Myeloid Sirtuin 2 expression does not impact long-term Mycobacterium tuberculosis control

Sirtuins (Sirts) regulate several cellular mechanisms through deacetylation of several transcription factors and enzymes. Recently, Sirt2 was shown to prevent the development of inflammatory processes and its expression favors acute Listeria monocytogenes infection. The impact of this molecule in the context of chronic infections remains unknown. We found that specific Sirt2 deletion in the myeloid lineage transiently increased Mycobacterium tuberculosis load in the lungs and liver of conditional mice. Sirt2 did not affect long-term infection since no significant differences were observed in the bacterial burden at days 60 and 120 post-infection. The initial increase in M. tuberculosis growth was not due to differences in inflammatory cell infiltrates in the lung, myeloid or CD4+ T cells. The transcription levels of IFN-?, IL-17, TNF, IL-6 and NOS2 were also not affected in the lungs by Sirt2-myeloid specific deletion. Overall, our results demonstrate that Sirt2 expression has a transitory effect in M. tuberculosis infection. Thus, modulation of Sirt2 activity in vivo is not expected to affect chronic infection with M. tuberculosis.

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.