msc in micro genetics of mycobacterium TB.pptx

rmt

1. INSTITUTE OF HEALTH FACULTY OF HEALTH SCIENCE SCHOOL OF MEDICAL LABORATORY SCIENCE DEPARTMENT OF MEDICAL MICROBIOLOGY Course : Microbial Genetics and Molecular Techniques seminar on Genetics of Mycobacterium tuberculosis by Desalegn Ashenafi presented for Dr .Mulualem Tadesse(PHD, Associate prof)

2. OBJECTIVE At the end of this presentation the participants are expected to ; Explain the characteristics of MTB, transmission, virulence factors, and clinical manifestation Genome composition, gene replication, expression, and regulation of MTB DNA damage, repair, and mutation in MTB Genes that code for virulence factors and antibiotic resistance Mechanism of gene transfer and genetic diversity Molecular diagnostic techniques of TB

3. OUTLINE INTRODUCTION GENOME OF TB DNA REPLICATION ,TRANSCRIPTION AND TRANSLATION GENE EXPRESSION AND REGULATION DNA DAMAGE ,REPAIR AND MUTATION IN TB GENES THAT CODE FOR VIRULENT FACTOR MECHANISM OF GENE TRANSFER GENETIC DIVERSITY OF TB APPLICATION OF MOLECULAR TECHNIQUES FOR DIAGNOSIS OF TB

4. Introduction Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis disease. It is Slender, acid fast bacilli and slow growing bacteria. They are non-motile, obligate aerobes and non-spores forming organism. Technically neither gram negative nor positive But structurally share common molecules with gram positive bacteria cell wall . Have lipid rich cell wall (mycolic acid) Impermeable to many dyes ,drugs and cations and anions

5. A total of 1.3 million people died from TB in 2022 TB is the second leading infectious killer after COVID-19 (above HIV and AIDS). In 2022, an estimated 10.6 million people fell ill with tuberculosis (TB) worldwide Multidrug-resistant TB (MDR-TB) remains a public health crisis and a health security threat . Estimated annual number of people who developed MDR/RR-TB in 2022 was 410 000 . Most of the cause of this resistant is attributed to gene mutation in bacteria Transmit by respiratory droplet and reproduce in host macrophage mycolic acid , lipoarabino mannan , early antigen-secreted protein 6 kDa (ESAT-6 ) and other enzymes are virulent factor of TB

6. Virulence factors

7. CLINICAL MANIFESTATION Primary Tuberculosis is either asymptomatic or manifest only by fever and malaise. Controlled by immune system in most of the cases In approximately 5% of patients, the primary disease is not controlled and merges into the reactivation type of tuberculosis, or disseminates to many organs. Reactivation is associated with immune suppression,coinfection and socioeconomic status Cough is the universal symptom of tuberculosis. It is initially dry, but as the disease progresses sputum is produced, which even later is mixed with blood (hemoptysis) Fever, Malaise , Fatigue , sweating, and weight loss are the sign and symptoms of the disease.

8. Genetic composition of TB Has genome of 4.4 Mb that contains ~4,000 genes. Has single, circular chromosome with a sequence of 4,411,529 base pairs with a G + C content of 65.6% . This represents the second-largest bacterial genome sequence currently available (after that of Escherichia coli). dnaA gene, a hallmark for the origin of replication, oriC, was chosen as the start point for numbering (Rv0001).

9. DNA replication, transcription and translation DNA replication starts at a unique site,  oriC The functional  oriC  in  M. tb  is an 814-bp region that is flanked by genes encoding DnaA (Rv0001), DnaN (Rv0002), and RecF (Rv0003) proteins that are involved in DNA replication or repair.  DnaA  and  dnaN  genes are separated by a 527-bp intergenic region and  recF  is located 19-bp downstream of  dnaN The initiation of bacterial DNA replication is mediated by DnaA, which interacts with DNA sequences referred to as DnaA boxes The  oriC  region of  M.tb  has 13 such non-perfect DnaA boxes of which 11 were shown to interact with DnaA   dnaN  codes for the β′ subunit of DNA polymerase III, the major bacterial replicase, that directs the bidirectional replication of the chromosome and the downstream gene,   recF , codes for a DNA-binding protein involved in recombination repair and in the resumption of DNA replication at disrupted replication forks

10. Genes Protein(enzymes) Their function dnaA DnaA Binds to the sequence called DNA A boxes and initiate replication dnaB DnaB(Helicase) Unwind the double strand of DNA dnaN DnaN (DNA polymerase beta subunit) It involves in nucleotide addition of the newly synthesized strands. dnaG RNA primase It synthesis RNA short RNA segment (RNA primer). recF DNA binding protein Involve in recombination repair ssb genes ( Rv0054) Single strand binding protein Prevent reannealing of unwound DNA gyrA(Rv0006) and gyrB(Rv0005) Alpha and beta subunit of DNA gyrase It reduce the torsion caused due to supercoiling ligA(Rv3014c) and ligB(Rv3062 Alpha and beta subunit of DNA ligase Seals the ogazaki fragments

11. Gene expression and regulation Gene expression encompasses the transcription and translation process. RNA polymerase is the primary enzymes that involve in transcription It can recognize the promoter site and then can synthesis the complementary RNA sequence of the DNA template strands. Genes rpoA ,rpoB,rpoC encode alpha ,beta subunit and beta prime subunit of RNA polymerase . Genes rpl,rpm and rps involves in transcription by encoding large and small subunit of ribosomal proteins. The mRNA binds to small ribosomal subunits and tRNA will binds to mRNA and translation will continue

12. Genes that codes for regulatory protein that involves in transcription and translation plays role in gene expression regulation Promoter regions that regulates gene expression in TB are Lac operator Ara operator Tet operator Emb operator Myc operator TetR is the repressor gene and it binds to tet operator when

13. DNA damage ,repair and mutation in TB E xtremely challenging and hostile environments during infection due to the host immune response and possible antibiotic treatment can cause DNA damage to TB. D ifferent endogenous and exogenous radicals and oxidants, in the form of reactive oxygen and nitrogen species can damage the DNA. These compounds can compromise the integrity of bacterial lipids, proteins and nucleic acids as part of the host defense response. Mycobacterium tuberculosis repair the damage by three main mechanisms

14. Cont … E xcision repair :it can be base excision or nucleotide excision repair . Base excision repair DNA damage reversal and recombinational repair , SOS repair and mutagenesis

15. Genes that code for virulent factor and drug resistance

16. Mechanism of gene transfer in TB Horizontal gene transfer is the common gene transfer mechanism in TB Transduction: Many phages that can infect mycobacteria are identified (e.g. phage k, Phage D29, phage TM4) The possibility of gene transfer among different species of mycobacteria is indicated Transformation and conjugation in mycobacterium tuberculosis are controversial among different scientists Slow growth of the TB and lipid-rich cell wall makes taking up genetic material from the environment difficult

17. Genetic diversity of TB MBTC has seven main lineages that are adapted to human only and this lineages have variation of geographical spread Some of them have more specified geographical areas whereas others are found worldwide with different degree of prevalence. Lineage 2 (East-Asian lineage) and lineage 4 (Euro-American) lineage occurs worldwide whereas lineage 5(West Africa 1) and lineage 6 (West Africa 2) are restricted to West Africa. Lineage 1(indo-oceanic lineage) and Lineage 3 (10)(central Asian lineage) shows intermediate distributions and lineage 7 (Ethiopian lineage) found exclusively in Ethiopia

18. Molecular techniques for diagnosis TB Nucleic acid amplification (NAAT) It detects the DNA (deoxyribonucleic acid) of Mycobacterium tuberculosis complex (MTBC) in a sputum or other respiratory sample. NAAT testing includes a step that amplifies (or copies) the genetic material. Polymerase Chain Reaction (PCR) is a common form of NAAT used in laboratory diagnosis

19. Line probe assay DNA strip-based tests that allow users to determine the drug resistance profile of an MTBC strain by interpreting a pattern of bands that represent lines of immobilized probes that are bound (or hybridized) to MTBC amplicons (DNA amplification products). Designed to target the most common mutations associated with resistance to first- and second-line anti-TB agents and specific MTBC wild-type (WT) DNA sequences. They can be used to test culture isolates (indirect testing) and for direct testing of acid-fast bacilli smear microscopy-positive specimens (first-line LPA) and both smear-positive and smear-negative sputum specimens (second-line LPA). Mutations are detected by binding of amplicons to probes that target the most common mutations (MUT probes) (with, e.g., first- and second-line LPAs) or by lack of amplicons binding (i.e., lack of hybridization) to the corresponding WT probes (e.g., PZA-LPA), defined as “inferred resistance”. The post-hybridization reaction leads to the development of Coloured bands on the strip at the site of probe binding

20. GeneXpert MTB/RIF An automated polymerase chain reaction (PCR) test (that is, a molecular test) utilizing the GeneXpert platform. Is a single test that can detect both Mycobacterium tuberculosis complex and rifampicin resistance within 2 hours after starting the assay, with minimal hands-on technical time. Unlike conventional PCR sample processing, PCR amplification and detection are integrated into a single self-enclosed test unit, which is the Xpert MTB/RIF cartridge. All steps in the assay are automated and contained within the cartridge. The assay’s sample reagent, used to liquefy sputum, is tuberculocidal (that is, it has the ability to kill TB bacteria. Requires an uninterrupted and stable electrical power supply, temperature control and yearly calibration of the instrument’s module

21. RESTRICTION FRAGMENT LENGTH POLYMORPHISM Analyses DNA fragments which are generated by restriction enzymes, which cut DNA at specific sequences. In RFLP the region of the bacterial genome called IS6110 is amplified using PCR and then digested with restriction enzymes. The fragments are separated based on their size through gel electrophoresis. The pattern of fragment distribution on the gel will be compared with the standard profiles and help to determine the bacterial strain. Spoligotyping Is a technique used to identify and classify strains of mycobacterium tuberculosis by focusing on the detection of specific DNA sequences known as spacers within the direct repeat(DR) region of the Mtb genome. First the DR region will be amplified by PCR and then hybridized with a set of oligonucleotide probes called spacers that correspond to different spacer sequences.

22. Mycobacterial inter-spread repetitive unit variable number tandem repeats (MIRU-VNTR): It focuses on the analysis of variable number tandem repeats within the Mtb genome. This region is highly variable among different strains, so that helps us to differentiate strains of the bacteria. Genotype MTBDRplus assay is a rapid test that can detect MDR-TB within 8 hours and includes the following steps: deoxyribonucleic acid (DNA) extraction, multiplex polymerase chain reaction (PCR), reverse hybridization, and resistance gene mutations detection. Almost all rifampicin resistance is caused due to mutation in rpoB gene while mutations in the katG and inhA genes confer high-level and low-level INH resistance, respectively. Genotype MTBDRplus assay detects all these mutations

23. Summary

24. References