Biology subjects

Sayes, F.

Publications and source records attributed to Sayes, F..

6 recordsLinked to original sources

bioRxiv2025

Improved Immune Responses and Tuberculosis Protection by Aerosol Vaccination with recombinant BCG expressing ESX-1 from Mycobacterium marinum

The currently licensed anti-tuberculosis (TB) vaccine, Mycobacterium bovis BCG, provides limited protection against pulmonary TB in adolescents and adults, the main cause of TB transmission and mortality. To obtain an improved BCG-based vaccine candidate with increased immune signaling but still low virulence, we have previously generated a recombinant BCG strain named BCG::ESX-1Mmar, which is heterologously expressing ESX-1 functions of Mycobacterium marinum and thereby modulates the host innate immune responses via phagosomal rupture-associated induction of type I interferon production and enhanced inflammasome activity, leading to superior protection against TB disease in murine infection models. As protection may also vary with the route of vaccination, here, we have explored aerosol vaccination relative to subcutaneous vaccination, using BCG Pasteur and BCG::ESX-1Mmar. We found that mice vaccinated via the aerosol route with BCG Pasteur or BCG::ESX-1Mmar both yielded higher frequencies of CD4+ and CD8+ T effector memory (TEM) cells in the lungs compared to subcutaneously immunised mice, whereas comparable poly-functional Th1 (IL-2, TNF- and IFN-{gamma}) cytokine-producing subsets were observed in the spleens of all vaccinated mice. Significantly higher IL-17 responses without severe lung pathology were seen in the lungs of aerosol-vaccinated mice associated to local and transient inflammatory cytokine responses and immune cell infiltrations. Aerosol vaccination also elicited high amounts of humoral IgG and IgM responses in the bronchoalveolar lavage fluid and induced substantial lung CD69+ CD103+ T resident memory (TRM) cells, containing both CD4+ and CD8+ T subsets, in the airways of immunised mice, whereas this was not the case for subcutaneous vaccination. These effects led to significant improved protection against M. tuberculosis and reduced lung pathology in aerosol-vaccinated mice compared to subcutaneously vaccinated mice. Moreover, BCG::ESX-1Mmar induced enhanced T-cell immunity and superior protection compared to parental BCG Pasteur for both vaccination routes and thereby represents an interesting candidate for developing improved vaccination strategies against TB. Author summaryAnti-tuberculosis vaccine efficacy is influenced by multiple parameters, including the immunogenicity of the vaccine strain, the type of preclinical host model used, and the route of vaccination. Given recent advances in the field of mucosal vaccination, in the current study we were particularly interested to explore and compare aerosol-based vaccination with standard subcutaneous vaccination in a C57BL/6J mouse model using our recently developed recombinant BCG::ESX-1Mmar vaccine candidate in comparison with parental BCG Pasteur. Our results show that in this setting the protective efficacy of mucosal vaccination was superior to subcutaneous vaccination for both vaccine strains, whereby the use of BCG::ESX-1Mmar induced additional benefits in terms of bacterial load reduction compared to standard BCG Pasteur. Taken together, we propose that aerosol vaccination using BCG::ESX-1Mmar as live-attenuated vaccine candidate is a promising and powerful combination for obtaining improved protection against an M. tuberculosis challenge, a concept that can now be tested in other animal models in a perspective of a putative clinical trial.

microbiology↗

bioRxiv2025

Nitrate-responsive Mycobacterial Intracytoplasmic Membranes dampen Inflammation during Mycobacterium tuberculosis Infection

Subcellular compartmentalization of metabolic processes is a main feature of prokaryotic and eukaryotic architecture. Environmental bacteria generate intracytoplasmic membranes (ICMs) as a crucial strategy to adapt their metabolism to environmental changes. While pathogenic intracellular bacteria also perceive various stressful stimuli during host interactions, the subsequent re-organization of their internal architecture has not been explored. Using cryo-electron tomography, we show that Mycobacterium tuberculosis (Mtb), a major human pathogen, is able to form ICMs outside and inside host cells in a strain-dependent manner. We characterize these Mycobacterial intracytoplasmic Membranes (MIMs) as nitrate-induced structures involved in regulation of metabolism. Furthermore, we uncover that MIM formation during macrophage infection correlates with the ability of Mtb to dampen cellular inflammatory responses. Our findings reveal a previously uncharacterized cytoplasmic structure in Mtb and link it to a functional mechanism that enables the bacterium to adapt to its intracellular niche.

microbiology↗

bioRxiv2025

Evaluation of the role of whiB6 and kdpDE in the dominant multidrug resistant clone Mycobacterium tuberculosis B0/W148

Multidrug resistant (MDR) strains of Mycobacterium tuberculosis represent an obstacle to eradicating tuberculosis (TB) due to the low treatment success rate of MDR TB. Among them, the MDR B0/W148 clone has recently evolved from the M. tuberculosis Beijing lineage 2 and widely disseminated in Russia and Europe. To get more insights into the genetic factors underlying the evolutionary success of the MDR M. tuberculosis B0/W148 clone in addition to environmental and patient-related features, we focused on two mutations specific from this clone that are found in the transcriptional regulators WhiB6 and KdpDE and investigated in a H37Rv strain background the transcriptional profile associated with these mutations and their impact on the in vitro and in vivo growth characteristics. Through the construction and use of H37Rv{Delta}whiB6, H37Rv{Delta}kdpDE, and complemented strains, we found that both mutations did not impair the in vitro growth of M. tuberculosis in standard mycobacterial growth media. The mutation T51P in whiB6 prevented the upregulation of 9 genes in the esx-1 core region and 44 genes elsewhere in the genome, while the deletion of two nucleotides in kdpD leads to a fusion protein of KdpD with KdpE that inhibits the transcriptional activity of KdpE. Both mutations did not lead to hypervirulence in a mouse infection model. These results point to the role of other MDR B0/W148 specific mutations in the wide geographic diffusion of this clone, and/or put in question a hypothesized hypervirulence as driving factor for this large dissemination. ImportanceHuman tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, remains a global public health issue estimated to have been responsible for 1.25 million deaths in 2023. Multidrug resistant (MDR) strains of M. tuberculosis, resistant to rifampicin and isoniazid, lead to lower treatment success. Among them, the MDR B0/W148 clone has widely disseminated in Russia and Europe. To get more insights into the genetic factors underlying the evolutionary success of this clone, we investigated two strain-specific mutations found in the transcriptional regulators WhiB6 and KdpDE. By constructing and analysing laboratory M. tuberculosis strains carrying these specific mutations, we found numerous changes in their transcriptional profiles, whereas we observed only little impact of these mutations on virulence of M. tuberculosis in a mouse infection model. Our study provides new insights into the transcriptional landscape of the selected MDR strains, although no direct connection to virulence could be established.

microbiology↗

bioRxiv2024

A marine sponge-associated mycobacterium closely related to Mycobacterium tuberculosis

Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis.

microbiology↗

bioRxiv2020

Phthiocerol dimycocerosates from Mycobacterium tuberculosis increase the membrane activity of bacterial effectors and host receptors

Mycobacterium tuberculosis (Mtb) synthesizes a variety of atypical lipids that are exposed at the cell surface and help the bacterium infect macrophages and escape elimination by the cells immune responses. In the present study, we investigate the mechanism of action of one family of hydrophobic lipids, the phthiocerol dimycocerosates (DIM/PDIM), major lipid virulence factors. DIM are transferred from the envelope of Mtb to host membranes during infection. Using the polarity-sensitive fluorophore C-Laurdan, we visualized that DIM increase the membrane polarity of a supported lipid bilayer put in contact with mycobacteria, even beyond the site of contact. We observed that DIM activate the complement receptor 3, a predominant receptor for phagocytosis of Mtb by macrophages. DIM also increased the activity of membrane-permeabilizing effectors of Mtb, among which the virulence factor EsxA. This is consistent with previous observations that DIM help Mtb disrupt host cell membranes. Taken together, our data show that transferred DIM spread within the target membrane, remodel lipid organization and increase the activity of host cell receptors and bacterial effectors, diverting in a nonspecific manner host cell functions. We therefore bring new insight into the molecular mechanisms by which DIM increase Mtbs capability to escape the cells immune responses.

microbiology↗

bioRxiv2018

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

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 {Delta}ppe38/71-associated secretion defect therefore also occurs in the M. bovis-derived tuberculosis vaccine BCG and could be 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.

microbiology↗