Lipid Phenotype of Two Distinct Subpopulations of Mycobacterium bovis Bacillus Calmette-Guérin Tokyo 172 Substrain

Takashi Naka,Shinji Maeda,Mamiko Niki,Naoya Ohara,Saburo Yamamoto,Ikuya Yano,Jun-Ichi Maeyama,Hisashi Ogura,Kazuo Kobayashi,Nagatoshi Fujiwara

doi:10.1074/jbc.m111.310037

Abstract

Bacillus Calmette-Guérin (BCG) Tokyo 172 is a predominant World Health Organization Reference Reagent for the BCG vaccine. Recently, the BCG Tokyo 172 substrain was reported to consist of two subpopulations with different colony morphologies, smooth and rough. Smooth colonies had a characteristic 22-bp deletion in Rv3405c of the region of difference (RD) 16 (type I), and rough colonies were complete in this region (type II). We hypothesized that the morphological difference is related to lipid phenotype and affects their antigenicity. We determined the lipid compositions and biosynthesis of types I and II. Scanning electron microscopy showed that type I was 1.5 times longer than type II. Phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) were found only in type I. Although it has been reported that the RD16 is involved in the expression of PGL, type II did not possess PGL/PDIM. We examined the ppsA-E gene responsible for PGL/PDIM biosynthesis and found that the existence of PGL/PDIM in types I and II is caused by a ppsA gene mutation not regulated by the RD16. PGL suppressed the host recognition of total lipids via Toll-like receptor 2, and this suggests that PGL is antigenic and involved in host responses, acting as a cell wall component. This is the first report to show the difference between lipid phenotypes of types I and II. It is important to clarify the heterogeneity of BCG vaccine substrains to discuss and evaluate the quality, safety, and efficacy of the BCG vaccine.

Highlights

The heterogeneity of substrains of Bacillus Calmette-Guérin (BCG), the only available tuberculosis vaccine, was studied
To produce a stable and safe vaccine, it is important to clarify the differences of each substrain and estimate whether BCG substrains have similar protective potency
BCG substrains obtained from the Pasteur Institute before 1927 produced methoxyMA, but those obtained after 1931 failed to synthesize methoxy-Mycolic acids (MAs) [2]

Summary

Background

The heterogeneity of substrains of Bacillus Calmette-Guérin (BCG), the only available tuberculosis vaccine, was studied. PGL suppressed the host recognition of total lipids via Toll-like receptor 2, and this suggests that PGL is antigenic and involved in host responses, acting as a cell wall component This is the first report to show the difference between lipid phenotypes of types I and II. More than 14 BCG substrains are used throughout the world, and the heterogeneity of their genomics has been studied These strains are different from each other and the original BCG strain and possibly affect the efficacy of the vaccine [2,3,4,5]. The smooth colonies showed a 22-bp deletion in Rv3405c of RD16 (type I), and rough colonies were complete in this region (type II) Both genotypes and colony morphologies have been found in common BCG Tokyo 172 vaccine preparations, but the major population was always type I. We demonstrate the heterogeneity of lipid components in types I and II and clarify the lipid phenotypes, their biosynthesis, and their functions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION