Åke Forsberg

The cytotoxic protein YopE of Yersinia obstructs the primary host defence

It has previously been shown that the plasmid‐encoded YopE protein of Yersinia pseudotubercuiosis is a virulence determinant. In this study, HeLa cells, macrophages and mice were used as different model systems to determine the actual role of YopE in the virulence process. The YopE protein mediates a cytotoxic response on a confluent layer of HeLa cells. A prerequisite of this activity is that the pathogen binds to the cell surface. YopE also induces a cytotoxic response on mouse macrophages where it influences the ability of the pathogen to resist phagocytosis. Bacterial mutants defective in their ability to express YopE are avirulent after oral or Intra peritonea I infection but virulent following intravenous injection. On the basis of these results, we propose a role for YopE in the virulence process of Yersinia.

Role of Fraction 1 Antigen of Yersinia pestis in Inhibition of Phagocytosis

ABSTRACT Yersinia pestis, the causative agent of plague, expresses a capsule-like antigen, fraction 1 (F1), at 37°C. F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is unique to Y. pestis. F1 is a surface polymer composed of a protein subunit, Caf1, with a molecular mass of 15.5 kDa. The secretion and assembly of F1 require the caf1M and caf1A genes, which are homologous to the chaperone and usher protein families required for biogenesis of pili. F1 has been implicated to be involved in the ability of Y. pestis to prevent uptake by macrophages. In this study we addressed the role of F1 antigen in inhibition of phagocytosis by the macrophage-like cell line J774. The Y. pestis strain EV76 was found to be highly resistant to uptake by J774 cells. An in-frame deletion of the caf1M gene of the Y. pestis strain EV76 was constructed and found to be unable to express F1 polymer on the bacterial surface. This strain had a somewhat lowered ability to prevent uptake by J774 cells. Strain EV76C, which is cured for the virulence plasmid common to the pathogenic Yersinia species, was, as expected, much reduced in its ability to resist uptake. A strain lacking both the virulence plasmid and caf1M was even further hampered in the ability to prevent uptake and, in this case, essentially all bacteria (95%) were phagocytosed. Thus, F1 and the virulence plasmid-encoded type III system act in concert to make Y. pestis highly resistant to uptake by phagocytes. In contrast to the type III effector proteins YopE and YopH, F1 did not have any influence on the general phagocytic ability of J774 cells. Expression of F1 also reduced the number of bacteria that interacted with the macrophages. This suggests that F1 prevents uptake by interfering at the level of receptor interaction in the phagocytosis process.

The V-antigen of Yersinia is surface exposed before target cell contact and involved in virulence protein translocation

Type III‐mediated translocation of Yop effectors is an essential virulence mechanism of pathogenic YersiniaLcrV is the only protein secreted by the type III secretion system that induces protective immunity. LcrV also plays a significant role in the regulation of Yop expression and secretion. The role of LcrV in the virulence process has, however, remained elusive on account of its pleiotropic effects. Here, we show that anti‐LcrV antibodies can block the delivery of Yop effectors into the target cell cytosol. This argues strongly for a critical role of LcrV in the Yop translocation process. Additional evidence supporting this role was obtained by genetic analysis. LcrV was found to be present on the bacterial surface before the establishment of bacteria target cell contact. These findings suggest that LcrV serves an important role in the initiation of the translocation process and provides one possible explanation for the mechanism of LcrV‐induced protective immunity.

The surface‐located YopN protein is involved in calcium signal transduction in Yersinia pseudotuberculosis

The low‐calcium response (Icr) is strongly conserved among the pathogenic Yersinia species and is observed when the pathogen is grown at 37°C in Ca2+‐depleted medium. This response is characterized by a general metabolic downshift and by a specific induction of virulence‐plasmid‐encoded yop genes. Regulation of yop expression is exerted at transcriptional level by a temperature‐regulated activator and by Ca2+‐regulated negative elements. The yopN gene was shown to encode a protein (formerly also designated Yop4b) which is surface‐located when Yersmia is grown at 37°C. yopN was found to be part of an operon that is induced during the low‐calcium response. Insertional inactivation of the yopN gene resulted in derepressed transcription of yop genes. A hybrid plasmid containing the yopN gene under the control of the tac promoter fully restored the wild‐type phenotype of the yopN mutant. Thus the surface‐located YopN somehow senses the calcium concentration and transmits a signal to shut off yop transcription when the calcium concentration is high.

Intracellular targeting of exoenzyme S of Pseudomonas aeruginosa via type III-dependent translocation induces phagocytosis resistance, cytotoxicity and disruption of actin microfilaments.

Exoenzyme S (ExoS) is an ADP‐ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. The amino‐terminal half of ExoS exhibits homology to the YopE cytotoxin of pathogenic Yersinia. Recently, YopE was found to be translocated into the host cell by a bacteria–cell contact‐dependent mechanism involving the ysc‐encoded type III secretion system. By using an approach in which exoS was expressed in different strains of Yersinia, including secretion and translocation mutants, we could demonstrate that ExoS was secreted and translocated into HeLa cells by a similar mechanism to that described previously for YopE. Similarly to YopE, the presence of ExoS in the host cell elicited a cytotoxic response, correlating with disruption of the actin microfilament structure. A similar cytotoxic response was also induced by a mutated form of ExoS with a more than 2000‐fold reduced ADP‐ribosyltransferase activity. However, the enzymatically active ExoS elicited a more definite rounding up of the HeLa cells, which also correlated with decreased viability of the cells after prolonged infection compared with cells infected with strains expressing mutated ExoS or YopE. This suggests that ExoS can act through two different mechanisms on the host cell. The expression of ExoS by Yersinia also mediated an anti‐phagocytic effect on macrophages. In addition, we present evidence that extracellularly located P. aeruginosa is able to target ExoS into eukaryotic cells. Taken together, our data suggest that P. aeruginosa, by analogy with Yersinia, targets virulence proteins into the eukaryotic cytosol via a type III secretion‐dependent mechanism as part of an anti‐phagocytic strategy.

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.

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.

The chaperone‐like protein YerA of Yersinia pseudotuberculosis stabilizes YopE in the cytoplasm but is dispensible for targeting to the secretion loci

The virulence plasmid‐encoded YopE cytotoxin of Yersinia pseudotuberculosis is secreted across the bacterial membranes and subsequently translocated into the eukaryotic cell. Translocation of YopE into target cells was recently shown to be polarized and only occurred at the zone of contact between the pathogen and the eukaryotic cell.

YscP and YscU Regulate Substrate Specificity of the Yersinia Type III Secretion System

Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37 degrees C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.

Regulation of type III secretion systems

Type III secretion systems are utilised by numerous Gram-negative bacteria to efficiently interact with a host. Appropriate expression of type III genes is achieved through the integration of several regulatory pathways that ultimately co-ordinate the activity of a central transcriptional activator usually belonging to the AraC family. The complex regulatory cascades allow this virulence strategy to be utilised by different bacteria even if they occupy diverse niches that define a unique set of environmental cues. Simulating the appropriate combination of signals in vitro to allow a meaningful interpretation of the type III assembly and secretion regulatory cascade remains a common goal for researchers. Pieces of the puzzle slowly emerge to provide insightful views into the complex regulatory networks that allow bacteria to assemble and utilise type III secretion to efficiently colonise a host.