Differences in virulence and immune response induced in a murine model by isolates of Mycobacterium ulcerans from different geographic areas.

Buruli ulcer (BU) is the third most common mycobacterial disease in immunocompetent hosts. BU is caused by Mycobacterium ulcerans, which produces skin ulcers and necrosis at the site of infection. The principal virulence factor of M. ulcerans is a polyketide-derived macrolide named mycolactone, which has cytotoxic and immunosuppressive activities. We determined the severity of inflammation, histopathology and bacillary loads in the subcutaneous footpad tissue of BALB/c mice infected with 11 different M. ulcerans isolates from diverse geographical areas. Strains from Africa (Benin, Ghana, Ivory Coast) induced the highest inflammation, necrosis and bacillary loads, whereas the strains collected from Australia, Asia (Japan, Malaysia, New Guinea), Europe (France) and America (Mexico) induced mild inflammation. Subsequently, animals were infected with the strain that exhibited the highest (Benin) or lowest (Mexico) level of virulence in order to analyse the local immune response generated. The Mexican strain, which does not produce mycolactone, induced a predominantly T helper type 1 (Th1) cytokine profile with constant high expression of the anti-microbial peptides beta defensins 3 and 4, in co-existence with low expression of the anti-inflammatory cytokines interleukin (IL)-10, IL-4 and transforming growth factor (TGF)-beta. The highly virulent strain from Benin which produces mycolactone A/B induced the opposite pattern. Thus, different local immune responses were found depending on the infecting M. ulcerans strain.

Immunogenicity and protection induced by Mycobacterium tuberculosis mce-2 and mce-3 mutants in a Balb/c mouse model of progressive pulmonary tuberculosis.

Mycobacterial proteins coded by the mammalian cell entry (mce) genes allow for cell invasion into the host. The Mycobacterium tuberculosismce-2 and mce-3 mutants have impaired synthesis of mce proteins and are attenuated in BALB/c mice. Intra-tracheal infection of Balb/c mice with either mce mutant induced lower but progressive production of IFN-gamma and TNF-alpha, as well as larger delayed type hypersensitivity (DTH) reactions, than their parental H37Rv strain. When used as a subcutaneous vaccine and, before challenge, both mutants were more attenuated than BCG in Balb/c and immunodeficient nude mice. Cell suspensions from lymph nodes and spleen from mce mutant vaccinated mice stimulated with mycobacterial culture filtrate antigens (CFA) or immunodominant antigens (ESAT-6, Ag85) produced more INF-gamma than BCG-vaccinated animals. Used as subcutaneous vaccines, 60 days before intra-tracheal challenge with the hypervirulent strain of M. tuberculosis (Beijing code 9501000), both mutants induced a higher level of protection than BCG; 72% and 63% of the mice vaccinated with the mce-2 and mce-3 mutants, respectively, survived for 16 weeks after the challenge as compared to 30% of those vaccinated with BCG. Likewise, there was less tissue damage (pneumonia) and lower colony forming units (CFU) in the mice vaccinated with either of the two mutants as compared to the findings in mice vaccinated with BCG. These data suggest that lack of mce-2 and -3 gene expression decreases virulence and increases immunogenicity of live vaccines, favouring their ability to protect against tuberculosis, which was better than the protection conferred by BCG.

Immunogenicity and protective efficacy of the Mycobacterium tuberculosis fadD26 mutant.

The Mycobacterium tuberculosis fadD26 mutant has impaired synthesis of phthiocerol dimycocerosates (DIM) and is attenuated in BALB/c mice. Survival analysis following direct intratracheal infection confirmed the attenuation: 60% survival at 4 months post-infection versus 100% mortality at 9 weeks post-infection with the wild-type strain. The fadD26 mutant induced less pneumonia and larger DTH reactions. It induced lower but progressive production of interferon (IFN)-gamma, interleukin (IL)-4 and tumour necrosis factor (TNF)-alpha. Used as a subcutaneous vaccine 60 days before intratracheal challenge with a hypervirulent strain of M. tuberculosis (Beijing code 9501000), the mutant induced a higher level of protection than did Bacille Calmette-Guérin (BCG). Seventy per cent of the mice vaccinated with the fadD26 mutant survived at 16 weeks after challenge compared to 30% of those vaccinated with BCG. Similarly, there was less tissue damage (pneumonia) and lower colony-forming units (CFU) in the mice vaccinated with the fadD26 mutant compared to the findings in mice vaccinated with BCG. These data suggest that DIM synthesis is important for the pathogenicity of M. tuberculosis, and that inactivation of DIM synthesis can increase the immunogenicity of live vaccines, and increase their ability to protect against tuberculosis.

Local production of nitric oxide in patients with tuberculosis.

Nitric oxide (NO), produced by the inducible nitric oxide synthase (iNOS), is important in host defence against Mycobacterium tuberculosis in rodents, but the presence of high-output NO production in human tuberculosis has been controversial. We investigated iNOS and nitrotyrosine (Ntyr) expression in pleural (n = 7), pulmonary (n = 5) and lymph node biopsies (n = 5) from untreated, newly diagnosed tuberculosis patients. Many iNOS and Ntyr reactive macrophages were observed in granulomas, including Langhans giant cells, indicating high-output NO production at the primary site of disease in tuberculosis.