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Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, [LpxE.sub.HP] (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by [EptA.sub.HP] (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated [LpxE.sub.Hp]-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into [lpxE.sub.HP] and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of [LpxE.sub.HP] activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of [LpXE.sub.HP]. Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 [micro]g/ml). However, disruption of [lpXE.sub.HP] in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 [micro]g/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.

The Chlamydia trachomatis divalent cation-dependent regulator (DorA), encoded by open reading frame CT296, is a distant relative of the ferric uptake regulator (Fur) family of iron-responsive regulators. Chlamydial DcrA specifically binds to a consensus Escherichia coli Fur box and is able to complement an E. coli Fur mutant. In this report, the E. coli Fur titration assay (FURTA) was used to locate chlamydial genomic sequences that are recognized by E. coli Fur. The predictive regulatory regions of 28 C. trachomatis open reading frames contained sequences functionally recognized by E. coli Fur; targets include components of the type III secretion pathway, elements involved in envelope and cell wall biogenesis, predicted transport proteins, oxidative defense enzymes, and components of metabolic pathways. Selected FURTA-positive sequences were subsequently examined for recognition by C. trachomatis DcrA using an electrophoretic mobility shift assay. The resultant data show that C. trachomatis DcrA binds to native chlamydial genomic sequences and, overall, substantiate a functional relationship between chlamydial DorA and the Fur family of regulators.

Chlamydia trachomatis, the most common bacterial sexually transmitted disease agent worldwide, enters a viable, non-dividing and non-infectious state (historically termed persistence and more recently referred to as the chlamydial stress response) when exposed to penicillin G in culture. Notably, penicillin G-exposed chlamydiae can reenter the normal developmental cycle upon drug removal and are resistant to azithromycin-mediated killing. Because penicillin G is less frequently prescribed than other β-lactams, the clinical relevance of penicillin G-induced chlamydial persistence/stress has been questioned. The goal of this study was to determine whether more commonly used penicillins also induce C. trachomatis serovar E persistence/stress. All penicillins tested, as well as clavulanic acid, induced formation of aberrant, enlarged reticulate bodies (RB) (called aberrant bodies or AB) characteristic of persistent/stressed chlamydiae. Exposure to the penicillins and clavulanic acid also reduced chlamydial infectivity by >95%. None of the drugs tested significantly reduced chlamydial unprocessed 16S rRNA or genomic DNA accumulation, indicating that the organisms were viable, though non-infectious. Finally, recovery assays demonstrated that chlamydiae rendered essentially non-infectious by exposure to ampicillin, amoxicillin, carbenicillin, piperacillin, penicillin V, and clavulanic acid recovered infectivity after antibiotic removal. These data definitively demonstrate that several commonly used penicillins induce C. trachomatis persistence/stress at clinically relevant concentrations. [ABSTRACT FROM AUTHOR]

Mast cells are bone marrow-derived cells that are widely distributed in the tissue. They are found predominantly in the subepithelial tissue near blood vessels and nerves and usually are sprinkled diffusely without forming clusters. In tissue sections stained with hematoxylin and eosin, normal mast cells usually display a round-to-oval nucleus with clumped chromatin and indistinct or no nucleoli. They have moderately abundant cytoplasm and are oval, spindle, or polygonal in shape. The cytoplasm is amphophilic, and sometimes small slightly eosinophilic granules may be visible. Hematoxylin and eosin staining is not a specific or reliable method for detecting mast cells in tissue sections because of variable cellular morphology. For confirmation of mast cells, special stains, such as mast cell tryptase or CD117, are required.