http://edoc.rki.de/176904/44 2026-09-23T08:46:26Z http://edoc.rki.de/176904/13925 A Plasmodium cysteine protease required for efficient transition from the liver infection stage Putrianti, Elyzana Dewi; Schmidt-Christensen, Anja; Heussler, Volker; Matuschewski, Kai; Ingmundson, Alyssa The transitions between developmental stages are critical points in the Plasmodium life cycle. The development of Plasmodium in the livers of their mammalian hosts bridges malaria transmission and the onset of clinical symptoms elicited by red blood cell infection. The egress of Plasmodium parasites from the liver must be a carefully orchestrated process to ensure a successful switch to the blood stage of infection. Cysteine protease activity is known to be required for liver-stage Plasmodium egress, but the crucial cysteine protease(s) remained unidentified. Here, we characterize a member of the papain-like cysteine protease family, Plasmodium berghei serine repeat antigen 4 (PbSERA4), that is required for efficient initiation of blood-stage infection. Through the generation PbSERA4-specific antisera and the creation of transgenic parasites expressing fluorescently tagged protein, we show that PbSERA4 is expressed and proteolytically processed in the liver and blood stages of infection. Targeted disruption of PbSERA4 results in viable and virulent blood-stage parasites. However, upon transmission from mosquitoes to mice, Pbsera4(-) parasites displayed a reduced capacity to initiate a new round of asexual blood-stage replication. Our results from cultured cells indicate that this defect results from an inability of the PbSERA4-deficient parasites to egress efficiently from infected cells at the culmination of liver-stage development. Protection against infection with wildtype P. berghei could be generated in animals in which Pbsera4(-) parasites failed to establish infection. Our findings confirm that liver-stage merozoite release is an active process and demonstrate that this parasite-encoded cysteine protease contributes to parasite escape from the liver.; Plasmodium parasites cause over 200 million cases of malaria every year. When parasites are transmitted by mosquito bite, they initially colonize the liver before they move into the blood and cause disease. During successful transition from the liver into the blood, Plasmodium cloak themselves in host plasma membrane as they egress from the liver cells. Although some aspects of how Plasmodium exit their host hepatocytes appear unique, certain attributes are shared across diverse pathogens. For example, protease activity is required not only for multiple stages of Plasmodium exit, but is also involved in the egress of some bacteria and other protozoan. Here we characterize a protease in Plasmodium berghei that is expressed in the liver and conserved across Plasmodium species. Through gene targeting, we found PbSERA4 is required for efficient egress of Plasmodium from the liver. In the absence of this protease the transition between the liver and blood stages of growth is prolonged due to inefficient parasite release from liver cells. These findings provide new insights into the function of a conserved Plasmodium protease and into the process of Plasmodium escape from the liver. 2020-09-21T00:00:00Z http://edoc.rki.de/176904/13924 Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose Richter, Anja M.; Possling, Alexandra; Malysheva, Nadezhda; Yousef, Kaveh P.; Herbst, Susanne; von Kleist, Max; Hengge, Regine In many bacteria, the biofilm-promoting second messenger c-di-GMP is produced and degraded by multiple diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. High target specificity of some of these enzymes has led to theoretical concepts of “local” c-di-GMP signaling. In Escherichia coli K-12, which has 12 DGCs and 13 PDEs, a single DGC, DgcC, is specifically required for the biosynthesis of the biofilm exopolysaccharide pEtN-cellulose without affecting the cellular c-di-GMP pool, but the mechanistic basis of this target specificity has remained obscure. DGC activity of membrane-associated DgcC, which is demonstrated in vitro in nanodiscs, is shown to be necessary and sufficient to specifically activate cellulose biosynthesis in vivo. DgcC and a particular PDE, PdeK (encoded right next to the cellulose operon), directly interact with cellulose synthase subunit BcsB and with each other, thus establishing physical proximity between cellulose synthase and a local source and sink of c-di-GMP. This arrangement provides a localized, yet open source of c-di-GMP right next to cellulose synthase subunit BcsA, which needs allosteric activation by c-di-GMP. Through mathematical modeling and simulation, we demonstrate that BcsA binding from the low cytosolic c-di-GMP pool in E. coli is negligible, whereas a single c-di-GMP molecule that is produced and released in direct proximity to cellulose synthase increases the probability of c-di-GMP binding to BcsA several hundred-fold. This local c-di-GMP signaling could provide a blueprint for target-specific second messenger signaling also in other bacteria where multiple second messenger producing and degrading enzymes exist. 2020-07-24T00:00:00Z http://edoc.rki.de/176904/13923 Development of a novel selective agar for the isolation and detection of Bacillus anthracis Rohde, Alexander; Papp, Stefanie; Feige, P.; Grunow, R.; Kaspari, O. Aims The aim of this study was to develop a novel selective agar for the specific isolation and detection of Bacillus anthracis. Methods and Results Based on published data on antibiotic resistance and susceptibility of B. anthracis and other closely related species of the Bacillus cereus sensu lato group, a new selective agar formulation termed CEFOMA (Bacillus CEreus sensu lato group‐specific antibiotics, FOsfomycin, MAcrolides) was developed and evaluated. All tested strains of B. anthracis were able to grow on CEFOMA with the same colony number as on non‐selective media, whereas CEFOMA inhibited the growth of the other species within the B. cereus sensu lato group. In comparison to other selective agars, CEFOMA had a superior performance and considerably reduced the total amount of accompanying flora in soil. Furthermore, B. anthracis was successfully isolated from deliberately spiked soil samples. Conclusions CEFOMA is a highly promising selective agar for the efficient isolation of B. anthracis from environmental samples with a large bacterial background flora. Significance and Impact of the Study The isolation of B. anthracis from environmental samples is severely impaired by the lack of adequate selective agars which suppress the growth of other bacteria. CEFOMA agar represents an important improvement and suitable alternative to currently used selective agars. 2020-08-01T00:00:00Z http://edoc.rki.de/176904/13922 Resurgence of an international hepatitis A outbreak linked to imported frozen strawberries, Germany, 2018 to 2020 Ruscher, Claudia; Faber, Mirko; Werber, Dirk; Stark, Klaus; Bitzegeio, Julia; Michaelis, Kai; Sagebiel, Daniel; Wenzel, Jürgen J.; Enkelmann, Julia Following outbreaks linked to frozen strawberries in Sweden and Austria in 2018, 65 cases linked to the same hepatitis A virus strain were detected in Germany between October 2018 and January 2020, presenting in two waves. Two case–control studies and a comparison of cases’ consumption frequencies with purchase data from a large consumer panel provided strong evidence for frozen strawberry cake as the main vehicle of transmission. Of 46 cases interviewed, 27 reported consuming frozen strawberry cake and 25 of these identified cake(s) from brand A spontaneously or in product picture-assisted recall. Trace back investigations revealed that the Polish producer involved in the previous outbreaks in Sweden and Austria had received frozen strawberries from Egypt via a wholesaler that also delivered frozen strawberries to manufacturer of brand A. Phylogenetic analyses linked the outbreak strain to similar strains formerly isolated from sewage, stool and strawberries in Egypt. Complete trace back and timely recall of products with strong evidence of contamination is important to control an outbreak and prevent later resurgence, particularly for food items with a long shelf life. Continued molecular surveillance of hepatitis A is needed to identify outbreaks and monitor the success of food safety interventions. 2020-09-17T00:00:00Z