Mycobacterium tuberculosis - the 10 years of epidemiological and diagnostics studies

Tuberculosis (ТВ) is the main bacterial pathogen that causes more deaths than AIDS, malaria and all infectious diseases. The unusual long doubling time (about 24h), highly hydrophobic cell envelope resistant to chemical lysis was the reason to delay the molecular study of this bacteria. Fifteen years ago, we did not have any molecular tools and methods for genetic manipulation or isolation and analysis of intracellular protein and nucleic acids. Today we have many useful shuttle or integration vectors for basic study of mycobacteria. The full sequence of M. tuberculosis genome is already known. At the present time the diagnosis of tuberculosis is supported with fast-culture system BACTEC and molecular techniques based on PCR and DNA hybridization. The mechanisms of resistance to antituberculosis drugs were described, and first identification of resistance profile is available by using PCR and sequencing or real time PCR methods. In our group in the Center for Microbiology and Virology Polish Academy of Sciences and in the Dept, of Genetics of Microorganisms, University Łódź we have characterized new insertion sequences from M. tuberculosis complex- 18990 and IS1607. In diagnostic studies we have proposed the DIG-PCR ELISA assay as a reliable, specific and sensitive test to identify M. tuberculosis directly in clinical samples. We have performed wide epidemiological studies of M. tuberculosis strains isolated from Polish ТВ patients including drug - and multidrug - resistant strains. Finally we identified the most frequently present mutations responsible for drug resistance of polish clinical isolates of M. tuberculosis.Zadanie pt. „Digitalizacja i udostępnienie w Cyfrowym Repozytorium Uniwersytetu Łódzkiego kolekcji czasopism naukowych wydawanych przez Uniwersytet Łódzki” nr 885/P-DUN/2014 dofinansowane zostało ze środków MNiSW w ramach działalności upowszechniającej naukę

Molecular genotyping methods in epidemiological investigations of TB infections

Gruźlica pozostaje jednym z najistotniejszych problemów zdrowotnych na świecie [...

Evaluation of NAD(+)-dependent DNA ligase of mycobacteria as a potential target for antibiotics

Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD+-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD+-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD+-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD+-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria

Recent Developments in Mycobacteriology: A Clinical and Diagnostic Perspective

Editorial for a special issue of the journal "BioMed Research International" with Table of contents.International audienceMycobacteria comprise a diverse group of bacteria that are widely distributed in nature, some of which cause significant disease in humans. The most prominent representative of this group is the Mycobacterium tuberculosis complex, which includes the etiological agents of tuberculosis (TB), which, with over 8 million new cases and nearly 2 million deaths annually, continues to cause one of the major health burdens for humans. This complex also includes the causative agents of TB in animals, of which Mycobacterium bovis, the causative agent of bovine TB is the most prominent. Whereas the prevalence of TB has been closely monitored in most parts of the world, the epidemiology of nontuberculous mycobacterial (NTM) infections remains poorly defined; in spite of the fact that the importance of NTMs as a cause of opportunistic infections of humans has been increasingly recognized over the last two decades, particularly in areas where the incidence of TB is in decline. The purpose of this special issue is to provide a reader with some recent achievements in mycobacteriology, with particular emphasis on the developments which have direct relevance to the clinical practice and diagnostic performance. The articles within this issue differ considerably from each other with respect to their research scopes and methodologies, thus reflecting the multidirectional character of the research in the ever-expanding field of mycobacteriology. We face an exciting era in mycobacteriology, where greater understanding of the mycobacteria leads to technical improvements that change clinical practice and these techniques in turn help to curb the epidemic of mycobacterial diseases of humans and animals

Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses

Institutional Review Board Statement: The experimental procedures were approved and conducted according to guidelines of the appropriate Polish Local Ethics Commission for Experiments on Animals No. 9 in Lodz (Agreement 9/ŁB87/2018). Acknowledgments: We thank Jeremy Rock and Sarah Fortune for providing us with the pLJR965 vector and detailed instructions for the generation of Cas9-regulated strains in M. tuberculosis. The authors thank the mass spectrometry service at the Institute of Biochemistry and Biophysics PAS in Warsaw for MS analysis. The MS analysis equipment used for the analysis was sponsored in part by the Centre for Preclinical Research and Technology (CePT), a project cosponsored by the European Regional Development Fund and Innovative Economy, the National Cohesion Strategy of Poland.Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level.A.B. was supported by grant “OPUS” from the National Science Centre, Poland, UMO2015/19/B/NZ6/02978. P.P. was supported by grant “OPUS” from the National Science Centre, Poland, UMO-2019/33/B/NZ1/02770

Draft Genome Sequences of Mycobacterium kansasii Strains 1010001454, 1010001458, 1010001468, 1010001493, 1010001495, and 1010001469, Isolated from Environmental Sources

Mycobacterium kansasii belongs to the nontuberculous mycobacteria (NTM) and causes opportunistic infections with both pulmonary and extrapulmonary manifestations. Here, we report the draft genome sequences of six environmental M. kansasii strains, designated 1010001495 (type I), 1010001469 (type II), 1010001468 (type III), 1010001458 (type IV), 1010001454 (type V), and 1010001493 (type V), originally isolated in five different European countries.The study was financed by the National Centre for Research and Development: “LIDER” Program (contract no. LIDER/044/457/L-4/12/NCBR/2013)

Nontuberculous mycobacteria - why treatment is so difficult?

Zakażenia prątkami niegruźliczymi stanowią w dzisiejszych czasach znaczący problem. Infekcje te dotyczą najczęściej osób z obniżoną odpornością. Atypowe prątki są często przyczyną mykobakteriozy płucnej, ale także pozapłucnej, w przypadkach kiedy infekcja rozwija się w obrębie skóry, tkanek miękkich czy kości. Nowoczesna diagnostyka wykorzystująca narzędzia biologii molekularnej pozwala na szybką identyfikację gatunku MOTT, jednakże wykrycie atypowych prątków nie zawsze świadczy o mykobakteriozie i konieczności terapii, która ze względu na liczne mechanizmy oporności MOTT może okazać się nieskuteczna. Interesującym zjawiskiem jest także zdolność prątków atypowych do tworzenia biofilmów, trójwymiarowych struktur wielokrotnie zmniejszających wrażliwość tych drobnoustrojów na antybiotyki.Nontuberculous mycobacteria (NTM) is a group of opportunistic species of mycobacteria other than Mycobacterium tuberculosis complex and Mycobacterium leprae, which are widespread in the environment occurring in soil, water and dust. Therefore, it is common to localize them in the respiratory, gastrointestinal tract and skin. In the past two decades, increasing number of infections caused by atypical mycobacteria was reported worldwide. Development of molecular biology and new diagnostic tests enables faster distinction of atypical mycobacteria from Mycobacterium tuberculosis complex and more accurate identification of the species. Most atypical mycobacteria are naturally resistant to antibiotics commonly used for treatment of both mycobacteriosis and tuberculosis. The drug resistance of NTM involves nonspecific mechanisms, which also occur in other bacteria, and specific mechanisms characteristic for mycobacteria only. Resistance can be innate, determined by the bacterial genome, or acquired, as the result of mutational changes