Julia Harwood

Characterization of monospecies biofilm formation by Helicobacter pylori.

As all bacteria studied to date, the gastric pathogen Helicobacter pylori has an alternate lifestyle as a biofilm. H. pylori forms biofilms on glass surfaces at the air-liquid interface in stationary or shaking batch cultures. By light microscopy, we have observed attachment of individual, spiral H. pylori to glass surfaces, followed by division to form microcolonies, merging of individual microcolonies, and growth in the third dimension. Scanning electron micrographs showed H. pylori arranged in a matrix on the glass with channels for nutrient flow, typical of other bacterial biofilms. To understand the importance of biofilms to the H. pylori life cycle, we tested the effect of mucin on biofilm formation. Our results showed that 10% mucin greatly increased the number of planktonic H. pylori while not affecting biofilm bacteria, resulting in a decline in percent adherence to the glass. This suggests that in the mucus-rich stomach, H. pylori planktonic growth is favored over biofilm formation. We also investigated the effect of specific mutations in several genes, including the quorum-sensing gene, luxS, and the cagE type IV secretion gene. Both of these mutants were found to form biofilms approximately twofold more efficiently than the wild type in both assays. These results indicate the relative importance of these genes to the production of biofilms by H. pylori and the selective enhancement of planktonic growth in the presence of gastric mucin.

Molecular analysis of the virulence locus of the Salmonella dublin plasmid pSDL2

The virulence properties of various non‐typhoid Salmonella serotypes depend on the presence of large plasmids 60–100 kb in size. We have shown previously that the virulence region on the 80 kb plasmid pSDL2 of Salmonella dublinLane maps within a 14kb SalI fragment. In this report we show that an 8.2kb region within this fragment is sufficient to express lethal disease in BALB/c mice. Sequence analysis of this segment revealed six sequential open reading frames designated vsdA–F, which encode putative proteins of 13–65kDa. Deletion analysis and location of Tn5‐oriT inserts which abolish virulence suggest that vsdA, vsdC, vsdD and vsdE are essential for virulence expression. Downstream of vsdF we discovered a locus involved in stable plasmid maintenance. Deletion of that region resulted in plasmid multimerization and instability.

Role of Gamma Interferon in Helicobacter pylori Induction of Inflammatory Mediators during Murine Infection

ABSTRACT Gamma interferon (IFN-γ) has been proposed to play an important role in Helicobacter-related gastritis. Using the IFN-γ gene knockout (IFN-γ−/−) mouse model and a murine gastric epithelial cell line, GSM06, we demonstrated that Helicobacter pylori maximally induced macrophage inflammatory protein-2 (MIP-2) and inducible nitric oxide synthase (iNOS) mRNA only in wild-type mice. MIP-2 and iNOS mRNA were also induced by H. pylori in GSM06 cells. Induction of cyclooxygenase 2 mRNA through IFN-γ was demonstrated in GSM06 cells. These data indicate that IFN-γ mediates the induction of MIP-2 and iNOS mRNA expression by H. pylori in mice.

Differential expression of HLA-DQA1 alleles associated with promoter polymorphism.

Abstract The promoter regions of DQA1 alleles are polymorphic, with some of the nucleotide sequence polymorphisms being located within X and Y motifs essential for the regulation of HLA class II gene transcription. In the present study, we demonstrate that polymorphisms in the promoter regions of three DQA1 alleles influence constitutive and inducible transcription of reporter gene constructs in epithelial, B-, and T-cell lines. Promoter strength in transient transfection assays generally followed a pattern of DQA1*03011>DQA1*0101>DQA1*05011, and was not affected by the endogenous HLA haplotype of the recipient cells. TNFα significantly activated the DQA1*05011 promoter, but had little effect on the DQA1*03011 promoter. Mutagenesis of the X and Y motifs of DQA1*03011 and DQA1*05011 demonstrated that nucleotide sequence polymorphisms in the Y element had the greatest effect on the promoter strength and differential TNFα inducibility. The finding that alleles of the DQA1 gene differ in promoter strength and responsiveness to TNFα suggests a mechanism for the association of certain alleles of this gene with susceptibility to autoimmune disease.

Structure and evolution of the promoter regions of the DQA genes

HLA-DQ antigens are unique among the class II antigens in that their α chains are highly polymorphic. In the present study, we characterized the general structure of the promoter regions of the DQA genes derived from different DR haplotypes and defined their nucleotide sequence polymorphisms. The promoter of each DQA1 allele contains three sequence motifs which are not present in non-DQA related class II genes: one identical to a tumor necrosis factor (TNFα) response element, one similar to an NFκB binding element, and one similar to a W motif. All DQA alleles lack TATA and CCAAT boxes in the proximal promoter region but carry other sequence elements characteristic of MHC class II genes, including S, X, X2, and Y boxes, and a pyrimidine-rich tract upstream of the X box. Nucleotide sequence polymorphisms among the various DQA1 alleles were noted within the promoter region, with some of the differences mapping within, or close to, regulatory elements that are important for the expression of MHC class II genes. All DQA1 alleles carry an unrearranged, full length, Alu-Sx related repeat immediately upstream of the proximal promoter region. This repeat was not present in the DQA2 (DXA) genes analyzed, confirming that DQ locus duplication probably occurred before integration of the Alu repeat into the primordial DQA1 locus, some 31–43 million years (myr) ago. The DQA2 promoter region is highly conserved between DR4 and DR3 haplotypes, with the degree of conservation exceeding that expected from the neutral mutation rate.