Engineered phage-based therapeutic materials inhibit Chlamydia trachomatis intracellular infection

Abstract

Developing materials that are effective against sexually transmitted pathogens such as Chlamydia trachomatis (Ct) and HIV-1 is challenging both in terms of material selection and improving bio-membrane and cellular permeability at desired mucosal sites. Here, we engineered the prokaryotic bacterial virus (M13 phage) carrying two functional peptides, integrin binding peptide (RGD) and a segment of the polymorphic membrane protein D (PmpD) from Ct, as a phage-based material that can ameliorate Ct infection. Ct is a globally prevalent human pathogen for which there are no effective vaccines or microbicides. We show that engineered phage stably express both RGD motifs and Ct peptides and traffic intracellularly and into the lumen of the inclusion in which the organism resides within the host cell. Engineered phage were able to significantly reduce Ct infection in both HeLa and primary endocervical cells compared with Ct infection alone. Polyclonal antibodies raised against PmpD and co-incubated with constructs prior to infection did not alter the course of infection, indicating that PmpD is responsible for the observed decrease in Ct infection. Our results suggest that phage-based design approaches to vector delivery that overcome mucosal cellular barriers may be effective in preventing Ct and other sexually transmitted pathogens.

Fig. 1

Schematic of the M13 phage peptide library and gene construction. The phage express high density signaling RGD motifs (~1.5×10¹³ epitopes/cm²) on pVIII and PmpD on pIII.

Fig. 2

M13-RGD₈ uptake in HeLa 229 cells is significantly higher than for WT M13 phage. (A) Quantitated titers of internalized M13-RGD₈ (RGD modification on pVIII protein of M13 phage) in HeLa cells compared with wild type (WT, unmodified M13 phage) show that the recovered M13-RGD₈ was viable and able to infect the bacterial host cells (E. Coli) up to day 2 and then decreased dramatically after that time to day 10 (pfu, plaque forming unit; *, p<0.0001). (B) Uptake is significantly higher in M13-RGD₈ infected cells and peaks at day 1 compared with WT M13 phage. Blue, DAPI; Green, M13-RGD₈; 40x; inset, 100x. Data represent three independent experiments.

Fig. 3

M13-RGD₈ is efficiently internalized by HeLa cells prior to or during infection with Ct. (A) Infection of HeLa cells with Ct reference strain L₂ at an MOI of one; (B) Infection of HeLa cells with M13-RGD₈ at a concentration of 10⁹ pfu/mL; (C) Pre-treatment of HeLa cells with M13-RGD₈ for 2 h prior to Ct infection using the same MOI and pfu as in A and B, respectively; and (D) Co-treatment of HeLa cells with M13-RGD₈ and Ct. (D, Inset) Ct inclusion surrounded by M13-RGD₈. Blue, DAPI (nuclei); Green, M13-RGD₈; Red, Ct (red, magenta). 40x; inset, 100x. (E) Quantitated titers of M13-RGD₈ uptake (y-axis, left) and quantitated Ct IFU (y-axis, right) at 36 h post infection show dramatically increased phage and Ct viability. Data represent three independent experiments.

Fig. 4

M13-RGD₈-PmpD₃ phage are effective in reducing Ct infection in HeLa and primary endocervical (PEC) cells. HeLa and PEC cells were treated with M13-RGD₈-PmpD₃ (10¹¹ pfu/mL) with or without Ct strain L₂ at an MOI of one (see Methods). (A) HeLa cells: Ct infection alone; M13-RGD₈-PmpD₃ alone; Pre-treatment at 2 h with M13-RGD₈-PmpD₃, and then infection with Ct; and Co-infection with M13-RGD₈-PmpD₃ and Ct. Inset, phage surrounding the nucleus; (B) PEC cells: same experimental conditions as for HeLa cells. Inset, co-treatment experiment with M13-RGD₈-PmpD₃ phage surrounding the inclusion of Ct. Blue, DAPI; Green, RGD₈-PmpD₃; Red/magenta, Ct. 40x; insert, 100x. Data represent three independent experiments; (C) Quantitated results of pre-treatment vs co-infection with M13-RGD₈-PmpD₃ on Ct infection show significant reduction in Ct infection in HeLa (black) cells and PEC cells (red hatched) compared with Ct infection alone (*, p<0.001; **, p<0.0001, respectively). Data represent three independent experiments.

Fig. 5

PEC cells co-treated with M13-RGD₈-PmpD₃ and Ct show trafficking of phage into the inclusion. (A) Timeline from 12 h to 23 h; PEC cells infected with M13-RGD₈-PmpD₃ at 12 h; inclusion surrounded by phage at 20 h; invasion of the inclusion by M13-RGD₈-PmpD₃ begins at 24 h and 30 h; disruption of inclusion and dispersal of contents at 36 h. (B) Same timeline as in A except there is no infection with M13-RGD₈-PmpD_3; the inclusions are visibly larger than in A throughout the developmental cycle of the organism. (C and D) Laser scanning microscopy with orthogonal and 3D projection of Z-stack (x–z and y–z plane) imaging using a Zeiss LSM 710 confocal microscope; height of field displays an orthogonal slice through a 3D grid showing M13-RGD₈-PmpD₃ inside the inclusion. (E) laser scanning microscopy with orthogonal Z-stack (x–z and y–z plane) imaging using a Zeiss LSM 710 confocal microscope; height of field displays an orthogonal slice through a 3D grid showing that M13-RGD₈ does not translocate into the lumen of the inclusion. Data represent three independent experiments.

Fig. 6

Polyclonal antibody (Poly-Ab) raised against the PmpD peptide and applied to HeLa cells significantly reduces Ct infection. Infection was reduced by 26.6% using the Poly-Ab compared to Ct infection alone (*, p=0.008). The Poly-Ab complexed with M13-RGD₈PmpD₃ and purified prior to co-infection with Ct failed to impact infection compared with M13-RGD₈PmpD₃ alone (**, p=0.024). Data represent three independent experiments.