State Medical and Pharmaceutical University Chisinau, Moldova Lecture for students Immunology to tuberculosis Koch-MetSChnikoW-Forum МEЧНИКОВ-КОХ-ФОРУМ Timo Ulrichs Vice president and head of the section of tuberculosis, Koch-Metchnikov-Forum, Berlin
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State Medical and Pharmaceutical UniversityChisinau, Moldova
Lecture for students
Immunology to tuberculosis
Koch-MetSChnikoW-Forum МEЧНИКОВ-КОХ-ФОРУМ
Timo Ulrichs
Vice president and head of the section of tuberculosis, Koch-Metchnikov-Forum,
Berlin
Intracellular bacteriaMycobacteria and Tuberculosis
Mycobacteriamycobacterial cell wall
Tuberculosis epidemiologypathogenesispersistencedrug therapyconventional anti-TB drugs and MDRunconventional approaches of new drug design
Overview Mycobacteria
•There are >70 species of mycobacteria
•Of these, two are major pathogens: 1.Mycobacterium tuberculosis (Koch, 1882) 2.Mycobacterium leprae (Hansen, 1874)
•The remaining mycobacteria are environmental organisms-collectively known as MOTTS (Mycobacteria Other Than Tuberculosis)
•MOTT organisms are responsible for opportunistic infections, especially in people with AIDS
All mycobacteria are: 1.ACID FAST- i.e. they do not destain with acid and alcohol once stained with arylmethane dyes 2.AEROBIC 3.CONTAIN MYCOLIC ACIDS 4.THEIR GENOMES HAVE A 59-66% GC CONTENT
Intracellular bacteriaMycobacteria and Tuberculosis
Mycobacteriamycobacterial cell wall
Tuberculosis epidemiologypathogenesispersistencedrug therapyconventional anti-TB drugs and MDRunconventional approaches of new drug design
Mycobacterial cell wall
Proteins
Man-capped lipoarabino-mannan
Mycolic acid
Glycolipids
Arabinogalactan
Peptidoglycan
PM
Cytosol
CellWall
Mycobacterial cell wall
Unique cell wall: waxy, hydrophobic and high lipid content Up to 60% of the dry weight of the organisms may be mycolic acids- long chain, branched fatty acids. The type of mycolic acid can be used to distinguish different mycobacteria. The mycolic acids and short chain fatty acids form a pseudo outer membrane and are responsible for the unusual staining characteristics of the cells.
Mycobacterial cell wall
The wall is also responsible for the hydrophobicity of these organisms. The wall has adjuvant properties and may be responsible for the development of delayed type hypersensitivity (DTH). All mycobacterial pathogens are intracellular pathogens - the wall helps the organism to survive within the macrophage by resisting oxydative damage.
Intracellular bacteriaMycobacteria and Tuberculosis
Mycobacteriamycobacterial cell wall
Tuberculosis epidemiologypathogenesispersistencedrug therapyconventional anti-TB drugs and MDRunconventional approaches of new drug design
Threat to mankind already in ancient Egypt:
Tuberculosis
Famous victims of TB
•Anton Checkov •Branwell Bronté •Emily Bronté •Frédéric Chopin •John Keats •D.H. Lawrence •Vivien Leigh •George Orwell •Paganini •Edgar Allan Poe •Jean J. Rousseau •Sir Walter Scott •P.B.Shelly •R.L. Stevenson •Simonetta Vespucci girl friend of Guilano de‘ Medici
He asks Sandro Botticelli to create a painting of her...
Sandro Botticelli, Die Geburt der Venus (1485/86), Uffizien, Florenz
Tuberculosis
Intracellular bacteriaMycobacteria and Tuberculosis
Mycobacteriamycobacterial cell wall
Tuberculosis epidemiologypathogenesispersistencedrug therapyconventional anti-TB drugs and MDRunconventional approaches of new drug design
Tuberculosis
Global numbers of tuberculosis (WHO, 2011)
• 1.6 million deaths per year• 9.4 million new infections per year
• Every third of the human World population is infected.• 5 to 10 % of the infected will develop TB during their life.
Tuberculosis
Global numbers of tuberculosis (WHO, 2011)
If TB cannot be brought under control within the next 30 years:
• 1000 million new infections• 200 million new disease cases• 35 million deaths
Tuberculosis
Global numbers of tuberculosis (WHO, Geneva)
Mycobacterium tuberculosis - morphology
Slender, straight or slightly curved bacillus, non-motile, non-encapsulated and does not form spores Acid fast bacillus (AFB) Aerobic Slow growing- divides every 18-24 hr. Resistant to drying and chemical disinfectants Sensitive to heat (Pasteurization) and UV light
Mycobacterium tuberculosis - genome
The M.tb genome has been sequenced
•First major pathogen to be sequenced •4,411,522 bp •3 924 open reading frames •GC content of 65.6% •+/- 70% of the genes can be identified at this stage, the remainder are unique and encode proteins with unknown functions •59 % of genes are transcribed in the same direction as chromosomal replication
Intracellular bacteriaMycobacteria and Tuberculosis
Mycobacteriamycobacterial cell wall
Tuberculosis epidemiologypathogenesispersistencedrug therapyconventional anti-TB drugs and MDRunconventional approaches of new drug design
Pathogenesis of infection with Mycobacterium tuberculosis
Pathogenesis of infection with Mycobacterium tuberculosis
Pathogenesis of infection with Mycobacterium tuberculosis
Pathogenesis of infection with Mycobacterium tuberculosis
Pathogenesis of infection with Mycobacterium tuberculosis
Pathogenesis of infection with Mycobacterium tuberculosis
The human tuberculous granuloma
The human tuberculous granuloma
Intracellular bacteriaMycobacteria and Tuberculosis
Mycobacteriamycobacterial cell wall
Tuberculosis epidemiologypathogenesispersistencedrug therapyconventional anti-TB drugs and MDRunconventional approaches of new drug design
The human tuberculous granuloma
The human tuberculous granuloma
Intracellular bacteriaMycobacteria and Tuberculosis
Mycobacteriamycobacterial cell wall
Tuberculosis epidemiologypathogenesispersistencedrug therapyconventional anti-TB drugs and MDRunconventional approaches of new drug design
Tuberculosis – laboratory diagnosis
Laboratory diagnosis is based on the demonstration of M. tuberculosis in a clinical specimen.
Types of Clinical specimens: sputum pleural biopsy broncho-alveolar washings - fibre-optic bronchoscopy biopsy specimen of lung tissueCSF gastric washing biopsy from other anatomical sites
Tuberculosis – laboratory diagnosis
Microbiological diagnosis Microscopic investigation:- Ziehl-Neelsen or Auramine O stain (AFB) Culture- may take 2-6 weeks for the isolation of M. tuberculosis
Decontamination (to kill contaminating organisms) and concentration of the specimen
Molecular diagnosis depends on the demonstration of M. tuberculosis DNA or RNA in the specimen. RNA and mRNA, in particular, may be a better indication of mycobacterial cell viability
Tuberculosis – tuberculin skin test
Tuberculin is a partially purified extract of M. tuberculosis proteins (PPD) PPD evokes a delayed hypersensitivity (DTH) response when injected into the skin- this forms the basis of the Tuberculin Skin Test Tuberculin skin testing can be used to identify individuals, especially children, with active tuberculosis. It can be used to trace contacts of patients with active tuberculosis A positive tuberculin test may be an indication for INH prophylaxis Mantoux and Heaf tests are different types of tuberculin skin tests. Individual M. tuberculosis proteins are being identified and isolated- these may form the basis of a single, defined protein tuberculin skin test that does not cross-react with proteins from other mycobacteria such as M. bovis BCG.
Anti-TB chemotherapy
1944: Waksman and colleagues discovered Strepto-mycin:„a revolution in the treatment of TB disease“
monotherapy, resistance, failure
1950s: Isoniazid, Pyrazinamide: combination therapy„the complete eradication of TB disease is in sight“
1993: WHO declares of TB as a global health emergency
multi-drug-resistance (MDR) increasing
Multi-drug resistance (MDR)
490,000 new MDR cases every year; more than 110,000 deaths
> 200,000 cases in Russia
> 5.3% of TB cases caused by MDR-strains
> 10% MDR-TB in Baltic states, Eastern Europe, several provinces in Russia, China
1/106 mutation rate: triple treatment has 10-18 risk of resistance, if strain is susceptible for all agents;10-6 risk of resistance, if strain is already resistant to two drugs>108 organisms/lesion during active TB
What went wrong?
Lack of compliance
WHO: directly observed therapy (DOT) with remarkable success
Effectiveness of TB drugs differs between in vitro and in vivo conditions
possible reasons:drug availability in M.tb.-infected lung lesionsphysiologic heterogenicity of M.tb. during persistence
(adapted from McKinney, Nature Medicine, special focus tuberculosis, 2001)
Conventional TB drugs
target processes of mycobacterial cell growth and division
are bacteriostatic, not bacteriocidal
have been selected for effectiveness against M.tb. cultures in vitro, not in vivo
often have to be activated in vivo (what is not done by dormant M.tb.)
are inactive within the phagosome in most cases
Intracellular bacteriaMycobacteria and Tuberculosis
Mycobacteriamycobacterial cell wall
Tuberculosis epidemiologypathogenesispersistencedrug therapyconventional anti-TB drugs and MDRunconventional approaches of new drug
design
1. target the genetic requirement for in vivo growth and persistence
2. circumvent mycobacterial resistance strategies
3. increase vulnerability to host defense
Unconventional approaches for new TB drugs
Isocitrate lyase, the key enzyme for the gyloxylate shunt, activated only for fatty acid metabolism in vivo
Target the genetic requirement
Circumvent mycobacterial resistance
Drug-enhancing drugs (like -lactams + -lactamase-inhibtors)mutant SigF M.tb. is hypersensitive to RMP
SigF inhibitors promote killing by rifampin
• target the genetic requirement for in vivo growth and persistence• circumvent mycobacterial resistance strategies• increase vulnerability to host defense
by means of:
• information on M.tb. genome and proteome• better understanding of mycobacterial strategies for persistence• modern high throughput screening: proteomics, transcriptomics for identifying novel targets, test in animal models using chronic vs. acute vs. dormant infection
Unconventional approaches for new TB drugs
Tuberculosis
Global numbers of tuberculosis (WHO, Geneva)
Tuberculosis
Directly observed therapy, DOTS
New drugs for TB control
„Uncivilized peoples not only rub over their arrow with one kind of poison, but with two or three totally different kinds of poison.“
Paul Ehrlich, 1913
If you want to efficiently fight your enemy, first get to know every detail of him!