Acute SIV Infection in Sooty Mangabey Monkeys Is Characterized by Rapid Virus Clearance from Lymph Nodes and Absence of Productive Infection in Germinal Centers

Lymphoid tissue immunopathology is a characteristic feature of chronic HIV/SIV infection in AIDS-susceptible species, but is absent in SIV-infected natural hosts. To investigate factors contributing to this difference, we compared germinal center development and SIV RNA distribution in peripheral lymph nodes during primary SIV infection of the natural host sooty mangabey and the non-natural host pig-tailed macaque. Although SIV-infected cells were detected in the lymph node of both species at two weeks post infection, they were confined to the lymph node paracortex in immune-competent mangabeys but were seen in both the paracortex and the germinal center of SIV-infected macaques. By six weeks post infection, SIV-infected cells were no longer detected in the lymph node of sooty mangabeys. The difference in localization and rate of disappearance of SIV-infected cells between the two species was associated with trapping of cell-free virus on follicular dendritic cells and higher numbers of germinal center CD4+ T lymphocytes in macaques post SIV infection. Our data suggests that fundamental differences in the germinal center microenvironment prevent productive SIV infection within the lymph node germinal centers of natural hosts contributing to sustained immune competency

Mycobacterium tuberculosis releases an antacid that remodels phagosomes

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Mycobacterial Metabolic Syndrome: Triglyceride Accumulation Decreases Growth Rate and Virulence of Mycobacterium Tuberculosis

Mycobacterium tuberculosis (Mtb) mutants lacking the operon Rv1411c-1410c encoding a lipoprotein, Rv1411c (LprG) and a putative transporter, Rv1410c (Rv1410) are dramatically attenuated for growth in mice. Previous work in our lab, using the model organism Mycobacterium smegmatis, suggested that this operon regulated the lipid content of the cell wall. Work in other laboratories characterizing LprG as a lipid-binding lipoprotein lead us to hypothesize that these bacteria grew poorly due to loss of a key lipid important in the host-pathogen interaction. Based on structural and biochemical studies we hypothesized that this attenuation was due to a lipid transport defect. Using whole cell lipidomic analysis, we found changes in LprG-1410 mutants including accumulation of triacylglyceride (TAG) species in the absence of the transport system. We have identified TAG in outer membrane fractions and supernatants of Mtb, have demonstrated the ability of LprG to transport TAG in an in vitro vesicle transfer assay, and have co-crystallized LprG with TAG. Moreover, accumulation of intracellular TAG substantially decreases growth under carbon stress in vitro and in vivo in the mouse model. Our results suggest a far different model – that TAG is ordinarily transported out of the cell and, in the absence of a transporter, limits cell proliferation independent of the host immune response. This suggests that TAG is a key metabolic regulator of cellular growth within the host

Lipidomic Analysis Links Mycobactin Synthase K to Iron Uptake and Virulence in M. tuberculosis

The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes

Molecular profiling of Mycobacterium tuberculosis identifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette–Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.

Lipidomic Analysis Links Mycobactin Synthase K to Iron Uptake and Virulence in <i>M</i>. <i>tuberculosis</i>

<div><p>The prolonged survival of <i>Mycobacterium tuberculosis</i> (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual <i>mbt</i> genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth <i>in vitro</i> and <i>in vivo</i>. The deletion mutant, Δ<i>mbtN</i>, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of <i>mbtK</i> ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The <i>mbtK</i> mutant showed markedly reduced iron scavenging and growth <i>in vitro</i>. Further, Δ<i>mbtK</i> was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.</p></div

<i>mbtK</i> is required for growth during iron-starvation and early virulence <i>in vivo</i>.

<p>(<b>A</b>) Liquid M. tb cultures grown in iron-depleted medium, supplemented or not with 50 μM ferric chloride. (<b>B</b>) Representative plates from three replicates of the strains shown in (A) grown for 3 weeks on iron-depleted plates supplemented or not with 50 μM ferric chloride. (<b>C</b>) Δ<i>mbtK</i> and complemented Δ<i>mbtK</i>, marked chromosomally with unique identifiers (q-tags), were mixed 50:50 and used to infect fifteen C57/B6 mice at ~1,000 CFU via aerosol. Five mice were sacrificed at each time point. Lung homogenates were plated for CFU, colonies were counted and collected from plates to prepare genomic DNA. Average total recovered CFU were 1,131, 68,355 and 655,650 at 24 hours, 1 week and 6 weeks, respectively. Quantitative PCR for the q-tag specific to each strain was performed in duplicate, resulting in chromosomal equivalents (CEQ) of each strain to the total CEQ recovered per lung [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004792#ppat.1004792.ref037" target="_blank">37</a>]. Log ratios were evaluated by unpaired T-tests.</p

<i>mbtK</i> deletion decreases phospholipid abundance during iron starvation.

<p>Mean intensities of representative lipids from 5 glycerophospholipid classes and mycobactin control were normalized to their value in triplicate iron-depleted wild type cultures grown and analyzed in triplicate. Dashed line indicates the ion intensity in iron-depleted wild type. Statistical comparisons are Student’s t-tests of ion intensities in iron-supplemented wild type and iron-depleted Δ<i>mbtK</i>. MB = mycobactin, PE = phosphotidylethanolamine, PI = phosphotidylinositol, CL = cardiolipin, PG = phosphatidylglycerol, TAG = triacylglyceride, FA = fatty acid.</p

<i>mbtK</i> deletion depletes phosphatidylinositol during iron starvation.

<p><b>(A)</b> Phosphatidylinositol C35:0, neutral mass 852.5728, was detected in the positive mode (<b>B</b>) as [M+H]⁺ (<i>m/z</i> 853.5802) from triplicate total lipid extracts that were normalized for mass. Chromatogram is representative of triplicate runs. (<b>C</b>) Collision of phosphatidylinositol C35:0 in the negative mode as the [M-H]^- ion, <i>m/z</i> 851.5655, showed the expected fragments confirming its structure.</p

Mycobactin synthases synthesize M. tb siderophores mycobactin and carboxymycobactin.

<p>(<b>A</b>) The <i>mbt-2</i> locus encodes mycobactin synthases that generate and modify the lipidic portion of mycobactin and carboxymycobactin. (<b>B</b>) M. tb mutants in <i>mbtN</i> and <i>mbtK</i> were generated by homologous recombination between pJM1::Δ<i>mbtN</i> or pJM1::Δ<i>mbtK</i> and flanking regions. Dots, <i>hyg</i> hygromycin resistance cassette and <i>sacB Bacillus subtilis</i> levansucrase gene. (<b>C</b>) The structures mycobactin T, monodeoxymycobactin, dideoxymycobactin and carboxymycobactin T are shown with the potential unsaturation indicated by a dashed line. In addition to the depicted core structures, known variations in the fatty acyl chain, listed below as <i>m/z</i> values of [M+H]⁺ or [M+Fe-2H]⁺ adducts, were calculated and used to search the lipidomics datasets. Monodeoxymycobactin forms both [M+H]⁺ and [M+Fe-2H]⁺ adducts. Apo-mycobactin was not detected. For carboxymycobactin, R = H or CH₃. Detected ions, highlighted in red, are highlighted in scatter plots in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004792#ppat.1004792.g002" target="_blank">Fig. 2A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004792#ppat.1004792.g003" target="_blank">3A</a>.</p