Comparative Proteomics Analysis of Human Macrophages Infected with Virulent Mycobacterium bovis

Abstract

Mycobacterium bovis (M. bovis), the most common pathogens of tuberculosis (TB), is virulent to human and cattle, and transmission between cattle and humans warrants reconsideration concerning food safety and public health. Recently, efforts have begun to analyze cellular proteomic responses induced by Mycobacterium tuberculosis (M. tb). However, the underlying mechanisms by which virulent M. bovis affects human hosts are not fully understood. For the present study, we utilized a global and comparative labeling strategy of isobaric tag for relative and absolute quantitation (iTRAQ) to assess proteomic changes in the human monocyte cell line (THP-1) using a vaccine strain and two virulent strains H37Rv and M. bovis. We measured 2,032 proteins, of which 61 were significantly differentially regulated. Ingenuity Pathway Analysis was employed to investigate the canonical pathways and functional networks involved in the infection. Several pathways, most notably the phagosome maturation pathway and TNF signaling pathway, were differentially affected by virulent strain treatment, including the key proteins CCL20 and ICAM1. Our qRT-PCR results were in accordance with those obtained from iTRAQ. The key enzyme MTHFD2, which is mainly involved in metabolism pathways, as well as LAMTOR2 might be effective upon M. bovis infection. String analysis also suggested that the vacuolar protein VPS26A interacted with TBC1D9B uniquely induced by M. bovis. In this study, we have first demonstrated the application of iTRAQ to compare human protein alterations induced by virulent M. bovis infections, thus providing a conceptual understanding of mycobacteria pathogenesis within the host as well as insight into preventing and controlling TB in human and animal hosts' transmission.

Keywords: M. bovis; M. tb; THP-1 cell; iTRAQ; pathway analysis; proteomics.

Figure 1

The general work flow.

Figure 2

Venn diagrams show the overlap of quantified proteins.

Figure 3

Venn diagrams display the overlap of differentially expressed proteins.

Figure 4

Comparison of GO term annotation for significantly regulated proteins. (A) Molecular Function, (B) biological process, (C) cellular component, and (D) protein class.

Figure 5

Functional characterization of significantly altered proteins in THP-1 cells infected with MTBC strain H37Rv, M. bovis, or BCG. (A) Top canonical pathways, (B) diseases and disorders, (C) molecular and cellular functions, (D) physiological system development and functions, and (E) toxicity functions.

Figure 6

The detailed view of the top-score networks. Red indicates significantly up-regulated proteins, and green indicates significantly down-regulated proteins. White indicates the proteins that were not identified in our data. The color depth reflects the fold-change of proteins. The shapes are indicative of the molecular class. Lines with arrows connecting between the molecules indicate molecular relationships. Solid lines indicate direct interactions, and dashed lines indicate indirect interactions. (A) The top-score IPA network of BCG infection: cellular development, cellular growth and proliferation, cell death and survival, (B) the top two IPA networks of H37Rv infection: cellular movement, hematological system development and function, and immune cell trafficking (left); cancer, cell death, and survival, and cellular movement (right), (C) the top two IPA networks of M. bovis infection: free radical scavenging, hereditary disorder, and immunological disease (left); cell death and survival, organismal injury and abnormalities, and tissue morphology (right). Additional networks are depicted in Table S4.

Figure 7

STRING analysis revealing the interaction partners of the significantly altered proteins. Red color indicates 31 proteins involved in vesicle category.

Figure 8

Real-time RT-PCR analysis of significantly altered proteins in THP-1 cells infected by MTBC (10 MOI) or a mock. (A) BCG in black bars, (B) H37Rv in gray bars, (C) M. bovis in dark gray bars.

Figure 9

STRING analysis revealing 27 interaction partners of the significantly altered proteins. The blue oval indicates unique proteins involved in M. bovis induced proteins; the red square indicates the common proteins involved in virulent strains-H37Rv and M. bovis induced proteins.