# Boosting Immunogenicity of a Recombinant Mycobacterium smegmatis Strain via Zinc-Dependent Ribosomal Proteins

## TL;DR

A recombinant strain of Mycobacterium smegmatis shows better protection against tuberculosis than BCG, with zinc and ribosomal proteins playing key roles in its effectiveness.

## Contribution

The study identifies a recombinant M. smegmatis strain with enhanced immunogenicity and explores the role of zinc and ribosomal proteins in vaccine efficacy.

## Key findings

- The IKEPLUS strain of M. smegmatis provides better survival benefits than BCG in a TB mouse model.
- Zinc is crucial for M. smegmatis biofilm formation and protective efficacy against Mtb.
- Variants of IKEPLUS induce higher macrophage apoptosis compared to wild-type M. smegmatis.

## Abstract

Tuberculosis (TB) continues to be a major global health burden and kills over a million people annually. New immunization strategies are required for the development of an efficacious TB vaccine that can potentially induce sterilizing immunity. In this study, we first confirmed that a live vaccine strain of Mycobacterium smegmatis, previously designated as IKEPLUS, conferred a higher survival benefit than the Bacillus Calmette-Guerin (BCG) in a murine model of intravenous Mycobacterium tuberculosis (Mtb) infection. We have shown that there was a significant increase in the expression of the Rv0282 gene, which is encoded in the esx-3 locus, which played an important role in iron uptake when IKEPLUS was grown in both low zinc and iron-containing Sauton medium. We then confirmed using in vitro assays of biofilm formation that zinc plays a vital role in the growth and formation of M. smegmatis biofilms. IKEPLUS grown in low zinc media led to the better protection of mice after intravenous challenge with a very high dosage of Mtb. We also showed that various variants of IKEPLUS induced apoptotic cell-death of infected macrophages at a higher rate than wild-type M. smegmatis. We next attempted to determine if zinc containing ribosomal proteins such as rpmb2 could contribute to protective efficacy against Mtb infection. Since BCG has an established role in anti-mycobacterial efficacy, we boosted BCG vaccinated mice with rmpb2, but this did not lead to an increment in the protection mediated by BCG.