Natural T Cell Epitope Containing Methyl Lysines on Mycobacterial Heparin-Binding Hemagglutinin.

T cell epitopes are mostly nonmodified peptides, although posttranslationally modified peptide epitopes have been described, but they originated from viral or self-proteins. In this study, we provide evidence of a bacterial methylated T cell peptide epitope. The mycobacterial heparin-binding hemagglutinin (HBHA) is a protein Ag with a complex C-terminal methylation pattern and is recognized by T cells from humans latently infected with Mycobacterium tuberculosis By comparing native HBHA with recombinant HBHA produced in Mycobacterium smegmatis (rHBHA-Ms), we could link antigenic differences to differences in the methylation profile. Peptide scan analyses led to the discovery of a peptide containing methyl lysines recognized by a mAb that binds to native HBHA ∼100-fold better than to rHBHA-Ms This peptide was also recognized by T cells from latently infected humans, as evidenced by IFN-γ release upon peptide stimulation. The nonmethylated peptide did not induce IFN-γ, arguing that the methyl lysines are part of the T cell epitope.

HBHA-Induced Polycytotoxic CD4+ T Lymphocytes Are Associated with the Control of Mycobacterium tuberculosis Infection in Humans.

Heparin-binding hemagglutinin (HBHA), a surface protein of Mycobacterium tuberculosis, is an attractive vaccine candidate and marker of protective immunity against tuberculosis, although the mechanisms underlying this protective immunity are not fully understood. Comparisons of the immune responses of latently M. tuberculosis-infected (LTBI) subjects to those of patients with active tuberculosis (aTB) may help to identify surrogate markers of protection, as LTBI subjects are most often lifelong protected against the disease. HBHA was shown to induce strong Th1 responses and cytotoxic CD8+ responses in LTBI subjects, but additional mechanisms of control of M. tuberculosis infection remain to be identified. In this study, using HBHA-induced blast formation as a readout of specific T lymphocyte activation, we report the presence in M. tuberculosis-infected subjects of HBHA-induced CD4+ T cell blasts that degranulate, as measured by surface capture of CD107a. This suggests the induction by HBHA of a CD4+ T cell subset with cytolytic function, and as nearly half of these cells also contained IFN-γ, they had both Th1 and cytotoxic characteristics. We further identified a CD4+ T lymphocyte subset producing IFN-γ together with a combination of mediators of cytotoxicity, i.e., perforin, granzymes, and granulysin, and we called them polycytotoxic CD4+ T lymphocytes. Interestingly, whereas purified protein derivative induced such cells in both LTBI subjects and patients with aTB, HBHA-specific polycytotoxic CD4+ T lymphocytes were detected in LTBI subjects and not in patients with pulmonary aTB. To our knowledge, we thus identified a new HBHA-induced CD4+ T cell subset that may contribute to the control of M. tuberculosis infection.

Capturing the mutational landscape of the beta-lactamase TEM-1.

Adaptation proceeds through the selection of mutations. The distribution of mutant fitness effect and the forces shaping this distribution are therefore keys to predict the evolutionary fate of organisms and their constituents such as enzymes. Here, by producing and sequencing a comprehensive collection of 10,000 mutants, we explore the mutational landscape of one enzyme involved in the spread of antibiotic resistance, the beta-lactamase TEM-1. We measured mutation impact on the enzyme activity through the estimation of amoxicillin minimum inhibitory concentration on a subset of 990 mutants carrying a unique missense mutation, representing 64% of possible amino acid changes in that protein reachable by point mutation. We established that mutation type, solvent accessibility of residues, and the predicted effect of mutations on protein stability primarily determined alone or in combination changes in minimum inhibitory concentration of mutants. Moreover, we were able to capture the drastic modification of the mutational landscape induced by a single stabilizing point mutation (M182T) by a simple model of protein stability. This work thereby provides an integrated framework to study mutation effects and a tool to understand/define better the epistatic interactions.

Sunscreen use during recreational activities on a French Atlantic beach: release of UV filters at sea and influence of air temperature.

Organic UV filters are emerging contaminants in personal care products such as sunscreens. The toxicity of numerous of these UV filter compounds has been demonstrated in several marine taxa. However, whilst the biological impact has already been largely demonstrated, the anthropogenic drivers leading to UV filter contamination still need to be identified. In this work, a survey was conducted on a site of the French Atlantic Coast (i) to describe beachgoers' behaviours (sunscreen use and beach frequentation), (ii) provide an estimation of the UV filters released at sea and (iii) highlight the effect of air temperature on these behaviours and on the release of UV filters. In parallel with these estimations of the UV filters released at sea, in situ chemical measurements were performed. By comparing the results of both approaches, this interdisciplinary work provides an insight of how the observations of beachgoers' behaviour modulations and attendance level fluctuations could be used to prevent UV filter contaminations and ultimately manage the ecotoxicological risk.

Combined regulation of pro-inflammatory cytokines production by STAT3 and STAT5 in a model of B. pertussis infection of alveolar macrophages.

Bordetella pertussis is a highly contagious respiratory pathogen responsible for whooping-cough or pertussis. Despite high vaccination coverage worldwide, this gram-negative bacterium continues to spread among the population. B. pertussis is transmitted by aerosol droplets from an infected individual to a new host and will colonize its upper respiratory tract. Alveolar macrophages (AMs) are effector cells of the innate immune system that phagocytose B. pertussis and secrete both pro-inflammatory and antimicrobial mediators in the lungs. However, understanding their role in B. pertussis pathogenesis at the molecular level is hampered by the limited number of primary AMs that can be collected in vivo. In order to decipher the regulation of innate response induced by B. pertussis infection, we used for the first time self-renewing, non-transformed cells, called Max Planck Institute (MPI) cells, which are phenotypically and functionally very close to pulmonary AMs. Using optimized infection conditions, we characterized the entry and the clearance of B. pertussis within MPI macrophages. We showed that under these conditions, MPI cells exhibit a pro-inflammatory phenotype with the production of TNF, IL-1ß, IL-6 and MIP-2α, similarly to primary AMs purified from broncho-alveolar fluids of mice. In addition, we explored the yet uncharacterized role of the signal transduction activator of transcription (STAT) proteins family in the innate immune response to B. pertussis infection and showed for the first time the parallel regulation of pro-inflammatory cytokines by STAT3 and STAT5 in MPI macrophages infected by B. pertussis. Altogether, this work highlights the interest of using MPI cells for experiments optimization and preliminary data acquisition to understand B. pertussis interaction with AMs, and thus significantly reduce the number of animals to be sacrificed.

Analysis of a Combined HBHA and ESAT-6-Interferon-γ-Release Assay for the Diagnosis of Tuberculous Lymphadenopathies.

BACKGROUND AND OBJECTIVES: The incidence of tuberculosis lymphadenopathy (TBLA) is increasing, and diagnostic procedures lack sensitivity and are often highly invasive. TBLA may be asymptomatic, and differential diagnosis with other adenopathies (ADPs) is difficult. We evaluated a blood-cell interferon-γ release assay (IGRA) with two different stage-specific mycobacterial antigens for the differential diagnosis of ADP suspected of mycobacterial origin. METHODS: Twenty-one patients were included and divided into three groups: (1) cervical/axillar ADP (n = 8), (2) mediastinal ADP (n = 10), and (3) disseminated ADP (n = 3). The mycobacterial antigens used for the IGRA were the heparin-binding haemagglutinin (HBHA) and the early-secreted antigenic target-6 (ESAT-6), a latency-associated antigen and a bacterial replication-related antigen, respectively. Diagnosis of TBLA based on microbiological results and/or response to anti-TB treatment was obtained for 15 patients. RESULTS: An IGRA profile highly suggestive of active TB (higher IFN-γ response to ESAT-6 compared to HBHA) was found for 3/6 TBLA patients from group 1, and for all the TBLA patients from groups 2 and 3, whereas this profile was not noticed in patients with a final alternative diagnosis. CONCLUSION: These results highlight the potential value of this combined HBHA/ESAT-6 IGRA as a triage test for the differential diagnosis of ADP.

Male Differentiation in the Marine Copepod Oithona nana Reveals the Development of a New Nervous Ganglion and Lin12-Notch-Repeat Protein-Associated Proteolysis.

Copepods are among the most numerous animals, and they play an essential role in the marine trophic web and biogeochemical cycles. The genus Oithona is described as having the highest density of copepods. The Oithona male paradox describes the activity states of males, which are obliged to alternate between immobile and mobile phases for ambush feeding and mate searching, respectively, while the female is less mobile and feeds less. To characterize the molecular basis of this sexual dimorphism, we combined immunofluorescence, genomics, transcriptomics, and protein-protein interaction approaches and revealed the presence of a male-specific nervous ganglion. Transcriptomic analysis showed male-specific enrichment for nervous system development-related transcripts. Twenty-seven Lin12-Notch Repeat domain-containing protein coding genes (LDPGs) of the 75 LDPGs identified in the genome were specifically expressed in males. Furthermore, some LDPGs coded for proteins with predicted proteolytic activity, and proteases-associated transcripts showed a male-specific enrichment. Using yeast double-hybrid assays, we constructed a protein-protein interaction network involving two LDPs with proteases, extracellular matrix proteins, and neurogenesis-related proteins. We also hypothesized possible roles of the LDPGs in the development of the lateral ganglia through helping in extracellular matrix lysis, neurites growth guidance, and synapses genesis.

Investigating population-scale allelic differential expression in wild populations of Oithona similis (Cyclopoida, Claus, 1866).

Acclimation allowed by variation in gene or allele expression in natural populations is increasingly understood as a decisive mechanism, as much as adaptation, for species evolution. However, for small eukaryotic organisms, as species from zooplankton, classical methods face numerous challenges. Here, we propose the concept of allelic differential expression at the population-scale (psADE) to investigate the variation in allele expression in natural populations. We developed a novel approach to detect psADE based on metagenomic and metatranscriptomic data from environmental samples. This approach was applied on the widespread marine copepod, Oithona similis, by combining samples collected during the Tara Oceans expedition (2009-2013) and de novo transcriptome assemblies. Among a total of 25,768 single nucleotide variants (SNVs) of O. similis, 572 (2.2%) were affected by psADE in at least one population (FDR < 0.05). The distribution of SNVs under psADE in different populations is significantly shaped by population genomic differentiation (Pearson r = 0.87, p = 5.6 × 10-30), supporting a partial genetic control of psADE. Moreover, a significant amount of SNVs (0.6%) were under both selection and psADE (p < .05), supporting the hypothesis that natural selection and psADE tends to impact common loci. Population-scale allelic differential expression offers new insights into the gene regulation control in populations and its link with natural selection.

Proportions of interferon-γ-producing ascites lymphocytes in response to mycobacterial antigens: A help for early diagnosis of peritoneal tuberculosis in a low TB incidence country.

BACKGROUND: Peritoneal tuberculosis (TB) remains difficult to diagnose because of its non-specific clinical features and the lack of efficient microbiological tests. As delayed diagnosis is associated with high mortality rates, new diagnostic tools are needed. METHODS AND FINDINGS: We investigated for 24 patients prospectively enrolled with a possible diagnosis of peritoneal TB, the diagnostic value of the analysis of IFN-γ production by peritoneal fluid lymphocytes in response to a short in vitro stimulation with mycobacterial antigens. The patients were classified in two groups: non-TB and confirmed or highly probable TB. Diagnosis of TB was based on microbiological and histopathological criteria and/or a favorable response to anti-TB treatment. The IFN-γ production by peritoneal CD4+ T lymphocytes was analyzed by flow cytometry after an overnight in vitro stimulation with three different mycobacterial antigens, purified protein derivative (PPD), heparin-binding haemagglutinin (HBHA) or early-secreted-antigen-target-6 (ESAT-6). The percentages of PPD-, HBHA- or ESAT-6-induced IFN-γ-producing peritoneal fluid CD4+ T lymphocytes were higher in the TB group than in the non-TB group (p = 0.0007, p = 0.0004, and p = 0.0002 respectively). Based on cut-off values determined by ROC curve analysis of the results from TB and highly probable TB compared to those of non-TB patients, the sensitivity of these three tests was 100% with a specificity of 92%. CONCLUSIONS: The analysis of mycobacterial-induced IFN-γ production by peritoneal lymphocytes is a promising tool to reliably and rapidly diagnose peritoneal TB. Further studies should be performed on larger cohorts of patients in high-TB-incidence countries to confirm the clinical value of this new diagnostic approach for peritoneal TB.

Translocation path of a substrate protein through its Omp85 transporter.

TpsB proteins are Omp85 superfamily members that mediate protein translocation across the outer membrane of Gram-negative bacteria. Omp85 transporters are composed of N-terminal POTRA domains and a C-terminal transmembrane ß-barrel. In this work, we track the in vivo secretion path of the Bordetella pertussis filamentous haemagglutinin (FHA), the substrate of the model TpsB transporter FhaC, using site-specific crosslinking. The conserved secretion domain of FHA interacts with the POTRA domains, specific extracellular loops and strands of FhaC and the inner ß-barrel surface. The interaction map indicates a funnel-like pathway, with conformationally flexible FHA entering the channel in a non-exclusive manner and exiting along a four-stranded ß-sheet at the surface of the FhaC barrel. This sheet of FhaC guides the secretion domain of FHA along discrete steps of translocation and folding. This work demonstrates that the Omp85 barrel serves as a channel for translocation of substrate proteins.