H. pylori Inhibits Heat Shock Protein Production


Source: https://www.medicalnewstoday.com/articles/325119.php#1

During stressful conditions, cells have the ability to produce heat shock proteins or HSP which function as a companion that play a role in aggregation, assembly, transport, and folding in proteins (Science Direct). New evidence suggests that cells also produce HSP which regulate the immune system when exposed to pathogens. Almost half of the world’s population is infected with Helicobacter pylori bacteria which cause chronic gastritis. H. pylori infection may lead to peptic ulcer or in worst case, stomach cancer. Study suggests that H. pylori is capable of reducing the expression of HSP. However, only a few percentage of the infected develop clinical illness and therefore, scientists are interested in bacterial and host factors that may be related with illness. A small group of researchers from University of California Davis, Wendy S. Axsen, Cathy M. Styer, and Jay V. Solnick, further investigated this fact by examining the role of various H. pylori strains and identified virulence factors in cell culture and mouse model. The results of these experiment include evidence showing the reduction of HSP expression using co-culture of H. pylori with two gastric carcinoma cell lines. Mice was also infected with H. pylori which resulted in the reduction of HSP. It has been thought that the reduction of HSP expression by H. pylori may be an immune evading mechanism that causes chronic infection since HSP functions as a warning signal during bacterial infection.

Cytotoxin associated gene pathogenicity island or Cag PAI is one of the bacterial factors in H. pylori related with disease. Cag PAI is a group of gene that codes a type IV secretion system necessary for the virulence and survival of the bacteria. It is often found in H. pylori that causes the symptomatic illnesses (peptic ulcer, stomach cancer) than illnesses that do not show any symptoms. When H. pylori attaches to the gastric epithelial cells, the type IV secretion is activated and the bacteria utilize a syringe-like pilus that injects into the stomach cells and transfers cytotoxin-associated gene A or CagA, peptidoglycan, and other bacterial elements. CagA is a 120-145kDa protein encoded on the 40kb Cag PAI which is thought to be associated in stomach cancer development (Hatakeyama, 2005). Vacuolating cytotoxin or VacA is another toxin from H. pylori associated with disease. It is an 88-kDa pore-forming protein synthesize by approximately half of the H. pylori strains and has the ability to induce vacuolation in stomach cells (Science Direct).

Under normal circumstances, the total protein content of cells contains about 5 to 10% of heat shock proteins. In healthy cells, HSP has many intracellular functions including aid in protein transport, prevention of protein aggregation, and protection of newly formed polypeptides or proteins. HSP can be activated by different exposures including high temperature, oxidative stress, deficiency in nutrients, UV radiation, chemicals, ethanol, and bacterial infection. HSPs are categorized by their molecular weight (HSP60, HSP90, HSP27, HSP70, HSP40). They are regulated at transcriptional level and have been thought to serve as an early danger signal which activates the immune system. Some types of HSP bind to the receptors of antigen-presenting cells and activates the production of cytokines during inflammation. Generally, HSP expression is increased during bacterial infection but new study suggests that H. pylori can reduce the expression of HSP. Cell culture and mouse model was used to understand the role of H. pylori in HSP expression. Isogenic mutants were also examined to discover whether genetic diversity and bacterial components such as Cag PAI and VacA play a role in reducing the HSP expression.

Determining the reduction of HSP expression by H. pylori requires culturing the bacteria with stomach epithelial cells for several hours and performing Western blots with antibodies that will react specifically with HSP70, HSP27, and HSP60. Western blot is a technique used in research to detect, separate, and analyze proteins based on their molecular weights (Mahmood, 2012). One of the H. pylori strains reduced the HSP70 expression (Figure 1). The same strain also reduced the HSP60 expression of only one type of stomach cancer cell and was observed only on the 6- and 12-hour time period.

Text Box: Figure 1. Western blot result of two types of stomach cancer cells infected with H. pylori. At all time points, the expression of HSP70 was reduced by H. pylori. The first type of stomach cancer cell shows no reduction in HSP60 expression but shows reduction on the second type of stomach cancer cell only at the 6- and 12-hour time period.
Figure 1. Western blot result of two types of stomach cancer cells infected with H. pylori. At all time points, the expression of HSP70 was reduced by H. pylori. The first type of stomach cancer cell shows no reduction in HSP60 expression but shows reduction on the second type of stomach cancer cell only at the 6- and 12-hour time period.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494282/

H. pylori are highly diverse and another type of experiment was performed to determine if the reduction of HSP expression is caused by a specific strain of the bacteria. Three types of H. pylori strains were cultured together with the stomach cancer cells and the expression of HSP70 and HSP60 was observed. After the 12-hour culture, the result shows that all three strains of the bacteria reduced the HSP70 expression and two of the strains also reduced the HSP60 expression (Figure 2).

Text Box: Figure 2. Western blot result of the three types of H. pylori strains (J166, J99, 26695). All strains reduced the HSP70 expression and two strains reduced the HSP60 expression.
Figure 2. Western blot result of the three types of H. pylori strains (J166, J99, 26695). All strains reduced the HSP70 expression and two strains reduced the HSP60 expression.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494282/

Another type of experiment was performed to determine if the primary virulence factors of the H. pylori are associated with the reduction of the HSP expression. H. pylori mutants in VacA, Cag PAI, and CagA were created and observed if they influence the HSP expression reduction. The result shows that the normal strain and all mutated strains reduced the HSP70 expression suggesting that the virulence factors of H. pylori are not associated with the reduction of HSP expression.

Groups of mice were infected with H. pylori and stomach tissue samples were taken at two different time points, after 1- and 12-week period. The tissue samples were used to determine the total RNA isolation and the number of viable H. pylori bacteria. The result shows that at 1 week, the infected mice were colonized with the bacteria and at 12 weeks, the number of H. pylori bacteria reduced (Figure 3).

Text Box: Figure 3. Number of viable H. pylori bacteria at the two time periods. At 1 week, a good number of bacteria colonized the stomach tissues of the infected mice. At 12 weeks, the average number of viable H. pylori reduced.
Figure 3. Number of viable H. pylori bacteria at the two time periods. At 1 week, a good number of bacteria colonized the stomach tissues of the infected mice. At 12 weeks, the average number of viable H. pylori reduced.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494282/

To determine the amount of HSP expression in mice infected with the bacteria, cDNA from the stomach tissue samples was amplified using polymerase chain reaction or PCR. PCR is a technique used to quantify small segments of DNA (Genome.gov). The result shows that the HSP70 expression was reduced at both time points between the uninfected control mice and infected mice, and the number of viable H. pylori affects the amount of HSP70 expression and in this case, the more bacteria, the less HSP70 expression (Figure 4).

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Figure 4. HSP70 expression in mice infected with H. pylori. At 1 week where the number of viable bacteria is at most, HSP70 expression was significantly reduced. At 12 weeks, the reduction in the number of bacteria amplified the expression of HSP70.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494282/

The experiments confirmed that H. pylori reduces the expression of HSP, and the reduction is not influenced by the virulence factors of the bacteria. One study results in the reduction of HSP70 expression by adding CagA from another source but the physiological relevance is not fully understood. The reduction of HSP70 expression is most notable in cell culture compared with the group of mice infected with H. pylori. HSP expression reduction has also been found in other types of study such as proteomic and microarray in epithelial cell cultures, and in other animal models including studies associated with a species of monkey. The reduction of HSP expression by H. pylori is a very powerful phenomenon that can be shown across different cell lines, animal models, and different strains of H. pylori and the reduction seems not dependent on the virulence factors of the bacteria. This article was chosen because it demonstrates how bacteria can affect the physiological processes of the cells and cause disease. According to World Health Organization, stomach cancer is the third leading cause cancer death and it is interesting to know from this article that a cancer may originate from a foreign pathogens that can inhibit protein production related to immunity. The experiments involve only one species of bacteria and observed only one protein inhibition. Who knows what other protein production that an H. pylori can inhibit and who knows if the other species of bacteria affect a mechanism in a cell. The article gives us one reason to study microbiology and not to underestimate any microorganisms around us.

References:

Axsen, W. et. Al. (2009) Inhibition of Heat Shock Protein by Helicobater pylori. Microbial Pathogenesis. Volume 47. Page 231-236. October 2009. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494282/

Hatakeyama, M. et. Al. (2005). Helicobacter pylori CagA: A New Paradigm for Bacterial Carcinogenesis. Cancer Science. 96: 835-843. Accessed November 17, 2019

Science Direct. Vacuolating Toxin. Accessed November 17, 2019. https://www.sciencedirect.com/topics/medicine-and-dentistry/vacuolating-toxin

Science Direct. Heat Shock Proteins. Accessed November 17, 2019. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/heat-shock-proteins

Mahmood, T. et. Al. (2012). Western Blot: Technique, Theory, and Trouble Shooting. North American Journal of Medical Sciences. Vol 4. Page 429-434. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/

Genome.gov. (2015). Polymerase Chain Reaction (PCR) Fact Sheet. National Human Genome Research Institute. Accessed November 17, 2019. https://www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet

World Health Organization. (2018). Cancer. Accessed November 17, 2019. https://www.who.int/news-room/fact-sheets/detail/cancer


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