Modulation of membrane traffic between endoplasmic reticulum, ERGIC and Golgi to generate compartments for the replication of bacteria and viruses

Several bacteria and viruses remodel cellular membranes to form compartments specialised for replication. Bacteria replicate within inclusions which recruit membrane vesicles from the secretory pathway to provide nutrients for microbial growth and division. Viruses generate densely packed membrane vesicles called viroplasm which provide a platform to recruit host and viral proteins necessary for replication. This review describes examples where both intracellular bacteria (Salmonella, Chlamydia and Legionella) and viruses (picornaviruses and hepatitis C) recruit membrane vesicles to sites of replication by modulating proteins that control the secretory pathway. In many cases this involves modulation of Rab and Arf GTPases.     

The efficiency of protein compartmentalization into the secretory pathway

Numerous proteins targeted for the secretory pathway are increasingly implicated in functional or pathological roles at alternative cellular destinations. The parameters that allow secretory or membrane proteins to reside in intracellular locales outside the secretory pathway remain largely unexplored. In this study, we have used an extremely sensitive and quantitative assay to measure the in vivo efficiency of signal sequence-mediated protein segregation into the secretory pathway. Our findings reveal that segregation efficiency varies tremendously among signals, ranging from >95 to <60%. The nonsegregated fraction is generated by a combination of mechanisms that includes inefficient signal-mediated translocation into the endoplasmic reticulum and leaky ribosomal scanning. The segregation efficiency of some, but not other signal sequences, could be influenced in cis by residues in the mature domain or in trans by yet unidentified cellular factors. These findings imply that protein compartmentalization can be modulated in a substrate-specific manner to generate biologically significant quantities of cytosolically available secretory and membrane proteins.     

Secretive bacterial pathogens and the secretory pathway

Eukaryotic cells possess two extensive endomembrane systems, each consisting of several sub-compartments connected by vesicular trafficking. One of these systems, the endocytic pathway, serves incoming traffic, and the other system, the secretory pathway (SP), is responsible for surface-bound traffic of intracellularly formed vesicles. Compartments derived of either system can be colonized by intracellular pathogens. In this review, we discuss the interactions between the SP and prominent intracellular bacterial pathogens of the genera Legionella, Brucella, Chlamydia and Salmonella. We emphasize secreted bacterial effector proteins, which directly manipulate host components of this pathway.     

病原细菌效应蛋白靶向宿主细胞核研究进展*

细胞核是细胞遗传与代谢的控制中心,调控细胞对外界的响应、代谢、生长和分化等细胞活动。在细菌感染宿主细胞过程中,个别细菌来源的效应蛋白能够靶向进入宿主细胞核,影响细胞核内基因的转录、RNA剪切、DNA修复以及染色质重组等生命活动,将这些能够进入细胞核的细菌效应蛋白称之为核调节蛋白。对病原菌分泌的核调节蛋白进入宿主细胞核的方式,以及不同病原菌的核调节蛋白调控宿主细胞的生命过程进行归纳总结,从而为深入探究病原细菌感染宿主细胞的致病机理提供理论基础。     

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.     

Targeting of host Rab GTPase function by the intravacuolar pathogen Legionella pneumophila

The intracellular pathogen Legionella pneumophila replicates in a vacuole that recruits material from the host cell endoplasmic reticulum (ER). Biogenesis of this unique vacuole depends on the bacterial Dot/Icm type IV secretion system that translocates proteins across host cell membranes. Here, we show that two translocated substrates, SidM and LidA, target host cell Rab1, a small GTPase regulating ER-to-Golgi traffic. SidM is a guanosine nucleotide exchange factor for Rab1 that recruits Rab1 to Legionella-containing vacuoles, a process that is enhanced by LidA. Expression of sidM in mammalian cells interferes with the secretory pathway and causes Golgi fragmentation. Consistent with a collaborative relationship between the two proteins, immobilized SidM and LidA synergize to promote Rab1-dependent binding of early secretory vesicles. These results indicate that proteins translocated into the host cell by the intravacuolar pathogen L. pneumophila are able to recapitulate events involved in host secretory trafficking.     

The trans Golgi Network Is Lost from Cells Infected with African Swine Fever Virus

The cellular secretory pathway is important during the assembly and envelopment of viruses and also controls the transport of host proteins, such as cytokines and major histocompatibility proteins, that function during the elimination of viruses by the immune system. African swine fever virus (ASFV) encodes at least 26 proteins with stretches of hydrophobic amino acids suggesting entry into the secretory pathway (R. J. Yanez, J. M. Rodriguez, M. L. Nogal, L. Yuste, C. Enriquez, J. F. Rodriguez, and E. Vinuela, Virology 208:249-278, 1995). To predict how and where these potential membrane proteins function, we have studied the integrity of the secretory pathway in cells infected with ASFV. Remarkably, ASFV caused complete loss of immunofluorescence signal for the trans Golgi network (TGN) marker protein TGN46 and dispersed the AP1 TGN adapter complex. Loss of TGN46 signal was not due to degradation of TGN46, suggesting redistribution of TGN46 to other membrane compartments. ASFV markedly slowed transport of cathepsin D to lysosomes, demonstrating that loss of TGN structure correlated with loss of TGN function. ASFV shows a tropism for macrophages, and it is possible that ASFV compromises TGN function to augment the activity of viral membrane proteins or to suppress the function of host immunoregulatory proteins.     

Armed and dangerous: Toxoplasma gondii uses an arsenal of secretory proteins to infect host cells

Toxoplasma gondii is a protozoan parasite that infects a wide variety of warm-blooded animals and humans, in which it causes opportunistic disease. As an obligate intracellular parasite, T. gondii must invade a host cell to survive and replicate during infection. Recent studies suggest that T. gondii secretes a variety of proteins that appear to function during invasion or intracellular replication. These proteins originate from three distinct regulated secretory organelles called micronemes, rhoptries and dense granules. By discharging the contents of its secretory organelles at precise steps in invasion, T. gondii appears to timely deploy secretory proteins to their correct target destinations. Based on the timing of secretion and the characteristics of secretory proteins, an emerging theme is that T. gondii compartmentalizes its secretory proteins according to general function. Thus, it appears that micronemal proteins may function during parasite attachment to host cells, rhoptry proteins may facilitate parasite vacuole formation and host organellar association, and dense granule proteins likely promote intracellular replication, possibly by transporting and processing nutrients from the host cell. However, as more T. gondii secretory proteins are identified and characterized, it is likely that additional functions will be ascribed to each class of proteins secreted- by this fascinating invasive parasite.     

Tracing Putative Trafficking of the Glycolytic Enzyme Enolase via SNARE-Driven Unconventional Secretion

Glycolytic enzymes are cytosolic proteins, but they also play important extracellular roles in cell-cell communication and infection. We used Saccharomyces cerevisiae to analyze the secretory pathway of some of these enzymes, including enolase, phosphoglucose isomerase, triose phosphate isomerase, and fructose 1,6-bisphosphate aldolase. Enolase, phosphoglucose isomerase, and an N-terminal 28-amino-acid-long fragment of enolase were secreted in a sec23-independent manner. The enhanced green fluorescent protein (EGFP)-conjugated enolase fragment formed cellular foci, some of which were found at the cell periphery. Therefore, we speculated that an overview of the secretory pathway could be gained by investigating the colocalization of the enolase fragment with intracellular proteins. The DsRed-conjugated enolase fragment colocalized with membrane proteins at the cis-Golgi complex, nucleus, endosome, and plasma membrane, but not the mitochondria. In addition, the secretion of full-length enolase was inhibited in a knockout mutant of the intracellular SNARE protein-coding gene TLG2. Our results suggest that enolase is secreted via a SNARE-dependent secretory pathway in S. cerevisiae.     

Cargo selection in the early secretory pathway of African trypanosomes

Membrane and secretory proteins are synthesized by ribosomes and then enter the endoplasmic reticulum (ER) where they undergo glycosylation and quality control for proper folding. Subsequently, proteins are transported to the Golgi apparatus and then sorted to the plasma membrane or intracellular organelles. Transport vesicles are formed at ER-exit sites (ERES) on the ER with several coat protein complexes. Cargo proteins loaded into the vesicles are selected by specific interactions with cargo receptors and/or adaptors during vesicle formation. p24 family and intracellular lectin ERGIC-53-membrane proteins are the known cargo receptors acting in the early secretory pathway (ER-Golgi). Oligomerization of the cargo receptors have been suggested to play an important role in cargo selection and sorting via posttranslational modifications in fungi and metazoans. On the other hand, the mechanisms involved in the early secretory pathway in protozoa remain unclear. In this review, we focus on Trypanosoma brucei as a representative of protozoan and discuss differences and commonalities in the molecular mechanisms of its early secretory pathway compared with other organisms.     

A conserved subtilisin-like protein TgSUB1 in microneme organelles of Toxoplasma gondii

Proteolytic processing plays a significant role in the process of invasion by the obligate intracellular parasite Toxoplasma gondii. We have cloned a gene, TgSUB1, encoding for a subtilisin-type serine protease found in T. gondii tachyzoites. TgSUB1 protein is homologous to other Apicomplexan and bacterial subtilisins and is processed within the secretory pathway of the parasite. Initial cleavage occurs in the endoplasmic reticulum, after which the protein is transported to micronemes, vesicles that secrete early during host cell invasion. Upon stimulation of microneme secretion, TgSUB1 is cleaved into smaller products that are secreted from the parasite. This secondary processing is inhibited by brefeldin A and serine protease inhibitors. TgSUB1 is a candidate processing enzyme for several microneme proteins cleaved within the secretory pathway or during invasion.     

Beauty and the yeast: compartmental organization of the secretory pathway

Our perception of intracellular organelles and cellular architecture was initially based on striking light and electron micrographs of animal and plant cells. The high degree of compartmental organization within specalized mammalian secretory cells aided early efforts to track the movement of proteins through the organelles of the secretory pathway. In contrast, the morphological detail of the yeast Saccharomyces cerevisiae appeared superficially simple, even primitive, by comparison with the higher eukaryotic cells. However, the combination of genetic tools and the development of assays reconstituting vesicular traffic in yeast have facilitated the identification and characterization of individual proteins that function in the secretory pathway. Analogies between the function of yeast and mammalian proteins in vesicular traffic are being drawn with increasing frequency. In this review, the combination of genetic, biochemical, molecular and cell biological approaches used to study compartmental organization in the yeast secretory pathway will be discussed. The rapid progress in our understanding of yeast membrane traffic has revealed the beauty of working with this organism.     

Inside or outside: detecting the cellular location of bacterial pathogens

Salmonella are intracellular pathogens that infect and multiply inside macrophages. Although Salmonella are some of the best-studied pathogens, it is difficult to determine quickly and reliably whether the bacteria are intracellular or extracellular. We have developed a novel method using differential fluorescence of two fluorescent proteins to determine the cellular location of pathogenic bacteria in macrophage infection assays. Using the differential expression of two unique fluorescent proteins that are expressed under specific conditions, we have developed a real-time assay for macrophage infections. The critical advantages of this system are that it does not alter the bacterial surface, it is not toxic to either the bacteria or the host cell, and it may be used in real-time quantitative assays. This assay can be readily applied to any other model pathogenic systems such as Listeria, Mycobacteria, and Legionella in which intracellular gene expression has been characterized.     

At the crossroads: communication of bacteria‐containing vacuoles with host organelles

Invasive bacterial pathogens are engulfed upon host cell entry in a vacuolar environment called the bacteria‐containing vacuole (BCV). BCVs directly contact with numerous host compartments, mainly vesicles of the endocytic pathway, such as endosomes or lysosomes. In addition, they also interact with the endoplasmic reticulum and endomembranes of the secretory pathway. These connections between the pathogen and the host occur either through heterotypic membrane fusions or through membrane contact sites. The precise regulation of BCV contacts with host compartments defines the constitution of the intracellular bacterial niche. It emerges that the associated pathways may control the stability of the BCV resulting either in vacuolar or cytoplasmically growing bacteria. Here, we will portray how the usage of novel proteomics and imaging technologies allows comparison of the communication of different host cell compartments with four relevant intracellular human pathogens, namely Salmonella enterica, Legionella pneumophila, Shigella flexneri and Francisella tularensis. The first two remain mainly within the BCV, and the latter two escape into the cytoplasm.     

Modulation of the host innate immune and inflammatory response by translocated bacterial proteins

Bacterial secretion systems play a central role in interfering with host inflammatory responses to promote replication in tissue sites. Many intracellular bacteria utilize secretion systems to promote their uptake and survival within host cells. An intracellular niche can help bacteria avoid killing by phagocytic cells, and may limit host sensing of bacterial components. Secretion systems can also play an important role in limiting host sensing of bacteria by translocating proteins that disrupt host immune signalling pathways. Extracellular bacteria, on the other hand, utilize secretion systems to prevent uptake by host cells and maintain an extracellular niche. Secretion systems, in this case, limit sensing and inflammatory signalling which can occur as bacteria replicate and release bacterial products in the extracellular space. In this review, we will cover the common mechanisms used by intracellular and extracellular bacteria to modulate innate immune and inflammatory signalling pathways, with a focus on translocated proteins of the type III and type IV secretion systems.     

Vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes

During the development of the asexual stage of the malaria parasite, Plasmodium falciparum, the composition, structure and function of the host cell membrane is dramatically altered, including the ability to adhere to vascular endothelium. Crucial to these changes is the transport of parasite proteins, which become associated with or inserted into the erythrocyte membrane. Protein and membrane targeting beyond the parasite plasma membrane must require unique pathways, given the parasites intracellular location within a parasitophorous vacuolar membrane and the lack of organelles and biosynthetic machinery in the host cell necessary to support a secretory system. It is not clear how these proteins cross the parasitophorous vacuolar membrane or how they traverse the erythrocyte cytosol to reach their final destinations. The identification of: (1) a P. falciparum homologue of the protein Sar1p, which is an essential component of the COPII-based secretory system in mammalian cells and yeast and (2) electron-dense, possibly coated, secretory vesicles bearing P. falciparum erythrocyte membrane protein 1 and P. falciparum erythrocyte membrane protein 3 in the host cell cytosol of P. falciparum infected erythrocytes recently provided the first direct evidence of a vesicle-mediated pathway for the trafficking of some parasite proteins to the erythrocyte membrane. The major advance in uncovering the parasite-induced secretory pathway was made by incubating infected erythrocytes with aluminium tetrafluoride, an activator of guanidine triphosphate-binding proteins, which resulted in the accumulation of the vesicles into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminium fluoride treatment, making their capture by electron microscopy possible. It appears that malaria parasites export proteins into the host cell cytosol to support a vesicle-mediated protein trafficking pathway.     

乙酰化修饰在嗜肺军团菌致病过程中的作用

乙酰化修饰是由乙酰基转移酶、去乙酰化酶介导的可逆的蛋白质翻译后修饰。其中,乙酰基转移酶将乙酰辅酶A的乙酰基团转移至底物蛋白的氨基酸残基,而乙酰基团的去除由去乙酰化酶完成。乙酰化修饰参与许多基本生物学过程的调节作用,越来越多的研究表明,蛋白质乙酰化修饰在病原菌的致病过程中具有重要作用。病原菌,如引起非典型性肺炎的嗜肺军团菌,可以通过分泌具有乙酰基转移酶活性的效应蛋白靶向宿主细胞信号通路的关键蛋白质因子,干扰宿主细胞信号通路及免疫反应。本文主要从嗜肺军团菌的致病机制、乙酰化修饰及乙酰化修饰在病原体致病过程中的调控作用进行综述,突出已知的乙酰化毒力蛋白的例子,并讨论它们如何影响与宿主的相互作用,为理解乙酰化修饰在嗜肺军团菌致病过程中的作用机制提供参考。     

Expression of calreticulin P-domain results in impairment of secretory pathway in Leishmania donovani and reduced parasite survival in macrophages

The secretory proteins of Leishmania are thought to be involved in the parasite survival inside the insect vector or mammalian host. It is clear from studies in higher eukaryotes that proper folding in the endoplasmic reticulum and targeting out of the endoplasmic reticulum is critical for the function of secretory proteins. The endoplasmic reticulum chaperones such as calreticulin play an important role in the quality control of secretory proteins. However, very little is known about the secretory pathway of trypanosomatid parasites such as Leishmania. In the present study, we show that overexpression of the P-domain of Leishmania donovani calreticulin in transfected L. donovani resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases. This effect is associated with an intracellular accumulation of active enzyme in these transfected parasites. In addition, parasites expressing the P-domain calreticulin showed a significant decrease in survival inside human macrophages. This study suggests that altering the function of an endoplasmic reticulum chaperone such as calreticulin in Leishmania may affect the targeting of proteins that are associated with the virulence of the parasite during their trafficking through the parasite secretory pathway.     

Secretory trafficking in neuronal dendrites

The neuronal secretory pathway represents the intracellular route for proteins involved in synaptic transmission and plasticity, as well as lipids required for outgrowth and remodelling of dendrites and axons. Although neurons use the same secretory compartments as other eukaryotic cells, the enormous distances involved, as well as the unique morphology of the neuron and its signalling requirements, challenge canonical models of secretory pathway organization. Here, we review evidence for a distributed secretory pathway in neurons, suggest mechanisms that may regulate secretory compartment distribution, and discuss the implications of a distributed secretory pathway for neuronal morphogenesis and neural-circuit plasticity.     

Viruses and the autophagy machinery

Autophagy is a major intracellular pathway for degradation and recycling of long-lived proteins and cytoplasmic organelles that plays an essential role in maintenance of homeostasis in response to starvation and other cellular stresses. Autophagy is also important for a variety of other processes including restriction of intracellular pathogen replication. Our understanding of the fascinating relationship between viruses and the autophagy machinery is still in its infancy but it is clear that autophagy is a newly recognized facet of innate and adaptive immunity against viral infection. Although the autophagy pathway is emerging as a component of host defense, certain viruses have developed strategies to counteract these antiviral mechanisms, and others appear to have co-opted the autophagy machinery as proviral host factors favoring viral replication. The complex interplay between autophagy and viral infection will be discussed in this review.