Abstract
Francisella tularensis is the causative agent of tularemia and has gained recent interest as it poses a significant biothreat risk. F. novicida is commonly used as a laboratory surrogate for tularemia research due to genetic similarity and susceptibility of mice to infection. Currently, there is no FDA-approved tularemia vaccine, and identifying therapeutic targets remains a critical gap in strategies for combating this pathogen. Here, we investigate the soluble lytic transglycosylase or Slt in F. novicida, which belongs to a class of peptidoglycan-modifying enzymes known to be involved in cell division. We assess the role of Slt in biology and virulence of the organism as well as the vaccine potential of the slt mutant. We show that the F. novicida slt mutant has a significant growth defect in acidic pH conditions. Further microscopic analysis revealed significantly altered cell morphology compared to wild-type, including larger cell size, extensive membrane protrusions, and cell clumping and fusion, which was partially restored by growth in neutral pH or genetic complementation. Viability of the mutant was also significantly decreased during growth in acidic medium, but not at neutral pH. Furthermore, the slt mutant exhibited significant attenuation in a murine model of intranasal infection and virulence could be restored by genetic complementation. Moreover, we could protect mice using the slt mutant as a live vaccine strain against challenge with the parent strain; however, we were not able to protect against challenge with the fully virulent F. tularensis Schu S4 strain. These studies demonstrate a critical role for the Slt enzyme in maintaining proper cell division and morphology in acidic conditions, as well as replication and virulence in vivo. Our results suggest that although the current vaccination strategy with F. novicida slt mutant would not protect against Schu S4 challenges, the Slt enzyme could be an ideal target for future therapeutic development.
Highlights
Francisella tularensis is a Gram-negative coccobacillus which causes the disease tularemia, or “rabbit fever” in humans
To determine the role of the peptidoglycan-modifying soluble lytic transglycosylase (Slt) enzyme in virulence of Francisella, we initially focused on mutagenesis of the slt gene in live vaccine strain (LVS) and the fully virulent F. tularensis Schu S4 strains
These results suggested slt is an essential gene in LVS and F. tularensis
Summary
Francisella tularensis is a Gram-negative coccobacillus which causes the disease tularemia, or “rabbit fever” in humans. Francisella primarily infects macrophages, which are thought to be the major reservoir for the bacteria in vivo and critical for its ability to cause disease in animals and humans (McLendon et al, 2006). Francisella tularensis is comprised of two subspecies that cause disease in humans, tularensis (type A) and holarctica (type B). A third subspecies, novicida, rarely causes disease in humans, but is commonly used as a surrogate strain for fully virulent F. tularensis. It has a high degree of genetic similarity, can infect macrophages, produces disease in mice, and can be handled under BSL-2 conditions (Kingry and Petersen, 2014)
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