Translocation of surface-localized effectors in type III secretion.

Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells. Upon host cell contact, purified YopH coated on Y. pseudotuberculosis was specifically and rapidly translocated across the target-cell membrane, which led to a physiological response in the infected cell. In addition, translocation of externally added YopH required a functional T3SS and a specific translocation domain in the effector protein. Efficient, T3SS-dependent translocation of purified YopH added in vitro was also observed when using coated Salmonella typhimurium strains, which implies that T3SS-mediated translocation of extracellular effector proteins is conserved among T3SS-dependent pathogens. Our results demonstrate that polarized T3SS-dependent translocation of proteins can be achieved through an intermediate extracellular step that can be reconstituted in vitro. These results indicate that translocation can occur by a different mechanism from the assumed single-step conduit model.

The membrane localization domain is required for intracellular localization and autoregulation of YopE in Yersinia pseudotuberculosis.

Recent work has shown that a domain of YopE of Yersinia pseudotuberculosis ranging from amino acids 54 to 75 (R. Krall, Y. Zhang, and J. T. Barbieri, J. Biol. Chem. 279:2747-2753, 2004) is required for proper localization of YopE after ectopic expression in eukaryotic cells. This domain, called the membrane localization domain (MLD), has not been extensively studied in Yersinia. Therefore, an in cis MLD deletion mutant of YopE was created in Y. pseudotuberculosis. The mutant was found to secrete and translocate YopE at wild-type levels. However, the mutant was defective in the autoregulation of YopE expression after the infection of HeLa cells. Although the mutant translocated YopE at wild-type levels, it showed a delayed HeLa cell cytotoxicity. This delay was not caused by a change in GTPase activating protein (GAP) activity, since the mutant showed wild-type YopE GAP activity toward Rac1 and RhoA. The MLD mutant displayed a changed intracellular localization pattern of YopE in HeLa cells after infection, and the YopEDeltaMLD protein was found to be dispersed within the whole cell, including the nucleus. In contrast, wild-type YopE was found to localize to the perinuclear region of the cell and was not found in the nucleus. In addition, the yopEDeltaMLD mutant was avirulent. Our results suggest that YopE must target proteins other than RhoA and Rac1 and that the MLD is required for the proper targeting and hence virulence of YopE during infection. Our results raise the question whether YopE is a regulatory protein or a "true" virulence effector protein.

Induction of the Yersinia type 3 secretion system as an all-or-none phenomenon.

Pathogenic Yersinia spp. possess a protein secretion system, designated as type 3, that plays a clear role in promoting their survival vis-à-vis the macrophage. Inductive expression of the Yersinia type 3 secretion system (T3SS), triggered either by host cell contact, or, in the absence of host cells, by a reduction in extracellular calcium ion levels, is accompanied by a withdrawal from the bacterial division cycle. Here, we analyzed Ca(2+)-dependent induction of the T3SS at the single-cell level to understand how Yersinia coordinates pro-survival and growth-related activities. We utilized a novel high-throughput quantitative microscopy approach as well as flow cytometry to determine how Ca(2+) levels, T3SS expression, and cellular division are interrelated. Our analysis showed that there is a high degree of homogeneity in terms of T3SS expression levels among a population of Y. pseudotuberculosis cells following the removal of Ca(2+), and that T3SS expression appears to be independent of the cellular division cycle. Unexpectedly, our analysis showed that Ca(2+) levels are inversely related to the initiation of inductive T3SS expression, and not to the intensity of activation once initiated, thus providing a basis for the seemingly graded response of T3SS activation observed in bulk-level analyses. The properties of the system described here display both similarities to and differences from that of the lac operon first described 50 years ago by Novick and Weiner.

Regulation of Yersinia Yop-effector delivery by translocated YopE.

The bacterial pathogen Yersinia pseudotuberculosis uses a type III secretion (T3S) system to translocate Yop effectors into eukaryotic cells. Effectors are thought to gain access to the cytosol via pores formed in the host cell plasma membrane. Translocated YopE can modulate this pore formation through its GTPase-activating protein (GAP) activity. In this study, we analysed the role of translocated YopE and all the other known Yop effectors in the regulation of effector translocation. Elevated levels of Yop effector translocation into HeLa cells occurred by YopE-defective strains, but not those defective for other Yop effectors. Only Yersinia devoid of YopK exhibits a similar hyper-translocation phenotype. Since both yopK and yopE mutants also failed to down-regulate Yop synthesis in the presence of eukaryotic cells, these data imply that translocated YopE specifically regulates subsequent effector translocation by Yersinia through at least one mechanism that involves YopK. We suggest that the GAP activity of YopE might be working as an intra-cellular probe measuring the amount of protein translocated by Yersinia during infection. This may be a general feature of T3S-associated GAP proteins, since two homologues from Pseudomonas aeruginosa, exoenzyme S (ExoS) and exoenzyme T (ExoT), can complement the hyper-translocation phenotypes of the yopE GAP mutant.

The host-encoded Heme Regulated Inhibitor (HRI) facilitates virulence-associated activities of bacterial pathogens.

Here we show that cells lacking the heme-regulated inhibitor (HRI) are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S) system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria. For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2α kinase) are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.

Cell division in the cerebral cortex of adult rats after photothrombotic ring stroke.

Neurogenesis has been shown to occur in the cerebral cortex in adult rats after ischemic stroke. The origin of the newborn neurons is largely unknown. This study aimed to explore cell division in the poststroke penumbral cortex. Adult male Wistar rats were subjected to photothrombotic ring stroke. After repeated delivery of the DNA duplication marker BrdU, the animals were sacrificed at various times poststroke. BrdU was detected by immunohistochemistry/immunofluorescence labeling, as was the M-phase marker Phos H3 and the spindle components alpha-tubulin/gamma-tubulin. DNA damage was examined by TUNEL staining. Cell type was ascertained by double immunolabeling with the neuronal markers Map-2ab/beta-tubulin III and NeuN/Hu or the astrocyte marker GFAP. From 16h poststroke, BrdU-immunolabeled cells appeared in the penumbral cortex. From 24h, Phos H3 was colocalized with BrdU in the nuclei. Mitotic spindles immunolabeled by alpha-tubulin/gamma-tubulin appeared inside the cortical cells containing BrdU-immunopositive nuclei. Unexpectedly, the markers of neuronal differentiation, Map-2ab/beta-tubulin III/NeuN/Hu, were expressed in the Phos H3-immunolabeled cells, and NeuN was detected in some cells containing spindles. This study suggests that in response to a sublethal ischemic insult, endogenous cells with neuronal immunolabeling may duplicate their nuclear DNA and commit cell mitosis to generate daughter neurons in the penumbral cortex in adult rats.

Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis.

YopE of Yersinia pseudotuberculosis inactivates three members of the small RhoGTPase family (RhoA, Rac1 and Cdc42) in vitro and mutation of a critical arginine abolishes both in vitro GTPase-activating protein (GAP) activity and cytotoxicity towards HeLa cells, and renders the pathogen avirulent in a mouse model. To understand the functional role of YopE, in vivo studies of the GAP activity in infected eukaryotic cells were conducted. Wild-type YopE inactivated Rac1 as early as 5 min after infection whereas RhoA was down regulated about 30 min after infection. No effect of YopE was found on the activation state of Cdc42 in Yersinia-infected cells. Single-amino-acid substitution mutants of YopE revealed two different phenotypes: (i) mutants with significantly lowered in vivo GAP activity towards RhoA and Rac1 displaying full virulence in mice, and (ii) avirulent mutants with wild-type in vivo GAP activity towards RhoA and Rac1. Our results show that Cdc42 is not an in vivo target for YopE and that YopE interacts preferentially with Rac1, and to a lesser extent with RhoA, during in vivo conditions. Surprisingly, we present results suggesting that these interactions are not a prerequisite to establish infection in mice. Finally, we show that avirulent yopE mutants translocate YopE in about sixfold higher amount compared with wild type. This raises the question whether YopE's primary function is to sense the level of translocation rather than being directly involved in downregulation of the host defence.

Role of fraction 1 antigen of Yersinia pestis in inhibition of phagocytosis.

Yersinia pestis, the causative agent of plague, expresses a capsule-like antigen, fraction 1 (F1), at 37 degrees 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.

Visualisation of zebrafish infection by GFP-labelled Vibrio anguillarum.

Vibrio anguillarum is an invasive pathogen of fish causing a septicaemia called vibriosis. In this work, transparent zebrafish were immersed in water containing green fluorescent protein labelled V. anguillarum. The infection was visualised at the whole fish and single bacterium levels using microscopy. The gastrointestinal tract was the first site where the pathogen was detected. This enteric localisation occurred independently of the flagellum or motility. On the other hand, chemotactic motility was essential for association of the pathogen with the fish surface. In conclusion, the zebrafish infection model provides evidence that the intestine and skin represent sites of infection by V. anguillarum and suggests a host site where chemotaxis may function in virulence.

In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity.

The YopE cytotoxin of Yersinia is an essential virulence determinant that is translocated into the eukaryotic target cell via a plasmid-encoded type III secretion system. YopE possess a GTPase activating protein activity that in vitro has been shown to down regulate RhoA, Rac1, and Cdc42. Translocated YopE induces de-polymerisation of the actin microfilament structure in the eukaryotic cell which results in a rounding up of infected cells described as a cytotoxic effect. Here, we have investigated the importance of different regions of YopE for induction of cytotoxicity and in vitro GAP activity. Sequential removal of the N- and C-terminus of YopE identified the region between amino acids 90 and 215 to be necessary for induction of cytotoxicity. Internal deletions containing the essential arginine at position 144 resulted in a total loss of cytotoxic response. In-frame deletions flanking the arginine finger defined a region important for the cytotoxic effect to amino acids 166-183. Four triple-alanine substitution mutants in this region, YopE166-8A, 169-71A, 175-7A and 178-80A were still able to induce cytotoxicity on HeLa cells although they did not show any in vitro GAP activity towards RhoA, Rac1 or Cdc42. A substitution mutant in position 206-8A showed the same phenotype, ability to induce cytotoxic response but no in vitro GAP activity. We speculate that YopE may have additional unidentified targets within the eukaryotic cell.