Comparison of the transcriptome, lipidome, and c-di-GMP production between BCGΔBCG1419c and BCG, with Mincle- and Myd88-dependent induction of proinflammatory cytokines in murine macrophages.

We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.

Lipoarabinomannan mediates localized cell wall integrity during division in mycobacteria.

The growth and division of mycobacteria, which include clinically relevant pathogens, deviate from that of canonical bacterial models. Despite their Gram-positive ancestry, mycobacteria synthesize and elongate a diderm envelope asymmetrically from the poles, with the old pole elongating more robustly than the new pole. The phosphatidylinositol-anchored lipoglycans lipomannan (LM) and lipoarabinomannan (LAM) are cell envelope components critical for host-pathogen interactions, but their physiological functions in mycobacteria remained elusive. In this work, using biosynthetic mutants of these lipoglycans, we examine their roles in maintaining cell envelope integrity in Mycobacterium smegmatis and Mycobacterium tuberculosis. We find that mutants defective in producing mature LAM fail to maintain rod cell shape specifically at the new pole and para-septal regions whereas a mutant that produces a larger LAM becomes multi-septated. Therefore, LAM plays critical and distinct roles at subcellular locations associated with division in mycobacteria, including maintenance of local cell wall integrity and septal placement.

Detergent-induced quantitatively limited formation of diacyl phosphatidylinositol dimannoside in Mycobacterium smegmatis.

Mycobacterial plasma membrane, together with the peptidoglycan-arabinogalactan cell wall and waxy outer membrane, creates a robust permeability barrier against xenobiotics. The fact that several anti-tuberculosis drugs target plasma membrane-embedded enzymes underscores the importance of the plasma membrane in bacterial physiology and pathogenesis. Nevertheless, its accurate phospholipid composition remains undefined, with conflicting reports on the abundance of phosphatidylinositol mannosides (PIMs), physiologically important glycolipids evolutionarily conserved among mycobacteria and related bacteria. Some studies indicate cardiolipin, phosphatidylethanolamine, and phosphatidylinositol as dominant structural phospholipids. Conversely, some suggest PIMs dominate the plasma membrane. A striking example of the latter is the use of reverse micelle extraction, showing diacyl phosphatidylinositol dimannoside (Ac2PIM2) as the most abundant phospholipid in a model organism, Mycobacterium smegmatis. Our recent work reveals a rapid response mechanism to membrane-fluidizing stress in mycobacterial plasma membrane: monoacyl phosphatidylinositol dimannoside and hexamannoside (AcPIM2 and AcPIM6), are converted to diacyl forms (Ac2PIM2 and Ac2PIM6). Given the dynamic nature of PIMs, we aimed to resolve the conflicting data in the literature. We show that unstressed M. smegmatis lacks an Ac2PIM2-dominated plasma membrane. Ac2PIM2 accumulation is induced by experimental conditions involving sodium docusate, a component of the reverse micellar solution. Using chemically synthesized PIMs as standards, we accurately quantified phospholipid ratio in M. smegmatis through liquid chromatography-mass spectrometry, revealing that mycobacterial plasma membrane is dominated by cardiolipin, phosphatidylethanolamine, and phosphatidylinositol. Thus, PIMs are quantitatively minor but responsive to environmental stresses in M. smegmatis. Our study paves the way for accurate modeling of mycobacterial plasma membrane.

A cell wall synthase accelerates plasma membrane partitioning in mycobacteria.

Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced both by bilayer-intrinsic factors like molecular composition and by bilayer-extrinsic factors such as interactions with other membranes and solid supports. While cellular membranes can departition in response to bilayer-intrinsic or -extrinsic disruptions, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis spatially and biochemically departitions in response to the fluidizing agent benzyl alcohol, then repartitions upon fluidizer washout. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth during recovery from benzyl alcohol exposure. PonA2's role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. Active cell wall polymerization promotes de novo membrane partitioning and the completed cell wall polymer helps to maintain membrane partitioning. Our work highlights the complexity of membrane-cell wall interactions and establishes a facile model system for departitioning and repartitioning cellular membranes.

Lipoarabinomannan regulates septation in Mycobacterium smegmatis.

The growth and division of mycobacteria, which include several clinically relevant pathogens, deviate significantly from that of canonical bacterial models. Despite their Gram-positive ancestry, mycobacteria synthesize and elongate a diderm envelope asymmetrically from the poles, with the old pole elongating more robustly than the new pole. In addition to being structurally distinct, the molecular components of the mycobacterial envelope are also evolutionarily unique, including the phosphatidylinositol-anchored lipoglycans lipomannan (LM) and lipoarabinomannan (LAM). LM and LAM modulate host immunity during infection, but their role outside of intracellular survival remains poorly understood, despite their widespread conservation among non-pathogenic and opportunistically pathogenic mycobacteria. Previously, Mycobacterium smegmatis and Mycobacterium tuberculosis mutants producing structurally altered LM and LAM were shown to grow slowly under certain conditions and to be more sensitive to antibiotics, suggesting that mycobacterial lipoglycans may support cellular integrity or growth. To test this, we constructed multiple biosynthetic lipoglycan mutants of M. smegmatis and determined the effect of each mutation on cell wall biosynthesis, envelope integrity, and division. We found that mutants deficient in LAM, but not LM, fail to maintain cell wall integrity in a medium-dependent manner, with envelope deformations specifically associated with septa and new poles. Conversely, a mutant producing abnormally large LAM formed multiseptated cells in way distinct from that observed in a septal hydrolase mutant. These results show that LAM plays critical and distinct roles at subcellular locations associated with division in mycobacteria, including maintenance of local cell envelope integrity and septal placement.

Mycobacterium smegmatis: The Vanguard of Mycobacterial Research.

The genus Mycobacterium contains several slow-growing human pathogens, including Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium. Mycobacterium smegmatis is a nonpathogenic and fast growing species within this genus. In 1990, a mutant of M. smegmatis, designated mc2155, that could be transformed with episomal plasmids was isolated, elevating M. smegmatis to model status as the ideal surrogate for mycobacterial research. Classical bacterial models, such as Escherichia coli, were inadequate for mycobacteria research because they have low genetic conservation, different physiology, and lack the novel envelope structure that distinguishes the Mycobacterium genus. By contrast, M. smegmatis encodes thousands of conserved mycobacterial gene orthologs and has the same cell architecture and physiology. Dissection and characterization of conserved genes, structures, and processes in genetically tractable M. smegmatis mc2155 have since provided previously unattainable insights on these same features in its slow-growing relatives. Notably, tuberculosis (TB) drugs, including the first-line drugs isoniazid and ethambutol, are active against M. smegmatis, but not against E. coli, allowing the identification of their physiological targets. Furthermore, Bedaquiline, the first new TB drug in 40 years, was discovered through an M. smegmatis screen. M. smegmatis has become a model bacterium, not only for M. tuberculosis, but for all other Mycobacterium species and related genera. With a repertoire of bioinformatic and physical resources, including the recently established Mycobacterial Systems Resource, M. smegmatis will continue to accelerate mycobacterial research and advance the field of microbiology.

Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins in Mycobacterium smegmatis.

Mycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD). The IMD is concentrated in the polar regions of growing cells and becomes less polarized under nongrowing conditions. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized biosynthesis of envelope components. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a few of these have been verified by independent experimental methods. Furthermore, some IMD-associated proteins may have escaped proteomic identification and remain to be identified. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains, each producing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed polar fluorescence patterns characteristic of IMD proteins. Twenty of these had previously been suggested to localize to the IMD based on proteomic data. Of the nine remaining IMD candidate proteins, three were confirmed by biochemical methods to be associated with the IMD. Taken together, this new colocalization strategy is effective in verifying the IMD association of proteins found by proteomic analyses while facilitating the discovery of additional IMD-associated proteins. IMPORTANCE The intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis. However, proteomics on its own is unlikely to detect every IMD-associated protein because of technical and biological limitations. Here, we describe fluorescent protein colocalization as an alternative, independent approach. Using a combination of fluorescence microscopy, proteomics, and subcellular fractionation, we identified three new proteins associated with the IMD. Such a robust method to rigorously define IMD proteins will benefit future investigations to decipher the synthesis, maintenance, and functions of this membrane domain and help delineate a more general mechanism of subcellular protein localization in mycobacteria.

Cell Walls and Membranes of Actinobacteria.

Actinobacteria is a group of diverse bacteria. Most species in this class of bacteria are filamentous aerobes found in soil, including the genus Streptomyces perhaps best known for their fascinating capabilities of producing antibiotics. These bacteria typically have a Gram-positive cell envelope, comprised of a plasma membrane and a thick peptidoglycan layer. However, there is a notable exception of the Corynebacteriales order, which has evolved a unique type of outer membrane likely as a consequence of convergent evolution. In this chapter, we will focus on the unique cell envelope of this order. This cell envelope features the peptidoglycan layer that is covalently modified by an additional layer of arabinogalactan . Furthermore, the arabinogalactan layer provides the platform for the covalent attachment of mycolic acids , some of the longest natural fatty acids that can contain ~100 carbon atoms per molecule. Mycolic acids are thought to be the main component of the outer membrane, which is composed of many additional lipids including trehalose dimycolate, also known as the cord factor. Importantly, a subset of bacteria in the Corynebacteriales order are pathogens of human and domestic animals, including Mycobacterium tuberculosis. The surface coat of these pathogens are the first point of contact with the host immune system, and we now know a number of host receptors specific to molecular patterns exposed on the pathogen's surface, highlighting the importance of understanding how the cell envelope of Actinobacteria is structured and constructed. This chapter describes the main structural and biosynthetic features of major components found in the actinobacterial cell envelopes and highlights the key differences between them.

Small Molecule Chelators Reveal That Iron Starvation Inhibits Late Stages of Bacterial Cytokinesis.

Bacterial cell division requires identification of the division site, assembly of the division machinery, and constriction of the cell envelope. These processes are regulated in response to several cellular and environmental signals. Here, we use small molecule iron chelators to characterize the surprising connections between bacterial iron homeostasis and cell division. We demonstrate that iron starvation downregulates the transcription of genes encoding proteins involved in cell division, reduces protein biosynthesis, and prevents correct positioning of the division machinery at the division site. These combined events arrest the constriction of the cell during late stages of cytokinesis in a manner distinct from known mechanisms of inhibiting cell division. Overexpression of genes encoding cell division proteins or iron transporters partially suppresses the biological activity of iron chelators and restores growth and division. We propose a model demonstrating the effect of iron availability on the regulatory mechanisms coordinating division in response to the nutritional state of the cell.