Streptococcus pneumoniae, as illustrated by the authors
Have you or someone you know ever had a sinus or middle ear infection? Maybe then you’ve unknowingly had a run-in with the star of today’s show: Streptococcus pneumoniae. S. pneumoniae is a bacteria that causes infection in many human body systems. It can cause many common infections like sinus or ear infections most of us are familiar with, as well as serious illnesses like bacteremia and pneumonia. In some cases, it can even cross the blood-brain barrier and cause meningitis. According to the World Health Organization, meningitis causes inflammation of the spinal cord and brain, and can result in many serious complications such as long-term speech, language, and learning problems, limb amputation, and death. S. pneumoniae is one of the four main causes of bacterial meningitis.
This bacteria is amazing in its ability to survive the defense mechanisms set forth by the brain to prevent external organisms from entering the intensely regulated brain environment. The human brain wants to keep out pathogens and keep in cerebral fluid, so we’ve evolved some pretty fail-safe tactics to ensure that organisms such as S. pneumoniae stay outside. One way our central nervous system is protected is through the blood-brain barrier, a membrane between brain interstitium and the blood that is selectively semipermeable. This means that it regulates molecule and ion movement to shelter the brain from toxins but allow in necessary things like nutrients. The blood-brain barrier has been an important component of mainstream science conversations such as microplastics and treatment of diseases of the brain like Alzheimers or A.L.S. The selective permeability of the blood-brain barrier means that it can be challenging to get medication to the brain through injections into other areas of the body. S. pneumoniae, however, has been able to cross this barrier and cause infection within the brain and spinal fluid.
Inside the brain, brain microvascular endothelial cells, or BMECs for short, contain tiny organelles called endocytic vacuoles, which are like the gatekeepers of the brain. These little vacuoles are responsible for exactly what matter can enter and exit the most critical area of the human body, and can rapidly acidify organisms such as S. pneumoniae when they get too close. This acidification is intended to kill unwanted organisms. Much of S. pneumoniae will end up in these vacuoles in as little as 2 hours post-infection. In most cases, the infringing bacteria will degrade and die, but an incredible new study examined the circumstances under which they survive. An ability to resist lethal pH contributes to S. pneumoniae’s ability to cross the blood-brain barrier and cause serious infection.
Scientists researching this bacteria in the lab at the Indian Institute of Technology Bombay discovered that the key factor to survival of S. pneumoniae within this unfriendly environment is a sugar-metabolizing enzyme called Pyruvate Oxidase, or SpxB. Enzymes such as this one catalyze some sort of chemical reaction, and SpxB is critical as it is able to produce both hydrogen peroxide and acetyl phosphate. The hydrogen peroxide created by SpxB temporarily disables the digestive lysosomal enzymes within the endocytic vacuoles, which prevents further bacterial degradation. Acetyl phosphate is able to trigger an acid-tolerance response within the bacteria through the CiaR protein, and both of the chemicals work together to allow for S. pneumoniae to safely cross the blood-brain barrier and avoid the little gatekeeping vacuoles that are working so hard to keep them out. Sneaky, right?
Diving in a little further, let’s discuss exactly how the acidic degradation of bacteria is conducted by the endocytic vacuoles in normal-functioning and non-mutated bacteria. The vacuoles must go through a maturation process in order to reach their full potency. They’ll start off with markers of PI3P and Rab 5, which indicate early stages of development. Mature endosomes are marked by PI3, 5P2, and Rab7. These markers are different sorts of molecules, but you can just think of them like tags that help scientists identify different endosome stages. Once in late stages, the endocytic vacuoles will fuse with lysosomes, allowing for acidic degradation.
Figure 1: Researchers visualized endosomal markers association with S. pneumoniae using immunofluorescence microscopy. This allowed researchers to understand if the bacteria could survive in the acidic lysosome environment.
Although hard to see in the main image, green dyed pneumococci are present within acidified vacuoles of endothelial cells. This is clearer in the “WT” box. Endothelial nuclei are stained blue, with pink showing a lysosomal marker and green showing wild type S. pneumoniae. Lysosome presence represents the final stage of maturation and acidification of the vacuoles, where they fuse with lysosomes. This indicates a pH sufficient to degrade many unwanted pathogens such as S. pneumoniae. The presence of S. pneumoniae demonstrates its ability to survive acidic degradation through acid tolerance. The study also identified S. pneumoniae present in other stages of vacuole maturation, demonstrating their ability to survive early and late stages. This figure does not demonstrate the mechanism through which it survives, rather just proves its presence within the lysosomes.
Now that we’ve established its presence inside what should be a lethally acidic environment, we can explore how exactly it manages this. The formal name for the mechanism by which S. pneumoniae is able to survive acidic degradation is the Acid Tolerance Response, or ATR. The CiaR protein that contributes to the ATR must be regulated as a two-component system, and must be phosphorylated to activate the genes necessary for the ATR. This phosphorylation of CiaR can be conducted by Acetyl phosphate, a by-product of pyruvate oxidase (SpxB) activity. Hydrogen peroxide is produced as a by-product of the catalyzation of SpxB to Acetyl phosphate. Basically, S. pneumoniae is able to sense and respond to the environmental changes of the lysosomes through the ATR. If bacteria do not contain either the CiaR or SpxB enzymes, their survival is significantly reduced under the conditions present in the acidic endocytic vacuoles.
The experiment on S. pneumoniae demonstrated that the loss of either enzyme decreases the intracellular bacterial survival rate. This results in a lower expression of CiaR genes. The researchers confirmed that SpxB supports the function of CiaR through the production of Acetyl phosphate by finding that double mutants with mutations in both SpxB and CiaH (a CiaR-related gene) have the lowest rate of gene activation and survival compared to its peers. In short, S. pneumoniae activates CiaR through a SpxB-generated Acetyl phosphate to trigger the acid tolerance response, allowing for the survival of the bacteria when faced with endocytic lysosomal vacuoles through passage of the blood-brain barrier. That’s quite the mouthful.
So what does that mean for broader science, and in our context of understanding this complex and interesting bacteria and its impact on the human body? It’s critical to understand how a disease functions for future treatment options. Understanding how the biology of the CiaR protein could allow future treatments to be created, perhaps by targeting the ability of S. pneumoniae to survive the acidic environment. It’s quite possible that this new research on bacteria will pave the way for future studies on disease prevention and treatment, offering life saving opportunities for people everywhere. Further, an increased understanding of how one organism is able to cross the blood-brain barrier could open the door for researchers to learn from S. pneumoniae and utilize its techniques to deploy therapeutic interventions across the blood-brain barrier.
About the Authors:
Mads Hurley ‘25 and True Usiatynski’25 are seniors, roommates, and best friends in the Biological Sciences department at Mount Holyoke. Mads also has an Art History minor, and True is a double major with International Relations. When they’re not studying, they can be found riding for the varsity hunt seat or western team, going for a run, or watching The Last of Us. Mads hopes to work in the field of oceanography and True aspires to be an infectious disease physician.
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