Disruption of Mycobacterial AftB Results in Complete Loss of Terminal β(1 → 2) Arabinofuranose Residues of Lipoarabinomannan

Lipoarabinomannan (LAM) and arabinogalactan (AG) are the two major mycobacterial cell wall (lipo)­polysaccharides, which contain a structurally similar arabinan domain that is highly branched and assembled in a stepwise fashion by variety of arabinofuranosyltransferases (Ara<i>f</i>T). In addition to playing an essential role in mycobacterial physiology, LAM and its biochemical precursor lipomannan possess potent immunomodulatory activities that affect the host immune response. In the search of additional mycobacterial Ara<i>f</i>Ts that participate in the synthesis of the arabinan segment of LAM, we disrupted <i>aftB</i> (<i>MSMEG_6400</i>) in <i>Mycobacterium smegmatis</i>. The deletion of chromosomal <i>aftB</i> locus could only be achieved in the presence of a rescue plasmid carrying a functional copy of <i>aftB</i>, strongly suggesting that it is essential for the viability of <i>M. smegmatis</i>. Isolation and detailed structural characterization of a LAM molecule derived from the conditional mutant deficient in AftB revealed the absence of terminal β(1 → 2)-linked arabinofuranosyl residues. Furthermore, we demonstrated that truncated LAM displays proinflammatory activity, which is due to its ability to activate Toll-like receptor 2. All together, our results indicate that AftB is an essential mycobacterial Ara<i>f</i>T that plays a role in the synthesis of the arabinan domain of LAM

Image_1_A humanized mouse model for in vivo evaluation of invariant Natural Killer T cell responses.tif

Invariant natural killer T (iNKT) cells mediate immune responses when stimulated by glycolipid agonists presented by CD1d. In extensive studies of synthetic analogues of α-galactosyl ceramides, we identified numerous examples of significant differences in the recognition of specific glycolipids in wild type mice versus human iNKT cell clones or PBMC samples. To predict human iNKT cell responses more accurately in a mouse model, we derived a mouse line in which compound genetic modifications were used to express a human-like iNKT cell TCR along with human CD1d in place of the endogenous mouse proteins. Detailed transcriptional and phenotypic profiling demonstrated that these partially humanized mice developed an expanded population of T cells recognizing CD1d-presented glycolipid antigens, among which a subset characterized by expression of chemokine receptor CXCR6 had features characteristic of authentic iNKT cells. Responses to iNKT cell activating glycolipids in these mice generated cytokine production in vitro and in vivo that showed a pattern of fine specificity that closely resembled that of cultured human iNKT cell clones. Anti-tumor responses to variants of α-galactosyl ceramide in VαKI mice also correlated with their potency for stimulating human iNKT cells. This genetically modified mouse line provides a practical model for human presentation and recognition of iNKT cell activators in the context of a normally functioning immune system, and may furnish valuable opportunities for preclinical evaluation of iNKT cell-based therapies.</p

DataSheet_1_A humanized mouse model for in vivo evaluation of invariant Natural Killer T cell responses.pdf

Invariant natural killer T (iNKT) cells mediate immune responses when stimulated by glycolipid agonists presented by CD1d. In extensive studies of synthetic analogues of α-galactosyl ceramides, we identified numerous examples of significant differences in the recognition of specific glycolipids in wild type mice versus human iNKT cell clones or PBMC samples. To predict human iNKT cell responses more accurately in a mouse model, we derived a mouse line in which compound genetic modifications were used to express a human-like iNKT cell TCR along with human CD1d in place of the endogenous mouse proteins. Detailed transcriptional and phenotypic profiling demonstrated that these partially humanized mice developed an expanded population of T cells recognizing CD1d-presented glycolipid antigens, among which a subset characterized by expression of chemokine receptor CXCR6 had features characteristic of authentic iNKT cells. Responses to iNKT cell activating glycolipids in these mice generated cytokine production in vitro and in vivo that showed a pattern of fine specificity that closely resembled that of cultured human iNKT cell clones. Anti-tumor responses to variants of α-galactosyl ceramide in VαKI mice also correlated with their potency for stimulating human iNKT cells. This genetically modified mouse line provides a practical model for human presentation and recognition of iNKT cell activators in the context of a normally functioning immune system, and may furnish valuable opportunities for preclinical evaluation of iNKT cell-based therapies.</p

MALDI-TOF mass spectroscopy analysis of purified LOS’s from <i>M</i>.<i>kansasii</i> WT and Δ<i>MKAN27435</i> strains in positive ion mode.

<p>All molecular ions are [M+Na]⁺ and species indicated with an asterisk (*) correspond to <i>m/z</i> values with an intact acyl group.</p

MALDI-TOF mass spectroscopy analyses tabulated to list LOS species with corresponding <i>m/z</i> values in <i>M</i>. <i>kansasii</i> WT and Δ<i>MKAN27435</i> strains.

<p>All molecular ions are [M+Na]⁺and an asterisk (*) indicates species with an intact acyl group. Presence (+) or absence (-) of each species is indicated for each strain.</p><p>MALDI-TOF mass spectroscopy analyses tabulated to list LOS species with corresponding <i>m/z</i> values in <i>M</i>. <i>kansasii</i> WT and Δ<i>MKAN27435</i> strains.</p

Structures of different LOS subclasses from <i>M</i>. <i>kansasii</i>

<p>(A) LOS-I-III (n = 1–3), (B) LOS-IV-VII (n = 2–4), (C) LOS-VII; R1, R2 and R3 represents the acyl chain attached to tetra glucose core of LOS. (D) Schematic showing predicted domains and topology of <i>MKAN27435</i>. The amino and carboxy terminals are indicated as ‘N’ and ‘C’ respectively, and numbers represent predicted borders of domains which are depicted as grey areas. The regions corresponding to the predicted transmembrane domains are indicated with black bars.</p

Autoradiograph of 2-D TLC analysis of polar lipids extracted from <i>M</i>. <i>kansasii</i> WT, Δ<i>MKAN27435</i>, Δ<i>MKAN27435</i>-C strains.

<p>Arrows indicate the different LOS species. I and II indicate system E dimension 1 and 2 respectively. Dimension I: Chloroform: Methanol: H₂O (60:30:6); Dimension II: Chloroform: Acetic acid: Methanol: H₂O (40:25:3:6).</p

MALDI Spiral-TOF Mass Spectroscopy analysis of total MAMES purified from the Δ<i>MsdesA1</i> mutant grown in the presence or absence of acetamide.

<p>MALDI Spiral-TOF Mass Spectroscopy analysis of total MAMES purified from the Δ<i>MsdesA1</i> mutant grown in the presence or absence of acetamide.</p

2D-argentation TLC of fatty acyl methyl esters extracted from polar and apolar lipid extracts of the Δ<i>MsdesA1</i> mutant grown in the presence or absence of acetamide.

<p>(A) Direction-I separated in petroleum ether:acetone (95:5, v/v); direction-II separated thrice in AgNO₃ treated silica gel in petroleum ether:ethyl acetate (9:1, v/v). Mycolate species from apolar extracts are indicated and are also present in lesser amounts in the polar extracts. F; fatty acyl methyl esters.</p

Argentation TLC of ¹⁴C-labelled methyl esters of cell wall mycolic acids (MAMES) extracted from the Δ<i>MsdesA1</i> mutant grown in the presence or absence of acetamide.

<p>(A) Direction-I separated in petroleum ether:acetone (95:5, v/v); direction-II separated thrice in AgNO₃ treated silica gel in petroleum ether:ethyl acetate (9:1, v/v). The cyclopropanated derivative is indicated as X₁ and the faster-migrating species that co-migrated with α-MAMES in direction-I are indicated as X₂. The clear ‘imprint’ appearing superimposed on the comet shaped X₂ spot in the 8h –acetamide TLC is likely due to unlabelled α₂ mycolates synthesised at the early stages of DesA1 depletion. (B) Bar graph showing the relative amounts of each ¹⁴C-labelled mycolate subspecies indicated as a percentages of the total amounts of ¹⁴C-labelled mycolic acids detected on the TLC plates, as determined by densitometry.</p