On the stability of mycobacterial ribosomes during stasis

Latent tuberculosis is estimated to account for over 99% of Mycobacterium tuberculosis infections globally. Bacteria are believed to enter a non-­replicating persistent (NRP) state to counter the effect of oxygen and nutrient limitation during latent infection. While NRP bacteria are believed to retain some metabolic activity, it is not known how the stability and functionality of the biosynthetic apparatus is maintained. Using the Wayne hypoxia model, we focused on mycobacterial ribosomes and found that, in contrast to enteric bacteria, no higher order structures (e.g. ribosomal dimers) are formed upon entry into stasis. We devised a strategy incorporating microfluidic, proteomic and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during NRP. We compared the stability of wild-type ribosomes to those of mutants in the transcriptional regulator DosR, which cannot survive prolonged oxygen starvation. While stability was comparable under conditions of active growth and normoxic stasis, ΔdosR mutants showed a marked decrease in levels of 70S ribosomes and 30S ribosomal subunits under hypoxia. Microfluidic analyses were consistent with these observations and pointed to a progressive degradation of rRNA during prolonged hypoxia, with evidence of discrete rRNA cleavage. Proteomic analysis also supported a gradual degradation of ribosomes and led to the identification of S30AE proteins as possible ribosome stabilisation factors. There is an S30AE domain protein is the DosR regulon and it may contribute to the observed destabilisation of the ribosome during hypoxia in ΔdosR mutants. Macromolecular stability is further compromised during infection by the presence of reactive oxygen and nitrogen species. We used mass spectrometry to assess the impact of oxidative stress on mycobacterial nucleic acids. We found that RNA was more susceptible to damage. Since only limited RNA synthesis occurs in NRP, the ability of cells to preserve intact ribosomes could be crucial for long-­term survival

Antimicrobial resistance in Mycobacterium tuberculosis : mechanistic and evolutionary perspectives

Antibiotic-resistant Mycobacterium tuberculosis strains are threatening progress in containing the global tuberculosis epidemic. Mycobacterium tuberculosis is intrinsically resistant to many antibiotics, limiting the number of compounds available for treatment. This intrinsic resistance is due to a number of mechanisms including a thick, waxy, hydrophobic cell envelope and the presence of drug degrading and modifying enzymes. Resistance to the drugs which are active against M. tuberculosis is, in the absence of horizontally transferred resistance determinants, conferred by chromosomal mutations. These chromosomal mutations may confer drug resistance via modification or overexpression of the drug target, as well as by prevention of prodrug activation. Drug resistance mutations may have pleiotropic effects leading to a reduction in the bacterium's fitness, quantifiable e.g. by a reduction in the in vitro growth rate. Secondary so-called compensatory mutations, not involved in conferring resistance, can ameliorate the fitness cost by interacting epistatically with the resistance mutation. Although the genetic diversity of M. tuberculosis is low compared to other pathogenic bacteria, the strain genetic background has been demonstrated to influence multiple aspects in the evolution of drug resistance. The rate of resistance evolution and the fitness costs of drug resistance mutations may vary as a function of the genetic background

The genetic background modulates the evolution of fluoroquinolone-resistance in Mycobacterium tuberculosis

Fluoroquinolones (FQ) form the backbone in experimental treatment regimens against drug-susceptible tuberculosis. However, little is known on whether the genetic variation present in natural populations of Mycobacterium tuberculosis (Mtb) affects the evolution of FQ-resistance (FQ-R). To investigate this question, we used nine genetically distinct drug-susceptible clinical isolates of Mtb and measured their frequency of resistance to the FQ ofloxacin (OFX) in vitro. We found that the Mtb genetic background led to differences in the frequency of OFX-resistance (OFX-R) that spanned two orders of magnitude and substantially modulated the observed mutational profiles for OFX-R. Further, in vitro assays showed that the genetic background also influenced the minimum inhibitory concentration and the fitness effect conferred by a given OFX-R mutation. To test the clinical relevance of our in vitro work, we surveyed the mutational profile for FQ-R in publicly available genomic sequences from clinical Mtb isolates, and found substantial Mtb lineage-dependent variability. Comparison of the clinical and the in vitro mutational profiles for FQ-R showed that 51% and 39% of the variability in the clinical frequency of FQ-R gyrA mutation events in Lineage 2 and Lineage 4 strains, respectively, can be attributed to how Mtb evolves FQ-R in vitro. As the Mtb genetic background strongly influenced the evolution of FQ-R in vitro, we conclude that the genetic background of Mtb also impacts the evolution of FQ-R in the clinic

Reference set of Mycobacterium tuberculosis clinical strains: a tool for research and product development

The Mycobacterium tuberculosis complex (MTBC) causes tuberculosis (TB) in humans and various other mammals. The human-adapted members of the MTBC comprise seven phylogenetic lineages that differ in their geographical distribution. There is growing evidence that this phylogeographic diversity modulates the outcome of TB infection and disease. For decades, TB research and development has focused on the two canonical MTBC laboratory strains H37Rv and Erdman, both of which belong to Lineage 4. Relying on only a few laboratory-adapted strains can be misleading as study results might not be directly transferrable to clinical settings where patients are infected with a diverse array of strains, including drug-resistant variants. Here, we argue for the need to expand TB research and development by incorporating the phylogenetic diversity of the MTBC. To facilitate such work, we have assembled a group of 20 genetically well-characterized clinical strains representing the seven known human-adapted MTBC lineages. With the "MTBC clinical strains reference set" we aim to provide a standardized resource for the TB community. We hope it will enable more direct comparisons between studies that explore the physiology of MTBC beyond the laboratory strains used thus far. We anticipate that detailed phenotypic analyses of this reference strain set will increase our understanding of TB biology and assist in the development of new control tools that are broadly effective

Isolation of Bacterial Ribosomes with Monolith Chromatography

We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns. Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis (iTRAQ). The speed and simplicity of this approach could accelerate the study of many different aspects of ribosomal biology

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.We thank S. Lecher, S. Li and J. Zallet for technical support. Calculations were performed at the sciCORE scientific computing core facility at the University of Basel. This work was supported by the Swiss National Science Foundation (grants 310030_166687 (S.G.) and 320030_153442 (M.E.) and Swiss HIV Cohort Study grant 740 to L.F.), the European Research Council (309540-EVODRTB to S.G.), TB-PAN-NET (FP7-223681 to S.N.), PathoNgenTrace projects (FP7-278864-2 to S.N.), SystemsX.ch (S.G.), the German Center for Infection Research (DZIF; S.N.), the Novartis Foundation (S.G.), the Natural Science Foundation of China (91631301 to Q.G.), and the National Institute of Allergy and Infectious Diseases (5U01-AI069924-05) of the US National Institutes of Health (M.E.)

On the stability of mycobacterial ribosomes during stasis

Latent tuberculosis is estimated to account for over 99% of Mycobacterium tuberculosis infections globally. Bacteria are believed to enter a non-­replicating persistent (NRP) state to counter the effect of oxygen and nutrient limitation during latent infection. While NRP bacteria are believed to retain some metabolic activity, it is not known how the stability and functionality of the biosynthetic apparatus is maintained. Using the Wayne hypoxia model, we focused on mycobacterial ribosomes and found that, in contrast to enteric bacteria, no higher order structures (e.g. ribosomal dimers) are formed upon entry into stasis. We devised a strategy incorporating microfluidic, proteomic and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during NRP. We compared the stability of wild-type ribosomes to those of mutants in the transcriptional regulator DosR, which cannot survive prolonged oxygen starvation. While stability was comparable under conditions of active growth and normoxic stasis, ΔdosR mutants showed a marked decrease in levels of 70S ribosomes and 30S ribosomal subunits under hypoxia. Microfluidic analyses were consistent with these observations and pointed to a progressive degradation of rRNA during prolonged hypoxia, with evidence of discrete rRNA cleavage. Proteomic analysis also supported a gradual degradation of ribosomes and led to the identification of S30AE proteins as possible ribosome stabilisation factors. There is an S30AE domain protein is the DosR regulon and it may contribute to the observed destabilisation of the ribosome during hypoxia in ΔdosR mutants. Macromolecular stability is further compromised during infection by the presence of reactive oxygen and nitrogen species. We used mass spectrometry to assess the impact of oxidative stress on mycobacterial nucleic acids. We found that RNA was more susceptible to damage. Since only limited RNA synthesis occurs in NRP, the ability of cells to preserve intact ribosomes could be crucial for long-­term survival.EThOS - Electronic Theses Online ServiceWellcome TrustGBUnited Kingdo

Napoved obnašanja pilota med dinamičnim obremenilnim testom z uporabo tehnologije vgrajenih senzorjev deformacij

A standard dynamic loading test of the pile was performed on the highway section Slivnica - Hajdina near Maribor, Slovenia. Parallel to standard testing procedures the new monitoring technology based on specially developed strain sensors installed inside the pile body along the pile axis was introduced. On the basis of the measured results the normal strains along the pile axis were measured. Taking into consideration the elastic modulus of the concrete the normal stresses in the axial direction of the pile were also calculated and afterwards the shear stresses along the pile shaft have been estimated as well as the normal stresses below the pile toe. The estimation was made by considering a constant value for the pile diameter. The measured results were also compared with the computer simulation of the pile and the soil behaviour during all the successive test phases. The strain measurements inside the pile body during the standard dynamic loading test in present case did not have the purpose of developing an alternative method of pile loading tests. The presented monitoring technology proved itself as a very accurate and consistent. It gave in the first place the possibility of a closer look at the strains and stresses of the most unapproachable parts of different types of concrete structure elements especially piles and other types of deep foundations.Na avtocestni povezavi Slivnica – Hajdina v bližini Maribora je bil na testnem pilotu izveden standardni dinamični obremenilni preizkus. Poleg ustaljenega postopka izvedbe preiskave je bila dodatno testirana na novo razvita tehnologija monitoringa namenjena spremljanju deformacij znotraj težko dostopnih betonskih konstrukcijskih elementov, še posebej pilotov in drugih vrst globokih temeljev. V testni pilot je bilo zato dodatno vgrajenih devet posebej izdelanih senzorjev za meritve specifičnih deformacij, ki so bili razporejeni na enakomernih razdaljah dveh metrov vzdolž osi pilota. Z omenjenimi senzorji smo v testnem pilotu spremljali deformacijsko stanje v betonu pilota med samim preizkusom ter na osnovi izmerjenih rezultatov ocenili normalne napetosti vzdolž osi pilota in trenje vzdolž plašča pilota ter normalne napetosti pod nogo pilota. Med preizkusom je bilo izvedenih sedem udarcev s prostopadno utežjo določene mase, pri čemer je bila utež spuščena z različnih višin. Pri vsakem od sedmih obtežnih primerov so bile med drugim izmerjene tudi specifične deformacije vzdolž celotne osi pilota povzročene s primarnim udarcem uteži ob pilot ter po odbojih uteži še s sekundarnim in terciarnim udarcem. Za vsakega od naštetih dogodkov so bile poiskane maksimalne vrednosti za vsa merilna mesta. Prav tako pa so bile po umiritvi sistema izvrednotene preostale oziroma trajne vrednosti specifičnih deformacij, ki so bile po prvem obtežnem primeru največje, pri ostalih šestih pa so bile bistveno manjše. Krivulje, ki povezujejo omenjene skupine maksimalnih vrednosti kažejo pričakovani trend upadanja vrednosti v smeri od vrha pilota proti nogi. Iz izmerjenih vrednosti so bile izvrednotene tudi največje vrednosti nateznih specifičnih deformacij, ki so posledica prehoda valovne fronte mimo posameznega senzorja. Ob upoštevanju elasticitetnega modula betona so bile izvrednotene normalne napetosti v betonu pilota vzdolž njegove osi. Na osnovi sprememb rezultante normalnih napetosti na območju med posameznimi merskimi mesti vzdolž pilota pa je bil ocenjen tudi potek rezultante strižnih napetosti oziroma specifičnega trenja ob plašču pilota. Ocena je bila narejena ob predpostavki konstantne debeline pilota. Rezultate meritev smo primerjali tudi z računalniško simulacijo napetostnega in deformacijskega stanja v pilotu in tleh, zato je bila neposredno ob pilotu izvedena tudi vrtina, ki je omogočila natančno klasifikacijo posameznih slojev zemljine in določitev njihovih mehanskih lastnosti

Evolution of drug resistance in tuberculosis : recent progress and implications for diagnosis and therapy

Drug-resistant tuberculosis is a growing threat to global public health. Recent efforts to understand the evolution of drug resistance have shown that changes in drug-target interactions are only the first step in a longer adaptive process. The emergence of transmissible drug-resistant Mycobacterium tuberculosis is the result of a multitude of additional genetic mutations, many of which interact, a phenomenon known as epistasis. The varied effects of these epistatic interactions include compensating for the reduction of the biological cost associated with the development of drug resistance, increasing the level of resistance, and possibly accommodating broader changes in the physiology of resistant bacteria. Knowledge of these processes and our ability to detect them as they happen informs the development of diagnostic tools and better control strategies. In particular, the use of whole genome sequencing combined with surveillance efforts in the field could provide a powerful instrument to prevent future epidemics of drug-resistant tuberculosis