Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for a number of host-pathogen interactions, including phagolysosomal escape and invasion of erythrocytes. One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an opiA-null mutant in the live vaccine strain, F. tularensis LVS. OpiA was not required for erythrocyte invasion; however, deletion of opiA affected growth of F. tularensis LVS in broth cultures in a medium-dependent manner. We also found that opiA influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of F. tularensis. A fluorescently tagged OpiA (OpiA–emerald-green fluorescent protein [EmGFP]) accumulated at the cell poles of F. tularensis, which is consistent with the location of the T6SS. However, OpiA-EmGFP also exhibited a highly dynamic localization, and this fusion protein was detected in erythrocytes and THP-1 cells in vitro, further supporting that OpiA is secreted. Similar to previous reports with F. novicida, our data demonstrated that opiA had a minimal effect on intracellular replication of F. tularensis in host immune cells in vitro. However, THP-1 cells infected with the opiA mutant produced modestly (but significantly) higher levels of the proinflammatory cytokine tumor necrosis factor alpha compared to these host cells infected with wild-type bacteria. We conclude that, in addition to its role in host-pathogen interactions, our results reveal that the function of opiA is central to the biology of F. tularensis bacteria. IMPORTANCE F. tularensis is a pathogenic intracellular pathogen that is of importance for public health and strategic defense. This study characterizes the opiA gene of F. tularensis LVS, an attenuated strain that has been used as a live vaccine but that also shares significant genetic similarity to related Francisella strains that cause human disease. The data presented here provide the first evidence of a T6SS effector protein that affects the physiology of F. tularensis, namely, the growth, cell size, viability, and aminoglycoside resistance of F. tularensis LVS. This study also adds insight into our understanding of OpiA as a determinant of virulence. Finally, the fluorescence fusion constructs presented here will be useful tools for dissecting the role of OpiA in infection.

Francisella tularensis is the cause of the disease tularemia. This bacterium usually infects the host via arthropod bites. F. tularensis is able to invade mammalian erythrocytes which increases tick colonization following a blood meal. We hypothesized that bacterial genes involved in erythrocyte invasion would be upregulated in the presence of red blood cells. mRNA profiling experiments revealed that FTL_0885 was one of the most highly induced genes in the presence of erythrocytes.  We initially attempted to generate deletion and disruption mutants of FTL_0885.  However, these attempts were unsuccessful likely due to the proximity FTL_0885 to a neighboring essential gene.  A transposon insertion mutation was available in the homolog to FTL_0885 in a related bacterium, F. novicida.  F. novicida is not a human pathogen and its ability to invade erythrocytes is poorly characterized.  Therefore, we are reconstructing this mutation in the F. tularensis LVS background so that we may study the effects of this gene on erythrocyte invasion.  Also, because erythrocytes may contain cues important for pathogenesis, we also were interested in determining whether the FTL_0885 mutant is attenuated. To test this, the mutant bacteria will be evaluated for their ability to replicate in host macrophages in vitro.  In addition, the chicken embryo infection model will be used to evaluate in vivo pathogenesis.

Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for a number of host-pathogen interactions including phagolysosomal escape and invasion of erythrocytes.    One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an opiA-null mutant in the live vaccine strain, F. tularensis LVS. OpiA was not required for erythrocyte invasion, however, deletion of opiA affected growth of F. tularensis LVS in broth cultures in a medium-dependent manner.    We also found that opiA influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of F. tularensis.  A fluorescently tagged OpiA (OpiA-EmGFP) accumulated at the cell poles of F. tularensis which is consistent with the location of the T6SS.  However, OpiA-EmGFP also exhibited a highly dynamic localization and this fusion protein was detected in erythrocytes and THP-1 cells in vitro further supporting that OpiA is secreted. Similar to previous reports using F. novicida, our data demonstrated that opiA had a minimal effect on intracellular replication of F. tularensis in host immune cells in vitro. However, THP-1 cells infected with the opiA mutant produced modestly (but significantly) higher levels of the pro-inflammatory cytokine TNF-α compared to these host cells infected with wild-type bacteria.   Together, our results support previous observations that implicate opiA in infection and virulence, but also reveal an additional role for opiA central to the biology of F. tularensis bacteria. (This research was made possible by NASA West Virginia Space Grant Consortium Training Grant #NNX15A101H and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

Francisella tularensis is a gram-negative, intracellular pathogen capable of establishing a lethal infection at a dose of <10 CFU. Fluorescence microscopy is a vital tool used to study F. tularensis host-pathogen interactions. However, F. tularensis is only weakly fluorescent when expressing fluorescent proteins such as emGFP and BFP using previously described molecular tools. In contrast, our laboratory previously described robust expression of tdTomato while under the control of the Francisella glucose-repressible promoter (FGRp; plasmid construct pTC3D). However, F. tularensis bacteria were unable to produce substantial expression of tdTomato or other fluorescent proteins, such as emGFP, while genes encoding these proteins were under the control of the robust groE promoter (groEp). Therefore, we analyzed the DNA upstream of tdtomato in pTC3D and identified an in-frame N-terminal fusion of the first 36 codons of FTL_0580 (580N), followed by code for a three amino linker domain (PAT), and subsequently the start codon of tdTomato potentially producing a stable hybrid protein. The previously identified FGRp alone could not produce robust expression of fluorescent proteins. Moreover, a frame-shift disrupting the coding sequence of 580N resulted in poor fluorescent protein expression. Inclusion of 580N in-frame controlling expression of Emerald GFP, emGFP, under the control of FGRp or groEp, however, resulted in robust fluorescence. We hypothesize that 580N stabilizes the fluorescent protein to which it is fused within F. tularensis. Application of this new biotechnology will expand our ability to investigate F. tularensis through the use of fluorescence microscopy.

Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for a number of host-pathogen interactions including phagolysosomal escape and invasion of erythrocytes.    One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an opiA-null mutant in the live vaccine strain, F. tularensis LVS. OpiA was not required for erythrocyte invasion, however, deletion of opiA affected growth of F. tularensis LVS in broth cultures in a medium-dependent manner.    We also found that opiA influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of F. tularensis.  A fluorescently tagged OpiA (OpiA-EmGFP) accumulated at the cell poles of F. tularensis which is consistent with the location of the T6SS.  However, OpiA-EmGFP also exhibited a highly dynamic localization and this fusion protein was detected in erythrocytes and THP-1 cells in vitro further supporting that OpiA is secreted. Similar to previous reports using F. novicida, our data demonstrated that opiA had a minimal effect on intracellular replication of F. tularensis in host immune cells in vitro. However, THP-1 cells infected with the opiA mutant produced modestly (but significantly) higher levels of the pro-inflammatory cytokine TNF-α compared to these host cells infected with wild-type bacteria.   Together, our results support previous observations that implicate opiA in infection and virulence, but also reveal an additional role for opiA central to the biology of F. tularensis bacteria. (This research was made possible by NASA West Virginia Space Grant Consortium Training Grant #NNX15A101H and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).