OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability inFrancisella tularensisLVS

Abstract

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.

PdpC, a secreted effector protein of the type six secretion system, is required for erythrocyte invasion by Francisella tularensis LVS

“…appear to exert cumulative effects and are likely to work in combination for full virulence. This does not appear to the be case for erythrocyte invasion, as we have recently characterized the function of OpiA in F. tularensis LVS and it did not have any effect on erythrocyte invasion (Cantlay et al, 2020). OpiA was of interest because it has been identified as a PI (3) kinase and had been shown to act on host macrophage membranes (Ledvina et al, 2018).…”

“…To create a construct in which a pdpC-emgfp allele was expressed from the strong Francisella FGRp promoter ( Horzempa et al., 2008b ), pdpC was cloned into pSC18 ( Cantlay et al., 2020 ), a vector containing emgfp and FGRp . The full-length pdpC gene, amplified from chromosomal DNA with SC4/SC5, was cloned into the Eco RI- Sal I site of pFNLTP8 ( Maier et al., 2004 ) to generate pSC10.…”

“…The plasmid pKHEG ( Cantlay et al., 2020 ) was mobilized into the pdpC -null mutant strain by electroporation. THP-1 human monocytes (ATCC) were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gemini Bioproducts), 25 mM HEPES (Corning), 2 mM Glutagro (Corning), 1mM Sodium Pyruvate (Gibco) and 0.05 mM 2-mercaptoethanol at 37°C with 5% CO 2 to a density of 5x10 8 cells ml -1 , washed once in warm growth medium and incubated in 35-mm dishes at 37°C with 5% CO 2 with wild type and mutant F. tularensis LVS strains containing emgfp on the plasmid pKHEG at an MOI of 100 for 24 hours.…”

An Overview of Anti-Eukaryotic T6SS Effectors

“…The protein is translocated into phagocytic cells and reduces the levels of TNF-α, a pro-inflammatory cytokine from monocytes required to block intracellular replication. OpiA contributes to the pathogenesis of F. tularensis, as demonstrated using a chicken embryo infection model (Cantlay et al, 2020).…”

“…It belongs to a family of bacterial PI3K enzymes and also plays a role in evasion of innate immunity in host cells by reducing the levels of TNF-α. Eshraghi et al, 2016;Ledvina et al, 2018;Cantlay et al, 2020 OpiB Contributes to intracellular growth in phagocytic cells. The C-terminus is homologous to the ankyrin repeat domains and the N-terminus corresponds to an evolutionarily conserved cysteine protease.…”