Mycobacterium tuberculosis and Tuberculosis (page 3)
(This chapter has 4 pages)
© Kenneth Todar, PhD
Virulence Mechanisms and Virulence Factors
The virulence of Mycobacterium
is extraordinarily complicated and multifaceted. Although the organism
apparently does not produce any toxins, it possesses a huge repertoire
of structural and physiological properties
that have been recognized for their contribution
to mycobacterial virulence and to pathology of tuberculosis. Some of the
general properties of Mycobacterium
tuberculosis that render it virulent are discussed below. This section
is followed by a more specific discussion of the complex array of
virulence determinants exhibited by this pathogen. This should not surprising for
one of the most successful human pathogens to have evolved.
Some general properties of Mycobacterium tuberculosis that
contribute to its virulence.
Special mechanisms for cell entry.
The tubercle bacillus can bind directly to mannose receptors on
macrophages via the cell wall-associated mannosylated glycolipid, LAM,
or indirectly via certain complement receptors or Fc receptors.
Intracellular growth. MTB can
grow intracellularly. This is an effective means of evading the immune
system. In particular, antibodies and complement are ineffective. Once
MTB is phagocytosed, it can inhibit phagosome-lysosome fusion by
secretion of a protein that
modifies the phagosome membrane. It may remain in the
phagosome or escape from the phagosome, in either case, finding a
protected environment for growth in the macrophage.
Detoxification of oxygen radicals.
MTB interferes with the toxic effects of reactive oxygen intermediates
produced in the process of phagocytosis by three mechanisms:
1. Compounds including glycolipids, sulfatides and LAM down regulate
the oxidative cytotoxic mechanism.
2. Macrophage uptake via complement receptors may bypass the activation
of a respiratory burst.
3. The oxidative burst may be counteracted by production of
catalase and superoxide dismutase enzymes.
Antigen 85 complex. This
complex is composed of a group of proteins secreted by MTB that are
known to bind fibronectin. These proteins may aid in walling off the
bacteria from the immune system and may facilitate tubercle formation.
Slow generation time. Because
of MTB's slow generation time, the immune system may not readily
recognize the bacteria or may not be triggered sufficiently to
eliminate them. Many other chronic disease are caused by bacteria with
slow generation times, for example, slow-growing M. leprae causes leprosy, Treponema pallidum causes syphilis,
and Borrelia burgdorferi
causes Lyme disease.
High lipid concentration in cell wall.
This accounts for impermeability and resistance to
antimicrobial agents, resistance to killing by acidic and alkaline
compounds in both the intracellular and extracellular environment, and
resistance to osmotic lysis via complement deposition and attack by
Cord factor. Cord factor
(trehalose 6, 6' dimycolate) is a glycolipid found in the cell walls of
mycobacteria, which causes the cells to grow in serpentine cords. It is
primarily associated with virulent strains of MTB. It is known to be
toxic to mammalian cells and to be an inhibitor of PMN migration. Its
exact role in MTB virulence is unclear, although it has been shown to
induce granulomatous reactions identical to those seen in TB.
M. tuberculosis virulence is
studied both in tissue culture,
using macrophages, dendritic cells or pneumocytes, and in animal
models, primarily mice. Tissue culture models are easier and more humane
work with and give faster results, but they are limited to studying
early stages of infection. Ultimately, only in animal models can all the stages
of TB be studied.
Since sequencing the Mycobacterium
tuberculosis genome in 1998, genetic methods are more commonly
used to study
the bacterium's virulence. The usual genetic approaches to study
virulence are to disrupt, inactivate, modify, delete or complement a
gene and assess the effects in the macrophage or mouse model.
In an exhaustive and comprehensive review of possible virulence
determinants of Mycobacterium
tuberculosis, Issar Smith (Clinical Microbiology Reviews. Vol.
16, No. 3, p 463-496. 2003) identified four culture filtrate
enzymes, eleven cell surface proteins, four groups of enzymes involved
in cellular metabolism (including lipid metabolism, amino acid and
purine biosynthesis, metal uptake, anaerobic respiration and oxidative
stress) and three sets
of transcriptional regulators to which some degree of the organism's
virulence might be attributed.
Of approximately 4,000 genes in the Mycobacterium
tuberculosis genome, 525 are involved in
cell wall and "cell processes", 188 genes encode regulatory proteins, and 91
genes are involved in "virulence, detoxification and adaptation". Over
200 genes are identified as encoding enzymes for the metabolism of
acids. This large number of M.
tuberculosis enzymes that putatively use fatty acids may be
related to the ability of the pathogen to grow in the tissues of the
infected host, where fatty acids may be the major carbon source. This
thought to be an important aspect of
M. tuberculosis physiology during infection. The point is that
these genes, as well as those from other classes, may be directly or indirectly
involved in virulence. Some of these genes and their products or
activities are described below.
19-kDa protein. The 19-kDa
protein is a secreted antigenic protein that is immunologically
recognized by T cells and sera from TB patients. When M. tuberculosis
enters macrophages and other phagocytic cells, this
surface-exposed glyco-lipoprotein is thought to cause host
signaling events as it interacts with its receptor, TLR2. There
is suggestive but inconclusive evidence that mutant strains of Mycobacterium tuberculosis
deficient in production of the 19-kDa protein are rapidly cleared from
the lungs and spleen of infected mice. Adding back the wild-type M. tuberculosis gene to these
strains allowed growth in lungs that was similar to the growth of
standard wild-type strains, which suggested that the 19-kDa protein is
essential for virulence. It has been reported that addition of the
purified 19-kDa protein to human macrophages causes upregulation of the
Th1 cytokine IL-12. Similarly, the 19-kDa protein can activate human
Glutamine synthase. The enzyme
is not secreted into culture filtrates during growth but results from
cell leakage and lysis. glnA1
mutations have not been made in M.
tuberculosis, but the specific glutamine synthase inhibitor,
L-methionine-SR-sulfoximine (MSO), inhibits the growth of Mycobacterium tuberculosis in vitro
and in macrophages but has no effect on
nonpathogenic mycobacteria. In addition to its essential role in
nitrogen metabolism, glutamine synthase is involved in the synthesis of
a poly-L-glutamate-glutamine cell wall component found in pathogenic
mycobacteria. These findings have led to the suggestion that
this enzyme is a possible
target for the development of new drugs which have less toxicity than MSO for
Erp. Erp is a surface-located
protein secreted by M. tuberculosis.
The protein is similar to an exported 28kDa antigen (the PLGTS
antigen) in M. leprae and is
not found in nonpathogenic mycobacteria. Its function in virulence is
Mas. Mas encodes
mycocerosic aid synthase, an enzyme that catalyzes the synthesis of
long-chain, multiply methylated branched fatty acids, called
mycocerosic acids, that are found only in pathogenic mycobacteria.
FbpA. Mycobacteria have three
mycolyl-transferase enzymes, encoded by three genes, fbpA, fbpB, and fbpC, that transfer long-chain
mycolic acids to trehalose derivatives. The proteins can also bind
the cell matrix protein fibronectin. The Fbp proteins are also found in
the culture filtrate and are known as the antigen 85A, 85B, and
85C complex (Antigen 85 complex).
The three fbp genes
have been separately inactivated, but only the M. tuberculosis fbpA mutant showed
severely attenuated growth in human and murine macrophages.
The observation that these proteins produce a formidable immunologic
response led to
the creation of a new live vaccine that was made by introducing the M. tuberculosis fbpB gene into M. bovis BCG. This recombinant
strain shows better protection against virulent M. tuberculosis infection than does
the parent BCG strain in a guinea pig model.
OmpA. OmpA is a porin-like
protein that can form pores in liposomes, a general property of porin family
expression is induced by low pH, as well as by engulfment into
macrophages. An ompA mutant
following phenotypes: although it showed delayed growth at acid pH, it
ultimately grew at wild-type levels; it could not take up small
molecules like serine at low pH; it showed reduced ability to grow in
both human and murine macrophages. This suggested that the environment
encountered by M. tuberculosis
during infection is acidic, and that OmpA played a role in the bacterial
response to this condition.
HbhA. HbhA is a heparin-binding
hemagglutin protein that is localized on the surface of virulent
mutants exhibited wild-type
ability to be phagocytosed by and to grow in murine or human
macrophages. They are taken up poorly by pneumocytic cells,
although the bacterial generation time was normal intracellularly. The
mutant grew normally in the lungs of infected mice but had a longer
generation time and reached a lower bacterial load in spleens compared
to the wild-type. The properties of this mutant indicate that
HbhA is important for M. tuberculosis
interaction with pneumocytes and that this interaction may play a
role in extrapulmonary dissemination.
LAM. LAM is a complex
that contains repeating arabinose-mannose disaccharide subunits. It is
a major component of the M.
tuberculosis cell wall. It is known to be
an immunomodulator analogous to the 19-kDa protein. Addition of LAM to
murine macrophages depresses IFN-gamma production. LAM can also
scavenge oxygen radicals, in vitro, and inhibits the host protein
kinase C. It has been suggested that LAM functions to downregulate host immune
responses to M. tuberculosis infection,
to protect the bacterium from potentially lethal mechanisms like
the respiratory burst.
MbtB. The mbt operon, consisting of mbtA through mbtJ, encodes enzymes whose
function is to synthesize mycobactin and carboxymycobactin, the major
siderophores produced by M. tuberculosis.
The operon is part of a regulon that is repressed in high iron conditions. MbtB is an enzyme that
catalyzes an essential step in mycobactin synthesis.
Like many other bacterial pathogens, during infection, Mycobacterium tuberculosis requires
acquisition system consisting of siderophores,
iron from host iron-containing proteins such as transferrin and
lactoferrin. Iron is essential for growth of the bacteria and so the element must
be extracted from protein-bound iron in the host or from largely insoluble ferric salts in the
environment. Iron uptake systems
are required to mobilize these forms of iron
for transport into the cell. In bacteria, siderophores
usually perform this chelation and solubilization function, and the
iron they carry is taken into cells by high-affinity iron transport systems.
When the mbtB gene of M. tuberculosis is inactivated the
resulting mutant strain is unable to synthesize the two
siderophores. The mutant showed wild-type growth in iron-rich media,
but grew poorly when iron is limited. It also grew more slowly than the
wild type in human macrophages indicating that the phagosome containing mycobacteria may be low
in iron. Furthermore, excess iron exacerbates the progression of TB in
humans and animal models. These observations suggest that iron
availability, made possible through the activities of siderophores,
promotes the growth and virulence of the M. tuberculosis.
Oxidative stress proteins. Most aerobic organisms have enzymes
that degrade peroxides and
superoxide, which are normal byproducts of aerobic respiration, but also
are toxic oxygen radicals. These
enzymes, generally superoxide dismutases, catalases and peroxidases, are also important for the response to various
external oxidative stresses. Since phagocytic cells produce oxygen
radicals during the respiratory burst to kill invading bacteria, it is
not surprising that these enzymes may contribute to M. tuberculosis virulence. Enzymes
found in M. tuberculosis that
combat oxygen radicals include AhpC,
an alkyl hydroperoxide
reductase that detoxifies organic hydroxyperoxides, and SodA and SodC,
two species of superoxide dismutase that degrade superoxides, which are
normal by-products of aerobic respiration and are also produced
by the phagocytic respiratory burst.
Nitrate reductase. . M.
tuberculosis was originally thought to be an obligate aerobe,
but there are numerous experimental indicators that the bacterium can
grow in microareophilic environments, especially during the later stages
of infection, e.g., in lung granulomas. Wild type M. tuberculosis has been shown to
possess an inducible nitrate reductase (NarG encoded by narG) which allows respiration
using NO3 as a final electron acceptor. If anaerobic or
growth is an important feature of M.
tuberculosis physiology during infection, the existence of
nitrate reductase could be a significant factor in sustaining growth
under these conditions.
The specific bacterial adhesins involved in the complex interaction
between M. tuberculosis
and the human host are largely unknown. Nevertheless, a few potential
adherence factors have been considered, including the heparin-binding
hemagglutin (HbhA), a
fibronectin-binding protein, and a polymorphic
acidic, glycine-rich protein, called PE-PGRS.
HbhA is a surface-exposed
protein that is involved in binding Mycobacterium
tuberculosis to epithelial cells but not to
phagocytes. It could be involved in extrapulmonary spread after the
initial long-term colonization of the host. Fibronectin-binding
proteins (FbpA), first
identified as the α-antigen (Antigen
85 complex), can bind to the
extracellular matrix protein fibronectin in vitro. This property may
represent a mechanism of tissue colonization. The surface-exposed
PE-PGRS proteins found in M.
tuberculosis and Mycobacterium
show fibronectin-binding properties.
It has been shown recently that Mycobacterium
tuberculosis produces pili
during human infection, which
could be involved in initial colonization of the host (Alteri et al.,
Proc Natl Acad Sci U S A. 2007 March 20; 104(12): 5145–5150). On the
basis of electron microscopic evidence, M. tuberculosis produces a dense
fibrillar meshwork composed of thin coiled, aggregated fibers
resembling pili that extend many microns away from the bacterial
surface. These structures have been named Mycobacterium tuberculosis pili or
MTP. Additionally, biochemical
and genetic data demonstrate that M.
tuberculosis produces pili,
whose pilin subunit is encoded by the Rv3312A
gene. The serum of
tuberculosis patients with active TB has been shown to contain IgG
antibodies to MTP, which suggests that the structures are produced in
vivo during human infection. Furthermore, isolated MTP bind to the
extracellular matrix protein laminin in vitro, suggesting that they act
as an adhesin and may be an important host colonization factor of M. tuberculosis.
Because MTP are produced in vivo, and the M. tuberculosis habitat is
the human body from which it is transmitted directly from person to
person, it is
likely that pili play an important role in some aspect of human TB
infection. If MTP are proven essential for M. tuberculosis to establish
infection, as in certain other microbes, then the purified MTP could be
considered as a vaccine candidate.
Clinical Identification and Diagnosis of
The diagnosis of tuberculosis requires detection of acid-fast
in sputum via the Ziehl-Neelsen method as previously described.
The organisms must then be cultured from sputum. First, the
sample is treated with NaOH. This kills other contaminating
but does not kill the MTB present because cells are resistant to
compounds by virtue of their lipid layer.
The media used for growth of MTB and the the resulting colony
been described previously. However, methods of culturing can take 4-6
to yield visible colonies. As a result, another method is commonly used
call the BACTEC System. The media used in the BACTEC system
radio-labeled palmitate as the sole carbon source. As MTB multiplies,
it breaks down the palmitate and liberates radio-labeled CO2.
Using the BACTEC system, MTB growth can be detected in 9-16 days vs
weeks using conventional media.
Skin Testing is performed as the tuberculin or Mantoux
test. PPD (purified protein derivative) is employed
the test antigen in the Mantoux test. PPD is generated by
a culture of MTB, specifically Old Tuberculin (OT). 5 TU (tuberculin
units), which equals 0.000lmg of PPD, in a 0.1 ml volume is
injected in the forearm. The test is read within 48-72 hours.
Administering the Mantoux test. CDC.
The test is considered positive if the diameter of the resulting
is 10 mm or greater. The lesion is characterized by erythema (redness)
and swelling and induration (raised and hard). 90% of people that have
a lesion of 10 mm or greater are currently infected with MTB or have
been previously exposed to MTB. 100% of people that have a lesion of
mm or greater are currently infected with MTB or have been previously
exposed to MTB.
False positive tests usually manifest themselves as lesser
These lesser reactions could indicate prior exposure or infection with
other mycobacteria or vaccination with BCG. However, in places were the
vaccine is not used, lesser reactions should be regarded as highly
False negatives are more rare than false positives but are
common in AIDS patients as they have an impaired CMI response. Other
such as malnutrition, steroids, etc., can rarely result in a false
Because administration of a single drug often leads to the
of a bacterial population resistant to that drug, effective
for the treatment of TB must contain multiple drugs to which the
are susceptible. When two or more drugs are used simultaneously,
helps prevent the emergence of tubercle bacilli resistant to the
However, when the in vitro susceptibility of a patient's isolate is not
known, which is generally the case at the beginning of therapy,
two agents to which the patient's isolate is likely to be susceptible
be difficult, and improper selection of drugs may subsequently result
the development of additional drug-resistant organisms.
Hence, tuberculosis is usually treated with four different
agents The course of drug therapy usually lasts from 6-9 months.
The most commonly used drugs are rifampin (RIF) isoniazid (INH),
(PZA ) and ethambutol (EMB) or streptomycin (SM). When adherence with
regimen is assured, this four-drug regimen is highly effective. Based
on the prevalence and characteristics of drug-resistant
at least 95% of patients will receive an adequate regimen (at least two
drugs to which their organisms are susceptible) if this four-drug
is used at the beginning of therapy.
a patient who is treated with the four-drug regimen, but who defaults
is more likely to be cured and not relapse when compared with a patient
treated for the same length of time with a three-drug regimen.
Drugs used to treat TB disease. From left
to right isoniazid, rifampin, pyrazinamide, and ethambutol.
(not shown) is given by injection. CDC.
A vaccine against MTB is available. It is called BCG
of Calmette and Guerin, named after the two Frenchmen that developed
consists of a live attenuated strain derived from Mycobacterium
This strain of Mycobacterium has remained avirulent for over 60
The vaccine is not 100% effective. Studies suggest a 60-80%
rate in children.
The vaccine is not administered in the U.S. for several reasons:
� The vaccine cannot circumvent disease reactivation in previously
� The vaccine does not prevent infection, only disease. Therefore,
the entire population would have to be vaccinated if the vaccine was to
be considered efficacious.
� Vaccination may complicate the way the tuberculin skin test is read
in this country. In places that do not vaccinate, the skin test may be
used to monitor the effectiveness of antibiotic therapy.
Tuberculosis (MDR TB) and Extensively
Drug-Resistant Tuberculosis (XDR TB)
Resistance to anti-TB drugs can occur when these drugs are misused or
mismanaged. Examples include when patients do not complete their full
course of treatment; when health-care providers prescribe the wrong
treatment, the wrong dose, or length of time for taking the drugs; when
the supply of drugs is not always available; or when the drugs are of
Multidrug-resistant tuberculosis (MDR
TB) is TB that is resistant to at
least two of the best anti-TB drugs, isoniazid and rifampicin. These
drugs are considered first-line drugs and are used to treat all persons
with TB disease.
Extensively drug resistant TB (XDR TB)
is a relatively rare type of MDR
TB. XDR TB is defined as TB which is resistant to isoniazid and
rifampin, plus resistant to any fluoroquinolone and at least one of
three injectable second-line drugs (i.e., amikacin, kanamycin, or
capreomycin). Because XDR TB is resistant to first-line and second-line
drugs, patients are left with less effective treatment options, and
cases often have worse treatment outcomes.
Both MDR TB and XDR TB are more common in TB patients that do not take
their medicines regularly or as prescribed, or who experience
reactivation of TB disease after having taken TB medicine in the past.
Persons with HIV infection or other conditions that can compromise the
immune system are at highest risk for MDR TB and XDR TB. They are more
likely to develop TB disease once infected and have a higher risk of
death from disease.
The risk of acquiring XDR TB in the United States appears to be
relatively low. However, it is important to acknowledge the ease at
which TB can spread. But long as XDR TB exists, the U.S. is at some
The risk of acquiring MDR TB in the United States is < 0.7% in
U.S.-born persons but higher in foreign-born persons. Since
1998, the percentage of U.S.-born patients with MDR TB has remained
fairly constant. However, of the total number of reported primary MDR
cases, the proportion occurring in foreign-born persons increased from
25% (103 of 407) in 1993 to 80% (73 of 91) in 2006.
The Centers for Disease Control (CDC) Division
Elimination advises how to prevent or minimize the development
strains of M. tuberculosis.
� The most important thing a patient can do is to take all of their
medications exactly as prescribed by a health care provider. No doses
should be missed and treatment should not be stopped early. Patients
should tell their health care provider if they are having trouble
taking the medications. If patients plan to travel, they should talk to
their health care providers and make sure they have enough medicine to
last while away.
� Health care providers can help prevent MDR TB by quickly
cases, following recommended treatment guidelines, monitoring patients�
response to treatment, and making sure therapy is completed.
� Another way to prevent getting MDR TB is to avoid exposure to known
TB patients in closed or crowded places such as hospitals, prisons, or
homeless shelters. persons who work in hospitals or health-care
settings where TB patients are likely to be seen should consult
infection control or occupational health experts. Ask about
administrative and environmental procedures for preventing exposure to
TB. Once those procedures are implemented, additional measures could
include using personal respiratory protective devices.
In February 2008, the World Health Organization released its fourth
global report on anti-TB drug resistance, which indicated that the
number of MDR TB cases worldwide was the highest ever reported (489,139
cases in 2006) and that XDR TB had been reported in 45 countries. A
critical need exists for new drugs and new drug regimens to address
this growing challenge.