Elucidation of a mechanism of oxidative stress regulation in Francisella tularensis live vaccine strain

Abstract

Summary Francisella tularensis causes a lethal human disease known as tularemia. As an intracellular pathogen, Francisella survives and replicates in phagocytic cells, such as macrophages. However, to establish an intracellular niche, Francisella must overcome the oxidative stress posed by the reactive oxygen species (ROS) produced by the infected macrophages. OxyR and SoxR/S are two well-characterized transcriptional regulators of oxidative stress responses in several bacterial pathogens. Only the OxyR homolog is present in F. tularensis, while the SoxR homologs are absent. The functional role of OxyR has not been established in F. tularensis. We demonstrate that OxyR regulates oxidative stress responses and provides resistance against ROS, thereby contributing to the survival of the F. tularensis subsp. holarctica live vaccine strain (LVS) in macrophages and epithelial cells and contributing to virulence in mice. Proteomic analysis reveals the differential production of 128 proteins in the oxyR gene deletion mutant, indicating its global regulatory role in the oxidative stress response of F. tularensis. Moreover, OxyR regulates the transcription of the primary antioxidant enzyme genes by binding directly to their putative promoter regions. This study demonstrates that OxyR is an important virulence factor and transcriptional regulator of the oxidative stress response of the F. tularensis LVS.

Lack of OxyR and KatG Results in Extreme Susceptibility of Francisella tularensis LVS to Oxidative Stress and Marked Attenuation In vivo

“…Overall, it appears that OxyR, as expected, regulates katG in the LVS strain, however, the regulation does not completely abolish its expression as is the case observed for various other bacterial species, e.g., E. coli (Michán et al, 1999), Salmonella enterica (Morgan et al, 1986), Haemophilus influenza (Whitby et al, 2012), or Moraxella catarrhalis (Hoopman et al, 2011). In both the present study, and in the previous study, it was observed that the lack of OxyR led to marked suppression of ahpC2 (Ma et al, 2016). In addition Ma et al demonstrated suppressed expression of both katG and sodB in Δ oxyR by real-time PCR and demonstrated that OxyR binds to the upstream promoter regions of each gene.…”

“…A similar investigation has been performed recently by Ma et al which studied the role of OxyR in LVS (Ma et al, 2016). It was found that OxyR controlled transcription of katG and the findings agree with the reduced catalase activity of Δ oxyR observed in the present study.…”

“…The F. tularensis AhpC enzyme is important for the detoxification of ${\text{O}}_2^{-}$ and peroxynitrite (ONOO − ), but not of H 2 O 2 , in the highly virulent SCHU S4 strain (Binesse et al, 2015), but the importance in the LVS strain is yet unknown. F. tularensis also encodes an oxyR homolog, the role of which has been studied recently (Ma et al, 2016). It was found that the absence of OxyR rendered LVS defective for oxidative stress defense, growth in macrophages and epithelial cells, and virulence in mice.…”

“…As aforementioned, antioxidant enzymes, such as catalase, AhpC, SodC, and SodB, all contribute to the virulence of F. tularensis in mice, although each appears to render the bacterium only moderately attenuated and this indicates that the antioxidant system of F. tularensis is complex and may in part possess overlapping functions (Lindgren et al, 2007; Ma et al, 2016). Indeed, a double deletion mutant of katG and ahpC has not been possible to generate in F. tularensis (Binesse et al, 2015) and this demonstrates that the cooperative functions of these enzymes are crucial, although either one is not essential.…”

“…The aim of the present study was to better understand this interconnecting web of antioxidants in F. tularensis . To this end, a double deletion mutant, Δ oxyR/ Δ katG , was generated since this mutant, besides lack of catalase activity, should have a repressed expression of OxyR-regulated antioxidant genes, one of which is AhpC (Ma et al, 2016). We hypothesized that the lack of both KatG and OxyR would lead to a severely impaired phenotype of F. tularensis LVS.…”