreover, Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. Specific inhibitors are commonly used to assess cellular functions of proteolytic systems as well as to identify their protein substrates. Here, we present and discuss known proteasome and Lon protease inhibitors. To date, very few inhibitors of Lon have been described and no specific inhibitors of this protease are available. The current knowledge on both catalytic mechanisms and inhibitors of these two proteases is first described and attempts to define specific non-peptidic inhibitors of the human Lon protease are presented.

Aeromonas Exoenzyme T of Aeromonas salmonicida Is a Bifunctional Protein That Targets the Host Cytoskeleton

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.

Free Oligosaccharides in the Cytosol of Caenorhabditis elegans Are Generated through Endoplasmic Reticulum-Golgi Trafficking

Free oligosaccharides (FOSs) in the cytosol of eukaryotic cells are mainly generated during endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded glycoproteins. We analyzed FOS of the nematode Caenorhabditis elegans to elucidate its detailed degradation pathway. The major FOSs were high mannose-type ones bearing 3-9 Man residues. About 94% of the total FOSs had one GlcNAc at their reducing end (FOS-GN1), and the remaining 6% had two GlcNAc (FOS-GN2). A cytosolic endo-beta-N-acetylglucosaminidase mutant (tm1208) accumulated FOS-GN2, indicating involvement of the enzyme in conversion of FOS-GN2 into FOS-GN1. The most abundant FOS in the wild type was Man(5)GlcNAc(1), the M5A' isomer (Manalpha1-3(Manalpha1-6)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAc), which is different from the corresponding M5B' (Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAc) in mammals. Analyses of FOS in worms treated with Golgi alpha-mannosidase I inhibitors revealed decreases in Man(5)GlcNAc(1) and increases in Man(7)GlcNAc(1). These results suggested that Golgi alpha-mannosidase I-like enzyme is involved in the production of Man(5-6)-GlcNAc(1), which is unlike in mammals, in which cytosolic alpha-mannosidase is involved. Thus, we assumed that major FOSs in C. elegans were generated through Golgi trafficking. Analysis of FOSs from a Golgi alpha-mannosidase II mutant (tm1078) supported this idea, because GlcNAc(1)Man(5)GlcNAc(1), which is formed by the Golgi-resident GlcNAc-transferase I, was found as a FOS in the mutant. We concluded that significant amounts of misfolded glycoproteins in C. elegans are trafficked to the Golgi and are directly or indirectly retro-translocated into the cytosol to be degraded.

Type III secretion system translocator has a molten globule conformation both in its free and chaperone‐bound forms

Type III secretion systems of Gram-negative pathogenic bacteria allow the injection of effector proteins into the cytosol of host eukaryotic cells. Crossing of the eukaryotic plasma membrane is facilitated by a translocon, an oligomeric structure made up of two bacterial proteins inserted into the host membrane during infection. In Pseudomonas aeruginosa, a major human opportunistic pathogen, these proteins are PopB and PopD. Their interactions with their common chaperone PcrH in the cytosol of the bacteria are essential for the proper function of the injection system. The interaction region between PopD and PcrH was identified using limited proteolysis, revealing that the putative PopD transmembrane fragment is buried within the PopD/PcrH complex. In addition, structural features of PopD and PcrH, either individually or within the binary complex, were characterized using spectroscopic methods and 1D NMR. Whereas PcrH possesses the characteristics of a folded protein, PopD is in a molten globule state either alone or in the PopD/PcrH complex. The molten globule state is known to enable the membrane insertion of translocation/pore-forming domains of bacterial toxins. Therefore, within the bacterial cytoplasm, PopD is preserved in a state that is favorable to secretion and insertion into cell membranes.

Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain

Vibrio cholerae RTX is a large multifunctional bacterial toxin that causes actin crosslinking. Due to its size, it was predicted to undergo proteolytic cleavage during translocation into host cells to deliver activity domains to the cytosol. In this study, we identified a domain within the RTX toxin that is conserved in large clostridial glucosylating toxins TcdB, TcdA, TcnA, and TcsL; putative toxins from V. vulnificus, Yersinia sp., Photorhabdus sp., and Xenorhabdus sp.; and a filamentous/hemagglutinin-like protein FhaL from Bordetella sp. In vivo transfection studies and in vitro characterization of purified recombinant protein revealed that this domain from the V. cholerae RTX toxin is an autoprocessing cysteine protease whose activity is stimulated by the intracellular environment. A cysteine point mutation within the RTX holotoxin attenuated actin crosslinking activity suggesting that processing of the toxin is an important step in toxin translocation. Overall, we have uncovered a new mechanism by which large bacterial toxins and proteins deliver catalytic activities to the eukaryotic cell cytosol by autoprocessing after translocation.

Systematic analysis of the regulation of type three secreted effectors in Salmonella enterica serovar Typhimurium

BackgroundThe type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions.ResultsWe performed a systematic analysis of the regulation of type III effectors during growth in vitro. We have tested the ability of seven regulatory genes to regulate ten effector genes. Each regulator was expressed in the absence of the other six to avoid cascade effects. Our results confirm and extend the previously reported regulation of TTSS1 and TTSS2 effectors by InvF-SicA and SsrB respectively.ConclusionThe set of strains constructed for this study can be used to quickly and systematically study the regulation of newly identified effector genes of Salmonella enterica. The approach we have used can also be applied to study complex regulatory cascades in other bacterial species.

Minimal YopB and YopD translocator secretion by Yersinia is sufficient for Yop-effector delivery into target cells

Pathogenic Yersinia sp. utilise a common type III secretion system to translocate several anti-host Yop effectors into the cytosol of target eukaryotic cells. The secreted YopB and YopD translocator proteins are essential for this process, forming pores in biological membranes through which the effectors are thought to gain access to the cell interior. The non-secreted cognate chaperone, LcrH, also plays an important role by ensuring pre-secretory stabilisation and efficient secretion of YopB and YopD. This suggests that LcrH-regulated secretion of the translocators could be used by Yersinia to control effector translocation levels. We collected several LcrH mutants impaired in chaperone activity. These poorly bound, stabilised and/or secreted YopB and YopD in vitro. However, these mutants generally maintained stable substrates during a HeLa cell infection and these infected cells were intoxicated by translocated effectors. Surprisingly, this occurred in the absence of detectable YopB- and YopD-dependent pores in eukaryotic membranes. A functional type III translocon must therefore only require minuscule amounts of secreted translocator proteins. Based on these observations, LcrH dependent control of translocation via regulated YopB and YopD secretion would need to be exquisitely tight.

The discovery of SycO highlights a new function for type III secretion effector chaperones

Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone-binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation-prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.

Substrate Transfer from the Chaperone Hsp70 to Hsp90

Hsp90 is an essential chaperone protein in the cytosol of eukaryotic cells. It cooperates with the chaperone Hsp70 in defined complexes mediated by the adaptor protein Hop (Sti1 in yeast). These Hsp70/Hsp90 chaperone complexes play a major role in the folding and maturation of key regulatory proteins in eukaryotes. Understanding how non-native client proteins are transferred from one chaperone to the other in these complexes is of central importance. Here, we analyzed the molecular mechanism of this reaction using luciferase as a substrate protein. Our experiments define a pathway for luciferase folding in the Hsp70/Hsp90 chaperone system. They demonstrate that Hsp70 is a potent capture device for unfolded protein while Hsp90 is not very efficient in this reaction. When Hsp90 is absent, in contrast to the in vivo situation, Hsp70 together with the two effector proteins Ydj1 and Sti1 exhibits chaperone activity towards luciferase. In the presence of the complete chaperone system, Hsp90 exhibits a specific positive effect only in the presence of Ydj1. If this factor is absent, the transferred luciferase is trapped on Hsp90 in an inactive conformation. Interestingly, identical results were observed for the yeast and the human chaperone systems although the regulatory function of human Hop is completely different from that of yeast Sti1.

Fluorescent reporters for the ubiquitin–proteasome system

Regulated turnover of proteins in the cytosol and nucleus of eukaryotic cells is primarily performed by the ubiquitin-proteasome system (UPS). The UPS is involved in many essential cellular processes. Alterations in this proteolytic system are associated with a variety of human pathologies, such as neurodegenerative diseases, cancer, immunological disorders and inflammation. The precise role of the UPS in the pathophysiology of these diseases, however, remains poorly understood. Detection of UPS aberrations has been a major challenge because of the complexity of the system. Most studies focus on various aspects of the UPS, such as substrate recognition, ubiquitination, deubiquitination or proteasome activity, and do not provide a complete picture of the UPS as an integral system. To monitor the efficacy of the UPS, a number of reporter substrates have been developed based on fluorescent proteins, such as the green fluorescent protein and its spectral variants. These fluorescent UPS reporters contain specific degradation signals that target them with high efficiency and accuracy for proteasomal degradation. Several studies have shown that these reporters can probe the functionality of the UPS in cellular and animal models and provide us with important information on the status of the UPS under various conditions. Moreover, these reporters can aid the identification and development of novel anti-cancer and anti-inflammatory drugs based on UPS inhibition.

Fluorescent reporters for the ubiquitin–proteasome system

Regulated turnover of proteins in the cytosol and nucleus of eukaryotic cells is primarily performed by the ubiquitin–proteasome system (UPS). The UPS is involved in many essential cellular processes. Alterations in this proteolytic system are associated with a variety of human pathologies, such as neurodegenerative diseases, cancer, immunological disorders and inflammation. The precise role of the UPS in the pathophysiology of these diseases, however, remains poorly understood. Detection of UPS aberrations has been a major challenge because of the complexity of the system. Most studies focus on various aspects of the UPS, such as substrate recognition, ubiquitination, deubiquitination or proteasome activity, and do not provide a complete picture of the UPS as an integral system. To monitor the efficacy of the UPS, a number of reporter substrates have been developed based on fluorescent proteins, such as the green fluorescent protein and its spectral variants. These fluorescent UPS reporters contain specific degradation signals that target them with high efficiency and accuracy for proteasomal degradation. Several studies have shown that these reporters can probe the functionality of the UPS in cellular and animal models and provide us with important information on the status of the UPS under various conditions. Moreover, these reporters can aid the identification and development of novel anti-cancer and anti-inflammatory drugs based on UPS inhibition.

Type III secretion: The bacteria-eukaryotic cell express

Type III secretion (T3S) is an export pathway used by Gram-negative pathogenic bacteria to inject bacterial proteins into the cytosol of eukaryotic host cells. This pathway is characterized by (i) a secretion nanomachine related to the bacterial flagellum, but usually topped by a stiff needle-like structure; (ii) the assembly in the eukaryotic cell membrane of a translocation pore formed by T3S substrates; (iii) a non-cleavable N-terminal secretion signal; (iv) T3S chaperones, assisting the secretion of some substrates; (v) a control mechanism ensuring protein delivery at the right place and time. Here, we review these different aspects focusing in open questions that promise exciting findings in the near future.

The UBA2 Domain Functions as an Intrinsic Stabilization Signal that Protects Rad23 from Proteasomal Degradation

The proteasome-interacting protein Rad23 is a long-lived protein. Interaction between Rad23 and the proteasome is required for Rad23's functions in nucleotide excision repair and ubiquitin-dependent degradation. Here, we show that the ubiquitin-associated (UBA)-2 domain of yeast Rad23 is a cis-acting, transferable stabilization signal that protects Rad23 from proteasomal degradation. Disruption of the UBA2 domain converts Rad23 into a short-lived protein that is targeted for degradation through its N-terminal ubiquitin-like domain. UBA2-dependent stabilization is required for Rad23 function because a yeast strain expressing a mutant Rad23 that lacks a functional UBA2 domain shows increased sensitivity to UV light and, in the absence of Rpn10, severe growth defects. The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and the human Rad23 homolog hHR23A have similar protective activities. Thus, the UBA2 domain of Rad23 is an evolutionarily conserved stabilization signal that allows Rad23 to interact with the proteasome without facing destruction.

Yersinia Outer Protein P Inhibits CD8 T Cell Priming in the Mouse Infection Model

Pathogenic yersiniae translocate a mixture of effector proteins called Yersinia outer proteins (Yops) into the cytosol of eukaryotic cells by their type III secretion system. YopP is one of the best characterized of these effector proteins and known to inhibit the proinflammatory response of the host by interfering with NF-kappaB signal transduction and inducing apoptosis of macrophages. The effects of YopP on the immune response were studied by a Yersinia Ag-independent approach using bacteria that translocate the well-characterized model Ag listeriolysin O of Listeria monocytogenes via their type III secretion system. In this study we demonstrate a novel function for YopP in vivo. It is shown for the first time that YopP not only counteracts the innate immune defense but also inhibits the adaptive immune system by suppressing the development of an effective CD8 T cell response in a mouse model. A possible mechanism for this could be the inhibition of Ag presentation by dendritic cells (DC). In vitro this is shown to be due to the rapid induction of programmed DC death and to inhibition of DC maturation. Using this approach we could further show that the listeriolysin O-specific CD8 T cells generated in vivo by the yopP mutant are functional and are able to protect mice against a lethal challenge with wild type Listeria.

Measure for measure in the control of type III secretion hook and needle length

Bacterial flagella and injectisomes are supramolecular structures that are responsible for motility and for delivering toxic proteins into the cytosol of eukaryotic cells, respectively. They look very similar to each other. Both systems are called type III secretion pathways, and their components share substantial sequence similarities. One remarkable feature of the type III systems is that the length of their substructure is fairly well controlled by a secretion switching machinery, which consists of at least two proteins, a length control protein and an integral membrane secretion component. Here, we review how and why the length of these structures must be accurately controlled.

Structural and functional studies of the enteropathogenic Escherichia coli type III needle complex protein EscJ

The type III secretion system (TTSS) is a macromolecular structure that spans the cell wall of Gram-negative bacterial pathogens, enabling delivery of virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. TTSS consists of a conserved needle complex (NC) that is composed of sets of inner and outer membranes rings connected by a periplasmic rod. Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrhoeagenic pathogen that uses TTSS to induce actin polymerization and colonizes the intestinal epithelium. In EPEC, EscJ is predicted to be targeted to the periplasm, in a sec-dependent manner, and to bridge the TTSS membrane-associated rings. In this study we determined the global fold of EscJ using Nuclear Magnetic Resonance spectroscopy. We show that EscJ comprises two subdomains (D1 - amino acid residues 1-55 in the mature protein, and D2 - amino acid residues 90-170), each comprising a three-stranded beta-sheet flanked by two alpha-helices. A flexible region (residues 60-85) couples the structured regions D1 and D2. Periplasmic overexpression of EscJ(D1) and EscJ(D2) in a single escJ mutant bacterium failed to restore protein secretion activity, suggesting that the flexible linker is essential for the rod function. In contrast, periplasmic overexpression of EscJ(D1) and EscJ(D2) in the same wild-type bacterium had a dominant-negative phenotype suggesting defective assembly of the TTSS and protein translocation.