Interconnection of the mycobacterial heparin‐binding hemagglutinin with cholesterol degradation and heme/iron pathways identified by proximity‐dependent biotin identification inMycobacterium smegmatis

Abstract

Deciphering protein-protein interactions is a critical step in the identification and the understanding of biological mechanisms deployed by pathogenic bacteria. The development of in vivo technologies to characterize these interactions is still in its infancy, especially for bacteria whose subcellular organization is particularly complex, such as mycobacteria. In this work, we used the proximity-dependent biotin identification (BioID) to define the mycobacterial heparin-binding hemagglutinin (HbhA) interactome in the saprophytic bacterium Mycobacterium smegmatis. M. smegmatis is a commonly used model to study and characterize the physiology of pathogenic mycobacteria, such as Mycobacterium tuberculosis. Here, we adapted the BioID technology to in vivo protein-protein interactions studies in M. smegmatis, which presents several advantages, such as maintaining the complex organization of the mycomembrane, offering the possibility to study membrane or cell wall-associated proteins, including HbhA, in the presence of cofactors and post-translational modifications, such as the complex methylation pattern of HbhA. Using this technology, we found that HbhA is interconnected with cholesterol degradation and heme/iron pathways. These results are in line with previous studies showing the dual localization of HbhA, associated with the cell wall and intracytoplasmic lipid inclusions, and its induction under high iron growth conditions. AbbreviationsAPEX ascorbic acid peroxidase BioID proximity-dependent biotin identification BirA E. coli biotin ligase CW cell wall HbhA heparin-binding hemagglutinin HRP horseradish peroxidase ILI intracytoplasmic lipid inclusions Mtb Mycobacterium tuberculosis PPI protein-protein interactions

Proximity‐dependent biotin identification links cholesterol catabolism with branched‐chain amino acid degradation in Mycobacterium smegmatis

“…To identify proteins that interact with HsaD, each sample of biotinylated proteins from three independent biological replicates was analyzed by mass spectrometry (Table S3). From our previous study 16 and as confirmed by western blot analyses (Figure 1C), overexpression of birA alone may result in nonspecific biotinylation of mycobacterial proteins. Therefore, candidate proteins, as quantified using spectral counting, were selected by comparing their enrichment in M. smegmatis producing the bait of interest, BirA‐HsaD or HsaD‐BirA, over M. smegmatis producing BirA alone.…”

“…Surprisingly, all the interactants identified with MSMEG_1634‐BirA or BirA‐MSMEG_1634 were also enriched (ratio ≥7.8) when MSMEG_1634 was produced alone (Table S6). These proteins were not overrepresented when HsaC or HsaD were produced alone (Table S4 and S5), nor in our previous study with HbhA 16 . Moreover, BkdA, BkdB, BkdC, HrpA, and MSMEG_3726 were not overrepresented in the M. smegmatis strain overproducing MSMEG_1634‐BirA (Table S6).…”

“…In our previous BioID study, we identified an interaction between HsaC and HbhA, when HbhA-BirA was overproduced in M. smegmatis. 16 However, we were not able to detect the reverse interaction using HsaC fused to BirA. This may be due to the low abundance of endogenous HbhA in M. smegmatis.…”

“…For each condition, the number of spectra for one protein was normalized against Replicate 1 and was calculated as follows: Normalized number of spectra (Protein X) = number of spectra (Protein X) in replicate 1 + [number of spectra (Protein X) in replicate 2 × total number of spectra in replicate 1/total number of spectra in replicate 2] + [number of spectra (Protein X) in replicate 3 × total number of spectra in replicate 1/total number of spectra in replicate 3]. Furthermore, as the expression of birA alone may result in nonspecific biotinylation of mycobacterial proteins, 16 the ratio was calculated as follows: Ratio (protein X) = normalized number of spectra with HsaC, HsaD, or MSMEG_1634 in fusion with BirA/normalized number of spectra with BirA alone. For calculation purpose, when the Normalized number of spectra with BirA alone for Protein X was zero, it was arbitrarily set to 0.5 (between 0 and 1 spectrum) in order to calculate a ratio.…”

“…We have recently shown that proximity-dependent biotin identification (BioID) is a powerful tool to detect and characterize protein-protein interactions (PPI) in mycobacteria by defining the heparin-binding hemagglutinin HbhA interactome. 16 Briefly, after generating a fusion protein between the protein of interest and the Escherichia coli biotin ligase (BirA), proximity labelling allows the identification of proteins that strongly, weakly, or transiently interact or are closely associated (within 10-20 nm) with the fusion protein, 17 allowing to define its proxisome (or proximal interactome). 18 In addition, this technology can help to identify multi-partner complexes directly in the organism of interest, preserving the intact subcellular structures, the presence of enzyme cofactors and posttranslational modifications (for review).…”