Biology subjects

Vilcheze, C.

Publications and source records attributed to Vilcheze, C..

5 recordsLinked to original sources

bioRxiv2026

Polyketide synthase 12 is an in vivo essential source of novel mycolyl lipids in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is a major pathogen worldwide that infects and transmits only among humans, yet nearly all in vivo virulence research occurs in non-human hosts. To overcome this central challenge in tuberculosis research, we leveraged a dataset of more than 50,000 sequenced isolates to identify Mtb genes that that are functionally preserved during natural infection, disease causation, and transmission between humans. This whole-genome ranking identified polyketide synthase 12 (pks12 ) as an exceptionally in vivo essential gene in human tuberculosis, which we validated in zebrafish and mouse models. Though Pks12 produces a mycoketide lipid in only trace amounts, pks12 deletion severely altered the host-facing surface and arabinoglycan architecture of Mtb. This amplified effect was explained through the discovery of mannosyl-{beta}-1-phosphomycoketide monomycolate (MPMMM), which is synthesized from the known Pks12 product at higher abundance by antigen 85 mycolyltransferases. Thus, we used a new host-facing genomic-metabolomic-phenotypic approach to discover the functions of an in vivo essential Mtb gene, which controls the physical structure of the Mtb-host interface.

microbiology↗

bioRxiv2026

Visualizing early Mycobacterium tuberculosis interactions with murine lung macrophages using intravital imaging

Intravital microscopy enables direct visualization of dynamic cellular processes within intact tissues, but its application to Mycobacterium tuberculosis (Mtb) has been limited by Biosafety Level 3 (BSL-3) containment requirements and the technical challenges of stabilizing the lung for high-resolution imaging. Here, we present a protocol that combines the thoracic Window for High-Resolution Imaging of the murine Lung (WHRIL) with a genetically defined, triple-auxotrophic Mtb strain (mc27902) approved for use under BSL-2 conditions. We describe the construction of a tdTomato-expressing derivative (mc28471) preparation of bacteria for intravenous infection and intravital imaging in reporter mice. This system enables visualization of rapid bacterial entry into the pulmonary vasculature, subsequent aggregation, and vascular occlusion, dissemination into the lung parenchyma, and macrophage uptake over three days post-infection. This protocol provides the first practical platform for real-time intravital imaging of mycobacteria in the lung and establishes a foundation for mechanistic studies of bacterial physiology, host recognition, and immune-mediated clearance using safe Mtb surrogates. SummaryThis protocol describes a biosafety level 2 (BSL-2)-compatible intravital imaging platform for visualizing Mycobacterium tuberculosis (Mtb) in the intact murine lung at single cell resolution. By combining the Window for High-Resolution Imaging of the murine Lung (WHRIL) with a fluorescently labeled, genetically defined triple auxotrophic Mtb strain (mc27902), this approach overcomes long-standing biosafety and technical barriers that have prevented real-time imaging of mycobacterial infection in vivo. The method enables direct visualization of early bacterial localization, aggregation, vascular interactions, and macrophage uptake during the initial hours to days following infection, providing a practical foundation for mechanistic studies of host-pathogen interactions under safe experimental conditions.

immunology↗

bioRxiv2025

Loss of the ESX-5 secretion locus in Mycobacterium tuberculosis reshapes the mycomembrane and enhances ESX-1 substrate secretion

The ESX-5 secretion system, uniquely found in slow-growing mycobacteria, is predicted to secrete over 150 proteins across the inner membrane of Mycobacterium tuberculosis (M.tb). Although many of these substrates are believed to promote M.tb virulence, most remain poorly characterized. Here, we use a complete locus deletion strain of ESX-5 in M.tb to examine the molecular changes caused by a broad loss in ESX-5 secretory substrates. We confirmed the selective loss of PE/PPE proteins secreted by ESX-5 into both the culture filtrate (CF) and outer mycomembrane (OMM) fractions of the M.tb {Delta}esx5 mutant. In examining other ESX systems, we found that ESX-1 substrate levels were increased in both the CF and OMM fractions of the {Delta}esx5 mutant. Conversely, the ESX-3 locus was transcriptionally repressed upon ESX-5 deletion. We noted that the {Delta}esx5 mutant had altered morphology in the form of wrinkled distortions of the bacterial surface. Likewise, we identified increased susceptibility of the {Delta}esx5 mutant to a variety of large (molecular weight >550 g/mol) antimicrobial compounds, suggesting that an intact ESX-5 system is required for M.tb to exclude such molecules. Our findings suggest that removing the ESX-5 system from M.tb fundamentally alters the properties of the mycobacterial OMM and impacts the expression and secretion activity of other ESX systems. Significance StatementMycobacterium tuberculosis (M.tb) uses the ESX-5 secretion system to export numerous proteins that shape host-pathogen interactions. Here, we found that deleting ESX-5 from M.tb not only prevented the secretion of many ESX-5 substrates but also impacted other ESX systems. The M.tb {Delta}esx5 mutant had increased ESX-1 substrate secretion but reduced ESX-3 expression. In addition, the M.tb {Delta}esx5 mutant displayed altered cell surface morphology and increased vulnerability to large antibiotic drugs, suggesting a critical role for ESX-5 for maintaining outer membrane integrity. These findings highlight ESX-5 as a central modulator of secretion and cell envelope composition with implications for drug targeting and vaccine development.

microbiology↗

bioRxiv2024

Intravenous Immunization with Triple Auxotrophs of Mycobacterium tuberculosis: A novel vaccine strategy against tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a leading infectious cause of mortality worldwide despite widespread use of the BCG vaccine and the availability of sterilizing pharmacopoeia. Recent research indicates that the intravenous administration of BCG confers sterilizing immunity against Mtb pulmonary challenge in non-human primates. However, while BCG is relatively safe, complications such as disseminated BCGosis have been observed in immunocompromised individuals. Double auxotrophic mutants of Mtb lacking the ability to synthesize leucine and pantothenate are safe and sterilized in immunocompromised mice and SIV-infected Rhesus macaques. We examined how immunization with a Mtb triple auxotrophic strain, mc27902, which cannot synthesize leucine, pantothenate, and arginine, protects immunocompetent mice from a virulent Mtb infection. The route of immunization was a crucial factor for protection with mc27902 with intravenous immunization being 100 times more effective in protecting immunocompetent mice from Mtb challenge when compared to conventional subcutaneous vaccination with BCG. To further increase the safety of the attenuated auxotroph for vaccine purposes, the type VII secretion system Esx1 responsible for BCG attenuation was deleted in mc27902. When tested by prime-boost immunization of immunocompetent mice followed by aerosol challenge with virulent Mtb, mc27902 {Delta}esx1 provided similar protection to mc27902. This robust protection against Mtb infection conferred by mc27902 and mc27902 {Delta}esx1 in a mouse model paves the way for new TB vaccine development using highly attenuated, auxotrophic Mtb strains.

microbiology↗

bioRxiv2023

The PDIM paradox of Mycobacterium tuberculosis: new solutions to a persistent problem

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous mutations that cause PDIM loss leading to virulence attenuation and increased cell wall permeability. We discovered that PDIM loss is due to a metabolic deficiency of methylmalonyl-CoA that impedes the growth of PDIM-producing bacilli. This can be remedied by supplementation with odd-chain fatty acids, cholesterol, or vitamin B12. We developed a much-needed facile and scalable routine assay for PDIM production and show that propionate supplementation enhances the growth of PDIM-producing bacilli and selects against PDIM-negative mutants, analogous to in vivo conditions. Our results solve a major issue in tuberculosis research and exemplify how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

microbiology↗