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. 2017 May 9:7:173.
doi: 10.3389/fcimb.2017.00173. eCollection 2017.

The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

Deanna M Schmitt  1 , Rebecca Barnes  1 , Taylor Rogerson  1 , Ashley Haught  1 , Leanne K Mazzella  1 , Matthew Ford  1 , Tricia Gilson  1 , James W-M Birch  1 , Anders Sjöstedt  2 , Douglas S Reed  3 , Jonathan M Franks  4 , Donna B Stolz  4 , James Denvir  5 , Jun Fan  5 , Swanthana Rekulapally  5 , Donald A Primerano  5 , Joseph Horzempa  1
Affiliations

The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

Deanna M Schmitt et al. Front Cell Infect Microbiol. .

Abstract

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.

Keywords: erythrocyte invasion; spectrin; tick borne disease; tularemia; type VI secretion system.

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Figures

Figure 1
Figure 1
Residing within erythrocytes enhances the colonization of ticks by F. tularensis by protecting them from low pH. (A,B) Erythrocytes incubated with F. tularensis LVS bacteria to allow for invasion were either left intact or lysed to liberate intracellular bacteria. Each group was fed to A. americanum (A) or I. scapularis (B) ticks using a glass capillary tube. After feeding, the ticks were incubated for 24 h, and then homogenized and plated on media selective for Francisella. Increased colonization occurred with intact erythrocytes in comparison to lysed erythrocytes for both I. scapularis and A. americanum ticks (**p < 0.01; unpaired Student's t-test). (C) F. tularensis LVS bacteria were incubated with human erythrocytes for invasion to occur. Erythrocytes were either left intact or lysed to liberate intracellular bacteria. Cells were then incubated in media containing 10% serum at the pH indicated. At designated time points, viable F. tularensis bacteria were enumerated and statistically significant differences were determined by two-way ANOVA followed by Sidak's multiple comparisons test (*p < 0.05). Data shown are mean ± SD. For all panels, Data are representative of at least three independent experiments in which each individual iteration showed significant differences between the same groups. Each experiment contained at least four separate wells per group.
Figure 2
Figure 2
Bacterial factors are secreted into erythrocytes prior to invasion. F. tularensis Schu S4 was incubated with human erythrocytes for 4 h and processed for immunogold transmission electron microscopy. Ultra-thin sections (70 nm) were probed with anti-F. tularensis rabbit antisera and then a 5 nm gold-labeled anti-rabbit secondary antibody. The black arrows designate the location of F. tularensis antigen within the erythrocyte. Scale bars represent 100 nm.
Figure 3
Figure 3
Erythrocyte invasion by F. tularensis is mediated by components of the type VI secretion system. (A–C) Invasion of human erythrocytes by wild-type F. tularensis LVS, ΔmglA, the type VI secretion system mutants iglC null and ΔdotU, and their corresponding complemented mutant strains, iglC null::pTG28 and ΔdotU::pDotU, was measured by a gentamicin protection assay. F. tularensis bacteria and erythrocytes (“+RBC”) were co-cultured for 3 h. Cells were then treated with gentamicin, washed, lysed, diluted, and plated to enumerate CFUs (mean ± SEM). Control wells excluded erythrocytes (“−RBC”). Data are representative of five or more independent experiments. For each experiment, we observed significant differences between the same groups. Each iteration contained at least four separate wells per group. *p < 0.05 for LVS vs. ΔmglA (A), LVS vs. iglC null (B), and LVS vs. ΔdotU (C); two-way ANOVA followed by Sidak's multiple comparisons test. (D,E) Human erythrocytes incubated with F. tularensis LVS, iglC null, or iglC null::pTG28 were subjected to DIFM. Representative images (D) or a quantification of at least five fields of view per group exhibiting bacteria interacting with erythrocytes (E) indicate that iglC is important for erythrocyte invasion (χ2, p < 0.0001). Ft, F. tularensis; perm, permeabilization; RBC, erythrocyte (red blood cell).
Figure 4
Figure 4
Actin is not required for invasion of erythrocytes by F. tularensis. Erythrocytes were treated with 50 μg/mL cytochalasin D (Cyt.) for 30 min (A) or 16 μM phalloidin for 20 min (B) prior to infection with F. tularensis LVS and then subjected to a gentamicin protection assay to measure invasion. Wells lacking erythrocytes served as controls. Data are expressed as mean CFU ± SD for at least three independent experiments. For each experiment, we observed significant differences between the same groups. Each iteration contained at least 4 separate wells per group. RBC, red blood cell; Veh, vehicle, chloroform; Cyt, cytochalasin D.
Figure 5
Figure 5
Spectrin is required for erythrocyte invasion by F. tularensis. (A) Addition of P. guttatus venom reduces invasion of erythrocytes by F. tularensis LVS. Erythrocytes were treated with 25 μg/mL P. guttatus venom for 20 min prior to infection. Bacteria were co-incubated with the erythrocytes and were subjected to gentamicin protection assay to measure invasion. To confirm that altered membrane fluidics were not responsible for the decreased invasion observed in panel A, erythrocytes were treated with 50 μg/mL mitoxantrone for 30 min prior to infection and subjected to gentamicin protection assay (B). Wells lacking erythrocytes served as controls. For both panels, Data (CFU ± SD) are representative of at least three independent experiments in which each individual iteration showed significant differences between the same groups. Each experiment contained at least 4 separate wells per group. Snake venom (P. guttatus); Mtx, mitoxantrone. ***p < 0.001; two-way ANOVA followed by Sidak's multiple comparisons test.
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