Fate of Francisella tularensis capsule and O-antigen mutants in human macrophages

Francisella tularensis is the causative agent of tularemia and is categorized by the CDC as a Tier 1 select agent. This gram-negative, facultative-intracellular bacterium infects macrophages by escaping the phagosome and replicating with high efficacy in the cytosol. Multiple virulence factors, including capsule and lipopolysaccharide (LPS), are expressed by F. tularensis. Biosynthesis of capsule and LPS O-antigen requires the same O-antigen biosynthesis gene cluster and, together, expression of capsule and O-antigen confer serum resistance. Mutations in the O-antigen biosynthesis gene cluster not only result in serum sensitivity, but also attenuate the ability to cause disease in vivo. In addition to changes in F. tularensis virulence, individual capsule and O-antigen mutants appear to have distinct intracellular phenotypes in macrophages. As previously shown by Lindemann et al. (2011), the capsule and O-antigen mutants FTT1236, FTT1237, and FTT1238 all replicated in human monocyte derived macrophages (MDMs) up to 16 hr and then ceased to replicate after that. This is hypothesized to be due to MDM cytotoxicity. In contrast, Raynaud et al. (2007) showed that the capsule and O-antigen mutant wbtA completely lacked replication in J774 macrophages, the reason for which has not been identified. A potential explanation for the loss of F. tularensis capsule and O-antigen mutant replication is capture and degradation by the host cell's autophagy pathway.

Capture and degradation by autophagy is an accepted innate immune response to many intracellular pathogens. When small subpopulations of bacteria that normally replicate in membrane-bound vacuoles become cytosolic, such as Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium, they are targeted to forming autophagosomes through ubiquitination and binding of autophagy receptors. Pathogens have also developed methods to circumvent recognition and degradation by autophagy. Since F. tularensis replicates in the cytosol, it stands to reason that it has a means of evading detection by autophagy. We propose that expression of capsule and O-antigen acts as a mechanism used by F. tularensis to protect itself in an extracellular environment, as well as during intracellular infection.

In this thesis we characterized nine different capsule and O-antigen mutants, and found different replication phenotypes in MDMs and varying degrees of MDM cytotoxicity. Also, only a subset of the mutants was detected by the autophagy marker, ubiquitin, supporting our hypothesis that different capsule and O-antigen mutants have diverse fates in MDMs. We also show that LVS and Schu S4 wbtA mutants had similar phenotypes. Upon further evaluation, we found that LVS wbtA more readily colocalized with ubiquitin, autophagy receptors, and the autophagy membrane protein LC3B, but not Beclin-1 or LAMP-1. This supports our hypothesis that capsule and O-antigen mutants are more susceptible to recognition by autophagy. Yet, because we did not observe LAMP-1 colocalization, there may be defects in the maturation of autophagosomes to degradative autolysosomes. Finally, we found that the fate of LVS wbtA in MDMs is dissimilar from J774 macrophages, suggesting macrophage species affect mutant fate. This thesis shows that different capsule and O-antigen mutants have multiple fates in MDMs, and suggest that F. tularensis capsule and O-antigen act as protective virulence factors that limit detection by autophagy.