Tc Toxin Complex Research Articles

Involvement of N-glycans in binding of Photorhabdus luminescens Tc toxin.

Photorhabdus luminescens Tc toxins are large tripartite ABC-type toxin complexes, composed of TcA, TcB and TcC proteins. Tc toxins are widespread and have shown a tropism for a variety of targets including insect, mammalian and human cells. However, their receptors and the specific mechanisms of uptake into target cells remain unknown. Here, we show that the TcA protein TcdA1 interacts with N-glycans, particularly Lewis X/Y antigens. This is confirmed using N-acetylglucosamine transferase I (Mgat1 gene product)-deficient Chinese hamster ovary (CHO) Lec1 cells, which are highly resistant to intoxication by the Tc toxin complex most likely due to the absence of complex N-glycans. Restoring Mgat1 gene activity, and hence complex N-glycan biosynthesis, recapitulated the sensitivity of these cells to the toxin. Exogenous addition of Lewis X trisaccharide partially inhibits intoxication in wild-type cells. Additionally, sialic acid also largely reduced binding of the Tc toxin. Moreover, proteolytic activation of TcdA1 alters glycan-binding and uptake into target cells. The data suggest that TcdA1-binding is most likely multivalent, and carbohydrates probably work cooperatively to facilitate binding and intoxication.

Structure of a Tc holotoxin pore provides insights into the translocation mechanism

Tc toxins are modular toxin systems of insect and human pathogenic bacteria. They are composed of a 1.4-MDa pentameric membrane translocator (TcA) and a 250-kDa cocoon (TcB and TcC) encapsulating the 30-kDa toxic enzyme (C terminus of TcC). Binding of Tc toxins to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. A high-resolution structure of a holotoxin embedded in membranes is missing, leaving open the question of whether TcB-TcC has an influence on the conformational transition of TcA. Here we show in atomic detail a fully assembled 1.7-MDa Tc holotoxin complex from Photorhabdus luminescens in the membrane. We find that the 5 TcA protomers conformationally adapt to fit around the cocoon during the prepore-to-pore transition. The architecture of the Tc toxin complex allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Importantly, assembly of the holotoxin at the membrane results in spontaneous translocation of the toxic enzyme, indicating that this process is not driven by a proton gradient or other energy source. Mammalian lipids with zwitterionic head groups are preferred over other lipids for the integration of Tc toxins. In a nontoxic Tc toxin variant, we can visualize part of the translocating toxic enzyme, which transiently interacts with alternating negative charges and hydrophobic stretches of the translocation channel, providing insights into the mechanism of action of Tc toxins.

Tc Toxin Complexes: Assembly, Membrane Permeation, and Protein Translocation.

Tc toxin complexes are virulence factors of many bacteria, including insect and human pathogens. Tc toxins are composed of three subunits that act together to perforate the host membrane, similar to a syringe, and translocate toxic enzymes into the host cell. The reactions of the toxic enzymes lead to deterioration and ultimately death of the cell. We review recent high-resolution structural and functional data that explain the mechanism of action of this type of bacterial toxin at an unprecedented level of molecular detail. We focus on the steps that are necessary for toxin activation and membrane permeation. This is where the largest conformational transitions appear. Furthermore, we compare the architecture and function of Tc toxins with those of anthrax toxin and vertebrate teneurin.

Secretion Systems and Secreted Proteins in Gram-Negative Entomopathogenic Bacteria: Their Roles in Insect Virulence and Beyond.

Many Gram-negative bacteria have evolved insect pathogenic lifestyles. In all cases, the ability to cause disease in insects involves specific bacterial proteins exported either to the surface, the extracellular environment, or the cytoplasm of the host cell. They also have several distinct mechanisms for secreting such proteins. In this review, we summarize the major protein secretion systems and discuss examples of secreted proteins that contribute to the virulence of a variety of Gram-negative entomopathogenic bacteria, including Photorhabdus, Xenorhabdus, Serratia, Yersinia, and Pseudomonas species. We also briefly summarize two classes of exported protein complexes, the PVC-like elements, and the Tc toxin complexes that were first described in entomopathogenic bacteria.

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 Å structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 Å structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

Structural Insights into Tc Toxin Action

Tripartite Tc toxin complexes of bacterial pathogens perforate the host membrane by forming channels that translocate toxic enzymes into the host, including humans. The underlying mechanism is complex but poorly understood. I will present the first high-resolution structure of a complete 1.7 MDa Tc toxin complex composed of TcA, TcB and TcC. TcB and TcC form a large cocoon, in which the toxic domain resides and is autoproteolytically cleaved. Binding of TcB/TcC to the pore-forming and receptor-binding TcA opens the cocoon and results in a continuous protein translocation channel, in which the toxic domain is secreted. Our results allow us for the first time to understand key steps of infections involving Tc toxins at molecular level and shed new light on the interaction of bacterial pathogens, such as the plague pathogen Yersinia pestis, with their hosts.

Photorhabdus luminescens Toxins TccC3 and TccC5 Affect the Interaction of Actin with Actin-Binding Proteins Essential for Treadmilling.

Actin is one of the most abundant cellular proteins and an essential constituent of the actin cytoskeleton, which by its dynamic behavior participates in many cellular activities. The organization of the actin cytoskeleton is regulated by a large number of proteins and represents one of the major targets of bacterial toxins. A number of bacterial effector proteins directly modify actin: Clostridial bacteria produce toxins, which ADP-ribosylate actin at Arg177 leading to inhibition of actin polymerization. The bacterium Photorhabdus luminescens produces several types of protein toxins, including the high molecular weight Tc toxin complex, whose component TccC3 ADP-ribosylates actin at Thr148 promoting polymerization and aggregation of intracellular F-actin leading to inhibition of several cellular functions, such as phagocytosis. Here, we review recent findings about the functional consequences of these actin modifications and for the Thr148-ADP-ribosylated actin the subsequent alterations in the interaction with actin-binding proteins . In addition, we describe the effects of ADP-ribosylation of Rho GTPases by the TccC5 component.

Mechanism of Tc toxin action revealed in molecular detail

Tripartite Tc toxin complexes of bacterial pathogens perforate the host membrane and translocate toxic enzymes into the host cell, including in humans. The underlying mechanism is complex but poorly understood. Here we report the first, to our knowledge, high-resolution structures of a TcA subunit in its prepore and pore state and of a complete 1.7 megadalton Tc complex. The structures reveal that, in addition to a translocation channel, TcA forms four receptor-binding sites and a neuraminidase-like region, which are important for its host specificity. pH-induced opening of the shell releases an entropic spring that drives the injection of the TcA channel into the membrane. Binding of TcB/TcC to TcA opens a gate formed by a six-bladed β-propeller and results in a continuous protein translocation channel, whose architecture and properties suggest a novel mode of protein unfolding and translocation. Our results allow us to understand key steps of infections involving Tc toxins at the molecular level.

Actin as target for modification by bacterial protein toxins

Various bacterial protein toxins and effectors target the actin cytoskeleton. At least three groups of toxins/effectors can be identified, which directly modify actin molecules. One group of toxins/effectors causes ADP-ribosylation of actin at arginine-177, thereby inhibiting actin polymerization. Members of this group are numerous binary actin-ADP-ribosylating exotoxins (e.g. Clostridium botulinum C2 toxin) as well as several bacterial ADP-ribosyltransferases (e.g. Salmonella enterica SpvB) which are not binary in structure. The second group includes toxins that modify actin to promote actin polymerization and the formation of actin aggregates. To this group belongs a toxin from the Photorhabdus luminescens Tc toxin complex that ADP-ribosylates actin at threonine-148. A third group of bacterial toxins/effectors (e.g. Vibrio cholerae multifunctional, autoprocessing RTX toxin) catalyses a chemical crosslinking reaction of actin thereby forming oligomers, while blocking the polymerization of actin to functional filaments. Novel findings about members of these toxin groups are discussed in detail.

Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes

Photorhabdus luminescens secretes both high molecular weight insecticidal toxin complexes and also a range of extracellular proteases into culture broth. Previous studies by others have suggested that insecticidal activity of the broth is associated with these proteases. However, by gene cloning and targeted knock-out, we have previously shown that oral insecticidal activity is associated with high molecular weight ‘toxin complexes’ (Tc) encoded by toxin complex or tc genes. Here we further clarify this distinction by biochemically separating the protease fractions away from the oral insecticidal activity of the Tc proteins. We purified three distinct protease fractions from the broth: one consisting of a single species of 55 kDa and two of several putatively related species of ∼40 kDa. All of these clearly separate from the oral insecticidal activity associated with the high molecular weight Tc proteins and also show no effect on insect weight gain following injection into the haemocoel. Here we examine the substrate preferences and inhibitor profiles of these protease fractions and discuss their relationship with those previously described from other P. luminescens strains and phase variants.