Koch-MetSChnikoW-Forum E--

State Medical and Pharmaceutical University Chisinau, Moldova Lecture for students

Immunology to tuberculosis
Timo Ulrichs
Vice president and head of the section of tuberculosis, Koch-Metchnikov-Forum, Berlin

Intracellular bacteria Mycobacteria and Tuberculosis

Mycobacteria mycobacterial cell wall Tuberculosis epidemiology pathogenesis persistence drug therapy conventional anti-TB drugs and MDR unconventional 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 organismscollectively 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 bacteria Mycobacteria and Tuberculosis

Mycobacteria mycobacterial cell wall Tuberculosis epidemiology pathogenesis persistence drug therapy conventional anti-TB drugs and MDR unconventional approaches of new drug design

Mycobacterial cell wall

Proteins Man-capped lipoarabinomannan Mycolic acid Glycolipids PM Arabinogalactan Cytosol Peptidoglycan

Cell Wall

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 acidslong 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 bacteria Mycobacteria and Tuberculosis

Mycobacteria mycobacterial cell wall Tuberculosis epidemiology pathogenesis persistence drug therapy conventional anti-TB drugs and MDR unconventional approaches of new drug design

Tuberculosis
Threat to mankind already in ancient Egypt:

Famous victims of TB
Anton Checkov Branwell Bront Emily Bront Frdric 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...

Tuberculosis

Sandro Botticelli, Die Geburt der Venus (1485/86), Uffizien, Florenz

Intracellular bacteria Mycobacteria and Tuberculosis

Mycobacteria mycobacterial cell wall Tuberculosis epidemiology pathogenesis persistence drug therapy conventional anti-TB drugs and MDR unconventional 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, nonencapsulated 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 bacteria Mycobacteria and Tuberculosis

Mycobacteria mycobacterial cell wall Tuberculosis epidemiology pathogenesis persistence drug therapy conventional anti-TB drugs and MDR unconventional 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 bacteria Mycobacteria and Tuberculosis

Mycobacteria mycobacterial cell wall Tuberculosis epidemiology pathogenesis persistence drug therapy conventional anti-TB drugs and MDR unconventional approaches of new drug design

The human tuberculous granuloma

The human tuberculous granuloma

Intracellular bacteria Mycobacteria and Tuberculosis

Mycobacteria mycobacterial cell wall Tuberculosis epidemiology pathogenesis persistence drug therapy conventional anti-TB drugs and MDR unconventional 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 tissue CSF 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 isolatedthese 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 Streptomycin: 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 lesions physiologic heterogenicity of M.tb. during persistence

Persistence of M. tuberculosis in vivo

active tuberculous lesion
intact granuloma

fast growing, metabolically active

slow growing, metabolically inactive, antimicrobial tolerant

Development of resistance

No eradication possible!

Intracellular bacteria Mycobacteria and Tuberculosis

Mycobacteria mycobacterial cell wall Tuberculosis epidemiology pathogenesis persistence drug therapy conventional anti-TB drugs and MDR unconventional 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.

FAS-I fatty acid synt. arabinosyltransferase D-ala-D-ala ligase RNA polymerase 16S rRNA DNA gyrase

Pyrazinamide, PZA cell wall fatty acid biosynthesis Ethambutol, ETB cell wall arabinogalactan synthesis Cycloserine cell wall peptidoglycan synthesis Rifampin, RMP Streptomycin, SM Fluoroquinolones RNA synthesis polypeptide synthesis DNA synthesis

(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 bacteria Mycobacteria and Tuberculosis

Mycobacteria mycobacterial cell wall Tuberculosis epidemiology pathogenesis persistence drug therapy conventional anti-TB drugs and MDR unconventional approaches of new drug design

Unconventional approaches for new TB drugs

1. target the genetic requirement for in vivo growth and persistence 2. circumvent mycobacterial resistance strategies

3. increase vulnerability to host defense

Target the genetic requirement

Isocitrate lyase, the key enzyme for the gyloxylate shunt, activated only for fatty acid metabolism in vivo

Circumvent mycobacterial resistance

Drug-enhancing drugs (like -lactams + -lactamase-inhibtors) mutant SigF M.tb. is hypersensitive to RMP

SigF inhibitors promote killing by rifampin

Unconventional approaches for new TB drugs

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

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!