Abstract
Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in magnesium uptake, we demonstrated that FmvB was outer membrane-localized. Finally, ΔfmvB was found to be attenuated in mice and cytokine analyses revealed that ΔfmvB-infected mice produced lower levels of pro-inflammatory cytokines, including GM-CSF, IL-3, and IL-10, compared with mice infected with wild-type F. tularensis. Taken together, although the function of FmvA remains unknown, FmvB appears to play a role in magnesium uptake and F. tularensis virulence. These results may provide new insights into the importance of magnesium for intracellular pathogens.
Highlights
Francisella tularensis is an intracellular Gram-negative bacterium and the etiological agent of lethal disease tularemia
When the SchuS4 genome was published in 2005 [21], the authors noted the presence of five orthologs of unknown function and these orthologs had no homologs in other bacteria
Because of the complex nature of iron uptake by F. tularensis, including functional compensation among the three previously-characterized paralogs [23, 26], our studies required the use of single and double-gene deletion mutants, growth studies in several different iron-limiting media, and transcriptional analyses to confirm that fmvA and fmvB were not involved in F. tularensis iron uptake
Summary
Francisella tularensis is an intracellular Gram-negative bacterium and the etiological agent of lethal disease tularemia. Holarctica cause human disease [2]. Subspecies tularensis, known as Type A, is the most virulent and is found exclusively in North America [1, 3]. Type A strains are extremely infectious, with fewer than 25 organisms capable of causing severe disease and death in humans [4]. Holarctica, known as Type B, causes similar clinical disease in humans but has a higher infectious dose (
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