Usefulness of the IgA and IgG Responses to Macrophage Migration Inhibitory Factor for the Diagnosis of Tuberculosis.

Serological tests offer the potential in order to improve the diagnosis of tuberculosis (TB). Macrophage migration inhibitory factor (MIF) plays a protective role in infection control in TB; however, to date, no studies on antibody responses to MIF have been reported. We measured immunoglobulin (Ig)A and IgG responses to MIF in individuals with either active tuberculosis (ATB; n = 65), latent tuberculosis (LTBI; n = 53), or in non-infected individuals (NI; n = 62). The QuantiFERON-TB Gold In-Tube (QFT-GIT) assay was used in order to screen for LTBI. The level of IgA against MIF was significantly lower in LTBI and ATB patients than in NI individuals, was significantly related to LTBI and ATB diagnosis, and it could discriminate between LTBI and ATB. In contrast, the level of IgG against MIF was significantly lower in LTBI patients than in NI individuals and was significantly related to LTBI diagnosis. Anti-MIF IgG levels were significantly lower in AFB-negative TB, minimal TB, and new ATB patients, than in the NI group. IgA and IgG levels against MIF both showed significant negative correlations with IFN-γ levels, as assessed using the QFT-GIT test. Although none of the antibodies could achieve high diagnostic predictive power individually, our results suggest the possibility of using IgA antibody responses to MIF in the diagnosis of LTBI and ATB.

Diagnostic Potential of IgG and IgA Responses to Mycobacterium tuberculosis Antigens for Discrimination among Active Tuberculosis, Latent Tuberculosis Infection, and Non-Infected Individuals.

Tuberculosis remains a major public health problem. Conventional tests are inadequate to distinguish between active tuberculosis (ATB) and latent tuberculosis infection (LTBI). We measured antibody responses to Mycobacterium tuberculosis antigens (Mycobacterium tuberculosis chorismate mutase (TBCM), antigen 85B (Ag85B), early secreted antigen-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) in ATB, LTBI, and non-infected (NI) individuals. Serum immunoglobulin G (IgG) and immunoglobulin A (IgA) levels were measured and the QuantiFERON-TB Gold In-Tube assay was used to diagnose LTBI. IgG levels against TBCM were significantly higher in LTBI than NI subjects. IgG and IgA levels against Ag85B and IgG levels against CFP-10 were significantly higher in ATB, followed by LTBI, and then NI. When the ATB group was subdivided, IgG levels against Ag85B and CFP-10 were significantly higher in each subgroup compared with those in LTBI and NI groups. Positive correlation trends between interferon-gamma and IgG levels against Ag85B, TBCM, and CFP-10 and IgA levels against Ag85B in LTBI and NI subjects were observed. Age- and sex-adjusted models showed that IgG against TBCM and CFP-10 was independently related to LTBI diagnosis, and IgG against Ag85B was independently related to the diagnosis of ATB and could distinguish between LTBI and ATB. Overall, IgG antibody responses to TBCM, Ag85B, and CFP-10 can discriminate among ATB, LTBI, and NI groups.

Mycobacterium abscessus infection leads to enhanced production of type 1 interferon and NLRP3 inflammasome activation in murine macrophages via mitochondrial oxidative stress.

Mycobacterium abscessus (MAB) is a rapidly growing mycobacterium (RGM), and infections with this pathogen have been increasing worldwide. Recently, we reported that rough type (MAB-R) but not smooth type (MAB-S) strains enhanced type 1 interferon (IFN-I) secretion via bacterial phagosome escape, contributing to increased virulence. Here, we sought to investigate the role of mitochondrial oxidative stress in bacterial survival, IFN-I secretion and NLRP3 inflammasome activation in MAB-infected murine macrophages. We found that live but not heat-killed (HK) MAB-R strains increased mitochondrial ROS (mtROS) and increased release of oxidized mitochondrial DNA (mtDNA) into the cytosol of murine macrophages compared to the effects of live MAB-S strains, resulting in enhanced NLRP3 inflammasome-mediated IL-1ß and cGAS-STING-dependent IFN-I production. Treatment of the infected macrophages with mtROS-modulating agents such as mito-TEMPO or cyclosporin A reduced cytosolic oxidized mtDNA, which inhibited the MAB-R strain-induced production of IL-1ß and IFN-I. The reduced cytosolic oxidized mtDNA also inhibited intracellular growth of MAB-R strains via cytosolic escape following phagosomal rupture and via IFN-I-mediated cell-to-cell spreading. Moreover, our data showed that mtROS-dependent IFN-I production inhibited IL-1ß production, further contributing to MAB-R intracellular survival in murine macrophages. In conclusion, our data indicated that MAB-R strains enhanced IFN-I and IL-1ß production by inducing mtROS as a pathogen-associated molecular pattern (PAMP). These events also enhance bacterial survival in macrophages and dampen inflammation, which contribute to the pathogenesis of MAB-R strains.

Heat-killed Mycobacterium paragordonae therapy exerts an anti-cancer immune response via enhanced immune cell mediated oncolytic activity in xenograft mice model.

A therapeutic strategy capable of skewing toward a Th1-type immune response is crucial for cancer treatment. Recently, we reported Mycobacterium paragordonae (Mpg) as a potential live vaccine for mycobacterium infections. In this study, we explored the immunotherapeutic potential of heat-killed Mpg (HK-Mpg) in a mouse tumor xenograft model and elucidated its underlying antitumor mechanisms. MC38 cells derived from murine colon adenocarcinoma were implanted by subcutaneously injecting mice. The anticancer effects of HK-Mpg therapy were compared with HK-M. bovis BCG, an effective adjuvant for cancer immunotherapy. HK-Mpg treatment enhanced tumor reduction and mouse survival. Furthermore, HK-Mpg treatment synergistically enhanced the anticancer therapeutic effect of cisplatin. In addition, HK-Mpg enhanced inflammatory cytokine production and recruitment of immune cell into tumor-infiltrating sites and splenocytes in vaccinated mice. Our mechanistic study demonstrates that HK-Mpg therapy elicits a strong antitumor immune response in mice, mainly through natural killer cell-mediated oncolytic activity via the activation of dendritic cells (DCs) and by enhancing inflammatory cytokines production such as IL-12 from DC. Hence, HK-Mpg can be a potential immunotherapy adjuvant, enhancing the effect of cancer chemotherapy.

Potential of recombinant Mycobacterium paragordonae expressing HIV-1 Gag as a prime vaccine for HIV-1 infection.

Recombinant Mycobacterium strains such as recombinant BCG (rBCG) have received considerable attention for the HIV-1 vaccine development. Recently, we described a temperature-sensitive Mycobacterium paragordonae (Mpg) strain as a novel live tuberculosis vaccine that is safer and showed an enhanced protective effect against mycobacterial infection compared to BCG. We studied the possibility of developing a vaccine against HIV-1 infection using rMpg strain expressing the p24 antigen (rMpg-p24). We observed that rMpg-p24 can induce an increased p24 expression in infected antigen presenting cells (APCs) compared to rBCG-p24. We also observed that rMpg-p24 can induce enhanced p24 specific immune responses in vaccinated mice as evidenced by increased p24-specific T lymphocyte proliferation, gamma interferon induction, antibody production and cytotoxic T lymphocyte (CTL) responses. Furthermore, an rMpg-p24 prime and plasmid DNA boost showed an increased CTL response and antibody production compared to rBCG or rMpg alone. In summary, our study indicates that a live rMpg-p24 strain induced enhanced immune responses against HIV-1 Gag in vaccinated mice. Thus, rMpg-p24 may have potential as a preventive prime vaccine in a heterologous prime-boost regimen for HIV-1 infection.

New Mycobacteroides abscessus subsp. massiliense strains with recombinant hsp65 gene laterally transferred from Mycobacteroides abscessus subsp. abscessus: Potential for misidentification of M. abscessus strains with the hsp65-based method.

It has been reported that lateral gene transfer (LGT) events among Mycobacteroides abscessus strains are prevalent. The hsp65 gene, a chronometer gene for bacterial phylogenetic analysis, is resistant to LGT events, particularly among mycobacterial strains, rendering the hsp65-targeting method the most widely used method for mycobacterial detection. To determine the prevalence of M. abscessus strains that are subject to hsp65 LGT, we applied rpoB typing to 100 clinically isolated Korean strains of M. abscessus that had been identified by hsp65 sequence analysis. The analysis indicated the presence of 2 rough strains, showing a discrepancy between the 2 typing methods. MLST analysis based on the partial sequencing of seven housekeeping genes, erm(41) PCR and further hsp65 PCR-restriction enzyme and polymorphism analysis (PRA) were conducted to identify the two strains. The MLST results showed that the two strains belong to M. abscessus subsp. massiliense and not to M. abscessus subsp. abscessus, as indicated by the rpoB-based analysis, suggesting that their hsp65 genes are subject to LGT from M. abscessus subsp. abscessus. Further analysis of these strains using the hsp65 PRA method indicated that these strains possess a PRA pattern identical to that of M. abscessus subsp. abscessus and distinct from that of M. abscessus subsp. massiliense. In conclusion, we identified two M. abscessus subsp. massiliense rough strains from Korean patients with hsp65 genes that might be laterally transferred from M. abscessus subsp. abscessus. To the best of our knowledge, this is the first demonstration of possible LGT events associated with the hsp65 gene in mycobacteria. Our results also suggest that there is the potential for misidentification when the hsp65-based protocol is used for mycobacterial identification.

rt269I Type of Hepatitis B Virus (HBV) Leads to HBV e Antigen Negative Infections and Liver Disease Progression via Mitochondrial Stress Mediated Type I Interferon Production in Chronic Patients With Genotype C Infections.

Hepatitis B virus infection is a serious global health problem and causes life-threatening liver disease. In particular, genotype C shows high prevalence and severe liver disease compared with other genotypes. However, the underlying mechanisms regarding virological traits still remain unclear. This study investigated the clinical factors and capacity to modulate Type I interferon (IFN-I) between two HBV polymerase polymorphisms rt269L and rt269I in genotype C. This report compared clinical factors between rt269L and rt269I in 220 Korean chronic patients with genotype C infections. The prevalence of preC mutations between rt269L and rt269I was compared using this study's cohort and the GenBank database. For in vitro and in vivo experiments, transient transfection using HBV genome plasmid and HBV virion infection using HepG2-hNTCP-C4 and HepaRG systems and hydrodynamic injection of HBV genome into mice tails were conducted, respectively. This report's clinical data indicated that rt269I vs. rt269L was more significantly related to HBV e antigen (HBeAg) negative serostatus, lower levels of HBV DNA and HBsAg, and disease progression. Our epidemiological study showed HBeAg negative infections of rt269I infections were attributed to a higher frequency of preC mutations at 1896 (G to A). Our in vitro and in vivo studies also found that rt269I could lead to mitochondrial stress mediated STING dependent IFN-I production, resulting in decreasing HBV replication via the induction of heme-oxygenase-1. In addition, we also found that rt269I could lead to enhanced iNOS mediated NO production in an IFN-I dependent manner. These data demonstrated that rt269I can contribute to HBeAg negative infections and liver disease progression in chronic patients with genotype C infections via mitochondrial stress mediated IFN-I production.

Insights From the Genome Sequence of Mycobacterium paragordonae, a Potential Novel Live Vaccine for Preventing Mycobacterial Infections: The Putative Role of Type VII Secretion Systems for an Intracellular Lifestyle Within Free-Living Environmental Predators.

Mycobacterium paragordonae (Mpg) is a temperature-sensitive Mycobacterium species that can grow at permissive temperatures but fails to grow above 37°C. Due to this unique growth trait, Mpg has recently been proposed as a novel live vaccine candidate for the prevention of mycobacterial infections. Furthermore, the increasing frequency of the isolation of Mpg from water supply systems led us to hypothesize that the free-living amoeba system is the natural reservoir of Mpg. In this study, we report the complete 6.7-Mb genome sequence of Mpg and show that this genome comprises four different plasmids with lengths of 305 kb (pMpg-1), 144 kb (pMpg-2), 26 kb (pMpg-3), and 17 kb (pMpg-4). The first two plasmids, pMpg-1 and -2, encode distinct Type VII secretion systems (T7SS), ESX-P5 and ESX-2, respectively. Genome-based phylogeny indicated that Mpg is the closest relative to M. gordonae, which has a 7.7-Mb genome; phylogenetic analysis revealed an average of 86.68% nucleotide identity between these two species. The most important feature of Mpg genome is the acquisition of massive genes related to T7SS, which may have had effect on adaptation to their intracellular lifestyle within free-living environmental predators, such as amoeba. Comparisons of the resistance to bacterial killing within amoeba indicated that Mpg exhibited stronger resistance to amoeba killing compared to M. gordonae and M. marinum, further supporting our genome-based findings indicating the special adaptation of Mpg to free-living amoeba. We also determined that, among the strains studied, there were more shared CDS between M. tuberculosis and Mpg. In addition, the presence of diverse T7SSs in the Mpg genome, including an intact ESX-1, may suggest the feasibility of Mpg as a novel tuberculosis vaccine. Our data highlight a significant role of lateral gene transfer in the evolution of mycobacteria for niche diversification and for increasing the intracellular survival capacity.

Phagosome Escape of Rough Mycobacterium abscessus Strains in Murine Macrophage via Phagosomal Rupture Can Lead to Type I Interferon Production and Their Cell-To-Cell Spread.

Mycobacterium abscessus complex (MAB) is a rapidly growing mycobacterium(RGM) whose clinical significance as an emerging human pathogen has been increasing worldwide. It has two types of colony morphology, a smooth (S) type, producing high glycopeptidolipid (GPL) content, and a rough (R) type, which produces low levels of GPLs and is associated with increased virulence. However, the mechanism responsible for their difference in virulence is poorly known. By ultrastructural examination of murine macrophages infected, we found that MAB-R strains could replicate more actively in the macrophage phagosome than the S variants and that they could escape into cytosol via phagosomal rupture. The cytosolic access of MAB-R strains via phagosomal rupture led to enhanced Type I interferon (IFN) production and cell death, which resulted in their cell-to-cell spreading. This behavior can provide an additional niche for the survival of MAB-R strains. In addition, we found that their enhancement of cell death mediated cell spreading are dependent on Type I IFN signaling via comparison of wild-type and IFNAR1 knockout mice. In conclusion, our data indicated that a transition of MAB-S strains into MAB-R variants increased their virulence via enhanced Type I IFN production, which led to enhanced survival in infected macrophage via cell death mediated cell-to-cell spreading. This result provides not only a novel insight into the difference in virulence between MAB-R and -S variants but also hints to their treatment strategy.

A description of Mycobacterium chelonae subsp. gwanakae subsp. nov., a rapidly growing mycobacterium with a smooth colony phenotype due to glycopeptidolipids.

Three rapidly growing mycobacterial strains, MOTTH4W, MOTT36WT and MOTT68W, were isolated from the sputa of three independent Korean patients co-infected with Mycobacterium yongonense Type II strains. The 16S rRNA gene sequences of all three strains were unique, which were closest to that of Mycobacterium chelonae subsp. bovis KCTC 39630T (99.9 % similarity). Multilocus sequence typing analysis targeting 10 housekeeping genes including hsp65 and rpoB revealed the distinct phylogenetic location of these strains, which were clustered with M. chelonae subsp. chelonae ATCC 35752T and M. chelonae subsp. bovis KCTC 39630T. Phylogenetic analysis based on whole genome sequences revealed a 95.89 % average nucleotide identity (ANI) value with M. chelonae subsp. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, phenotypic characteristics such as a smooth colony morphology and growth inhibition at 37 °C, distinct MALDI-TOF MS profiles of extracted total lipids due to surface glycopeptidolipids, and distinct drug susceptibility profiles further supported the taxonomic characterization of these strains as representing a novel subspecies of Mycobacterium chelonae. Mycobacterium chelonae subsp. gwanakae subsp. nov. is proposed and the type strain is MOTT36WT (=KCTC 29127T=JCM 32454T).

Development of a Live Recombinant BCG Expressing Human Immunodeficiency Virus Type 1 (HIV-1) Gag Using a pMyong2 Vector System: Potential Use As a Novel HIV-1 Vaccine.

Even though the rate of new human immunodeficiency virus type 1 (HIV-1) infections is gradually decreasing worldwide, an effective preventive vaccine for HIV-1 is still urgently needed. The recombinant Mycobacterium bovis BCG (rBCG) is promising for the development of an HIV-1 vaccine. Recently, we showed that a recombinant Mycobacterium smegmatis expressing HIV-1 gag in a pMyong2 vector system (rSmeg-pMyong2-p24) increased the efficacy of a vaccine against HIV-1 in mice. Here, we evaluated the potential of an rBCG expressing HIV-1 p24 antigen Gag in pMyong2 (rBCG-pMyong2-p24) in a vaccine application for HIV-1 infection. We found that rBCG-pMyong2-p24 elicited an enhanced HIV-1 p24 Gag expression in rBCG and infected antigen-presenting cells. We also found that compared to rBCG-pAL-p24 in a pAL5000 derived vector system, rBCG-pMyong2-p24 elicited enhanced p24-specific immune responses in vaccinated mice as evidenced by higher levels of HIV-1 Gag-specific CD4 and CD8 T lymphocyte proliferation, gamma interferon ELISPOT cell induction, antibody production, and cytotoxic T lymphocytes (CTL) responses. Furthermore, rBCG-pMyong2-p24 showed a higher level of p24-specific Ab production than rSmeg-pMyong2-p24 in the same pMyong2 vector system. In conclusion, our data indicated that a live recombinant BCG expressing HIV-1 Gag using a pMyong2 vector system, rBCG-pMyong2-p24 elicited an enhanced immune response against HIV-1 infections in a mouse model system. So, rBCG-pMyong2-p24 may have the potential as a prime vaccine in a heterologous prime-boost vaccine strategy for HIV-1 infection.

Patterns of rpoC Mutations in Drug-Resistant Mycobacterium tuberculosis Isolated from Patients in South Korea.

BACKGROUND: Rifampicin (RFP) is one of the principal first-line drugs used in combination chemotherapies against Mycobacterium tuberculosis, and its use has greatly shortened the duration of chemotherapy for the successful treatment of drug-susceptible tuberculosis. Compensatory mutations have been identified in rpoC that restore the fitness of RFP-resistant M. tuberculosis strains with mutations in rpoB. To investigate rpoC mutation patterns, we analyzed 93 clinical M. tuberculosis isolates from patients in South Korea. METHODS: Drug-resistant mycobacterial isolates were cultured to determine their susceptibility to anti-tubercular agents. Mutations in rpoC were identified by sequencing and compared with the relevant wild-type DNA sequence. RESULTS: In total, 93 M. tuberculosis clinical isolates were successfully cultured and tested for drug susceptibilities. They included 75 drug-resistant tuberculosis species, of which 66 were RFP-resistant strains. rpoC mutations were found in 24 of the 66 RFP-resistant isolates (36.4%). Fifteen different types of mutations, including single mutations (22/24, 91.7%) and multiple mutations (2/24, 8.3%), were identified, and 12 of these mutations are reported for the first time in this study. The most frequent mutation involved a substitution at codon 452 (nt 1356) resulting in amino acid change F452L. CONCLUSION: Fifteen different types of mutations were identified and were predominantly single-nucleotide substitutions (91.7%). Mutations were found only in dual isoniazid- and RFP-resistant isolates of M. tuberculosis. No mutations were identified in any of the drug-susceptible strains.

A temperature sensitive Mycobacterium paragordonae induces enhanced protective immune responses against mycobacterial infections in the mouse model.

Recently, we introduced a temperature sensitive Mycobacterium spp., Mycobacterium paragordonae (Mpg). Here, we checked its potential as a candidate for live vaccination against Mycobacterium tuberculosis and Mycobacterium abscessus. Intravenous infections of mice with Mpg led to lower colony forming units (CFUs) compared to infection with BCG, suggesting its usefulness as a live vaccine. The analyses of immune responses indicated that the highly protective immunity elicited by Mpg was dependent on effective dendritic maturation, shift of cytokine patterns and antibody production toward a Th1 phenotype, and enhanced cytotoxic T cell response. Compared to BCG, Mpg showed a more effective protective immune response in the vaccinated mice against challenges with 2 different mycobacterial strains, M. tuberculosis H37Ra or M. abscessus Asan 50594. Our data suggest that a temperature sensitive Mpg may be a potentially powerful candidate vaccine strain to induce enhanced protective immune responses against M. tuberculosis and M. abscessus.

Description of Mycobacterium chelonae subsp. bovis subsp. nov., isolated from cattle (Bos taurus coreanae), emended description of Mycobacterium chelonae and creation of Mycobacterium chelonae subsp. chelonae subsp. nov.

Three rapidly growing mycobacterial strains, QIA-37T, QIA-40 and QIA-41, were isolated from the lymph nodes of three separate Korean native cattle, Hanwoo (Bos taurus coreanae). These strains were previously shown to be phylogenetically distinct but closely related to Mycobacterium chelonae ATCC 35752T by taxonomic approaches targeting three genes (16S rRNA, hsp6 and rpoB) and were further characterized using a polyphasic approach in this study. The 16S rRNA gene sequences of all three strains showed 99.7 % sequence similarity with that of the M. chelonae type strain. A multilocus sequence typing analysis targeting 10 housekeeping genes, including hsp65 and rpoB, revealed a phylogenetic cluster of these strains with M. chelonae. DNA-DNA hybridization values of 78.2 % between QIA-37T and M. chelonae indicated that it belongs to M. chelonae but is a novel subspecies distinct from M. chelonae. Phylogenetic analysis based on whole-genome sequences revealed a 95.44±0.06 % average nucleotide identity (ANI) value with M. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, distinct phenotypic characteristics such as positive growth at 37 °C, at which temperature M. chelonae does not grow, further support the taxonomic status of these strains as representatives of a novel subspecies of M. chelonae. Therefore, we propose an emended description of Mycobacterium chelonae, and descriptions of M. chelonae subsp. chelonae subsp. nov. and M. chelonae subsp. bovis subsp. nov. are presented; strains ATCC 35752T(=CCUG 47445T=CIP 104535T=DSM 43804T=JCM 6388T=NCTC 946T) and QIA-37T (=KCTC 39630T=JCM 30986T) are the type strains of the two novel subspecies.

Effect of amikacin on cell wall glycopeptidolipid synthesis in Mycobacterium abscessus.

Cultivation of the smooth colony Mycobacterium abscessus at the sub-minimum inhibitory concentration (MIC) of amikacin changed its growth pattern including its colony morphology (smooth to rough) and cell arrangement (dispersed to cord formation). In addition, reduced sliding motility and biofilm formation were observed. The amount of glycogpetidolipid (GPL) and mRNA expression of key genes involved in GPL synthesis were decreased in the amikacin-treated M. abscessus strain. An in vitro infection assay revealed that the amikacin-treated smooth M. abscessus strain induced more pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) than that of the smooth strain in murine macrophage cells. These results suggest that long-term exposure to a low concentration of amikacin causes a physical change in the cell wall which may increase its virulence.

Phylogenetic analysis of Mycobacterium massiliense strains having recombinant rpoB gene laterally transferred from Mycobacterium abscessus.

Recent multi locus sequence typing (MLST) and genome based studies indicate that lateral gene transfer (LGT) events in the rpoB gene are prevalent between Mycobacterium abscessus complex strains. To check the prevalence of the M. massiliense strains subject to rpoB LGT (Rec-mas), we applied rpoB typing (711 bp) to 106 Korean strains of M. massiliense infection that had already been identified by hsp65 sequence analysis (603 bp). The analysis indicated 6 smooth strains in M. massiliense Type I (10.0%, 6/60) genotypes but no strains in M. massiliense Type II genotypes (0%, 0/46), showing a discrepancy between the 2 typing methods. Further MLST analysis based on the partial sequencing of seven housekeeping genes, argH, cya, glpK, gnd, murC, pta and purH, as well as erm(41) PCR proved that these 6 Rec-mas strains consisted of two distinct genotypes belonging to M. massiliense and not M. abscessus. The complete rpoB sequencing analysis showed that these 6 Rec-mas strains have an identical hybrid rpoB gene, of which a 478 bp partial rpoB fragment may be laterally transferred from M. abscessus. Notably, five of the 6 Rec-mas strains showed complete identical sequences in a total of nine genes, including the seven MLST genes, hsp65, and rpoB, suggesting their clonal propagation in South Korea. In conclusion, we identified 6 M. massiliense smooth strains of 2 phylogenetically distinct genotypes with a specific hybrid rpoB gene laterally transferred from M. abscessus from Korean patients. Their clinical relevance and bacteriological traits remain to be elucidated.

Identification of Novel A2/C2 Inter-Genotype Recombinants of Hepatitis B Virus from a Korean Chronic Patient Co-Infected with Both Genotype A2 and C2.

Nearly all cases of Hepatitis B virus (HBV) infections in South Korea have the C2 genotype. Here, we have identified a chronically infected patient who was co-infected with HBV of both the A2 and C2 genotypes by screening 135 Korean chronically infected patients using direct sequencing protocols targeting the 1032-bp polymerase reverse transcriptase (RT) region. Further polymerase chain reaction (PCR)-cloning analysis (22 clones) of the RT showed that this patient had genotype C2 (12 clones), genotype A2 (six clones) and A2/C2 inter-genotype HBV recombinants (four clones). BootScan analysis showed that three of the four recombinants have different types of recombination breakpoints in both the RT and overlapping hepatitis B surface antigen (HBsAg) region. Given the significance of HBsAg as a diagnostic or vaccination target against HBV infection, clinical implications of these identified recombinants should be studied in the future. To our knowledge, this is the first report on A2/C2 inter-genotype HBV recombinants.

Recombinant Mycobacterium smegmatis with a pMyong2 vector expressing Human Immunodeficiency Virus Type I Gag can induce enhanced virus-specific immune responses.

Recently, we have developed a novel Mycobacterium-Escherichia coli shuttle vector system using pMyong2, which can provide an enhanced expression of heterologous genes in recombinant Mycobacterium smegmatis (rSmeg). To investigate the usefulness of rSmeg using pMyong2 in vaccine application, we vaccinated M. smegmatis with pMyong2 system expressing Human Immunodeficiency Virus Type I (HIV-1) Gag p24 antigen (rSmeg-pMyong2-p24) into mice and examined its cellular and humoral immune responses against HIV gag protein. We found that rSmeg-pMyong2-p24 expressed higher levels of Gag protein in bacteria, macrophage cell line (J774A.1) and mouse bone marrow derived dendritic cells (BMDCs) compared to rSmeg strains using two other vector systems, pAL5000 derived vector (rSmeg-pAL-p24) and the integrative plasmid, pMV306 (rSmeg-pMV306-p24). Inoculation of mice with rSmeg-pMyong2-p24 elicited more effective immunity compared to the other two rSmeg strains, as evidenced by higher levels of HIV-1 Gag-specific CD4 and CD8 T lymphocyte proliferation, interferon gamma ELISPOT cell induction, and antibody production. Furthermore, rSmeg-pMyong2-p24 showed a higher level of cytotoxic T cell response against target cells expressing Gag p24 proteins. Our data suggest that Mycobacterium-Escherichia coli shuttle vector system with pMyong2 may provide an advantage in vaccine application of rSmeg over other vector systems.

Role of the DNA Mismatch Repair Gene MutS4 in Driving the Evolution of Mycobacterium yongonense Type I via Homologous Recombination.

We recently showed that Mycobacterium yongonense could be divided into two genotypes: Type I, in which the rpoB gene has been transferred from Mycobacterium parascrofulaceum, and Type II, in which the rpoB gene has not been transferred. Comparative genome analysis of three M. yongonense Type I, two M. yongonense Type II and M. parascrofulaceum type strains were performed in this study to gain insight into gene transfer from M. parascrofulaceum into M. yongonense Type I strains. We found two genome regions transferred from M. parascrofulaceum: one contained 3 consecutive genes, including the rpoBC operon, and the other contained 57 consecutive genes that had been transferred into M. yongonense Type I genomes via homologous recombination. Further comparison between the M. yongonense Type I and II genomes revealed that Type I, but not Type II has a distinct DNA mismatch repair gene (MutS4 subfamily) that was possibly transferred via non-homologous recombination from other actinomycetes. We hypothesized that it could facilitate homologous recombination from the M. parascrofulaceum to the M. yongonense Type I genomes. We therefore generated recombinant Mycobacterium smegmatis containing a MutS4 operon of M. yongonense. We found that the M. tuberculosis rpoB fragment with a rifampin resistance-conferring mutation was more frequently inserted into recombinant M. smegmatis than the wild type, suggesting that MutS4 is a driving force in the gene transfer from M. parascrofulaceum to M. yongonense Type I strains via homologous recombination. In conclusion, our data indicated that MutS4 in M. yongonense Type I genomes may drive gene transfer from M. parascrofulaceum via homologous recombination, resulting in division of M. yongonense into two genotypes, Type I and II.

Mycobacterium paraintracellulare sp. nov., for the genotype INT-1 of Mycobacterium intracellulare.

Three mycobacterial strains, isolated from independent Korean patients with pulmonary infections, belonging to the Mycobacterium intracellulare genotype 1 (INT-1) were characterized using a polyphasic approach. The sequences of the 16S rRNA gene and internal transcribed spacer 1 (ITS1) of the INT-1 strains were identical to those of Mycobacterium intracellulare ATCC 13950T. However, multilocus sequence typing (MLST) analysis targeting five housekeeping genes (hsp65, rpoB, argG, gnd and pgm) revealed the phylogenetic separation of these strains from M. intracellulare ATCC 13950T. DNA-DNA hybridization values of >70 % confirmed that the three isolates belong to the same species, while the values of <70 % between one of them and the type strains of M. intracellulare and Mycobacterium chimaera confirmed their belonging to a distinct species. In addition, phenotypic characteristics such as positive growth on MacConkey agar and in acidic broth culture, unique matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS profiles of lipids, and unique mycolic acids profiles further supported the taxonomic status of these strains as representatives of a novel species of the Mycobacterium avium complex named Mycobacterium paraintracellulare. The type strain is MOTT64T (=KCTC 29084T=JCM 30622T).