Kachiko Sekiya

Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure

Enteropathogenic Escherichia coli (EPEC) secretes several Esp proteins via the type III secretion system (secreton). EspA, EspB, and EspD are required for translocation of the effector proteins into host cells, in which EspB and EspD are thought to form a pore in the host membrane. Recent study has shown that EspA forms a filamentous structure that assembles as a physical bridge between bacteria and host cell surfaces, which then functions as a conduit for the translocation of bacterial effectors into host cells. To investigate the supermolecular structure of the type III secreton in EPEC, we partially purified it from the bacteria membrane and observed it via transmission electron microscopy. The EPEC type III secreton was composed of a basal body and a needle part and was similar to those of Salmonella and Shigella, except for a sheath-like structure at the tip of the needle. The length of sheath-like structures varied; it extended more than 600 nm and was 10 times longer than the Shigella needle part. The putative major needle component, EscF, was required for both secretion of Esp proteins and needle complex formation. Interestingly, elongation of the sheath-like structure was observed under constitutive expression of EspA but not of EscF. Furthermore, the transmission electron microscopy view with immunogold labeled anti-EspA antibodies clearly showed that EspA is a component of the sheath-like structure. This study revealed, to our knowledge for the first time, the supermolecular structure of the EPEC type III secreton and its direct association with the EspA-sheath-like structure.

Assembly of the Type III Secretion Apparatus of Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.

Effects of Bordetella pertussis components on IgE and IgG1 responses.

The effect of dermonecrotic toxin (DNT), fimbrial hemagglutinin (FHA), K‐agglutinogen, lipopolysaccharide (LPS), and pertussigen from Bordetella pertussis on the production of IgE and IgG1 antibodies to hen egg albumin (Ea) was investigated in C57BL/6 mice. The IgE antibody contents were determined by passive cutaneous anaphylaxis (PCA) in the skin of Lewis rats, while the IgG1 antibody contents were determined by PCA reactions on the skin of mice using sera that had been heated for 3 hr at 56 C to destroy the IgE antibodies. Among the B. pertussis components tested, pertussigen was the most effective adjuvant for increasing the IgE and IgG1 antibodies to Ea. LPS also moderately increased both types of antibodies, and FHA slightly increased the IgG1 titers. When LPS was given 5 days before Ea, it suppressed both IgE and IgG1 titers while FHA had only slight adjuvant action on both type of antibodies.

Azithromycin Inhibits the Formation of Flagellar Filaments without Suppressing Flagellin Synthesis in Salmonella enterica Serovar Typhimurium

ABSTRACT The present study shows that a sub-MIC of the macrolide antibiotic azithromycin (AZM) diminishes the virulence function of Salmonella enterica serovar Typhimurium. We first constructed an AZM-resistant strain (MS248) by introducing ermBC, an erythromycin ribosome methylase gene, into serovar Typhimurium. The MIC of AZM for MS248 exceeded 100 μg/ml. Second, we managed to determine the efficacy with which a sub-MIC of AZM reduced the virulence of MS248 in vitro. On the one hand, AZM (10 μg/ml) in the culture medium was unable to inhibit the total protein synthesis, growth rate, or survival within macrophages of MS248. On the other hand, AZM (10 μg/ml) reduced MS248s swarming and swimming motilities in addition to its invasive activity in Henle-407 cells. Electron micrographs revealed no flagellar filaments on the surface of MS248 after overnight growth in L broth supplemented with AZM (10 μg/ml). However, immunoblotting analysis showed that flagellin (FliC) was fully synthesized within the bacterial cells in the presence of AZM (10 μg/ml). In contrast, the same concentration of AZM reduced the export of FliC to the culture medium. These results indicate that a sub-MIC of AZM was able to affect the formation of flagellar filaments, specifically by reducing the amount of flagellin exported from bacterial cells, but it was not involved in suppressing the synthesis of flagellin. Unfortunately, AZM treatment was ineffective against murine salmonellosis caused by MS248.

Association of a Regulatory Gene, slyA with a Mouse Virulence of Salmonella Serovar Choleraesuis

The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated by using an slyA‐defective mutant. The defective mutant was constructed by the insertion of a kanamycin‐resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 105 higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild‐type strain of S. serovar Choleraesuis was 40‐fold higher than that of S. serovar Typhimurium. The time course of infection observed in the mice organs also proved the clear difference of the virulence between the parent and the mutant strains. These results suggested that the slyA gene product functions as a virulence‐associated regulator also in S. serovar Choleraesuis.

Electron Microscopic Observations on Tracheal Epithelia of Mice Infected with Bordetella bronchiseptica

To clarify the pathogenesis of Bordetella in vivo infection, the tracheal epithelia of mice were examined in detail by electron microscopy at various intervals after intranasal inoculation with graded doses of phase I Bordetella bronchiseptica. In mice infected with a lethal dose (6 to 7 × 107 CFU), a remarkable rupture of the cell membranes of cilia and microvilli of the middle trachea was found on day I postinfection. The rupture of the membrane was observed over the entire tracheal epithelia, on day 2 after infection. The affected cilia were constricted at the transitional region and were broken off. In the ciliated cells the adherence of organisms to ciliary apexes and colonization in the interciliary spaces were also remarkable. In both the ciliated and nonciliated epithelial cells, the cytoplasmic vacuolation and pyknosis or karyorrehexis were also notable. In mice infected with one‐tenth of the lethal dose, similar findings were seen, but appeared more slowly and the bacteria were not seen attaching to ciliary apexes. In mice receiving one‐hundredth of the lethal dose, only mild cilial abnormality such as aggregation of cilia, and slight cytoplasmic vacuolation were found 6 days postinfection. Based on these findings, a possible mechanism of the ciliary damages produced by B. bronchiseptica was postulated.

Electron Microscopic Observations on Ciliated Epithelium of Tracheal Organ Cultures Infected with Bordetella bronchiseptica

Using mouse tracheal organ cultures, the pathogenic effect of Bordetella bronchiseptica to epithelial cells was studied by electron microscopy. The ultrastructure of epithelial cells in uninfected tracheal rings was preserved well for longer than 3 days. In mouse tracheal rings infected with graded doses (3 × 105 to 107 CFU/ml) of phase I B. bronchiseptica, the colonization in the interciliary spaces of ciliated epithelial cells was observed after a 20‐hr infection period. The infected tracheal rings showed swelling of nonciliated cells as well as ciliated cells, rupture of cell membrane of cilia, swelling and disappearance of cilia, and atrophic cytomorphosis of epithelial cells. The severity of these changes occurred depending on the infection doses. These changes were essentially similar to those observed previously in the tracheal epithelia of the B. bronchiseptica‐infected mice. The usefulness of this in vitro model was suggested for studying the pathogenesis of Bordetella infection.