The unique PE/PPE multigene family of proteins occupies almost 10% of the coding sequence of Mycobacterium tuberculosis (M.tb), the causative agent of human tuberculosis. Although some members of this family have been shown to be involved in pathways essential to M.tb pathogenesis, their precise physiological functions remain largely undefined. Here, we investigate the roles of the conserved members of the ‘PE only’ subfamily Rv0285 (PE5) and Rv1386 (PE15) in mediating host-pathogen interactions. Recombinant Mycobacterium smegmatis strains expressing PE5 and PE15 showed enhanced survival vs controls in J774.1 and THP-1 macrophages - this increase in viable counts was correlated with a reduction in transcript levels of inducible nitric oxide synthase. An up-regulation of anti- and down-regulation of pro-inflammatory cytokine levels was also observed in infected macrophages implying an immuno-modulatory function for these proteins. Induction of IL-10 production upon infection of THP-1 macrophages was associated with increased phosphorylation of the MAP Kinases p38 and ERK1/2, which was abolished in the presence of the pharmacological inhibitors SB203580 and PD98059. The PE5-PPE4 and PE15-PPE20 gene pairs were observed to be co-operonic in M.tb, hinting at an additional level of complexity in the functioning of these proteins. We conclude that M.tb exploits the PE proteins to evade the host immune response by altering the Th1 and Th2 type balance thereby favouring in vivo bacillary survival.

Members of the Mycobacterium avium complex (MAC) are characterized as nontuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact the infection outcome. The current study aimed to characterize the genomic changes within M. avium isolates collected from single patients over time and test the host immune responses to these clinical isolates. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on 40 MAC isolates isolated from 15 patients at the Department of Medical Microbiology at St. Olavs Hospital in Trondheim, Norway. Isolates from patients (patients 4, 9, and 13) for whom more than two isolates were available were selected for further analysis. These isolates exhibited extensive sequence variation in the form of single-nucleotide polymorphisms (SNPs), suggesting that M. avium accumulates mutations at higher rates during persistent infections than other mycobacteria. Infection of murine macrophages and mice with sequential isolates from patients showed a tendency toward increased persistence and the downregulation of inflammatory cytokines by host-adapted M. avium strains. The study revealed the rapid genetic evolution of M. avium in chronically infected patients, accompanied by changes in the virulence properties of the sequential mycobacterial isolates.

Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the Pseudomonas putida-derived positively regulated XylS/Pm expression system to control inducible gene expression in Mycobacterium smegmatis and Mycobacterium tuberculosis, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/Pm-regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer m-toluate, we achieve a robust, time- and dose-dependent reversible induction of Pm-mediated expression in mycobacteria, with low background expression levels. XylS/Pm is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance.

Mycobacterium avium (Mav) complex is increasingly reported to cause non-tuberculous infections in individuals with a compromised immune system. Treatment is complicated and no vaccines are available. Previous studies have shown some potential of using genetically modified Mycobacterium smegmatis (Msm) as a vaccine vector to tuberculosis since it is non-pathogenic and thus would be tolerated by immunocompromised individuals. In this study, we used a mutant strain of Msm disrupted in EspG 3 , a component of the ESX-3 secretion system. Infection of macrophages and dendritic cells with Msm espG 3 showed increased antigen presentation compared to cells infected with wild-type Msm. Vaccination of mice with Msm espG 3 , expressing the Mav antigen MPT64, provided equal protection against Mav infection as the tuberculosis vaccine, Mycobacterium bovis BCG. However, upon challenge with Mav, we observed a high frequency of IL-17-producing CD4+ (Th17 cells) and CD8+ (Tc17 cells) T cells in mice vaccinated with Msm espG 3 ::mpt64 that was not seen in BCG-vaccinated mice. Adoptive transfer of cells from Msm espG 3-vaccinated mice showed that cells from the T cell compartment contributed to protection from Mav infection. Further experiments revealed Tc17-enriched T cells did not provide prophylactic protection against subsequent Mav infection, but a therapeutic effect was observed when Tc17-enriched cells were transferred to mice already infected with Mav. These initial findings are important, as they suggest a previously unknown role of Tc17 cells in mycobacterial infections. Taken together, Msm espG 3 shows promise as a vaccine vector against Mav and possibly other (myco)bacterial infections.

2Mycobacterium avium (Mav) complex (MAC) are characterized as non-tuberculosis 2 3 3 2 form of single nucleotide polymorphisms (SNPs), suggesting that Mav accumulates mutations at 3 3 high rates during persistent infections. Infection of murine macrophages and mice with sequential 3 4 isolates from patients showed a tendency towards increased persistence and down-regulation of 3 5 inflammatory cytokines by host-adapted Mav strains. The study revealed rapid genetic evolution 3 6 of Mav in chronically infected patients accompanied with change in virulence properties of the 3 7 sequential mycobacterial isolates. 3 8 IMPORTANCE 3 9 MAC are a group of opportunistic pathogens, consisting of Mav and M. intracellulare species. 4 0 Mav is found ubiquitously in the environment. In Mav infected individuals, Mav has been known 4 1 to persist for long periods of time, and anti-mycobacterial drugs are unable to effectively clear the 4 2infection. The continued presence of the bacteria, could be attributed to either a single persistent 4 3 strain or reinfection with the same or different strain. We examined sequential isolates collected 4 4 3 over time from Mav infected individuals and observed that most patients carried the same strain 4 5 overtime and were not re infected. We observed high rates of mutation within the serial isolates, 4 6