Many of the current antimycobacterial agents require some form of cellular activation unmasking reactive groups, which in turn will bind to their specific targets. Therefore, understanding the mechanisms of activation of current antimycobacterials not only helps to decipher mechanisms of drug resistance but may also facilitate the development of alternative activation strategies or of analogues that do not require such processes. Herein, through the use of genetically defined strains of Mycobacterium bovis BCG we provide evidence that EthA, previously shown to activate ethionamide, also converts isoxyl (ISO) and thiacetazone (TAC) into reactive species. These results were further supported by the development of an in vitro assay using purified recombinant EthA, which allowed direct assessment of the metabolism of ISO. Interestingly, biochemical analysis of [ 14 C]acetate-labeled cultures suggested that all of these EthA-activated drugs inhibit mycolic acid biosynthesis via different mechanisms through binding to specific targets. This report is also the first description of the molecular mechanism of action of TAC, a thiosemicarbazone antimicrobial agent that is still used in the treatment of tuberculosis as a second-line drug in many developing countries. Altogether, the results suggest that EthA is a common activator of thiocarbamide-containing drugs. The broad specificity of EthA can now be used to improve the activation process of these drugs, which may help overcome the toxicity problems associated with clinical thiocarbamide use.Despite the availability of effective therapies, tuberculosis (TB), caused by Mycobacterium tuberculosis, is still a leading cause of death (11). The human immunodeficiency virus pandemic, which contributes substantially to the morbidity and mortality from TB, and the emergence of multidrug-resistant strains of M. tuberculosis (23) have compounded the problem. Although infections by drug-sensitive strains can be successfully cured (7), the emergence of drug resistance has prompted new drug research, particularly the search for new drug targets and the definition of mechanisms of drug resistance (16). When TB cases cannot be treated by first-line protocols due to resistance issues, the last resort for combating multidrug-resistant infections relies on the action of second-line antitubercular drugs.Work from the last decade has revealed M. tuberculosis to be unique among bacteria in that several drugs require activation in situ to become inhibitory. Drugs such as isoniazid (INH), ethionamide (ETH), and pyrazinamide (PZA) all require activation for activity against M. tuberculosis, and resistance can be mediated by mutations that eliminate the activation step. Such inactivation has been demonstrated for the catalase-peroxidase KatG in INH resistance (33), the nicotinamidase-peroxidase PncA in PZA resistance (24), and the flavin adenine dinucleotide (FAD)-containing Baeyer-Villiger monooxygenase EthA in ETH resistance (3, 9, 30). Interestingly, both activated forms of INH and ETH targe...

The low level of available iron in vivo is a major obstacle for microbial pathogens and is a stimulus for the expression of virulence genes. In this study, Mycobacterium tuberculosis H37Rv was grown aerobically in the presence of limited iron availability in chemostat culture to determine the physiological response of the organism to iron-limitation. A previously unidentified wax ester accumulated under iron-limited growth, and changes in the abundance of triacylglycerol and menaquinone were also observed between iron-replete and iron-limited chemostat cultures. DNA microarray analysis revealed differential expression of genes involved in glycerolipid metabolism and isoprenoid quinone biosynthesis, providing some insight into the underlying genetic changes that correlate with cell-wall lipid profiles of M. tuberculosis growing in an iron-limited environment.

The unusual and complex cell wall of pathogenic mycobacteria plays a major role in pathogenesis, with specific complex lipids acting as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids. Recent work in several laboratories has established that the Mycobacterium tuberculosis fadD26-mmpL7 (Rv2930-Rv2942) locus plays a major role in PDIM biosynthesis and secretion and that PDIM is required for virulence. Here we describe two independent transposon mutants (WAg533 and WAg537) of Tuberculosis, caused by closely related members of the Mycobacterium tuberculosis complex, continues to have a major impact on human and animal health worldwide and is responsible for the death of approximately two million people each year, primarily in developing nations (14). Mycobacterium bovis, the pathogen responsible for bovine tuberculosis, is a broad-host-range member of the M. tuberculosis complex, and its transmission to humans is probably responsible for some 5% of human tuberculosis deaths (15). The current tuberculosis vaccine, M. bovis bacillus Calmette-Guérin (BCG), has shown highly variable efficacy, and a significantly better vaccine is urgently required.In New Zealand, traditional test and slaughter approaches to eradication of bovine tuberculosis from domestic livestock have been frustrated by the presence of introduced wildlife, particularly the Australian brushtail possum (Trichosurus vulpecula), which maintains a reservoir of infection (23). Extensive wildlife culling operations over many years have failed to eliminate infected possums from many parts of the country, and vaccination of wildlife against tuberculosis is being investigated. Any vaccine developed for tuberculosis control in the New Zealand environment must be compatible with largescale vaccination of animals, and this requirement has directed research towards the development of rationally attenuated strains of M. bovis with vaccine efficacy (9,11,12). Moredetailed investigation of the attenuation of some of these strains (10, 38) is also contributing to our understanding of the molecular determinants required for tuberculosis pathogenesis.Among the known determinants required for virulence in pathogenic mycobacteria are complex lipid components of the mycobacterial cell wall that act as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids produced by members of the M. tuberculosis complex and closely related species (17). PDIMs are built upon polyketide scaffolds and comprise multimethyl-branched long-chain mycocerosic acids diesterified with long-chain phthiocerol or phthiodiolone diols (28) (Fig. 1). Additional PDIM variants include the phenol-and glycosylphenol-PDIMs (Fig. 1). Recent work in several laboratories has established that proteins encoded by genes at the M. tuberculosis fadD26-mmpL7 locus (fa...

It is a long held belief that the strong immunostimulatory activity of the Mycobacterium bovis bacillus Calmette-Guérin vaccine and Freund’s complete adjuvant is due to specific mycobacterial cell envelope components, such as lipids and polysaccharides. Implicated mycobacterial lipids include, among others, the so-called cord factor or trehalose dimycolate, but limited information is available regarding the precise molecular nature of the stimulatory components responsible for the interaction with human APCs. In this regard, the majority of research aimed at identifying and characterizing individual immunostimulatory mycobacterial lipids has been performed in the murine system using bone marrow-derived dendritic cells. In this study, it is documented that potent immunostimulatory activity lies within the bacillus Calmette-Guérin nonpolar lipid class. This activity can be narrowed down to a remarkably simple monomycolyl glycerol (MMG) with the ability to stimulate human dendritic cells as assessed by enhanced expression of activation markers and the release of proinflammatory cytokines. A synthetic analog of MMG based on 32 carbons (C32) was found to exhibit comparable levels of immunostimulatory activities. Immunization of mice with the tuberculosis vaccine candidate, Ag85B-ESAT-6, in MMG or the synthetic analog using cationic liposomes as the delivery vehicle was found to give rise to a prominent Th1 response characterized by significant levels of IFN-γ. Together, this development opens up the possibility of producing a novel class of chemically defined lipid adjuvants to enhance the activity of new vaccine formulations, directed against infectious agents including tuberculosis.

The immunostimulatory activity of lipids associated with the mycobacterial cell wall has been recognized for several decades and exploited in a large variety of different adjuvant preparations. Previously, we have shown that a mycobacterial lipid extract from Mycobacterium bovis bacillus Calmette-Guérin delivered in cationic liposomes was a particular efficient Th1-inducing adjuvant formulation effective against tuberculosis. Herein, we have dissected the adjuvant activity of the bacillus Calmette-Guérin lipid extract showing that the majority of the activity was attributable to the apolar lipids and more specifically to a single lipid, monomycoloyl glycerol (MMG), previously also shown to stimulate human dendritic cells. Delivered in cationic liposomes, MMG induced the most prominent Th1-biased immune response that provided significant protection against tuberculosis. Importantly, a simple synthetic analog of MMG, based on a 32 carbon mycolic acid, was found to give rise to comparable high Th1-biased responses with a major representation of polyfunctional CD4 T cells coexpressing IFN-γ, TNF-α, and IL-2. Furthermore, comparable activity was shown by an even simpler monoacyl glycerol analog, based on octadecanoic acid. The use of these synthetic analogs of MMG represents a promising new strategy for exploiting the immunostimulatory activity and adjuvant potential of components from the mycobacterial cell wall without the associated toxicity issues observed with complex mycobacterial preparations.

Aims:  The aims of this study were to identify analogues of L‐proline which inhibit the growth of Escherichia coli in both laboratory culture media and normal human urine and to study their mechanisms of uptake. Methods and Results:  The susceptibility of E. coli to L‐proline analogues was studied by radial streak assays on agar plates and by minimal inhibitory concentration determinations in liquid media. Only L‐selenaproline (SCA) inhibited growth in Mueller–Hinton medium and human urine as well as in glucose minimal medium. L‐Proline did not prevent the inhibition of growth by SCA and strains defective in L‐proline transport were as susceptible to SCA as wild‐type strains. However, E. coli was resistant to SCA in the presence of L‐cysteine and L‐cystine. Spontaneous mutants selected for resistance to SCA or L‐selenocystine were resistant to the other compound and had reduced growth in minimal medium containing L‐cysteine or L‐cystine as the sole sulfur source. Conclusions:  L‐selenaproline inhibited the growth of E. coli under conditions that may occur in the urinary tract and appeared to be taken up by the L‐cystine transport system. Significance and Impact of the Study:  Although urinary tract infections caused by E. coli can be treated with sulfamethoxazole/trimethoprim and quinolones, resistance to these antibiotics has been increasing. These results suggest that L‐selenaproline may represent a new class of compounds that could be used to treat these infections.

No abstract

2 Background 3 Trichomonas vaginalis is an amitochondrial parasitic that causes human trichomoniasis, 4 the most common non-viral sexually transmitted infection in the world. The therapy of 5 choice is metronidazole (MTZ). Despite MTZ effectiveness, resistant cases are 6 becoming more frequent. Another point to emphasize are the side effects that may result 7 in treatment discontinuation, leading to further spread of infection and emergence of 8 resistant strains. This scenario reveals the need to develop new therapeutic options. 9 Photodynamic therapy (PDT) is an experimental treatment that involves the activation 10 of photosensitive substances and the generation of cytotoxic oxygen species and free 11 radicals to promote the selective destruction of target tissues. A previous study, from 12 our group, identified an excellent in vitro PDT activity using methylene blue and light 13 emitting diode against MTZ sensitive and resistant strains of T. vaginalis. The aim of 14 this study was to evaluate the efficacy of PDT in vivo and clarify its high 15 trichomonicidal potential by evaluating its action upon T. vaginalis trophozoites through 16 transmission electron microscopy (TEM). 17 18 Methodology 19 Seven-week-old female Balb/c mice were infected intravaginally with T. vaginalis 20 trophozoites. On the third day of infection, methylene blue was introduced into the 21 vaginal canal of the animals, which then received 68.1 J / cm 2 of radiation for 35.6 sec. 22 Control groups without infection and infected, treated with metronidazole were also 23 included for comparison. Twenty-four hours after treatment the vaginal canal of the 24 animals was scraped and the samples processed by the immunocytochemistry technique.3 3 25 After in vitro photodynamic treatment, T. vaginalis trophozoites were processed for 26 TEM. Ultrathin sections were collected in 400-mesh copper grids, contrasted with 5% 27 uranyl acetate and 3% lead citrate, in aqueous solutions for 20 and 5 min., respectively 28 and observed in a Jeol JEM 230 transmission electron microscope. 29 30 Results 31 TEM showed morphological changes such as centripetal displacement of organelles, 32 cannibalism, hydrogenosomal damage, intense cytoplasmic vacuolization, dilated 33 endoplasmic reticulum cisternae and membrane discontinuity, in both resistant and 34 sensitive strains, suggesting that trichomonicidal activity is mainly due to necrosis. 35 PDT significantly reduced infection in animals treated with a single therapy session, 36 compared to control groups, being statistically as efficient as MTZ. 37 38 Conclusions 39 Our results demonstrated high trichomonicidal activity of PDT with morphological 40 alterations compatible with necrosis. Therefore these results indicate that PDT 41 represents not only an alternative therapy for refractory trichomoniasis, but also 42 routinely for this important neglected parasitic disease.43 44