Pseudomonas Bacteria Escape Immunity by Disrupting Energy Production in Macrophages todayheadline

Found lurking in freshwater, hot tubs, and pools, the bacterium Pseudomonas aeruginosa can cause blindness, rashes, and a constellation of other symptoms when it crosses into humans.¹ Often a hospital-acquired pathogen, it tends to infect people with burns or weakened immunity, and it has evolved to resist multiple antibiotics and counteract the immune system, rendering it difficult to treat.^2,3 In a recent publication in eLife, Harvard University molecular microbiologists Laurence Rahme and Arijit Chakraborty found that these bacteria release a chemical that inhibits energy generation in the mitochondria of macrophages, thus dampening the immune response.⁴ This work identified a new tactic that P. aeruginosa uses to subvert host immunity, and it intimated a new approach for treating the recalcitrant infection.

One of the chemicals produced by Pseudomonas, called 2-aminoacetophenone (2-AA), is a useful biomarker for Pseudomonas infections in the clinic, but many of its functions, including its effects on innate immune cells, remain unexplored.⁵ Previously, the Harvard researchers found that macrophages don’t engulf and dispose of P. aeruginosa—an energy intensive process—in the presence of 2-AA.⁶ In the present study, they explored which mechanisms 2-AA might use to interfere with macrophage functions, focusing on how this molecule dampens macrophage bioenergetics.

The team discovered that laboratory cultures of mouse macrophages inoculated with 2-AA produced less adenosine triphosphate (ATP), the molecule that cells use as an “energy currency” to fund energy-demanding biochemical reactions.⁷ This confirmed their suspicion that 2-AA dampens energy production in the cell. However, multiple pathways produce ATP. Since some pathways produce more ATP than others, they had to pinpoint the one 2-AA blocks to work out the magnitude of its impact.

There are two main pathways that cells use to convert glucose into energy. The first is glycolysis, which occurs in the cytoplasm and produces two molecules of ATP per molecule of glucose. Pyruvate, the breakdown product of glycolysis, is then imported into the mitochondria where it fuels other energy-generating pathways, namely the Krebs cycle and oxidative phosphorylation. These produce approximately 30 additional ATP copies.⁸ Since only oxidative phosphorylation consumes oxygen, the researchers conducted a Seahorse assay to measure oxygen uptake by the cells using a probe that fluoresces in the presence of this gas molecule.⁹ Oxygen consumption dropped in cells exposed to 2-AA, revealing that the more-profitable energy-generating pathway crashed.

They also measured the levels of pyruvate in the cell.⁸ 2-AA’s presence was correlated with higher levels of pyruvate in the cytoplasm, suggesting pyruvate couldn’t travel into the mitochondria. “So, we don’t have the energy production we are expecting,” Rahme said. 

Because in vitro experiments don’t reflect the complexity of the immune system, Rahme and her team sought to validate these findings in living animals. They infected mice with either wild type P. aeruginosa or a mutant that lacked the multiple virulence factor regulator (MvfR)—a transcription factor required to express the enzymes that synthesize 2-AA.¹⁰ In the spleen—an organ abundant in immune cells—ATP levels dropped within 24 hours in mice infected with the wild type bacteria but not in mice that received the 2-AA-lacking mutant.¹¹ They observed a drop in the level of acetyl-cofactor A, a breakdown product of pyruvate formed after it enters mitochondria, confirming that the drop in ATP was due to energy-generating pathways shutting down. They also assessed the impact of 2-AA on bacterial burden in the spleen; by day 10, mice had an easier time killing off the bacteria in the absence of this chemical.

As Pseudomonas bacteria grow increasingly resistant to antibiotics, researchers need to develop different types of therapeutics to treat them.³ Kayeen Vadakkan, a microbiologist at St. Mary’s College, Thrissur who was not involved with the work, suggested that 2-AA could serve as a new bull’s eye that drugs could target. “We can complement our immune system,” he said, proposing that drugs that block 2-AA’s effects could give macrophages a boost. Rahme’s laboratory is working on this therapeutic approach. “We’re very excited because the inhibitor of MvfR that we developed is working pretty well,” she said, referring to further research not included in this study. However, more research must take place to assess its efficacy and safety before it can be used in the clinic.

Besides blocking 2-AA to fight bacteria, researchers could theoretically harness it to stave off autoimmune diseases. In some disorders, such as rheumatoid arthritis and lupus, overactive macrophages exacerbate inflammation.¹² “2-AA is a molecule which is anti-inflammatory in nature,” Chakraborty said, suggesting that it may have potential as an immunosuppressive drug.

Disclosure of Conflict of Interest: Study coauthor Laurence Rahme has a financial interest in Spero Therapeutics, a company developing therapies to treat bacterial infections.