Human beings have a peaceful coexistence with the trillions of microorganisms that line the mouth, skin, nose, and intestine.
But some bacteria from the air or from food invade the cells of the body and can cause fatal diseases, such as food poisoning, pneumonia, tuberculosis, and bubonic plague. These invasive bacteria don’t always make toxins nor do they always kill the cells they inhabit. How these microbes enter cells, hijack cellular machinery, and cause disease remains a mystery.
For the past 25 years, Ralph Isberg and his colleagues have been using genetic tools to elucidate the steps invasive microbes use to grow inside human cells and spread through the body. They also analyze how infected human cells are co-opted to help the bacteria evade immune detection. The contributions from his laboratory group might someday lead to better treatments for these diseases.
Isberg first became interested in microbiology—the study of microorganisms—from a microbiology course he took as an Oberlin College undergraduate. A requirement was to give a presentation about a research subject from the current literature. A topic he addressed in the talk ultimately became the subject of his Ph.D. thesis from Harvard in 1984. He also went on to do a postdoctoral fellowship at Stanford with an author from one of the papers he discussed as an undergraduate.
His doctoral work focused on DNA snippets, called transposable elements, that move around the chromosome of the bacterium Escherichia coli. Isberg showed that a transposable element could enter the bacterial chromosome only if the latter were super-coiled, a novel finding at that time because scientists were only beginning to focus on the three dimensional properties of DNA.
Isberg also revealed how the transposable elements contain genes that encode two proteins, one active and one inactive, that either permit or prevent the transposon from jumping into the bacterial chromosome. When both proteins form a complex, the complex is inactive and transposition does not occur because the inactive protein “dominantly inhibits” the active protein. Transposition, a rare event, happens only when active proteins, which are far less numerous than inactive proteins, form a complex.
“At the time, the importance of dominant inhibition wasn't so clear,” Isberg says. “In fact, I was worried that I had made a mistake. Now, molecular biologists routinely use dominant inhibition in experiments to evaluate the importance of individual proteins for performing specific processes in the cell."
As a postdoctoral fellow, Isberg decided to use the molecular genetics methods he learned in graduate school to figure out how bacterial pathogens enter host cells. The mechanism of entry was then unknown and was ripe for investigation, he says.
Isberg soon found a gene, called invasin, that allows entry into host cells by Yersinia pseudotuberculosis, a microbe that causes intestinal disease and often results in the infection of multiple organs. He also found that the invasin protein binds to receptors, members of the integrin family, located on the surface of intestinal cells, called M cells.
Additionally, with clever experiments devised by an M.D. fellow in his laboratory, his group clarified how Y. pseudotuberculosis spreads throughout the body. It had been thought the bacteria needed to grow within intestinal lymph nodes to enter the bloodstream and cause systemic disease. His group showed that the bacterium could spread to deep organs without ever entering into the lymph nodes.
Because Isberg was interested in how bacteria invade cells and because Y. pseudotuberculosis lives only transiently in a host cell, he decided in the 1990s to begin studying the intracellular microbe Legionella pneumophilia, the causative agent of Legionnaire’s pneumonia.
He soon found genes the L. pneumophilia microbe uses to grow inside a membrane-bound compartment in the host cell and to evade destruction by not entering into the lysosome, a cellular waste disposal organelle that normally digests other microbes.
“The bacterium goes into the cell and uses a membrane-bound compartment as a ‘starter house’,” Isberg explains. “But as it has more kids, it needs more room and has to expand the house.” L. pneumophilia commandeers membranes from the endoplasmic reticulum, an organelle involved in protein synthesis and transport, to provide building materials to enlarge this house.
Different invasive pathogens use other membranes in the cell to form their compartments. Chlamydia, a sexually transmitted microbe, uses membranes from the Golgi apparatus organelle to reproduce, explains Isberg describing others’ work. Mycobacterium tuberculosis exploits small vesicles called early endosomes.
Isberg and others have identified almost 30 genes that encode a channel that L. pneumophilia forms to move proteins inside the cell to help it grow. Additionally, his group found a large number of proteins that move through this channel, enabling the bacteria to reproduce and evade lysosomal destruction. He believes there may be more than 200 proteins that move through this channel.
Isberg’s group is trying to determine how these hundreds of bacterial proteins interact with each other and with cellular proteins to permit the invasion process. Eliminating one protein at a time does not prevent the microbe from reproducing, he says. Therefore, the microbe may have multiple mechanisms that allow it to grow inside the cell, which Isberg is also attempting to decipher. This was a level of complexity that he did not expect when he started to work on this organism.
When he first found the invasin protein, Isberg says he was disappointed, thinking that the project he chose to pursue was too simple. Now, Isberg is a bit more careful what he wishes for. He realizes that the complexity of bacterial pathogenesis might not be fully understood for another 25 years. Luckily, Isberg welcomes the challenge.