Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide–phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F‐actin. In the present study, we have characterized the dynamics of F‐actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F‐actin accumulated progressively around phagosomes containing wild‐type L. donovani promastigotes during the first hour of phagocytosis. Using LPG‐defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG‐dependent accumulation of periphagosomal F‐actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCα to the phagosome. Accumulation of periphagosomal F‐actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.

Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and that incorporation of mannosecapped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor. LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase, contradicting some previous suggestions.Mycobacterium tuberculosis, the etiological agent of tuberculosis, spreads by aerosol, mainly infecting alveolar macrophages, by which the bacterium is ingested (17). Through inhibition of macrophage functions, the bacterium modulates the host immune response (25). The best-characterized virulence factor of M. tuberculosis, lipoarabinomannan (LAM), is an abundant glycolipid, which is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and extends through the cell wall of the bacterium (5). During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage via specific receptors, including the macrophage mannose receptor and complement receptor 3 (13,14,19), and can then be detected at multiple sites in the cell (27). The host cell exports LAM from the phagosome in an exocytosis-like manner, eliciting responses in bystander cells (1-3). The M. tuberculosis surface harbors mannose-capped LAM (ManLAM), whereas other, less pathogenic, mycobacteria contain LAM either with a phospho-myo-inositol cap (PILAM) or no cap (6). The type of capping and the presence of the GPI anchor is crucial for virulence (13,14). The effects of Man-LAM on the host cell are multiple, but the interference of ManLAM with phagosomal maturation is the best characterized (21) and has been demonstrated in murine and human macrophages (10,12,14).Studies on human lymphocytes showed that LAM localizes to membrane rafts of the lymphocyte membrane, thereby interfering with signaling affecting cytokine production (20).Membrane rafts are cholesterol-and glycosphingolipid-rich domains that act as platforms for cell signaling processes (16). The aim of this study was to investigate whether the effects of LAM are due to incorporation of LAM into membrane rafts of human macrophages. We establish that LAM is incorporated into membrane rafts of the macrophage cell membrane via its GPI anchor, resulting in reduced phagosomal maturation. MATERIALS AND METHODSAntigens and antibodies. ManLAM, cell wall fractions (CWF), and phosphatidylinositol mannosides (PIM) from the virulen...

Lipophosphoglycan (LPG), the major surface glycoconjugate on Leishmania donovani promastigotes, is crucial for the establishment of infection inside macrophages. LPG comprises a polymer of repeating Galβ1,4Manα-PO 4 attached to a lysophosphatidylinositol membrane anchor. LPG is transferred from the parasite to the host macrophage membrane during phagocytosis and induces periphagosomal F-actin accumulation correlating with an inhibition of phagosomal maturation. The biophysical properties of LPG suggest that it may be intercalated into membrane rafts of the host cell membrane. The aim of this study was to investigate if the effects of LPG on phagosomal maturation are mediated via action on membrane rafts. We show that LPG accumulates in rafts during phagocytosis of L. donovani and that disruption of membrane rafts abolished the effects of LPG on periphagosomal F-actin and phagosomal maturation, indicating that LPG requires intact membrane rafts to manipulate host cell functions. We conclude that LPG associates with membrane rafts in the host cell and exert its actions on host-cell actin and phagosomal maturation through subversion of raft function.2 3

DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, DNA samples from crime scenes often contain PCR-inhibitory substances, which may generate blank or incomplete DNA profiles. Extensive DNA purification can be required to rid the sample of these inhibitors, although these procedures increase the risk of DNA loss. Most forensic laboratories use commercial DNA amplification kits (e.g., AmpFlSTR SGM Plus) with the DNA polymerase AmpliTaq Gold as the gold standard. Here, we show that alternative DNA polymerase-buffer systems can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition in crime scene samples, without additional sample preparation. DNA profiles from 20 of 32 totally or partially inhibited crime scene saliva samples were significantly improved using Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity instead of AmpliTaq Gold. A statistical model for unbiased quality control of forensic DNA profiles was developed to quantify the results. Our study demonstrates the importance of adjusting the chemistry of the PCR to enhance forensic DNA analysis and diagnostic PCR, providing an alternative to laborious sample preparation protocols.

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