Gene knock-out in Mycobacterium abscessus using Streptococcus thermophilus CRISPR/Cas.

The CRISPRi system using dCas9Sth1 from Streptococcus thermophilus developed for Mycobacterium tuberculosis and M. smegmatis was modified to allow gene knock-out in M. abscessus. Efficacy of the knock-out system was evaluated by applying deletions and insertions to the mps1 gene. A comparative genomic analysis of mutants and wild type validated the target specificity.

Genetic diversification of persistent Mycobacterium abscessus within cystic fibrosis patients.

Mycobacterium (M.) abscessus infections in Cystic Fibrosis (CF) patients cause a deterioration of lung function. Treatment of these multidrug-resistant pathogens is associated with severe side-effects, while frequently unsuccessful. Insight on M. abscessus genomic evolvement during chronic lung infection would be beneficial for improving treatment strategies. A longitudinal study enrolling 42 CF patients was performed at a CF center in Berlin, Germany, to elaborate phylogeny and genomic diversification of in-patient M. abscessus. Eleven of the 42 CF patients were infected with M. abscessus. Five of these 11 patients were infected with global human-transmissible M. abscessus cluster strains. Phylogenetic analysis of 88 genomes from isolates of the 11 patients excluded occurrence of M. abscessus transmission among members of the study group. Genome sequencing and variant analysis of 30 isolates from 11 serial respiratory samples collected over 4.5 years from a chronically infected patient demonstrated accumulation of gene mutations. In total, 53 genes exhibiting non-synonymous variations were identified. Enrichment analysis emphasized genes involved in synthesis of glycopeptidolipids, genes from the embABC (arabinosyltransferase) operon, betA (glucose-methanol-choline oxidoreductase) and choD (cholesterol oxidase). Genetic diversity evolved in a variety of virulence- and resistance-associated genes. The strategy of M. abscessus populations in chronic lung infection is not clonal expansion of dominant variants, but to sustain simultaneously a wide range of genetic variants facilitating adaptation of the population to changing living conditions in the lung. Genomic diversification during chronic infection requires increased attention when new control strategies against M. abscessus infections are explored.

MALDI Spectra Database for Rapid Discrimination and Subtyping of Mycobacterium kansasii.

Mycobacterium kansasii is an emerging non-tuberculous mycobacterial (NTM) pathogen capable of causing severe lung disease. Of the seven currently recognized M. kansasii genotypes (I-VII), genotypes I and II are most prevalent and have been associated with human disease, whereas the other five (III-VII) genotypes are predominantly of environmental origin and are believed to be non-pathogenic. Subtyping of M. kansasii serves as a valuable tool to guide clinicians in pursuing diagnosis and to initiate the proper timely treatment. Most of the previous rapid diagnostic tests for mycobacteria employing the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology focused on species-level identification. The purpose of this study was to establish MALDI-TOF MS reference spectra database for discrimination of M. kansasii at the genotype level. A panel of 32 strains, representatives of M. kansasii genotypes I-VI were selected, whole cell proteins extracted and measured with MALDI-TOF MS. A unique main spectra (MSP) library was created using MALDI Biotyper Compass Explorer software. The spectra reproducibility was assessed by computing composite correlation index and MSPs cross-matching. One hundred clinical M. kansasii isolates used for testing of the database resulted in 90% identification at genus-level, 7% identification at species-level and 2% identification was below the threshold of log score value 1.7, of which all were correct at genotype level. One strain could not be identified. On the other hand, 37% of strains were identified at species level, 40% at genus level and 23% was not identified with the manufacturer's database. The MALDI-TOF MS was proven a rapid and robust tool to detect and differentiate between M. kansasii genotypes. It is concluded that MALDI-TOF MS has a potential to be incorporated into the routine diagnostic workflow of M. kansasii and possibly other NTM species.

Environmental Mycobacterium avium subsp. hominissuis have a higher probability to act as a recipient in conjugation than clinical strains.

Mycobacterium avium subsp. hominissuis (MAH) is a widespread opportunistic pathogen that can be isolated from environment (dust, soil and water) and patients with lung or lymphnode infection. In our previous research we revealed the pronounced genetic diversity in MAH by identifying eight different types of a newly described genomic island. In order to identify mechanisms of such horizontal gene transfer we now analyzed the ability of 47 MAH isolates to inherit the conjugative plasmid pRAW from M. marinum. A higher percentage of environmental isolates (22.7%) compared to clinical isolates (8%) had the capacity to function as recipient in conjugal plasmid transfer. Genetic analysis showed additionally that environmental isolates contained more genes homologous to genes present on conjugative mycobacterial plasmids than clinical isolates. Comparative analysis of the genomes of the isolates pointed to a possible association between the ability to act as recipient in conjugation and the structure of a genomic region containing the radC gene and a type I restriction/modification system. Finally we found that uptake of pRAW decreased the resistance against various antibiotics.

Dormancy Associated Translation Inhibitor (DATIN/Rv0079) of Mycobacterium tuberculosis interacts with TLR2 and induces proinflammatory cytokine expression.

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 proteins encoded by the dormancy survival regulon (DosR regulon) are mainly responsible for survival of the bacilli in a latent form. To maintain latency, mycobacteria orchestrate a balanced interplay of different cytokines secreted by immune cells during the granulomatous stage. The function of most of the DosR regulon proteins of M. tuberculosis is unknown. In this study, we have shown that one of the DosR regulon proteins, DATIN, encoded by the gene Rv0079, can stimulate macrophages and peripheral blood mononuclear cells (PBMC) to secrete important cytokines that may be significant in granuloma formation and its maintenance. The expression level of DATIN in Mycobacterium bovis BCG was found to be upregulated in pH stress and microaerobic conditions. Computational modeling, docking and simulation study suggested that DATIN might interact with TLR2. This was further confirmed through the interaction of recombinant DATIN with TLR2 expressed by HEK293 cells. When in vitro differentiated THP-1 cells were treated with recombinant DATIN, increased secretion of TNF-α, IL-1ß and IL-8 was observed in a dose dependent manner. When differentiated THP-1 cells were infected with a modified BCG strain that overexpressed DATIN, augmented secretions of TNF-α, IL-1ß and IL-8 were observed as compared to a reference BCG strain containing empty vector. Similarly, human PBMCs when infected with M. bovis BCG that overexpressed DATIN, upregulated secretion of proinflammatory cytokines IFN-γ, TNF-α, IL-1ß and IL-8. The cytokine profiles dissected herein point to a possible role of DATIN in maintenance of latency with the help of the proinflammatory responses.

Illegitimate recombination: an efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis.

BACKGROUND: The 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. RESULTS: We 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). CONCLUSIONS: We 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.

The role of the mycobacterial DNA-binding protein 1 (MDP1) from Mycobacterium bovis BCG in host cell interaction.

BACKGROUND: Mycobacterium tuberculosis differs from most pathogens in its ability to multiply inside monocytes and to persist during long periods of time within granuloma in a status of latency. A class of proteins called mycobacterial histone-like proteins has been associated with regulation of replication and latency, but their precise role in the infection process has yet to be uncovered. Our study aimed at defining the impact of the histone-like protein MDP1 from M. bovis BCG (mycobacterial DNA-binding protein 1, corresponding to Rv2986c from M. tuberculosis) on early steps of infection. RESULTS: Previously, a BCG (Bacillus Calmette Guérin) strain had been generated by antisense-technique exhibiting reduced MDP1 expression. This strain was now used to analyse the impact of reduced amount of MDP1 on the interaction with human blood monocytes, macrophage lines and PBMC (peripheral blood mononuclear cells). MDP1 was revealed to be required for growth at acidic pH and for intracellular replication in human blood monocytes. Down-regulation of MDP1 resulted in reduced secretion of the cytokine IL-1ß by infected human PBMC. In addition, a reduction of MDP1 expression had a major impact on the formation of fused multi-nucleated macrophages. In monocyte preparations from human blood as well as in human and mouse macrophage cell lines, both the percentage of multi-nucleated cells and the number of nuclei per cell were much enhanced when the monocytes were infected with BCG expressing less MDP1. CONCLUSION: MDP1 from M. bovis BCG affects the growth at acidic pH and the intracellular replication in human monocytes. It furthermore affects cytokine secretion by host cells, and the formation of fused multi-nucleated macrophages. Our results suggest an important role of MDP1 in persistent infection.

Integrated microRNA-mRNA-analysis of human monocyte derived macrophages upon Mycobacterium avium subsp. hominissuis infection.

BACKGROUND: Many 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 FINDINGS: We 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/SIGNIFICANCE: We 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.

The mycobacterial DNA-binding protein 1 (MDP1) from Mycobacterium bovis BCG influences various growth characteristics.

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. RESULTS: The expression rate of the MDP1 protein in the recombinant M. bovis BCG containing the MDP1-antisense plasmid was reduced by about 50% compared to the reference strain M. bovis BCG containing the empty vector. In comparison to this reference strain, the recombinant M. bovis BCG grew faster in broth culture and reached higher cell masses in stationary phase. Likewise its intracellular growth in mouse and human macrophages was ameliorated. Bacterial clumping in broth culture was reduced by the antisense plasmid. The antisense plasmid increased the susceptibility of the bacteria towards Ampicillin. 2-D protein gels of bacteria maintained under oxygen-poor conditions demonstrated a reduction in the number and the intensity of many protein spots in the antisense strain compared to the reference strain. CONCLUSION: The MDP1 protein has a major impact on various growth characteristics of M. bovis BCG. It plays an important role in virulence-related traits such as aggregate formation and intracellular multiplication. Its impact on the protein expression in a low-oxygen atmosphere indicates a role in the adaptation to the hypoxic conditions present in the granuloma.