DC-SIGN Is the Major Mycobacterium tuberculosis Receptor on Human Dendritic Cells

Early interactions between lung dendritic cells (LDCs) and Mycobacterium tuberculosis, the etiological agent of tuberculosis, are thought to be critical for mounting a protective anti-mycobacterial immune response and for determining the outcome of infection. However, these interactions are poorly understood, at least at the molecular level. Here we show that M. tuberculosis enters human monocyte-derived DCs after binding to the recently identified lectin DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). By contrast, complement receptor (CR)3 and mannose receptor (MR), which are the main M. tuberculosis receptors on macrophages (Mφs), appeared to play a minor role, if any, in mycobacterial binding to DCs. The mycobacteria-specific lipoglycan lipoarabinomannan (LAM) was identified as a key ligand of DC-SIGN. Freshly isolated human LDCs were found to express DC-SIGN, and M. tuberculosis–derived material was detected in CD14−HLA-DR+DC-SIGN+ cells in lymph nodes (LNs) from patients with tuberculosis. Thus, as for human immunodeficiency virus (HIV), which is captured by the same receptor, DC-SIGN–mediated entry of M. tuberculosis in DCs in vivo is likely to influence bacterial persistence and host immunity

Is Adipose Tissue a Place for Mycobacterium tuberculosis Persistence?

BACKGROUND: Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), has the ability to persist in its human host for exceptionally long periods of time. However, little is known about the location of the bacilli in latently infected individuals. Long-term mycobacterial persistence in the lungs has been reported, but this may not sufficiently account for strictly extra-pulmonary TB, which represents 10–15% of the reactivation cases. METHODOLOGY/PRINCIPAL FINDINGS: We applied in situ and conventional PCR to sections of adipose tissue samples of various anatomical origins from 19 individuals from Mexico and 20 from France who had died from causes other than TB. M. tuberculosis DNA could be detected by either or both techniques in fat tissue surrounding the kidneys, the stomach, the lymph nodes, the heart and the skin in 9/57 Mexican samples (6/19 individuals), and in 8/26 French samples (6/20 individuals). In addition, mycobacteria could be immuno-detected in perinodal adipose tissue of 1 out of 3 biopsy samples from individuals with active TB. In vitro, using a combination of adipose cell models, including the widely used murine adipose cell line 3T3-L1, as well as primary human adipocytes, we show that after binding to scavenger receptors, M. tuberculosis can enter within adipocytes, where it accumulates intracytoplasmic lipid inclusions and survives in a non-replicating state that is insensitive to the major anti-mycobacterial drug isoniazid. CONCLUSIONS/SIGNIFICANCE: Given the abundance and the wide distribution of the adipose tissue throughout the body, our results suggest that this tissue, among others, might constitute a vast reservoir where the tubercle bacillus could persist for long periods of time, and avoid both killing by antimicrobials and recognition by the host immune system. In addition, M. tuberculosis-infected adipocytes might provide a new model to investigate dormancy and to evaluate new drugs for the treatment of persistent infection

The Hydrophobic Domain of the Mycobacterial Erp Protein Is Not Essential for the Virulence of Mycobacterium tuberculosis

Erp (exported repetitive protein) is a member of a mycobacterium-specific family of extracellular proteins. A hydrophobic region that is localized at the C-terminal domain and that represents a quarter of the protein is highly conserved across species. Here we show that this hydrophobic region is not essential for restoring the virulence and tissue damage of an erp::aph mutant strain of M. tuberculosis as assessed by bacterial counts and lung histology analysis in a mouse model of tuberculosis

<i>M. tuberculosis</i> persists in a non-replicating state inside adipocytes.

<div><p> <b>A.</b> 3T3-L1 mouse (grey bars) and primary human (black bars) adipocytes were infected with <i>M. tuberculosis</i>, clinical isolate MT103, at a MOI of 1.</p> <p>Mycobacterial binding to the cells was measured after 4 h at 4°C in the presence of various inhibitors.</p> <p>Man, yeast mannan; LAM, lipoarabinomannan; pA, polyadenosinic acid; pI, polyinosinic acid; Fuc, fucoidan; PC, PAz-PC, a predominant form of oxidized low-density lipoprotein.</p> <p>Results are expressed as % of the binding without inhibitor (Ø).</p> <p> <b>B.</b> 3T3-L1 adipocytes (grey bars) and pre-adipocytes (black bars) were infected as described in A. in the presence of Fuc or PAz-PC.</p> <p>Mycobacterial binding was assessed and analysed as in A.</p> <p> <b>C.</b> 3T3-L1 pre-adipocytes (black squares) and adipocytes (open squares) were pulsed with <i>M. tuberculosis</i> MT103 at a MOI of 1 for 4 h at 37°C, then chased with fresh medium.</p> <p>Mycobacterial survival, expressed as % of the bacterial load at day 0, was monitored on a 10-day period.</p> <p>The results are standardized as % of the bacterial load at day 0 because of the differential <i>M. tuberculosis</i> binding to adipocytes and pre-adipocytes.</p> <p> <b>D.</b> 3T3-L1 adipocytes were infected as described in C.</p> <p>Viable counts (CFU) were quantified by plating serial dilutions of cell lysates (open bars), and total counts (CEQ) were quantified by qPCR (grey bars).</p> <p> <b>E.</b> 3T3-L1 adipocytes were infected as described in C.</p> <p>In order to kill extra-cellular bacteria cells were pre-incubated with 200 µg/ml amikacin 2 h prior lysis and plating.</p> <p>Results are expressed as % of bacterial load at day 0.</p> <p> <b>F.</b> 3T3-L1 adipocytes and pre-adipocytes were infected as described in C.</p> <p>Cells were treated with 1 µg/ml rifampicin (Rif), 0.1 µg/ml isoniazid (Inh), 25 µg/ml pyrazinamide (Pza) or 5 µg/ml ethambutol (Emb) at day 3, and bacterial loads were quantified at day 5.</p> <p>Results are expressed as % of bacterial load at day 5 in the absence of antibiotics (Ø).</p> <p>In all panels, results are the mean of three independent experiments and bars indicate ± sd.</p> <p>NT, not tested; NS, not significant, and *, p<0.05, as assessed by Mann-Whitney test of median comparison.</p></div

Multicenter quality control of hepatitis C virus protease inhibitor resistance genotyping.

International audienceHepatitis C virus (HCV) protease inhibitor resistance-associated substitutions are selected during triple-therapy breakthrough. This multicenter quality control study evaluated the expertise of 23 French laboratories in HCV protease inhibitor resistance genotyping. A panel of 12 well-defined blinded samples comprising two wild-type HCV strains, nine transcripts from synthetic NS3 mutant samples or from clinical strains, and one HCV RNA-negative sample was provided to the participating laboratories. The results showed that any laboratory with expertise in sequencing techniques should be able to provide reliable HCV protease inhibitor resistance genotyping. Only a 0.7% error rate was reported for the amino acid sites studied. The accuracy of substitution identification ranged from 75% to 100%, depending on the laboratory. Incorrect results were mainly related to the methodology used. The results could be improved by changing the primers and modifying the process in order to avoid cross-contamination. This study underlines the value of quality control programs for viral resistance genotyping, which is required prior to launching observational collaborative multicenter studies on HCV resistance to direct-acting antiviral agents

Naturally Occurring Resistance-Associated Variants of Hepatitis C Virus Protease Inhibitors in Poor Responders to Pegylated Interferon-Ribavirin

International audienceThe pretherapeutic presence of protease inhibitor (PI) resistance-associated variants (RAVs) has not been shown to be predictive of triple-therapy outcomes in treatment-naive patients. However, they may influence the outcome in patients with less effective pegylated interferon (pegIFN)-ribavirin (RBV) backbones. Using hepatitis C virus (HCV) population sequence analysis, we retrospectively investigated the prevalence of baseline nonstructural 3 (NS3) RAVs in a multicenter cohort of poor IFN-RBV responders (i.e., prior null responders or patients with a viral load decrease of 3-fold shift in 50% inhibitory concentration (IC50) for telaprevir or boceprevir, T54S was the most frequently detected mutation (3.9%), followed by A156T, R155K (0.7%), V36M, and V55A (0.35%). Mutations were more frequently found in patients infected with genotype 1a (7.5 to 23.6%) than 1b (3.3 to 19.8%) (P = 0.03). No other sociodemographic or viroclinical characteristic was significantly associated with a higher prevalence of RAVs. No obvious effect of baseline RAVs on viral load was observed. In this cohort of poor responders to IFN-RBV, no link was found with a sustained virological response to triple therapy, regardless of the algorithm used for the detection of mutations. Based on a cross-study comparison, baseline RAVs are not more frequent in poor IFN-RBV responders than in treatment-naive patients and, even in these difficult-to-treat patients, this study demonstrates no impact on treatment outcome, arguing against resistance analysis prior to treatment

Intra-host demographic dynamics for NS5A-dup (A) and wild (B) strains of HCV1-b derived from Bayesian Skyline Plotting at the NS5A locus.

<p>When temporal sampling was available, time was scaled in years; otherwise time is given in coalescence time.</p