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.

Impact of Mycobacteroides abscessus colony morphology on biofilm formation and antimicrobial resistance.

Mycobacteroides abscessus is one of the most resistant bacteria so far known and causes severe and hard to treat lung infections in predisposed patients such as those with Cystic Fibrosis (CF). Further, it causes nosocomial infections by forming biofilms on medical devices or water reservoirs. An eye-catching feature of M. abscessus is the growth in two colony morphotypes. Depending on the presence or absence of glycopeptidolipids on the cell surface, it forms smooth or rough colonies. In this study, a porous glass bead biofilm model was used to compare biofilm formation, biofilm organization and biofilm matrix composition in addition to the antimicrobial susceptibility of M. abscessus biofilms versus suspensions of isogenic (smooth and rough) patient isolates. Both morphotypes reached the same cell densities in biofilms. The biofilm architecture, however, was dramatically different with evenly distributed oligo-layered biofilms in smooth isolates, compared to tightly packed, voluminous biofilm clusters in rough morphotypes. Biofilms of both morphotypes contained more total biomass of the matrix components protein, lipid plus DNA than was seen in corresponding suspensions. The biofilm mode of growth of M. abscessus substantially increased resistance to the antibiotics amikacin and tigecycline. Tolerance to the disinfectant peracetic acid of both morphotypes was increased when grown as biofilm, while tolerance to glutaraldehyde was significantly increased in biofilm of smooth isolates only. Overall, smooth colony morphotypes had more pronounced antimicrobial resistance benefit when growing as biofilm than M. abscessus showing rough colony morphotypes.

Impact of climate change on waterborne infections and intoxications.

Progressive climate change holds the potential for increasing human health risks from waterborne infections and intoxications, e. g. through an increase in pathogen concentrations in water bodies, through the establishment of new pathogens or through possible changes in pathogen properties. This paper presents some examples of potential impacts of climate change in Germany. Non-cholera Vibrio occur naturally in seawater, but can proliferate significantly in shallow water at elevated temperatures. In the case of Legionella, climate change could lead to temporary or longer-term increased incidences of legionellosis due to the combination of warm and wet weather. Higher temperatures in piped cold water or lower temperatures in piped hot water may also create conditions conducive to higher Legionella concentrations. In nutrient-rich water bodies, increased concentrations of toxigenic cyanobacteria may occur as temperatures rise. Heavy rainfall following storms or prolonged periods of heat and drought can lead to increased levels of human pathogenic viruses being washed into water bodies. Rising temperatures also pose a potential threat to human health through pathogens causing mycoses and facultatively pathogenic micro-organisms: increased infection rates with non-tuberculous mycobacteria or fungi have been documented after extreme weather events.

Molecular Epidemiology of Mycobacterium abscessus Isolates Recovered from German Cystic Fibrosis Patients.

Infections due to Mycobacterium abscessus are a major cause of mortality and morbidity in cystic fibrosis (CF) patients. Furthermore, M. abscessus has been suspected to be involved in person-to-person transmissions. In 2016, dominant global clonal complexes (DCCs) that occur worldwide among CF patients have been described. To elucidate the epidemiological situation of M. abscessus among CF patients in Germany and to put these data into a global context, we performed whole-genome sequencing of a set of 154 M. abscessus isolates from 123 German patients treated in 14 CF centers. We used MTBseq pipeline to identify clusters of closely related isolates and correlate those with global findings. Genotypic drug susceptibility for macrolides and aminoglycosides was assessed by characterization of the erm(41), rrl, and rrs genes. By this approach, we could identify representatives of all major DCCs (Absc 1, Absc 2, and Mass 1) in our cohort. Intrapersonal isolates showed higher genetic relatedness than interpersonal isolates (median 3 SNPs versus 16 SNPs; P < 0.001). We further identified four clusters with German patients from same centers clustering with less than 25 SNPs distance (range 3 to 18 SNPs) but did not find any hint for in-hospital person-to-person transmission. This is the largest study investigating phylogenetic relations of M. abscessus isolates in Germany. We identified representatives of all reported DCCs but evidence for nosocomial transmission remained inconclusive. Thus, the occurrence of genetically closely related isolates of M. abscessus has to be interpreted with care, as a direct interhuman transmission cannot be directly deduced. IMPORTANCE Mycobacterium abscessus is a major respiratory pathogen in cystic fibrosis (CF) patients. Recently it has been shown that dominant global clonal complexes (DCCs) have spread worldwide among CF patients. This study investigated the epidemiological situation of M. abscessus among CF patients in Germany by performing whole-genome sequencing (WGS) of a set of 154 M. abscessus from 123 German patients treated in 14 CF centers. This is the largest study investigating the phylogenetic relationship of M. abscessus CF isolates in Germany.

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.

Critical Role of Zur and SmtB in Zinc Homeostasis of Mycobacterium smegmatis.

Zinc homeostasis is crucial for bacterial cells, since imbalances affect viability. However, in mycobacteria, knowledge of zinc metabolism is incomplete. Mycobacterium smegmatis (MSMEG) is an environmental, nonpathogenic Mycobacterium that is widely used as a model organism to study mycobacterial metabolism and pathogenicity. How MSMEG maintains zinc homeostasis is largely unknown. SmtB and Zur are important regulators of bacterial zinc metabolism. In mycobacteria, these regulators are encoded by an operon, whereas in other bacterial species, SmtB and Zur are encoded on separate loci. Here, we show that the smtB-zur operon is consistently present within the genus Mycobacterium but otherwise found only in Nocardia, Saccharothrix, and Corynebacterium diphtheriae By RNA deep sequencing, we determined the Zur and SmtB regulons of MSMEG and compared them with transcriptional responses after zinc starvation or excess. We found an exceptional genomic clustering of genes whose expression was strongly induced by zur deletion and zinc starvation. These genes encoded zinc importers such as ZnuABC and three additional putative zinc transporters, including the porin MspD, as well as alternative ribosomal proteins. In contrast, only a few genes were affected by deletion of smtB and zinc excess. The zinc exporter ZitA was most prominently regulated by SmtB. Moreover, transcriptional analyses in combination with promoter and chromatin immunoprecipitation assays revealed a special regulation of the smtB-zur operon itself: an apparently zinc-independent, constitutive expression of smtB-zur resulted from sensitive coregulation by both SmtB and Zur. Overall, our data revealed yet unknown peculiarities of mycobacterial zinc homeostasis.IMPORTANCE Zinc is crucial for many biological processes, as it is an essential cofactor of enzymes and a structural component of regulatory and DNA binding proteins. Hence, all living cells require zinc to maintain constant intracellular levels. However, in excess, zinc is toxic. Therefore, cellular zinc homeostasis needs to be tightly controlled. In bacteria, this is achieved by transcriptional regulators whose activity is mediated via zinc-dependent conformational changes promoting or preventing their binding to DNA. SmtB and Zur are important antagonistically acting bacterial regulators in mycobacteria. They sense changes in zinc concentrations in the femtomolar range and regulate transcription of genes for zinc acquisition, storage, and export. Here, we analyzed the role of SmtB and Zur in zinc homeostasis in Mycobacterium smegmatis Our results revealed novel insights into the transcriptional processes of zinc homeostasis in mycobacteria and their regulation.

Mutation on lysX from Mycobacterium avium hominissuis impacts the host-pathogen interaction and virulence phenotype.

The lysX gene from Mycobacterium avium hominissuis (MAH) is not only involved in cationic antimicrobial resistance but also regulates metabolic activity. An MAH lysX deficient mutant was shown to exhibit a metabolic shift at the extracellular state preadapting the bacteria to the conditions inside host-cells. It further showed stronger growth in human monocytes. In the present study, the LysX activity on host-pathogen interactions were analyzed. The lysX mutant from MAH proved to be more sensitive toward host-mediated stresses such as reactive oxygen species. Further, the lysX mutant exhibited increased inflammatory response in PBMC and multinucleated giant cell (MGC) formation in human macrophages during infection studies. Coincidentally, the lysX mutant strain revealed to be more reproductive in the Galleria mellonella infection model. Together, these data demonstrate that LysX plays a role in regulating the bacillary load in host organisms and the lack of lysX gene facilitates MAH adaptation to intracellular host-habitat, thereby suggesting an essential role of LysX in the modulation of host-pathogen interaction.

Type I interferon induced by TLR2-TLR4-MyD88-TRIF-IRF3 controls Mycobacterium abscessus subsp. abscessus persistence in murine macrophages via nitric oxide.

Mycobacterium abscessus (MAB) is an emerging, rapidly growing non-tuberculous Mycobacterium causing therapy-resistant pulmonary disease especially in patients with cystic fibrosis (CF). Smooth and rough colony type MAB can be isolated from infected patients whereby rough colony type MAB are more often associated with severe disease. Disease severity is also associated with an alternated type I interferon (IFN-I) response of the MAB-infected patients. However the relevance of this response for the outcome of MAB infection is still unknown. In this study, we analyzed the IFNß expression of murine macrophages infected with a MAB rough colony strain (MAB-R) isolated from a patient with progressive CF and compared it to macrophages infected with the MAB smooth colony type reference strain (MAB-S). We found that MAB-R infected macrophages expressed significantly more IFNß mRNA and protein than MAB-S infected macrophages. Higher IFNß induction by MAB-R was associated with higher TNF expression and intracellular killing while low IFNß induction was associated with lower TNF expression and persistence of MAB-S. IFNß induction was independent of the intracellular cGAS-STING recognition pathway. MAB appeared to be recognized extracellularly and induced IFNß expression via TLR2-TLR4-MyD88-TRIF-IRF3 dependent pathways. By using macrophages lacking the IFN-I receptor we demonstrate that MAB induced IFN-I response essentially contributed to restricting MAB-R and MAB-S infections by activating macrophage Nos2 expression and nitric oxide production. Thus IFN-I seem to influence the intrinsic ability of macrophages to control MAB infections. As MAB persists over long time periods in susceptible patients, our findings suggest that virulence of MAB strains is promoted by an insufficient IFN-I response of the host.

Analysis of long non-coding RNA and mRNA expression in bovine macrophages brings up novel aspects of Mycobacterium avium subspecies paratuberculosis infections.

Paratuberculosis is a major disease in cattle that severely affects animal welfare and causes huge economic losses worldwide. Development of alternative diagnostic methods is of urgent need to control the disease. Recent studies suggest that long non-coding RNAs (lncRNAs) play a crucial role in regulating immune function and may confer valuable information about the disease. However, their role has not yet been investigated in cattle with respect to infection towards Paratuberculosis. Therefore, we investigated the alteration in genomic expression profiles of mRNA and lncRNA in bovine macrophages in response to Paratuberculosis infection using RNA-Seq. We identified 397 potentially novel lncRNA candidates in macrophages of which 38 were differentially regulated by the infection. A total of 820 coding genes were also significantly altered by the infection. Co-expression analysis of lncRNAs and their neighbouring coding genes suggest regulatory functions of lncRNAs in pathways related to immune response. For example, this included protein coding genes such as TNIP3, TNFAIP3 and NF-κB2 that play a role in NF-κB2 signalling, a pathway associated with immune response. This study advances our understanding of lncRNA roles during Paratuberculosis infection.

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.

Proteome Analysis of a M. avium Mutant Exposes a Novel Role of the Bifunctional Protein LysX in the Regulation of Metabolic Activity.

Lysyl-phosphatidylglycerol is one of the components of the mycobacterial membrane that contributes to the resistance to cationic antimicrobial peptides, a host-induced frontline defense against invading pathogens. Its production is catalyzed by LysX, a bifunctional protein with lysyl transferase and lysyl transfer RNA synthetase activity. Comparative proteome analysis of a lysX mutant of Mycobacterium avium strain 104 and the wild type indicated that the lysX mutant strain undergoes a transition in phenotype by switching the carbon metabolism to ß-oxidation of fatty acids, along with accumulation of lipid inclusions. Surprisingly, proteins associated with intracellular survival were upregulated in the lysX mutant, even during extracellular growth, preparing bacteria for the conditions occurring inside host cells. In line with this, the lysX mutant exhibited enhanced intracellular growth in human-blood-derived monocytes. Thus, our study exposes the significance of lysX in the metabolism and virulence of the environmental pathogen M. avium hominissuis.

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.

Metabolic phenotype of clinical and environmental Mycobacterium avium subsp. hominissuis isolates.

BACKGROUND: Mycobacterium avium subsp. hominissuis (MAH) is an emerging opportunistic human pathogen. It can cause pulmonary infections, lymphadenitis and disseminated infections in immuno-compromised patients. In addition, MAH is widespread in the environment, since it has been isolated from water, soil or dust. In recent years, knowledge on MAH at the molecular level has increased substantially. In contrast, knowledge of the MAH metabolic phenotypes remains limited. METHODS: In this study, for the first time we analyzed the metabolic substrate utilization of ten MAH isolates, five from a clinical source and five from an environmental source. We used BIOLOG Phenotype MicroarrayTM technology for the analysis. This technology permits the rapid and global analysis of metabolic phenotypes. RESULTS: The ten MAH isolates tested showed different metabolic patterns pointing to high intra-species diversity. Our MAH isolates preferred to use fatty acids such as Tween, caproic, butyric and propionic acid as a carbon source, and L-cysteine as a nitrogen source. Environmental MAH isolates resulted in being more metabolically active than clinical isolates, since the former metabolized more strongly butyric acid (p = 0.0209) and propionic acid (p = 0.00307). DISCUSSION: Our study provides new insight into the metabolism of MAH. Understanding how bacteria utilize substrates during infection might help the developing of strategies to fight such infections.

A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany.

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n=41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A-H (sized 6.2-73.3kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.

Identification and comparative analysis of a genomic island in Mycobacterium avium subsp. hominissuis.

Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacterium causing opportunistic infections. The objective of this study was to identify flexible genome regions in MAH isolated from different sources. By comparing five complete and draft MAH genomes we identified a genomic island conferring additional flexibility to the MAH genomes. The island was absent in one of the five strains and had sizes between 16.37 and 84.85kb in the four other strains. The genes present in the islands differed among strains and included phage- and plasmid-derived genes, integrase genes, hypothetical genes, and virulence-associated genes like mmpL or mce genes.

Genetic characterization of German Mycobacterium avium strains isolated from different hosts and specimens by multilocus sequence typing.

Infections caused by Mycobacterium avium and its subspecies are reported as emerging disease in many countries worldwide. In our study we applied the multilocus sequence typing technology to 98 German M. avium strains originating from different hosts and specimens to examine the degree of the genetic diversity. By MLST, 80% of strains were identified as subspecies 'M. avium hominissuis', and 20% as subspecies M. avium avium/M. avium silvaticum. Distinctly different MLST profiles were identified for both subspecies. Based on the analysis of 4 and 5 loci, 87 and 106 SNPs and 1 codon deletion could be detected, respectively, resulting in 40 different strain profiles. Twelve out of these have recently been described for strains coming from different countries, yet in our study, additional new strain profiles (n=28) were found. The high degree of diversity within 'M. avium subsp. hominissuis' as well as the relatedness of human, porcine and environmental strains could be confirmed by IS1245 RFLP fingerprinting. The detection of ISMav6 and hsp65 code 15 in one adult patient strain being positive for IS901, but displaying 'M. avium subsp. hominissuis' MLST profile revealed that PCR for detection of IS901 is not a definitive proof of M. avium subsp. avium/M. avium subsp. silvaticum.

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.

Novel Mycobacterium tuberculosis complex isolate from a wild chimpanzee.

Tuberculosis (TB) is caused by gram-positive bacteria known as the Mycobacterium tuberculosis complex (MTBC). MTBC include several human-associated lineages and several variants adapted to domestic and, more rarely, wild animal species. We report an M. tuberculosis strain isolated from a wild chimpanzee in Côte d'Ivoire that was shown by comparative genomic and phylogenomic analyses to belong to a new lineage of MTBC, closer to the human-associated lineage 6 (also known as M. africanum West Africa 2) than to the other classical animal-associated MTBC strains. These results show that the general view of the genetic diversity of MTBC is limited and support the possibility that other MTBC variants exist, particularly in wild mammals in Africa. Exploring this diversity is crucial to the understanding of the biology and evolutionary history of this widespread infectious disease.

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.