RD5-mediated lack of PE_PGRS and PPE-MPTR export in BCG vaccine strains results in strong reduction of antigenic repertoire but little impact on protection

Abstract

Tuberculosis is the deadliest infectious disease worldwide. Although the BCG vaccine is widely used, it does not efficiently protect against pulmonary tuberculosis and an improved tuberculosis vaccine is therefore urgently needed. Mycobacterium tuberculosis uses different ESX/Type VII secretion (T7S) systems to transport proteins important for virulence and host immune responses. We recently reported that secretion of T7S substrates belonging to the mycobacteria-specific Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins of the PGRS (polymorphic GC-rich sequences) and MPTR (major polymorphic tandem repeat) subfamilies required both a functional ESX-5 system and a functional PPE38/71 protein for secretion. Inactivation of ppe38/ 71 and the resulting loss of PE_PGRS/PPE-MPTR secretion were linked to increased virulence of M. tuberculosis strains. Here, we show that a predicted total of 89 PE_PGRS/PPE-MPTR surface proteins are not exported by certain animal-adapted strains of the M. tuberculosis complex including M. bovis. This Δ ppe38/71-associated secretion defect therefore also occurs in the M. bovis-derived tuberculosis vaccine BCG and could be partially restored by introduction of the M. tuberculosis ppe38-locus. Epitope mapping of the PPE-MPTR protein PPE10, further allowed us to monitor T-cell responses in splenocytes from BCG/ M. tuberculosis immunized mice, confirming the dependence of PPE10-specific immune-induction on ESX-5/PPE38-mediated secretion. Restoration of PE_PGRS/PPE-MPTR secretion in recombinant BCG neither altered global antigenic presentation or activation of innate immune cells, nor protective efficacy in two different mouse vaccination-infection models. This unexpected finding stimulates a reassessment of the immunomodulatory properties of PE_PGRS/PPE-MPTR proteins, some of which are contained in vaccine formulations currently in clinical evaluation.

Author summary

One of the major findings of the pioneering Mycobacterium tuberculosis H37Rv genome sequencing project was the identification of the highly abundant PE and PPE proteins, named after their N-terminal motifs Pro–Glu (PE) or Pro–Pro–Glu (PPE). Within the 20 years of research since then, many claims were made that PE/PPE proteins, including the two large subgroups encoded by repetitive sequences with very high GC content (PE_PGRS and PPE-MPTR families), are exported to the bacterial surface or beyond, and show broad immunomodulatory impact on host-pathogen interaction. We thus screened strains from different branches of the M. tuberculosis complex, including the attenuated Mycobacterium bovis BCG vaccine strains, for their capacity to export PE_PGRS/PPE-MPTR proteins. Strikingly, we found that BCG strains were unable to export the plethora of PE_PGRS/PPE-MPTR proteins due to the absence of the region of difference RD5, which in M. tuberculosis encodes PPE38, required for PE_PGRS/PPE-MPTR export. Surprisingly, the restoration of PE_PGRS/PPE-MPTR export by genetic complementation in recombinant BCG did not result in immunomodulatory changes or altered protection in mouse models. Our results thus put into perspective the numerous reports on virulence-associated immunomodulatory impact of individual PE_PGRS and PPE-MPTR proteins and open novel questions on their biological function(s).