ulrichs, tuberculosis, immunology, 111216

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

Classification

Mycobacteria belong to the

•Order: ACTINOMYCETALES •Family: MYCOBACTERIACEAE •Genus: MYCOBACTERIUM

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

Solid medium- Lowenstein-Jensen Liquid medium-(Middlebrook)

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

Persistence of M. tuberculosis in vivo

slow growing, metabolically inactive,antimicrobial tolerant

No eradication possible!

intact granuloma

fast growing, metabolically active

Development of resistance

active tuberculous lesion

Intracellular bacteriaMycobacteria and Tuberculosis

Mycobacteriamycobacterial cell wall

Tuberculosis epidemiologypathogenesispersistencedrug therapyconventional anti-TB drugs and MDRunconventional approaches of new drug design

Conventional TB drugs

Drug cellular process targeted molecular target

Isonizid, INH cell wall mycolic acid synthesis enoyl-ACP reductase

b-ketoacyl ACP synt.

Pyrazinamide, PZA cell wall fatty acid biosynthesis FAS-I fatty acid synt.Ethambutol, ETB cell wall arabinogalactan synthesisarabinosyltransferaseCycloserine cell wall peptidoglycan synthesis D-ala-D-ala ligase

Rifampin, RMP RNA synthesis RNA polymeraseStreptomycin, SM polypeptide synthesis 16S rRNA

Fluoroquinolones DNA synthesis DNA gyrase

(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!