Invasion of Red Blood Cells by Francisella tularenesis
By Amesh A. Adalja MD, FACP, August 5, 2011
Francisella tularensis subspecies tularensis, a category A bioweapon, is endemic in many regions of the world, including in the United States. This highly infectious bacterium is capable of causing fatal tularemia, and its virulence is thought to be partly related to its ability to invade and replicate itself within host cells (primarily phagocytes). Although F.tularensis is known to invade other cell types in addition to phagocytes, the susceptibility of erythrocytes (RBCs)—the most numerous cell in the blood—to infection with this bacteria is not known. A research team led by Dr. Gerard Nau at the University of Pittsburgh has recently demonstrated that F.tularensis has the capacity to invade RBCs in a mouse model.1
Evidence of RBC Invasion and Transmission by Transfusion
The researchers infected mice intratracheally to model inhalational tularemia, the most likely form in which the bacteria would be delivered as a weapon. After inoculation, to assess whether invasion of RBCs occurred, blood smears from the mice were examined with Wright-Giemsa stains. At 4 days post-infection, dark bodies were apparent in and outside the RBCs. Using double-immunofluorescence microscopy analysis and a gentamicin protection assay (as intracellular bacteria would be protected from the effects of gentamicin), the researchers determined that the bacteria were definitively intracellular. Transfusion of infected RBCs to naïve mice produced rapid disease, confirming the virulence of intraerythrocytic F.tularensis.1
Human RBCs Infected In Vitro
F.tularensis was co-cultivated with human RBCs to determine whether they, like mice RBCs, were capable of becoming infected. Using double-immunofluorescence microscopy and gentamicin protection assays, intracellular presence of F.tularensis was confirmed. However, the bacteria did not demonstrate evidence of replication within the RBCs. Results were further confirmed with scanning electron microscopy, transmission electron microscopy, and flow cytometry. The research team next determined that it is likely that the complement receptor is used by the bacteria to gain entry into the RBC. There are other noncomplement related factors involved as well.1,2
Clinical Implications of RBC Invasion
There are several clinical implications of this important finding.1,2
1. If RBC invasion is a significant occurrence in vivo, it may help to explain such phenomenon as relapse after treatment. The recommended treatment duration with antimicrobials is 14 days.3 This is much shorter than the 120-day lifespan of an RBC, which may serve as a sanctuary site for the bacteria. Before recommending the extension of antimicrobial therapy, though, another question needs to be answered: What proportion of F.tularensis-infected RBCs would be cleared by the spleen? The lack of replication may forestall damage to the RBC and prevent removal by the splenic macrophages.
2. Can F.tularensis be transmitted via blood transfusion, as was clearly demonstrated in a mouse model?
3. What role do infected host-erythrocytes play in vector species such as ticks, deer flies, and mosquitoes? Do RBCs protect the bacteria from digestion in the vector? Do infected RBCs play a role in transmission from vector species?
4. RBCs lack the ability to pinocytize the first line treatment (aminoglycosides),1 and doxycycline has poor penetration as well,4 but other antimicrobials active against tularemia (eg, chloramphenicol, fluoroquinolones) do penetrate RBCs. Would this difference in RBC penetration produce a discernible clinical effect, and should it influence treatment guidelines?
Horzempa J, O'Dee DM, Stolz DB, Franks J, Clay D, Nau GJ. Invasion of erythrocytes by Francisella tularensis. J Infect Dis 2011;204:51-59.
Conlan JW. Francisella tularensis: A red-blooded pathogen. J Infect Dis 2011;204:6-8.
Dennis DT, Inglesby TV, Henderson DA, et al. Consensus statement: Tularemia as a biological weapon: Medical and public health management. JAMA 2001;285:2763-2773.
Deshmukh PV, Badgujar PC, Gatne MM. In-vitro red blood cell partitioning of doxycycline. Indian J Pharmacol. 2009;41:173-5.
Kornguth ML, Kunin CM. Uptake of antibiotics by human erythrocytes. J. Infect. Dis. 1976;133:175–184.