Mycobacterium tuberculosis AtsG (Rv0296c), GlmU (Rv1018c) and SahH (Rv3248c) Proteins Function as the Human IL-8-Binding Effectors and Contribute to Pathogen Entry into Human Neutrophils

Abstract

Mycobacterium tuberculosis is an extremely successful intracellular pathogen that has evolved a broad spectrum of pathogenic mechanisms that enable its manipulation of host defense elements and its survival in the hostile environment inside phagocytes. Cellular influx into the site of mycobacterial entry is mediated by a variety of chemokines, including interleukin-8 (IL-8), and the innate cytokine network is critical for the development of an adaptive immune response and infection control. Using affinity chromatography, liquid chromatography electrospray ionization tandem mass spectrometry and surface plasmon resonance techniques, we identified M. tuberculosis AtsG arylsulphatase, bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyl transferase (GlmU) and S-adenosyl-L-homocysteine hydrolase (SahH) as the pathogen proteins that bind to human IL-8. The interactions of all of the identified proteins (AtsG, GlmU and SahH) with IL-8 were characterized by high binding affinity with KD values of 6.83x10-6 M, 5.24x10-6 M and 7.14x10-10 M, respectively. Furthermore, the construction of Mtb mutant strains overproducing AtsG, GlmU or SahH allowed determination of the contribution of these proteins to mycobacterial entry into human neutrophils. The significantly increased number of intracellularly located bacilli of the overproducing M. tuberculosis mutant strains compared with those of "wild-type" M. tuberculosis and the binding interaction of AtsG, GlmU and SahH proteins with human IL-8 may indicate that these proteins participate in the modulation of the early events of infection with tubercle bacilli and could affect pathogen attachment to target cells.

Fig 1. SDS-PAGE analysis of the affinity chromatography-purified M. tuberculosis proteins interacting with human IL-8.

(A) Mycobacterial whole-cell extract applied onto the agarose resin (lane 1). (B) Affinity chromatography-purified Mtb proteins binding human IL-8: lane 1-mycobacterial whole-cell lysate, lane 2-flowthrough fraction collected after the washing step, lane 3-eluate fraction 1, lane 4-eluate fraction 2, lane 5-eluate fraction 3. M-protein molecular weight standard.

Fig 2. Western blotting analysis of recombinant M. tuberculosis rAtsG, rGlmU, rSahH, rSerA proteins developed in E. coli and their interactions with human IL-8.

(A) Immunodetection of recombinant Mtb rAtsG (lane 1), rGlmU (lane 2), rSahH (lane 3) and rSerA (lane 4) with anti-His Tag mouse monoclonal IgG1 antibodies. (B) Human IL-8 binding by recombinant Mtb rAtsG (lane 5), rGlmU (lane 6), rSahH (lane 7) and rSerA (lane 8). M-protein molecular weight standard.

Fig 3. Surface plasmon resonance evaluation of protein-protein interplay between recombinant M. tuberculosis rAtsG, rGlmU, rSahH proteins and human IL-8.

Sensorgrams of the binding interaction between IL-8 immobilized onto the CM5 sensor chip and (A) Mtb rAtsG, (B) Mtb rGlmU or (C) Mtb rSahH protein at final concentrations of 2.5 μg, 5 μg, 10 μg and 20 μg at an analyte flow rate of 5 μl/min. The presented kinetic data were calculated from at least three independent runs of each concentration.

Fig 4. Analysis of M. tuberculosis overproducing mutants.

Western blotting determination of the level of AtsG, GlmU or SahH protein biosynthesis by MtbAtsG↑, MtbGlmU↑ or MtbSahH↑ overproducing mutant strain, respectively, compared with the “wild-type” Mtb strain. M-protein molecular weight standard.

Fig 5. Overproduction of AtsG, GlmU or SahH protein increases the attachment/entry of M. tuberculosis into human neutrophils.

(A) Evaluation of the engulfment of the Mtb mutant strain overproducing AtsG (MtbAtsG↑), GlmU (MtbGlmU↑) or SahH (MtbSahH↑) protein by human neutrophils compared with that of the “wild-type” Mtb strain. (B) Effect of prior exposure to IL-8 on the attachment/entry of MtbAtsG↑, MtbGlmU↑, MtbSahH↑ overproducing mutants into human neutrophils compared with IL-8-pre-incubated and IL-8-untreated “wild-type” Mtb strain. Data are presented as mean values (±SD) from two independent experiments comprising of nine or five repetitions, respectively.