Identification of sensitive indicators in immune response for leprosy affected patients: An observational clinical study of safety and immunogenicity of influenza vaccine.

ABSTRACT: Cured leprosy patients have special physical conditions, which could pose challenges for safety and immunogenicity after immunization. We performed an observational clinical study aimed to identify the safety and immunogenicity of influenza vaccine in cured leprosy patients. A total of 65 participants from a leprosarium were recruited into leprosy cured group or control group, and received a 0.5 ml dose of the inactivated split-virion trivalent influenza vaccine and a follow-up 28 days proactive observation of any adverse events. Hemagglutination and hemagglutination inhibition test was performed to evaluate serum antibody titer, flow cytometry was conducted to screen of cytokines level. The total rate of reactogenicity was 0.0% [0/41] in leprosy cured group and 37.5% [9/24] in control group. The seroconversion rate for H1N1 was difference between leprosy cured group and control group (41.83% vs 79.17%, P = .0082), but not for H3N2 (34.25% vs 50.00%, P = .4468). At day 0, leprosy cured group have relatively high concentration of interleukin-6, interleukin-10, tumor necrosis factor, interferon-γ, and interleukin-17 compared to control group. The interleukin-2 concentration increased 2 weeks after vaccination compared to pre-vaccination in leprosy cured group, but declined in control group (0.92 pg/ml vs -0.02 pg/ml, P = .0147). Leprosy cured group showed a more rapid down-regulation of interleukin-6 when influenza virus was challenged compared to control group (-144.38 pg/ml vs -11.52 pg/ml, P < .0001). Subgroup analysis revealed that the immunization administration declined interleukin-17 concentration in Tuberculoid type subgroup, but not in Lepromatous type subgroup or control group. Clinically cured leprosy patients are relatively safe for influenza vaccine. Leprosy cured patient have immune deficit in producing antibody. Interleukin-6 and interleukin-17 were 2 sensitive indicators in immune response for leprosy affected patients. The identification of indicators might be help management of leprosy and used as predictive markers in leprosy early symptom monitoring.

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria.

Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc.IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential.

Flavonoids as Novel Efflux Pump Inhibitors and Antimicrobials Against Both Environmental and Pathogenic Intracellular Mycobacterial Species.

Therapeutic treatment options for opportunistic non-tuberculous mycobacterial (NTM) infection and/or serious mycobacterial infections such as tuberculosis (TB) and leprosy are limited due to the spread of antimicrobial resistance mechanism. Plant-derived natural compounds as prospective efflux pump inhibitors may present a promising adjunct to conventional chemotherapy by enhancing mycobacterial susceptibility to antibiotics. This study served to evaluate the antimicrobial and resistance-modifying profile of a range of plant-derived flavonoids against the mycobacterial model strains: M. smegmatis, M. aurum, and M. bovis BCG. The minimum inhibitory concentrations (MICs) of the compounds against the mycobacterial strains were determined using both agar dilution and broth dilution assays, while their efflux inhibitory activity was investigated via an ethidium bromide-based fluorometric assay. All compounds were screened for their synergistic effects with ethidium bromide (EtBr) and rifampicin (RIF) against M. smegmatis. Skullcapflavone II (5,2'-dihydroxy-6,7,8,6'-tetramethoxyflavone, 1) exerted potent antimicrobial activity against M. aurum and M. bovis BCG and considerably increased the susceptibility of M. smegmatis to EtBr and RIF. Nobiletin (5,6,7,8,3',4'-hexamethoxyflavone, 2) was determined to be the most potent efflux-inhibitor in M. aurum and M. smegmatis. However, a connection between strong modulatory and putative efflux activity of the compounds could not be observed. Nevertheless, the results highlight two polymethoxyflavones, skullcapflavone II and nobiletin, with potent antimycobacterial and antibiotic resistance modulating activities as valuable adjuvants in anti-mycobacterial therapies.

Methods for determining the antimicrobial susceptibility of mycobacteria. / Métodos de determinación de sensibilidad a los antimicrobianos en micobacterias.

Mycobacteria are a large group of microorganisms, multiple species of which are major causes of morbidity and mortality, such as tuberculosis and leprosy. At present, the emergence and spread of multidrug-resistant strains of Mycobacterium tuberculosis complex are one of the most serious health problems worldwide. Furthermore, in contrast to M. tuberculosis and Mycobacterium leprae, non-tuberculous mycobacteria (NTM) are more frequently isolated and, in many cases, treatment is based on drug susceptibility testing. This article is a review of the different methods to determine the in vitro drug susceptibility of M. tuberculosis complex and the most relevant NTM isolates. The molecular techniques currently used for rapid detection of resistance of clinical specimens are also analysed.

Non-Tuberculous Mycobacteria: Classification, Diagnostics, and Therapy.

Non-tuberculous mycobacteria (NTM) are species other than those belonging to the Mycobacterium tuberculosis complex and do not cause leprosy. NTM are generally free-living organisms that are ubiquitous in the environment. There have been more than 140 NTM species identified to-date. They can cause a wide range of infections, with pulmonary infections being the most frequent (65-90 %). There is growing evidence that the incidence of NTM lung diseases and associated hospitalizations are on the rise, mainly in regions with a low prevalence of tuberculosis. A crucial clinical problem remains the evaluation of NTM significance in relation to the disease, especially in regard to the colonization of the respiratory tract in patients with residual lesions after tuberculosis or bronchiectasis. Clinical and radiographic pictures of mycobacteriosis, as well as therapy, have often similarities to those of tuberculosis. The treatment regimen should be individualized. In addition to antituberculotics, antibiotics are used more frequently. The most common mycobacteria causing lung disease in Slovakia are Mycobacterium avium and Mycobacterium abscessus.

Consensus numbering system for the rifampicin resistance-associated rpoB gene mutations in pathogenic mycobacteria.

The rpoB gene codes for the RNA polymerase ß subunit, which is the target of rifampicin, an essential drug in the treatment of tuberculosis and other mycobacterial infections. This gene is present in all bacteria, but its length and nucleotide sequence vary between bacterial species, including mycobacteria. Mutations in the rpoB gene alter the structure of this protein and cause drug resistance. To describe the resistance-associated mutations, the scientific and medical communities have been using, since 1993, a numbering system based on the Escherichia coli sequence annotation. Using E. coli reference for describing mutations in mycobacteria leads to misunderstandings, particularly with the increasing use of whole genome sequencing, which brought an alternative numbering system based on the Mycobacterium tuberculosis rpoB sequence. We propose using a consensus numbering system for the reporting of resistance mutations based on the reference genomes from the species interrogated (such as strain H37Rv for M. tuberculosis). This manuscript provides the necessary figures and tables allowing researchers, microbiologists and clinicians to easily convert other annotation systems into one common language.

Host Immune Responses Differ between M. africanum- and M. tuberculosis-Infected Patients following Standard Anti-tuberculosis Treatment.

Epidemiological differences exist between Mycobacterium africanum (Maf)- and Mycobacterium tuberculosis (Mtb)-infected patients, but to date, contributing host factors have not been characterised. We analysed clinical outcomes, as well as soluble markers and gene expression profiles in unstimulated, and ESAT6/CFP-10-, whole-Maf- and Mtb-stimulated blood samples of 26 Maf- and 49 Mtb-HIV-negative tuberculosis patients before, and after 2 and 6 months of anti-tuberculosis therapy. Before treatment, both groups had similar clinical parameters, but differed in few cytokines concentration and gene expression profiles. Following treatment the body mass index, skinfold thickness and chest X-ray scores showed greater improvement in the Mtb- compared to Maf-infected patients, after adjusting for age, sex and ethnicity (p = 0.02; 0.04 and 0.007, respectively). In addition, in unstimulated blood, IL-12p70, IL12A and TLR9 were significantly higher in Maf-infected patients, while IL-15, IL-8 and MIP-1α were higher in Mtb-infected patients. Overnight stimulation with ESAT-6/CFP-10 induced significantly higher levels of IFN-γ and TNF-α production, as well as gene expression of CCL4, IL1B and TLR4 in Mtb- compared to Maf-infected patients. Our study confirms differences in clinical features and immune genes expression and concentration of proteins associated with inflammatory processes between Mtb- and Maf-infected patients following anti-tuberculosis treatment These findings have public health implications for treatment regimens, and biomarkers for tuberculosis diagnosis and susceptibility.

Synthesis and biological activity of alkynoic acids derivatives against mycobacteria.

2-Alkynoic acids have bactericidal activity against Mycobacterium smegmatis but their activity fall sharply as the length of the carbon chain increased. In this study, derivatives of 2-alkynoic acids were synthesized and tested against fast- and slow-growing mycobacteria. Their activity was first evaluated in M. smegmatis against their parental 2-alkynoic acids, as well as isoniazid, a first-line antituberculosis drug. The introduction of additional unsaturation or heteroatoms into the carbon chain enhanced the antimycobacterial activity of longer chain alkynoic acids (more than 19 carbons long). In contrast, although the modification of the carboxylic group did not improve the antimycobacterial activity, it significantly reduced the toxicity of the compounds against eukaryotic cells. Importantly, 4-(alkylthio)but-2-ynoic acids, had better bactericidal activity than the parental 2-alkynoic acids and on a par with isoniazid against the slow-grower Mycobacterium bovis BCG. These compounds had also low toxicity against eukaryotic cells, suggesting that they could be potential therapeutic agents against other types of topical mycobacterial infections causing skin diseases including Mycobacterium abscessus, Mycobacterium ulcerans, and Mycobacterium leprae. Moreover, they provide a possible scaffold for future drug development.

The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae.

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

Mycolic acids: structures, biosynthesis, and beyond.

Mycolic acids are major and specific lipid components of the mycobacterial cell envelope and are essential for the survival of members of the genus Mycobacterium that contains the causative agents of both tuberculosis and leprosy. In the alarming context of the emergence of multidrug-resistant, extremely drug-resistant, and totally drug-resistant tuberculosis, understanding the biosynthesis of these critical determinants of the mycobacterial physiology is an important goal to achieve, because it may open an avenue for the development of novel antimycobacterial agents. This review focuses on the chemistry, structures, and known inhibitors of mycolic acids and describes progress in deciphering the mycolic acid biosynthetic pathway. The functional and key biological roles of these molecules are also discussed, providing a historical perspective in this dynamic area.

The structure, function, and regulation of Mycobacterium FtsZ.

FtsZ is a widely distributed major cytoskeletal protein involved in the archaea and bacteria cell division. It is the most critical component in the division machinery and similar to tubulin in structure and function. Four major roles of FtsZ have been characterized: cell elongation, GTPase, cell division, and bacterial cytoskeleton. FtsZ subunits can be assembled into protofilaments. Mycobacteria consist of a large family of medical and environmental important bacteria, such as M. leprae, M. tuberculosis, the pathogen of leprosy, and tuberculosis. Structure, function, and regulation of mycobacteria FtsZ are summarized here, together with the implication of FtsZ as potential novel drug target for anti-tuberculosis therapeutics.

Synthesis and antileprosy activity of some dialkyldithiocarbamates.

OBJECTIVES: To investigate the antileprosy potential of a set of original compounds with antimycobacterial activity. METHODS: We developed a facile synthesis of 2-chloro-3-cyano-5-nitropyridine and synthesized a series of 3-cyano-2-dialkyldithiocarbamoyl-5-nitropyridine derivatives. In vivo therapeutic efficacy against Mycobacterium leprae was assessed in the infected mouse footpad model. RESULTS: The compounds were active in vitro against Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium vaccae and Mycobacterium fortuitum, with MICs generally in the range of 0.4-6.25 mg/L. Reduction of the bacterial load in vivo in the mouse footpad and toxic side effects were dependent on the individual structure of the compounds and on the doses applied. Compounds 2a, 3a and 3b reduced the number of M. leprae by two orders of magnitude, comparable to the effect of dapsone. Co-administration of compounds 2a and 3a with dapsone synergistically enhanced the activity. In addition, these compounds were well tolerated over the treatment period of 7.5 months. CONCLUSIONS: Individual synthetic dithiocarbamate derivatives have promising antileprosy activity.

Controlling gene expression in mycobacteria.

The genus mycobacterium contains some of the most important human pathogens, including Mycobacterium tuberculosis, which causes tuberculosis in approximately 8 million people annually; Mycobacterium leprae, the etiologic agent of leprosy, which affects millions of people in Asia, Africa and Latin America; and Mycobacterium bovis, which causes tuberculosis in animals and people. Genetic tools for mycobacteria have been developed during the last 15 years and have helped to improve our understanding of the biology and pathogenesis of mycobacteria. However, genetic switches have only recently been developed that allow control of mycobacterial gene expression. Such systems have been used to facilitate protein overexpression in mycobacteria and to analyze gene function, in particular functions of essential genes. Some of the recently developed systems will allow controlling gene expression during animal infections and may therefore become invaluable tools for drug target validation. This article will review the features of available mycobacterial genetic switches and discuss their applications.

The evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from Psoralea corylifolia and Sanguinaria canadensis.

Extracts from forty-three plant species were selected on account of reported traditional uses for the treatment of TB and/or leprosy. These were assayed for antimycobacterial activities. A simple in vitro screening assay was employed using two model species of mycobacteria, M. aurum and M. smegmatis. Crude methanolic extracts from three of the plants, C. mukul, P. corylifolia and S. canadensis, were found to have significant antimycobacterial activity against M. aurum only (MIC=62.5 microg/ml). Bioassay guided fractionation led to the isolation of two known benzophenanthridine alkaloids, sanguinarine (1) and chelerythrine (2), from the roots S. canadensis and the known phenolic meroterpene, bakuchiol (3) from the seeds of P. corylifolia. The fractionation of the resin of C. mukul lead to a decrease in antimycobacterial activity and hence further work was not pursued. Compound (2) was the most active against M. aurum and M. smegmatis (IC(50)=7.30 microg/ml [19.02 microM] and 29.0 microg/ml [75.56 microM], respectively). M. aurum was the most susceptible organism to all three compounds. No significant difference in antimycobacterial activity was observed when the two alkaloids were tested for activity in media of differing pH values. The activities of the pure compounds against M. aurum were comparable with those against M. bovis BCG with compound (2) being the most active (M. bovis BCG, IC(50)=14.3 microg/ml [37.3 microM]). These results support the use of these plants in traditional medicine.

[Comparative characterization of the antigenic composition of M. leprae and M. lufu]. / Sravnitel'naia kharakteristika antigennogo sostava M. leprae i M.lufu.

The antigenic structure of M. leprae and M. lufu was comparatively studied for the first time. M. lufu was found to have M. leprae-specific protein with a molecular weight of 36 kDa. M. leprae and M. lufu were similar in their fractional composition of proteins and an antibody response to determinants with equal molecular weights in patients with different forms of leprosy and its varying severity. The findings may improve a diagnostic system in leprosy by using M. lufu antigens as an alternative.

[The comparative cultural-biochemical and immunochemical identification of laboratory strains of cultured mycobacteria from leprous patient lesions and from soil]. / Sranitel'naia kul'tural'no-biokhimicheskaia i immunokhimicheskaia identifikatsiia laboratornykh shtammov kul'tiviruemykh mikobakterii iz leproznykh porazhenii bol'nykh i pochvy.

The paper presents the results of studied of cultural biochemical and immunochemical properties of the cultured mycobacteria isolated from patients with leprous lesions of (M.01, M.011) and the soil (M. lufu) in compared to other well-known mycobacteria and between them. M.01, M.011, and M. lufu differ from other representatives of the genus Mycobacterium, but they are close to each other in their cultural biochemical and immunochemical properties, antigenic composition, and protein spectra. The findings are an additional characteristic of the biological properties of M.01, M.011, and M. lufu.

Bactericidal action of ampicillin/sulbactam against intracellular mycobacteria.

The resistance of mycobacteria to beta-lactam antibiotics is attributed to their ability to synthesize beta-lactamase. In our previous studies, beta-lactam/beta-lactamase-inhibitor combinations suppressed the growth of several mycobacteria in axenic cultures and ampicillin/sulbactam was bactericidal to Mycobacterium tuberculosis H37Rv in vitro, and to Mycobacterium leprae multiplying in mouse foot-pads. Since both these organisms multiply in phagocytic cells in the host, it is important to know whether the drug combination is active against mycobacteria multiplying in macrophages. We tested the action of ampicillin/sulbactam against four potentially pathogenic (to humans or to animals) mycobacteria, M. simiae, M. haemophilum, M. avium, M. microti, when phagocytosed by mouse macrophages. Bacteria were exposed to monolayers of peritoneal macrophages harvested from BALB/c mice. Unphagocytosed bacilli were removed and three concentrations of ampicillin/sulbactam were tested. Optimum activity was observed at 100 mg/l which killed 58-97% of the mycobacteria within macrophages, as determined by the CFU. beta-Lactam/beta-lactamase-inhibitors, especially ampicillin/sulbactam, might provide an effective alternative therapy against infections caused by mycobacteria resistant to other drugs.

Activity of quinolones against mycobacteria.

The fluoroquinolones have been shown to be active in vitro against many mycobacterial species, including most strains of Mycobacterium tuberculosis complex and M. fortuitum, and some strains of M. kansasii, M. avium-intracellulare (MAI) complex and M. leprae. Ciprofloxacin, ofloxacin and sparfloxacin are the best studied of these agents to date, and are among the most active of this group against M. tuberculosis and other mycobacteria. Treatment of patients with multidrug-resistant pulmonary tuberculosis using ofloxacin has resulted in the selection of quinolone-resistant mutants in a few patients. Many strains of MAI, however, are resistant to fluoroquinolones, and structure-activity relationships and DNA gyrase studies have been undertaken to identify the moieties associated with activity and the lack thereof. The genetic and molecular basis of quinolone resistance in mycobacteria has revealed both the recent progress made in these areas and the limitations of the quinolones against this genus. Considerable progress will need to be made in resolving these issues in order for the quinolones to become clinically useful antimycobacterial agents.

Antimycobacterial activities of riminophenazines.

Riminophenazines were specifically developed as drugs active against Mycobacterium tuberculosis but extensive research over several decades has shown that these compounds are also active against many other mycobacterial infections, particularly those caused by Mycobacterium leprae and the Mycobacterium avium complex (MAC). Clofazimine, the lead compound in this series, is included in the regimens that are approved by the WHO for the treatment of leprosy and has contributed significantly to the control of that disease, particularly that caused by dapsone-resistant bacteria. Despite early problems, clofazimine has shown clinical efficacy in tuberculosis, in particular that caused by multiple drug resistant strains. Clofazimine does not induce resistance and also inhibits emergence of resistance to isoniazid in M. tuberculosis. The efficacy of clofazimine against MAC is more varied and the availability of better drugs has limited its use. Newer riminophenazines, such as B746 and B4157, not only showed increased anti-mycobacterial activity but also produced less skin pigmentation, which is the main drawback of this group of compounds. The most important virtues of riminophenazines, such as intracellular accumulation in mononuclear phagocytic cells, anti-inflammatory activity, a low incidence of drug resistance and slow metabolic elimination, make them attractive candidates for the treatment of mycobacterial infections. It is essential, however, to investigate the newer analogues clinically, while continuing the pursuit of alternate candidates that demonstrate higher anti-mycobacterial activity and lower rates of skin pigmentation.