Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and ...lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF(-/-)) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b(+) macrophages and CD4(+) T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF(-/-) mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF(-/-) mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF(-/-) mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.
Host lipid metabolism is an important target for subversion by pathogenic mycobacteria such as Mycobacterium tuberculosis. The appearance of foam cells within the granuloma are well-characterised ...effects of chronic tuberculosis. The zebrafish-Mycobacterium marinum infection model recapitulates many aspects of human-M. tuberculosis infection and is used as a model to investigate the structural components of the mycobacterial granuloma. Here, we demonstrate that the zebrafish-M. marinum granuloma contains foam cells and that the transdifferentiation of macrophages into foam cells is driven by the mycobacterial ESX1 pathogenicity locus. This report demonstrates conservation of an important aspect of mycobacterial infection across species.
•Foam cells are identified by Oil Red O staining in zebrafish adults and embryos infected with Mycobacterium marinum.•The transdifferentiation of macrophages into foam cells is driven by the mycobacterial pathogenicity locus ESX1.•Zebrafish are an emerging model for studying mycobacterial infection-induced abnormalities in lipid metabolism.
Inhaled delivery of drugs incorporated into poly (lactic-co-glycolic acid) (PLGA) microparticles allows a sustained lung concentration and encourages phagocytosis by alveolar macrophages that ...harboring Mycobacterium tuberculosis. However, limited data are available on the effects of physicochemical properties of PLGA, including the monomer ratio (lactide:glycide) and molecular weight (MW) on the aerosol performance, macrophage uptake, and toxicity profile. The present study aims to address this knowledge gap, using PLGAs with monomer ratios of 50:50, 75:25 and 85:15, MW ranged 24 – 240kDa and an anti-tuberculosis (TB) drug, rifapentine. The PLGA-rifapentine powders were produced through a solution spray drying technique. The particles were spherical with a smooth surface and a volume median diameter around 2μm (span ~2). When the powders were dispersed using an Osmohaler® at 100L/min for 2.4s, the fine particle fraction (FPFtotal, wt.% particles in aerosol <5μm relative to the total recovered drug mass) was ranged between 52 and 57%, with no significant difference between the formulations. This result suggests that the monomer ratio and MW are not crucial parameters for the aerosol performance of PLGA. The phagocytosis analysis was performed using Thp-1 monocyte-derived macrophages. The highest rate of uptake was observed in PLGA 85:15 followed by 75:25 and 50:50 with about 90%, 80% and 70%, respectively phagocytosis over 4h of exposure. Furthermore, the cytotoxicity analysis on Thp-1 and human lung adenocarcinoma epithelial cells demonstrated that PLGA concentration up to 1.5mg/mL, regardless of the monomer composition and MW, were non-toxic. In conclusion, the monomer ratio and MW are not crucial in determining the aerosol performance and cytotoxicity profile of PLGA however, the particles with high lactide composition have a superior tendency for macrophage uptake.
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The ohmyungsamycin and ecumicin natural product families are structurally related cyclic depsipeptides that display potent antimycobacterial activity. Herein the total syntheses of ohmyungsamycin A, ...deoxyecumicin, and ecumicin are reported, together with the direct biological comparison of members of these natural product families against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). The synthesis of each of the natural products employed a solid‐phase strategy to assemble the linear peptide precursor, involving a key on‐resin esterification and an optional on‐resin dimethylation step, before a final solution‐phase macrolactamization between the non‐proteinogenic N‐methyl‐4‐methoxy‐l‐tryptophan amino acid and a bulky N‐methyl‐l‐valine residue. The synthetic natural products possessed potent antimycobacterial activity against Mtb with MIC90’s ranging from 110–360 nm and retained activity against Mtb in Mtb‐infected macrophages. Deoxyecumicin also exhibited rapid bactericidal killing against Mtb, sterilizing cultures after 21 days.
A unified strategy towards antimycobacterial natural products: The total syntheses of the cyclic depsipeptide natural products ohmyungsamycin A, deoxyecumicin, and ecumicin are described. Synthesis was achieved through a unified solid‐phase synthetic strategy and a late‐stage, pre‐organized macrolactamization at an unusually hindered junction. Access to these natural products enabled a detailed comparison on their antimycobacterial activity.
Tuberculosis remains a leading cause of mortality among infectious diseases worldwide, prompting the need to discover new drugs to fight this disease. We report here the design, synthesis, and ...antimycobacterial activity of isatin-mono/bis-isoniazid hybrids. Most of the compounds exhibited very high activity against Mycobacterium tuberculosis, with MICs in the range of 0.195 to 0.39 μg/ml, and exerted a more potent bactericidal effect than the standard antitubercular drug isoniazid (INH). Importantly, these compounds were found to be well tolerated at high doses (>200 μg/ml) on Vero kidney cells, leading to high selectivity indices. Two of the most promising hybrids were evaluated for activity in THP-1 macrophages infected with M. tuberculosis, among which compound 11e was found to be slightly more effective than INH. Overexpression of InhA along with cross-resistance determination of the most potent compounds, selection of resistant mutants, and biochemical analysis, allowed us to decipher their mode of action. These compounds effectively inhibited mycolic acid biosynthesis and required KatG to exert their biological effects. Collectively, this suggests that the synthesized isatin-INH hybrids are promising antitubercular molecules for further evaluation in preclinical settings.
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Endolysins are bacteriophage-derived protein molecules highly effective for bacterial killing. Cpl-1 and ClyJ-3 are native and chimeric endolysins, respectively, having antimicrobial ...activity against Streptococcus pneumoniae which causes lung infections. We conducted the first feasibility study on nebulisation of Cpl-1 and ClyJ-3, with a focus on the antimicrobial activity, structural changes of the proteins and aerosol performance. Bacterial colony counts, live cell imaging and Fourier-transform infrared(FTIR) spectroscopy were used to evaluate the proteins before and after jet or vibrating mesh nebulisation. These nebulised aerosols were inhalable with a volume median size of 3.8–4.2 µm (span 1.1–2.3) measured by laser diffraction. However, nebulisation caused almost complete loss in bioactivity of ClyJ-3, which were corroborated with the live cell imaging observation and protein structural damage with a large intensity reduction in the amide absorption bands between 1300 and 1700 cm−1. In contrast, the bactericidal activity of Cpl-1 showed no significant difference (p ≥ 0.05) before and after mesh nebulisation with 4.9 and 4.6-log10 bacterial count reduction, respectively. However, jet nebulisation reduced the bioactivity of Cpl-1 and the effect was time-dependent showing 1.7, 1.0-log10 bacterial count reduction at 7 and 14 min with complete loss of antimicrobial activity at 21 min after nebulisation, respectively. The results were consistent with time-dependent changes in live cell images and FTIR amide band changes at 1655, 1640, 1632 and 1548 cm−1. In conclusion, it is feasible to nebulise endolysins for inhalation delivery but it depends on both the protein and the nebuliser, with the mesh nebuliser being the preferred choice.
Macrophages display marked plasticity with functions in both inflammation and tissue repair. Evidence demonstrates that this spectrum of macrophage phenotypes is influenced by their local ...microenvironment and tissue origin. However, in vitro macrophage experiments often do not or cannot readily use macrophages from the most relevant tissue of origin. This study investigated if the origin of two C57BL/6 mouse macrophage cell lines of alveolar (AMJ2‐C11) and peritoneal (IC‐21) origin may influence their response to mycobacterial infection. Both cell lines equally controlled the growth of Mycobacterium bovis BCG and Mycobacterium tuberculosis, although the expression of all proinflammatory cytokines and chemokines measured (TNF, IL‐6, MCP‐1, MIP‐1α, MIP‐1β, and RANTES) was significantly higher in AMJ2‐C11 cells than in IC‐21 cells. During M. tuberculosis infection, IL‐6, MCP‐1, and RANTES expression increased 5‐fold, and MIP‐1β expression increased 30‐fold. Additionally, AMJ2‐C11 cells exhibited significantly higher inducible nitric oxide synthase activity than IC‐21 cells, indicative of a more polarized M1 response. The expression of multiple surface markers was also assessed by flow cytometry. CD80 and CD86 were significantly upregulated in AMJ2‐C11 cells and downregulated in IC‐21 cells during M. tuberculosis infection. The results support the notion that the origin of tissue‐resident macrophages influences their phenotype and antimicrobial response and demonstrate hereto unrecognized potential for these cell lines in in vitro studies.
This study demonstrates that macrophages of differing tissue origin exhibit contrasting responses to mycobacterial infection. Alveolar macrophages demonstrated enhanced cytokine expression, nitric oxide activity, and upregulation of inflammatory surface receptors compared with peritoneal macrophages following Mycobacterium tuberculosis infection. The results suggest hereto unrecognized potential for these cell lines in in vitro studies.
Tuberculosis (TB) remains a staggering burden on global public health. Novel preventative tools are desperately needed to reach the targets of the WHO post-2015 End-TB Strategy. Peptide or ...protein-based subunit vaccines offer potential as safe and effective generators of protection, and enhancement of local pulmonary immunity may be achieved by mucosal delivery. We describe the synthesis of a novel subunit vaccine via native chemical ligation. Two immunogenic epitopes, ESAT61–20 and TB10.43–11 from Mycobacterium tuberculosis (Mtb), were covalently conjugated to the TLR2-ligand Pam2Cys to generate a self-adjuvanting lipopeptide vaccine. When administered mucosally to mice, the vaccine enhanced pulmonary immunogenicity, inducing strong Th17 responses in the lungs and multifunctional peripheral T-lymphocytes. Mucosal, but not peripheral vaccination, provided substantial protection against Mtb infection, emphasizing the importance of delivery route for optimal efficacy.
The hallmark of tuberculosis (TB) is the formation of immune cell-enriched aggregates called granulomas. While granulomas are pathologically diverse, their tissue-wide heterogeneity has not been ...spatially resolved at the single-cell level in human tissues. By spatially mapping individual immune cells in every lesion across entire tissue sections, we report that in addition to necrotizing granulomas, the human TB lung contains abundant non-necrotizing leukocyte aggregates surrounding areas of necrotizing tissue. These cellular lesions were more diverse in composition than necrotizing lesions and could be stratified into four general classes based on cellular composition and spatial distribution of B cells and macrophages. The cellular composition of non-necrotizing structures also correlates with their proximity to necrotizing lesions, indicating these are foci of distinct immune reactions adjacent to necrotizing granulomas. Together, we show that during TB, diseased lung tissue develops a histopathological superstructure comprising at least four different types of non-necrotizing cellular aggregates organized as satellites of necrotizing granulomas.
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This study aimed to develop inhalable powders containing phages active against antibiotic-resistant Pseudomonas aeruginosa for pulmonary delivery. A Pseudomonas phage, PEV2, was spray ...dried into powder matrices comprising of trehalose (0–80%), mannitol (0–80%) and l-leucine (20%). The resulting powders were stored at various relative humidity (RH) conditions (0, 22 and 60% RH) at 4°C. The phage stability and in vitro aerosol performance of the phage powders were examined at the time of production and after 1, 3 and 12 months storage. After spray drying, a total of 1.3 log titer reduction in phage was observed in the formulations containing 40%, 60% and 80% trehalose, whereas 2.4 and 5.1 log reductions were noted in the formulations containing 20% and no trehalose, respectively. No further reduction in titer occurred for powders stored at 0 and 22% RH even after 12 months, except the formulation containing no trehalose. The 60% RH storage condition had a destructive effect such that no viable phages were detected after 3 and 12 months. When aerosolised, the total lung doses for formulations containing 40%, 60% and 80% trehalose were similar (in the order of 105 pfu). The results demonstrated that spray drying is a suitable method to produce stable phage powders for pulmonary delivery. A powder matrix containing ≥40% trehalose provided good phage preservation and aerosol performances after storage at 0 and 22% RH at 4°C for 12 months.
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