Virulence protein export systems in Salmonella and Shigella: a new family or lost relatives?

A number of Gram-negative bacterial pathogens secrete 'virulence determinants' directly into the extracellular medium, where they interact with host cells to promote disease. The study of the secretion machinery used by these organisms to transport specific virulence determinants out to the cell surface and beyond is of growing importance in the field of bacterial pathogenesis. Elements of the secretion machinery are shared by several pathogens. These homologous elements may lead to a better understanding of how the machinery works, but the unique elements will tell us more about what distinguishes one bacterial pathogen from another.     

IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formation

Invasion of epithelial cells by Shigella flexneri involves entry and intercellular dissemination. Entry of bacteria into non-phagocytic cells requires the IpaA-D proteins that are secreted by the Mxi-Spa type III secretion machinery. Type III secretion systems are found in several Gram-negative pathogens and serve to inject bacterial effector proteins directly into the cytoplasm of host cells. In this study, we have analysed the IpgD protein of S. flexneri, the gene of which is located on the virulence plasmid at the 5' end of the mxi-spa locus. We have shown that IpgD (i) is stored in the bacterial cytoplasm in association with a specific chaperone, IpgE; (ii) is secreted by the Mxi-Spa type III secretion system in amounts similar to those of the IpaA-D proteins; (iii) is associated with IpaA in the extracellular medium; and (iv) is involved in the modulation of the host cell response after contact of the bacterium with epithelial cells. This suggests that IpgD is an effector that might be injected into host cells to manipulate cellular processes during infection.     

Secretion of proteins and assembly of bacterial surface organelles: shared pathways of extracellular protein targeting

Extracellular or surface localization of virulence determinants is an important attribute of pathogenic microorganisms. The past decade has seen significant research advances in defining the steps and identifying the necessary machinery for protein secretion from bacterial cells. In Gram-negative pathogens, four distinct classes of secretion pathways have been identified that deliver virulence factors to their sites of action. These pathways are responsible for the delivery of soluble extracellular enzymes into the surrounding medium, or for specifically targeting proteins to the host cell. In several instances protein secretion pathways are similar to those involved in assembly of bacterial appendages. Combination of biochemical and genetic analyses has recently revealed that the pathways of protein secretion and surface localization of various organelles are mechanistically similar which was not apparent simply by comparing amino acid sequences of related proteins. The choice of the pathway that a protein will utilize may not be dictated only by the specific requirement of the secreted protein to traverse the cell envelope in the functional form, but also by the need to assure its delivery to the correct site of action outside the bacterial cell.     

Direct detection of bacterial protein secretion using whole colony proteomics

Bacteria use a variety of secretion systems to transport proteins beyond their cell membrane to interact with their environment. For bacterial pathogens, these systems are key virulence determinants that transport bacterial proteins into host cells. Genetic screens to identify bacterial genes required for export have relied on enzymatic or fluorescent reporters fused to known substrates to monitor secretion. However, they cannot be used in analysis of all secretion systems, limiting the implementation across bacteria. Here, we introduce the first application of a modified form of whole colony MALDI-TOF MS to directly detect protein secretion from intact bacterial colonies. We show that this method is able to specifically monitor the ESX-1 system protein secretion system, a major virulence determinant in both mycobacterial and Gram-positive pathogens that is refractory to reporter analysis. We validate the use of this technology as a high throughput screening tool by identifying an ESAT-6 system 1-deficient mutant from a Mycobacterium marinum transposon insertion library. Furthermore, we also demonstrate detection of secreted proteins of the prevalent type III secretion system from the Gram-negative pathogen, Pseudomonas aeruginosa. This method will be broadly applicable to study other bacterial protein export systems and for the identification of compounds that inhibit bacterial protein secretion.     

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.     

Structural microbiology at the pathogen-host interface

Bacterial pathogens achieve the internalization of a multitude of virulence factors into eukaryotic cells. Some secrete extracellular toxins which bring about their own entry, usually by hijacking cell surface receptors and endocytic pathways. Others possess specialized secretion and translocation systems to directly inject bacterial proteins into the host cytosol. Recent advances in the structural biology of these virulence factors has begun to reveal at the molecular level how these bacterial proteins are delivered and modulate host activities ranging from cytoskeletal structure to cell cycle progression.     

Bacterial Type IV secretion systems: versatile virulence machines

Many bacterial pathogens employ multicomponent protein complexes to deliver macromolecules directly into their eukaryotic host cell to promote infection. Some Gram-negative pathogens use a versatile Type IV secretion system (T4SS) that can translocate DNA or proteins into host cells. T4SSs represent major bacterial virulence determinants and have recently been the focus of intense research efforts designed to better understand and combat infectious diseases. Interestingly, although the two major classes of T4SSs function in a similar manner to secrete proteins, the translocated 'effectors' vary substantially from one organism to another. In fact, differing effector repertoires likely contribute to organism-specific host cell interactions and disease outcomes. In this review, we discuss the current state of T4SS research, with an emphasis on intracellular bacterial pathogens of humans and the diverse array of translocated effectors used to manipulate host cells.     

Sweet host revenge: Galectins and GBPs join forces at broken membranes

Most bacterial pathogens enter and exit eukaryotic cells during their journey through the vertebrate host. In order to endure inside a eukaryotic cell, bacterial invaders commonly employ bacterial secretion systems to inject host cells with virulence factors that co‐opt the host's membrane trafficking systems and thereby establish specialised pathogen‐containing vacuoles (PVs) as intracellular niches permissive for microbial growth and survival. To defend against these microbial adversaries hiding inside PVs, host organisms including humans evolved an elaborate cell‐intrinsic armoury of antimicrobial weapons that include noxious gases, antimicrobial peptides, degradative enzymes, and pore‐forming proteins. This impressive defence machinery needs to be accurately delivered to PVs, in order to fight off vacuole‐dwelling pathogens. Here, I discuss recent evidence that the presence of bacterial secretion systems at PVs and the associated destabilisation of PV membranes attract such antimicrobial delivery systems consisting of sugar‐binding galectins as well as dynamin‐like guanylate‐binding proteins (GBPs). I will review recent advances in our understanding of intracellular immune recognition of PVs by galectins and GBPs, discuss how galectins and GBPs control host defence, and highlight important avenues of future research in this exciting area of cell‐autonomous immunity.     

Type III protein secretion in Pseudomonas syringae

The type III secretion system is an essential virulence system used by many Gram-negative bacterial pathogens to deliver effector proteins into host cells. This review summarizes recent advancements in the understanding of the type III secretion system of Pseudomonas syringae, including regulation of the type III secretion genes, assembly of the Hrp pilus, secretion signals, the putative type III effectors identified to date, and their virulence action after translocation into plant cells.     

Bacterial pathogenesis: the answer to virulence is in the pore.

A wide variety of Gram-negative bacterial pathogens use a 'type III' protein secretion system to deliver bacterial virulence factors into host cells. Recent results suggest that Gram-positive pathogens may employ similar methods to deliver virulence factors into host cells.     

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.     

Structural biology of bacterial pathogenesis

Recent years have seen a rapid increase in structural information on proteins implicated in bacterial pathogenesis. The different modes by which bacteria establish contact with their host tissues are exemplified by the structures of bacterial adhesins in complex with their cognate host receptor. A more detailed structural understanding of the various Gram-negative secretion systems has emerged with the determination of the structures of type I and type IV secretion system components, and with the elucidation of the mechanism of fibre formation in the chaperone-usher pathway of pilus biogenesis. Finally, the structures of complexes of secreted virulence factors bound to their host targets have unravelled the mechanisms by which bacterial pathogens exploit cellular processes to their advantage.     

AlgW regulates multiple Pseudomonas syringae virulence strategies

Gram-negative bacterial pathogens have evolved a number of virulence-promoting strategies including the production of extracellular polysaccharides such as alginate and the injection of effector proteins into host cells. The induction of these virulence mechanisms can be associated with concomitant downregulation of the abundance of proteins that trigger the host immune system, such as bacterial flagellin. In Pseudomonas syringae, we observed that bacterial motility and the abundance of flagellin were significantly reduced under conditions that induce the type III secretion system. To identify genes involved in this negative regulation, we conducted a forward genetic screen with P. syringae pv. maculicola ES4326 using motility as a screening phenotype. We identified the periplasmic protease AlgW as a key negative regulator of flagellin abundance that also positively regulates alginate biosynthesis and the type III secretion system. We also demonstrate that AlgW constitutes a major virulence determinant of P. syringae required to dampen plant immune responses. Our findings support the conclusion that P. syringae co-ordinately regulates virulence strategies through AlgW in order to effectively suppress host immunity.     

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.     

Show me the substrates: modulation of host cell function by type IV secretion systems

Evidence for the involvement of type IV protein secretion systems in bacterial virulence is accumulating. Many of the substrate proteins secreted by type IV systems either hijack or interfere with specific host cell pathways. These substrates can be injected directly into host cells via the type IV apparatus or are secreted by the type IV machinery in a state that allows them to gain access to cellular targets without the further assistance of the type IV system. Arguably, the protein substrates of most type IV secretion systems remain undiscovered. Here, we review the activities of known type IV substrates and discuss the putative roles of unidentified substrates.     

Control of epithelial cell structure and developmental fate: lessons from Helicobacter pylori

Valuable insights into eukaryotic regulatory circuits can emerge from studying interactions of bacterial pathogens such as Helicobacter pylori with host tissues. H. pylori uses a type IV secretion system (T4SS) to deliver its CagA virulence protein to epithelial cells, where much of it becomes phosphorylated. CagA's phosphorylated and non-phosphorylated forms each interact with host regulatory proteins to alter cell structure and cell fate. Kwok and colleagues showed that CagA destined for phosphorylation is delivered using host integrin as receptor and H. pylori's CagL protein as an integrin-specific adhesin, and that CagL-integrin-binding activates the kinase cascade responsible for CagA phosphorylation. This research contributes to understanding infectious disease and the control of cell fates.     

Pathogens reWritE Rho's rules

Many bacterial pathogens use a specialized "type III" secretion system to deliver virulence effector proteins into host mammalian cells. In this issue of Cell, Alto et al. (2006) describe a new family of effectors that share a WxxxE sequence motif. These effectors directly stimulate host signaling pathways by mimicking activated Ras-like cellular GTPases.     

The type III secretion system needle tip complex mediates host cell sensing and translocon insertion

Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow 'needle' protruding from the bacterial surface. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which proteins that facilitate host cell invasion are translocated. As the needle is implicated in host cell sensing and secretion regulation, its tip should contain components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we reveal tip complexes of differing compositions and functional states, which appear to represent the molecular events surrounding host cell sensing and pore formation. Our studies indicate that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.     

A bacterial effector targets host DH‐PH domain RhoGEFs and antagonizes macrophage phagocytosis

Bacterial pathogens often harbour a type III secretion system (TTSS) that injects effector proteins into eukaryotic cells to manipulate host processes and cause diseases. Identification of host targets of bacterial effectors and revealing their mechanism of actions are crucial for understating bacterial virulence. We show that EspH, a type III effector conserved in enteric bacterial pathogens including enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli and Citrobacter rodentium, markedly disrupts actin cytoskeleton structure and induces cell rounding up when ectopically expressed or delivered into HeLa cells by the bacterial TTSS. EspH inactivates host Rho GTPase signalling pathway at the level of RhoGEF. EspH directly binds the DH‐PH domain in multiple RhoGEFs, which prevents their binding to Rho and thereby inhibits nucleotide exchange‐mediated Rho activation. Consistently, infection of mouse macrophages with EPEC harbouring EspH attenuates phagocytosis of the bacteria as well as FcγR‐mediated phagocytosis. EspH represents the first example of targeting RhoGEFs by bacterial effectors, and our results also reveal an unprecedented mechanism used by enteric pathogens to counteract the host defence system.