Process of protein transport by the type III secretion system.

The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.     

Process of Protein Transport by the Type III Secretion System

The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.     

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.     

Recognition and ubiquitination of Salmonella type III effector SopA by a ubiquitin E3 ligase, HsRMA1

Salmonella translocate bacterial effectors into host cells to confer bacterial entry and survival. It is not known how the host cells cope with the influx of these effectors. We report here that the Salmonella effector, SopA, interacts with host HsRMA1, a ubiquitin E3 ligase with a previously unknown function. SopA is ubiquitinated and degraded by the HsRMA1-mediated ubiquitination pathway. A sopA mutant escapes out of the Salmonella-containing vacuoles less frequently to the cytosol than wild type Salmonella in HeLa cells in a HsRMA1-dependent manner. Our data suggest that efficient bacterial escape into the cytosol of epithelial cells requires HsRMA1-mediated SopA ubiquitination and contributes to Salmonella-induced enteropathogenicity.     

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.     

Phosphatases and kinases delivered to the host cell by bacterial pathogens

The gram-negative type III secretion pathway translocates bacterial proteins directly into eukaryotic host cells, thus allowing a pathogen to interfere directly with host signalling pathways. Protein and inositol phosphatases and protein kinases have been identified as delivered effectors in three bacterial pathogens, Salmonella, Shigella and Yersinia, and it is expected that several more such type III effectors will be found.     

细菌三型分泌系统效应蛋白转运的研究进展

三型分泌系统(Type 3 secretion system,T3SS)作为存在于革兰氏阴性菌中的分泌系统之一,对革兰氏阴性菌的致病有重要作用。T3SS的致病作用体现在T3SS能直接将效应蛋白转运至宿主细胞,进而通过效应蛋白调控细胞的一系列通路,促进细菌定殖于细胞。而效应蛋白的转运受到两方面因素的调控,一方面是效应蛋白本身的信号序列,另一方面是T3SS相关蛋白的辅助。本文围绕近年来T3SS的构成、效应蛋白转运机制方面的最新进展进行概要综述。     

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.     

Bacterial EPIYA effectors – Where do they come from? What are they? Where are they going?

Recent studies have revealed a distinct class of bacterial effectors defined by the presence of EPIYA or EPIYA‐related motif. These bacterial EPIYA effectors are delivered into host cells via type III or IV secretion, where they undergo tyrosine phosphorylation at the EPIYA motif and thereby manipulate host signalling by promiscuously interacting with multiple SH2 domain‐containing proteins. Up to now, nine EPIYA effectors have been identified from various bacteria. These effectors do not share sequence homology outside the EPIYA motif, arguing against the idea that they have common ancestors. A search of mammalian proteomes revealed the presence of a mammalian EPIYA‐containing protein, Pragmin, which potentiates Src family kinase (SFK) activity by binding and sequestrating the SFK inhibitor Csk upon EPIYA phosphorylation. As several bacterial EPIYA effectors also target Csk, they may have evolved through generation of sequences that mimic the Pragmin EPIYA motif. EPIYA motifs are often diverged through multiple duplications in each bacterial effector. Such a structural plasticity appears to be due to intrinsic disorder of the EPIYA‐containing region, which enables the bacterial effectors to undergo efficient phosphorylation and mediate promiscuous interaction with multiple host proteins. Given the functional versatility of the EPIYA motif, many more bacterial EPIYA effectors will soon be emerging.     

The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication

Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication.     

The inflammation-associated Salmonella SopA is a HECT-like E3 ubiquitin ligase

Salmonella translocate a group of type III effectors into the host cells to induce entry, promote survival and cause intestinal inflammation. Although the biochemical and cellular mechanisms of how bacterial effectors function inside host cells remain largely unknown, studies have indicated that a likely strategy is to exploit host cellular pathways through functional mimicry. We report here that SopA, a Salmonella type III effector, mimics the mammalian HECT E3 ubiquitin ligase. SopA preferentially uses the host UbcH5a, UbcH5c and UbcH7 as E2s, which are involved in inflammation. Both the wild-type SopA and the mutant SopAC753S were expressed and translocated at similar levels during the infection of HeLa cells. A Salmonella strain expressing a catalytically incompetent SopAC753S mutant had reduced Salmonella-induced polymorphonuclear leukocytes transepithelial migration. We speculate that SopA ubiquitinate bacterial/host proteins involved in Salmonella-induced intestinal inflammation.     

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.     

Subversion of membrane transport pathways by vacuolar pathogens

Mammalian phagocytes control bacterial infections effectively through phagocytosis, the process by which particles engulfed at the cell surface are transported to lysosomes for destruction. However, intracellular pathogens have evolved mechanisms to avoid this fate. Many bacterial pathogens use specialized secretion systems to deliver proteins into host cells that subvert signaling pathways controlling membrane transport. These bacterial effectors modulate the function of proteins that regulate membrane transport and alter the phospholipid content of membranes. Elucidating the biochemical function of these effectors has provided a greater understanding of how bacteria control membrane transport to create a replicative niche within the host and provided insight into the regulation of membrane transport in eukaryotic cells.     

Prenylation: From bacteria to eukaryotes

For their protection from host cell immune defense, intracellular pathogens of eukaryotic cells developed a variety of mechanisms, including secretion systems III and IV which can inject bacterial effectors directly into eukaryotic cells. These effectors may function inside the host cell and may be posttranslationally modified by host cell machinery. Recently, prenylation was added to the list of possible posttranslational modifications of bacterial proteins. In this work we describe the current state of the knowledge about the prenylation of eukaryotic and prokaryotic proteins and prenylation inhibitors. The bioinformatics analyses suggest the possibility of prenylation for a number of Francisella genus proteins.     

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.     

Bacterial remodelling of the host epigenome: functional role and evolution of effectors methylating host histones

The modulation of the chromatin organization of eukaryotic cells plays an important role in regulating key cellular processes including host defence mechanisms against pathogens. Thus, to successfully survive in a host cell, a sophisticated bacterial strategy is the subversion of nuclear processes of the eukaryotic cell. Indeed, the number of bacterial proteins that target host chromatin to remodel the host epigenetic machinery is expanding. Some of the identified bacterial effectors that target the chromatin machinery are ‘eukaryotic‐like’ proteins as they mimic eukaryotic histone writers in carrying the same enzymatic activities. The best‐studied examples are the SET domain proteins that methylate histones to change the chromatin landscape. In this review, we will discuss SET domain proteins identified in the Legionella, Chlamydia and Bacillus genomes that encode enzymatic activities targeting host histones. Moreover, we discuss their possible origin as having evolved from prokaryotic ancestors or having been acquired from their eukaryotic hosts during their co‐evolution. The characterization of such bacterial effectors as modifiers of the host chromatin landscape is an exciting field of research as it elucidates new bacterial strategies to not only manipulate host functions through histone modifications but it may also identify new modifications of the mammalian host cells not known before.     

Anchoring of bacterial effectors to host membranes through host-mediated lipidation by prenylation: a common paradigm

Post-translational lipidation by prenylation of the CaaX-box C-terminal motif in eukaryotic proteins facilitates anchoring of hydrophilic proteins, such as Ras and Rab, to membranes. A large cadre of bacterial effectors injected into host cells is anchored to host membranes by unknown mechanisms. As already documented for Legionella and Salmonella, we propose a common paradigm of microbial exploitation of the host prenylation machinery for anchoring of injected effectors to host membranes. This is supported by numerous potential microbial CaaX-box-containing proteins identified using refined bioinformatic tools. We also propose utilization of the CaaX motif as a membrane-targeting tag for proteins expressed in eukaryotic cells to facilitate deciphering of biological function.     

Identifying type III effectors of plant pathogens and analyzing their interaction with plant cells

Many bacterial pathogens cause disease by injecting virulence proteins (effectors) into host cells via the specialized type III secretion system. Recently, exceptional progress in identifying effectors was made in the phytopathogen Pseudomonas syringae using a novel genetic screen and bioinformatic approach. These studies, along with localization experiments, suggest that most P. syringae effectors function by targeting the plasma membrane, chloroplasts or mitochondria of host cells. The type III secretome of P. syringae is highly variable and dynamic, a lesson gleaned from a comparative genomic analysis. Variation in the effector repertoire is likely to facilitate the adaptation of P. syringae to different hosts.     

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.