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

Synthesis of α‑Glucan in Mycobacteria Involves a Hetero-octameric Complex of Trehalose Synthase TreS and Maltokinase Pep2

Recent evidence established that the cell envelope of <i>Mycobacterium tuberculosis</i>, the bacillus causing tuberculosis (TB), is coated by an α-glucan-containing capsule that has been implicated in persistence in a mouse infection model. As one of three known metabolic routes to α-glucan in mycobacteria, the cytoplasmic GlgE-pathway converts trehalose to α(1 → 4),α(1 → 6)-linked glucan in 4 steps. Whether individual reaction steps, catalyzed by trehalose synthase TreS, maltokinase Pep2, and glycosyltransferases GlgE and GlgB, occur independently or in a coordinated fashion is not known. Here, we report the crystal structure of <i>M. tuberculosis</i> TreS, and show by small-angle X-ray scattering and analytical ultracentrifugation that TreS forms tetramers in solution. Together with Pep2, TreS forms a hetero-octameric complex, and we demonstrate that complex formation markedly accelerates maltokinase activity of Pep2. Thus, complex formation may act as part of a regulatory mechanism of the GlgE pathway, which overall must avoid accumulation of toxic pathway intermediates, such as maltose-1-phosphate, and optimize the use of scarce nutrients

RT-PCR expression of the sRNA downstream of <i>bd0108</i> and expression of a control gene <i>dnaK</i>.

<p>Expression of a small, non-coding RNA downstream of <i>bd0108</i> was shown to be different in HI strains carrying different mutations in <i>bd0108</i> and attack phase HD100. Expression was highest in HID13 (ATA->ATG start codon mutation) and the strains with the markerless deletion of <i>bd0108</i>. Strain HID22 (<i>bd0108∆</i>42bp), had slightly lower expression, while those strains with a wild-type <i>bd0108</i>; HD100 and HID2, show much lower expression of the sRNA. Expression of <i>dnaK</i> was uniform across the samples indicating a matched amount of total RNA was used for the experiment.</p

Predation of HI strains compared to predatory HD100.

<div><p>Time-lapse microscopy of individual wild-type HD100 cells (<b>A</b>(<b>a</b>)) and cells of the markerless deletion mutant of <i>bd0108</i> (A(b)) preying upon the <i>E. coli</i> strain pMAL-p2_mCherry with a fluorescent periplasm. At T=0 there is attachment of free swimming <i>Bdellovibrio</i> in both strains, with invasion occurring within 2 frames (5 minutes) for most cells observed. Arrows indicate both free swimming and attached <i>Bdellovibrio</i> cells. By 1 hour the <i>Bdellovibrio</i> is established in the periplasm and begun to grow as a filament. By 8 hours the growing filaments of both the wild-type and deletion strains have septated to form single progeny and the prey bursts releasing them. Predation was carried out on a 1% Agarose pad with images acquired every 2.5 minutes.</p> <p><b>B</b>. Reduction of <i>E. coli</i> numbers in a predatory lysate comparing wild-type HD100 or spontaneously generated HIs with the markerless deletion of <i>bd0108</i> strains. The spontaneously generated HI strains included some with a variety of point mutations or the common 42bp deletion in <i>bd0108</i> and some with a wild-type copy of <i>bd0108</i>. HI cultures readily prey upon <i>E. coli</i> in liquid cultures, reducing prey numbers by several logs in 2 days with predation by the population as a whole appearing to be slightly slower than wild-type HD100. All HI strains were grown axenically, independently of prey cells before the experiment. </p></div

Alignment of Bd0109 with other RHS elements.

<p>Multiple sequence alignment constructed using Clustal W showing that the main regions of homology between the <i>Bdellovibrio</i> Bd0109 predicted protein and other RHS elements is the core pFAM RHS repeat (PF05593) region which consists of a repeating YD element. The whole Bd0109 sequence contains 13 YD or YE sequences and several other Y residues. The sequences aligned are from the genera indicated with the names, with the second <i>Dickeya</i> sequence being RhsB. </p

Plaque assay of predation by Host Independent mutants compared to HD100.

<p>Dilution of predatory cultures of <i>Bdellovibrio</i> strains on double layer YPSC overlay plates containing prey <i>E. coli</i> S17-1 in the top layer. Dilution was carried out in Ca/HEPES with 10 µl of the resulting dilution series spotted at positions indicated in the diagram. Plates show there is no significant effect on HD100 having extra copies of the <i>bd0108</i> gene or the 42 bp deletion variant of <i>bd0108</i> on plaquing ability. Complementation tests for the HID13 strain (ATG->ATA mutation in <i>bd0108</i>) with the plasmid pSUP404.2 containing the wild-type <i>bd0108</i> gene showed an increase in plaquing, however this was not observed for the Δ<i>bd0108</i> deletion strain. Plates are representative of at least three independent repeats.</p

PFU to CFU ratio for pooled spontaneous HI strains and the markerless <i>bd0108</i> deletion HI mutants of <i>Bdellovibrio</i>.

<p>In the spontaneous generated HI strains; HID2 and HID26 (wild-type for <i>bd0108</i>) HID6, HID13, HID18, (point mutations in <i>bd0108</i>) and HID22 (<i>bd0108∆</i>42bp), and the markerless deletion mutants of <i>bd0108</i>, complementation tests shows that these strains carrying the pSUP404.2 plasmid encoding the wild-type <i>bd0108</i> is significantly deficient in the capacity to form HI colonies on PY agar plates compared to the capacity to form plaques on double layer overlay plates. This was significant compared to cells containing the pSUP404.2 plasmid alone, or the pSUP404.2-Δ42 with the 42 bp deletion variant of <i>bd0108</i>. The ratio of CFU:PFU was reduced from 2.16 x 10^-1 in strains containing the pSUP404.2 alone to 3.21 x 10^-2 in strains containing pSUP404.2-108 (p = 0.026) but were not significantly reduced relative to strains containing pSUP404.2-Δ42, 2.56 x 10^-1 (p = 0.31). The difference between strains carrying the pSUP404.2-108 and the pSUP404.2-Δ42 was also significant (P<<0.01).</p

Model for possible interactions of Bd0108/Bd0109 controlling the extrusion and retraction of pili.

<div><p>A. Operonal structure of the <i>bd0108 </i><i>hit</i> locus and surrounding genes, predicted to have a role in the formation of a Type IVb pilus. Genes are colour coded to correspond to their predicted function in the pilus diagrams underneath.</p> <p><b>B</b>. In wild-type cells <i>bd0108</i> and <i>bd0109</i> are co-expressed, the mRNA is then translated into proteins containing a signal sequence recognised by the Sec system, the signal is cleaved, and the proteins are transported into the periplasm where the mature Bd0108 protein transiently interacts with Bd0109 to sequester it. When Bd0109 is unbound, it could then anchor at either the cell wall, or with the mature pilus fibre. Both scenarios are possible due to Bd0109’s structural cleft binding carbohydrate that is present in both cell wall and the mature and glycosylated pili. Bd0109 mediates successful pilus extrusion/retraction and signal back into the cytoplasm. In wild-type pilus formation Bd1290 pre-pilins are held in the inner membrane and are assembled into the pilus fibre possibly by the flp pilus ATPases Bd0110 and Bd0111. The balance of sequestering and release of Bd0109 by Bd0108 in the periplasm permits to successful extrusion and retraction of the pilus fibre upon environmental cues.</p> <p><b>C</b>. In the absence of Bd0108 protein, Bd0109 is not sequestered and is free to mediate more frequently with pilus extrusion and retraction, resulting in very few pili extruded beyond the cell surface and cues for HI growth signalled to the cell.</p> <p><b>D</b>. In HI strains containing the 42 bp deletion variant of <i>bd0108</i>, the gene is still expressed. The truncated form of Bd0108 alters the dynamics of the Bd0109 functionalisation are altered (possibly by over-sequestration of Bd0109) and hyper-extruded pili are seen on the surface more frequently. Hyper-extruded pili or no pili send similar internal signals to regulate prey independent growth.</p></div

Luminescent prey assay of predation efficiency for Host Dependent and Host Independent strains.

<p>For Host Dependent cells (<b>A</b>) with a genomic copy of the wild-type <i>bd0108</i> gene, carrying the pSUP404.2 plasmid encoding either the wild-type <i>bd0108</i> gene or the 42 bp deletion variant of <i>bd0108</i> had no effect on predation compared to those carrying the empty pSUP404.2 plasmid. This assay shows the typical result of logarithmically faster reduction in luminescence with more <i>Bdellovibrio</i> initially added and shows that the extra copies of <i>bd0108</i> or the 42 bp deletion variant have no significant effect on this. For Host Independent cells (<b>B</b>) there is not the same proportional decrease in luminescence when more cells are added supporting the hypothesis that a proportion are growing axenically as HIs rather than predatorily. HI mutants carrying the pSUP404.2 plasmid encoding the wild-type <i>bd0108</i> gene, however, restore the typical initial-cell-number to drop in luminescence relationship seen in the HD wild-type. This suggests that the presence of a wild-type <i>bd0108</i>, but not the 42 bp deletion variant, <i>in </i><i>trans</i> restores the HI cells to a predatory lifestyle.</p

<i>In trans</i> expression of fluorescently tagged Bd0108 and Bd0109 in <i>E. coli</i> S17-1 heterologous host.

<div><p><b>A</b>. <i>E. coli</i> S17-1 expressing a C-terminal mCherry fusion of Bd0108 protein <i>in </i><i>trans</i>. Fluorescence can be seen in the periplasm of the cells, with large amounts towards the poles of the cell where cells are plasmolysed.</p> <p><b>B</b>. <i>E. coli</i> S17-1 expressing a C-terminal mTFP fusion of Bd0109 protein <i>in </i><i>trans</i>. Fluorescence can be seen in the periplasm of the cells, with large amounts towards the poles of the cell where cells are plasmolysed.</p></div