BackgroundMany efforts have been made to understand basal mechanisms of mycobacterial infections. Macrophages are the first line of host immune defence to encounter and eradicate mycobacteria. Pathogenic species have evolved different mechanisms to evade host response, e.g. by influencing macrophage apoptotic pathways. However, the underlying molecular regulation is not fully understood. A new layer of eukaryotic regulation of gene expression is constituted by microRNAs. Therefore, we present a comprehensive study for identification of these key regulators and their targets in the context of host macrophage response to mycobacterial infections.Methodology/Principal FindingsWe performed microRNA as well as mRNA expression analysis of human monocyte derived macrophages infected with several Mycobacterium avium hominissuis strains by means of microarrays as well as quantitative reverse transcription PCR (qRT-PCR). The data revealed the ability of all strains to inhibit apoptosis by transcriptional regulation of BCL2 family members. Accordingly, at 48 h after infection macrophages infected with all M. avium strains showed significantly decreased caspase 3 and 7 activities compared to the controls. Expression of let-7e, miR-29a and miR-886-5p were increased in response to mycobacterial infection at 48 h. The integrated analysis of microRNA and mRNA expression as well as target prediction pointed out regulative networks identifying caspase 3 and 7 as potential targets of let-7e and miR-29a, respectively. Consecutive reporter assays verified the regulation of caspase 3 and 7 by these microRNAs.Conclusions/SignificanceWe show for the first time that mycobacterial infection of human macrophages causes a specific microRNA response. We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs. This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response. In addition, functional relevance was confirmed by uncovering the control of major caspases 3 and 7 by let-7e and miR-29a, respectively.

Significance and Impact of the Study: This study was conducted to investigate the ecological abundance of the most prominent clinical nontuberculous mycobacteria (NTM) in Germany, the Mycobacterium avium ssp. hominissuis (MAH). Examination of soil, water, dust and biofilm samples revealed that MAH in Germany was predominant in soil and dust. No MAH was identified in water and biofilms. Our finding contributes to the identification of the environmental niche of this opportunistic pathogen and proposes soil and dust as sources of MAH infection in Germany. AbstractThe nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria found in soil, water and dust. The spread of NTM infection depends on the exposure to reservoirs with high proportions of mycobacteria, the virulence of the NTM strains, the enhanced sensitivity to infections such as those of immune-compromised hosts and patient risk factors such as Cystic Fibrosis. Since several decades, NTM lung disease has been increasingly observed in slender postmenopausal women. The most important NTM in Germany is Mycobacterium avium ssp. hominissuis (MAH). The routes of MAH infection are in almost all cases unknown, but water is often suspected as source of infection. We wanted to examine this hypothesis by determining the frequency of MAH in environmental samples of water, biofilms, soil and dust originating from Germany. We found MAH in 33% of the dust samples and 20% of the soil samples. No MAH could be isolated from water and biofilm. Dust and soil clearly presented more abundance of MAH in comparison with water and biofilms. Therefore, more attention should be paid to soil and dust in Germany as an important source of Myco. avium infections. IntroductionNTM are natural inhabitants of soil and water. More than 140 species of environmental mycobacteria are known today and at least 40 of them are associated with lung disorders (Griffith 2010). Mycobacterium avium plays a prominent role among NTM and is a clinically important NTM for being causal agent of pulmonary disease in immunecompromised patients, lymphadenitis in small children and disseminated disease in patients with acquired immunodeficiency syndrome (AIDS) (Reuss et al. 2009;Hoefsloot et al. 2013;Thomson et al. 2013). Over the last decades, slender elderly postmenopausal women have been increasingly diagnosed with NTM lung infection (Chan and Iseman 2010;Epson and Winthrop 2012).A study that analysed the hospitalizations associated with pulmonary nontuberculous mycobacterial infections in Germany from 2005 to 2011 found an average annual age-adjusted rate of 0Á91 hospitalizations per 100 000 of population. The hospitalization rates increased during the study period, and the most pronounced average increase of 6Á4% per year was observed among females (Ringshausen et al. 2013 The source of MAH infection remains unknown for most cases. MAH are opportunistic environmental mycobacteria and their reservoirs include drinking water distribution systems, household plumbing, peat rich soils, boreal soil...

Background: Pathogenic mycobacteria such as M. tuberculosis, M. bovis or M. leprae are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1) has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a M. bovis BCG derivative expressing a MDP1-antisense gene.

Mycobacterium tuberculosis, the cause of tuberculosis in humans, is present approximately in one third of the world's population, mostly in a dormant state. The dosR regulon proteins are mainly responsible for its dormancy survival. To maintain latency, mycobacteria achieve a

BackgroundThe genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed.ResultsWe developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS).ConclusionsWe established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.