Research Article: 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

Date Published: June 18, 2018

Publisher: Public Library of Science

Author(s): Louis S. Ates, Fadel Sayes, Wafa Frigui, Roy Ummels, Merel P. M. Damen, Daria Bottai, Marcel A. Behr, Jeroen W. J. van Heijst, Wilbert Bitter, Laleh Majlessi, Roland Brosch, David M. Lewinsohn.


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 Δppe38/71-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.

Partial Text

Tuberculosis is the deadliest infectious disease worldwide and is responsible for more than 1.7 million deaths per year [1]. Its causative agent, Mycobacterium tuberculosis, is a slow growing bacterium inherently resistant to many antibiotics. This problem is further exacerbated by rising levels of acquired drug resistance, resulting in multi-drug-resistant (MDR) and extensively-drug-resistant (XDR) strains of M. tuberculosis, which require treatment regimens of two years with low treatment success rates and severe side effects [1–3]. These worrying developments highlight the need for a successful vaccine, halting the transmission of tuberculosis [4]. The currently used vaccine is based on Mycobacterium bovis, attenuated through serial culture by Calmette and Guérin and therefore known as Bacille Calmette-Guérin (BCG) [5–7]. BCG is generally believed to protect relatively well against severe forms of disseminated tuberculosis in children, but is unable to induce full protection or halt transmission of M. tuberculosis in adolescents and adults [4,8,9]. Furthermore, even these protective traits are subject to controversy, which may be caused by the plethora of genomic mutations and recombination events that have accrued during the worldwide sub-culturing of the original BCG strain [5,6,10,11].

We previously demonstrated that loss-of-function mutations in the ppe38-locus of M. tuberculosis block PE_PGRS and PPE-MPTR secretion and increase virulence in a mouse model [37]. In this work, we examined the correlation of known ppe38 deletions in other lineages of the MTBC with a PE_PGRS/PPE-MPTR secretion defect. We hypothesized that the success of certain clinical isolates of Lineage 4 could perhaps be explained by their RD5-like deletion, which includes ppe38 [51,83]. However, secretion analysis of these Lineage 4 strains revealed that a single copy of PPE71 carrying a 21 bp deletion (corresponding to the loss of amino acids MGGAGAG), seems to be functional and sufficient to support PE_PGRS secretion. Similarly, although intriguing differences in protein secretion levels were observed between M. canettii strains, we found that all analyzed strains secreted PE_PGRS proteins. The anticipated polymorphisms in the ppe38-locus of selected M. canettii strains [48] were likely caused by a sequence assembly problem of repetitive sequences. These results highlight the difficulties of bio-informatic analyses of this locus, which is hampered by the high sequence similarity between ppe38 and ppe71, that seem to cause already some discrepancies between the reference genomes of M. tuberculosis H37Rv and CDC1551 [29,37,38,84].