Borrelia burgdorferi is a flat-wave, motile spirochete that causes Lyme disease. Motility is provided by periplasmic flagella (PFs) located between the cell cylinder and an outer membrane sheath. The structure of these PFs, which are composed of a basal body, a hook, and a filament, is similar to the structure of flagella of other bacteria. To determine if hook formation influences flagellin gene transcription in B. burgdorferi, we inactivated the hook structural gene flgE by targeted mutagenesis. In many bacteria, completion of the hook structure serves as a checkpoint for transcriptional control of flagellum synthesis and other chemotaxis and motility genes. Specifically, the hook allows secretion of the anti-sigma factor FlgM and concomitant late gene transcription promoted by 28 . However, the control of B. burgdorferi PF synthesis differs from the control of flagellum synthesis in other bacteria; the gene encoding 28 is not present in the genome of B. burgdorferi, nor are any 28 promoter recognition sequences associated with the motility genes. We found that B. burgdorferi flgE mutants lacked PFs, were rod shaped, and were nonmotile, which substantiates previous evidence that PFs are involved in both cell morphology and motility. Although most motility and chemotaxis gene products accumulated at wild-type levels in the absence of FlgE, mutant cells had markedly decreased levels of the flagellar filament proteins FlaA and FlaB. Further analyses showed that the reduction in the levels of flagellin proteins in the spirochetes lacking FlgE was mediated at the posttranscriptional level. Taken together, our results indicate that in B. burgdorferi, the completion of the hook does not serve as a checkpoint for transcriptional regulation of flagellum synthesis. In addition, we also present evidence that the hook protein in B. burgdorferi forms a high-molecular-weight complex and that formation of this complex occurs in the periplasmic space.Borrelia burgdorferi, the causative agent of Lyme disease, is a spirochete with a characteristic flat-wave morphology (24, 25; for a review, see reference 12). As a result of their unusual morphology, B. burgdorferi and other spirochete species have a highly specialized ability that allows them to traverse viscous gellike media (8). This unique swimming ability enables these bacteria to penetrate into specific host connective tissues and ecological niches (43). The importance of motility as a virulence factor has been implicated in several spirochete species, including Treponema denticola (42), Brachyspira hyodysenteriae (54), Borrelia garinii (60), and B. burgdorferi (10, 56).Motility in B. burgdorferi is provided by bundles of between 7 and 11 periplasmic flagella (PFs) that are subterminally attached near the cell ends. These PFs extend inward along the cell cylinder beneath an outer membrane sheath (6,24,28,46). B. burgdorferi PFs have a structure similar to that of flagella of other bacteria; a PF is composed of a basal body, a hook, and a filament containing a single major ...

Chlamydia spp. express a functional type III secretion system (T3SS) necessary for pathogenesis and intracellular growth. However, certain essential components of the secretion apparatus have diverged to such a degree as to preclude their identification by standard homology searches of primary protein sequences. One example is the needle subunit protein. Electron micrographs indicate that chlamydiae possess needle filaments, and yet database searches fail to identify a SctF homologue. We used a bioinformatics approach to identify a likely needle subunit protein for Chlamydia. Experimental evidence indicates that this protein, designated CdsF, has properties consistent with it being the major needle subunit protein. CdsF is concentrated in the outer membrane of elementary bodies and is surface exposed as a component of an extracellular needle-like projection. During infection CdsF is detectible by indirect immunofluorescence in the inclusion membrane with a punctuate distribution adjacent to membrane-associated reticulate bodies. Biochemical cross-linking studies revealed that, like other SctF proteins, CdsF is able to polymerize into multisubunit complexes. Furthermore, we identified two chaperones for CdsF, termed CdsE and CdsG, which have many characteristics of the Pseudomonas spp. needle chaperones PscE and PscG, respectively. In aggregate, our data are consistent with CdsF representing at least one component of the extended Chlamydia T3SS injectisome. The identification of this secretion system component is essential for studies involving ectopic reconstitution of the Chlamydia T3SS. Moreover, we anticipate that CdsF could serve as an efficacious target for anti-Chlamydia neutralizing antibodies.

The Lyme disease bacterium Borrelia burgdorferi is a motile spirochete with a flat-wave morphology. The periplasmic flagella, which are situated between the outer membrane sheath and cell cylinder, are essential for both the cell's wavy shape and motility. Here we focus on the structure and regulation of its periplasmic flagella. Previous studies have suggested that the periplasmic flagella consist of a polymer of the major filament protein FlaB and a minor protein, FlaA. We used immunoprecipitation methodology to present further evidence that FlaA is indeed a flagellar protein. In addition, in contrast to FlaA of the spirochete Brachyspira hyodysenteriae, B. burgdorferi FlaA did not impact the overall helical shape of the periplasmic flagella. We have previously shown that B. burgdorferi lacks the sigma factor-dependent cascade control of motility gene transcription found in other bacteria. To begin to understand motility gene regulation in B. burgdorferi, we examined the effects of an insertion mutation in flaB on the amounts of proteins encoded by motility genes. Of several motility gene-encoded proteins examined, only the amount of FlaA was decreased in the flaB mutant; it was 13% compared to the wild-type amount. Real-time reverse transcriptase PCR analysis indicated that this inhibition was not the result of a decrease in flaA mRNA. In addition, protein stability analysis suggested that FlaA was turned over in the flaB mutant. Our results indicate that the lack of FlaB negatively influences the amount of FlaA found in the cell and that this effect is at the level of either translational control or protein turnover.Borrelia burgdorferi is a motile spirochete that is the causative agent of Lyme disease. The organelles for motility, the periplasmic flagella (PFs), reside between the outer membrane sheath and protoplasmic cell cylinder. Approximately 7 to 11 PFs are subterminally attached at each end of the cell, and these filaments overlap in the center of the cell (4, 27, 31). Motility is likely to be an important virulence factor for these spirochetes, as B. burgdorferi penetrates into tissues where other organisms fail to invade (11,34). Moreover, motility has been postulated to be essential for the life cycle of these spirochetes in both the tick and the mammalian hosts (45, 55).We are beginning to understand the dynamics of B. burgdorferi motility (see references 11, 42, and 45 for recent reviews). Recent results indicate that the PFs have both skeletal and motility functions (50, 61). Cells of mutants that are targeted in flaB, which encodes the major PF protein, are nonmotile and are rod shaped instead of being a flat wave as are wild-type cells (50, 61). During translational motility, backward moving waves are generated that are responsible for cell displacement (28). These waves result from the rotation of the relatively rigid left-handed helically shaped PFs closely juxtaposed to and interacting with the flexible protoplasmic cell cylinder: the PFs act as a gear generating waves along the cell cylin...

Chlamydia trachomatis is a Gram-negative obligate intracellular bacterium that is the causative agent of common sexually transmitted diseases and the leading cause of preventable blindness worldwide. It has been observed that YtgA (CT067) is very immunogenic in patients with chlamydial genital infections. Homology analyses suggested that YtgA is a soluble periplasmic protein and a component of an ATP-binding cassette (ABC) transport system for metals such as iron. Since little is known about iron transport in C. trachomatis, biochemical assays were used to determine the potential role of YtgA in iron acquisition. 59Fe binding and competition studies revealed that YtgA preferentially binds iron over nickel, zinc or manganese. Western blot and densitometry techniques showed that YtgA concentrations specifically increased 3–5-fold in C. trachomatis, when cultured under iron-starvation conditions rather than under general stress conditions, such as exposure to penicillin. Finally, immuno-transmission electron microscopy provided evidence that YtgA is more concentrated in C. trachomatis during iron restriction, supporting a possible role for YtgA as a component of an ABC transporter.

The oestrogen receptor (ER) α-β+ HEC-1B and the ERα+β+ Ishikawa (IK) cell lines were investigated to dissect the effects of oestrogen exposure on several parameters of Chlamydia trachomatis infection. Antibody blockage of ERα or ERβ alone or simultaneously significantly decreased C. trachomatis infectivity (45-68%). Addition of the ERβ antagonist, tamoxifen, to IK or HEC-1B prior to or after chlamydial infection caused a 30-90% decrease in infectivity, the latter due to disrupted eukaryotic organelles. In vivo, endometrial glandular epithelial cells are stimulated by hormonally influenced stromal signals. Accordingly, chlamydial infectivity was significantly increased by 27% and 21% in IK and HEC-1B cells co-cultured with SHT-290 stromal cells exposed to oestrogen. Endometrial stromal cell/epithelial cell co-culture revealed indirect effects of oestrogen on phosphorylation of extracellular signal-regulated kinase and calcium-dependant phospholipase A2 and significantly increased production of interleukin (IL)-8 and IL-6 in both uninfected and chlamydiae-infected epithelial cells. These results indicate that oestrogen and its receptors play multiple roles in chlamydial infection: (i) membrane oestrogen receptors (mERs) aid in chlamydial entry into host cells, and (ii) mER signalling may contribute to inclusion development during infection. Additionally, enhancement of chlamydial infection is affected by hormonally influenced stromal signals in conjunction with direct oestrogen stimulation of the human epithelia.