SummaryIn the Gram-positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called s R in response to changes in the cytoplasmic thiol±disulphide status, and the activity of s R is controlled post-translationally by an anti-sigma factor, RsrA. In vitro, the antisigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol±disulphide redox status. Here, we investigate the function of RsrA in vivo. A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and s R -dependent transcription, confirming that RsrA is a negative regulator of s R and a key sensor of thiol±disulphide status. Targeted mutagenesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti-sigma factor activity and that a mutant RsrA protein containing only these three cysteines was active and still redox sensitive in vivo. We also show that RsrA is a metalloprotein, containing near-stoichiometric amounts of zinc. On the basis of these data, we propose that a thiol±disulphide redox switch is formed between two of C11, C41 and C44, and that all three residues play an essential role in anti-sigma factor activity in their reduced state, perhaps by acting as ligands for zinc. Unexpectedly, rsrA null mutants were blocked in sporulation, probably as a consequence of an increase in the level of free s R .

CLR induces greater erm(41) expression and thus higher macrolide resistance than AZM in M. abscessus infection. AZM may be more effective against M. abscessus, whereas both macrolides appear to be equally effective against M. massiliense.

SummaryAlternate sigma factors provide an effective way of diversifying bacterial gene expression in response to environmental changes. In Streptomyces coelicolor where more than 65 sigma factors are predicted, s R is the major regulator for response to thiol-oxidative stresses. s R becomes available when its bound antisigma factor RsrA is oxidized at sensitive cysteine thiols to form disulphide bonds. s R regulon includes genes for itself and multiple thiol-reducing systems, which constitute positive and negative feedback loops respectively. We found that the positive amplification loop involves an isoform of s R (s RЈ ) with an N-terminal extension of 55 amino acids, produced from an upstream start codon. A major difference between constitutive s R and inducible s RЈ is that the latter is markedly unstable (t1/2~10 min) compared with the former (> 70 min). The rapid turnover of s RЈ is partly due to induced ClpP1/P2 proteases from the s R regulon. This represents a novel way of elaborating positive and negative feedback loops in a control circuit. Similar phenomenon may occur in other actinomycetes that harbour multiple start codons in the sigR homologous gene. We observed that sigH gene, the sigR orthologue in Mycobacterium smegmatis, produces an unstable larger isoform of s H upon induction by thiol-oxidative stress.

Accumulating evidence indicates that latency-associated Mycobacterium tuberculosis (Mtb)-specific antigens from the dormancy survival regulator regulon (DosR) may be promising novel vaccine target antigens for the development of an improved tuberculosis vaccine. After transcriptional profiling of DosR-related genes in the hyper-virulent Beijing Mtb strain K and the reference Mtb strain H37Rv, we selected Rv3131, a hypothetical nitroreductase, as a vaccine antigen and evaluated its vaccine efficacy against Mtb K. Mtb K exhibited stable and constitutive up-regulation of rv3131 relative to Mtb H37Rv under three different growth conditions (at least 2-fold induction) including exponential growth in normal culture conditions, hypoxia, and inside macrophages. Mice immunised with Rv3131 formulated in GLA-SE, a well-defined TLR4 adjuvant, displayed enhanced Rv3131-specific IFN-γ and serum IgG2c responses along with effector/memory T cell expansion and remarkable generation of Rv3131-specific multifunctional CD4+ T cells co-producing TNF-α, IFN-γ and IL-2 in both spleen and lung. Following challenge with Mtb K, the Rv3131/GLA-SE-immunised group exhibited a significant reduction in bacterial number and less extensive lung inflammation accompanied by the obvious persistence of Rv3131-specific multifunctional CD4+ T cells. These results suggest that Rv3131 could be an excellent candidate for potential use in a multi-antigenic Mtb subunit vaccine, especially against Mtb Beijing strains.

The Beijing Mycobacterium tuberculosis family is widely distributed and is the most common M. tuberculosis strain in East Asia. The highly transmissible and predominant Beijing M. tuberculosis strain in Korea, M. tuberculosis K1, was characterized using an aerobic challenge mouse model and a latent tuberculosis model with M. tuberculosis H37Rv as a reference. M. tuberculosis K1 multiplied over ten times more rapidly than M. tuberculosis H37Rv during the early stage of infection and induced high levels of histopathology in the lung. Low levels of T helper cell (Th) Th1 [interferon (IFN)-c, interleukin (IL)-12p40] and Th2 cytokines (IL-4, IL-10) were induced in the lungs of M. tuberculosis K1-infected mice. In the latent model, mice infected with M. tuberculosis K1 exhibited more frequent relapse from the latent state than did mice infected with M. tuberculosis H37Rv. In conclusion, M. tuberculosis K1, a prevalent Beijing strain in Korea, is expected to spread due to its rapid growth during the early stages of infection, low-level induction of the immune response and high relapse rates from a latent state.