Toll-like receptor pathways in the immune responses to mycobacteria

The control of Mycobacterium tuberculosis infection depends on recognition of the pathogen and the activation of both the innate and adaptive immune responses. Toll-like receptors (TLR) were shown to play a critical role in the recognition of several pathogens. Mycobacterial antigens recognise distinct TLR resulting in rapid activation of cells of the innate immune system. Recent evidence from in vitro and in vivo investigations, summarised in this review demonstrates TLR-dependent activation of innate immune response, while the induction of adaptive immunity to mycobacteria may be TLR independent.     

Stress responses and genetic variation in bacteria

Under stressful conditions mechanisms that increase genetic variation can bestow a selective advantage. Bacteria have several stress responses that provide ways in which mutation rates can be increased. These include the SOS response, the general stress response, the heat-shock response, and the stringent response, all of which impact the regulation of error-prone polymerases. Adaptive mutation appears to be process by which cells can respond to selective pressure specifically by producing mutations. In Escherichia coli strain FC40 adaptive mutation involves the following inducible components: (i) a recombination pathway that generates mutations; (ii) a DNA polymerase that synthesizes error-containing DNA; and (iii) stress responses that regulate cellular processes. In addition, a subpopulation of cells enters into a state of hypermutation, giving rise to about 10% of the single mutants and virtually all of the mutants with multiple mutations. These bacterial responses have implications for the development of cancer and other genetic disorders in higher organisms.     

Stress responses to comparative handling procedures in sheep

The objective of this study was to compare some husbandry procedures on the base of physiological stress parameters and evaluate the welfare status in sheep. Forty ewes were used as the study material. Measurements were taken during several routine husbandry procedures such as milking, shearing, weighing, loading and hoof care. Data regarding time spent for each application, as well as heart and respiratory rates were recorded during the applications. Blood samples were taken 15 min before and after each application and malondialdehyde (MDA), glutathione-peroxidase (GSH-Px), cortisol T₃ and T₄ parameters were measured. In addition, changes in the same parameters between pre- and post-application periods were evaluated. According to the results, machine milking caused less stress than hand milking. No significant difference was seen between shearing methods for hand shearer or clipper; however, both applications caused stress in animals. The results for weighing methods of animals demonstrated significant differences in cortisol, T₃ and T₄ values in favor of traditional method. Cortisol, T₃ and T₄ levels were significantly higher in manual loading compared with loading by ramp. Regarding hoof care, all the examined parameters differed in favor of modern method. On the other hand, significant differences were determined between the stress parameters regarding pre- and post-applications. All values differed for hand milking while no significant difference was observed in MDA and T₃ values in machine milking group. Parameters in weighing groups changed significantly. For loading process, GSH, cortisol, T₃ and T₄ values differed in both treatment groups. With regard to hoof care, parameters except T₄ in laying group differed significantly. An increase occurred in minute-based measurements of heart and respiratory rates parallel to physiological data. The number of the respiratory rates during the applications differed except for the shearing process. All the parameters displayed significant differences between groups in terms of heart rates. Time spent for each application also differed between groups. Time saved for milking, shearing, weighing, loading and hoof care was 3.23 min, 4.37 min, 1.71 min, 7.85 s and 1.55 min, respectively. These results appear to provide a tangible advantage of using new husbandry methods to the breeders. It was concluded that using new methods in sheep husbandry procedures provided advantages in terms of saving time and reducing labor, as well as improved conditions for welfare of animals. In addition, it facilitated the routine works and flock husbandry.     

Esterases in mycobacteria

Esterases ofMycobacterium phlei (acetic ester acetyl hydrolase E.C.3.1.6 and carboxylic esterhydrolase E.C.3.1.1.1.) obtained after separation on Sephadex G-100 can be temporarily, for a short time interval, activated by adding calcium ions. The activation of esterases isolated from cells was non-repeteable, whereas the temporary activation of esterases from the culture filtrate could be repeated by increasing concentrations of calcium ions. However, the value of activation gradually decreased. Similarly with calcium ions strontium ions were also effective, however, higher concentrations were required and the activation was non-repeatable. Magnesium ions were practically without any effect. Possible mechanisms of the temporary activation of esterases ofMycobacterium phlei are discussed.     

Esterases in mycobacteria

Esterases ofMycobacterium phlei isolated by means of Sephadex G-100 chromatography could be temporarily activated by adding calcium ions. This activation could not be brought about in crude enzyme preparations from cells or in crude extracts from the culture filtrate. It was demonstrated that compounds (or a compound) without the esterase activity isolated after the separation of crude enzyme preparations on Sephadex G-100 (peak C) are responsible for the above difference. It was found that the suppression by compounds from the “C peak” of the temporary activation of esterases with calcium ions depends most probably on the ratio of these compounds to the quantity of the enzyme rather than on their concentration. In addition, the compounds of the C peak themselves were found to activate esterases. The activation was lower than the temporary activation with calcium ions. The possible mechanisms of the temporary activation of esterases and the importance of these findings, from the point of view of regulation of the activity of esterases during submerged cultivation ofMycobacterium phlei, are discussed.     

Esterases in mycobacteria

A procedure for the isolation of a crude enzyme preparation of esterases fromMycobacterium phlei was worked out. The procedure consists in breaking cells in 1% KCl by ultrasonication, ultracentrifugation at 40,000 r.p.m. and isolation of acetone and ether dried enzyme preparation. Specific activity increased 2.8-fold after completion of the procedure. Esterases fromMycobacterium phlei were separated on Sephadex of G series to two enzymes with different substrate specificity. The first enzyme, acetic ester acetyl-hydrolase (E.C. 3.1.1.6) was found to be relatively specific for ethylacetate, the second, carboxylic ester hydrolase (E.C. 3.1.1.1) for ethylbutyrate and tributyrine. Preparations of both enzymes were made from the crude extracts of cells and from a mixture of macromolecular compounds isolated from the culture filtrate ofMycobacterium phlei.     

Recombination in mycobacteria

Mycobacterium tuberculosis , the causative agent of tuberculosis (TB) is thought to infect a quarter of the world's population and accounts for 3 million deaths each year. Leprosy, caused by Mycobacterium leprae continues to afflict millions. In many countries, the incidence of TB is increasing due to its association with acquired immune deficiency syndrome (AIDS) and the emergence of multidrug resistance strains of tubercle bacilli. Genes that encode major antigens, enzymes, potential virulence determinants and drug resistance in mycobacteria have been isolated and characterized; however, further genetic analysis of pathogenic mycobacteria has been severely hampered by the difficulty in precisely defining the phenotype of both wild-type and mutant genes by utilizing homologous recombination to perform allele exchange. Recombination mechanisms have been intensely studied in Escherichia coli but it is unclear how far mechanistic pathways elucidated in this species are applicable to other organisms, such as mycobacteria. The aim of this review is to examine what is currently known about homologous recombination in mycobacteria. A model is proposed to account for both low levels of homologous recombination and high levels of illegitimate recombination found in the tubercle bacillus.     

Esterases in mycobacteria

It was found that a submerged culture ofMycobacterium phlei degrades simple esters (ethylacetate and ethylbutyrate) as well as synthetic lipids (triacetine and tributyrine). The effect of pH on the rate of degradation of tributyrine was investigated and the maximum activity of esterases found within a wide range of pH. The activity of esterases was followed during growth of a submerged culture ofMycobacterium phlei. Esterases were not released into the cultivation medium during growth or even during the early stationary phase. Only a low steady activity of esterases could be demonstrated in a filtrate of the cultivation liquid. The total activity of esterases reached its maximum after a 6–11 day incubation. The specific activity of esterases reached a maximum on the 6th day of incubation; its value decreased to about one half and did not change substantially on prolonged incubation. Changes in the specific activity of esterases were found to be time-related with changes of pH and a decrease of the specific activity was associated with a release of macromolecular compounds into the incubation medium. Esterases as well as other macromolecular compounds were isolated from the filtrate of the cultivation medium ofMycobacterium phlei. The isolated preparation contained 60–72% total activity of esterases present in the filtrate of the cultivation liquid.     

Surrounded by mycobacteria: nontuberculous mycobacteria in the human environment

A majority of the Mycobacterium species, called the nontuberculous mycobacteria (NTM), are natural inhabitants of natural waters, engineered water systems, and soils. As a consequence of their ubiquitous distribution, humans are surrounded by these opportunistic pathogens. A cardinal feature of mycobacterial cells is the presence of a hydrophobic, lipid-rich outer membrane. The hydrophobicity of NTM is a major determinant of aerosolization, surface adherence, biofilm-formation, and disinfectant- and antibiotic resistance. The NTM are oligotrophs, able to grow at low carbon levels [>50 μg assimilable organic carbon (AOC) l⁻¹], making them effective competitors in low nutrient, and disinfected environments (drinking water). Biofilm formation and oligotrophy lead to survival, persistence, and growth in drinking water distribution systems. In addition to their role as human and animal pathogens, the widespread distribution of NTM in the environment, coupled with their ability to degrade and metabolize a variety of complex hydrocarbons including pollutants, suggests that NTM may be agents of nutrient cycling.     

Stress responses and replication of plasmids in bacterial cells

Plasmids, DNA (or rarely RNA) molecules which replicate in cells autonomously (independently of chromosomes) as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage lambda that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.     

'Atypical' mycobacteria in milk

Seventy-seven isolates of 'atypical' mycobacteria were obtained from 288 raw milk samples but no isolates were made from 76 samples of pasteurized milk. Thirty-two clinically important 'atypical' mycobacteria were isolated.     

Sulfite reduction in mycobacteria

Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict anaerobic conditions [k(cat) = 1.0 (+/-0.1) electron consumed per second, and K(m(SO(3)(-2))) = 27 (+/-1) microM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (sirA, originally misannotated nirA) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed che1) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe(2+) into siroheme. Deletion of che1 causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery.     

Gene manipulation in mycobacteria

Gene manipulation in mycobacteria developed in two phases. In the first phase genes of mycobacteria were transferred into cells ofE. coli andStreptomyces lividans. In the second phase, heterologous genes were transferred into mycobacteria either with a shuttle phasmid or hybrid plasmids. A prerequisite for successful gene manipulation in mycobacteria was a thorough understanding of plasmids in mycobacteria. Construction of recombinant DNA molecules contributed not only to the fact that mycobacteria did not remain outside the mainstream of modern genetic research but also to their present practical importance.     

Sliding motility in mycobacteria

Mycobacteria are nonflagellated gram-positive microorganisms. Previously thought to be nonmotile, we show here that Mycobacterium smegmatis can spread on the surface of growth medium by a sliding mechanism. M. smegmatis spreads as a monolayer of cells which are arranged in pseudofilaments by close cell-to-cell contacts, predominantly along their longitudinal axis. The monolayer moves away from the inoculation point as a unit with only minor rearrangements. No extracellular structures such as pili or fimbriae appear to be involved in this process. The ability to translocate over the surface correlates with the presence of glycopeptidolipids, a mycobacterium-specific class of amphiphilic molecules located in the outermost layer of the cell envelope. We present evidence that surface motility is not restricted to M. smegmatis but is also a property of the slow-growing opportunistic pathogen M. avium. This form of motility could play an important role in surface colonization by mycobacteria in the environment as well as in the host.     

Structural differences in lipomannans from pathogenic and nonpathogenic mycobacteria that impact CD1b-restricted T cell responses

Mannosylated molecules on the Mycobacterium tuberculosis surface are important determinants in the immunopathogenesis of tuberculosis. To date, much attention has been paid to mannose-capped lipoarabinomannan, which mediates phagocytosis and intracellular trafficking of M. tuberculosis by engaging the macrophage mannose receptor and subsequently binds to intracellular CD1b molecules for presentation to T cells. Another important mannosylated lipoglycan on the M. tuberculosis surface is lipomannan (LM). Comparative structural detail of the LMs from virulent and avirulent strains is limited as is knowledge regarding their differential capacity to be recognized by the adaptive immune response. Here, we purified LM from the avirulent M. smegmatis and the virulent M. tuberculosis H(37)R(v), performed a comparative structural biochemical analysis, and addressed their ability to stimulate CD1b-restricted T cell clones. We found that M. tuberculosis H(37)R(v) produces a large neutral LM (TB-LM); in contrast, M. smegmatis produces a smaller linear acidic LM (SmegLM) with a high succinate content. Correspondingly, TB-LM was not as efficiently presented to CD1b-restricted T cells as SmegLM. Thus, here we correlate the structure-function relationships for LMs with CD1b-restricted T cell responses and provide evidence that the structural features of TB-LM contribute to its diminished T cell responsiveness.     

Stress responses mediated by the CBL calcium sensors in plants

Calcium ions (Ca²⁺) are involved as second messenger in plant responses to a broad array of environmental stimuli, including biotic and abiotic stresses. Therefore, understanding Ca²⁺-signaling mechanisms may lead to the development of transgenic crops with enhanced tolerance to adverse environmental conditions. In order to initiate the signaling cascades and give rise to relevant cellular and physiological responses, changes in the parameters of Ca²⁺ transients should be first detected by appropriate Ca²⁺ sensors in plant cells. In this regard, elucidations of plant Ca²⁺ sensors and their target molecules are critical steps for unraveling the Ca²⁺ signal transduction pathways. Recent studies have revealed that plants possess many Ca²⁺-binding proteins with different properties, which can serve as distinct Ca²⁺ sensors. This present review mainly focuses on a family of calcineurin B-like Ca²⁺ sensors which has been most recently identified from higher plants including Arabidopsis, rice, maize and pea.