Small-molecule inhibitors specifically targeting type III secretion.

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. A number of small organic molecules having this potential have recently been identified (A. M. Kauppi, R. Nordfelth, H. Uvell, H. Wolf-Watz, and M. Elofsson, Chem. Biol. 10:241-249, 2003). Using different reporter gene constructs, we showed that compounds that belong to a class of acylated hydrazones of different salicylaldehydes target the TTS system of Yersinia pseudotuberculosis. One of these compounds, compound 1, was studied in detail and was found to specifically block Yop effector secretion under in vitro conditions by targeting the TTS system. In this respect the drug mimics the well-known effect of calcium on Yop secretion. In addition, compound 1 inhibits Yop effector translocation after infection of HeLa cells without affecting the eukaryotic cells or the bacteria. A HeLa cell model that mimics in vivo conditions showed that compound 1 chemically attenuates the pathogen to the advantage of the eukaryotic cell. Thus, our results show proof of concept, i.e., that small compounds targeting the TTS system can be identified, and they point to the possible use of TTS inhibitors as a novel class of antibacterial agents.

Yersinia species in collagenous colitis: a serologic study.

BACKGROUND: The etiology of collagenous colitis is unknown. An infectious cause seems a possibility, and in a recent report three out of six patients with collagenous colitis were shown to have had an infection with Yersinia enterocolitica. The aim was to investigate the occurrence of Yersinia antibodies in collagenous colitis. METHODS: Sera from 32 collagenous colitis patients and 17 healthy controls were analysed for antibodies against Yersinia virulence proteins. RESULTS: Collagenous colitis patients had Yersinia antibodies more often than the controls, 9 having a positive and 4 an intermediate antibody score of the 32 patients. In comparison, I out of 17 controls had a positive and 2 an intermediate antibody score, which represents a strong, although not significant, trend (P = 0.078). CONCLUSION: The data showed that Yersinia antibodies are more common in collagenous colitis patients than in healthy controls. In some cases, Yersinia might have been the triggering event in the development of collagenous colitis.

YopB of Yersinia enterocolitica is essential for YopE translocation.

A previous study has shown that YopB of Yersinia spp. is essential for translocation of Yop effectors across the eucaryotic plasma membrane (M.-P. Sory and G. R. Cornelis, Mol. Microbiol. 14:583--594, 1994). However, this role was recently challenged (V. T. Lee and O. Schneewind, Mol. Microbiol. 31:1619--1629, 1999). Using protease protection and digitonin extraction, we reconfirm that YopB of Yersinia enterocolitica is essential for the translocation of YopE into HeLa cell monolayers.

LcrV is a channel size-determining component of the Yop effector translocon of Yersinia.

Delivery of Yop effector proteins by pathogenic Yersinia 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.

Localization of the Yersinia PTPase to focal complexes is an important virulence mechanism.

The protein tyrosine phosphatase YopH, produced by the pathogen Yersinia pseudotuberculosis, is an essential virulence determinant involved in antiphagocytosis. Upon infection, YopH is translocated into the target cell, where it recognizes focal complexes. Genetic analysis revealed that YopH harbours a region that is responsible for specific localization of this PTPase to focal complexes in HeLa cells and professional phagocytes. This region is a prerequisite for blocking an immediate-early Yersinia-induced signal within target cells. The region is also essential for antiphagocytosis and virulence, illustrating the biological significance of localization of YopH to focal complexes during Yersinia infection. These results also indicate that focal complexes play a role in the general phagocytic process.

Modulation of virulence factor expression by pathogen target cell contact.

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.

Cell-surface-bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into 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.