No abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) poses a major threat to the global public health. Importantly, latent tuberculosis infection (LTBI) still impedes the elimination of TB incidence since it has a substantial risk to develop active disease. A multi-stage subunit vaccine comprising active and latency antigens of Mtb has been raised as the promising vaccine to trigger immune protection against all stages of TB. Therefore, the discovery of new antigens that could trigger broad immune response is essential. While current development of TB vaccine mainly focuses on protective immunity mediated by adaptive immune response, the knowledge on triggering the innate immune response by antigens is still limited. We showed that recombinant dormancy-associated Mtb proteins Rv2659c and Rv1738 were recognized by human innate immune recognition molecules, Toll-like receptors (TLRs) 2 and 4 by using HEK-Blue™ hTLR2/hTLR4 systems. We further demonstrated that these two proteins activated phosphorylated NF-κB p65 (Ser536) in the human CD14+ blood cells. We also investigated that these two proteins significantly induced level of pro- and anti-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-10 and TNF-α) which were mediated through TLR2 and TLR4 pathways in human peripheral blood mononuclear cells (hPBMCs). These findings suggest that proteins Rv2659c and Rv1738 stimulated innate immune response targeting TLR2 and TLR4 to produce inflammatory cytokines, and their benefits would be valuable for the development of an effective prophylactic tuberculosis vaccine.

Mycoplasma contamination detrimentally affects cellular functions and the growth of intracellular pathogens in cell cultures. Although several mycoplasmacidal agents are commercially available for sterile cell cultures, they are not applicable to rickettsia-infected cells. In our attempt to find an anti-mycoplasma drug for contaminated rickettsial cultures, we determined the susceptibilities of three common Mycoplasma species to daptomycin. Mycoplasma orale and M. arginini showed low-level resistance to daptomycin (minimum inhibitory concentration, MIC = 2 mg/L), whereas M. hyorhinis was high-level resistant (MIC = 32 mg/L). However, some Mycoplasma isolates developed higher resistance to daptomycin after failed treatments with inadequate doses or durations. An aminoglycoside (gentamicin) was still active against M. hyorhinis and could be used in Orientia cultures. For complete eradication of mycoplasmas in Rickettsia cultures, we recommend a 3-week treatment with daptomycin at 256 mg/L. In contaminated Orientia cultures, daptomycin at 32 mg/L was effective in eradicating M. orale, whereas either gentamicin or amikacin (100 mg/L) was effective in eradicating M. hyorhinis. Unlike each drug alone, the combinations of daptomycin plus clindamycin and/or quinupristin/dalfopristin proved effective in eradicating M. hyorhinis. In summary, our study demonstrated the in vitro anti-mycoplasma activity of daptomycin and its application as a new mycoplasma decontamination method for Rickettsia and Orientia cultures.