Elastic ankle exoskeletons reduce soleus muscle force but not work in human hopping

This is the author accepted manuscript. The final version is available from the American Physiological Society via the DOI in this recordInspired by elastic energy storage and return in tendons of human leg muscle-tendon units (MTU), exoskeletons often place a spring in parallel with an MTU to assist the MTU. However, this might perturb the normally efficient MTU mechanics and actually increase active muscle mechanical work. This study tested the effects of elastic parallel assistance on MTU mechanics. Participants hopped with and without spring-loaded ankle exoskeletons that assisted plantar flexion. An inverse dynamics analysis, combined with in vivo ultrasound imaging of soleus fascicles and surface electromyography, was used to determine muscle-tendon mechanics and activations. Whole body net metabolic power was obtained from indirect calorimetry. When hopping with spring-loaded exoskeletons, soleus activation was reduced (30-70%) and so was the magnitude of soleus force (peak force reduced by 30%) and the average rate of soleus force generation (by 50%). Although forces were lower, average positive fascicle power remained unchanged, owing to increased fascicle excursion (+4-5 mm). Net metabolic power was reduced with exoskeleton assistance (19%). These findings highlighted that parallel assistance to a muscle with appreciable series elasticity may have some negative consequences, and that the metabolic cost associated with generating force may be more pronounced than the cost of doing work for these muscles.This study was in part funded by US Israel Binational Science Foundation Start Up Grant 2011152 awarded to G. S. Sawicki

Characterising resuscitation promoting factor fluorescent-fusions in mycobacteria

Background Resuscitation promoting factor proteins (Rpfs) are peptidoglycan glycosidases capable of resuscitating dormant mycobacteria, and have been found to play a role in the pathogenesis of tuberculosis. However, the specific roles and localisation of each of the 5 Rpfs in Mycobacterium tuberculosis remain mostly unknown. In this work our aim was to construct fluorescent fusions of M. tuberculosis Rpf proteins as tools to investigate their function. Results We found that Rpf-fusions to the fluorescent protein mCherry are functional and able to promote cell growth under different conditions. However, fusions to Enhanced Green Fluorescent Protein (EGFP) were non-functional in the assays used and none were secreted into the extracellular medium, which suggests Rpfs may be secreted via the Sec pathway. No specific cellular localization was observed for either set of fusions using time-lapse video microscopy. Conclusions We present the validation and testing of five M. tuberculosis Rpfs fused to mCherry, which are functional in resuscitation assays, but do not show any specific cellular localisation under the conditions tested. Our results suggest that Rpfs are likely to be secreted via the Sec pathway. We propose that such mCherry fusions will be useful tools for the further study of Rpf localisation, individual expression, and function. Keywords Rpfs, mycobacteria, tuberculosis, fluorescent fusions, microscopy

Deciphering the metabolic response of Mycobacterium tuberculosis to nitrogen stress.

© 2015 John Wiley & Sons Ltd.A key component to the success of Mycobacterium tuberculosis as a pathogen is the ability to sense and adapt metabolically to the diverse range of conditions encountered in vivo, such as oxygen tension, environmental pH and nutrient availability. Although nitrogen is an essential nutrient for every organism, little is known about the genes and pathways responsible for nitrogen assimilation in M. tuberculosis. In this study we have used transcriptomics and chromatin immunoprecipitation and high-throughput sequencing to address this. In response to nitrogen starvation, a total of 185 genes were significantly differentially expressed (96 up-regulated and 89 down regulated; 5% genome) highlighting several significant areas of metabolic change during nitrogen limitation such as nitrate/nitrite metabolism, aspartate metabolism and changes in cell wall biosynthesis. We identify GlnR as a regulator involved in the nitrogen response, controlling the expression of at least 33 genes in response to nitrogen limitation. We identify a consensus GlnR binding site and relate its location to known transcriptional start sites. We also show that the GlnR response regulator plays a very different role in M. tuberculosis to that in non-pathogenic mycobacteria, controlling genes involved in nitric oxide detoxification and intracellular survival instead of genes involved in nitrogen scavenging

Deciphering the response of Mycobacterium smegmatis to nitrogen stress using bipartite active modules.

Background The ability to adapt to environments with fluctuating nutrient availability is vital for bacterial survival. Although essential for growth, few nitrogen metabolism genes have been identified or fully characterised in mycobacteria and nitrogen stress survival mechanisms are unknown. Results A global transcriptional analysis of the mycobacterial response to nitrogen stress, showed a significant change in the differential expression of 16% of the Mycobacterium smegmatis genome. Gene expression changes were mapped onto the metabolic network using Active Modules for Bipartite Networks (AMBIENT) to identify metabolic pathways showing coordinated transcriptional responses to the stress. AMBIENT revealed several key features of the metabolic response not identified by KEGG enrichment alone. Down regulated reactions were associated with the general reduction in cellular metabolism as a consequence of reduced growth rate. Up-regulated modules highlighted metabolic changes in nitrogen assimilation and scavenging, as well as reactions involved in hydrogen peroxide metabolism, carbon scavenging and energy generation. Conclusions Application of an Active Modules algorithm to transcriptomic data identified key metabolic reactions and pathways altered in response to nitrogen stress, which are central to survival under nitrogen limiting environments

Targeting the chromosome partitioning protein ParA in tuberculosis drug discovery

CAUL read and publish agreement 2022Publishe

The Balance of Apoptotic and Necrotic Cell Death in Mycobacterium tuberculosis Infected Macrophages Is Not Dependent on Bacterial Virulence

An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission

Tetracycline-inducible gene regulation in mycobacteria

A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml(−1) of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells

A novel biosafety level 2 compliant tuberculosis infection model using a ΔleuDΔpanCD double auxotroph of Mycobacterium tuberculosis H37Rv and Galleria mellonella

Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialised animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output

Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability

Objectives The emergence of MDR-TB, coupled with shrinking antibiotic pipelines, has increased demands for new antimicrobials with novel mechanisms of action. Antimicrobial peptides have increasingly been explored as promising alternatives to antibiotics, but their inherent poor in vivo stability remains an impediment to their clinical utility. We therefore systematically evaluated unnatural amino acid-modified peptides to design analogues with enhanced anti-mycobacterial activities. Methods Anti-mycobacterial activities were evaluated in vitro and intracellularly against drug-susceptible and MDR isolates of Mycobacterium tuberculosis using MIC, killing efficacy and intracellular growth inhibition studies. Toxicity profiles were assessed against mammalian cells to verify cell selectivity. Anti-mycobacterial mechanisms were investigated using microfluidic live-cell imaging with time-lapse fluorescence microscopy and confocal laser-scanning microscopy. Results Unnatural amino acid incorporation was well tolerated without an appreciable effect on toxicity profiles and secondary conformations of the synthetic peptides. The modified peptides also withstood proteolytic digestion by trypsin. The all D-amino acid peptide, i(llkk)2i (II-D), displayed superior activity against all six mycobacterial strains tested, with a 4-fold increase in selectivity index as compared with the unmodified L-amino acid peptide in broth. II-D effectively reduced the intracellular bacterial burden of both drug-susceptible and MDR clinical isolates of M. tuberculosis after 4 days of treatment. Live-cell imaging studies demonstrated that II-D permeabilizes the mycobacterial membrane, while confocal microscopy revealed that II-D not only permeates the cell membrane, but also accumulates within the cytoplasm. Conclusions Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity