Could TIFA signaling render H. pylori not so tough?

Science Spotlight

Could TIFA signaling render H. pylori not so tough?

From the Salama Laboratory, Human Biology

Oct. 16, 2017

        Helicobacter pylori (H. pylori) is a common bacterium that infects and colonizes the stomach. In some cases, the inflammation caused by the infection can lead to gastric and duodenal ulcers and cancer. H. pylori is the primary identified cause of pathogen-associated gastric cancer globally. Antibiotic resistance is rapidly becoming a problem for treatment, and there is currently no vaccine. Both the innate and adaptive immune systems can recognize H. pylori but are rarely able to clear the bacteria. Because this bacterium is so successful at evading and manipulating the immune system, a deeper understanding of the immune response to H. pylori is critical for identifying new methods of treating the infection.

        When H. pylori enters the human digestive tract, bacteria attach to epithelial lining of the stomach, often triggering a robust immune response in the gastric epithelial cells which release proinflammatory cytokines. Strains of H. pylori carrying the cytotoxin-associated gene A (cagA+ strains) provoke a greater inflammatory response and more frequently lead to development of ulcers and cancer. The CagA protein is injected into the gastric cell from the bacterium through the type IV secretion system (T4SS) where it disrupts signaling and triggers the cytokine response. Other molecules are also injected through T4SS that activate the global transcription factor NF-KB, a major regulator of proinflammatory genes and cytokines such as the neutrophil recruiter, interleukin-8 (IL-8). Neutrophils infiltrate the stomach lining and recruit other immune cells, leading to chronic inflammation and tissue damage.

        Prior to the current publication, the consensus was that the inflammatory response outlined above was due to activation of NOD1, an intracellular receptor that mediates the immune response and activates NF-KB. In a study published in MBio, the Salama lab (Human Biology Division) determined that NOD1-dependent responses are not the only immune responses triggered by H. pylori; they identified tumor necrosis factor receptor-associated factor (TRAF) interacting protein with forkhead-associated domain (TIFA) as a vital innate signaling factor contributing to the inflammatory response.

        The researchers found that when they knocked out NOD1 in a gastric adenocarcinoma cell line (AGS cells) using CRISPR/Cas9, the cells produced less IL-8 in response to H. pylori, but they were still able to generate some IL-8, demonstrating that NOD1-mediated signaling is not the only immune response pathway activated. Furthermore, when they knocked out TIFA, the cells produced significantly less IL-8 in the presence of H. pylori, particularly early in infection. Knocking out both NOD1 and TIFA had an additive effect. These data suggest that each pathway contributes to H. pylori response, but they are independent of each other.

        Gall and colleagues further probed into the mechanism of the TIFA-mediated immune response and found that cag-T4SS is necessary both to stimulate an IL-8 response and also activate TIFA. A cag–T4SS mutant bacterial strain could not generate any IL-8 response in the AGS cells suggesting that the bacterial factor that stimulates TIFA is directly delivered to the host through the cag-T4SS. TIFA is activated by the bacterial metabolite heptose-1,7-biphosphate (HBP), a component necessary for the synthesis of the LPS of H. pylori (Gaudet et al. 2015). Therefore, the authors proposed the following model: delivery of the HBP metabolite into the gastric epithelial cell cytosol through cag-T4SS activates TIFA and leads to activation of NF-KB, which ultimately results in the production of IL-8. A summary of this model is provided in Figure 1. Upstream metabolites of HBP do not activate TIFA, implicating HBP as the crucial molecule for initiation of this pathway.

A model of TIFA signaling in H. pylori

Figure 1. Model of H. pylori cag-T4SS-dependent, NF-KB-driven innate immune response in gastric epithelial cells. The response is initiated by HBP injection into the cytosol that activates TIFA. TIFA activates TRAF6, which in turn activates NF-KB. NF-KB upregulates proinflammatory genes including the IL-8 gene. Following TIFA activation, peptidoglycan (PG) activates NOD1, which eventually also leads to NF-KB activation. CagA associates with TAK1 leading to its polyubiquitination mediated by TRAF6. Polyubiquinated TAK1 also activates NF-KB. HBP is generated through the ADP-heptose biosynthesis pathway in the bacteria. Four H. pylori enzymes are involved. GmhA and HldE are essential in the G27 H. pylori strain. Knockout of each of the others, GmhB and HldD, results in growth abnormalities.

This image was provided by Tina Gall.

 

        The authors further wondered if the heptose metabolites in the HBP synthesis pathway are imperative for the H. pylori bacteria to survive and colonize the host. They mutated a total of four heptose metabolism enzymes (hldD/E and gmhA/B) present in Gram-negative bacteria. Deletion of hldD and gmhB resulted in bacteria with a severe growth defect. Deletion of hldE and gmhA appeared to be lethal to the bacteria. Putting hldE under the control of an inducible promoter revealed that HldE is necessary for HBP synthesis. These results suggest another potential therapeutic strategy targeting the enzymes involved in heptose metabolism. First author Tina Gall says, “Targeting these enzymes is…a viable therapeutic targeting strategy. In fact, because these enzymes are highly conserved across most Gram-negative bacteria, they represent a broad-spectrum and relatively untapped antibiotic development strategy. There has been some effort by various groups to develop inhibitors to these enzymes. For example, the French pharmaceutical company Mutabilis performed a high throughput screen for HldE inhibitors, identified hits and showed that they potently inhibit E. coli growth (Desroy et al. 2013). It would be interesting to study these types of inhibitors in the context of H. pylori infection, not only to test if they indeed limit bacterial growth, but also as a means of studying the mechanisms underlying LPS synthesis in H. pylori.” 

        The discovery of a NOD1 independent innate immune response after H. pylori infection opens up new avenues to explore for therapeutic interventions. However, the relative contribution of each pathway during a natural infection cannot be definitively deciphered using cell lines. Tina Gall says, “Our goal is to gain a better understanding of the innate immune pathways that H. pylori triggers in the host. Using a gastric adenocarcinoma cell line we were able to uncover a previously unappreciated pathogen recognition pathway that gastric epithelial cells use to detect H. pylori. However, cancer cell lines have numerous limitations, for example, AGS cells do not express the full breadth of the pathogen recognition receptors that one would find in primary gastric epithelial cells. We are currently developing methods to CRISPR target human primary gastric epithelial cells and co-culture conditions that will allow us to test the relative contribution of various pathogen recognition pathways in this more physiologically relevant system.” The Salama Lab is also working with collaborators who developed a Tifa knockout mouse model. “We are very interested to see if these mice are more susceptible to H. pylori infection. We can also use gastric tissue from these mice to generate gastric organoids and ask questions about whether TIFA signaling in the epithelial cell compartment is sufficient to control bacterial infection and what the downstream consequences of TIFA signaling are on recruitment of immune cells to the site of infection,” says Tina Gall.


The National Institutes of Health provided the funding for this research.

 

References:

Gall A, Gaudet RG, Gray-Owen SD, Salama NR. 2017. TIFA Signaling in Gastric Epithelial Cells Initiates the cag Type 4 Secretion System-Dependent Innate Immune Response to Helicobacter pylori Infection. MBio, 8(4).

Gaudet RG, Sintsova A, Buckwalter CM, Leung N, Cochrane A, Li J, Cox AD, Moffat J, Gray-Owen SD. 2015. Cytosolic detection of the bacterial metabolite HBP activates TIFA-dependent innate immunity. Science, 348 (6240).

Desroy N, Denis A, Oliveira C, et al. 2013. Novel HldE-K inhibitors leading to attenuated Gram negative bacterial virulence. J Med Chem, 56(4).