Mechanisms of Phenotypic Rifampicin Tolerance in <i>Mycobacterium tuberculosis</i> Beijing Genotype Strain B0/W148 Revealed by Proteomics

The “successful” Russian clone B0/W148 of <i>Mycobacterium tuberculosis</i> Beijing is well-known for its capacity to develop antibiotic resistance. During treatment, resistant mutants can occur that have inheritable resistance to specific antibiotics. Next to mutations, <i>M. tuberculosis</i> has several mechanisms that increase their tolerance to a variety of antibiotics. Insights in the phenotypic mechanisms that contribute to drug tolerance will increase our understanding of how antibiotic resistance develops in <i>M. tuberculosis</i>. In this study, we examined the (phospho)­proteome dynamics in <i>M. tuberculosis</i> Beijing strain B0/W148 when exposed to a high dose of rifampicin; one of the most potent first-line antibiotics. A total of 2,534 proteins and 191 phosphorylation sites were identified, and revealed the differential regulation of DosR regulon proteins, which are necessary for the development of a dormant phenotype that is less susceptible to antibiotics. By examining independent phenotypic markers of dormancy, we show that persisters of <i>in vitro</i> rifampicin exposure entered a metabolically hypoactive state, which yields rifampicin and other antibiotics largely ineffective. These new insights in the role of protein regulation and post-translational modifications during the initial phase of rifampicin treatment reveal a shortcoming in the antituberculosis regimen that is administered to 8–9 million individuals annually

Comprehensive Analysis of the Mouse Brain Proteome Sampled in Mass Spectrometry Imaging

On-tissue enzymatic digestion is performed in mass spectrometry imaging (MSI) experiments to access larger proteins and to assign protein identities. Most on-tissue digestion MSI studies have focused on method development rather than identifying the molecular features observed. Herein, we report a comprehensive study of the mouse brain proteome sampled by MSI. Using complementary proteases, we were able to identify 5337 peptides in the matrix-assisted laser desorption/ionization (MALDI) matrix, corresponding to 1198 proteins. 630 of these peptides, corresponding to 280 proteins, could be assigned to peaks in MSI data sets. Gene ontology and pathway analyses revealed that many of the proteins are involved in neurodegenerative disorders, such as Alzheimer’s, Parkinson’s, and Huntington’s disease

HLA-I binding affinity of eluted peptides derived from the invariant chain.

*<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone-0034649-g002" target="_blank">Figure 2</a> for amino acid position in the invariant chain protein.</p>†<p>IC50 is the concentration used to obtain half maximal competition and represents the mean value of two independent experiments.</p>‡<p>Binding affinity is classified according to the following IC50 cut-off values: high affinity, ≤5 µM; intermediate (int) affinity, 5–15 µM; low affinity, 15–100 µM; no binding, >100 µM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>.</p

Surface display of the CLIP epitope on HLA-II-negative leukemic cells and influence of the invariant chain on HLA-I expression.

<p>(A) Surface expression of CLIP, HLA-DR, total HLA-II (‘HLA-DRPQ’) and invariant chain (‘CD74’) of myeloid cells from an acute promyelocytic leukemia (APL) patient, as determined by flow cytometry. Myeloid cells were defined as CD45^dim/SSC^low/int and expression thresholds were set according to unstained myeloid cells. (B) Quantitative analysis on frequencies of myeloid cells from APL patients that express CLIP (n = 9), HLA-DR (n = 9), total HLA-II (n = 6) and CD74 (n = 6). Frequencies indicate percentage tumor cells that reach threshold expression based on unstained leukemic cells. (C) The effect of invariant chain Ii down-modulation in KG-1 (CLIP^-) and THP-1 (CLIP⁺) leukemic cells on HLA-I expression at the cell surface. Intracellular staining (ICS) of Ii (PIN1.1) and surface staining of HLA-I (W6/32) were compared between Ii siRNA-transduced and non-transduced cells.</p

Promiscuous binding of CLIP-peptide to distinct HLA-I alleles, representing four supertypes.

<p>The CLIP peptide RMATPLLMQALPM (peptide 3) was tested for binding affinity in a competition-based cellular peptide binding assay. The four tested HLA-I alleles (HLA-A0201, -A0301, -B0702 and -B4002) harbor a completely distinct binding pocket and bind different peptide ligands. CLIP peptide shows intermediate to high binding affinity to all of these. Separate positive control peptides efficiently bind to their respective HLA allele: GILGFVFTL (A0201 peptide), QVPLRPMTYK (A0301 peptide), SPSVDKARAEL (B0702 peptide) and GEFGGFGSV (B4002 peptide) with IC50 values of 3.7, 0.2, 0.7 and 0.2, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>. The peptide concentration started at 100 µg/ml for HLA-A0201, -A0301 and -B4002 and 25 µg/ml for HLA-B0702, followed by a serial dilution of a factor two. Exact IC50 values of the CLIP peptides are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone-0034649-t002" target="_blank">Table 2</a>.</p

Invariant chain-derived peptides identified in isolated HLA-I molecules of B-LCLs.

<p>Peptide elutions of purified HLA-I molecules from EBV-transformed B-LCLs resulted in the identification of five peptides originating from the invariant chain. HLA-I purification and subsequent mass spectrometry analysis are described in <i>Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#s2" target="_blank">Methods</a></i>. Of note, peptide 3 and 4 are located in the CLIP region, known for universal binding to HLA-II molecules.</p

HLA-I binding affinity of peptide length variants located in the CLIP region of the invariant chain.

*<p>Peptide found with HLA-I elutions.</p>†<p>Amino acid (AA) position in the invariant chain protein.</p>‡<p>Predicted HLA-I allele to which the peptide binds. Peptide binding prediction was done with netMHC (<a href="http://www.cbs.dtu.dk/services/NetMHC" target="_blank">http://www.cbs.dtu.dk/services/NetMHC</a>). Binding predictions can be made for peptide lengths between 8 and 11 for all alleles with a novel approximation algorithm using artificial neural networks trained on 9-mer peptides <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Lundegaard1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Lundegaard2" target="_blank">[27]</a>. Only peptides are shown with a predicted binding affinity of 500 nM or stronger.</p>§<p>IC50 is the concentration used to obtain half maximal competition and represents the mean value of two independent experiments.</p>¶<p>Binding affinity is classified according to the following IC50 cut-off values: high affinity, ≤5 µM; intermediate (int) affinity, 5–15 µM; low affinity, 15–100 µM; no binding, >100 µM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>.</p

Proteasomal Degradation of Proinsulin Requires Derlin-2, HRD1 and p97

<div><p>Patients with type 1 diabetes (T1D) suffer from beta-cell destruction by CD8⁺ T-cells that have preproinsulin as an important target autoantigen. It is of great importance to understand the molecular mechanism underlying the processing of preproinsulin into these CD8⁺ T-cell epitopes. We therefore studied a pathway that may contribute to the production of these antigenic peptides: degradation of proinsulin via ER associated protein degradation (ERAD). Analysis of the MHC class I peptide ligandome confirmed the presentation of the most relevant MHC class I-restricted diabetogenic epitopes in our cells: the signal peptide-derived sequence A15-A25 and the insulin B-chain epitopes H29-A38 and H34-V42. We demonstrate that specific silencing of Derlin-2, p97 and HRD1 by shRNAs increases steady state levels of proinsulin. This indicates that these ERAD constituents are critically involved in proinsulin degradation and may therefore also play a role in subsequent antigen generation. These ERAD proteins therefore represent interesting targets for novel therapies aiming at the reduction and possibly also prevention of beta-cell directed auto-immune reactions in T1D.</p></div

Multimodal Mass Spectrometry Imaging of <i>N</i>‑Glycans and Proteins from the Same Tissue Section

On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the <i>in situ</i> multimodal analysis of <i>N</i>-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both <i>N</i>-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique <i>N</i>-glycan and peptide identities

Epitopes eluted from MHC class I.

<p>Epitopes eluted from MHC class I.</p