Type VII (ESX) secretion systems of pathogenic mycobacteria, such as Mycobacterium tuberculosis, are crucial for intracellular survival, host cell lysis, and the subsequent cell-to-cell spread. In this issue of Cell Host & Microbe, Rybniker et al. (2014) have used these characteristics to identify two classes of type VII secretion inhibitors

Bitter, Wilbert

Kuijl, Coen

Bitter, W.

Kuijl, C.P.

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Targeting bacterial virulence: the coming out of type VII secretion inhibitors.

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Targeting Bacterial Virulence: The Coming Out of Type VII Secretion Inhibitors 

Cell Host & Microbe
PreviewsTargeting Bacterial Virulence:
The Coming Out of Type VII
Secretion InhibitorsWilbert Bitter1,2,* and Coen Kuijl1
1Department of Medical Microbiology, VU University Medical Center, 1081 BT Amsterdam, the Netherlands
2Molecular Microbiology, VU University, 1081 HV Amsterdam, The Netherlands
*Correspondence: w.bitter@vumc.nl
http://dx.doi.org/10.1016/j.chom.2014.09.010
Type VII (ESX) secretion systems of pathogenic mycobacteria, such as Mycobacterium tuberculosis, are
crucial for intracellular survival, host cell lysis, and the subsequent cell-to-cell spread. In this issue of Cell
Host & Microbe, Rybniker et al. (2014) have used these characteristics to identify two classes of type VII
secretion inhibitors.Under the threat of the approaching
‘‘postantibiotic era,’’ new strategies to
combat bacterial infections are being
explored. One of these strategies is to
block virulence mechanisms of bacterial
pathogens (Rasko and Sperandio, 2010).
This strategy has several advantages,
one of which is prevention of resistance
development. Because compounds that
block virulence mechanisms do not affect
microbial growth directly, it is hypothe-
sized that they are not subjected to
(strong) selective pressure for resistance.
Although this outlook is exciting, this hy-
pothesis has not been thoroughly tested
thus far. Another advantage is the rapid
alleviation of disease symptoms by block-
ing toxins and, by extension, toxin deliv-
ery. This approach is already successfully
used to treat active tetanus with human
antitetanus immunoglobulins. Addition-
ally, by using virulence factors as targets,
new chemical classes that were over-
looked previously could turn out to be
very useful in the treatment of bacterial
infections. A final and perhaps most
important advantage is that the normal
microflora is generally not affected by
compounds acting against virulence
mechanisms, which has obvious benefits
for the patients, but also reduces the
potential accumulation of resistance
genes within the host microbiome.
Blocking bacterial virulence can be
achieved in different ways: by blocking
microbial attachment (adhesion blockers),
by preventing biofilm formation, by dis-
rupting the regulation of virulence factors,
and by blocking the function of secreted
toxins. In recent years special attention430 Cell Host & Microbe 16, October 8, 2014has been given to the so-called secretion
inhibitors. These are compounds that
block specialized secretion systems of
bacterial pathogens. In order to transport
crucial virulence effector proteins across
the bacterial cell envelope and some-
times also across the host cell membrane,
bacteria evolved different specialized
secretion systems. In particular, Gram-
negative bacteria and the mycolic acid-
producing bacteria, both protected by
a double membrane, are dependent
on these specialized secretion systems.
These secretion systems are generally
known as type I to type IX secretion sys-
tems (Figure 1). The most enigmatic of
these secretion systems is probably the
type III secretion (T3S) system that resem-
bles and functions like a small syringe,
which is used to secrete a specific subset
of effector proteins into host cells. Impor-
tantly, many of these T3S systems are
unique for bacterial pathogens and crucial
for bacterial virulence.
Different strategies have been success-
fully used for the isolation of secretion
system inhibitors (overview in Figure 1).
Most T3S inhibitors interfere with gene
regulation required for the different stages
of T3S system biogenesis (Marshall and
Finlay, 2014). Other compounds block
the assembly of the injection needle com-
plex or the actual secretion process itself.
Importantly, some of these inhibitors have
a relatively broad range of activity and are
active in several different pathogens. For
instance, the salicylidene acylhydrazide
compounds block T3S systems in both
Chlamydia trachomatis and the Entero-
bacteriacaea. Although several of theseª2014 Elsevier Inc.secretion inhibitors are active in animal
models, they have usually been adminis-
tered at the same time as the pathogen
itself. Therefore, more rigorous animal
testing and pharmacokinetic/pharmaco-
dynamic modeling of these compounds
is required. The most promising secretion
inhibitor is in fact an antibody fragment
directed against the T3S system tip pro-
tein PcrV of Pseudomonas aeruginosa.
A modified single-chain Fab version of
these antibodies has been used with
some success in clinical trials both for
P. aeruginosa sepsis and cystic fibrosis
patients (Milla et al., 2014). However, de-
livery of antibodies to the site of infection
is often challenging, and small molecules
might therefore be preferred.
The new kids on the block in this
field are the type VII secretion (T7S) inhib-
itors. The T7S system is crucial for
M. tuberculosis, the causative agent of
tuberculosis and estimated to be respon-
sible for the death of 1.3 million people
each year. In the last decades, there has
been a steady rise in the number of
tuberculosis cases caused by multidrug-
resistant and extensively drug-resistant
M. tuberculosis strains. Therefore, the
discovery of novel drugs to treat tubercu-
losis is a major priority. Fortunately, after
a long period of near inactivity in this
area, there has been a renaissance in
tuberculosis drug development, which re-
sulted in the identification of a number of
promising new drugs (reviewed in Zumla
et al., 2013). These recent breakthroughs
clearly show that new tuberculosis drugs
with a new mode of action can be
identified.
Figure 1. Overview of Inhibition of Bacterial Secretion Systems
Indicated are the availability of inhibitors, method of identification, method of action of the small molecule, and the species targeted (Baron, 2010; Rybniker et al.,
2014).
Cell Host & Microbe
PreviewsM. tuberculosis has five T7S systems,
designated ESX-1 through ESX-5 (Stoop
et al., 2012). Three of these T7S systems
are important for survival in the host;
ESX-3 is responsible for the uptake of
iron and zinc, and ESX-5 is responsible
for the secretion of immunomodulatory
effector proteins. However, the most
crucial T7S system for virulence is
ESX-1. ESX-1 was identified as the
crucial virulence factor missing in the
attenuated vaccine strainMycobacterium
bovis BCG. About a dozen different
effector proteins have been identified
that are secreted by ESX-1. Although the
exact function of these different effector
proteins still needs to be determined,
together these proteins are required for
the escape of M. tuberculosis from thephagosome of the macrophage into the
cytosol. In addition, the lysis of host cells
and the subsequent cell-to-cell spread is
also dependent on ESX-1 effector pro-
teins. Therefore, blocking ESX-1 is a
promising strategy for the control of
M. tuberculosis.
Rybniker et al. have taken up the
challenge and developed a cell-based
screen to identify ESX-1 inhibitors.
Instead of macrophages, lung fibroblasts,
which are highly susceptible to lysis by
M. tuberculosis in an ESX-1-dependent
fashion, were chosen to monitor ESX-1
secretion. Rybniker et al. developed a
plate assay where they infected fibro-
blast with M. tuberculosis and measured
cell viability after 72 hr of infection. Fifty-
five putative ESX-1 compounds sig-Cell Host & Microbe 1nificantly increased fibroblast survival,
without affecting bacterial growth in cul-
ture. Within this group the benzothio-
phenes and the benzyloxy benzylidene
hydrazines were the two most prominent
chemical classes, and from each entity
one compound, BPT15 and BBH7,
respectively, was chosen for further anal-
ysis. Both these compounds were active
at low doses (IC50 of 2 mM) and, as pre-
dicted, blocked the secretion of ESX-1
effector molecules. Interestingly, BPT15
seemed to be an ESX-1-specific inhibitor,
whereas BBH7 also blocked the secretion
of other, ESX-1-independent, proteins, as
was determined by proteomic analysis.
Perhaps because of this broader secre-
tion defect, BBH7 also affected intracel-
lular growth of bacteria.6, October 8, 2014 ª2014 Elsevier Inc. 431
Cell Host & Microbe
PreviewsNext, Rybniker et al. performed RNA-
seq experiments to identify the mode of
action for these compounds. Exposure
to BBH7 resulted in the upregulation of
many genes associated with a metal-ion
overload. However, further research did
not identify any causative relationship be-
tween metal-ion overload and reduced
protein secretion; in fact, addition of zinc
resulted in increased ESX-1 secretion.
For the BTP15 compound, the analysis
was more successful; Rybniker noticed
that the addition of this compound
affected a limited number of genes.
Most of these affected genes belonged
to both the hypoxia-induced DosR regu-
lon and the MprA regulon. Subsequent
experiments clearly showed that only
mprA levels were affected. MprAB is a
classical two-component signal trans-
duction system, in which MprA is the
response regulator in the cytosol and
MprB sensor histidine kinase in the cyto-
plasmic membrane (He et al., 2006).
MprAB is induced by different forms of
cell envelope stress and upon intracellular
growth within macrophages. Rybniker432 Cell Host & Microbe 16, October 8, 2014et al. showed in an in vitro kinase assay
that BPT15 directly inhibits the MprB his-
tidine kinase of the two-component signal
transduction system. The link between
MprAB and the ESX-1 system is the
espA operon, which is tightly regulated
bymultiple response regulators, including
MprA (Pang et al., 2013). EspA is not only
a substrate of ESX-1, but also required
for the secretion of most other effector
molecules. Disruption of espA regulation
usually results in reduced secretion of
ESX-1 effectors. The next step will be to
test whether these compounds are able
to block mycobacterial virulence in an
animal infection model.
Blocking the T7S system ESX-1 could
be a highly efficient strategy to disarm
M. tuberculosis. However, blocking multi-
ple T7S systems is also an interesting
option, because this pathogen does not
seem to have horizontal gene transfer
and acquires resistance usually by accu-
mulating point mutations. By blocking
multiple essential targets, we might
confront the tubercle bacilli with an
insurmountable task. Interestingly, BBH7ª2014 Elsevier Inc.could be the first more general T7S
blocker.
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Type VII (ESX) secretion systems of pathogenic mycobacteria, such as Mycobacterium tuberculosis, are crucial for intracellular survival, host cell lysis, and the subsequent cell-to-cell spread. In this issue of Cell Host & Microbe, Rybniker et al. (2014) have used these characteristics to identify two classes of type VII secretion inhibitors

Type VII (ESX) secretion systems of pathogenic mycobacteria, such as Mycobacterium tuberculosis, are crucial for intracellular survival, host cell lysis, and the subsequent cell-to-cell spread. In this issue of Cell Host &amp; Microbe, Rybniker et al. (2014) have used these characteristics to identify two classes of type VII secretion inhibitors

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Targeting Bacterial Virulence: The Coming Out of Type VII Secretion Inhibitors

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