Bacterial LPX motif-harboring virulence factors constitute a species-spanning family of cell-penetrating effectors

Effector proteins are key virulence factors of pathogenic bacteria that target and subvert the functions of essential host defense mechanisms. Typically, these proteins are delivered into infected host cells via the type III secretion system (T3SS). Recently, however, several effector proteins have been found to enter host cells in a T3SS-independent manner thereby widening the potential range of these virulence factors. Prototypes of such bacteria-derived cell-penetrating effectors (CPEs) are the Yersinia enterocolitica-derived YopM as well as the Salmonella typhimurium effector SspH1. Here, we investigated specifically the group of bacterial LPX effector proteins comprising the Shigella IpaH proteins, which constitute a subtype of the leucine-rich repeat protein family and share significant homologies in sequence and structure. With particular emphasis on the Shigella-effector IpaH9.8, uptake into eukaryotic cell lines was shown. Recombinant IpaH9.8 (rIpaH9.8) is internalized via endocytic mechanisms and follows the endo-lysosomal pathway before escaping into the cytosol. The N-terminal alpha-helical domain of IpaH9.8 was identified as the protein transduction domain required for its CPE ability as well as for being able to deliver other proteinaceous cargo. rIpaH9.8 is functional as an ubiquitin E3 ligase and targets NEMO for poly-ubiquitination upon cell penetration. Strikingly, we could also detect other recombinant LPX effector proteins from Shigella and Salmonella intracellularly when applied to eukaryotic cells. In this study, we provide further evidence for the general concept of T3SS-independent translocation by identifying novel cell-penetrating features of these LPX effectors revealing an abundant species-spanning family of CPE.     

The invasion-associated type III secretion system of Salmonella typhimurium: common and unique features

Several bacterial pathogens make use of a specialized protein secretion system to inject effector proteins into host cells. This system, commonly referred to as type III secretion, is always associated with phenotypes related to intimate interactions between the pathogen and its respective host cells. The enteric pathogen Salmonella typhimurium utilizes a type III secretion system to invade nonphagocytic intestinal epithelial cells. Whereas the invasion-associated type III system of S. typhimurium has evolved to perform a specific function, many of the components of this system are conserved among the type III systems of other bacterial pathogens. This review will discuss the common and unique features of the S. typhimurium system in relation to the type III systems of other human pathogens. Topics discussed include the phenotypes associated with various type III systems, the genetic loci encoding these systems, the components of the type III secretion apparatus, the effector proteins and the mechanisms by which they enter host cells as well as the mechanisms used to regulate the expression of type III systems.     

Cellular microbiology of intracellular <Emphasis Type="Italic">Salmonella enterica</Emphasis>: functions of the type III secretion system encoded by <Emphasis Type="Italic">Salmonella</Emphasis> pathogenicity island 2

The facultative intracellular pathogen Salmonella enterica resides in a special membrane compartment of the host cell and modifies its host to achieve intracellular survival and proliferation. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has a central role in the interference of intracellular Salmonella with host cell functions. SPI2 function affects antimicrobial defense mechanisms of the host, intracellular transport processes, integrity and function of the cytoskeleton and host cell death. These modifications are mediated by translocation of a large number of effector proteins by the SPI2 system. In this review, we summarize recent work on the cellular phenotypes related to SPI2 function and contribution of SPI2 effector proteins to these manipulations. These studies reveal a complex set of pathogenic interferences between intracellular Salmonella and its host cells.Received 11 June 2004; received after revision 8 July 2004; accepted 12 July 2004     

Timing is everything: the regulation of type III secretion

Type Three Secretion Systems (T3SSs) are essential virulence determinants of many Gram-negative bacteria. The T3SS is an injection device that can transfer bacterial virulence proteins directly into host cells. The apparatus is made up of a basal body that spans both bacterial membranes and an extracellular needle that possesses a channel that is thought to act as a conduit for protein secretion. Contact with a host-cell membrane triggers the insertion of a pore into the target membrane, and effectors are translocated through this pore into the host cell. To assemble a functional T3SS, specific substrates must be targeted to the apparatus in the correct order. Recently, there have been many developments in our structural and functional understanding of the proteins involved in the regulation of secretion. Here we review the current understanding of protein components of the system thought to be involved in switching between different stages of secretion.     

The long and winding road: virulence effector proteins of plant pathogenic bacteria

Plant pathogenic bacteria inject about 30 virulence effector proteins into the host cell using a specialized secretion apparatus. Bacteria which are unable to do this elicit host immunity and cannot grow inside living plant tissue. Thus, the primary function of the effectors is to suppress host immunity. The identity of individual effectors within each complement varies even between closely related bacterial strains, and effectors themselves act redundantly and are apparently interchangeable. Many effectors are known to target components of plant defense pathways, but it is difficult to study their role in molecular terms. For some of them, there is controversy about their mode of action. We propose that effectors act promiscuously by targeting host molecules with low specificity and affinity.     

Bacterial serine proteases secreted by the autotransporter pathway: classification,specificity, and role in virulence

Serine proteases exist in eukaryotic and prokaryotic organisms and have emerged during evolution as the most abundant and functionally diverse group. In Gram-negative bacteria, there is a growing family of high molecular weight serine proteases secreted to the external milieu by a fascinating and widely employed bacterial secretion mechanism, known as the autotransporter pathway. They were initially found in Neisseria, Shigella, and pathogenic Escherichia coli, but have now also been identified in Citrobacter rodentium, Salmonella, and Edwardsiella species. Here, we focus on proteins belonging to the serine protease autotransporter of Enterobacteriaceae (SPATEs) family. Recent findings regarding the predilection of serine proteases to host intracellular or extracellular protein-substrates involved in numerous biological functions, such as those implicated in cytoskeleton stability, autophagy or innate and adaptive immunity, have helped provide a better understanding of SPATEs’ contributions in pathogenesis. Here, we discuss their classification, substrate specificity, and potential roles in pathogenesis.     

Lactoferrin

The first function attributed to lactoferrin (Lf), an iron binding protein belonging to the non-immune natural defences, was antimicrobial activity that depended on its capacity to sequester iron. Iron-independent microbicidal activities, requiring direct interaction between this cationic protein and microbial surface components, were later demonstrated. Many other anti-microbial and anti-viral functions have since been ascribed to Lf. In mucosal secretions, iron and Lf modulate the motility and aggregation of pathogenic bacteria. Lf inhibits bacterial adhesion on abiotic surfaces through ionic binding to biomaterials, or specific binding to bacterial structures or both. Lf inhibition of bacterial adhesion to host cells requires Lf binding to bacteria and/or host cells. Lf hinders microbial internalization by binding to both glycosaminoglycans and bacterial proteins which can be degraded by Lf-mediated proteolysis. Moreover, Lf internalisation and localisation to the host cell nuclei could modulate bacterial entry into cells through gene regulation. Finally, the capability of Lf to exert antiviral activity, through its binding to host cells and/or viral particles, strengthens the idea that it is an important brick in the mucosal wall, effective against both microbial and viral attacks.     

Lytic transglycosylases in macromolecular transport systems of Gram-negative bacteria

The cell wall of Gram-negative bacteria is essential for the integrity of the bacterial cell but also imposes a physical barrier to trans-envelope transport processes in which DNA and/or proteins are taken up or secreted by complex protein assemblies. The presence of genes encoding lytic transglycosylases in macromolecular transport systems (bacteriophage entry, type II secretion and type IV pilus synthesis, type III secretion, type IV secretion) suggests an important role for these specialised cell-wall-degrading enzymes. Such enzymes are capable of locally enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly and anchoring of supramolecular transport complexes in the cell envelope. In this review, current knowledge on the role and distribution of these specialised murein-degrading enzymes in diverse macromolecular transport systems is summarised and discussed.Received 13 February 2003; received after revision 25 April 2003; accepted 12 May 2003     

Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action

Proline-rich antimicrobial peptides are a group of cationic host defense peptides of vertebrates and invertebrates characterized by a high content of proline residues, often associated with arginine residues in repeated motifs. Those isolated from some mammalian and insect species, although not evolutionarily related, use a similar mechanism to selectively kill Gram-negative bacteria, with a low toxicity to animals. Unlike other types of antimicrobial peptides, their mode of action does not involve the lysis of bacterial membranes but entails penetration into susceptible cells, where they then act intracellularly. Some aspects of the transport system and cytoplasmic targets have been elucidated. These features make them attractive both as anti-infective lead compounds and as a new class of potential cell-penetrating peptides capable of internalising membrane-impermeant drugs into both bacterial and eukaryotic cells     

Biogenesis of bacterial inner-membrane proteins

All cells must traffic proteins into and across their membranes. In bacteria, several pathways have evolved to enable protein transfer across the inner membrane, the periplasm, and the outer membrane. The major route of protein translocation in and across the cytoplasmic membrane is the general secretion pathway (Sec-pathway). The biogenesis of membrane proteins not only requires protein translocation but also coordinated targeting to the membrane beforehand and folding and assembly into their protein complexes afterwards to function properly in the cell. All these processes are responsible for the biogenesis of membrane proteins that mediate essential functions of the cell such as selective transport, energy conversion, cell division, extracellular signal sensing, and motility. This review will highlight the most recent developments on the structure and function of bacterial membrane proteins, focusing on the journey that integral membrane proteins take to find their final destination in the inner membrane.     

Immunoregulation by the gut microbiota

The human intestinal mucosa is constantly exposed to commensal microbiota. Since the gut microbiota is beneficial to the host, hosts have evolved intestine-specific immune systems to co-exist with the microbiota. On the other hand, the intestinal microbiota actively regulates the host’s immune system, and recent studies have revealed that specific commensal bacterial species induce the accumulation of specific immune cell populations. For instance, segmented filamentous bacteria and Clostridium species belonging to clusters XIVa and IV induce the accumulation of Th17 cells in the small intestine and Foxp3⁺ regulatory T cells in the large intestine, respectively. The immune cells induced by the gut microbiota likely contribute to intestinal homeostasis and influence systemic immunity in the host.     

Hsp70 in parasites: as an inducible protective protein and as an antigen

The heat shock (HS) response is a general homeostatic mechanism that protects cells and the entire organism from the deleterious effects of environmental stresses. It has been demonstrated that heat shock proteins (HSP) play major roles in many cellular processes, and have a unique role in several areas of cell biology, from chronic degenerative diseases to immunology, from cancer research to interaction between host and parasites. This review deals with thehsp70 gene family and with its protein product, hsp70, as an antigen when pathogens infect humans. Members of HSP have been shown to be major antigens of many pathogenic organisms when they experience a major temperature shift upwards at the onset of infection and become targets for host B and T cells.     

The IQGAP-related protein DGAP1 mediates signaling to the actin cytoskeleton as an effector and a sequestrator of Rac1 GTPases

Proteins are typically categorized into protein families based on their domain organization. Yet, evolutionarily unrelated proteins can also be grouped together according to their common functional roles. Sequestering proteins constitute one such functional class, acting as macromolecular buffers and serving as an intracellular reservoir ready to release large quantities of bound proteins or other molecules upon appropriate stimulation. Another functional protein class comprises effector proteins, which constitute essential components of many intracellular signal transduction pathways. For instance, effectors of small GTP-hydrolases are activated upon binding a GTP-bound GTPase and thereupon participate in downstream interactions. Here we describe a member of the IQGAP family of scaffolding proteins, DGAP1 from Dictyostelium, which unifies the roles of an effector and a sequestrator in regard to the small GTPase Rac1. Unlike classical effectors, which bind their activators transiently leading to short-lived signaling complexes, interaction between DGAP1 and Rac1-GTP is stable and induces formation of a complex with actin-bundling proteins cortexillins at the back end of the cell. An oppositely localized Rac1 effector, the Scar/WAVE complex, promotes actin polymerization at the cell front. Competition between DGAP1 and Scar/WAVE for the common activator Rac1-GTP might provide the basis for the oscillatory re-polarization typically seen in randomly migrating Dictyostelium cells. We discuss the consequences of the dual roles exerted by DGAP1 and Rac1 in the regulation of cell motility and polarity, and propose that similar signaling mechanisms may be of general importance in regulating spatiotemporal dynamics of the actin cytoskeleton by small GTPases.     

Enteropathogenic Escherichia coli: a pathogen that inserts its own receptor into host cells

Enteropathogenic Escherichia coli (EPEC) is a major cause of infant diarrhea, killing hundreds of thousands of children per year worldwide. Intimate attachment to the host cell leading to the formation of actin-rich pedestals beneath the adhering bacteria is an essential feature of EPEC pathogenesis. EPEC attaches to host cells via the outer membrane adhesin, intimin. It was recently shown that EPEC inserts its own receptor for intimate adherence, Tir (translocated intimin receptor) into the host cell membrane. The focus of this review is on the discovery and characterization of this novel receptor, and our current understanding of its role in pedestal formation. Gram-negative bacterial secretion systems, including type III secretion systems, are reviewed and discussed in the context of Tir delivery into the host cell membrane. The relationship and relevance of in vitro models compared to the actual in vivo situation is essential to understanding disease. We have critically reviewed the use of animal models in studying EPEC infection. Elucidating the function of Tir will contribute to our understanding of how EPEC mediates disease.     

Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages

Enterococci are commensal organisms in the alimentary tract. However, they can cause a variety of life-threatening infections, especially in nosocomial settings. We hypothesized that induction of cell death might enable these facultative pathogenic bacteria to evade the innate immune response and to cause infections of their host. We demonstrate that E. faecium when exposed to lysozyme induces cell death in macrophages in vitro and in vivo. Flow cytometric analyses of J774A.1 macrophages infected with E. faecium revealed loss of cell membrane integrity indicated by uptake of propidium iodide and decrease of the inner mitochondrial transmembrane potential ΔΨ_m. Inhibition of caspases, treatment of macrophages with cytochalasin D, or rifampicin did not prevent cells from dying, suggesting cell death mechanisms that are independent of caspase activation, bacterial uptake, and intracellular bacterial replication. Characteristics of necrotic cell death were demonstrated by both lack of procaspase 3 activation and cell shrinkage, electron microscopy, and release of lactate dehydrogenase. Pretreatment of E. faecium with lysozyme and subsequently with broad spectrum protease considerably reduced cell death, suggesting that a bacterial surface protein is causative for cell death induction. Moreover, in a mouse peritonitis model we demonstrated that E. faecium induces cell death of peritoneal macrophages in vivo. Altogether, our results show that enterococci, under specific conditions such as exposure to lysozyme, induce necrotic cell death in macrophages, which might contribute to disseminated infections by these facultative pathogenic bacteria.     

The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells

Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.     

Membrane-shed vesicles from the parasite <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>: characterization and their association with cell interaction

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor–ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.     

Bacterial antibiotic efflux systems of medical importance

Multidrug efflux systems endow on bacterial cells the ability to limit the access of antimicrobial agents to their targets. By actively pumping out antibiotic molecules, these systems prevent the intracellular accumulation necessary for antibiotics to exert their lethal activity. Drug efflux appears to be one of the most widespread antibiotic resistance mechanisms among microorganisms, since it has been demonstrated to occur in many Gram-positive and Gram-negative bacteria including medically important species like staphylococci, streptococci, enterobacteria and opportunistic pathogens like Pseudomonas aeruginosa. Efflux pumps can be specific for only one substrate or accommodate a more or less wide range of noxious products. Export of structurally unrelated compounds confers a multidrug-resistance phenotype on bacterial cells. Therapeutically critical levels of resistance can be achieved by overexpression of efflux systems, especially in those species such as P. aeruginosa which possess a low outer membrane permeability. It is suspected that the dual physiological function of active efflux systems is both the secretion of intracellular metabolites and the protection against a variety of harmful substances that the microorganism may encounter in its natural environment.     

The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation

A challenging task for the adaptive immune system of vertebrates is to identify and eliminate intracellular antigens. Therefore a highly specialized antigen presentation machinery has evolved to display fragments of newly synthesized proteins to effector cells of the immune system at the cell surface. After proteasomal degradation of unwanted proteins or defective ribosome products, resulting peptides are translocated into the endoplasmic reticulum by the transporter associated with antigen processing and loaded onto major histocompatibility complex (MHC) class I molecules. Peptide-MHC I complexes are transported via the secretory pathway to the cell surface where they are then inspected by cytotoxic T lymphocytes, which can trigger an immune response. This review summarizes the current view of the intracellular machinery of antigen processing and of viral immune escape mechanisms to circumvent destruction by the host. Received 4 October 2005; received after revision 19 November 2005; accepted 24 November 2005