David Blanco, PhD
CHS A2-087G
MIMG C106
Mycobacteria
Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, is the
world’s leading cause of death in adults by a single infectious agent. In 1995, >3
million people died from tuberculosis and it is estimated that one third of the
world’s human population is currently infected with M. tuberculosis.
The incidence of TB continues to increase in developing countries and, after a
century of decline in developed countries, it is now increasing in developed
countries as well. The largest factor contributing to spread of TB in the US in
recent years has been AIDS, because immunodeficiency greatly increases the
conversion of asymptomatic to symptomatic disease. Symptomatic disease
results in the spread of infection. The most frightening contribution to increased
incidence is the emergence of multiple drug resistant strains of Mycobacteria.
Tuberculosis is caused by members of the Mycobacterium tuberculosis complex
which includes Mycobacterium tuberculosis, Mycobacterium bovis,
Mycobacterium africanum, and Mycobacterium microtii. Mycobacteria are
aerobic, non-sporeforming bacilli with unique staining characteristics. They are
gram positive like in their structure but stain characteristically with a special stain
using carbol-fuchsin. While Gram negative and positive bacteria decolorize with
acid-alcohol after carbol-fuchsin staining, Mycobacteria are resistant to acidalcohol destaining and are thus referred to as “acid fast”. The name “acid fast
bacilli” or “AFB” is nearly synonymous with Mycobacteria.
Mycobacteria are extremely slow growing – both in vitro and in vivo. Doubling
times can be from 12 to 24 hours, depending on the strain. In Mycobacteria
research laboratories, this has resulted in significant limitations and slow
progress. In vivo, this is a major contributing factor in increased incidence:
prolonged doubling times translates to long duration drug therapy and in turn
results in poor patient compliance with these medications. Decreased
compliance contributes to increased disease (and hence spread) and also to
increased incidence of drug resistant strains.
Basic biology of Mycobacterial infection.
Tuberculosis is a prototype of infections that require cellular immune response
(CMI) for their control. Like diseases caused by the related species,
Mycobacterium leprae, the outcome of M. tuberculosis infection is dramatically
influenced by the nature of the host immune response. Key stages in the
disease process are:
1. M. tuberculosis enters the lungs in aerosol droplets. It’s taken up by
alveolar macrophages. Activated macrophages can kill M. tuberculosis
but if the bacteria enter resting macrophages they can survive, multiply
and spread to other resting macrophages. Like Salmonella, M.
tuberculosis is able to prevent phagosome-lysosome fusion.
2. Initial growth in alveolar macrophages results in the formation of small
necrotic lesions with a solid, caseous centers in which M tuberculosis
growth is probably restricted. An effective T cell response is essential for
protection. Cytotoxic T lymphocytes (CTLs) are required to release
intracellular bacteria from host cells so that they can be killed by activated
macrophages. Helper T cells are also required to recruit and activate
additional macrophages.
3. For the majority of people infected with M. tuberculosis, bacteria are either
cleared by the action of T cells before symptoms even develop, or bacteria
become enclosed within a caseous lesion and remain dormant for months
or even years. In this dormant state, the bacteria become surrounded by
lymphocytes, macrophages, Langhan’s giant cells and fibroblasts.
Langhan’s giant cells are macrophages that have surrounded the M.
tuberculosis and fused to form one very large cell with many peripherally
located nuclei. This organization of cells is called a “granuloma” and
contributes to the formation of hard tubercles (hence the name). This is a
successful tissue reaction with respect to the containment of infection,
healing with eventual fibrosis, encapsulation, and scar formation.
4. In response to factors that compromise the immune system, such as
malnutrition, alcoholism, drug abuse, and AIDS, reactivation can occur.
The solid, caseous foci progress to liquefied capsules in which the M.
tuberculosis multiply. The liquefied lesions, along with the bacteria, are
discharged throughout the bronchial tree producing a tuberculous cavity.
In these lesions, bacterial numbers are 5 to 6 logs higher than in noncavity lesions. Coughing leads to delivery of M. tuberculosis into the air in
respiratory droplets.
Treatment and Control.
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Vaccination: BCG (bacilli Calmette-Guerin; attenuated strain of M. bovis)
has been used in many countries; its efficacy has not been conclusively
proven. It is not used in the US because BCG vaccination causes
conversion of the commonly used diagnostic skin test (to positive).
PPD (purified protein derivative) skin test: In the US, this skin test is often
the first evidence of exposure, positive skin test is followed by chest X-ray
and prophylactic drug therapy that usually is prescribed for 6 months to 1
year.
Multiple drug therapy: isoniazid, rifampin, pyrazinamide, and ethambutol
for 6 to 12 months.
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Drug resistant strains: MDR (multiple drug resistant) strains have been
associated with outbreaks of disease characterized by rapid progression
and high mortality (up to 75%!).
Genetic approaches to understanding Mycobacterial pathogenesis.
Development of genetic tools:
 M. smegmatis (a non-pathogenic species of Mycobacteria that grows
much faster and easier in the laboratory than M. tuberculosis); shuttle
plasmids (to transfer DNA into and out of Mycobacteria and for
manipulations in vitro and in E. coli); transformation protocols;
mycobacteriophage L5; and reporter genes (xylE, phoA).
Isolation of avirulent and attenuated strains:
 Spontaneous mutants, transposon mutants.
Complementation of attenuated strains with DNA from virulent strains:
(Collins et al., 1995. PNAS 92:8036-8040)
Collins et al. made use of an integrating cosmid shuttle vector, pYUB178, to
clone a gene that conferred virulence upon an avirulent M. bovis strain. M. bovis
WAg200 is virulent in a guinea pig model system: lesions in the spleens, livers,
kidneys, and lungs can be detected 12 weeks after subcutaneous injection of 1 X
106 cfu. M. bovis strain ATCC35721 is avirulent – it does not cause lesions in
guinea pigs even after subcutaneous injection of >107 cfu.
The hypothesis was that the lack of virulence in ATC35721 was due to the loss
or inactivation of one or more virulence genes. Since these two strains are so
closely related, the authors reasoned that they might be able to identify the
missing or inactive gene by complementation using DNA from the virulent
WAg200 strain.
PYUB178 has several useful features:
 attP and int are from mycobaceriophage L5. L5 is similar to
bacteriophage  in that it inserts into a specific site in the bacterial
chromosome, called attB, and insertion into this site (via homologous
recombination) requires a bacteriophage encoded protein, integrase,
encoded by the int gene.
 oriE is an origin of replication that functions in E. coli allowing for
manipulation (cloning) and propagation of the plasmid in E. coli. (Since
the plasmid does not contain an ori that functions in Mycobacteria it is a
suicide plasmid, i.e., it must integrate into the chromosome in order to be
maintained).
 aph encodes kanamycin resistance for selection.
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A  cos site allows the plasmid to be packaged into  and therefore very
large fragments of DNA can be cloned for tranduction into E. coli.
The unique BcII site can be used to clone fragments that have been
digested with BamHI, BgIII, or Sau3A.
Construction and characterization of a M. bovis WAg200 DNA library in
pYUB178.
WAg200 (virulent Mycobacteria) genomic DNA was digested with Sau3A (limited
digestion to generate large DNA fragments), separated by agarose gel
electrophoresis, and DNA fragments of 30-50 kb were isolated. These fragments
were ligated into the BcII-digested site of PYUB178, packaged into  phage
heads, and transduced into E. coli. Cosmid DNA was then prepared and
electroporated into M. bovis ATCC35721. Cointegrates were selected using
kanamycin resistance.
~4000 Kmr M. bovis ATCC35721 cointegrates were obtained. To determine the
diversity of the clones, 12 random cointegrates were analyzed by Southern blot.
The probe used in the southern blots was the Sspl-Dral fragment from pYUB178
that contains the BcII cloning site. There is one PstI site between the Dral site
and the int gene on the plasmid and there is another PstI site on the
Mycobacterial chromosome adjacent to the attB site. If genomic DNA from
cointegrate strains is digested with PstI and probed with the SspI-Dral fragment
from pYUB178 either one or two DNA bands should be detected; 1) if there are
no PstI sites within the DNA fragment cloned into the BcII site in pYUB178, one
large DNA fragment will be detected, 2) if there is one or more PstI sites within
the DNA inserted in the BcII site, two DNA fragments will be detected, each one
containing one end of the inserted DNA. (If there are two PstI sites within the
inserted DNA, the “internal” fragment will not be detected in this Southern blot).
Southern blot analysis showed that all 12 of the randomly selected cointegrates
produced a different hybridization pattern, indicating the integrated cosmids
contained different DNA fragments inserted into the BcII sites. The library,
therefore is diverse with respect to cloned DNA fragments from WAg200..
Selection of virulent cointegrate strains using a guinea pig model.
Three guinea pigs were inoculated subcutaneously with 107 cfu of pooled
cointegrates and then sacrificed 12 weeks later. Spleens, livers, kidneys, and
lungs were examined for tuberculous lesions. All three guinea pigs inoculated
with the pooled cointegrate strains had macroscopic lesion in their spleens while
control guinea pigs (inoculated with ATCC35721) had no lesions.
The Mycobacteria isolated from the spleen lesions were examined genetically:
Southern blot analysis showed that 11 of 14 (80%) of the clones recovered from
the lesions now produced the same pattern when digested with PstI and probed
with the same probe (SspI-DraI) used before to assess the diversity of the library.
This result indicates that a predominant clone was selected in vivo.
Cloning the complementing locus.
Because there is a NotI site between oriE and attP, and no NotI sites between
that one and BcII site on pYUB178, at least part of the DNA contained on the
complementing cointegrated plasmid could be recovered. DNA from the
“virulent” cointegrate was digested with NotI, ligated, transformed into E. coli, and
transformants were selected using kanamycin. This plasmid contains at least
some of the WAg200 DNA that conferred upon ATCC35721 the ability to form
lesions in guinea pigs.
Since this plasmid probably does not contain all of the WAg200 DNA present in
the cosmid that was integrated in ATCC35721 which conferred virulence, and
may not even contain the fragment of DNA necessary for virulence, the WAg200
DNA fragment recovered from the chromosome was used to probe the original
library in E. coli to search for “full length” clones. 48 of the clones in the library
hybridized to the fragment. All contained a 2.3 kb MluI fragement.
Confirmation that the locus confers virulence on ATCC35721.
To determine if the 2.3 kb MluI fragment was sufficient to confer virulence on
ATCC35721, it was cloned into pYUB178, integrated into the chromosome of
ATCC35721, and the cointegrated strain was inoculated into guinea pigs. The
results of this experiment showed that macroscopic lesions were detected in
spleens within 8 weeks post-inoculation.
DNA sequence analysis of the complementing locus.
The nucleotide sequence of the 2.3 kb MluI fragment was determined and shown
to have the potential to encode a sigma factor. Sigma factors are small proteins
that provide specificity to RNA polymerase by functioning as highly specific DNA
binding proteins. The gene was named rpoV (for virulence). Comparison of the
nucleotide sequence of rpoV from WAg200 and ATCC35721 revealed a signal
nucleotide difference resulting in an Arg to His substitution at position 522 of the
protein’s amino acid sequence. This signal amino acid substitution apparently
renders the RpoV protein in ATCC35721 non-functional – or at least insufficiently
functional with respect to virulence regulated genes. The hypothesis is the RpoV
sigma factor directs RNA polymerase to the promoters of virulence related genes
and without RpoV, virulence genes are not expressed.