Proteomic analysis of infected primary human leucocytes revealed PSTK as potential treatment-monitoring marker for active and latent tuberculosis.

Markers for monitoring clearance of Mycobacterium tuberculosis (Mtb) infection during anti-TB drug treatment could facilitate management of tuberculosis (TB) treatment, but are lacking. We aimed to screen for Mtb clearance markers from in-vitro-infected leucocytes and to evaluate these markers in followed-up active TB (ATB) patients and latent TB (LTBI) cases after anti-TB drug treatment. Extracellular proteins from primary leucocytes infected with each of the Mtb lineages (East-Asian, Indo-Oceanic, Euro-American and the laboratory strain H37Rv) were screened as possible clearance markers. Leucocytes infected with Staphylococcus aureus acted as controls. The proteomic analysis was performed using GeLC-MS/MS. Several quantitative and qualitative candidate clearance markers were found. These proteins were suppressed during the infection stage of all Mtb lineages and re-expressed after bacillary clearance. PSTK, FKBP8 and MGMT were common clearance markers among the four Mtb lineages in our model. Only PSTK was a potential clearance marker based on western blot validation analysis from culture supernatants. The PSTK marker was further validated with western blot analysis using serum samples (n = 6) from ATB patients and LTBI cases during anti-TB drug treatment, and from healthy controls (n = 3). Time-dependent increase of PSTK was found both in ATB and LTBI patients during the course of anti-TB drug treatment, but not in healthy controls. We have demonstrated that PSTK is a potential treatment-monitoring marker for active and latent TB.

Comparative Proteomics of Activated THP-1 Cells Infected with Mycobacterium tuberculosis Identifies Putative Clearance Biomarkers for Tuberculosis Treatment.

Biomarkers for determining clearance of Mycobacterium tuberculosis (Mtb) infection during anti-tuberculosis therapy or following exposure could facilitate enhanced monitoring and treatment. We screened for biomarkers indicating clearance of Mtb infection in vitro. A comparative proteomic analysis was performed using GeLC MSI/MS. Intracellular and secreted proteomes from activated THP-1 cells infected with the Mtb H37Rv strain (MOI = 1) and treated with isoniazid and rifampicin for 1 day (infection stage) and 5 days (clearance stage) were analyzed. Host proteins associated with early infection (n = 82), clearance (n = 121), sustained in both conditions (n = 34) and suppressed by infection (n = 46) were elucidated. Of the potential clearance markers, SSFA2 and CAECAM18 showed the highest and lowest protein intensities, respectively. A western blot of CAECAM18 validated the LC MS/MS result. For three clearance markers (SSFA2, PARP14 and PSME4), in vivo clinical validation was concordantly reported in previous patient cohorts. A network analysis revealed that clearance markers were enriched amongst four protein interaction networks centered on: (i) CD44/CCND1, (ii) IFN-ß1/NF-κB, (iii) TP53/TGF-ß and (iv) IFN-γ/CCL2. After infection, proteins associated with proliferation, and recruitment of immune cells appeared to be enriched possibly reflecting recruitment of defense mechanisms. Counteracting proteins (CASP3 vs. Akt and NF-κB vs. TP53) associated with apoptosis regulation and its networks were enriched among the early and sustained infection biomarkers, indicating host-pathogen competition. The BRCA1/2 network was suppressed during infection, suggesting that cell proliferation suppression is a feature of Mtb survival. Our study provides insights into the mechanisms of host-Mtb interaction by comparing the stages of infection clearance. The identified clearance biomarkers may be useful in monitoring tuberculosis treatment.