Six patients from northern Minnesota and Wisconsin with a febrile illness accompanied by granulocytic cytoplasmic morulae suggestive of ehrlichial infection were identified. Two patients died, and splenic granulocytes of one patient contained cytoplasmic vacuoles with organisms ultrastructurally characteristic of ehrlichiae. From one patient, a 1.5-kb DNA product was amplified by PCR with universal eubacterial primers of 16S rDNA. Analysis of the nucleotide sequence of the amplified product revealed 99.9 and 99.8% similarities with E. phagocytophila and E. equi, respectively, neither of which has previously been known to infect humans. From the variable regions of the determined sequence, a forward primer specific for three organisms (human granulocytic ehrlichia, E. phagocytophila, and E. equi) and a reverse primer for these ehrlichiae and E. platys were designed. By nested PCR with amplification by the universal primers and then reamplification with the specific primers described above, the expected 919-bp product was generated from the blood of the index patient and three additional patients. Blood from these four patients and two more patients with granulocytic morulae contained DNA which was amplified by nested PCR involving a combination of a universal primer and the human granulocytic ehrlichia-E. phagocytophila-E. equi-E. platys group-specific primer. This apparently vector-borne human granulocytic ehrlichia has only 92.5% 16S rDNA homology with E. chaffeensis. Nested PCR with group-specific primers did not amplify E. chaffeensis DNA, and E. chalfeensis-specific primers did not amplify DNAs of the human granulocytic ehrlichia. Thus, six patients were shown to be infected by an Ehrlichia species never previously reported to infect humans.

Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB, an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system.The staphylococcal agr system is the best-understood cyclic peptide-mediated quorum-sensing system among gram-positive bacteria. In the agr system, an autoinducing peptide (AIP) is generated from its propeptide, AgrD, by its processing enzyme, AgrB, and is then sensed by a two-component regulatory system comprising a membrane histidine kinase, AgrC, and a response regulator, AgrA (10,14,18,22,23). The cognate gene cluster consisting of these four components has been identified in the genome databases of Listeria, Clostridium, Lactobacillus, and Bacillus spp., suggesting that cyclic peptide-mediated quorum sensing is widespread among gram-positive bacteria (14,18,21).The fsr system in Enterococcus faecalis controls the expression of pathogenicity-related extracellular proteases, gelatinase, and a serine protease via a quorum-sensing mechanism (11,16,17), and recent studies have suggested that it also regulates biofilm formation (7, 15) and other genes important for virulence (2). The fsr quorum-sensing system also mediates a cyclic peptide named gelatinase biosynthesis-activating pheromone (GBAP), although the ring is formed by lactone instead of the thiolactone found in other gram-positive AIPs (10, 11, 21). However, a small open reading frame corresponding to staphylococcal agrD has not been identified in the nucleotide sequence of the fsr gene cluster (16). The 11-amino-acid sequence of GBAP was identified in the C-terminal part of the 242-residue deduced fsrB product (11). As shown in Fig. 1, the N-terminal part of FsrB (189 residues) shows sequence similarity to staphylococcal AgrB, and the remaining C-terminal part of FsrB (53 residues) appears to be a GBAP propeptide like AgrD. Based on these observations, we have suspected that FsrB autoprocesses its C-terminal part to generate GBAP, which is a unique biosynthetic mechanism compared with those of other cyclic AIPs.In the present study, we demonstrate the existence of a small open reading frame, fsrD, corresponding to agrD, which is carried in frame with fsrB but is translated independently of fsrB. Here we propose a revised fsr system model sharing a common mechanism of AIP biosynthesis with the thiolactonemediated quorum-sensing systems of staphylococci and probably other gram-positive bacteria.Construction of a nisin-inducible expression system for wild-type and mutant fsrBD stra...

Summary. The ultrastructure of Ehrlichia chafeensis (Arkansas strain) was studied in nonirradiated and irradiated monolayers of mouse embryo, Vero, BGM and L929 cells, and in non-irradiated DH82 cells. Within the intracellular parasitophorous vacuoles (morulae), two types of ehrlichial cells were found regularly-those with uniformly dispersed nucleoid filaments and ribosomes (reticulate cells) and smaller ones with centrally condensed nucleoid filaments and ribosomes (dense-cored cells), which represent the normal life cycle of ehrlichiae. In addition, large reticulate cells were observed, forming long projections of the cell wall, protrusions 'of cytoplasmic membrane into the periplasmic space, or budding of protoplast fragments (minute forms) into the periplasmic space. Ehrlichiae with abnormalities of protoplast fission were found, apparently leading to formation of giant, multilobular or elongated rod-like ehrlichiae. Morulae were usually surrounded by cisterns of granular endoplasmic reticulum and mitochondria and often contained vesicles, long tubules 25 nm in diameter, probably originating from the ehrlichial cell wall, and fibrillar ehrlichial antigen apparently shed from the surface of the cell wall. Some cells contained, in addition to normal morulae, a whole morula that had become dense and contained degenerating ehrlichiae. These results indicate that as well as normal growth and reproduction, ehrlichiae exhibit pathological events : they can be remarkably damaged inside the host cell vacuoles, presumably phagolysosomes, or enter a process morphologically similar to bacterial L-transformation.

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