SummaryIntracellular bacterial pathogens generally express chaperones such as Hsp100s during multiplication in host cells, allowing them to survive potentially hostile conditions. Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. The ability of F. tularensis to multiply and survive in macrophages is considered essential for its virulence. Although previous mutant screens in Francisella have identified the Hsp100 chaperone ClpB as important for intracellular survival, no detailed study has been performed. We demonstrate here that ClpB of F. tularensis live vaccine strain (LVS) is important for resistance to cellular stress. Promoter analysis shows that the transcriptional start is preceded by a s 32-like promoter sequence and we demonstrate that expression of clpB is induced by heat shock. This indicates that expression of clpB is dependent on the heat-shock response mediated by s 32, the only alternative s-factor present in Francisella. Our studies demonstrate that ClpB contributes to intracellular multiplication in vitro, but is not essential. However, ClpB is absolutely required for Francisella to replicate in target organs and induce disease in mice. Proteomic analysis of membrane-enriched fractions shows that five proteins are recovered at lower levels in the mutant strain. The crucial role of ClpB for in vivo persistence of Francisella may be linked to its assumed function in reactivation of aggregated proteins under in vivo stress conditions.

The relationship between gpl40, the membrane C3d receptor (CR2) of human B lymphocytes, and the Epstein-Barr virus receptor (EBVR) was analyzed by using the polyclonal anti-gpl40, previously prepared by immunizing rabbits with highly purified gpl4O (isolated by some of us) from CR2/EBVR-positive Raji cells. Polyclonal anti-gp72, a C3-binding membrane component, not related to the EBVR but also expressed on the Raji cell surface, was used as a control. Binding of rabbit IgG and EBV on cells was assessed by using immunofluorescence techniques with analysis by flow cytofluorometry. A semiquantitative bioassay was also used to measure the EBV binding. Polyclonal monospecific anti-gpl4OIgG inhibits directly the binding of EBV to Raji cells at the same concentration that inhibits the binding of EC3d on cells, whereas a 35 times higher concentration of anti-gp72 IgG or preimmune serum IgG does not. Anti-gp140 IgG treatment also inhibits the induction of EBV-determined nuclear antigen in normal tonsil B lymphocytes or in EBV-negative Ramos cells, whereas high concentrations of anti-gp72 IgG or preimmune serum IgG have no effect. These data strongly suggest that gpl4O, the CR2 of human B lymphocytes, is also the EBVR.

Background: Francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yetunidentified receptor for F. tularensis internalization has been suggested.