Galectin-3 binds to Helicobacter pylori O-antigen: it is upregulated and rapidly secreted by gastric epithelial cells in response to H. pylori adhesion.

Helicobacter pylori causes gastritis and some infections result in peptic ulceration, gastric adenocarcinoma or gastric lymphoma. A critical step in the pathogenesis of these diseases is the ability of H. pylori to adhere to gastric epithelial cells. A role for the lipopolysaccharide O-antigen side-chain in this process has previously been identified. In this study, evidence is presented that the receptor recognized by the O-antigen side-chain is galectin-3, a beta-galactoside-binding lectin. A variety of functions have been ascribed to galectin-3 including modulation of extracellular adhesion and chemotaxis of monocytes and neutrophils. Expression of galectin-3 is upregulated by gastric epithelial cells following adhesion of H. pylori, suggesting that in addition to colonization this protein also plays a role in the host response to infection. Upregulation of galectin-3 is inhibited by treating gastric epithelial cells with the mitogen-activated protein kinase (MAPK) inhibitors U0126 or PD098059 and does not occur in cells infected with either H. pylori cagE or cagA isogenic mutants. This implies that H. pylori-mediated expression of galectin-3 is dependent on delivery of CagA into the host cell cytosol and the subsequent stimulation of MAPK signalling. A further consequence of H. pylori adhesion is that it elicits a rapid release of galectin-3 from infected cells. A role for this phenomenon in initiating the trafficking of phagocytic cells to the site of infection is discussed.

Analysis of the role of HP0208, a phase-variable open reading frame, and its homologues HP1416 and HP0159 in the biosynthesis of Helicobacter pylori lipopolysaccharide.

The roles of the three ORFs HP0208, HP0159 and HP1416 in the biosynthesis of Helicobacter pylori 26695 LPS were investigated in this study. These ORFs represent a paralogous family of genes with homology to the Salmonella enterica serovar Typhimurium (hereafter referred to as S. typhimurium) waaJ gene, which encodes an alpha-1,2-glycosyltransferase required for core LPS biosynthesis. HP0208 contains multiple tandem repeats of the dimer 5'GA at its 5' end and its expression is predicted to be subject to phase variation. The number of 5'GA repeats present in this ORF was found to be non-permissive for the expression of HP0208 in the majority of H. pylori strains examined. To determine a role for this ORF in LPS biosynthesis a non-phase-variable, constitutively expressed variant of HP0208 was constructed and introduced into the genome of H. pylori 26695. Analysis of the LPS profile of this strain by Tricine-SDS-PAGE and immunoblotting with anti-Lewis Y antigen (Le(y)) mAbs confirmed a role for HP0208 in the biosynthesis of core LPS. A role for HP0159 and HP1416 in the biosynthesis of core LPS was also established. Although homologous to waaJ, H. pylori HP0208, HP0159 and HP1416 failed to complement an S. typhimurium waaJ mutant, suggesting that these ORFs encode functionally different enzymes.

Bacterial coaggregation: an integral process in the development of multi-species biofilms.

Coaggregation is a process by which genetically distinct bacteria become attached to one another via specific molecules. Cumulative evidence suggests that such adhesion influences the development of complex multi-species biofilms. Once thought to occur exclusively between dental plaque bacteria, there are increasing reports of coaggregation between bacteria from other biofilm communities in several diverse habitats. A general role for coaggregation in the formation of multi-species biofilms is discussed.

The role of licA phase variation in the pathogenesis of invasive disease by Haemophilus influenzae type b.

LicA encodes the enzyme phosphorylcholine kinase which catalyses the incorporation of phosphorylcholine (ChoP) into H. influenzae LPS. Expression of this gene is subject to phase variation, resulting in the spontaneous loss, or gain of phosphorylcholine (ChoP)-decorated LPS structures. To investigate the role of this phenomenon in the pathogenesis of invasive disease an H. influenzae mutant was constructed which lacked the ability to phase vary licA. This was achieved by introducing an in-frame deletion of the 5'-CAAT-3' repeats into licA using polymerase chain reaction. The resultant mutant, licADelta5'-CAAT-3', was unable to switch off expression of licA and constitutively expressed ChoP-decorated LPS structures, as judged by colony immunoblotting with Mabs 12D9 and HAS. This resulted in increased synthesis of high molecular mass LPS structures and the absence of non-ChoP-decorated LPS species as determined by T-SDS-PAGE analysis. Inability to switch off the expression of licA reduced the virulence of H. influenzae in an infant rat model of invasive disease and resulted in increased sensitivity to the bactericidal activity of serum in the presence of CRP. The ability to switch off the expression of licA through phase variation is therefore concluded to enhance the systemic survival of H. influenzae.

Phylogenetic relationships and coaggregation ability of freshwater biofilm bacteria.

Nineteen numerically dominant heterotrophic bacteria from a freshwater biofilm were identified by 16S ribosomal DNA gene sequencing, and their coaggregation partnerships were determined. Phylogenetic trees showed that both distantly related and closely related strains coaggregated at intergeneric, intrageneric, and intraspecies levels. One strain, Blastomonas natatoria 2.1, coaggregated with all 18 other strains and may function as a bridging organism in biofilm development.

The extracytoplasmic sigma factor, final sigma(E), is required for intracellular survival of nontypeable Haemophilus influenzae in J774 macrophages.

Nontypeable Haemophilus influenzae (NTHi) causes a wide variety of respiratory tract infections in humans. It is capable of invading and surviving in epithelial cells and has also been shown to persist in macrophage-like cell line J774A.1. To determine the molecular mechanisms which enable NTHi to survive in an intracellular environment, differential display reverse transcriptase PCR was used to identify genes which were either induced or upregulated by NTHi residing in macrophages. Using this approach, we identified one transcript which was consistently amplified from intracellular NTHi cDNA. Nucleotide sequence analysis of this product revealed that it spanned the 3' and 5' ends of rpoE and rseB, respectively, which form part of the extracytoplasmic stress operon that encodes and regulates expression of alternate sigma factor sigma E (final sigma(E)). To confirm that expression of rpoE was upregulated following uptake of NTHi by macrophages, an rpoE-lacZ transcriptional fusion was constructed, and expression of beta-galactosidase activity in broth-grown NTHi was compared with expression of beta-galactosidase activity in intracellular NTHi. The level of beta-galactosidase activity in NTHi 4 h after phagocytosis by macrophages was found to be 100-fold higher than that of broth-grown organisms, suggesting that genes of the final sigma(E) regulon may be important for persistence of NTHi in mammalian cells. The hypothesis that final sigma(E) plays a role in the intracellular survival of NTHi was subsequently confirmed by the decreased ability of an rpoE insertion mutant to survive in macrophages.

Phase variation of lic1A, lic2A and lic3A in colonization of the nasopharynx, bloodstream and cerebrospinal fluid by Haemophilus influenzae type b.

The role of phase variation of lic1A, lic2A and lic3A in the ability of Haemophilus influenzae type b to colonize the nasopharynx, bloodstream and cerebrospinal fluid (CSF) of infants was investigated. This was achieved by using PCR to determine the number of 5'-CAAT-3' repeats present in each gene, which is indicative of whether each ORF can be expressed. Multiple PCR products of different intensities were amplified from all three genes at each site sampled. This indicated that the nasopharynx, bloodstream and CSF were colonized by a heterogeneous population of organisms, expressing different combinations of lic genes. At each site however, a predominant PCR product was amplified from each gene, indicating that organisms with this genotype were the most abundant. The number of 5'-CAAT-3' repeats in this predominant product varied depending upon whether organisms were isolated from the nasopharynx, bloodstream or CSF. These observations suggest that the expression of different combinations of lic genes may influence the efficiency with which H. influenzae colonizes the nasopharynx, bloodstream and CSF of infant rats.

An in silico evaluation of Tn916 as a tool for generalized mutagenesis in Haemophilus influenzae Rd.

The transposon Tn916 was evaluated as a tool for generalized mutagenesis of the genome of Haemophilus influenzae. This was achieved in silico by searching the genome sequence of H. influenzae Rd for the published Tn916 target site consensus sequence 5' TT/ATTTT(N)6AAAAAA/TA. This search identified 16 putative target sites. In subsequent experiments, integration of Tn916 did not occur at any of these sites. Using the nucleotide sequences of these observed integration sites, a new consensus sequence, 5' TTTTT(N)xAAAAA (4 < or = x < or = 7), was derived. This sequence reflects the curve-twist-curve DNA topology which is a feature common to all Tn916 integration sites. A search of the H. influenzae Rd genome using the new consensus sequence identified 167 potential target sites, representing approximately 1% of the total genome. Only 80 of these sites were located within ORFs. The presence of such a limited number of target sites places severe constraints on the use of Tn916 as a tool for generalized mutagenesis of the genome of H. influenzae.