The curious case of Neisseria and the seven-carbon sugar

A scanning electron micrograph showing Neisseria gonorrheae bacteria (blue) infecting a type of human immune cell called a neutrophil (gold). (Image: Ryan Gaudet)
A scanning electron micrograph showing Neisseria gonorrhoeae bacteria (blue) infecting a type of human immune cell called a neutrophil (gold). (Image: Ryan Gaudet)

Walking into the little lunchroom in the back of the lab, I am greeted by a row of empty wine bottles. Celebratory drinks in the lab usually mean a manuscript’s been accepted for publication and when there’s that many empty bottles, you know it must be a pretty good journal. As I attempt to work out a wine to impact factor conversion in my head, the lab door opens and Ryan Gaudet (pronounced good-ie) walks in. Gaudet, a PhD student in the lab of Dr. Scott Gray-Owen at the University of Toronto, is the lead author of a paper recently published in the journal Science about a new signalling molecule produced by the bacteria Neisseria gonorrhoeae. It was for this paper that he and his labmates were celebrating.

To fully appreciate and understand this paper, we need to go back more than a decade to the early 2000s, when Adrienne Chen, an undergraduate student working in the Gray-Owen lab, noticed something peculiar: when cells infected with HIV were exposed to N. gonorrhoeae, HIV genes suddenly turned on and normally silent genes became expressed. This was a compelling finding because co-infection of HIV with the sexually transmitted infection gonorrhoea (caused by N. gonorrhoeae) is known to increase HIV shedding and enhance male-to-female transmission. A few years later, the mysteries of the gonorrhoea-HIV relationship drew Dr. Rebecca Malott, a postdoctoral fellow, to the Gray-Owen lab where she began a project aimed at trying to figure out how Neisseria bacteria turned on HIV gene expression.

That’s when the detective work began.

In the lab, bacteria are often grown in nutrient broths, their dense numbers turning the once-clear broth into a cloudy concoction. By sedimenting the heavy bacterial cells to the bottom of the test tube, researchers can obtain a clear liquid called the culture supernatant, a more depleted form of the nutrient broth originally used to grow the bacteria. Importantly, the culture supernatant also contains many bacterial products that act as chemical messengers between different bacteria and host cells. Malott knew that the culture supernatant from Neisseria was enough to trigger HIV gene expression, which meant that the activating signal, a compound she now referred to as “The Factor”, was released by the bacteria either intentionally or by accident.

Working with the culture supernatant, Malott tried one biochemical test after another to identify The Factor. Was it a protein? DNA? One by one, her results came back negative. She could definitively rule out a few things it was not, but still did not have a good lead on what it was. By the time Gaudet joined the lab in 2010, Malott had exhausted all biochemical options and was turning to genetics to help identify the elusive factor. She generated a library of over 1,800 Neisseria mutants, each of which carried a mutation in a single, random gene. If the mutation occurred in a gene that was required to produce the activating signal, that mutant should not be able to turn on HIV gene expression. She isolated the culture supernatants from each of the 1,800 mutants and tested them individually to see whether they could activate HIV genes. As luck would have it, all but one were able to trigger high levels of HIV gene expression.

“She came up with one gene that didn’t activate HIV anymore,” recalls Gaudet. That gene turned out to be hldA. The hldA gene is required for making a seven-carbon sugar called heptose-bisphosphate (HBP), which is a critical ingredient in making the long sugar chains that decorate the outer surface of so-called Gram-negative bacteria. Using a combination of genetic and biochemical approaches, Gaudet confirmed that HBP was, in fact, the activating signal in the Neisseria culture supernatant that was causing HIV genes to be turned on. For example, when cells infected with HIV were exposed to purified HBP, HIV gene expression increased dramatically. Also, a Neisseria mutant that could no longer metabolize HBP, and therefore contained greater than normal amounts of the sugar, stimulated even higher levels of HIV gene expression.

With the mystery of The Factor solved, Gaudet turned his attention towards another perplexing question: Why do Neisseria release HBP? To solve this riddle, he realized he needed first to figure out just what exactly HBP did.

A human neutrophil (blue) taking up Neisseria gonorrheae. (Image: Ryan Gaudet)
A human neutrophil (blue) taking up Neisseria gonorrhoeae (green). (Image: Ryan Gaudet)

Your body is a fortress, heavily armed and fortified against invading microbial pathogens. As a first line of defence against harmful microbes, your body employs specific cells in the immune system to detect pathogen-specific signals, or PAMPs. PAMPs alert our bodies to the presence of microbial intruders and turn on the appropriate immune responses to contain and destroy the enemy. An important criterion for PAMPs is that they are uniquely found in microbes and not in the animals or plants they infect. “We need to recognize something that’s on the microbe [but] not on the host. HBP fits really nicely because it’s a sugar that’s only made by microbes,” says Gaudet. “There are no sugars in the host that have a similar structure and chemical make-up as HBP. It’s completely absent.” Indeed, Gaudet’s work is the first to show that HBP functions as a PAMP in mammalian cells.

The type and strength of the body’s immune response depends on what kind of PAMP is detected. Most PAMPs cause massive inflammation and pyroptosis, a kind of altruistic cell death where some cells sacrifice themselves to kill invading microbes and protect neighbouring cells. In comparison, Gaudet found that HBP led to a more dampened immune response, one in which the body is primed and ready to attack but hasn’t initiated the battle just yet. It is this HBP-driven immune response that causes the increase in HIV gene expression Chen initially observed. While he’s done a lot of work to figure out how the HBP signal turns on this milder inflammatory response, he is still trying to understand why.

One hypothesis is that by activating the innate immune response with HBP, Neisseria is also messing around with the adaptive immune response. The innate immune response is immediate and generic—it attacks all foreign microbes with equal gusto regardless of who they are. In contrast, during an adaptive immune response, your body develops an immunological memory of the infection so that when it encounters the same pathogen later, it can respond and kill it faster.

Neisseria is one of the only bacteria [where] you can get reinfected by the same strain multiple times because we don’t develop adaptive immune memory,” says Gaudet. “One of the reasons could be that you get such a strong innate immune response [with HBP] that the signals for the adaptive response just kind of go crazy.”

Gaudet is also interested in why only Neisseria bacteria release HBP. Neisseria is not unique in its ability make HBP—nearly all Gram-negative bacteria do. But when Gaudet tested the culture supernatants from other Gram-negative bacteria like E. coli and Salmonella, none of them could trigger HIV gene expression—unless you forced the bacteria cells to burst open and release their HBP-laden contents. These other bacteria seem to be much better at keeping their sugars to themselves and do not release HBP. He thinks it may have to do with the life cycle of the bacteria. For intestinal pathogens like E. coli and Salmonella, releasing HBP in the gut might not be a good strategy because the thick layer of mucus lining the gut would likely prevent HBP from ever reaching a host cell.

Now that the celebrations are over, Gaudet is back at work answering these questions and try to gain a more thorough understanding of HBP’s role in Neisseria infection. The bottles of wine may be empty but a scientist’s job is never done.

References: 
Malott, R., Keller, B., Gaudet, R., McCaw, S., Lai, C., Dobson-Belaire, W., Hobbs, J., St. Michael, F., Cox, A., Moraes, T., & Gray-Owen, S. (2013). Neisseria gonorrhoeae-derived heptose elicits an innate immune response and drives HIV-1 expression Proceedings of the National Academy of Sciences, 110 (25), 10234-10239 DOI: 10.1073/pnas.1303738110

Gaudet, R., Sintsova, A., Buckwalter, C., Leung, N., Cochrane, A., Li, J., Cox, A., Moffat, J., & Gray-Owen, S. (2015). Cytosolic detection of the bacterial metabolite HBP activates TIFA-dependent innate immunity Science, 348 (6240), 1251-1255 DOI: 10.1126/science.aaa4921

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