Characterization of a \u3ci\u3eHelicobacter pylori\u3c/i\u3e Small RNA by RT-PCR

Helicobacter pylori, a bacterial gastric pathogen infecting approximately 50% of the human population, produces gastritis, ulcers, and gastric cancers. Colonizing the inhospitable and fluctuating environment in the stomach requires tight genetic control. However, H. pylori lacks many genetic regulatory elements present in other bacteria to control gene expression. Instead, over 200 small RNAs (sRNAs; noncoding RNAs shorter than 300 nucleotides) have been found in this bacterium, but few have been fully characterized. Of those, many are antisense to virulence genes. Characterizing these sRNAs is important in understanding the mechanisms of molecular genetics and potentially supporting medical management of this pathogen. In the current study, a previously identified but as-yet uncharacterized sRNA was analyzed through reverse-transcription polymerase chain reaction (RT-PCR) utilizing primer walking, a technique employing custom oligonucleotides to experimentally determine the beginning and end regions of a transcript. RT-PCR results are unclear, representing longer-than-expected transcripts of variable lengths. This sRNA occurs downstream of another predicted sRNA. The results may represent two overlapping (possibly rather large) transcripts. Future work includes RT-PCR of two other antisense sRNAs and Northern blotting to analyze the size and gene boundaries of these sRNAs. Total results will explicate these three sRNAs and provide a foundation for further inquiry into the regulatory role these small but impactful molecules play in H. pylori

Overexpressing Two Helicobacter pylori Small RNAs from a Bacterial Pathogenicity-related Chromosomal Region to Investigate Their Regulation of Virulence Genes

The bacterial pathogen Helicobacter pylori infects the stomachs of approximately 50% of humanity, causing symptomatic disease (e.g., stomach ulcers, gastric cancer, and MALT lymphoma) in 10-15% of the infected. Colonizing the acidic, inhospitable stomach requires H. pylori to tightly regulate gene expression despite lacking many common bacterial genetic regulatory elements. The pathogen may compensate by using abundant non-protein-coding small RNAs (sRNAs) to regulate gene expression, including of infection-intensifying virulence genes. Additionally, severe disease and cancer correlate with infection by H. pylori strains that contain a nonessential chromosomal region, the cytotoxin-associated gene pathogenicity island (cagPAI). This encodes powerful virulence factors that include a mechanism for injecting a cancer-promoting protein (CagA) into host cells. Despite identification of multiple cagPAI sRNAs, regulatory effects of only one have been characterized. To investigate potential sRNA-mediated regulation of RNA gene expression in the cagPAI and other virulence genes, we are developing strains overexpressing (abundantly producing) two promising cagPAI sRNAs. We cloned experimental plasmids (circular DNA molecules) to contain an overexpression promoter element and the relevant sRNA. Subsequently, we will introduce these plasmids into H. pylori to generate two sRNA-overexpressing strains. Finally, we will compare total RNA harvested from these two strains and the unmodified strain using RNA sequencing and reverse transcription quantitative polymerase chain reaction (RT-qPCR). The resulting identification/quantification of any significant regulation by these two H. pylori cagPAI sRNAs could illuminate aspects of sRNA regulation of the cancer-associated cagPAI region and other virulence genes

Overexpressing two \u3ci\u3eHelicobacter pylori\u3c/i\u3e small RNAs from a bacterial pathogenicity-related chromosomal region to investigate their regulation of virulence genes

The bacterial pathogen Helicobacter pylori infects the stomachs of approximately 50% of humanity, causing symptomatic disease (e.g., stomach ulcers, gastric cancer, and MALT lymphoma) in 10-15% of the infected. Colonizing the acidic, inhospitable stomach requires H. pylori to tightly regulate gene expression despite lacking many common bacterial genetic regulatory elements. The pathogen may compensate by using abundant non-protein-coding small RNAs (sRNAs) to regulate gene expression, including infection-intensifying virulence genes. Additionally, severe disease and cancer correlate with infection by H. pylori strains that contain a nonessential chromosomal region, the cytotoxin-associated gene pathogenicity island (cagPAI). This encodes powerful virulence factors that include a mechanism for injecting a cancer-promoting protein (CagA) into host cells. Despite identification of multiple cagPAI sRNAs, regulatory effects of only one have been characterized. To investigate potential sRNA-mediated regulation of RNA gene expression in the cagPAI and other virulence genes, we are developing strains overexpressing (abundantly producing) two promising cagPAI sRNAs. We cloned experimental plasmids (circular DNA molecules) to contain an overexpression promoter element and the relevant sRNA. Subsequently, we will introduce these plasmids into H. pylori to generate two sRNA-overexpressing strains. Finally, we will compare total RNA harvested from these two strains and the unmodified strain using RNA sequencing and reverse transcription quantitative polymerase chain reaction (RT-qPCR). The resulting identification/quantification of any significant regulation by these two H. pylori cagPAI sRNAs could illuminate aspects of sRNA regulation of the cancer-associated cagPAI region and other virulence genes