COLL, C EDUCACIÓN Y TECNOLOGÍA LAS TICS EN EL PROCESO DE ENSEÑANZA­ APRENDIZAJE

In the infection studies, the mammalian cells COS7 were infected with either Y. enterocolitica

wild-type strain (WA-P) or a virulence plasmid-cured strain (WA-C) and the behaviour of the cytoskeleton was monitored by antibody labellings of selected cytoskeletal proteins of the

infected cells. Additionally, the in vivo dynamics of actin, cofilin and profilin II in infected cells were examined via fusion of these proteins to GFP with the aid of the highly resolving confocal laser scanning microscopy (CLSM).

4.4.1 Indirect immunofluorescence study of selected cytoskeletal proteins in Y. enterocolitica infected cells

Antibodies specific for human vinculin or talin showed no difference in their labellings in both the uninfected and the WA-P or WA-C infected mammalian cells, suggesting there is no alterations for example in the size or distribution of these focal adhesion proteins in the presence or absence of the Yop effectors. Similarly, an anti-ß-COP antibody revealed in the WA-P or WA-C infected cells Golgi stainings which resembled those of the uninfected cells, leading to the suggestion that the Golgi network is not affected by the Yops. So far, a translocated Yersinia effector, YopH has been known to dephosphorylate the focal adhesion proteins, p130Cas ( Sakai et al., 1994; Burnham et al., 1996) and FAK (Richardson and Parsons, 1995), leading to dissembly of focal contacts and impairing bacterial uptake by host cells. Using the antibody approach in this study did not reveal any disruption of the focal adhesion proteins, at least not for vinculin and talin. Alternatively, the remodelling of cytoskeletal proteins could be investigated by their fusions to GFP so that artefacts generated via immunolabelling could be avoided, and abovemost, GFP-tagged proteins allow direct observation of their dynamics in living cells which could be easily resolved with confocal microscopy.

4.4.2 Y. enterocolitica infection disrupts the actin cytoskeleton

The behaviours of GFP-tagged actin, cofilin and profilin II in mammalian cells during Y.

enterocolitica infection were investigated using CLSM. GFP-profilin was observed to have an uniform cytoplasmic distribution as supported by data obtained with various anti-profilin antibodies which labelled the cytoplasm of amoebas (Tseng et al., 1984; Kaiser et al., 1999), plant cells (Vidali and Hepler, 1997) and vertebrate cells (Buss et al., 1992; Watanabe et al., 1997). In addition, nuclear labels were observed for cells transfected with GFP-profilin. The significance of GFP-profilin in the nucleus is unclear, however, some profilin antibodies were found capable of staining the nuclei of fixed cells (Tseng et al., 1984; Mayboroda et al., 1997; Schlueter et al., 1998). Like the uninfected cells, mammalian cells infected with either a

major effect on the distribution of GFP-profilin and that profilin might not be the host’s target of Yops.

For the study of actin dynamics during Y. enterocolitica infection, the distribution of GFP- actin fluorescence of transfected mammalian cells was compared with the F-actin localization via phalloidin staining. Data showed similar actin labellings with both approaches in the uninfected cells and also in the WA-P or WA-C infected cells, indicating the authenticity of the GFP-actin staining. At the sites of bacterial attachment, actin-rich phagocytic cup-like structures were observed for both WA-P and WA-C infected cells, suggesting that the mammalian cells respond already upon adhesion of Y. enterocolitica, and that this is a non- specific reaction of the host cells in response to bacterial infection independent of the Yop effectors since similar F-actin structures were generated in the presence or absence of secreted Yops. This remodelling of the actin cytoskeleton could perhaps involve the Rho family of small GTPases, as Rac and Cdc42 have been shown to be required for phagocytic cup formation and membrane ruffling in macrophages and leukocytes (Cox et al., 1997; Dharmawardhane et al., 1999).

In addition, after about 30 min of infection with WA-P, the GFP-actin staining started to disappear and dispersed actin patches were observed whereby up to 2 h of infection, the actin cytoskeleton was completely disrupted and reorganized for the rounding up of cells, brought about by the cytotoxic effect of the Yops. On the contrary, cells infected with a Yop secretion minus strain exhibited similar actin labelling as the uninfected cells with a cytoplasmic distribution of actin concentrated at the cortical regions whereby up to 2 h of infection, the cells still retained their spreading, flattened phenotype. These data showing the actin- disrupting activity of Yops with the use of GFP-tagged actin are in agreement with that obtained with phalloidin staining of actin, whereby two particular secreted Yersinia proteins, YopE (Rosqvist et al., 1991) and YopT (Iriarte and Cornelis, 1998) have been shown to dissemble actin filaments with YopT being suggested to exert a stronger cytotoxic effect than YopE (Iriarte and Cornelis, 1998; Zumbihl et al., 1999). The existence of homology between YopE and exoenzyme S (ExoS), a Pseudomonas ADP-ribosylating exotoxin (Kreuger and Barbieri, 1995; Yahr et al., 1996) harbouring also actin disrupting activity (Frithz-Lindsten et al., 1997), suggests the likelihood that YopE targets the small GTPases since ExoS has been reported to modify these signalling molecules (Coburn et al., 1989b; Pederson et al., 1999). In fact, YopT of Y. enterocolitica has recently been shown to be the first intracellularly translocated cytotoxin to act via modification of the small GTP-binding protein RhoA

(Zumbihl et al., 1999) whereby the use of a YopE mutant still expressing the cytotoxic activity of YopT led to the disruption of RhoA-mediated actin stress fibres.

As for the small actin-binding protein, cofilin, which formed actin-cofilin rods as observed via GFP-cofilin fluorescence, it was found to have similar distribution both in the uninfected cells and cells infected with a Yop secretion minus strain. However, infection of mammalian cells with the wild-type Y. enterocolitica after 1 h revealed a decrease in the length of the actin- cofilin rods, leading to several possible proposals. Firstly, the actin dissembling activity of the translocated Yops might prevent formation of long actin-cofilin rods. Secondly, since cofilin has been known to reversibly mediate actin polymerization and depolymerization in a pH- dependent way (Yonezawa et al., 1985; Abe et al., 1989; Hawkins et al., 1993), the Yops could perhaps regulate cofilin’s ability to disrupt actin filaments via alteration of the host’s cellular pH, thus forming shorter actin-cofilin rods. Last but not least, it has been suggested that in higher vertebrates, the subcellular distribution of cofilin as well as its interaction with actin in vivo is mediated by its phosphorylation and dephosphorylation (Nagaoka et al., 1996; Obinata et al., 1997). The actin-binding activity of cofilin has been demonstrated to be negatively regulated by phosphorylation on its Ser 3 residue (Abe et al., 1992; Agnew et al., 1995; Moriyama et al., 1996; Nagaoka et al., 1996) and that phosphorylated cofilin failed to interact with actin and showed only diffuse cytoplasmic distribution. Hence the Yops could possibly lead to modification of cofilin via phosphorylation, whereby this phosphorylated form of cofilin could no longer associate with actin, leading to its diffuse distribution in the cytoplasm and the lack of visible long actin-cofilin rods.

Further studies of the influence of Yersinia infection on the actin cytoskeleton could include the use of selected Yop mutants for correlation of particular Yop effector with its target host’s cytoskeletal protein. It will also be useful to express simultaneously GFP-fused Yops with selected cytoskeletal proteins tagged with variants of GFP to allow direct observation of their interactions with Yops, and to examine the actin dynamics in the Dictyostelium amoeba system as its actin-based motile processes are similar to that of the leukocytes.