Analysis of the secretome and identification of novel constituents from culture filtrate of bacillus Calmette-Guerin using high-resolution mass spectrometry

Abstract

Tuberculosis (TB) is an infectious bacterial disease that causes morbidity and mortality, especially in developing countries. Although its efficacy against TB has displayed a high degree of variability (0%-80%) in different trials, Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been recognized as an important weapon for preventing TB worldwide for over 80 years. Because secreted proteins often play vital roles in the interaction between bacteria and host cells, the secretome of mycobacteria is considered to be an attractive reservoir of potential candidate antigens for the development of novel vaccines and diagnostic reagents. In this study, we performed a proteomic analysis of BCG culture filtrate proteins using SDS-PAGE and high-resolution Fourier transform mass spectrometry. In total, 239 proteins (1555 unique peptides) were identified, including 185 secreted proteins or lipoproteins. Furthermore, 17 novel protein products not annotated in the BCG database were detected and validated by means of RT-PCR at the transcriptional level. Additionally, the translational start sites of 52 proteins were confirmed, and 22 proteins were validated through extension of the translational start sites based on N-terminus-derived peptides. There are 103 secreted proteins that have not been reported in previous studies on BCG [corrected] secretome and are unique to our study. The physicochemical characteristics of the secreted proteins were determined. Major components from the culture supernatant, including low-molecular-weight antigens, lipoproteins, Pro-Glu and Pro-Pro-Glu family proteins, and Mce family proteins, are discussed; some components represent potential predominant antigens in the humoral and cellular immune responses.

Fig. 1.

Venn diagram of the number of identifications predicted to be secreted by different programs. The culture filtrate proteins were predicted by the algorithms SignalP 4.0, TatP 1.0, PRED-LIPO, and SecretomeP 2.0, respectively. The numbers of proteins predicted by each program and all possible combinations are indicated in the Venn diagram.

Fig. 2.

Distributions of total culture filtrate proteins and secreted proteins identified in this study. The distribution of identifications in different (A) pI ranges, (B) molecular weight (MW) ranges, (C) subcellular localizations, and (D) functional categories. The culture filtrate proteins identified are illustrated in the blue histogram, and the secreted proteins are in red.

Fig. 3.

N-terminal extension of two gene models using peptide mapping upstream of the annotated translational start sites. A, four unique peptides (red lines) mapped to the upstream region of the annotated gene BCG_1741c (blue box). The gene prediction programs FgeneSB and GeneMark predicted the presence of a longer gene model extending the N-terminus of the gene (yellow box). The orthologous protein from M. tuberculosis H37Rv supported this N-terminal extension (purple box). B, the protein sequence of BCG_1741c was extended by 53 aa at the N-terminus. The underlined sequence in red indicates 17 unique peptides, including 4 extended N-terminal peptides. C, a non-tryptic N-terminal peptide, A.MPATSVANNSGSMVALATIEACPALPSR.L, mapped to the upstream of the annotated translational start site of BCG_1317. An extension of this protein sequence by 63 aa was also supported by the FgeneSB and GeneMark programs and Blastp searching. The seven unique peptides are underlined. The peptide sequences mapped to the products of genes BCG_1741c and BCG_1317 are indicated in red.

Fig. 4.

Identification of novel gene models based on peptide mapping to the genomic region. A, two unique peptides (red lines) with minimal IonScores of 40 mapped to the genomic region corresponding to a novel protein, BCGRF059986. The presence of this novel gene model was also supported by the FgeneSB and GeneMark programs (yellow box). This novel protein was found to be similar to a hypothetical protein MT3222 in M. tuberculosis H37Ra (purple box). B, protein sequence of a novel gene product. The identified region is in red. C, validation of the novel gene model BCGRF059986 via an RT-PCR approach. The amplified RT-PCR product confirmed the expression of new mRNAs for the novel gene. The size of the product was determined by means of an E-Gel Electrophoresis System using a 2% E-Gel pre-cast agarose gel. DNA Ladder, 1 kb Plus DNA marker (Invitrogen). For BCGRF059986, PCR reaction was performed using the novel gene cDNA as a template. For the negative control, PCR reaction was performed with RNAs as the template. No product displayed in this lane indicated that the RNAs were free of any contaminating genomic DNA. For ß-actin cDNA, a positive control, PCR reaction was performed using human ß-actin cDNA as a template, and the amplified 353-bp product was visualized. D, the transcription of the remaining 16 novel gene models was also confirmed via RT-PCR. PCR fragments of the expected sizes were observed, indicating that the novel genes were transcribed. PCR reactions performed with RNAs as the templates were used as negative controls.