Fig. 4: Repurposing the bacterial Type III secretion system (T3SS) for PNA delivery and release to clear intracellular infections of mammalian cells.

a Schematic showing the experimental setup for comparing PNA delivery into HeLa cells for treating an intracellular infection of Salmonella enterica serovar Typhimurium, strain SL1344 expressing GFP (STm-GFP), using either the bacterial T3SS inherent to STm-GFP (Bottom, T3SS-PNA) or CPP facilitated uptake (Top, Naked-PNA). All intracellular infection treatment experiments follow the general protocol of 45-min infection of HeLa cells 24 h after seeding, 75-min gentamicin treatment to remove extracellular bacteria, and 18-h incubation with treatment in media containing gentamicin to ensure an only intracellular infection model. For comparing T3SS delivered PNA to naked-PNA infection was done using STm-GFP at an MOI of 10. For the T3SS-PNA condition 10 µM of PNA was added during this 45-min infection stage; during this time the PNA is allowed to enter the Salmonella and subsequently be transported into the HeLa cell as it infects via its T3SS. For the naked-PNA condition 10 µM of PNA is added to the gentamicin containing media that the HeLa cells are incubated in for 18 h. Post 18 h of treatment HeLa cells are fixed for imaging or lysed for colony forming unit (CFU) analysis. b Representative images of HeLa cells uninfected (top left), infected and without treatment (bottom left), infected and treated with Naked-PNA (top right), and infected and treated with T3SS-PNA (bottom right). HeLa cells are stained with the nuclear stain DAPI (blue), the membrane stain Phalloidin (pink), and green pixels represent intracellular STm-GFP (green). Images show an evident decrease in STm-GFP during T3SS-α-rpsD or T3SS-α-lexA treatment compared to no treatment or with naked-PNA treatment. c Percent infection (CFU/mL of treatment normalized to no treatment) in the presence or absence of treatment. Significant reduction in STm in HeLa cells when PNA is delivered using the T3SS. Naked-PNA treatment does not produce significant therapeutic effect. d Schematic showing the experimental setup comparing the delivery and release approach to no treatment of an intracellular infection of HeLa cells. The same procedure was followed as in part A with few modifications. Pretreatment of the delivery STm (STm-GFP modified with holin release switch) is done by incubating in either 10 µM of PNA (bottom, T3SS/holin-PNA) or PBS (top, no treatment) for 45 min to allow for PNA uptake. Then HeLa cells are infected with a 4:1 ratio of delivery STm (green) to target STm (red), making the total MOI 10. After removing extracellular bacteria, the infected HeLa cells are incubated for 18 h; during this time the delivery STm releases the PNA to treat the Target STm strain. After 18-h of incubation the HeLa cells are lysed to determine the number of intracellular colony forming units. e CFU ratio of normalized target STm to normalized delivery STm (see methods section for normalization) shows significant reduction compared to no treatment. Triangles represent individual biological replicates of HeLa cells. f Percent infection of target STm (Target STm CFU normalized to no treatment) when using a non-T3SS bacteria (E. coli MG1655) as the delivery strain shows no reduction (p > 0.05). g Lysed ESBL KPN (CFU/mL) from intracellular infection in RAW264.7 macrophages at 1 MOI and treated for 18 h with delivery STm at 10 MOI carrying α-rpsD. All data shown are the average of three biological replicates with standard deviation shown as error bars. Grey circles or blue triangles indicate individual biological replicates.