Key to Virulence of Meningitis Bacterium
By: Carol A. Rouzer, VICB Communications
Published: April 8, 2010
Structure of the PorB protein provides keys to the how Neisseria meningitidis causes disease and triggers the immune system.
Bacterial meningitis, an infection of the membranes covering of the brain, is a life-threatening medical emergency. Although a number of bacterial species may cause meningitis, Neisseria meningitidis (Figure 1) is the only species that can cause meningitis epidemics. There are 1000 to 4000 cases of N. meningitidis-associated meningitis reported in the United States annually, with the incidence rate highest among children under five years of age and teen agers. The mortality rate is 10%.
Figure 1. Neisseria meningitidis a gram negative bacterium causes epidemic meningitis, primarily in young children and teenagers. (Image courtesy of Wikimedia Commons under the GNU Free Documentation License.)
N. meningitidis is one of only two species of Neisseria that cause disease in humans, the other being N. gonorrhoeae, which causes the sexually transmitted disease gonorrhea. Other species of Neisseria colonize human skin and mucous membranes harmlessly, so there is an ongoing search to identify virulence factors in N. meningitidis and N. gonorrhoeae. Virulence factors are components that convert bacterial cells from harmless bystanders to invading pathogens. One such factor that has been in pathogenic Neisseria is the PorB protein. Gram negative bacteria are surrounded by two membrane layers, an arrangement that provides protection to the cells but also poses a challenge regarding nutrient transport. One solution to this challenge is the class of porin proteins, which form pores through the outer membrane. PorB is a member of this protein class, providing a transport pore for sugars and ions. It is the second most common protein in the outer membrane of N. meningitidis.
While PorB performs a basic nutritive function for the bacterial cell, it also plays a role in disease pathogenesis. When an invading bacterium attaches itself to a host cell, the PorB protein is transferred to the host cell and becomes localized to mitochondria, the cellular structures where most energy-generating chemical reactions occur. Like the bacterial cells, mitochondria have two membranes, and PorB becomes embedded in the outer one. However, in the mitochondrial environment, PorB remains open continuously, allowing unregulated leakage of components out of the mitochondria, and ultimately influencing cell viability. Now, to better understand how PorB operates in both the bacterial and mitochondrial environment, Tina Iverson and her laboratory have determined the detailed structure of this protein from N. meningitidis through the process of x-ray crystallography [Tanabe et al. (2010) Proc. Natl. Acad. Sci. U.S.A., published online Mar 29, DOI: 10.1073/pnas.0912115107].
Figure 2. PorB exists as a trimer of three identical protein subunits each of which forms a pore, as can be seen in the electron density map from the X-ray data on the left and the cartoon depiction on the right. Reproduced with permission from Tanabe et al. (2010) Proc. Natl. Acad. Sci. U.S.A., published online Mar 29, DOI: 10.1073/pnas.0912115107.
Figure 3. A cartoon of the PorB structure viewed from the side. The beta barrel is formed by strands of amino acids (cyan) lined up side-by-side joined by loops of amino acids (gray). One loop (L3) forms a helical structure (red) and lies across the pore. This loop is likely responsible for flow of materials through the pore. Reproduced with permission from Tanabe et al. (2010) Proc. Natl. Acad. Sci. U.S.A., published online Mar 29, DOI: 10.1073/pnas.0912115107.
The Iverson lab discovered that PorB exists as a trimer of three identical protein molecules (Figure 1), each of which is characterized by a cylindrical structure known as a beta barrel (Figure 2). Through the center of the beta barrel is the pore that allows the transfer of sugars and ions. A loop of amino acids that lies across the opening likely plays a role in regulating flow through the pore. The Iverson group was able to identify three distinct channels within the PorB structure, one to allow passage of sugars, one for negatively charged ions, and one for positively charged ions (Figure 4). These results show how PorB can serve to allow the transport of multiple, very diverse species into the bacterial cell.
Figure 4. Diagram of PorB from the top (left) and the side (right) showing the binding sites and/or channels for sugars (magenta and cyan circles), ATP (black rectangle), positive ions (orange circle) negative ions (orange shading) and antibiotics (red triangle). Reproduced with permission from Tanabe et al. (2010) Proc. Natl. Acad. Sci. U.S.A., published online Mar 29, DOI: 10.1073/pnas.0912115107.
When PorB becomes embedded in the outer mitochondrial membrane of a host cell, it is exposed to the energy rich environment of the mitochondrion, including a high concentration of the energy storage molecule adenosine triphosphate (ATP). Previous studies had shown that ATP binds to PorB and alters its function. The Iverson group successfully identified the binding site for ATP in the PorB channel, and they showed that binding of ATP in the channel alters the pore structure. The result is that the pore channel is smaller, but it can no longer be closed by the loop of amino acids. The result is a pore that remains constantly open, explaining how the mitochondrion can be drained of vital components.
When N. meningitidis infects a host, the immune defense system is triggered to fight the infection. One of the first lines of defense is white blood cells bearing membrane receptors that bind to bacterial cell components. It is through these receptors that the white blood cells recognize the foreign invader. One such receptor is the Toll-like receptor 2 (TLR-2), which binds to many bacterial components, including PorB. An interesting question in immunology has always centered on how receptors such as TLR-2 recognize a wide variety of different bacterial components. The structural data from the Iverson group shed light on this question. They have found a ring of positively charged amino acids around the outer edge of PorB that are the likely site of interaction with a series of negatively charged amino acids on TLR-2 (Figure 5). Thus, it appears that a simple attraction between opposite charges may be the basis of PorB recognition.
Figure 5. Diagram of the interaction of PorB with TLR2, showing 2 PorB trimers (green) making contact with two TLR2 binding domains. Reproduced with permission from Tanabe et al. (2010) Proc. Natl. Acad. Sci. U.S.A., published online Mar 29, DOI: 10.1073/pnas.0912115107.
It is clear that the PorB protein plays a key role, in the normal physiology of the N. meningitidis bacterium. But it is also important to every aspect of the pathogenesis of meningitis from the ability of the bacterium to kill host cells to the initiation of the immune response. Thus, detailed structural studies such as these from the Iverson group are critical to our understanding of how this protein functions and hold promise for the development of new ways to battle this potentially devastating disease.