Intracellular versus Extracellular Growth

Hello everyone,

F. tularensis is what's called an intracellular pathogen. That means that it is thought to reside mostly inside of host cells during the course of infection, at least in mammals. This assessment is based largely on (1) the microscopic observation of F. tularensis cells inside host cells during experimental infection and (2) the ability of F. tularensis to invade and survive within mammalian cells cultured in the lab.

We wanted to determine what percentage of F. tularensis cells resided within insect cells during our experiments. So, we used what is called a "gentamicin protection assay." Gentamicin is an antibiotic that kills only extracellular bacteria, leaving those present inside of host cells perfectly unharmed.

We were surprised (which is one of the great things about being a scientist!) that most of the F. tularensis cells are not found inside of host cells during our experimental infection. Between 90% and 99% of the bacterial cells were located extracellularly. The graph below shows the total number of bacteria (orange) and the number of intracellular bacteria (green) per milliliter of roach hemolymph (equivalent of the insect blood at different time points throughout the experiment. Notice that the graph is plotted on a logarithmic scale, so that relatively small changes on the Y-axis actually represent very large changes in value.

 

Perhaps we shouldn't have been surprised with this finding because it's actually very similar to what was observed for Francisella novicida  (a cousin of F. tularensis ) in fruit flies. There are several possible reasons that could contribute to this finding:

• F. tularensis may be able to survive and replicate in roach hemolymph better than it can in mammalian blood. Identifying the reason(s) for such a difference would be important to forming a better understanding of tularemia and could help in developing more effective treatments.
• The original assumptions about the relative balance of intracellular versus extracellular bacteria during an active infection may need to be refined. These insect studies may be telling us something about the pathogenesis of F. tularensis that we don't already know.
• Insect immune cells (called hemocytes) may be better at preventing intracellular invasion or growth of F. tularensis compared with their mammalian counterparts. Again, understanding the reason(s) for such a difference would be very useful in developing future treatments.
• The gentamicin protection assay, as we've done it here, only detects bacteria in free-flowing hemolymph and not any bacteria associated with the internal organs of the insect. It could be that F. tularensis does invade insect cells associated with other tissues.