Zuzana Palčeková

Colorado State University

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Mary Jackson

Colorado State University

Shiva K. Angala

Colorado State University

Juan M. Belardinelli

Colorado State University

Michael McNeil

Colorado State University

Mercedes González-Juarrero

Colorado State University

Martine Gilleron

Institut de Pharmacologie et de Biologie Structurale

John D. McKinney

École Polytechnique Fédérale de Lausanne

Jérôme Nigou

Université de Toulouse

Haig Alexander Eskandarian

University of California, San Francisco

William H. Wheat

Colorado State University

Cooperative Institutions

Colorado State University

Centre National de la Recherche Scientifique

École Polytechnique Fédérale de Lausanne

Université Toulouse III - Paul Sabatier

Université de Toulouse

University of Pavia

Inserm

University of Tennessee Health Science Center

St. Jude Children's Research Hospital

Comenius University Bratislava

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Mechanical morphotype switching as an adaptive response in mycobacteria

Science Advances (2024)

Haig Alexander Eskandarian Yuxiang Chen Chiara Toniolo Juan M. Belardinelli Zuzana Palčeková

Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report the discovery of a virulence mechanism by which changes to the mechanical stiffness of the mycobacterial cell surface confer refraction to killing during infection. Long-term time-lapse atomic force microscopy was used to reveal a process of “mechanical morphotype switching” in mycobacteria exposed to host intracellular stress. A “soft” mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Both pharmacologic treatment, with bedaquiline, and a genetic mutant lacking uvrA modified the basal mechanical state of mycobacteria into a soft mechanical morphotype, enhancing survival in macrophages. Our study proposes microbial cell mechanical adaptation as a critical axis for surviving host-mediated stressors.

10.1126/sciadv.adh7957

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Citations (3)

Role of succinyl substituents in the mannose-capping of lipoarabinomannan and control of inflammation in Mycobacterium tuberculosis infection

PLoS Pathogens (2023)

Zuzana Palčeková Andrés Obregón‐Henao Kavita De Amanda Walz H. Lam

The covalent modification of bacterial (lipo)polysaccharides with discrete substituents may impact their biosynthesis, export and/or biological activity. Whether mycobacteria use a similar strategy to control the biogenesis of its cell envelope polysaccharides and modulate their interaction with the host during infection is unknown despite the report of a number of tailoring substituents modifying the structure of these glycans. Here, we show that discrete succinyl substituents strategically positioned on Mycobacterium tuberculosis ( Mtb ) lipoarabinomannan govern the mannose-capping of this lipoglycan and, thus, much of the biological activity of the entire molecule. We further show that the absence of succinyl substituents on the two main cell envelope glycans of Mtb , arabinogalactan and lipoarabinomannan, leads to a significant increase of pro-inflammatory cytokines and chemokines in infected murine and human macrophages. Collectively, our results validate polysaccharide succinylation as a critical mechanism by which Mtb controls inflammation.

Lipoarabinomannan

Arabinogalactan

Tunicamycin

10.1371/journal.ppat.1011636

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Azido Inositol Probes Enable Metabolic Labeling of Inositol-Containing Glycans and Reveal an Inositol Importer in Mycobacteria

ACS Chemical Biology (2023)

Heather L. Hodges Kwaku Obeng Charlotte Avanzi Alex P. Ausmus Shiva K. Angala

Bacteria from the genus Mycobacterium include pathogens that cause serious diseases in humans and remain as difficult infectious agents to treat. Central to these challenges are the composition and organization of the mycobacterial cell envelope, which includes unique and complex glycans. Inositol is an essential metabolite for mycobacteria due to its presence in the structural core of the immunomodulatory cell envelope glycolipids phosphatidylinositol mannoside (PIM) and PIM-anchored lipomannan (LM) and lipoarabinomannan (LAM). Despite their importance to mycobacterial physiology and pathogenesis, many aspects of PIM, LM, and LAM construction and dynamics remain poorly understood. Recently, probes that allow metabolic labeling and detection of specific mycobacterial glycans have been developed to investigate cell envelope assembly and dynamics. However, these tools have been limited to peptidoglycan, arabinogalactan, and mycolic acid-containing glycolipids. Herein, we report the development of synthetic azido inositol (InoAz) analogues as probes that can metabolically label PIMs, LM, and LAM in intact mycobacteria. Additionally, we leverage an InoAz probe to discover an inositol importer and catabolic pathway in Mycobacterium smegmatis. We anticipate that in the future, InoAz probes, in combination with bioorthogonal chemistry, will provide a valuable tool for investigating PIM, LM, and LAM biosynthesis, transport, and dynamics in diverse mycobacterial organisms.

Lipoarabinomannan

Mycobacterium smegmatis

Cell envelope

10.1021/acschembio.2c00912

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Unique Features of Mycobacterium abscessus Biofilms Formed in Synthetic Cystic Fibrosis Medium

Frontiers in Microbiology (2021)

Juan M. Belardinelli Wěi Li Charlotte Avanzi Shiva K. Angala Elena Lian

Characterizing Mycobacterium abscessus complex (MABSC) biofilms under host-relevant conditions is essential to the design of informed therapeutic strategies targeted to this persistent, drug-tolerant, population of extracellular bacilli. Using synthetic cystic fibrosis medium (SCFM) which we previously reported to closely mimic the conditions encountered by MABSC in actual cystic fibrosis (CF) sputum and a new model of biofilm formation, we show that MABSC biofilms formed under these conditions are substantially different from previously reported biofilms grown in standard laboratory media in terms of their composition, gene expression profile and stress response. Extracellular DNA (eDNA), mannose-and glucose-containing glycans and phospholipids, rather than proteins and mycolic acids, were revealed as key extracellular matrix (ECM) constituents holding clusters of bacilli together. None of the environmental cues previously reported to impact biofilm development had any significant effect on SCFM-grown biofilms, most likely reflecting the fact that SCFM is a nutrient-rich environment in which MABSC finds a variety of ways of coping with stresses. Finally, molecular determinants were identified that may represent attractive new targets for the development of adjunct therapeutics targeting MABSC biofilms in persons with CF.

Extracellular polymeric substance

Mycobacterium abscessus

10.3389/fmicb.2021.743126

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Citations (21)

Disruption of the SucT acyltransferase in Mycobacterium smegmatis abrogates succinylation of cell envelope polysaccharides

Journal of Biological Chemistry (2019)

Zuzana Palčeková Shiva K. Angala Juan M. Belardinelli Haig Alexander Eskandarian Maju Joe

Similar to other prokaryotes, mycobacteria decorate their major cell envelope glycans with minor covalent substituents whose biological significance remains largely unknown. We report on the discovery of a mycobacterial enzyme, named here SucT, that adds succinyl groups to the arabinan domains of both arabinogalactan (AG) and lipoarabinomannan (LAM). Disruption of the SucT-encoding gene in Mycobacterium smegmatis abolished AG and LAM succinylation and altered the hydrophobicity and rigidity of the cell envelope of the bacilli without significantly altering AG and LAM biosynthesis. The changes in the cell surface properties of the mutant were consistent with earlier reports of transposon mutants of the closely related species Mycobacterium marinum and Mycobacterium avium harboring insertions in the orthologous gene whose ability to microaggregate and form biofilms were altered. Our findings point to an important role of SucT-mediated AG and LAM succinylation in modulating the cell surface properties of mycobacteria. Similar to other prokaryotes, mycobacteria decorate their major cell envelope glycans with minor covalent substituents whose biological significance remains largely unknown. We report on the discovery of a mycobacterial enzyme, named here SucT, that adds succinyl groups to the arabinan domains of both arabinogalactan (AG) and lipoarabinomannan (LAM). Disruption of the SucT-encoding gene in Mycobacterium smegmatis abolished AG and LAM succinylation and altered the hydrophobicity and rigidity of the cell envelope of the bacilli without significantly altering AG and LAM biosynthesis. The changes in the cell surface properties of the mutant were consistent with earlier reports of transposon mutants of the closely related species Mycobacterium marinum and Mycobacterium avium harboring insertions in the orthologous gene whose ability to microaggregate and form biofilms were altered. Our findings point to an important role of SucT-mediated AG and LAM succinylation in modulating the cell surface properties of mycobacteria.

Mycobacterium smegmatis

Succinylation

Cell envelope

Acyltransferases

10.1074/jbc.ra119.008585

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Citations (22)

Impact of Methylthioxylose Substituents on the Biological Activities of Lipomannan and Lipoarabinomannan in Mycobacterium tuberculosis

ACS Infectious Diseases (2024)

Zuzana Palčeková Kavita De Shiva K. Angala Martine Gilleron Sophie Zuberogoïtia

Two lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), play various, albeit incompletely defined, roles in the interactions of mycobacteria with the host. Growing evidence points to the modification of LM and LAM with discrete covalent substituents as a strategy used by these bacteria to modulate their biological activities. One such substituent, originally identified in Mycobacterium tuberculosis (Mtb), is a 5-methylthio-d-xylose (MTX) sugar, which accounts for the antioxidative properties of LAM. The widespread distribution of this motif across Mtb isolates from several epidemiologically important lineages have stimulated interest in MTX-modified LAM as a biomarker of tuberculosis infection. Yet, several lines of evidence indicate that MTX may not be restricted to Mtb and that this motif may substitute more acceptors than originally thought. Using a highly specific monoclonal antibody to the MTX capping motif of Mtb LAM, we here show that MTX motifs not only substitute the mannoside caps of LAM but also the mannan core of LM in Mtb. MTX substituents were also found on the LM and LAM of pathogenic, slow-growing nontuberculous mycobacteria. The presence of MTX substituents on the LM and LAM from Mtb enhances the pro-apoptotic properties of both lipoglycans on LPS-stimulated THP-1 macrophages. A comparison of the cytokines and chemokines produced by resting and LPS-activated THP-1 cells upon exposure to MTX-proficient versus MTX-deficient LM further indicates that MTX substituents confer anti-inflammatory properties upon LM. These findings add to our understanding of the glycan-based strategies employed by slow-growing pathogenic mycobacteria to alter the host immune response to infection.

Lipoarabinomannan

10.1021/acsinfecdis.4c00079

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Citations (2)

Polysaccharide Succinylation Enhances the Intracellular Survival of Mycobacterium abscessus

ACS Infectious Diseases (2020)

Zuzana Palčeková Martine Gilleron Shiva K. Angala Juan M. Belardinelli Michael McNeil

Lipoarabinomannan (LAM) and its biosynthetic precursors, phosphatidylinositol mannosides (PIMs) and lipomannan (LM) play important roles in the interactions of Mycobacterium tuberculosis with phagocytic cells and the modulation of the host immune response, but nothing is currently known of the impact of these cell envelope glycoconjugates on the physiology and pathogenicity of nontuberculous mycobacteria. We here report on the structures of Mycobacterium abscessus PIM, LM, and LAM. Intriguingly, these structures differ from those reported previously in other mycobacterial species in several respects, including the presence of a methyl substituent on one of the mannosyl residues of PIMs as well as the PIM anchor of LM and LAM, the size and branching pattern of the mannan backbone of LM and LAM, and the modification of the arabinan domain of LAM with both succinyl and acetyl substituents. Investigations into the biological significance of some of these structural oddities point to the important role of polysaccharide succinylation on the ability of M. abscessus to enter and survive inside human macrophages and epithelial cells and validate for the first time cell envelope polysaccharides as important modulators of the virulence of this emerging pathogen.

Lipoarabinomannan

Succinylation

Mycobacterium abscessus

Mannan

Glycoconjugate

Mycobacterium smegmatis

10.1021/acsinfecdis.0c00361

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Citations (16)

Acylation of glycerolipids in mycobacteria

Nature Communications (2023)

Shiva K. Angala Ana Carreras‐González Emilie Huc‐Claustre Itxaso Anso Davinder Kaur

We report on the existence of two phosphatidic acid biosynthetic pathways in mycobacteria, a classical one wherein the acylation of the sn-1 position of glycerol-3-phosphate (G3P) precedes that of sn-2 and another wherein acylations proceed in the reverse order. Two unique acyltransferases, PlsM and PlsB2, participate in both pathways and hold the key to the unusual positional distribution of acyl chains typifying mycobacterial glycerolipids wherein unsaturated substituents principally esterify position sn-1 and palmitoyl principally occupies position sn-2. While PlsM selectively transfers a palmitoyl chain to the sn-2 position of G3P and sn-1-lysophosphatidic acid (LPA), PlsB2 preferentially transfers a stearoyl or oleoyl chain to the sn-1 position of G3P and an oleyl chain to sn-2-LPA. PlsM is the first example of an sn-2 G3P acyltransferase outside the plant kingdom and PlsB2 the first example of a 2-acyl-G3P acyltransferase. Both enzymes are unique in their ability to catalyze acyl transfer to both G3P and LPA.

Acyltransferases

Acyl group

10.1038/s41467-023-42478-x

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Citations (4)

Covalent modifications of polysaccharides in mycobacteria

Nature Chemical Biology (2018)

Shiva K. Angala Zuzana Palčeková Juan M. Belardinelli Mary Jackson

10.1038/nchembio.2571

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Citations (26)

A multitarget approach to drug discovery inhibiting Mycobacterium tuberculosis PyrG and PanK

Scientific Reports (2018)

Laurent R. Chiarelli Giorgia Mori Béatrice Silvia Orena Marta Esposito Thomas R. Lane

Abstract Mycobacterium tuberculosis , the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a “double-tool” in order to find additional hit compounds.

10.1038/s41598-018-21614-4

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Citations (46)