Olivier Poupel

A Plasmodium falciparum novel gene encoding a coronin-like protein which associates with actin filaments

Plasmodium falciparum, the major causative agent of human malaria, is an Apicomplexa protozoan parasite which invades in its intermediate host hepatocytes and erythrocytes. The driving force underlying internalization into the host cell is thought to involve both polymerization of parasite actin, as entry is inhibited by the cytochalasins, and an actin motor‐associated protein. In the related Apicomplexa parasite, Toxoplasma gondii, the involvement of parasite actin during both processes of motility and host cell entry has been genetically established. In a search for molecules that can regulate actin dynamics within Apicomplexa parasites, we have identified a P. falciparum homologue of the actin associated protein called coronin originally described in the amoeba Dictyostelium discoideum. The single copy gene displays a strong homology with the amoeba sequence and with the bovine and human coronin homologues recently cloned. This homology lies not only within the N‐terminus containing the five WD repeats that characterize coronin but also extends in the C‐terminal part. Furthermore, using an affinity‐purified mouse monoclonal antibody against D. discoideum coronin, we have detected in extracts of P. falciparum young and mature schizonts a 42‐kDa polypeptide which binds this antibody and is present in a Triton insoluble fraction that also contains parasite actin filaments. In addition, the recombinant protein encoded by the homologue nucleotidic sequence of P. falciparum coronin is indeed recognized by the antibody against D. discoideum coronin. Finally, the cross‐reactive polypeptide displays the ability to cosediment with exogenous F‐actin, a property which fits with its involvement in actin dynamics.

Toxoplasma gondii motility and host cell invasiveness are drastically impaired by jasplakinolide, a cyclic peptide stabilizing F-actin.

Actin polymerization and actin-myosin coupling activity most likely provide the driving force that the protozoan parasite Toxoplasma gondii has to exert to propulse itself during gliding and host cell entry. Nevertheless, little information is available on T. gondii tachyzoite actin dynamics, and in particular, the presence of actin filaments remains largely uncharacterized. Here, we report that the marine sponge peptide jasplakinolide, known to bind to filamentous actin, does indeed stabilize a pool of a parasite detergent-insoluble actin. This pool is likely to be formed by a dynamic assembled actin complex: first, it is competent for assembly/disassembly and secondly, it is sensitive to nucleotide phosphate concentration. In addition, T. gondii tachyzoites contain molecules which inhibit actin assembly and destabilize actin filaments. Thus, these activities could account for the remarkably low amount of the myosin-containing F-actin pool we describe here. Furthermore, when parasites are treated with cell-permeant jasplakinolide, they display a significant loss of both motility and host cell invasiveness. These data suggest that in vivo, the detergent-insoluble pool of actin is dynamic.

Peptidoglycan Crosslinking Relaxation Plays an Important Role in Staphylococcus aureus WalKR-Dependent Cell Viability

The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl–glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.

The pleiotropic CymR regulator of Staphylococcus aureus plays an important role in virulence and stress response.

We have characterized a novel pleiotropic role for CymR, the master regulator of cysteine metabolism. We show here that CymR plays an important role both in stress response and virulence of Staphylococcus aureus. Genes involved in detoxification processes, including oxidative stress response and metal ion homeostasis, were differentially expressed in a ΔcymR mutant. Deletion of cymR resulted in increased sensitivity to hydrogen peroxide-, disulfide-, tellurite- and copper-induced stresses. Estimation of metabolite pools suggests that this heightened sensitivity could be the result of profound metabolic changes in the ΔcymR mutant, with an increase in the intracellular cysteine pool and hydrogen sulfide formation. Since resistance to oxidative stress within the host organism is important for pathogen survival, we investigated the role of CymR during the infectious process. Our results indicate that the deletion of cymR promotes survival of S. aureus inside macrophages, whereas virulence of the ΔcymR mutant is highly impaired in mice. These data indicate that CymR plays a major role in virulence and adaptation of S. aureus for survival within the host.

Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ.

Oral susceptibility of Aedes albopictus to dengue type 2 virus: a study of infection kinetics, using the polymerase chain reaction for viral detection

Female Aedes albopictus mosquitoes, aged 1 week, were infected with DEN‐2 dengue virus. The kinetics of infection in mosquito brain and mesenteron were monitored using DNA probes with polymerase chain reaction (PCR) amplification of target DNA sequences coding for DEN‐2 virus envelope protein, compared with the standard immunofluorescence assay technique (IFA).

Rous sarcoma virus SRC gene expression on the growth of quail embryo skin fibroblasts and the establishment of permanent cell lines

Permanent cell lines of Quail embryo fibroblasts appear in cultures of cells infected with a wild type strain of Rous sarcoma virus (SR-RSV) or with its temperature sensitive transformation mutants (ts-T) (NYts68 and PA101) following a three step process. In step one, infected cells grow twice as fast as the control. The second step consists of a crisis during which the cell population is stationary for four to five weeks. Towards the fourth week several foci of cell growth are observed in the flasks. Respreading of the content of these flasks yields permanent lines. This constitutes the third step of the population evolution. In step one the growth rate of the infected cells is the same irrespective of the incubation temperature (36 degrees C or 41 degrees C) whereas the level of the pp60v-src activity is considerably depressed at 41 degrees C for NYts68 and PA101. Foci do not appear at restrictive temperature in the ts infected population and permanent lines are not recovered under that condition. These lines grow ony at 36 degrees C. It can be shown that the virus which they produce is not modified with respect to the temperature sensitivity of the src gene expression since newly infected fibroblasts grow equally well in step one at both 36 degrees C and 41 degrees C, and stop after the same number of generations. This finding suggests that the events which, during the crisis period, lead to the establishment of permanent lines, take place at the cellular level but depend on the activity of the pp60v-src protein for their occurrence or their expression.