Sebastian Håkansson

The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across the target cell plasma membrane and displays a contact-dependent membrane disrupting activity

During infection of cultured epithelial cells, surface‐located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non‐polar yopB mutant strain displays a wild‐type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage‐like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia‐induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB‐dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell‐to‐cell transfer of Yop effector proteins.

The Yersinia YpkA Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane

Multiple yop mutant strains of Yersinia pseudotuberculosis not expressing several virulence effector Yop proteins (YopH, M, E, K and YpkA) were engineered. When high‐copy‐number plasmids carrying the ypkA or the yopE gene with their endogenous promoters were introduced into the engineered strains, the corresponding Yop protein was secreted at high levels in vitro. These multiple yop mutant strains, when harbouring the yopE gene in trans, behaved as the wild‐type strain with respect to YopB‐dependent translocation of YopE through the HeLa cell plasma membrane. Using these multiple yop mutant strains, it was demonstrated that the YpkA Ser/Thr protein kinase mediates morphological alterations of infected cultured HeLa cells different from those mediated by YopE and YopH. Furthermore, YpkA is shown to be translocated by a YopB‐dependent translocation mechanism from surface‐located bacteria and subsequently targeted to the inner surface of the target‐cell plasma membrane. The pattern of YpkA localization after infection suggests that this Yop effector is involved in interference with signal transduction.

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.

Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole

Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry‐derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein‐rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite‐containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre‐existing, parasite‐containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two‐step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by Toxoplasma. The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.

Functional conservation of the secretion and translocation machinery for virulence proteins of yersiniae, salmonellae and shigellae.

Virulent bacteria of the genera Yersinia, Shigella and Salmonella secrete a number of virulence determinants, Yops, Ipas and Sips respectively, by a type III secretion pathway. The IpaB protein of Shigella flexneri was expressed in Yersinia pseudotuberculosis and found to be secreted under the same conditions required for Yop secretion. Likewise, YopE was secreted by the wild‐type strain LT2 of Salmonella typhimurium, but YopE was not secreted by the isogenic invA mutant. Secretion of both IpaB and YopE required their respective chaperones, IpgC and YerA. In addition, yopE‐containing S. typhimurium expressed a YopE‐mediated cytotoxicity on cultured HeLa cells. YopE was detected in the cytosol of the infected HeLa cells and the amount of translocated YopE correlated with the degree of cytotoxicity. Both translocation and cytotoxicity were prevented by the addition of gentamicin. Treatment of HeLa cells with cytochalasin D prior to infection prevented internalization of bacteria, but translocation of YopE was still observed. These results favour the hypothesis that YopE is translocated through the plasma membrane by surface‐located bacteria. We propose that virulent Salmonella and Shigella deliver virulence effector molecules into the target cell through the utilization of a functionally conserved secretion/translocation machinery similar to that shown for Yersinia.

YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane.

Introduction of anti‐host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram‐negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia, the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK‐mutant strain was found to induce a more rapid YopE‐mediated cytotoxic response in HeLa cells as well as in MDCK‐1 cells compared to the wild‐type strain. We found that this was the result of a cell‐contact‐dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB‐dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK‐mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild‐type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell‐associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.

Signal transduction in Yersinia pseudotuberculosis

Virulent Yersinia possess a common 70kb virulence plasmid which encodes a number of indispensable Yops virulence determinants. Yops proteins are regulated by external stimuli temperature and calcium concentration. At 37° yop transcription is induced and the rate of transcription is regulated by the Ca2+ concentration of the growth medium. In parallel to this transcriptional regulation, Yops are secreted into the growth medium by a specific Ca2+ regulated plasmid-encoded secretion system. One mutant (yopN) has been isolated and studies using this mutant suggest that the surface-located YopN protein senses the Ca2+ concentration and transmits this signal accordingly. The final step of the regulatory hierarchy involves a yop transcriptional repressor. LcrH is suggested to be this repressor since overproduction of LcrH leads to repression of transcription of yop genes. Several Yop proteins have been shown to be essential virulence determinants. Two of these, YopH and YopE, act in concert to block phagocytosis by macrophages. YopH exhibits a protein tyrosine phosphatase activity suggesting that YopH dephosphorylates host proteins. YopE is a cytotoxin. Recent studies have shown that interaction between the target cell surfaces and the pathogen triggers YopE expression and its polarized transfer through the plasma membrane of the target cell. The previous findings with respect to Yop regulation and Yop secretion is in agreement with the polarized transfer of Yop proteins into the target cell.

Plasmid Encoded Virulence of Yersinia

The three virulent members of the genus Yersinia harbour related virulence Plasmids with a molecular weight of about 60-70 kb (11). When exponential phase cultures of these organisms growing in a Ca2+ free medium are shifted from 26C to 37C, growth ceases over a period of about 2 generations (10). If however 2.5 mM Ca2+ is present in the medium growth continues normally. These bacteria are referred to as being Ca2+ dependent (CD). Plasmid cured strains, however, do not show this dependency on Ca2+ and are thus, Ca2+ independent (CI). Such bacteria are always avirulent. By transposon insertion mutagenesis a 20 kb region of the virulence plasmid has been identified which is involved in this low calcium response (1cr). Such CI-mutants do not require Ca2+ for prolonged growth at 37C and they are not virulent. Although the plasmids of Y. enterocolitica. Y. pestis and Y. pseudotuberculosis have been subjected to rearrengements, the Ca2+ region, however, of the different plasmids is conserved (11).