Vitamin D signaling through its nuclear vitamin D receptor has emerged as a key regulator of innate immunity in humans. Here we show that hormonal vitamin D, 1,25-dihydroxyvitamin D(3), robustly stimulates expression of pattern recognition receptor NOD2/CARD15/IBD1 gene and protein in primary human monocytic and epithelial cells. The vitamin D receptor signals through distal enhancers in the NOD2 gene, whose function was validated by chromatin immunoprecipitation and chromatin conformation capture assays. A key downstream signaling consequence of NOD2 activation by agonist muramyl dipeptide is stimulation of NF-kappaB transcription factor function, which induces expression of the gene encoding antimicrobial peptide defensin beta2 (DEFB2/HBD2). Pretreatment with 1,25-dihydroxyvitamin D(3) synergistically induced NF-kappaB function and expression of genes encoding DEFB2/HBD2 and antimicrobial peptide cathelicidin in the presence of muramyl dipeptide. Importantly, this synergistic response was also seen in macrophages from a donor wild type for NOD2 but was absent in macrophages from patients with Crohn disease homozygous for non-functional NOD2 variants. These studies provide strong molecular links between vitamin D deficiency and the genetics of Crohn disease, a chronic incurable inflammatory bowel condition, as Crohn's pathogenesis is associated with attenuated NOD2 or DEFB2/HBD2 function.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54.5%) strains, no identification was given and for 69 (38.7%) strains, an incorrect identification was obtained. Our study demonstrates that both systems gave a high level (above 85%) of correct identification for a wide range of microorganisms. However, VITEK MS gave more misidentification when the microorganism analysed was not present in the database, compared to Bruker Biotyper. This should be taken into account when this technology is used alone for microorganism identification in a public health laboratory, where isolates received are often difficult to identify and/or unusual microorganisms.

Significance: Through an in-depth analysis of whole-genome sequencing data from Nunavik, Québec, we inferred the evolution of a single dominant strain ofMycobacterium tuberculosis. Our analyses suggest thatM. tuberculosiswas first introduced into this region in the early 20th century. Since this time,M. tuberculosishas spread extensively, predominantly within but also between villages. Despite a genomic profile that lacks features of a hypervirulent strain, this strain has thrived in this region and continues to cause outbreaks. This suggests that successful clones ofM. tuberculosisneed not be inherently exceptional; host or social factors conducive to transmission may contribute to the ongoing tuberculosis epidemic in this and other high-incidence settings.

Background. Between November 2011 and November 2012, a Canadian village of 933 persons had 50 culture-positive cases of tuberculosis, with 49 sharing the same genotype. Methods. We performed Illumina-based whole-genome sequencing on Mycobacterium tuberculosis isolates from this village, during and before the outbreak. Phylogenetic trees were generated using the maximum likelihood method. Results. Three distinct genotypes were identified. Strain I (n = 7) was isolated in 1991–1996. Strain II (n = 8) was isolated in 1996–2004. Strain III (n = 62) first appeared in 2007 and did not arise from strain I or II. Within strain III, there were 3 related but distinct clusters: IIIA, IIIB, and IIIC. Between 2007 and 2010, cluster IIIA predominated (11 of 22 vs 2 of 40; P < .001), whereas in 2011–2012 clusters IIIB (n = 18) and IIIC (n = 20) predominated over cluster IIIA (n = 11). Combined evolutionary and epidemiologic analysis of strain III cases revealed that the outbreak in 2011–2012 was the result of ≥6 temporally staggered events, spanning from 1 reactivation case to a point-source outbreak of 20 cases. Conclusions. After the disappearance of 2 strains of M. tuberculosis in this village, its reemergence in 2007 was followed by an epidemiologic amplification, affecting >5% of the population.

Phagosomes containing M. tuberculosis and M. bovis BCG interact normally with early endosomes but fail to fuse with late endosomes and lysosomes. Whereas many early events of mycobacterial phagosomes have been elucidated, the exact mechanism of the inhibition of fusion with lysosomes is still unclear. Several Rab GTPase proteins were shown to be involved in membrane fusion and vesicular transport. In particular, Rab7 associates with the phagosomal membrane and regulates the fusion between late endosomes and lysosomes. This function of Rab7 was shown to be mediated in epithelial cell models by the Rab7 effector RILP (Rab7-interacting lysosomal protein). However, the relevance of Rab7-RILP interaction to phagosome biogenesis in macrophage infected with mycobacteria is still unknown. In this study, cotransfection of RAW 264.7 cells with Rab7 and RILP revealed that Rab7-RILP interaction occurs in macrophages ingesting latex beads. Thereafter, this cell system model was used to demonstrate that infection with live but not killed M. bovis BCG inhibited RILP recruitment despite Rab7 acquisition by the phagosome. Further investigation using immobilized RILP to pull down active Rab7 (GTP-bound form) from macrophage lysates demonstrated that inactive Rab7 (GDP-bound form) predominates in cells infected with live BCG. In addition, cell-free system experiments demonstrated that BCG culture supernatant contains a factor that catalyzes the GTP/GDP switch on recombinant Rab7 molecules. Such a factor was shown to diffuse beyond BCG phagosomes and target other Rab7-positive compartments. These findings suggest that live mycobacteria express within the macrophage a Rab7 deactivating factor leading to abortion of RILP-mediated fusion with lysosomes.

A successful Th cell response to bacterial infections is induced by mature MHC class II molecules presenting specific Ag peptides on the surface of macrophages. In recent studies, we demonstrated that infection with the conventional vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) specifically blocks the surface export of mature class II molecules in human macrophages by a mechanism dependent on inhibition of cathepsin S (Cat S) expression. The present study examined class II expression in macrophages infected with a rBCG strain engineered to express and secrete biologically active human Cat S (rBCG-hcs). Cat S activity was completely restored in cells ingesting rBCG-hcs, which secreted substantial levels of Cat S intracellularly. Thus, infection with rBCG-hcs, but not parental BCG, restored surface expression of mature MHC class II molecules in response to IFN-γ, presumably as result of MHC class II invariant chain degradation dependent on active Cat S secreted by the bacterium. These events correlated with increased class II-directed presentation of mycobacterial Ag85B to a specific CD4⁺ T cell hybridoma by rBCG-hcs-infected macrophages. Consistent with these findings, rBCG-hcs was found to accelerate the fusion of its phagosome with lysosomes, a process that optimizes Ag processing in infected macrophages. These data demonstrated that intracellular restoration of Cat S activity improves the capacity of BCG-infected macrophages to stimulate CD4⁺ Th cells. Given that Th cells play a major role in protection against tuberculosis, rBCG-hcs would be a valuable tuberculosis vaccine candidate.