# M13 phages engineered with chlamydia phage φCPG1 protein IN5 and arginine-glycine-aspartic acid inhibits Chlamydia trachomatis intracellular growth

## TL;DR

Engineered M13 phages with specific proteins and peptides can reduce Chlamydia trachomatis infection by targeting virulence genes.

## Contribution

A novel recombinant M13 phage with RGD and IN5 proteins is shown to inhibit Chlamydia trachomatis growth more effectively than previous versions.

## Key findings

- M13-RGD8-IN53 phage significantly reduces Chlamydia trachomatis infection.
- The phage downregulates several C. trachomatis virulence-related genes.
- RGD enhances phage permeability into cells, improving treatment efficacy.

## Abstract

•The recombinant M13-RGD8-IN53 could significantly reduce C. t infection.•The M13-RGD8-IN53 phage was better than the M13-IN53 phage in ameliorating C.t infection.•Treatment with the recombinant M13-RGD8-IN53 and M13-IN53 phages downregulated C. t genes related to virulence.

The recombinant M13-RGD8-IN53 could significantly reduce C. t infection.

The M13-RGD8-IN53 phage was better than the M13-IN53 phage in ameliorating C.t infection.

Treatment with the recombinant M13-RGD8-IN53 and M13-IN53 phages downregulated C. t genes related to virulence.

Chlamydia trachomatis (C. t) is the most common causative agent of sexually transmitted bacterial urogenital infections worldwide. C. t treatment failure is increasing because antibiotic resistance has developed in recent years. Therefore, the development of novel therapeutic strategies is necessary. Here, we constructed an M13 phage carrying two functional peptides, including the integrin binding peptide arginine-glycine-aspartic acid (RGD) on pⅧ and the IN5 protein from Chlamydia caviae phage φCPG1 on pIII to reduce C. t infection. We called these phages M13-RGD8-IN53. The recombinant phages successfully expressed IN5 proteins. Confocal laser scanning microscopy confirmed that the recombinant phages were able to enter HeLa cells and C. t inclusion bodies. IN5 protein was responsible for the observed decrease in C. t infection, while RGD enhanced the permeability of phages into the cells. The M13-RGD8-IN53 phage was better than the M13-IN53 phage in ameliorating C. t infection. qPCR revealed that treatment with the recombinant phages downregulated several C. t genes related to virulence, such as CT_046 (Hc2), CT_443 (OmcB), CT_444 (OmcA), CT_456 (Tarp), CT_666 (Cdsf), CT_694, CT_743 (Hc1), and CT_875 (TepP). The only upregulated gene was CT_119 (IncA). The recombinant phages impacted the C. t mainly in the middle and late stages of the development cycle. Our results suggest that novel recombinant phages are promising as candidates to treat C. t infection.