Yuriko Ozeki

Control of cell wall assembly by a histone-like protein in Mycobacteria.

Bacteria coordinate assembly of the cell wall as well as synthesis of cellular components depending on the growth state. The mycobacterial cell wall is dominated by mycolic acids covalently linked to sugars, such as trehalose and arabinose, and is critical for pathogenesis of mycobacteria. Transfer of mycolic acids to sugars is necessary for cell wall biogenesis and is mediated by mycolyltransferases, which have been previously identified as three antigen 85 (Ag85) complex proteins. However, the regulation mechanism which links cell wall biogenesis and the growth state has not been elucidated. Here we found that a histone-like protein has a dual concentration-dependent regulatory effect on mycolyltransferase functions of the Ag85 complex through direct binding to both the Ag85 complex and the substrate, trehalose-6-monomycolate, in the cell wall. A histone-like protein-deficient Mycobacterium smegmatis strain has an unusual crenellated cell wall structure and exhibits impaired cessation of glycolipid biosynthesis in the growth-retarded phase. Furthermore, we found that artificial alteration of the amount of the extracellular histone-like protein and the Ag85 complex changes the growth rate of mycobacteria, perhaps due to impaired down-regulation of glycolipid biosynthesis. Our results demonstrate novel regulation of cell wall assembly which has an impact on bacterial growth.

Virulence of Mycobacterium avium complex strains isolated from immunocompetent patients

Mycobacterium avium complex (MAC) disease has been increasing worldwide not only in immunocompromised but also in immunocompetent humans. However, the relationship between mycobacterial strain virulence and disease progression in immunocompetent humans is unclear. In this study, we isolated 6 strains from patients with pulmonary MAC disease. To explore the virulence, we examined the growth in human THP-1 macrophages and pathogenicity in C57BL/6 mice. We found that one strain, designated 198, which was isolated from a patient showing the most progressive disease, persisted in THP-1 cells. In addition, strain 198 grew to a high bacterial load with strong inflammation in mouse lungs and spleens 16 weeks after infection. To our knowledge, strain 198 is the first isolated MAC strain that exhibits hypervirulence consistently for the human patient, human macrophages in vitro, and even for immunocompetent mice. Other strains showed limited survival and weak virulence both in macrophages and in mice, uncorrelated to disease progression in human patients. We demonstrated that there is a hypervirulent clinical MAC strain whose experimental virulence corresponds to the serious disease progression in the patients. The existence of such strain suggests the involvement of bacterial virulence in the pathogenesis of pulmonary MAC disease in immunocompetent status.

Transient role of CD4+CD25+ regulatory T cells in mycobacterial infection in mice

CD4(+)CD25(+) regulatory T (Treg) cells cause immune suppression by inhibiting T cell effector functions and play pivotal roles not only in self-tolerance but also in immune response to parasitic microbial pathogens. Mycobacteria are major parasitic bacterial pathogens, but the role of CD4(+)CD25(+) Treg cells in mycobacterial infection is not yet defined. In this study we found that, at the early stage of infection, depletion of CD25(+) cells reduced both bacterial load and granuloma formation in mice infected with Mycobacterium tuberculosis strains, such as M. tuberculosis Erdman or M. tuberculosis Kurono. However, at a later stage of infection, bacterial burden and histopathology were similar regardless of depletion of CD25(+) cells. Severe combined immunodeficient (SCID) mice reconstituted with CD4(+)CD25(-) T cells alone or a combination of CD4(+)CD25(+) and CD4(+)CD25(-) T cells showed similar bacterial loads and survival kinetics after infection with M. tuberculosis Erdman. Consistent with in vivo data, in vitro studies revealed that mycobacterial antigens, purified protein derivative of tuberculin (PPD), failed to induce the suppressive function of CD4(+)CD25(+) Treg cells to CD4(+)CD25(-) effector T cells, as demonstrated by the lack of response of CD4(+)CD25(+) T cells to PPD, in mice chronically infected with Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis. Our data show that CD4(+)CD25(+) Treg cells have a transient effect at the early stage of mycobacterial infection but, contrary to the expectation, have little impact on the overall course of infection.

A Novel Mechanism of Growth Phase-dependent Tolerance to Isoniazid in Mycobacteria

Background: The mechanism underlying mycobacterial phenotypic tolerance to isoniazid is unknown. Results: MDP1, a mycobacterial histone-like protein, down-regulates KatG expression. Conclusion: Down-regulation of KatG by MDP1 causes growth phase-dependent phenotypic tolerance to isoniazid in mycobacteria. Significance: Understanding the mechanism by which mycobacteria acquire tolerance to isoniazid is important for developing novel therapies. Tuberculosis remains one of the most deadly infectious diseases worldwide and is a leading public health problem. Although isoniazid (INH) is a key drug for the treatment of tuberculosis, tolerance to INH necessitates prolonged treatment, which is a concern for effective tuberculosis chemotherapy. INH is a prodrug that is activated by the mycobacterial enzyme, KatG. Here, we show that mycobacterial DNA-binding protein 1 (MDP1), which is a histone-like protein conserved in mycobacteria, negatively regulates katG transcription and leads to phenotypic tolerance to INH in mycobacteria. Mycobacterium smegmatis deficient for MDP1 exhibited increased expression of KatG and showed enhanced INH activation compared with the wild-type strain. Expression of MDP1 was increased in the stationary phase and conferred growth phase-dependent tolerance to INH in M. smegmatis. Regulation of KatG expression is conserved between M. smegmatis and Mycobacterium tuberculosis complex. Artificial reduction of MDP1 in Mycobacterium bovis BCG was shown to lead to increased KatG expression and susceptibility to INH. These data suggest a mechanism by which phenotypic tolerance to INH is acquired in mycobacteria.

DNA Augments Antigenicity of Mycobacterial DNA-Binding Protein 1 and Confers Protection against Mycobacterium tuberculosis Infection in Mice

Mycobacterium consists up to 7% of mycobacterial DNA-binding protein 1 (MDP1) in total cellular proteins. Host immune responses to MDP1 were studied in mice to explore the antigenic properties of this protein. Anti-MDP1 IgG was produced after infection with either bacillus Calmette-Guérin or Mycobacterium tuberculosis in C3H/HeJ mice. However, the level of Ab was remarkably low when purified MDP1 was injected. MDP1 is considered to be associated with DNA in nucleoid, which contains immunostimulatory CpG motif. Therefore, we examined coadministration of MDP1 and DNA derived from M. tuberculosis. Consequently, this procedure significantly enhanced the production of MDP1-specific IgG. Five nanograms of DNA was enough to enhance MDP1-specific IgG production in the administration of 5 μg of MDP1 into mice. Strong immune stimulation by such a small amount of DNA is noteworthy, because >1,000- to 100,000-fold doses of CpG DNAs are used for immune activation. A synthetic peptide-based study showed that B cell epitopes were different between mice administered MDP1 alone and those given a mixture of MDP1 and DNA, suggesting that DNA alters the three-dimensional structure of MDP1. Coadministration of DNA also enhanced MDP1-specific IFN-γ production and reduced the bacterial burden of a following challenge of M. tuberculosis, showing that MDP1 is a novel vaccine target. Finally, we found that MDP1 remarkably enhanced TLR9-dependent immune stimulation by unmethylated CpG oligo DNA in vitro. To our knowledge, MDP1 is the first protein discovered that remarkably augments the CpG-mediated immune response and is a potential adjuvant for CpG DNA-based immune therapies.

Mycobacteria Exploit Host Hyaluronan for Efficient Extracellular Replication

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.

Loss of anti-mycobacterial efficacy in mice over time following vaccination with Mycobacterium bovis bacillus Calmette-Guérin.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the most often used vaccine worldwide and sole vaccine against tuberculosis. BCG is protective against severe form of childhood tuberculosis but less or not protective to adult pulmonary tuberculosis. Therefore, improved vaccination strategies and development of new tuberculosis vaccines are urgent demands. For those purposes, appropriate animal models that reflect human are critically useful. However, in animal models, BCG vaccination protects well against subsequent challenge of Mycobacterium tuberculosis. In this study we evaluated the duration of protective efficacy of the BCG vaccination in mice over time and found that efficacy was diminished 40 weeks after vaccination. The aged mice older than 45 weeks are protected sufficiently after the vaccination with BCG, suggesting that loss of its efficacy is not dependent on the age of mice but rather depends on the period from vaccination. The loss of protection occurred in TH1 polarized STAT6 deficient mice despite the maintenance of interferon (IFN)-gamma production activity of lymph node cells and splenic CD4(+) T cells against M. tuberculosis antigens. Our data suggest that the duration from vaccination may explain the variation in BCG efficacy against adult pulmonary tuberculosis.

Relationship between Mycobacterium Tuberculosis and HookwormInfections among School Children in Mbita, Kenya

Tuberculosis (TB) remains a serious threat for human health. The majority of TB cases arise from latent Mycobacterium tuberculosis infection (LTBI). Therefore, latent M. tuberculosis infection (LTBI) is a major reservoir of the pathogen, and every effort thus should be made to diagnose LTBI to ensure completion of the treatment of it. TB is endemic throughout most of the tropics, in which parasitic infections are prevalent as well. It was reported that Helminth infection, including hookworm, is a risk of active TB, but its effect on the establishment of LTBI is unknown. In this study, we conducted a cross-sectional survey of LTBI and parasitic infections among 240 children from schools situated along the shores of Lake Victoria in Mbita district, Kenya. Blood samples were analyzed for LTBI and enteric parasite infections. Among the 240 children examined, 75 (31.3 %) were found to have LTBI. Of the 75 children with LTBI, 10 children (13.3%) were found to be positive for hookworm eggs (odds ratio: 3.02; 95% confidence interval: 1.14-7.99). Our study suggests for the first time that hookworm infection is associated with not only active TB but also LTBI.

Antigen 85A and mycobacterial DNA‐binding protein 1 are targets of immunoglobulin G in individuals with past tuberculosis

Development of accurate methods for predicting progression of tuberculosis (TB) from the latent state is recognized as vitally important in controlling TB, because a majority of cases develop from latent infections. Past TB that has never been treated has a higher risk of progressing than does latent Mycobacterium tuberculosis infection in patients who have previously received treatment. Antibody responses against 23 kinds of M. tuberculosis proteins in individuals with past TB who had not been medicated were evaluated. These individuals had significantly higher concentrations of antibodies against Antigen 85A and mycobacterial DNA‐binding protein 1 (MDP1) than did those with active TB and uninfected controls. In addition, immunohistochemistry revealed colocalization of tubercle bacilli, antigen 85 and MDP1 inside tuberculous granuloma lesions in an asymptomatic subject, showing that M. tuberculosis in lesions expresses both antigen 85 and MDP1. Our study suggests the potential usefulness of measuring antibody responses to antigen 85A and MDP1 for assessing the risk of TB progression.

Dominant Incidence of Multidrug and Extensively Drug-Resistant Specific Mycobacterium tuberculosis Clones in Osaka Prefecture, Japan

Infection and transmission of multidrug-resistant Mycobacterium tuberculosis (MDR-Mtb) and extensively drug-resistant M. tuberculosis (XDR-Mtb) is a serious health problem. We analyzed a total of 1,110 Mtb isolates in Osaka Prefecture and neighboring areas from April 2000 to March 2009. A total of 89 MDR-Mtb were identified, 36 (48.5%) of which were determined to be XDR-Mtb. Among the 89 MDR-Mtb isolates, 24 (27.0%) phylogenetically distributed into six clusters based on mycobacterial interspersed repetitive units-various number of tandem repeats (MIRU-VNTR) typing. Among these six clusters, the MIRU-VNTR patterns of four (OM-V02, OM-V03, OM-V04, and OM-V06) were only found for MDR-Mtb. Further analysis revealed that all isolates belonging to OM-V02 and OM-V03, and two isolates from OM-V04 were clonal. Importantly such genotypes were not observed for drug-sensitive isolates. These suggest that few but transmissible clones can transmit after acquiring multidrug resistance and colonize even in a country with a developed, well-organized healthcare system.