Bacterial Injection Machines

A. INTRODUCTION

The delivery of virulence factors directly into host cells is a fascinating aspect of pathogenesis. For Gram-negative bacteria to translocate virulence factors into host cells, at least three membranes must be passed (two bacterial and a host plasma membrane). Bacterial injection machines deliver virulence factors to a specific cellular location where they intersect and influence host mechanisms. This minireview focuses on the Gram-negative bacterial translocation systems that mediate type III and type IV secretion. Remarkably, although these systems are complex multiprotein structures, there is significant similarity and analogy in function, and thus a conserved mechanistic theme in pathogenicity emerges.

Currently there are seven identified types of macromolecular secretion systems in Gram-negative bacterial pathogens (1, 2). This minireview focuses on the two systems that deliver macromolecules directly into eukaryotic cells: type III secretion system (T3SS)¹ and type IV secretion system (T4SS). The delivered macromolecules are referred to as effectors, as they affect and alter the host cellular process. Gram-negative bacterial effectors cross several biochemically distinct barriers, including the bacterial inner membrane, peptidoglycan layer, and outer membrane as well as the host plasma membrane, and even potentially intracellular host membranes. Plant pathogen effectors have the additional complexity of crossing the plant cell wall. The biochemistry of these delivery systems will be discussed, including what is known about how they are assembled and how they function.

B. TYPE III SECRETIONS SYSTEM

In the 1980s and 1990s researchers studying Yersinia, a genus that causes human diseases ranging from bubonic plague to gastrointestinal disease, found that the bacteria produced proteins that were thought to be associated with the outer membrane called Yops. Yops lacked classical signal sequences and were not secreted via a sec-dependent pathway and thus were assumed to be delivered by a new type of secretion system, which later became known as a T3SS, representing its order of discovery in secretion systems. In the last 10 years T3SS have been identified in more than 20 bacterial pathogens that infect plants and animals (Table I). Although there is a high degree of conservation among the components of the type III apparatus in different bacterial species, the pathogens often carry a distinct set of virulence factors with a variety of functions that can be translocated into either animal or plant cells. The overall theme of these T3SS is the direct delivery of proteins that alter and in effect "hijack" the infected host cell for the pathogen (reviewed in Refs. 3 and 4).

C. TYPE III APPARATUS COMPONENTS

Over 20 proteins are proposed to form a functional T3SS (Fig. 1A) (4, 5). YscN is thought to energize the secretion machinery, as it shares homology with the F₀F₁-ATPase and has an ATP-binding site. YscN from Yersinia and its homologue InvC from Salmonella typhimurium have been shown to have ATPase activity as mutations in the catalytic domain cause a loss of secretion (6, 7). YscN homologues are predicted to be located in the cytoplasm where they interact with membrane-bound components of the type III secretion apparatus, thereby energizing the system (4). It has been speculated that the ATPase polymerizes, by itself or with other components, to form the lower part of the T3SS, but this has not been shown. Lending support to this model, YscN has been shown to form a complex with three other cytoplasmic and/or inner membrane-associated Ysc proteins (8).

Many of the proteins involved in forming the T3SS have been localized or are predicted to be inner membrane proteins with varying numbers of transmembrane domains. For example the Yersinia YscV (LcrD) contains eight transmembrane domains and a large cytoplasmic C-terminal domain (9, 10). YscJ family members carry sec-dependent signal sequences and are lipoproteins (4, 11). The Pseudomonas syringae homologue HrcJ is associated with both inner and outer membranes (12), suggesting that it spans the periplasmic space.

Homologues of YscC (e.g. InvG, HrcC) are the only components of the type III apparatus that are clearly found in the outer membrane (12–15). YscC belongs to a family of proteins (secretins) that are involved in transporting large molecules across the outer membrane probably by forming a channel (4). YscC and its homologues form a ring-shaped oligomeric complex in the outer membrane with approximately a 20-nm diameter. Experimentally it has been shown that InvG has a cleavable signal sequence at residue 25, indicating that secretins are exported by the sec-dependent pathway (16). It has been demonstrated that small outer membrane lipoproteins are required to increase the efficiency for the correct localization and functioning of the YscC homologues (13, 14, 17–19). Recently we have shown that correct insertion and function of enteropathogenic Escherichia coli's (EPEC) EscC secretin in the outer membrane requires cytoplasmic and inner membrane components of the type III apparatus, namely EscN and EscV (20).