No abstract

By creating mutations within the Shigella flexneri ipaB gene, we have demonstrated that the invasion of epithelial cells is a three‐step process encompassing adhesion on the cell surface, entry and lysis of the phagocytic vacuole allowing subsequent access to the cytoplasm. SC403, an insertion mutant which lacks expression of IpaB but still expresses downstream genes, has been particularly studied. It is non‐invasive, does not elicit actin polymerization, but binds to HeLa cells indicating that an adhesion step occurs immediately prior to the entry process. The consequence of the inactivation of ipaB on the intracellular behaviour of S.flexneri was investigated using the macrophage cell line J774. SC403 was unable to lyse the phagocytic vacuole; moreover, this strain did not display the contact mediated haemolytic activity characteristics of Shigella. In addition to being a major component of the invasion complex, IpaB acts as a membrane‐lysing toxin enabling escape to the cytoplasmic compartment.

Helicobacter pylori NCTC11637 expresses a lipopolysaccharide (LPS) that comprises an O antigen side‐chain with structural homology to the human blood group antigen Lewis X (Lex). The role of this molecule in adhesion of H. pylori to gastric epithelial cells was investigated. Mutants expressing truncated LPS structures were generated through insertional mutagenesis of rfbM and galE; genes encode GDP mannose pyrophosphorylase and galactose epimerase respectively. Compositional and structural analysis revealed that the galE mutant expressed a rough LPS that lacked an O antigen side‐chain. In contrast, an O antigen side‐chain was still synthesized by the rfbM mutant, but it lacked fucose and no longer reacted with anti‐Lex monoclonal antibodies (Mabs). The ability of these mutants to bind to paraffin‐embedded sections from the antrum region of a human stomach was assessed. Adhesion of the wild type was characterized by tropic binding to the apical surface of mucosal epithelial cells and cells lining gastric pits. In contrast, both the rfbM and galE mutants failed to demonstrate tropic binding and adhered to the tissue surface in a haphazard manner. These results indicate that LPS and, more specifically, LeX structures in the O antigen side‐chain play an important role in targeting H. pylori to specific cell lineages within the gastric mucosa. The role of LeX in this interaction was confirmed by the tropic binding of synthetic Lex, conjugated to latex beads, to gastric tissue. The observed pattern of adhesion was indistinguishable from that of wild‐type H. pylori.

Coaggregating strains of aquatic bacteria were identified by partial 16S rRNA gene sequencing. The coaggregation abilities of four strains of Blastomonas natatoria and one strain of Micrococcus luteus varied with culture age but were always maximum in the stationary phase of growth. Each member of a coaggregating pair carried either a heat-and protease-sensitive protein (lectin) adhesin or a saccharide receptor, as coaggregation was reversed by sugars.Coaggregation is the cell-to-cell recognition of genetically distinct partner cell types (13) and was first demonstrated for bacteria from dental plaque (6). Coaggregation between oral bacteria is mediated by lectin-saccharide interactions between cell surface molecules on the partner organisms (1,5,8,10). Coaggregation also occurs between members of the urogenital flora (16) and between strains of Lactobacillus from chicken crops (19). Most recently, coaggregation between aquatic biofilm-forming bacteria was described and found to be reversed by simple sugars (2), although involvement of surface proteins was not investigated. In addition, most of the aquatic strains were unidentified, and the coaggregation scores for some pairs of aquatic bacteria showed variation between different batch cultures. This study describes the identification of five aquatic coaggregating bacteria by using 16S rRNA gene sequencing and investigates the role of surface proteins in the coaggregation process. In addition, the relationship between coaggregation ability and phase of growth in batch culture is presented.Five coaggregating bacteria isolated from biofilm samples and previously designated as strains 2.1, 2.3, 2.6, 2.8, and 2.13 (2) were grown on R2A agar at 25°C (Difco) (15). Batch cultures were grown in 100 ml of liquid R2A broth, with shaking at 200 rpm at 25°C. All five strains were characterized by a combination of biochemical tests and light microscopy and by sequencing approximately 650 bases of the 16S rRNA gene. Bacterial genomic DNA from each strain was obtained by boiling a single bacterial colony, and the primers used for amplification and sequencing of 16S rRNA gene fragments were 8FPL (20) and 806R (22). The nucleotide sequence of each PCR product was compared to known sequences in the EMBL database, and the organism with the closest sequence similarity was identified.All five strains could be identified to the species level, as all had greater than 98.5% similarity with the closest sequence in the database. Four strains were identified as Blastomonas natatoria (strains 2.1, 2.3, 2.6, and 2.8) and one strain was identified as Micrococcus luteus (strain 2.13). B. natatoria strains were gram-negative, obligately aerobic, oxidase-and catalasepositive rods giving highly pigmented yellow colonies on R2A agar. All four strains divided asymmetrically to give daughter cells with a single polar flagellum. Comparison of the partial 16S rRNA gene sequence of each of the B. natatoria strains showed that they had 97.9 to 99.7% identity, indicating that they were very closely rela...