Wednesday, November 6, 2013

"Ready or Not, Here I Come!" How Shigella flexneri Hides in our Bodies

            Dysentery is a GI disease with which not many in the industrialized world are familiar, but which posed a severe problem only a hundred years ago and still causes serious illness and death in the developing world. The bacterium that causes dysentery is called Shigella flexneri, which infects the mucosal surfaces of the colon and rectum. Shigella is a gram-negative, intracellular bacterium. This means that it has a cell membrane but no cell wall, and that when it infects a host it establishes itself and replicates inside the cell rather than in the spaces and fluid between cells. Intracellular bacteria can often be more difficult for the immune system to find, especially if they have ways to make a cell look healthy instead of showing the danger signs of infection.
            The innate immune system, or first responder to any infection of your body by a foreign pathogen, is not specific to particular pathogens and relies generally on compounds that are part of bacteria, viruses, or fungi, but are not present in humans1. A distinguishing characteristic of gram-negative bacteria like Shigella is a compound called lipopolysaccharide, or LPS. LPS is a component of the bacterial cell membrane, and is highly toxic when separated from the membrane2. LPS is strongly recognized by the innate immune system as a danger signal, also known as a pathogen-associated molecular pattern (PAMP)1. LPS is made of a lipid called Lipid A, an oligosaccharide (a type of sugar polymer), and the O-polysaccharide, another sugar located on the bacterial surface3. Immune cells recognize the Lipid A part of bacterial LPS, and this causes the release of certain small molecules called cytokines. The specific cytokines released, particularly IL-1ß , TNF-alpha, IL-18, cause inflammation, which helps alert the rest of the immune system to start combating the infection, but also causes many of the symptoms associated with bacillary dysentery3.

1. Diagram of bacterial LPS

            One would think that having undergone so much evolution, we wouldn’t need to worry too much about these pathogens, as our bodies should be used to combating them by now. Every day, however, all of that evolution is rubbed in our faces by the tiny organisms that make us sick. This is because while the human immune system evolves to fight pathogens and retains memories of them in special memory cells, the microbes are also evolving, at a much faster rate than we are, to avoid our bodies’ defenses. The Shigella bacterium has several ways to elude our immune system, and new adaptations are being discovered all the time. Ida Paciello and her team provide insight into one of the previously unknown ways this bacterium escapes our immune system in their article “Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation”.
            The basic argument of this paper is that the Lipid A portion of the LPS in the bacterial cell membrane is very different when it infects a human cell than when it is grown in a test tube in a lab. When grown with everything it needs to live, the Lipid A has 6 acyl chains, or fatty acid chains that compose it. Our immune cells recognize this number of chains, leading to the previously described cytokine release and inflammation. The authors show in their research that when infecting a human cell, the Lipid A is hypoacylated, meaning that it has fewer than six chains. This makes it difficult for our immune cells to recognize and combat, allowing the bacteria to infect many cells before an immune response begins. Rather than giving a human bacillary dysentery, the researchers infected HeLa cells, which are cancerous human cells that replicate quickly and indefinitely, and are therefore used in research labs across the globe. First, the team grew Shigella both in HeLa cells and in nutrients outside of any cell. They found that the acellularly grown LPS (aLPS) from these cells had six acyl tails as expected, whereas the intracellularly grown LPS (iLPS) had far fewer, usually three or four tails. Then, they tested the levels of NF-kB activated by phagocytic immune cells called macrophages when exposed to aLPS or iLPS. NF-kB is a protein complex that causes secretion of cytokines, and so is a good marker of the level of immune response elicited by the two types of LPS. The iLPS caused much less NF-kB activation, which is exactly what the researches hypothesized would happen due to its lack of acyl tails. When the researches checked the amount of inflammation-causing cytokines, particularly TNF-alpha, they found that stimulation of the macrophage immune cells with iLPS caused significantly lower levels of cytokine production than the six-tailed aLPS. All of these factors support the hypothesis that the intracellular LPS can avoid detection by the body’s immune system by having hypoacylated A lipids.
            When macrophages phagocytose (take up) bacteria, usually they chop then up and then use their parts to activate more of an immune response. Shigella, however, is an intracellular bacterium, and prevents the macrophage from killing it. Instead, Shigella infects the macrophage, which undergoes a process called apoptosis (cell suicide). Whether the researchers stimulated the cells with iLPS or aLPS, there was the same amount of cell death. This means that it is not the LPS in Shigella that stimulates apoptosis, but that it is caused by other signs of infection. Finally, the researchers looked at the response of neutrophils stimulated with iLPS or aLPS. Neutrophils are another type of phagocytic immune cell that, unlike macrophages, are able to successfully take up and kill Shigella. These cells are very interesting because although they can combat the bacterium, they cause the inflammation that helps Shigella colonize and brings about some disease symptoms. The researchers found that iLPS stimulation caused much less of a reaction from the neutrophils than aLPSs, which in turn would lead to less inflammation.

2. Macrophage Engulfing Bacteria
        Ultimately, these results show that Shigella flexneri is capable of hypoacylating its LPS, allowing it to hide from the immune system until it has colonized a massive amount of cells. At this point, the proliferating cells fully acylate their LPS, allowing for the huge immune response accompanied by a large amount of harmful inflammation. This mechanism is not unique to Shigella; Y. pestis, the bacterium that causes plague, uses a similar mechanism to change its number of acyl tails in its vector (mode of transmission) the flea, and the human host. This fact makes the researchers findings even more relevant in the clinical realm.
            Although much of the so-called “Discussion” section was simply a restatement of various portions of the Results section, this paper is definitely worth a read. It sheds light on a development in the arms race between humans and their pathogens, and in the future research into methods of recognizing the intracellular Shigella before it is too late are promising. Furthermore, we can learn more about ourselves by learning about our pathogens, because for many evasion mechanisms developed by pathogens, our own cells have an equally ingenious method of detection waiting to catch them. This type of research, then, is doubly rewarding because it can help us understand both a foreign organism and ourselves.

1.     Mak, Tak W. & Saunders, Mary E: Primer to the Immune Response: Academic Cell Update edition. Academic Cell (2010).

2.     Slonczewski, J. L., & Foster, J. W. Microbiology: An Evolving Science. WW Norton and Company. (2010)

3.     Paciello, Ida et al. “Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation”.  Proceedings of the National Academy of Science 2013; published ahead of print October 28, 2013, doi:10.1073/pnas.1303641110.

Image Sources

1.     http://hermes.mbl.edu/marine_org/images/animals/Limulus/blood/lpstyl01.gif
2.     http://www.relfe.com/Images/macrophage.jpg

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