Identification of potential nucleomodulins of Mycoplasma bovis by direct biotinylation and proximity-based biotinylation approaches

Abstract

Mycoplasma bovis (M. bovis) is a significant bovine pathogen associated with various diseases, including bovine bronchopneumonia and mastitis resulting in substantial economic losses within the livestock industry. However, the development of effective control measures for M. bovis is hindered by a limited understanding of its virulence factors and pathogenesis. Nucleomodulins are newly identified secreted proteins of bacteria that internalize the host nuclei to regulate host cell gene expression and serve as critical virulence factors. Although recent reports have initiated exploration of mycoplasma nucleomodulins, the efficiency of conventional techniques for identification is very limited. Therefore, this study aimed to establish high-throughput methods to identify novel nucleomodulins of M. bovis. Using a direct biotinylation (DB) approach, a total of 289 proteins were identified including 66 high abundant proteins. In parallel, the use of proximity-based biotinylation (PBB), identified 28 proteins. Finally, seven nucleomodulins were verified to be nuclear by transfecting the bovine macrophage cell line BoMac with the plasmids encoding EGFP-fused proteins and observed with Opera Phenix, including the known nucleomodulin MbovP475 and six novel nucleomodulins. The novel nucleomodulins were four ribosomal proteins (MbovP599, MbovP678, MbovP710, and MbovP712), one transposase (MbovP790), and one conserved hypothetical protein (MbovP513). Among them, one unique nucleomodulin MbovP475 was identified with DB, two unique nucleomodulins (MbovP513 and MbovP710) with PBB, and four nucleomodulins by both. Overall, these findings established a foundation for further research on M. bovis nucleomodulin-host interactions for identification of new virulence factors.

Keywords: Mycoplasma bovis; direct biotinylation; nucleomodulin; proximity-based biotinylation; secreted proteins.

Figure 1

Schematic illustration of the DB (A) and PBB (B) approaches.

Figure 2

Identification of potential nucleomodulins by the DB approach. (A) After co-incubation with biotin-labeled M. bovis protein, the nuclei and cytoplasm were isolated and detected. (B) The ratio between the IBAQ of 66 proteins in DB approach and these proteins in the total protein of M. bovis. (C) The COG functional distribution of potential M. bovis nucleomodulins by DB approach.

Figure 3

Identification of nucleomodulins by the PBB approach. (A) Localization of V5-H2B and biotin observed by confocal microscopy. DAPI: the nuclei labeled with blue; V5-tag: V5-H2B labeled with Green; Biotin: biotin labeled with red. (B) Silver staining assay was used to detect the different bands between the control and the H2B groups. (C) The venn diagram shows the number of proteins identified in the control and H2B group by LC–MS/MS. (D) The COG functional distribution of potential M. bovis nucleomodulins by the PBB approach.

Figure 4

Verification of nucleomodulins by the DB approach. The localization of potential nucleomodulins encoded by pEGFP plasmid (pEGFP-C1, pEGFP-116, pEGFP-280, pEGFP-475, pEGFP-582, pEGFP-699, pEGFP-791, and pEGFP-798) in BoMac was detected by fluorescence microscopy at 24 h post-transfection. The cells transfected with pEGFP-C1 served as control.

Figure 5

Verification of nucleomodulins by the PBB approach. The localization of potential nucleomodulins encoded by pEGFP plasmids (pEGFP-C1, pEGFP-513, pEGFP-599, pEGFP-678, pEGFP-710, pEGFP-712, and pEGFP-790) in BoMac was detected by fluorescence microscopy at 24 h post-transfection. The cells transfected with pEGFP-C1 served as the control.

Figure 6

Comparative analysis on potential nucleomodulins identified by the PBB and DB (A) or DB-H (B) approaches. DB-H: direct biotinylation (high abundance proteins).