Establishing the Role of FTL_1042 in the Intracellular Growth and Survival of Francisella tularensis

Open Access
Author:
Avanzato, Victoria Alina
Area of Honors:
Immunology and Infectious Disease
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Girish Soorappa Kirimanjeswara, Thesis Supervisor
  • Pamela Hankey, Honors Advisor
Keywords:
  • Francisella tularensis
  • novel antibiotics
  • trans-translation
  • virulence factors
  • vaccines
Abstract:
Francisella tularensis, the causative agent of tularemia, is a gram-negative bacteria with a wide host range such as ticks, mites, rabbits, and deer. Humans can become infected through arthropod bites, or ingestion/inhalation of contaminated material. Tularemia has multiple clinical manifestations, including the cutaneous, ulceroglandular, and pulmonary forms. The pulmonary form is the most severe and the infectious dose in humans is very low. F. tularensis has been used as a bioweapon and hence, has been classified as a select agent by the CDC. Potential for emergence of antibiotic resistance, either natural or intentional, coupled with lack of a licensed vaccine makes it hard to treat tularemia in case of an epidemic or bioterror attack. This study pursues two important avenues in preventing and treating Francisella infections: development of novel antibiotics and determination of virulence factors for vaccine development. Virulence determinants of F. tularensis have not been clearly established and are the focus of active research of many labs. The first section of this study presents an evaluation of the activity of KKL-10, a broad-spectrum inhibitor of the trans-translation pathway, against Francisella tularensis. The trans-translational pathway has evolved as a means to protect against translational complexes that have stalled on mRNA during protein translation, and is essential for the survival of many bacteria species, presenting an optimal target for novel antibiotics. KKL-10 was effective in arresting the growth of Francisella tularensis spp in liquid cultures and intracellularly at nanomolar concentrations. The compound appeared to be bacteriostatic rather than bactericidal at the concentrations used. KKL-10 was also found to inhibit highly virulent strain SchuS4 at minimum inhibitory concentration similar to that observed for LVS. Together, these results suggest that trans-translation pathway is a promising target for developing new antibiotics and that KKL-10 has a broad-spectrum activity even in intracellular compartments of a host cell. The second section of this study aims to characterize a novel lipoprotein proposed to function in the virulence of Francisella. Francisella infectivity potentiator B (Fip B) has previously been shown to contribute to intracellular growth of a virulent strain of F. tularensis. FTL_1042, an ortholog of FipB, has been identified in the Live Vaccine Strain (LVS) of F. tularensis. However, the function of this gene has not been described. The objective of the study was to characterize the function of FTL_1042. An in-frame deletion mutant strain of LVS lacking FTL_1042 was created by using allele replacement and confirmed by sequencing. The wild type LVS and Δ1042 grew at similar rate in liquid culture. However, intracellular levels of Δ1042 were significantly lower than the wild type 24 hours post infection in macrophages. These results show that FTL_1042 is involved in the intracellular growth and survival of F. tularensis LVS strain.