Strong evidence supports the idea that fatty acids rather than carbohydrates are the main energy source of Mycobacterium tuberculosis during infection and latency. Despite that important role, a complete scenario of the bacterium’s metabolism when lipids are the main energy source is still lacking. Here we report the development of an in vitro model to analyze adaptation of M. tuberculosis during assimilation of long-chain fatty acids as sole carbon sources. The global lipid transcriptome revealed a shift toward the glyoxylate cycle, the overexpression of main regulators whiB3, dosR, and Rv0081, and the increased expression of several genes related to reductive stress. Our evidence showed that lipid storage seems to be the selected mechanism used by M. tuberculosis to ameliorate the assumed damage of reductive stress and that concomitantly the bacilli acquired a slowed-growth and drug-tolerant phenotype, all characteristics previously associated with the dormant stage. Additionally, intergenic regions were also detected, including the unexpected upregulation of tRNAs that suggest a new role for these molecules in the acquisition of a drug-tolerant phenotype by dormant bacilli. Finally, a set of lipid signature genes for the adaptation process was also identified. This in vitro model represents a suitable condition to illustrate the participation of reductive stress in drugs’ activity against dormant bacilli, an aspect scarcely investigated to date. This approach provides a new perspective to the understanding of latent infection and suggests the participation of previously undetected molecules.

Mycobacterium smegmatis has two rRNA (rm) operons designated rrnA, and rrnB,. Appropriate restriction fragments of genomic DNA containing sequences immediately upstream from the 165 rRNA genes were cloned. We now report the nucleotide sequence of 552 bp upstream from the 5'-end of the Box A, antitermination element of the leader region of the rrnAf operon. The 5'-end of this segment of DNA was found to comprise 113 codons of an ORF encoding a protein which is significantly similar to UDP-N-acetylglucosamine 1-carboxyvinyl-transferase (EC 2.5.1.7), which is important to cell wall synthesis. A homologous ORF is located immediately upstream from the single rrn (rrnAJ operons of Mycobacterium tuberculosis and Mycobacterium leprae. Primer-extension analysis of the RNA fraction of M. smegmatis revealed four products which were related to transcription start points; the rrnb?, operon appears to have a single promoter whereas the rrnA, operon has three (PI, P2 and P3). Analysis of M. tuberculosis RNA revealed two products corresponding to transcripts directed by promoters homologous with P I and P3 of the nnA, of M. smegmatis. Thus, the promoter and upstream regions of the mnA, operon of M. smegmatis and the rrnAs operon of M. tuberculosis are homologous. The presence of P2 in M. smegmatis and its absence from M. tuberculosis is attributable to insertionddeletions of 97 bp.

Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial infections. Main virulence determinants of K. pneumoniae are pili, capsular polysaccharide, lipopolysaccharide, and siderophores. The histone-like nucleoid-structuring protein (H-NS) is a pleiotropic regulator found in several gram-negative pathogens. It has functions both as an architectural component of the nucleoid and as a global regulator of gene expression. We generated a Δhns mutant and evaluated the role of the H-NS nucleoid protein on the virulence features of K. pneumoniae. A Δhns mutant down-regulated the mrkA pilin gene and biofilm formation was affected. In contrast, capsule expression was derepressed in the absence of H-NS conferring a hypermucoviscous phenotype. Moreover, H-NS deficiency affected the K. pneumoniae adherence to epithelial cells such as A549 and HeLa cells. In infection experiments using RAW264.7 and THP-1 differentiated macrophages, the Δhns mutant was less phagocytized than the wild-type strain. This phenotype was likely due to the low adherence to these phagocytic cells. Taken together, our data indicate that H-NS nucleoid protein is a crucial regulator of both T3P and CPS of K. pneumoniae.

One rRNA operon of all mycobacteria studied so far is located downstream from a gene thought to code for the enzyme UDP-N-acetylglucosamine carboxyvinyl transferase (UNAcGCT), which is important to cell wall synthesis. This operon has been designated rrnA f for fast-growing mycobacteria and rrnA s for slow growers. We have investigated the upstream sequences and promoter activities of rrnA f operons of typical fast growers which also possess a second rrn (rrnB f ) operon and of the rrnA operons of the fast growers Mycobacterium abscessus and Mycobacterium chelonae, which each have a single rrn operon per genome. These fast growers have a common strategy for increasing the efficiency of transcription of their rrnA operons, thereby increasing the cells' potential for ribosome synthesis. This strategy involves the use of multiple (three to five) promoters which may have arisen through successive duplication events. Thus we have identified a hypervariable multiple promoter region (HMPR) located between the UNAcGCT gene and the 16S rRNA coding region. Two promoters, P1 and PCL1, appear to play pivotal roles in mycobacterial rRNA synthesis; they are present in all of the species examined and are the only promoters used for rRNA synthesis by the pathogenic slow growers. P1 is located within the coding region of the UNAcGCT gene, and PCL1 has a characteristic sequence that is related to but distinct from that of the additional promoters. In fast-growing species, P1 and PCL1 produce less than 10% of rRNA transcripts, so the additional promoters found in the HMPR are important in increasing the potential for rRNA synthesis during rapid growth. In contrast, rrnB operons appear to be regulated by a single promoter; because less divergence has taken place, rrnB appears to be younger than rrnA.

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Tuberculosis (TB) is currently the number one killer among infectious diseases worldwide. Lipids are abundant molecules during the infectious cycle of Mycobacterium tuberculosis (Mtb) and studies better mimicking its actual metabolic state during pathogenesis are needed. Though most studies have focused on the mycobacterial lipid metabolism under standard culture conditions, little is known about the transcriptome of Mtb in a lipid environment. Here we determined the transcriptome of Mtb H37Rv in a lipid-rich environment (cholesterol and fatty acid) under aerobic and hypoxic conditions, using RNAseq. Lipids significantly induced the expression of 368 genes. A main core lipid response was observed involving efflux systems, iron caption and sulfur reduction. In co-expression with ncRNAs and other genes discussed below, may act coordinately to prepare the machinery conferring drug tolerance and increasing a persistent population. Our findings could be useful to tag relevant pathways for the development of new drugs, vaccines and new strategies to control TB.

Tuberculosis (TB) is a chronic infectious disease, considered as the second leading cause of death worldwide, caused by Mycobacterium tuberculosis. The limited efficacy of the bacillus Calmette-Guérin (BCG) vaccine against pulmonary TB and the emergence of multidrug-resistant TB warrants the need for more efficacious vaccines. Reverse vaccinology uses the entire proteome of a pathogen to select the best vaccine antigens by in silico approaches. M. tuberculosis H37Rv proteome was analyzed with NERVE (New Enhanced Reverse Vaccinology Environment) prediction software to identify potential vaccine targets; these 331 proteins were further analyzed with VaxiJen for the determination of their antigenicity value. Only candidates with values ≥0.5 of antigenicity and 50% of adhesin probability and without homology with human proteins or transmembrane regions were selected, resulting in 73 antigens. These proteins were grouped by families in seven groups and analyzed by amino acid sequence alignments, selecting 16 representative proteins. For each candidate, a search of the literature and protein analysis with different bioinformatics tools, as well as a simulation of the immune response, was conducted. Finally, we selected six novel vaccine candidates, EsxL, PE26, PPE65, PE_PGRS49, PBP1, and Erp, from M. tuberculosis that can be used to improve or design new TB vaccines.

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