SummaryEscherichia coli K-12 harbours a chromosomal gene, clyA (sheA, hlyE ), that encodes a haemolytic 34 kDa protein. Recombinant E. coli overexpressing the cloned clyA gene accumulated this haemolysin in the periplasm and released only very small amounts of it into the external medium. The secretion of ClyA was confined to the log phase and paralleled by the partial release of several other periplasmic proteins. Sequencing of ClyA revealed the translational start point of the clyA gene and demonstrated that the clyA gene product is not N-terminally processed during transport. The transcription of clyA from its native promoter region was positively controlled by SlyA, a regulatory protein found in E. coli, Salmonella typhimurium and other Enterobacteriaceae. SlyA-controlled transcription started predominantly 72 bp upstream from clyA, as shown by primer extension. The corresponding putative promoter contains an unusual ¹10 sequence (TATGAAT) that is separated from a conventional ¹35 sequence by a GC-rich spacer. Site-directed deletion of the G in the ¹10 sequence abrogated the SlyA requirement for strong ClyA production, whereas a reduction in the GþC content of the spacer diminished the capability of SlyA to activate the clyA expression. Osmotic protection assays and lipid bilayer experiments suggested that ClyA forms stable, moderately cation-selective transmembrane pores that have a diameter of about 2.5-3 nm.

InlA-but not InlB-mediated internalization of Listeria monocytogenes by non-phagocytic mammalian cells needs the support of other internalinsneeds the supportive functions of the other internalins to trigger phagocytosis. None of these internalins seems to be required for cell-to-cell spread by L. monocytogenes, as shown by microinjection of Caco-2 cells with appropriate inl mutants.

Both human herpes viruses and Chlamydia are highly prevalent in the human population and are detected together in different human disorders. Here, we demonstrate that co-infection with human herpes virus 6 (HHV6) interferes with the developmental cycle of C. trachomatis and induces persistence. Induction of chlamydial persistence by HHV6 is independent of productive virus infection, but requires the interaction and uptake of the virus by the host cell. On the other hand, viral uptake is strongly promoted under co-infection conditions. Host cell glutathione reductase activity was suppressed by HHV6 causing NADPH accumulation, decreased formation of reduced glutathione and increased oxidative stress. Prevention of oxidative stress restored infectivity of Chlamydia after HHV6-induced persistence. We show that co-infection with Herpes simplex virus 1 or human Cytomegalovirus also induces chlamydial persistence by a similar mechanism suggesting that Chlamydia -human herpes virus co-infections are evolutionary shaped interactions with a thus far unrecognized broad significance.

Prostate cancer is the most common malignant tumor in men and is normally associated with increased serum levels of prostatespecific antigen (PSA). Therefore, PSA is one potential target for a prostate cancer vaccine. In this study we analyzed the functionality of new bacterial PSA vaccines, expressed and secreted via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of Type I secretion systems (T1SS) using an attenuated Salmonella enterica serovar Typhimurium aroA strain as carrier. The data demonstrate that a bacterial live vaccine encompassing T1SS in combination with cholera toxin subunit B can be successfully used for delivery of PSA to induce cytotoxic CD8 þ T-cell responses resulting in an efficient prevention of tumor growth in mice.