Francisella tularensis uses cholesterol and clathrin-based endocytic mechanisms to invade hepatocytes

Abstract

Francisella tularensis are highly infectious microbes that cause the disease tularemia. Although much of the bacterial burden is carried in non-phagocytic cells, the strategies these pathogens use to invade these cells remains elusive. To examine these mechanisms we developed two in vitro Francisella-based infection models that recapitulate the non-phagocytic cell infections seen in livers of infected mice. Using these models we found that Francisella novicida exploit clathrin and cholesterol dependent mechanisms to gain entry into hepatocytes. We also found that the clathrin accessory proteins AP-2 and Eps15 co-localized with invading Francisella novicida as well as the Francisella Live Vaccine Strain (LVS) during hepatocyte infections. Interestingly, caveolin, a protein involved in the invasion of Francisella in phagocytic cells, was not required for non-phagocytic cell infections. These results demonstrate a novel endocytic mechanism adopted by Francisella and highlight the divergence in strategies these pathogens utilize between non-phagocytic and phagocytic cell invasion.

Figure 1. F. novicida colonization of murine hepatocytes.

(A) Immunofluorescent and phase micrographs showing wild-type F. novicida, albumin, and DNA DAPI localization in infected liver tissue section harvested from mice challenged via the intraperitoneal route. Arrows indicate uninfected albumin-negative cells, large arrowheads point to infected albumin-positive cells containing uncountable levels of F. novicida and the small arrowhead points to an albumin-positive cells cells with few internalized bacteria respectively. Scale bar = 10m. (B) Quantification of cells infected with F. novicida based on immunofluorescent images of tissue sections stained with anti-mouse albumin and anti F. novicida antibodies. n (below) is defined as the number of tissue section regions collected in which image stacks were taken (each image stack represents between 500 to 1500 cells). Mouse 1 (n = 17), Mouse 2 (n = 21), and Mouse 3 (n = 17). *** P<0.0001.

Figure 2. F. novicida invades and replicates within BNL CL.2 and NMuLi hepatocytes.

(A) Immunofluorescent images of F. novicida infections in cultured hepatocytes. Hepatocytes were fixed following 24 h F. novicida infections with both BNL CL.2 and NMuLi cells. Samples were labelled with anti-F. novicida antibodies (green), fluorescent phalloidin to indicate the cell boundaries (red) and DAPI (blue). Arrowheads indicate some of the bacteria within the infected cells. Scale bar = 10m. (B) Quantification of the proportion of infected hepatocytes as assessed by microscopic examination. NMuLi (n = 17) and BNL CL.2 (n = 16). (C) Titre of intracellular F. novicida at various timepoints following infection of BNL CL.2 and NMuLi cell infections by invasion assay (n = 3).

Figure 3. Internalization of F. novicida into BNL CL.2 cells is a clathrin- and cholesterol-dependent process that requires actin.

(AD) BNL. CL.2 cells were pre-treated with the indicated drugs and incubated with F. novicida for 22 h followed by a 2 h gentamicin treatment for CFU enumeration. (A) Clathrin inhibitors: 80M monodansylcadaverine (MDC) or 5.0M chlorpromazine (CPZ). (B) Cholesterol inhibitors: 2.5mM MCD or 80M MDC supplemented with 1mM cholesterol. (C) Individual or a combination of 2.5mM MCD and 80M MDC. (D) Cytoskeleton inhibitors: 5M cytochalasin D (CytD), 2.5M latrunculin A (LN A) or 2.5M nocodazole (ND), * P<0.01. ** P< 0.05. (E) Western blot shows that the overall level of clathrin heavy-chain (HC) protein remains unaltered after 24 h F. novicida infections of BNL CL.2 cells compared to uninfected cells. For immunolocalization experiments, untreated cells were infected with F. novicida for 8h, fixed and stained with (F) an anti-F. novicida antibody (green), anti-clathrin HC (red) and DAPI or (G) an anti-F. novicida antibody (green) and filipin (blue) as well as phase contrast. Arrowheads indicate some of the bacteria thatco-localize with the other labels. Scale bar = 5m.

Figure 4. Internalization of F. novicida into NMuLi cells is a clathrin- and cholesterol dependent process that requires actin.

NMuLi cells were pre-treated with the indicated drugs and incubated with F. novicida for 22 h followed by a 2 h gentamicin treatment for CFU enumeration. (A) Clathrin inhibitors: 80M monodansylcadaverine (MDC) or 5.0M chlorpromazine (CPZ). (B) Cholesterol inhibitors: 2.5mM MCD or 80M MDC supplemented with 1mM cholesterol. (C) Individual or combination of 2.5mM MCD and 80M MDC. (D) Cells were treated simultaneously with 2g/mL progesterone and 10M nystatin (Prog/Nys). Cytoskeleton inhibitors: 5M cytochalasin D (CytD), 2.5M latrunculin A (LN A) * P<0.01, ** P<0.05.

Figure 5. F. novicida entry does not utilize caveolin-dependent or macropinocytosis pathways.

(A) BNL CL.2 and NMuLi cells were treated with the caveolin-inhibitor filipin and infected with F. novicida for 22 h followed by 2 h gentamicin treatment for invasion assays. (B) Immunolocalization of caveolin-1 (green) and DAPI (blue) of uninfected BNL CL.2 cells or following 8 h, 16 h, and 24 h infections with F. novicida showed no co-localization between F. novicida and caveolin-1. Arrowheads indicate the localization of F. novicida. Scale bar = 5m. (C) 5mM of amiloride was used to pre-treat BNL CL.2 cells and HeLa cells for 15 min prior to infection with F. novicida and Salmonella Thyphimurium (SL1344), respectively. Untreated cells were treated with media containing DMSO. Cells infected with F. novicida for 6 h followed by 2 h gentamicin treatment did not show a decrease in invasion while cells infected with S. Typhimurium for 30 min followed by 1 h gentamicin treatment showed a significant decrease in invasion *P<0.01.

Figure 6. Clathrin-associated adaptor proteins Esp15 and AP-2 are crucial for F. novicida invasion into non-phagocytic cells.

(A, D) BNL CL.2 cells were infected with F. novicida for 8 h and immunolocalized with anti-F. novicida together with (A) anti-Eps15 (red) or (D) anti-AP-2 -adaptin (green) antibodies. Arrowheads indicate bacteria and protein co-localization. Scale bar = 5m. (B and E). Western blots show the overall protein levels of (B) Eps15 (145kDa) or (E) AP-2 -adaptin (50kDa) in 8 h F. novicida infected cells (untreated with RNAi) remained unchanged compared to uninfected control. Cells transiently transfected with siRNA (48 h) showed a significant reduction in the targeted Eps15 or AP-2 -adaptin proteins as compared to cells transfected with siRNA control pool (CP). (C and F) Reduced Eps15 and AP-2 -adaptin lead to a significant defect in F. novicida internalization 24 h post-infection. *P<0.05.