bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.10.30.685640

Genetic interference of distinctive Mycobacterium tuberculosis peptidoglycan modifications enhances β-lactam susceptibility and reveals expression-sensitive host immune dynamics

Silveiro, C.·Marques, M.·Olivenca, F.·Pires, D.·Anes, E.·Catalao, M. J.
View original source ↗Export metadata ↓

Abstract

The high mortality associated with tuberculosis (TB), alongside the lack of efficient therapeutics against emerging multidrug-resistant Mycobacterium tuberculosis (Mtb) strains, emphasizes the need to identify novel antitubercular targets. Mycobacterial peptidoglycan, displaying characteristic modifications comprising the amidation of D-iso-glutamate (D-iGlu) and the N-glycolylation of muramic acid, is a promising therapeutic target. The genes encoding the enzymes mediating these PG modifications (murT/gatD and namH) were silenced in Mtb using CRISPR interference (CRISPRi) to investigate their impact on {beta}-lactam susceptibility and host immune responses. First, qRT-PCR confirmed successful target mRNA knockdown, with variable repression efficiency based on the selected sgRNA, PAM strength, and target site. Phenotypic characterization through spotting dilution and growth curve assays corroborated the essentiality of D-iGlu amidation for mycobacterial survival, in contrast to the N-glycolylation of muramic acid. Moreover, susceptibility assays showed that both PG modifications contribute to {beta}-lactam resistance, with sgRNA2-mediated murT knockdown substantially increasing {beta}-lactam and isoniazid susceptibility. Furthermore, checkerboard assays showed reductions in the minimum fractional inhibitory concentration index (FICImin) value for AMX/MEM+CLA and EMB combinations following the depletion of both PG modifications, with significant differences observed upon namH knockdown. Additionally, D-iGlu amidation was uncovered as a determinant of Mtb survival within THP-1-derived macrophages at 6 days post-infection. Infection of THP-1-derived macrophages with MurT/GatD-depleted Mtb upregulated IL-1{beta} and downregulated IL-10, whereas NamH depletion caused upregulation of both IL-1{beta} and IL-10. Altogether, our findings unveiled the potential of targeting these PG modifications for the development of innovative therapeutic regimens against TB.

Source connections

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Silveiro, C., Marques, M., Olivenca, F., Pires, D., Anes, E., Catalao, M. J.. 2025-10-30. Genetic interference of distinctive Mycobacterium tuberculosis peptidoglycan modifications enhances β-lactam susceptibility and reveals expression-sensitive host immune dynamics. https://doi.org/10.1101/2025.10.30.685640

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Cryo-EM Structure of Mycobacteriophage D29 Reveals a Specialized Portal-Capsid Junction and a Unique 13-Subunit Baseplate Collar

Mycobacteriophages are promising agents against drug-resistant mycobacterial infections, yet their structural organization and host-recognition mechanisms remain poorly understood. Here, we determined cryo-EM structures of the capsid, connector, tail tube, and baseplate of the lytic, broad-host-range mycobacteriophage D29, revealing a near-complete virion architecture. At the capsid-portal interface, five copies of the previously uncharacterized protein gp19.1 bridge adjacent capsid subunits around the dodecameric portal, potentially reinforcing the symmetry-mismatched vertex. At the baseplate, 13 copies of gp6 assemble into a collar surrounding the sixfold-symmetric gp23-gp27 core. Remarkably, this 6:13 symmetry mismatch is accommodated through six distinct gp6- gp27 interfaces involving 12 gp6 subunits, leaving one subunit largely disengaged from the core. This asymmetric organization produces a tilted, outward-flaring collar that presents an extensive surface with the potential to engage the host cell. Whole-cell binding and plaque-competition assays further implicate the gp6 collar and the flexible trimeric central fiber gp31 in host recognition. Together, these findings reveal distinct structural adaptations at symmetry-mismatched interfaces and provide a structural framework for understanding mycobacteriophage assembly and host engagement.

microbiology↗
bioRxiv2026

A Luciferase Reporter of Ester Prodrug Delivery to the Cytosol of Intracellular Mycobacteria

The bacterial cell envelope restricts access to cytoplasmic targets and is a principal reason that otherwise potent antibacterial compounds fail in cells. Masking an anionic carboxylic acid as an ester is a common way to cross that barrier, but the strategy succeeds only if the target organism also hydrolyzes the ester once the compound arrives. These two requirements, permeation and intracellular unmasking, are rarely measured together in live bacterial cells, and existing reporters cannot specify where unmasking occurred. Here we describe a bioluminescent platform that resolves both steps in real time in live mycobacteria using carboxylate esters of D-luciferin. Luciferase requires that an ester both reach the bacterial cytoplasm and be hydrolyzed there, which makes light a compartment-specific readout of productive delivery. Across a series of alkyl esters in Mycobacterium smegmatis, chain length governed permeation while branching adjacent to the carbonyl governed esterase processing, indicating that the two parameters can be tuned independently. The n-butyl ester gave the strongest signal in broth, in infected macrophages, and in the pathogen Mycobacterium avium, whereas the tert-butyl ester was inert in Mycobacterium avium and unmasking of the whole series was attenuated at phagosomal pH. Ester activation is therefore both species- and compartment-dependent, and masking groups are best selected in the organism and environment in which they must act.

microbiology↗
bioRxiv2026

Flexible phosphorus allocation to the phosphoproteome underpins diatom regulatory adaptations to fluctuating phosphate supply

Diatoms are major drivers of marine phosphorus (P) cycling and are highly adapted to cope with variable P supply. P-starved diatoms can sense increases in P availability within seconds, accumulating this limiting resource through luxury polyphosphate (polyP) storage. Yet, how diatoms coordinate such responses is unknown. Protein phosphorylation is a core mechanism for environmental signal transduction in eukaryotes, but how cells balance investment in protein phosphorylation against competing physiological demands during P limitation is poorly understood. Here, we characterise system-wide remodelling of the Phaeodactylum tricornutum phosphoproteome in response to P deficiency and subsequent resupply. We identify widespread proteome dephosphorylation across 44% of phosphosites, revealing a previously unrecognised P-sparing strategy in marine phytoplankton. Following P resupply, diatoms orchestrate extensive phosphoproteome reprogramming within minutes. We characterise previously unrecognized components for early recovery from P limitation, including a novel environmentally prevalent intracellular H+/phosphate transporter strongly conserved in diverse diatoms. We also identify a vacuolar Ca2+-dependent protein kinase (CDPK) associated with polyP-storage granules. Mutant and inhibitor analyses demonstrate a role for Ca2+- and CDPK-signalling in the regulation of luxury polyP accumulation. Our findings reveal that recovery from P starvation is mediated by rapid phosphoregulatory control of intracellular P trafficking, organellar ATP generation coupled with luxury P storage, enabling diatoms to exploit newly available P and thrive in fluctuating P regimes. Collectively, our study uncovers the phosphoproteome as a flexible intracellular P reservoir and establishes mechanistic links between intracellular P budgeting, signalling and the marine P cycle.

microbiology↗