Virulence factors in brucellosis: implications for aetiopathogenesis and treatment

Brucella species are responsible for the global zoonotic disease brucellosis. These intracellular pathogens express a set of factors - including lipopolysaccharides, virulence regulator proteins and phosphatidylcholine - to ensure their full virulence. Some virulence factors are essential for invasion of the host cell, whereas others are crucial to avoid elimination by the host. They allow Brucella spp. to survive and proliferate within its replicative vacuole and enable the bacteria to escape detection by the host immune system. Several strategies have been used to develop animal vaccines against brucellosis, but no adequate vaccine yet exists to cure the disease in humans. This is probably due to the complicated pathophysiology of human Brucella spp. infection, which is different than in animal models. Here we review Brucella spp. virulence factors and how they control bacterial trafficking within the host cell.     

Pathogen trafficking pathways and host phosphoinositide metabolism

Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila , Brucella abortus , Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI-modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells.     

布鲁氏菌胞内生存机制研究进展

布鲁氏菌是一种胞内寄生菌,可以在专业和非专业吞噬细胞内生存和复制。当布鲁氏菌与细胞接触时,细菌可以通过受体分子进入细胞。布鲁氏菌在细胞内首先定位于早期吞噬体,然后,在胞内改变其运输方向,最终抵达其胞内复制部位内质网,开始大量复制。这种复制既不影响细胞的基本功能,也不诱导细胞的损伤。主要综述了布鲁氏菌对细胞的侵袭、胞内运输和复制的相关研究进展。     

“Make way”: Pathogen exploitation of membrane traffic

Intracellular pathogens have evolved numerous strategies to manipulate their host cells to survive and replicate in a hostile environment. They often exploit membrane trafficking pathways to enter the cell, establish a replicative niche, avoid degradation and immune response, acquire nutrients and lastly, egress. Recent studies on membrane trafficking exploitation by intracellular pathogens have led to the discovery of novel and fascinating cell biology, including a noncanonical mechanism of ubiquitination and a novel mitophagy receptor. Thus, studying how pathogens target host cell membrane trafficking pathways is not only important for the development of new therapeutics, but also helps understanding fundamental mechanisms of cell biology.     

What is the nature of the replicative niche of a stealthy bug named Brucella?

Brucella spp. are facultatively intracellular bacteria that persist and multiply in the macrophages of their mammalian hosts. The so-called phagosome to which they have adapted is their natural living niche. Characterization of this niche would facilitate an understanding of the true relationship between the host cell and the intracellular bacteria. This Opinion analyses and discusses the characteristic properties and genesis of this vacuole during phagocytosis as deduced from the virulence factors necessary for intracellular multiplication of the pathogen. We conclude that the replicative niche of Brucella spp.--the 'brucellosome'--differs from all other cellular organelles, and that it isolates the pathogen from certain cytoplasmic nutrients. Adaptation to the stress conditions encountered and the use of anaerobic respiration enable brucellae to replicate in the compartment they create.     

Mammalian membrane trafficking as seen through the lens of bacterial toxins

A fundamental question of eukaryotic cell biology is how membrane organelles are organised and interact with each other. Cell biologists address these questions by characterising the structural features of membrane compartments and the mechanisms that coordinate their exchange. To do so, they must rely on variety of cargo molecules and treatments that enable targeted perturbation, localisation, and labelling of specific compartments. In this context, bacterial toxins emerged in cell biology as paradigm shifting molecules that enabled scientists to not only study them from the side of bacterial infection but also from the side of the mammalian host. Their selectivity, potency, and versatility made them exquisite tools for uncovering much of our current understanding of membrane trafficking mechanisms. Here, we will follow the steps that lead toxins until their intracellular targets, highlighting how specific events helped us comprehend membrane trafficking and establish the fundamentals of various cellular organelles and processes. Bacterial toxins will continue to guide us in answering crucial questions in cellular biology while also acting as probes for new technologies and applications.     

Manipulation of rab GTPase function by intracellular bacterial pathogens.

Intracellular bacterial pathogens have evolved highly specialized mechanisms to enter and survive within their eukaryotic hosts. In order to do this, bacterial pathogens need to avoid host cell degradation and obtain nutrients and biosynthetic precursors, as well as evade detection by the host immune system. To create an intracellular niche that is favorable for replication, some intracellular pathogens inhibit the maturation of the phagosome or exit the endocytic pathway by modifying the identity of their phagosome through the exploitation of host cell trafficking pathways. In eukaryotic cells, organelle identity is determined, in part, by the composition of active Rab GTPases on the membranes of each organelle. This review describes our current understanding of how selected bacterial pathogens regulate host trafficking pathways by the selective inclusion or retention of Rab GTPases on membranes of the vacuoles that they occupy in host cells during infection.     

Adaptation of the Brucellae to their intracellular niche

Members of the bacterial genus Brucella are facultative intracellular pathogens that reside predominantly within membrane-bound compartments within two host cell types, macrophages and placental trophoblasts. Within macrophages, the brucellae route themselves to an intracellular compartment that is favourable for survival and replication, and they also appear to be well-adapted from a physiological standpoint to withstand the environmental conditions encountered during prolonged residence in this intracellular niche. Much less is known about the interactions of the Brucella with placental trophoblasts, but experimental evidence suggests that these bacteria use an iron acquisition system to support extensive intracellular replication within these host cells that is not required for survival and replication in host macrophages. Thus, it appears that the brucellae rely upon the products of distinct subsets of genes to adapt successfully to the environmental conditions encountered within the two cell types within which they reside in their mammalian hosts.     

Salmonella type III secretion effectors: pulling the host cell's strings

The enteric pathogen Salmonella employs type III secretion systems to transport a cocktail of effector proteins directly into its host cell. These effectors act in concert to control a variety of host cell processes to successfully invade intestinal cells and to establish an intracellular, replication-permissive niche. Recent studies reveal new insights into the molecular mechanisms that underlie effector protein injection, host cell invasion, and manipulation of vesicle trafficking induced by the interplay between multiple effectors and host systems. These findings corroborate the importance of spatio-temporal regulation of effector protein function for fine-tuned modulation of the host cell machinery.     

Host cell processes that influence the intracellular survival of Legionella pneumophila

Key to the pathogenesis of intracellular pathogens is their ability to manipulate host cell processes, permitting the establishment of an intracellular replicative niche. In turn, the host cell deploys defence mechanisms that limit intracellular infection. The bacterial pathogen Legionella pneumophila, the aetiological agent of Legionnaire's Disease, has evolved virulence mechanisms that allow it to replicate within protozoa, its natural host. Many of these tactics also enable L. pneumophila's survival and replication inside macrophages within a membrane-bound compartment known as the Legionella-containing vacuole. One of the virulence factors indispensable for L. pneumophila's intracellular survival is a type IV secretion system, which translocates a large repertoire of bacterial effectors into the host cell. These effectors modulate multiple host cell processes and in particular, redirect trafficking of the L. pneumophila phagosome and mediate its conversion into an ER-derived organelle competent for intracellular bacterial replication. In this review, we discuss how L. pneumophila manipulates host cells, as well as host cell processes that either facilitate or impede its intracellular survival.     

Rab蛋白家族在神经类疾病中的作用

细胞内膜囊泡运输是一个复杂的通路网络,Rab GTPases是膜囊泡运输的主要调节剂,通常被认为是细胞内吞和分泌系统中各种细胞器和囊泡的特异性标记和识别物。与Rab蛋白相关的轴突运输、内体运输发生障碍是造成神经退行性疾病的重要原因之一。本文主要介绍了Rab蛋白在多种神经退行性疾病病理机制中的作用机理与调控机制,同时讨论了线粒体和胶质细胞功能异常与Rab蛋白之间的关联。深入探究Rab蛋白的作用机制对人类神经性疾病的早期诊断和治疗具有潜在的指导意义。     

Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.     

Functional analysis of host factors that mediate the intracellular lifestyle of Cryptococcus neoformans

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.     

RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1alpha in supporting Brucella replication

Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1alpha(-/-) murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1alpha, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.     

Homing-associated molecules CD73 and VAP-1 as targets to prevent harmful inflammations and cancer spread

Homing-associated molecules are of fundamental importance for proper functioning of our immune system as they direct the cells to sites of inflammation to create an immune response. However, they are also responsible for harmful cell trafficking, which takes place in acute and chronic inflammations as well as in tumor progression and metastatic spread of cancer. Therefore, these molecules are potential targets for developing drugs to prevent harmful inflammation and metastases of cancer. In this review, we will discuss about the most recent advances in studies elucidating the role of two homing-associated ecto-enzymes in physiological and pathological cell trafficking and their use as drug targets.     

Pore-forming activity is not sufficient for Legionella pneumophila phagosome trafficking and intracellular growth

Bacterial pathogens often subvert eukaryotic cellular processes in order to establish a replicative niche and evade host immunity. Inhibition of phagosome lysosome fusion is a strategy used by several intracellular bacteria that grow within mammalian cells. It was shown recently that Legionella pneumophila possesses a cytolytic activity that results from the insertion of pores in the macrophage membrane upon contact, and that this activity requires the dot/icm gene products, which are necessary for intracellular growth and phagosome trafficking. Other bacteria that inhibit phagosome lysosome fusion, such as Mycobacterium tuberculosis , demonstrate similar cytolytic activities, which suggests that formation of pores in the phagosome membrane may account for the defects observed in phagosome trafficking. In this study, we identify a new class of L. pneumophila mutant that retains the pore-forming activity found in virulent bacteria, but is defective in phagosome lysosome fusion inhibition and intracellular growth. These data indicate that cytolytic activity is not sufficient for L. pneumophila -induced alterations in phagosome trafficking. Rather, the pore may be a vehicle that facilitates delivery of bacterial-derived effector molecules to the host cell cytoplasm.     

Sitting in the driver's seat: Manipulation of mammalian cell Rab GTPase functions by apicomplexan parasites

Many intracellular microbial pathogens subvert, disrupt or otherwise modulate host membrane trafficking pathways to establish a successful infection. Among them, bacteria that are trapped in a phagosome during mammalian cell invasion, disengage the programmed degradation process by altering the identity of their replicative niche through the exclusion or recruitment of specific Rab GTPases to their vacuole. Many viruses co-opt essential cellular trafficking pathways to perform key steps in their lifecycles. Among protozoan parasites, Apicomplexa are obligate intracellular microbes that invade mammalian cells by creating a unique, nonfusogenic membrane-bound compartment that protects the parasites straightaway from lysosomal degradation. Recent compelling evidence demonstrates that apicomplexan parasites are master manipulators of mammalian Rab GTPase proteins, and benefit or antagonise Rab functions for development within host cells. This review covers the exploitation of mammalian Rab proteins and vesicles by Apicomplexa, focusing on Toxoplasma, Neospora, Plasmodium and Theileria parasites.     

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.     

Lipid acquisition by intracellular Chlamydiae

Chlamydia species are obligate intracellular pathogens that are important causes of human genital tract, ocular and respiratory infections. The bacteria replicate within a specialized membrane-bound compartment termed the inclusion and require host-derived lipids for intracellular growth and development. Emerging evidence indicates that Chlamydia has evolved clever strategies to fulfil its lipid needs by interacting with multiple host cell compartments and redirecting trafficking pathways to its intracellular niche. In this review, we highlight recent findings that have significantly expanded our understanding of how Chlamydia exploit lipid trafficking pathways to ensure the survival of this important human pathogen.