Coordinated delivery and function of bacterial MARTX toxin effectors

Bacteria often coordinate virulence factors to fine‐tune the host response during infection. These coordinated events can include toxins counteracting or amplifying effects of another toxin or though regulating the stability of virulence factors to remove their function once it is no longer needed. Multifunctional autoprocessing repeats‐in toxin (MARTX) toxins are effector delivery toxins that form a pore into the plasma membrane of a eukaryotic cell to deliver multiple effector proteins into the cytosol of the target cell. The function of these proteins includes manipulating actin cytoskeletal dynamics, regulating signal transduction pathways and inhibiting host secretory pathways. Investigations into the molecular mechanisms of these effector domains are providing insight into how the function of some effectors overlap and regulate one another during infection. Coordinated crosstalk of effector function suggests that MARTX toxins are not simply a sum of all their parts. Instead, modulation of cell function by effector domains may depend on which other effector domain are co‐delivered. Future studies will elucidate how these effectors interact with each other to modulate the bacterial host interaction.     

Molecular weaponry: diverse effectors delivered by the Type VI secretion system

The Type VI secretion system is a widespread bacterial nanomachine, used to deliver toxins directly into eukaryotic or prokaryotic target cells. These secreted toxins, or effectors, act on diverse cellular targets, and their action provides the attacking bacterial cell with a significant fitness advantage, either against rival bacteria or eukaryotic host organisms. In this review, we discuss the delivery of diverse effectors by the Type VI secretion system, the modes of action of the so‐called ‘anti‐bacterial’ and ‘anti‐eukaryotic’ effectors, the mechanism of self‐resistance against anti‐bacterial effectors and the evolutionary implications of horizontal transfer of Type VI secretion system‐associated toxins. Whilst it is likely that many more effectors remain to be identified, it is already clear that toxins delivered by this secretion system represent efficient weapons against both bacteria and eukaryotes.     

The targeting of plant cellular systems by injected type III effector proteins

The battle between phytopathogenic bacteria and their plant hosts has revealed a diverse suite of strategies and mechanisms employed by the pathogen or the host to gain the higher ground. Pathogens continually evolve tactics to acquire host resources and dampen host defences. Hosts must evolve surveillance and defence systems that are sensitive enough to rapidly respond to a diverse range of pathogens, while reducing costly and damaging inappropriate misexpression. The primary virulence mechanism employed by many bacteria is the type III secretion system, which secretes and translocates effector proteins directly into the cells of their plant hosts. Effectors have diverse enzymatic functions and can target specific components of plant systems. While these effectors should favour bacterial fitness, the host may be able to thwart infection by recognizing the activity or presence of these foreign molecules and initiating retaliatory immune measures. We review the diverse host cellular systems exploited by bacterial effectors, with particular focus on plant proteins directly targeted by effectors. Effector–host interactions reveal different stages of the battle between pathogen and host, as well as the diverse molecular strategies employed by bacterial pathogens to hijack eukaryotic cellular systems.     

Quantitative mass spectrometry catalogues Salmonella pathogenicity island-2 effectors and identifies their cognate host binding partners

Gram-negative bacterial pathogens have developed specialized secretion systems to transfer bacterial proteins directly into host cells. These bacterial effectors are central to virulence and reprogram host cell processes to favor bacterial survival, colonization, and proliferation. Knowing the complete set of effectors encoded by a particular pathogen is the key to understanding bacterial disease. In addition, the identification of the molecular assemblies that these effectors engage once inside the host cell is critical to determining the mechanism of action of each effector. In this work we used stable isotope labeling of amino acids in cell culture (SILAC), a powerful quantitative proteomics technique, to identify the proteins secreted by the Salmonella pathogenicity island-2 type three secretion system (SPI-2 T3SS) and to characterize the host interaction partners of SPI-2 effectors. We confirmed many of the known SPI-2 effectors and were able to identify several novel substrate candidates of this secretion system. We verified previously published host protein-effector binding pairs and obtained 11 novel interactions, three of which were investigated further and confirmed by reciprocal co-immunoprecipitation. The host cell interaction partners identified here suggest that Salmonella SPI-2 effectors target, in a concerted fashion, cellular processes such as cell attachment and cell cycle control that are underappreciated in the context of infection. The technology outlined in this study is specific and sensitive and serves as a robust tool for the identification of effectors and their host targets that is readily amenable to the study of other bacterial pathogens.     

A simple yeast-based strategy to identify host cellular processes targeted by bacterial effector proteins

Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.     

Bacterial protein toxins that modify host regulatory GTPases

Many bacterial pathogens produce protein toxins to outmanoeuvre the immune system of the host. Some of these proteins target regulatory GTPases such as those belonging to the RHO family, which control the actin cytoskeleton of the host cell. In this Review, I discuss a diversity of mechanisms that are used by bacterial effectors and toxins to modulate the activity of host GTPases, with a focus on covalent modifications such as ADP-ribosylation, glucosylation, adenylylation, proteolysis, deamidation and transglutamination.     

Bacterial effector interplay: a new way to view effector function

Bacterial pathogens are dependent on virulence factors to efficiently colonize and propagate within their hosts. Many Gram-negative bacterial pathogens rely on specialized proteinaceous secretion systems that inject virulence factors, termed effectors, directly into host cells. These bacterial effector proteins perform various functions within host cells; however, regulation of their function within the host cell is highly enigmatic. It is becoming increasingly apparent that many of these effectors directly influence and regulate each other and their mechanisms within the host cell. We discuss the emerging theme of bacterial effector interplay impacting infection and the importance of investigating this topic.     

Pathogen effectors: What do they do at plasmodesmata?

Plants perceive an assortment of external cues during their life cycle, including abiotic and biotic stressors. Biotic stress from a variety of pathogens, including viruses, oomycetes, fungi, and bacteria, is considered to be a substantial factor hindering plant growth and development. To hijack the host cell's defence machinery, plant pathogens have evolved sophisticated attack strategies mediated by numerous effector proteins. Several studies have indicated that plasmodesmata (PD), symplasmic pores that facilitate cell-to-cell communication between a cell and neighbouring cells, are one of the targets of pathogen effectors. However, in contrast to plant-pathogenic viruses, reports of fungal- and bacterial-encoded effectors that localize to and exploit PD are limited. Surprisingly, a recent study of PD-associated bacterial effectors has shown that a number of bacterial effectors undergo cell-to-cell movement via PD. Here we summarize and highlight recent advances in the study of PD-associated fungal/oomycete/bacterial effectors. We also discuss how pathogen effectors interfere with host defence mechanisms in the context of PD regulation.     

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.     

Plant pathogenic bacterial type III effectors subdue host responses

Like animals, plants sense bacterial pathogens through surface-localized pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat proteins (NB-LRR) and trigger defense responses. Many plant-pathogenic bacteria secrete a large repertoire of effector proteins into host cells to modulate host responses, enabling successful infection and multiplication in plants. A number of these effector proteins target plant innate immunity signaling pathways, while others induce specific host genes to enhance plant susceptibility. Substantial progress has been made in the past two years concerning biochemical function of effectors and their host targets. These advances provide new insights into regulatory mechanisms of plant immunity and host-pathogen co-evolution.     

Diversity of bacterial manipulation of the host ubiquitin pathways

Ubiquitination is generally considered as a eukaryotic protein modification, which is catalysed by a three‐enzyme cascade and is reversed by deubiquitinating enzymes. Ubiquitination directs protein degradation and regulates cell signalling, thereby plays key roles in many cellular processes including immune response, vesicle trafficking and cell cycle. Bacterial pathogens inject a series of virulent proteins, named effectors, into the host cells. Increasing evidence suggests that many effectors hijack the host ubiquitin pathways to benefit bacterial infection. This review summarizes the known functions and mechanisms of effectors from human bacterial pathogens including enteropathogenic Escherichia coli, Salmonella, Shigella, Chlamydia and Legionella, highlighting the diversity in their mechanisms for manipulating the host ubiquitin pathways. Many effectors adopt the molecular mimicry strategy to harbour similar structures or functional motifs with those of the host E3 ligases and deubiquitinases. On the other hand, a few of effectors evolve novel structures or new enzymatic activities to modulate various steps of the host ubiquitin pathways. The diversity in the mechanisms enhances the efficient exploitation of the host ubiquitination signalling by bacteria.     

Chemical engineering of bacterial effectors for regulating cell signaling and responses

Bacteria have evolved a variety of effector proteins to facilitate their survival and proliferation within the host environment. Continuous competition at the host–pathogen interface has empowered these effectors with unique mechanism and high specificity toward their host targets. The rich repertoire of bacterial effectors has thus provided us an attractive toolkit for investigating various cellular processes, such as signal transductions. With recent advances in protein chemistry and engineering, we now have the capability for on-demand control of protein activity with high precision. Herein, we review the development of chemically engineered bacterial effectors to control kinase-mediated signal transductions, inhibit protein translation, and direct genetic editing within host cells. We also highlight future opportunities for harnessing diverse prokaryotic effectors as powerful tools for mechanistic investigation and therapeutic intervention of eukaryotic systems.     

The role of interactions between phage and bacterial proteins within the infected cell: a diverse and puzzling interactome

Interactions between bacteriophage proteins and bacterial proteins are important for efficient infection of the host cell. The phage proteins involved in these bacteriophage–host interactions are often produced immediately after infection. A survey of the available set of published bacteriophage–host interactions reveals the targeted host proteins are inhibited, activated or functionally redirected by the phage protein. These interactions protect the bacteriophage from bacterial defence mechanisms or adapt the host-cell metabolism to establish an efficient infection cycle. Regrettably, a large majority of bacteriophage early proteins lack any identified function. Recent research into the antibacterial potential of bacteriophage–host interactions indicates that phage early proteins seem to target a wide variety of processes in the host cell – many of them non-essential. Since a clear understanding of such interactions may become important for regulations involving phage therapy and in biotechnological applications, increased scientific emphasis on the biological elucidation of such proteins is warranted.     

ExoS controls the cell contact-mediated switch to effector secretion in Pseudomonas aeruginosa

Type III secretion is used by many gram-negative bacterial pathogens to directly deliver protein toxins (effectors) into targeted host cells. In all cases, secretion of effectors is triggered by host cell contact, although the mechanism is unclear. In Pseudomonas aeruginosa, expression of all type III secretion-related genes is up-regulated when secretion is triggered. We were able to visualize this process using a green fluorescent protein reporter system and to use it to monitor the ability of bacteria to trigger effector secretion on cell contact. Surprisingly, the action of one of the major type III secreted effectors, ExoS, prevented triggering of type III secretion by bacteria that subsequently attached to cells, suggesting that triggering of secretion is feedback regulated. Evidence is presented that translocation (secretion of effectors across the host cell plasma membrane) of ExoS is indeed self-regulated and that this inhibition of translocation can be achieved by either of its two enzymatic activities. The translocator proteins PopB, PopD, and PcrV are secreted via the type III secretion system and are required for pore formation and translocation of effectors across the host cell plasma membrane. Here we present data that secretion of translocators is in fact not controlled by calcium, implying that triggering of effector secretion on cell contact represents a switch in secretion specificity, rather than a triggering of secretion per se. The requirement for a host cell cofactor to control effector secretion may help explain the recently observed phenomenon of target cell specificity in both the Yersinia and P. aeruginosa type III secretion systems.     

Plasma membrane association of three classes of bacterial toxins is mediated by a basic-hydrophobic motif

Plasma membrane targeting is essential for the proper function of many bacterial toxins. A conserved fourhelical bundle membrane localization domain (4HBM) was recently identified within three diverse families of toxins: clostridial glucosylating toxins, MARTX toxins and Pasteurella multocida-like toxins. When expressed in tissue culture cells or in yeast, GFP fusions to at least one 4HBM from each toxin family show significant peripheral membrane localization but with differing profiles. Both in vivo expression and in vitro binding studies reveal that the ability of these domains to localize to the plasma membrane and bind negatively charged phospholipids requires a basic-hydrophobic motif formed by the L1 and L3 loops. The different binding capacity of each 4HBM is defined by the hydrophobicity of an exposed residue within the motif. This study establishes that bacterial effectors utilize a normal host cell mechanism to locate the plasma membrane where they can then access their intracellular targets.     

Salmonella effectors: important players modulating host cell function during infection

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative facultative food-borne pathogen that causes gastroenteritis in humans. This bacterium has evolved a sophisticated machinery to alter host cell function critical to its virulence capabilities. Central to S. Typhimurium pathogenesis are two Type III secretion systems (T3SS) encoded within pathogenicity islands SPI-1 and SPI-2 that are responsible for the secretion and translocation of a set of bacterial proteins termed effectors into host cells with the intention of altering host cell physiology for bacterial entry and survival. Thus, once delivered by the T3SS, the secreted effectors play critical roles in manipulating the host cell to allow for bacteria invasion, induction of inflammatory responses, and the assembly of an intracellular protective niche created for bacterial survival and replication. Emerging evidence indicates that these effectors are modular proteins consisting of distinct functional domains/motifs that are utilized by the bacteria to activate intracellular signalling pathways modifying host cell function. Also, recently reported are the dual functionality of secreted effectors and the concept of 'terminal reassortment'. Herein, we highlight some of the nascent concepts regarding Salmonella effectors in the context of infection.     

A Legionella effector acquired from protozoa is involved in sphingolipids metabolism and is targeted to the host cell mitochondria

Legionella pneumophila infects alveolar macrophages and protozoa through establishment of an intracellular replication niche. This process is mediated by bacterial effectors translocated into the host cell via the Icm/Dot type IV secretion system. Most of the effectors identified so far are unique to L. pneumophila ; however, some of the effectors are homologous to eukaryotic proteins. We performed a distribution analysis of many known L. pneumophila effectors and found that several of them, mostly eukaryotic homologous proteins, are present in different Legionella species. In-depth analysis of LegS2, a L. pneumophila homologue of the highly conserved eukaryotic enzyme sphingosine-1-phosphate lyase (SPL), revealed that it was most likely acquired from a protozoan organism early during Legionella evolution. The LegS2 protein was found to translocate into host cells using a C-terminal translocation domain absent in its eukaryotic homologues. LegS2 was found to complement the sphingosine-sensitive phenotype of a Saccharomyces serevisia SPL-null mutant and this complementation depended on evolutionary conserved residues in the LegS2 catalytic domain. Interestingly, unlike the eukaryotic SPL that localizes to the endoplasmic reticulum, LegS2 was found to be targeted mainly to host cell mitochondria. Collectively, our results demonstrate the remarkable adaptations of a eukaryotic protein to the L. pneumophila pathogenesis system.     

The Salmonella effector protein SopB protects epithelial cells from apoptosis by sustained activation of Akt

Invasion of epithelial cells by Salmonella enterica is mediated by bacterial "effector" proteins that are delivered into the host cell by a type III secretion system. Although primarily known for their roles in actin rearrangements and membrane ruffling, translocated effectors also affect host cell processes that are not directly associated with invasion. Here, we show that SopB/SigD, an effector with phosphoinositide phosphatase activity, has anti-apoptotic activity in Salmonella-infected epithelial cells. Salmonella induced the sustained activation of Akt/protein kinase B, a pro-survival kinase, in a SopB-dependent manner. Failure to activate Akt resulted in increased levels of apoptosis after infection with a sopB deletion mutant (DeltasopB). Furthermore, cells infected with wild type bacteria, but not the DeltasopB strain, were protected from camptothecin-induced cleavage of caspase-3 and subsequent apoptosis. The anti-apoptotic activity of SopB was dependent on its phosphatase activity, because a catalytically inactive mutant was unable to protect cells from the effects of camptothecin. Finally, small interfering RNA was used to demonstrate the essential role of Akt in SopB-mediated protection against apoptosis. These results provide new insights into the mechanisms of apoptosis and highlight how bacterial effectors can intercept signaling pathways to manipulate host responses.     

Plant-bacterial pathogen interactions mediated by type III effectors

Effectors secreted by the bacterial type III system play a central role in the interaction between Gram-negative bacterial pathogens and their host plants. Recent advances in the effector studies have helped cementing several key concepts concerning bacterial pathogenesis, plant immunity, and plant-pathogen co-evolution. Type III effectors use a variety of biochemical mechanisms to target specific host proteins or DNA for pathogenesis. The identifications of their host targets led to the identification of novel components of plant innate immune system. Key modules of plant immune signaling pathways such as immune receptor complexes and MAPK cascades have emerged as a major battle ground for host-pathogen adaptation. These modules are attacked by multiple type III effectors, and some components of these modules have evolved to actively sense the effectors and trigger immunity.