Mycobacterial resuscitation promoting factors: roles and mechanisms in infected macrophages

Members of the Mycobacterium tuberculosis complex are the causative agents of tuberculosis, a major global health threat to human populations. The majority of infected individuals harbour a latent infection where bacteria persist in a non-replicating, dormant state. Resuscitation promoting factors (Rpfs) are secreted proteins whose peptidoglycan hydrolyzing abilities have been correlated with the reactivation of dormant bacteria; however, the precise molecular mechanisms underlying this remain poorly understood. The individual roles, localisation and expression patterns of Mycobacterium marinum Rpf homologues during macrophage infection were investigated by immunofluorescence using custom Rpf-specific polyclonal antibodies and confocal microscopy. We found Rpfs to associate to the polar ends of bacteria, along the lengths of the bacterial surface and free in the cytoplasm of infected macrophages suggesting a role for these proteins in apical extension and peptidoglycan biosynthesis. No distinct patterns of Rpf localisation were observed in relation to phagosomal mycobacteria. Microscopy based semi-quantification of Rpf expression levels during macrophage infection, and analysis of in vitro grown bacteria from logarithmic phase by flow cytometry identified that only a subset of mycobacteria produce Rpf at detectable levels. Significant protein sequence homology between RpfA and Listeria monocytogenes ActA suggested that RpfA could play a similar role in actin polymerisation and bacterial motility in the macrophage cytoplasm as ActA. However, under the conditions tested, ΔrpfA M. marinum did not show any differences to wildtype during infection of murine macrophages. Recombinant ActA was demonstrated to have muralytic activity, a previously uncharacterised property of this protein suggesting a function beyond the polymerisation of host cell actin

Humans in a Dish: The Potential of Organoids in Modeling Immunity and Infectious Diseases

For many decades, human infectious diseases have been studied in immortalized cell lines, isolated primary cells from blood and a range of animal hosts. This research has been of fundamental importance in advancing our understanding of host and pathogen responses but remains limited by the absence of multicellular context and inherent differences in animal immune systems that result in altered immune responses. Recent developments in stem cell biology have led to the in vitro growth of organoids that faithfully recapitulate a variety of human tissues including lung, intestine and brain amongst many others. Organoids are derived from human stem cells and retain the genomic background, cellular organization and functionality of their tissue of origin. Thus they have been widely used to characterize stem cell development, numerous cancers and genetic diseases. We believe organoid technology can be harnessed to study host–pathogen interactions resulting in a more physiologically relevant model that yields more predictive data of human infectious diseases than current systems. Here, we highlight recent work and discuss the potential of human stem cell-derived organoids in studying infectious diseases and immunity

Retention of EsxA in the Capsule-Like Layer of Mycobacterium tuberculosis Is Associated with Cytotoxicity and Is Counteracted by Lung Surfactant

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, primarily infects macrophages but withstands the host cells bactericidal effects. EsxA, also called virulence factor 6-kDa early secretory antigenic target (ESAT-6), is involved in phagosomal rupture and cell death. We provide confocal and electron microscopy data showing that M. tuberculosis bacteria grown without detergent retain EsxA on their surface. Lung surfactant has detergent-like properties and effectively strips off this surface-associated EsxA, which advocates a novel mechanism of lung surfactant-mediated defense against pathogens. Upon challenge of human macrophages with these M. tuberculosis bacilli, the amount of surface-associated EsxA rapidly declines in a phagocytosis-independent manner. Furthermore, M. tuberculosis bacteria cultivated under exclusion of detergent exert potent cytotoxic activity associated with bacterial growth. Together, this study suggests that the surface retention of EsxA contributes to the cytotoxicity of M. tuberculosis and highlights how cultivation conditions affect the experimental outcome.Funding Agencies|ZON-MW MKMD program (The Netherlands Organisation for Health Research and Development); Swedish Research Council/Swedish International Cooperation Agency (SIDA) [2012-3349, 2015-02593]; Swedish Heart Lung/the Oskar II Jubilee Foundations [20130685, 20150709]; County Council of Ostergotland</p

Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus

Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca2+-mediated signalling. Ca2+ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca2+ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuBKU80, were engineered to express the bioluminescent Ca2+-reporter aequorin. An aequorin-mediated method for routine Ca2+ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca2+]c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca2+, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The 'signatures' of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response. Concordant with the involvement of Ca2+-calmodulin complexes in these Ca2+-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca2+-signatures to all the challenges. The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses

Zebrafish embryo model for assessment of drug efficacy on mycobacterial persisters

Tuberculosis continues to kill millions of people each year. The main difficulty in eradication of the disease is the prolonged duration of treatment, which takes at least 6 months. Persister cells have long been associated with failed treatment and disease relapse because of their phenotypical, though transient, tolerance to drugs. By targeting these persisters, the duration of treatment could be shortened, leading to improved tuberculosis treatment and a reduction in transmission. The unique in vivo environment drives the generation of persisters; however, appropriate in vivo mycobacterial persister models enabling optimized drug screening are lacking. To set up a persister infection model that is suitable for this, we infected zebrafish embryos with in vitro-starved Mycobacterium marinum. In vitro starvation resulted in a persister-like phenotype with the accumulation of stored neutral lipids and concomitant increased tolerance to ethambutol. However, these starved wild-type M. marinum organisms rapidly lost their persister phenotype in vivo. To prolong the persister phenotype in vivo, we subsequently generated and analyzed mutants lacking functional resuscitation-promoting factors (Rpfs). Interestingly, the ΔrpfAB mutant, lacking two Rpfs, established an infection in vivo, whereas a nutrient-starved ΔrpfAB mutant did maintain its persister phenotype in vivo. This mutant was, after nutrient starvation, also tolerant to ethambutol treatment in vivo, as would be expected for persisters. We propose that this zebrafish embryo model with ΔrpfAB mutant bacteria is a valuable addition for drug screening purposes and specifically screens to target mycobacterial persisters

Zebrafish embryo model for assessment of drug efficacy on mycobacterial persisters

Tuberculosis continues to kill millions of people each year. The main difficulty in eradication of the disease is the prolonged duration of treatment, which takes at least 6 months. Persister cells have been long associated with failed treatment and disease relapse because of their phenotypical, though transient, tolerance to drugs. By targeting these persisters, the duration of treatment could be shortened, leading to improved tuberculosis treatment and a reduction in transmission. The unique in vivo environment drives generation of persisters, however appropriate in vivo mycobacterial persister models enabling optimized drug screening are lacking. To set up a persister infection model that is suitable for this, we infected zebrafish embryos with in vitro starved Mycobacterium marinum. In vitro starvation resulted in a persister-like phenotype with the accumulation of stored neutral lipids and concomitant increased tolerance to ethambutol. However, these starved wild-type M. marinum rapidly lost their persister phenotype in vivo. To prolong the persister phenotype in vivo we subsequently generated and analyzed mutants lacking functional resuscitation-promoting factors (Rpfs). Interestingly, the ΔrpfAB mutant, lacking two Rpfs, established an infection in vivo, whereas a nutrient-starved ΔrpfAB mutant did maintain its persister phenotype in vivo. This mutant was, after nutrient starvation, also tolerant to ethambutol treatment in vivo, as would be expected for persisters. We propose that this zebrafish embryo model with ΔrpfAB mutant bacteria is a valuable addition for drug screening purposes and specifically screens to target mycobacterial persisters

Pretreatment with the Ca²⁺-chelator BAPTA or the calmodulin inhibitor TFP differentially impacts upon Ca²⁺-signalling and homeostasis during <i>A</i>. <i>fumigatus</i> responses to stressors.

<p>After growth for 20.5 h at 25°C, <i>A</i>. <i>fumigatus</i> AEQ^CEA10 cultures were pre-treated for 30 min with either 5 mM BAPTA or 50 μM TFP prior to challenge with stressors. [Ca²⁺]_c amplitudes (measured simultaneously to stress application) and post-stimulatory [Ca²⁺]_c resting levels (10 min after stress) are represented as [Ca²⁺]_c values (A-B) and as fold change (Log₂ ratios of treated/untreated) with the modulators BAPTA (C-D) or TFP (E-F) under the different stress conditions indicated. For clarity, average values ± SD of the [Ca²⁺]_c amplitudes and post-stimulatory [Ca²⁺]_c resting levels are shown; however full Ca²⁺-signatures using the pretreatment with these modulators are plotted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138008#pone.0138008.s010" target="_blank">S9 Fig</a> Statistical analysis was performed using a 1-way ANOVA. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.005, **** <i>p</i> < 0.001.</p

Oligonucleotides used in this study.

<p>Recognition sites for restriction enzymes are indicated by bold, underlined, italicised text.</p><p>Oligonucleotides used in this study.</p

Ca²⁺-signatures in response to mechanical perturbation and hypo-osmotic shock are growth dependent.

<p>(A-C) The aequorin expressing strain AEQ^CEA10 was subjected to each stressor at various time-points of growth (18 to 24 h) at 25°C. Cultures were also microscopically analysed in order to compare the stage of conidial germination and germ tube growth with the [Ca²⁺]_c response. For clarity, average values for six technical replicates are shown without error bars; however the data is plotted with SD error bars in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138008#pone.0138008.s007" target="_blank">S6 Fig</a> for comparison. The arrows indicate the point at which each stress was applied via the injectors of the plate reader. Bar: 10 μm.</p

Plasmids used and generated in this study.

<p><i>amdS</i> = acetamidase gene of <i>A</i>. <i>nidulans</i>. Selection of transformants was performed onto minimal medium with only 0.01 M acetamide or acrylamide as a nitrogen source and lacking uridine.</p><p>Plasmids used and generated in this study.</p