Acid-Induced Gene Expression in Helicobacter pylori : Study in Genomic Scale by Microarray

Abstract

To understand the RNA expression in response to acid stress of Helicobacter pylori in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used. Total RNAs of H. pylori under growth conditions of pH 7.2 and 5.5 were extracted, reverse transcribed into cDNA, and labeled with biotin. Each microarray membrane was hybridized with cDNA probe from the same strain under two different pH conditions and developed by a catalyzed reporter deposition method. Gene expression of all ORFs was measured by densitometry. Among the 1,534 ORFs, 53 ORFs were highly expressed (м30% of rRNA control in densitometry ratios). There were 445 ORFs which were stably expressed (<30% of rRNA in densitometry) under both pH conditions without significant variation. A total of 80 ORFs had significantly increased expression levels at low pH, while expressions of 4 ORFs were suppressed under acidic condition. The remaining 952 ORFs were not detectable under either pH condition. These data were highly reproducible and comparable to those obtained by the RNA slot blot method. Our results suggest that microarray can be used in monitoring prokaryotic gene expression in genomic scale.Helicobacter pylori is the causative agent of chronic superficial gastritis in humans, and the presence of this organism increases risk of development of peptic ulcer disease and adenocarcinoma and mucosa-associated lymphoid tissue lymphoma of the distal stomach (2,3,10,19,21). The mechanism of pathogenesis remains largely unknown. The ability to survive under acidic stomach conditions might be one of the virulence mechanisms. Monitoring the response of H. pylori genes during acid stress may be helpful to understand the pathogenesis.Expression of many bacterial genes is induced in response to environmental stimuli (17). For technical reasons, only a limited subset of genes could be simultaneously analyzed until recently. Differential display of subsets of mRNA on a sequencing gel allows a broad search for expression differences (11, 15), but the method has been difficult to standardize. An rRNA subtraction approach has been used to identify differentially expressed genes in Mycobacterium avium (23); however, removal of rRNA by subtraction caused loss of mRNA. Chuang et al. (6) have analyzed differentially expressed genes in Escherichia coli by a method based on hybridization to spot overlapping clones. This method required subcloning and subsequent sequencing for the identification of relevant genes.Recently, a system for monitoring of a large number of gene expressions has been developed in eukaryotes using DNA microarray (5, 23, 24) or oligonucleotide microarray (16,22,29). Labeled cDNA, or in vitro-transcribed mRNA, was hybridized to the high-density probe microarray. Microarrays are able to analyze the expression of hundreds of genes in a single hybridization experiment. DNA array has also been recently adopted for monitoring gene expressions in bacteria. Unfortunately, since bacterial mRNA could not be separated from r...

TheKlebsiella pneumoniaeYfgL (BamB) lipoprotein contributes to outer membrane protein biogenesis, type-1 fimbriae expression, anti-phagocytosis, andin vivovirulence

“…Total RNA (40 mg) labeling, microarray hybridization, colorimetric detection, and densitometry were performed and analyzed as described previously. 11,46 Expression level of 23S rRNA on each membrane was used as an internal standard for normalization. Processed microarray data files have been deposited in the National Center for Biotechnology Information under GEO accession number GSE69327.…”

Identification of Campylobacter jejuni Genes Contributing to Acid Adaptation by Transcriptional Profiling and Genome-Wide Mutagenesis

“…A number of studies have sought to examine the transcriptional profile of bacteria grown under acidic and/or basic conditions (2,16,18,37,53,84). The small degree of overlap between these studies affirms the importance of using multiple approaches to characterize these responses as thoroughly as possible.…”

“…These studies revealed that low pH changes the expression of genes involved in energy generation and metabolism, reflecting a switch to metabolic processes that minimize acid production (18,37,53). In addition, pH affects the expression of genes involved in motility and chemotaxis (2,16,37,53,84) as well as genes that encode products that take up and/or export H ϩ (e.g., ATPase and electron transport chain components) (2,16,18,37,53). These studies also have shown that bacterial acid stress responses show some overlap with oxidative stress (16,53,67) and heat shock (2,18,37,67) responses.…”

The impact of parietal cells onHelicobacter pyloritropism and host pathology: An analysis using gnotobiotic normal and transgenic mice

“…8 and 9). Whole genome genotyping of Hp1 indicated that it contains the 93 of 95 H. pylori genes reported to be regulated by acid, suggesting that it is equipped to respond to the presence or absence of acid in its microenvironments (9)(10)(11). Our results demonstrate that this bacterium seeks out niches with sparse parietal cell populations and underscore the point that understanding the forces that shape the destiny of an infection requires a commitment to defining host and microbial responses as a function of the various gastric milieus that coexist within a single host.…”