Upon contact with the eukaryotic cell, Yersinia pseudotuberculosis increased the rate of transcription of virulence genes (yop), as determined by in situ monitoring of light emission from individual bacteria expressing luciferase under the control of the yopE promoter. The microbe-host interaction triggered export of LcrQ, a negative regulator of Yop expression, via the Yop-type III secretion system. The intracellular concentration of LcrQ was thereby lowered, resulting in increased expression of Yops. These results suggest a key role for the type III secretion system of pathogenic bacteria to coordinate secretion with expression of virulence factors after physical contact with the target cell.

YopH is translocated by cell-surface-bound bacteria through the plasma membrane to the cytosol of the HeLa cell. The transfer mechanism is contact dependent and polarizes the translocation to only occur at the contact zone between the bacterium and the target cell. More than 99% of the PTPase activity is associated with the HeLa cells. In contrast to the wild-type strain, the yopBD mutant cannot deliver YopH to the cytosol. Instead YopH is deposited in localized areas in the proximity of cell-associated bacteria. A yopN mutant secretes 40% of the total amount of YopH to the culture medium, suggesting a critical role of YopN in regulation of the polarized translocation. Evidence for a region in YopH important for its translocation through the plasma membrane of the target cell but not for secretion from the pathogen is provided.

The type III secretion (TTS) system is used by several animal and plant pathogens to deliver effector proteins into the cytosol of the eukaryotic target cell as a strategy to evade the defense reactions elicited by the infected organism. The fact that these systems are highly homologous implies that novel antibacterial agents that chemically attenuate the pathogens via a specific interaction with the type III secretion mechanism can be identified. Type III secretion (TTS) constitutes a common virulence system present in many gram-negative species, including Yersinia spp., Salmonella spp., Shigella spp., Pseudomonas aeruginosa, entheropathogenic Escherichia coli, enterohemoragic E. coli, and Chlamydia spp. (11,24). The bacteria depend on their respective TTS system to invade the host, resist phagocytosis, grow in deep tissues, and cause disease. Furthermore, studies have revealed that several components of the TTS systems are conserved between different species (11, 42). These findings offer a possibility to develop novel antibacterial agents that target TTS-based virulence (32, 50). Moreover, small molecules that interfere with TTS can be utilized as tools in efforts aiming at increasing our understanding of complex bacterial virulence systems by using a chemical genetics approach (29,50). The strategy of identifying and using small molecules in functional studies of microbial virulence is attractive and complements current methods in the field, as illustrated by some recent publications (7,26,27,47).The well-studied, 70-kb-plasmid-encoded Ysc (for Yersinia secretion) TTS system of Yersinia (51) represents a suitable target for both drug development (32) and a small-molecule approach to address protein function (50). Of the 11 known species of Yersinia, Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis are pathogenic to mammals (51). The Ysc TTS apparatus is essential for the bacteria to evade the host immune defense, and compounds targeting this mechanism will result in attenuation without affecting bacterial growth. Interestingly 10 of the Ysc proteins have counterparts in almost all TTS systems, and it has been shown that some components of the secretion systems are interchangeable among different species (20), demonstrating evolutionary conservation. Since the TTS systems are conserved among the gram-negative bacteria utilizing this virulence mechanism it is likely that compounds targeting TTS machinery in Yersinia will also affect the TTS system in other species and that data generated with one species would also be valid for others. The importance of TTS studies is further stressed by the fact that the number of multiresistant strains in different species that utilize this virulence system is rising (38). Moreover, multiresistant strains of Y. pestis, a potential weapon in biological warfare and bioterrorism (25), have been isolated (18).During the progress of an infection the Yersinia bacterium adheres to eukaryotic cells, e.g., macrophages, and injects a set of effector proteins, called Yops (for Y...

Summary Delivery of Yop effector proteins by pathogenicYersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact-dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127±195 of LcrV and to amino acids 106±173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size-determining structural component of the Yop translocon.

Agents that target bacterial virulence without detrimental effect on bacterial growth are useful chemical probes for studies of virulence and potential candidates for drug development. Several gram-negative pathogens employ type III secretion to evade the innate immune response of the host. Screening of a chemical library with a luciferase reporter gene assay in viable Yersinia pseudotuberculosis furnished several compounds that inhibit the reporter gene signal expressed from the yopE promoter and effector protein secretion at concentrations with no or modest effect on bacterial growth. The selectivity patterns observed for inhibition of various reporter gene strains indicate that the compounds target the type III secretion machinery at different levels. Identification of this set of inhibitors illustrates the approach of utilizing cell-based assays to identify compounds that affect complex bacterial virulence systems.