Mycolates of Mycobacterium tuberculosis modulate the flow of cholesterol for bacillary proliferation in murine macrophages

The differentiation of macrophages into lipid-filled foam cells is a hallmark of the lung granuloma that forms in patients with active tuberculosis (TB). Mycolic acids (MAs), the abundant lipid virulence factors in the cell wall of Mycobacterium tuberculosis (Mtb), can induce this foam phenotype possibly as a way to perturb host cell lipid homeostasis to support the infection. It is not exactly clear how MAs allow differentiation of foam cells during Mtb infection. Here we investigated how chemically synthetic MAs, each with a defined stereochemistry similar to natural Mtb-associated mycolates, influence cell foamy phenotype and mycobacterial proliferation in murine host macrophages. Using light and laser-scanning-confocal microscopy, we assessed the influence of MA structure first on the induction of granuloma cell types, second on intracellular cholesterol accumulation, and finally on mycobacterial growth. While methoxy-MAs (mMAs) effected multi-vacuolar giant cell formation, keto-MAs (kMAs) induced abundant intracellular lipid droplets that were packed with esterified cholesterol. Macrophages from mice treated with kMA were permissive to mycobacterial growth, whereas cells from mMA treatment were not. This suggests a separate yet key involvement of oxygenated MAs in manipulating host cell lipid homeostasis to establish the state of TB

Differential spontaneous folding of mycolic acids from Mycobacterium tuberculosis

AbstractMycolic acids are structural components of the mycobacterial cell wall that have been implicated in the pathogenicity and drug resistance of certain mycobacterial species. They also offer potential in areas such as rapid serodiagnosis of human and animal tuberculosis. It is increasingly recognized that conformational behavior of mycolic acids is very important in understanding all aspects of their function. Atomistic molecular dynamics simulations, in vacuo, of stereochemically defined Mycobacterium tuberculosis mycolic acids show that they fold spontaneously into reproducible conformational groupings. One of the three characteristic mycolate types, the keto-mycolic acids, behaves very differently from either α-mycolic acids or methoxy-mycolic acids, suggesting a distinct biological role. However, subtle conformational behavioral differences between all the three mycolic acid types indicate that cooperative interplay of individual mycolic acids may be important in the biophysical properties of the mycobacterial cell envelope and therefore in pathogenicity

When worry about climate change leads to climate action:How values, worry and personal responsibility relate to various climate actions

The IPCC's report on Global Warming of 1.5°C positioned climate change as one of the most worrying issues mankind has ever faced. Although many people worry about climate change, there is still much unknown about the origins and outcomes of worry about climate change; particularly, whether and how it can motivate specific and personal climate actions. The current paper investigates this critical relationship with data from the European Social Survey Round 8 (44,387 respondents from 23 countries). As expected, the more individuals worried about climate change, the more likely they were to take and support climate action. Yet, the process through which this association occurred differed between actions. Specifically, worry was both directly and indirectly, via feelings of personal responsibility to reduce climate change, associated with climate policy support; whereas worry was mostly indirectly associated with personal climate mitigation behaviours, via personal responsibility. In addition, worry about climate change appears partly rooted in biospheric values (i.e., caring about nature and the environment), and biospheric values were also clearly, directly and positively related to personal climate mitigation behaviours. The relationships were highly consistent across countries but varied somewhat in size. The results show how generic feelings about climate change can directly and indirectly affect both climate policy support and personal climate mitigation behaviours, thereby providing critical insights for science and policy making

Immune Requirements of Post-Exposure Immunization with Modified Vaccinia Ankara of Lethally Infected Mice

Current prophylactic vaccines work via the induction of B and T cell mediated memory that effectively control further replication of the pathogen after entry. In the case of therapeutic or post-exposure vaccinations the situation is far more complex, because the pathogen has time to establish itself in the host, start producing immune-inhibitory molecules and spread into distant organs. So far it is unclear which immune parameters have to be activated in order to thwart an existing lethal infection. Using the mousepox model, we investigated the immunological mechanisms responsible for a successful post-exposure immunization with modified vaccinia Ankara (MVA). In contrast to intranasal application of MVA, we found that intravenous immunization fully protected mice infected with ectromelia virus (ECTV) when applied three days after infection. Intravenous MVA immunization induced strong innate and adaptive immune responses in lethally infected mice. By using various gene-targeted and transgenic mouse strains we show that NK cells, CD4 T cells, CD8 T cells and antibodies are essential for the clearance of ECTV after post-exposure immunization. Post-exposure immunization with MVA is an effective measure in a murine model of human smallpox. MVA activates innate and adaptive immune parameters and only a combination thereof is able to purge ECTV from its host. These data not only provide a basis for therapeutic vaccinations in the case of the deliberate release of pathogenic poxviruses but possibly also for the treatment of chronic infections and cancer

The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antibodies

<div><p>Mycolic acids (MA) are major, species-specific lipid components of Mycobacteria and related genera. In <i>Mycobacterium tuberculosis</i>, it is made up of alpha-, methoxy- and keto-MA, each with specific biological functions and conformational characteristics. Antibodies in tuberculosis (TB) patient sera respond differently towards the three MA classes and were reported to cross-react with cholesterol. To understand the antigenicity and cholesterol cross-reactivity of MA, we generated three different chicken -derived phage-displayed single-chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA, but the first recognized all three classes of chemically synthetic MAs, the second only the two oxygenated types of MAs and the third only methoxy MA. The cholesterol cross-reactivity was investigated after grafting each of the three scFv types onto two configurations of constant chain domains–CH1-4 and CH2-4. Weak but significant cross-reactivity with cholesterol was found only with CH2-4 versions, notably those two that were also able to recognize the <i>trans</i>-keto MA. The cholesteroid nature of mycobacterial mycolic acids therefore seems to be determined by the <i>trans</i>-keto MA subclass. The significantly weaker binding to cholesterol in comparison to MA confirms the potential TB diagnostic application of these antibodies.</p></div

NK- and T-cell granzyme B and K expression correlates with age, CMV infection and influenza vaccine-induced antibody titres in older adults

Granzymes are a family of serine-proteases that act as critical mediators in the cytolytic and immunomodulatory activities of immune cells such as CD8+ T-cells and natural killer (NK) cells. Previous work indicates that both granzyme B (GZB) and K (GZK) are increased with age in CD8+ T-cells, and in the case of GZB, contribute to dysfunctional immune processes observed in older adults. Here, we sought to determine how GZB and GZK expression in NK-cells, and CD4+, CD8+, and gamma-delta T-cells, quantified in terms of positive cell frequency and mean fluorescence intensity (MFI), differed with age, age-related health-traits and the antibody response to high-dose influenza vaccine. We found that the frequency and MFI of GZB-expressing NK-cells, and CD8+ and Vδ1+ T-cells, and GZK-expressing CD8+ T-cells was significantly higher in older (66–97 years old; n = 75) vs. younger (24–37 years old; n = 10) adults by up to 5-fold. There were no significant associations of GZB/GZK expression with sex, frailty or plasma levels of TNF or IL-6 in older adults, but those who were seropositive for cytomegalovirus (CMV) exhibited significantly higher frequencies of GZB+ NK-cells, and CD4+, CD8+ and Vδ1+ T-cells, and GZK+ CD8+ T-cells (Cohen’s d = .5–1.5). Pre-vaccination frequencies of GZB+ NK-cells were positively correlated with vaccine antibody responses against A/H3N2 (d = .17), while the frequencies of GZK+ NK and CD8+ T-cells were inversely associated with A/H1N1 (d = −0.18 to −0.20). Interestingly, GZK+ NK-cell frequency was inversely correlated with pre-vaccination A/H1N1 antibody titres, as well as those measured over the previous 4 years, further supporting a role for this subset in influencing vaccine antibody-responses. These findings further our understanding of how granzyme expression in different lymphoid cell-types may change with age, while suggesting that they influence vaccine responsiveness in older adults

The first syntheses of single enantiomers of the major methoxymycolic acid of Mycobacterium tuberculosis

Mycolic acids (Scheme 1) are major constituents of the cell envelope of Mycobacterium tuberculosis and other mycobacteria, some of which are pathogenic to animals and humans.1 and 2 Their presence is thought to be linked with the resistance of these organisms to most current antibiotics and other chemotherapeutic agents.3 Mycolic acids can be divided into two parts, meromycolate and mycolic motif (Scheme 1). The latter is common to each mycobacterial mycolic acid, except for minor variations in the length of the chain(d)We thank Prof. D. E. Minnikin (Univ. of Birmingham) for providing a sample of natural methoxymycolic acid extracted from M. tuberculosis

The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antibodies

Mycolic acids (MA) are major, species-specific lipid components of Mycobacteria and related genera. In Mycobacterium tuberculosis, it is made up of alpha-, methoxy- and keto- MA, each with specific biological functions and conformational characteristics. Antibodies in tuberculosis (TB) patient sera respond differently towards the three MA classes and were reported to cross-react with cholesterol. To understand the antigenicity and cholesterol cross-reactivity of MA, we generated three different chicken -derived phage-displayed single- chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA, but the first recognized all three classes of chemically synthetic MAs, the second only the two oxygenated types of MAs and the third only methoxy MA. The cholesterol cross-reactivity was investigated after grafting each of the three scFv types onto two configurations of constant chain domains±CH1-4 and CH2-4. Weak but significant cross-reactivity with cholesterol was found only with CH2-4 versions, notably those two that were also able to recognize the trans-keto MA. The cholesteroid nature of mycobacterial mycolic acids therefore seems to be determined by the trans-keto MA subclass. The significantly weaker binding to cholesterol in comparison to MA confirms the potential TB diagnostic application of these antibodies.S1 Fig. Sequences of gallibody clones produced by antibody engineering. 12) Anti-MA 12, 16) Anti-MA 16, 18) Anti-MA 18, CH1-4 = full length constant region, CH2-4 = truncated constant region, VH = variable heavy chain, VL = variable light chain.S2 Fig. SDS-PAGE analysis illustrating gallibody purification using Ni-NTA affinity columns. A) 12CH1-4, B) 16CH1-4, C) 18CH1-4, D) 12CH2-4, E) 16CH2-4, F) 18CH2-4. Gel lanes 1) Marker, 2) Culture supernatant, 3) Flow through 1, 4) Flow through 2, 5) Washes, 6) Elution 1, 7) Elution 2, 8) Elution 3, 9) Elution 4. Successful purification is demonstrated by the comparable thickness of the 67 kDa band obtained with the culture supernatant (2) and the elutions (6±9).S1 Dataset. Experimental data used for producing Figs 3 and 4.S2 Dataset. Experimental data used for producing Fig 5.S3 Dataset. Experimental data used for producing Fig 6.The Council for Scientific and Industrial Research (CSIR) parliamentary grants (YL) and the National Research Foundation of South Africa for the grants, unique grant numbers: 99386 (HR), 88622, 80577 (YL) and TTK1206281756 (LN).http://www.plosone.orgam2018Biochemistr

Thiol modified mycolic acids

Patient serum antibodies to mycolic acids have the potential to be surrogate markers of active tuberculosis (TB) when they can be distinguished from the ubiquitously present cross-reactive antibodies to cholesterol. Mycolic acids are known to interact more strongly with antibodies present in the serum of patients with active TB than in patients with latent TB or no TB. Examples of single stereoisomers of mycolic acids with chain lengths corresponding to major homologues of those present in Mycobacterium tuberculosis have now been synthesised with a sulfur substituent on the terminal position of the -chain; initial studies have established that one of these binds to a gold electrode surface, offering the potential to develop second generation sensors for diagnostic patient antibody detection.MMS and ADS wish to acknowledge support from the Government of Iraq through the award of PhD studentships.http://www.elsevier.com/locate/chemphysliphb2017BiochemistryChemistr