Genetic engineering employing MPB70 and its promoter enables efficient secretion and expression of foreign antigen in bacillus Calmette Guérin (BCG) Tokyo.

Vaccination is an important factor in public health. The recombinant bacillus Calmette Guérin (rBCG) vaccine, which expresses foreign antigens, is expected to be a superior vaccine against infectious diseases. Here, we report a new recombination platform in which the BCG Tokyo strain is transformed with nucleotide sequences encoding foreign protein fused with the MPB70 immunogenic protein precursor. By RNA-sequencing, mpb70 was found to be the most transcribed among all known genes of BCG Tokyo. Small oligopeptide, namely, polyhistidine tag, was able to be expressed in and secreted from rBCG through a process in which polyhistidine tag fused with intact MPB70 were transcribed by an mpb70 promoter. This methodology was applied to develop an rBCG expressing the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2. Immunoblotting images and mass spectrometry data showed that RBD was also secreted from rBCG. Sera from mice vaccinated with the rBCG showed a tendency of weak neutralizing capacity. The secretion was retained even after a freeze-drying process. The freeze-dried rBCG was administered to and recovered from mice. Recovered rBCG kept secreting RBD. Collectively, our recombination platform offers stable secretion of foreign antigens and can be applied to the development of practical rBCGs.

CD8 T Cells Show Protection against Highly Pathogenic Simian Immunodeficiency Virus (SIV) after Vaccination with SIV Gene-Expressing BCG Prime and Vaccinia Virus/Sendai Virus Vector Boosts.

Toward development of a dual vaccine for human immunodeficiency virus type 1 (HIV-1) and tuberculosis infections, we developed a urease-deficient bacillus Calmette-Guérin (BCG) strain Tokyo172 (BCGΔurease) to enhance its immunogenicity. BCGΔurease expressing a simian immunodeficiency virus (SIV) Gag induced BCG antigen-specific CD4+ and CD8+ T cells more efficiently and more Gag-specific CD8+ T cells. We evaluated its protective efficacy against SIV infection in cynomolgus monkeys of Asian origin, shown to be as susceptible to infection with SIVmac251 as Indian rhesus macaques. Priming with recombinant BCG (rBCG) expressing SIV genes was followed by a boost with SIV gene-expressing LC16m8Δ vaccinia virus and a second boost with SIV Env-expressing Sendai virus. Eight weeks after the second boost, monkeys were repeatedly challenged with a low dose of SIVmac251 intrarectally. Two animals out of 6 vaccinees were protected, whereas all 7 control animals were infected without any early viral controls. In one vaccinated animal, which had the most potent CD8+ T cells in an in vitro suppression activity (ISA) assay of SIVmac239 replication, plasma viremia was undetectable throughout the follow-up period. Protection was confirmed by the lack of anamnestic antibody responses and detectable cell-associated provirus in various organs. Another monkey with a high ISA acquired a small amount of SIV, but it later became suppressed below the detection limit. Moreover, the ISA score correlated with SIV acquisition. On the other hand, any parameter relating anti-Env antibody was not correlated with the protection.IMPORTANCE Because both AIDS and tuberculosis are serious health threats in middle/low-income countries, development of a dual vaccine against them would be highly beneficial. To approach the goal, here we first assessed a urease-deficient bacillus Calmette-Guérin (BCG) for improvement of immunogenicity against both Mycobacterium tuberculosis and SIV. Second, we demonstrated the usefulness of Asian-origin cynomolgus monkeys for development of a preclinical AIDS vaccine by direct comparison with Indian rhesus macaques as the only validated hosts that identically mirror the outcomes of clinical trials, since the availability of Indian rhesus macaques is limited in countries other than the United States. Finally, we report the protective effect of a vaccination regimen comprising BCG, the highly attenuated vaccinia virus LC16m8Δ strain, and nontransmissible Sendai virus as safe vectors expressing SIV genes using repeated mucosal challenge with highly pathogenic SIVmac251. Identification of CD8+ T cells as a protective immunity suggests a future direction of AIDS vaccine development.

Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis.

Mycobacterium avium subspecies hominissuis (MAH) is a pathogen that causes various non-tuberculous mycobacterial diseases in humans and animals worldwide. Among the genus, MAH is characterized by relatively slow growth. Here, we isolated a rapidly growing variant of the MAH 104 strain. The variant strain (named N104) exhibited an enhanced growth rate and higher motility compared to the parent MAH 104 strain (P104). Whole-genome sequencing analysis of N104 revealed the loss of the stop codon of MAV_RS14660 due to a single nucleotide replacement, resulting in the substitution of the codon for tryptophan. Notably, exclusion of the stop codon ligated the open reading frames and caused the fusion of two adjacent proteins. A revertant parent strain, in which a mutation was introduced to restore the stop codon, revealed that elimination of the stop codon in MAV_RS14660 was responsible for the N104 phenotype. Furthermore, we analysed the phenotypes of the parent and mutated strains by determining the functions of the MAV_RS14660 and MAV_RS14655 coding regions flanking the stop codon. The mutant strains, expected to express a fusion protein, exhibited increased resistance to antimicrobial drugs and exogenous copper toxicity compared to that of the parent strains. These findings suggest that the fusion of the MAV_RS14660- and MAV_RS14655-encoding regions in the mutant N104 strain could be related to the modified functions of these intrinsic proteins.

Altered cytokine profiles in relapsed paucibacillary leprosy: a case report.

BACKGROUND: Individuals with relapses of leprosy should be monitored carefully, however, with respect to paucibacillary (PB) leprosy, it is sometimes difficult to make a definitive diagnosis of relapse, because the bacillary index is often negative. To evaluate the usefulness of cytokine profiling in a patient with relapsed PB leprosy who tested negative for anti-phenolic glycolipid-I antibodies, we analyzed the Mycobacterium leprae protein-induced cytokine expression in peripheral blood mononuclear cells of the patient. CASE PRESENTATION: An 89-year-old-male relapsed PB patient, first treated for leprosy over 50 years prior, was examined. In April 2012, he noticed three skin lesions consisting of annular erythema in the thighs. Slit skin smear tests were negative, and skin biopsies revealed a pathology of indeterminate-to-borderline tuberculoid leprosy. He received 600 mg of rifampicin once per month and 75 mg of dapsone daily for 12 months. The annular erythemas disappeared after starting treatment. Before treatment, and 6 and 12 months after starting treatment, the Th1/Th2 cytokine profiles in the supernatant of mononuclear cells from the patient before and after stimulation with Mycobacterium leprae soluble protein (MLS) were examined using a Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II. The CBA Enhanced Sensitivity Flex Set system was applied to detect small amounts of cytokines in the serum just before treatment and one year before relapse. In the culture supernatant, just before treatment, increases in IFN-γ level and the IFN-γ/IL-10 ratio and a decreased IL-6 level were observed without stimulation. Upon stimulation with MLS, just before treatment, both the IFN-γ and TNF levels increased markedly, and twelve months after starting treatment, the IFN-γ and TNF levels decreased greatly. In the serum, just before treatment, increases in IFN-γ and TNF levels and the IFN-γ/IL-10 ratio were evident compared with those measured one year before relapse. CONCLUSIONS: Cytokine profiling using culture supernatants and serum samples may be useful for the diagnosis of relapsed PB leprosy.

WQ-3810 inhibits DNA gyrase activity in ofloxacin-resistant Mycobacterium leprae.

BACKGROUND: Mycobacterium leprae causes leprosy and ofloxacin is used to control this bacterium. However, specific amino acid substitutions in DNA gyrases of M. leprae interferes with the effect of ofloxacin. METHODOLOGY/PRINCIPAL FINDINGS: Here we tested the inhibitory effect of WQ-3810 on DNA gyrases in M. leprae, using recombinant gyrases. We theorized that WQ-3810 and DNA gyrases interacted, which was tested in silico. Compared with control drugs like ofloxacin, WQ-3810 showed a better inhibitory effect on ofloxacin-resistant DNA gyrases. The in-silico study showed that, unlike control drugs, a specific linkage between a R1 group in WQ-3810 and aspartic acid at position 464 in the subunit B of DNA gyrases existed, which would enhance the inhibitory effect of WQ-3810. This linkage was confirmed in a further experiment, using recombinant DNA gyrases with amino acid substitutions in subunits B instead. CONCLUSIONS/SIGNIFICANCE: The inhibitory effect of WQ-3810 was likely enhanced by the specific linkage between a R1 group residue in its structure and DNA gyrases. Using interactions like the one found in the present work may help design new fluoroquinolones that contribute to halt the emergence of antibiotic-resistant pathogens.

[Leprosy awareness-raising summer seminar: results of participant survey during 2012 and 2015].

In order to assess the effectiveness of this leprosy awareness-raising program, we surveyed 123 participants between 2012 and 2015. They were asked about their satisfaction with the program and what impact it had on them. In the past four years 80% to 100% have reported being "very satisfied" with the seminar. Many participants were grateful for the opportunity to be able to learn about leprosy from multiple perspectives and interact with people affected by leprosy. Interaction and sharing of opinions between participants were also regarded as important. These findings elucidated the importance of this seminar to provide opportunities for knowing the right information about leprosy, interacting with people affected by leprosy, coming to know of their experience and thoughts, and gaining exposure to other participants' opinions.

Polyclonal activation of naïve T cells by urease deficient-recombinant BCG that produced protein complex composed of heat shock protein 70, CysO and major membrane protein-II.

BACKGROUND: Mycobacterium bovis bacillus Calmette-Guérin (BCG) is known to be only partially effective in inhibiting M. tuberculosis (MTB) multiplication in human. A new recombinant (r) urease-deficient BCG (BCG-dHCM) that secretes protein composed of heat shock protein (HSP)70, MTB-derived CysO and major membrane protein (MMP)-II was produced for the efficient production of interferon gamma (IFN-γ) which is an essential element for mycobacteriocidal action and inhibition of neutrophil accumulation in lungs. METHODS: Human monocyte-derived dendritic cells (DC) and macrophages were differentiated from human monocytes, infected with BCG and autologous T cells-stimulating activity of different constructs of BCG was assessed. C57BL/6 mice were used to test the effectiveness of BCG for the production of T cells responsive to MTB-derived antigens (Ags). RESULTS: BCG-dHCM intracellularly secreted HSP70-CysO-MMP-II fusion protein, and activated DC by up-regulating Major Histcompatibility Complex (MHC), CD86 and CD83 molecules and enhanced various cytokines production from DC and macrophages. BCG-dHCM activated naïve T cells of both CD4 and CD8 subsets through DC, and memory type CD4+ T cells through macrophages in a manner dependent on MHC and CD86 molecules. These T cell activations were inhibited by the pre-treatment of Ag-presenting cells (APCs) with chloroquine. The single and primary BCG-dHCM-inoculation produced long lasting T cells responsive to in vitro secondarily stimulation with HSP70, CysO, MMP-II and H37Rv-derived cytosolic protein, and partially inhibited the replication of aerosol-challenged MTB. CONCLUSIONS: The results indicate that introduction of different type of immunogenic molecules into a urease-deficient rBCG is useful for providing polyclonal T cell activating ability to BCG and for production of T cells responsive to secondary stimulation.

Amino acid substitutions at position 95 in GyrA can add fluoroquinolone resistance to Mycobacterium leprae.

Amino acid substitutions at position 89 or 91 in GyrA of fluoroquinolone-resistant Mycobacterium leprae clinical isolates have been reported. In contrast, those at position 94 in M. tuberculosis, equivalent to position 95 in M. leprae, have been identified most frequently. To verify the possible contribution of amino acid substitutions at position 95 in M. leprae to fluoroquinolone resistance, we conducted an in vitro assay using wild-type and mutant recombinant DNA gyrases. Fluoroquinolone-mediated supercoiling activity inhibition assay and DNA cleavage assay revealed the potent contribution of an amino acid substitution of Asp to Gly or Asn at position 95 to fluoroquinolone resistance. These results suggested the possible future emergence of quinolone-resistant M. leprae isolates with these amino acid substitutions and the usefulness of detecting these mutations for the rapid identification of fluoroquinolone resistance in leprosy.

Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein.

To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.

[Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein].

To activate naïve T cells convincingly using Mycobacterium bovis BCG (BCG), rBCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4+ and CD8+ subsets of naïve T cells than rBCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DC) to induce cytokine production and phenotypic changes, and activated CD4+ T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pre-treatment of DC with chloroquine inhibited both surface expression of MMP-II on DC and the activation of T cells by BCG-D70M-infected APCs. The naïve CD8+ T cell activation was inhibited by treatment of DC with brefeldin A and lactacystin so that the T cells was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naïve CD8+ T cells, effector T cells producing perforin and memory T cells having migration markers, were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70, and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II and urease depletion may provide useful tool for inducing better activation of naïve T cells.

Induction of cross-priming of naive CD8+ T lymphocytes by recombinant bacillus Calmette-Guerin that secretes heat shock protein 70-major membrane protein-II fusion protein.

Because Mycobacterium bovis bacillus Calmette-Guérin (BCG) unconvincingly activates human naive CD8(+) T cells, a rBCG (BCG-70M) that secretes a fusion protein comprising BCG-derived heat shock protein (HSP)70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed to potentiate the ability of activating naive CD8(+) T cells through dendritic cells (DC). BCG-70M secreted HSP70-MMP-II fusion protein in vitro, which stimulated DC to produce IL-12p70 through TLR2. BCG-70M-infected DC activated not only memory and naive CD8(+) T cells, but also CD4(+) T cells of both types to produce IFN-gamma. The activation of these naive T cells by BCG-70M was dependent on the MHC and CD86 molecules on BCG-70M-infected DC, and was significantly inhibited by pretreatment of DC with chloroquine. Both brefeldin A and lactacystin significantly inhibited the activation of naive CD8(+) T cells by BCG-70M through DC. Thus, the CD8(+) T cell activation may be induced by cross-presentation of Ags through a TAP- and proteosome-dependent cytosolic pathway. When naive CD8(+) T cells were stimulated by BCG-70M-infected DC in the presence of naive CD4(+) T cells, CD62L(low)CD8(+) T cells and perforin-producing CD8(+) T cells were efficiently produced. MMP-II-reactive CD4(+) and CD8(+) memory T cells were efficiently produced in C57BL/6 mice by infection with BCG-70M. These results indicate that BCG-70M activated DC, CD4(+) T cells, and CD8(+) T cells, and the combination of HSP70 and MMP-II may be useful for inducing better T cell activation.

Coexpression of MmpS5 and MmpL5 Contributes to Both Efflux Transporter MmpL5 Trimerization and Drug Resistance in Mycobacterium tuberculosis.

The increasing occurrence of multidrug-resistant Mycobacterium tuberculosis (Mtb) is a serious threat to global public health. Among the many mechanisms of drug resistance, only resistance-nodulation-division (RND)-type multidrug efflux systems can simultaneously render bacteria tolerant to numerous toxic compounds, including antibiotics. The elevated expression of RND-type xenobiotic efflux transporter complexes, which consist of an inner membrane transporter, membrane fusion protein, and outer membrane channel, plays a major role in multidrug resistance. Among the 14 mycobacterial membrane protein large (MmpL) proteins identified as inner membrane transporters of Mtb, MmpL5 is known to participate in the acquisition of resistance to bedaquiline and clofazimine. MmpL5 exports these drugs by forming a complex with the membrane fusion protein mycobacterial membrane protein small 5 (MmpS5). However, the role of MmpS5 in the efflux of antituberculous drugs by MmpL5 remains unclear. In this study, we focused on the in vivo dynamics of MmpL5 using green fluorescent protein (GFP). Single-molecule observations of MmpL5 showed substantial lateral displacements of MmpL5-GFP without the expression of MmpS5. Nondiffusing MmpL5-GFP foci typically showed three-step photobleaching, suggesting that MmpL5 formed a homotrimeric functional complex on the inner membrane in the presence of MmpS5. These results suggest that the expression of MmpS5 facilitates the assembly of monomeric MmpL5 into a homotrimer that is anchored to the inner membrane to transport various antimycobacterial drugs.IMPORTANCE It has been reported that mycobacterial membrane protein large 5 (MmpL5), a resistance-nodulation-division (RND)-type inner membrane transporter in Mycobacterium tuberculosis (Mtb), is involved in the transport of antimycobacterial drugs. However, the functional roles of the membrane fusion protein mycobacterial membrane protein small 5 (MmpS5), organized as an operon with MmpL5, are unclear. Via the single-molecule imaging of MmpL5, we uncovered the maintenance of the functional trimeric complex structure of MmpL5 in the presence of MmpS5. These findings demonstrate that the assembly mechanisms of mycobacterial RND efflux systems are the dynamically regulated process through interactions among the components. This represents the first report of the single-molecule observation of Mtb efflux transporters, which may enhance our understanding of innate antibiotic resistance.

Production of antibiotic resistance gene-free urease-deficient recombinant BCG that secretes antigenic protein applicable for practical use in tuberculosis vaccination.

Mycobacterium bovis BCG has been the only practical vaccine for tuberculosis. However, BCG cannot fully prevent adult pulmonary tuberculosis. Therefore, the improvement of BCG vaccine is necessary. We previously produced recombinant (r) BCG (BCG-PEST) for the better control of tuberculosis. BCG-PEST was developed by introducing PEST-Heat Shock Protein (HSP)70-Major Membrane Protein (MMP)-II-PEST fusion gene into urease-deficient rBCG using antibiotic-resistant gene for the selection of rBCG. HSP70-MMPII fusion protein is highly immunogenic and PEST sequence was added to enhance processing of the fusion protein. Although BCG-PEST effectively inhibited intrapulmonary growth of Mycobacterium tuberculosis (MTB), BCG with antibiotic-resistant gene is not appropriate for human use. Therefore, we produced antibiotic-resistant gene-free rBCG. We generated leucine-biosynthetic gene (leuD)-deficient BCG and introduced the fusion gene with leuD as the selection marker and named this rBCG as BCG-LeuPH. BCG-LeuPH activated human naïve T cells of both CD4 and CD8 subsets and efficiently inhibited aerosol-challenged MTB in mice. These results indicate that leuD can replace antibiotic-resistant gene for the selection of vaccine candidates of rBCG for human use.

Interaction of Quinolones Carrying New R1 Group with Mycobacterium leprae DNA Gyrase.

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and the treatment of choice is ofloxacin (OFX). Specific amino acid substitutions in DNA gyrase of M. leprae have been reported leading to resistance against the drug. In our previous study, WQ-3810, a fluoroquinolone with a new R1 group (6-amino-3,5-difluoropyridin-2-yl) was shown to have a strong inhibitory activity on OFX-resistant DNA gyrases of M. leprae, and the structural characteristics of its R1 group was predicted to enhance the inhibitory activity. Methodology/Principal Finding: To further understand the contribution of the R1 group, WQ-3334 with the same R1 group as WQ-3810, WQ-4064, and WQ-4065, but with slightly modified R1 group, were assessed on their activities against recombinant DNA gyrase of M. leprae. An in silico study was conducted to understand the molecular interactions between DNA gyrase and WQ compounds. WQ-3334 and WQ-3810 were shown to have greater inhibitory activity against M. leprae DNA gyrase than others. Furthermore, analysis using quinolone-resistant M. leprae DNA gyrases showed that WQ-3334 had greater inhibitory activity than WQ-3810. The R8 group was shown to be a factor for the linkage of the R1 groups with GyrB by an in silico study. Conclusions/Significance: The inhibitory effect of WQ compounds that have a new R1 group against M. leprae DNA gyrase can be enhanced by improving the binding affinity with different R8 group molecules. The information obtained by this work could be applied to design new fluoroquinolones effective for quinolone-resistant M. leprae and other bacterial pathogens.

Novel rhamnosyltransferase involved in biosynthesis of serovar 4-specific glycopeptidolipid from Mycobacterium avium complex.

Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1→4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.

Cloning and Protein Expression of eccB5 Gene in ESX-5 System from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis in human. One of the major M. tuberculosis virulence factors is early secretory antigenic target of 6-kDa (ESAT-6), and EccB5 protein encoded by eccB5 is one of its components. EccB5 protein is a transmembrane protein in ESX-5 system. The aim of this study is to explore the characteristics of wild-type EccB5 and its mutant form N426I. We expressed the EccB5 protein by cloning the mutant and wild-type eccB5 gene in Escherichia coli (E. coli). We compared the protein structure of wild type and mutant form of EccB5 and found changes in structure around Asn426 (loop structure) in wild type and around Ile426 (ß-strand) in the mutant. The truncated recombinant protein of EccB5 was successfully cloned and expressed using plasmid pCold I in E. coli DH5α and E. coli strain Rosetta-gami B (DE3) and purified as a 38.6 kDa protein by using the affinity column. There was no detectable adenosine triphosphatase activity in truncated forms of EccB5 and its mutant. In conclusion, our study reveals successful cloning and protein expression of truncated form of eccB5 gene of M. tuberculosis. EccB5 protein in ESX-5 system may be an important membrane component involved in the transport machinery of type VII secretion system, which is essential for growth and virulence.

WQ-3810: A new fluoroquinolone with a high potential against fluoroquinolone-resistant Mycobacterium tuberculosis.

Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb), caused by amino acid substitutions in DNA gyrase, has been increasingly reported worldwide. WQ-3810 is a newly developed FQ that is highly active against FQ-resistant pathogens; however, its activity against Mtb has not been evaluated. Herein we examined the efficacy of WQ-3810 against Mtb through the use of recombinant Mtb DNA gyrases. In addition, in vitro antimycobacterial activity of WQ-3810 was evaluated against recombinant Mtb var. bovis Bacille Calmette-Guérin strains in which gyrase-coding genes were replaced with Mtb variants containing resistance-conferring mutations. WQ-3810 showed a higher inhibitory activity than levofloxacin against most recombinant DNA gyrases with FQ-resistance mutations. Furthermore, WQ-3810 showed inhibition even against a DNA gyrase variant harboring a G88C mutation which is thought to confer the highest resistance against FQs in clinical Mtb isolates. In contrast, the FQ susceptibility test showed that WQ-3810 had relatively weak mycobactericidal activity compared with moxifloxacin. However, the combination of WQ-3810 and ethambutol showed the greatest degree of synergistic activity against recombinant strains. Since FQs and ethambutol have been used in multi-drug therapy for tuberculosis, WQ-3810 might represent a new, potent anti-tuberculosis drug that can be effective even against FQ-resistant Mtb strains.

Detection of serum antibodies to M. leprae major membrane protein-II in leprosy patients from Indonesia.

BACKGROUND: Sero-diagnostic methods are the easiest way of diagnosing an infectious disease in developing countries. In leprosy, phenolic glycolipid-1 (PGL-I) based methods for the detection of leprosy are currently available, but the use of these methods has been hindered due to the inherent problems of sensitivity. We previously showed that antibodies to Major Membrane Protein-II (MMP-II) derived from Mycobacterium leprae could be used to diagnose leprosy in Japan. METHODS: Sera from patients and healthy individuals were collected with informed consent and the anti-MMP-II antibody levels of the sera were measured by enzyme-linked immunosorbent assay. The study was conducted at South Sulawesi and Bali, in Indonesia. The study population included 40 each of multibacillary leprosy and paucibacillary leprosy patients, 30 tuberculosis and 16 patients with typhoid. RESULTS: We evaluated the anti-MMP-II antibody levels in Indonesian individuals. The cut-off value was determined from receiver operator characteristic curve as 0.124 using the O.D. titers for patients with multibacillary leprosy, so that the sensitivity of the test was 97.5% and the specificity taking healthy individuals as controls was 984%. Using the determined cut-off values, 98% of multibacillary (MB) leprosy and 48% of paucibacillary (PB) leprosy patients had positive levels of anti-MMP-II antibodies, 13% of patients with typhoid and 22% of the household contacts of MB leprosy had positive levels of anti-MMP-II antibodies. CONCLUSIONS: Our results suggest that measuring anti-MMP-II antibody levels could facilitate the detection of leprosy in endemic countries.

The Mycobacterium avium complex gtfTB gene encodes a glucosyltransferase required for the biosynthesis of serovar 8-specific glycopeptidolipid.

Mycobacterium avium complex (MAC) is one of the most common opportunistic pathogens widely distributed in the natural environment. The 28 serovars of MAC are defined by variable oligosaccharide portions of glycopeptidolipids (GPLs) that are abundant on the surface of the cell envelope. These GPLs are also known to contribute to the virulence of MAC. Serovar 8 is one of the dominant serovars isolated from AIDS patients, but the biosynthesis of serovar 8-specific GPL remains unknown. To clarify this, we compared gene clusters involved in the biosynthesis of several serovar-specific GPLs and identified the genomic region predicted to be responsible for GPL biosynthesis in a serovar 8 strain. Sequencing of this region revealed the presence of four open reading frames, three unnamed genes and gtfTB, the function of which has not been elucidated. The simultaneous expression of gtfTB and two downstream genes in a recombinant Mycobacterium smegmatis strain genetically modified to produce serovar 1-specific GPL resulted in the appearance of 4,6-O-(1-carboxyethylidene)-3-O-methyl-glucose, which is unique to serovar 8-specific GPL, suggesting that these three genes participate in its biosynthesis. Furthermore, functional analyses of gtfTB indicated that it encodes a glucosyltransferase that transfers a glucose residue via 1-->3 linkage to a rhamnose residue of serovar 1-specific GPL, which is critical to the formation of the oligosaccharide portion of serovar 8-specific GPL. Our findings might provide a clue to understanding the biosynthetic regulation that modulates the biological functions of GPLs in MAC.

CD4+ T-cell activation by antigen-presenting cells infected with urease-deficient recombinant Mycobacterium bovis bacillus Calmette-Guérin.

We constructed a recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG-Delta UT) that lacks urease, providing acidic intraphagosomal conditions to drive an effective human immune T-cell response. BCG-Delta UT-infected macrophages stimulated autologous CD4+ T cells more efficiently than parent BCG-infected macrophages. For further T-cell activation, BCG-Delta UT-infected macrophages required pretreatment with exogenous recombinant granulocyte-macrophage colony-stimulating factor or costimulation with either CD40 ligand or interferon-gamma. By contrast, BCG-Delta UT-infected dendritic cells induced significant activation of naïve CD4+ T cells without costimulating signals. C57BL/6 mice intradermally inoculated with BCG-Delta UT more efficiently produced memory T cells that responded to recall antigen. Therefore, the depletion of urease from BCG is useful for the activation of T cells.