Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection

Abstract

BackgroundPichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein.ResultsThe recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the α-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to ~ 46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O-mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb.ConclusionsrPstS-1 (98.9% sequence identity, O-mannosylated, and without tags) was produced and secreted by P. pastoris, demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1059-3) contains supplementary material, which is available to authorized users.

Integrative proteomic and glycoproteomic profiling of Mycobacterium tuberculosis culture filtrate

“…Another interesting protein in terms of its proposed role as active infection biomarker is PstS1, a periplasmic lipoprotein involved in phosphate transport across the membrane. It has been identified as a ConA interacting protein [18,20] and its immunoreactivity was proposed to be related to O-mannosylation [66], but direct evidence of its O-glycosylation in culture filtrate in first provided here (S5 Fig). Interestingly, the O-glycosylation site assignation differs from what was observed in the mycobacterial cassette expression system [20] or in a P. pastoris recombinant version of this protein [66].…”

Potential Plasticity of the Mannoprotein Repertoire Associated to Mycobacterium tuberculosis Virulence Unveiled by Mass Spectrometry-Based Glycoproteomics

“…Although mannoproteins are of promising interest, the biologic functions of the protein glycan chains in Mtb still remain elusive [30,64]. Most of our knowledge is inferred from the studies dedicated to the immuno-dominant antigen Apa-an alanine and proline rich mannoprotein-specifically secreted by live bacilli [26,[65][66][67]. Several Apa's major biologic properties have been clearly ascribed to the presence of the mannosidic appendages.…”

A potent subset of Mycobacterium tuberculosis glycoproteins as relevant candidates for vaccine and therapeutic target

“…According to global tuberculosis report in 2022, the estimated number of deaths from tuberculosis, increased between 2019 and 2021 (including 187,000 people with HIV ) and also the burden of resistant to at least one first line anti TB (DR-TB) is estimated to have enhanced from 2020 to 2021 2 . These reports demonstrate the urgency in perception the pathogenicity of M. tuberculosis , as well as the inevitability to promote novel therapeutic strategies for the treatment or prevent the disease 3 . Therefore, contrastive analyses of M. tuberculosis isolates have determined pathogenicity and virulence factors required for M. tuberculosis survival and distribution inside the host, as well candidate proteins (genes) for the development of new TB vaccines or drugs 4 .…”