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Chapter 3
ESX-5-deficient Mycobacterium marinum is hypervirulent in adult zebrafish
Weerdenburg EM, Abdallah AM, Mitra S, de Punder K, van der Wel NN, Bird S, Appelmelk BJ,
Bitter W, van der Sar AM
This work was published in Cellular Microbiology 2012; May;14(5):728-39; and published online
before print February 12, 2012, doi 10.1111/j.1462-5822.2012.01755
The original article can be accessed at: http://onlinelibrary.wiley.com/doi/10.1111/j.14625822.2012.01755.x/full
3
ESX-5-DEFICIENT
MYCOBACTERIUM MARINUM
IS HYPERVIRULENT IN ADULT
ZEBRAFISH
Eveline M. Weerdenburg1, Abdallah M. Abdallah1,2, Suman Mitra3, Karin de Punder2,
Nicole N. van der Wel2, Steve Bird3, Ben J. Appelmelk1, Wilbert Bitter1
and Astrid M. van der Sar1
Department of Medical Microbiology and Infection Control, VU
University Medical Center, Amsterdam, The Netherlands,
2
Division of Cell Biology-B6, Netherlands Cancer Institute-Antoni van Leeuwenhoek
Hospital (NKI-AVL), Amsterdam, The Netherlands,
3
Scottish Fish Immonology Research Centre, School of Biological Sciences,
University of Aberdeen, Aberdeen, UK
1
Cellular Microbiology, 2012 May;14(5):728-39
SUMMARY
ESX-5 is a mycobacterial type VII protein secretion system responsible for transport
of numerous PE and PPE proteins. It is involved in the induction of host cell death
and modulation of the cytokine response in vitro. In this work, we studied the effects
of ESX-5 in embryonic and adult zebrafish using Mycobacterium marinum. We found
that ESX-5-deficient M. marinum was slightly attenuated in zebrafish embryos.
Surprisingly, the same mutant showed highly increased virulence in adult zebrafish,
characterized by increased bacterial loads and early onset of granuloma formation
with rapid development of necrotic centres. This early onset of granuloma formation
was accompanied by an increased expression of proinflammatory cytokines and tissue
remodelling genes in zebrafish infected with the ESX-5 mutant. Experiments using
RAG-1-deficient zebrafish showed that the increased virulence of the ESX-5 mutant
was not dependent on the adaptive immune system. Mixed infection experiments with
wild-type and ESX-5 mutant bacteria showed that the latter had a specific advantage
in adult zebrafish and outcompeted wild-type bacteria. Together our experiments
indicate that ESX-5-mediated protein secretion is used by M. marinum to establish a
moderate and persistent infection.
48
INTRODUCTION
3
HYPERVIRULENCE OF ESX-5-DEFICIENT M. MARINUM
Mycobacterium tuberculosis is a very successful intracellular pathogen that is able
to persist for decades within its human host. An important factor that determines
pathogenicity of this infectious agent is secretion of virulence factors. In order to transport
proteins across their complex cell envelope, mycobacteria have a set of homologous
secretion systems which are also known as type VII secretion systems (T7SS) and are
encoded by the ESX loci in their genomes [1,2]. ESX-1 was the first T7SS to be identified
and is the most studied one. It is required for secretion of several virulence factors, such
as ESAT-6 and CFP-10 [3]. Pathogenic mycobacteria of the M. tuberculosis cluster that
lack ESX-1, such as Mycobacterium microti, the natural mutant Mycobacterium bovis
BCG and a deletion mutant of Mycobacterium tuberculosis, display reduced virulence
in vivo when compared to the ESX-1 knock-in or wildtype M. tuberculosis strains [4,5].
Recently, also ESX-5 was identified as an active protein secretion system [6,7].
Unlike ESX-1, the ESX-5 gene cluster is restricted to slow-growing, mostly pathogenic
mycobacteria such as M. tuberculosis and the fish pathogen Mycobacterium marinum.
In these species, ESX-5 has been shown to be responsible for the transport of different
members of the PE and PPE protein families [6-8]. For example, almost all members
of the PE_PGRS subfamily of PE proteins that are expressed during in vitro growth
of M. marinum, were found to be secreted in an ESX-5 dependent manner [6]. This
indicates that this pathogen may secrete more than 100 different proteins via ESX-5.
PE and PPE proteins are specific for mycobacteria and partially associated with the
ESX loci. In several mycobacterial species that contain ESX-5, PE and PPE genes are
highly expanded throughout the whole genome [9]. For instance, in M. tubercolosis,
about 10% of the coding potential of the genome is devoted to PE and PPE proteins
[10]. Together, these characteristics suggest that these ESX-5-secreted proteins are
important for virulence. However, their exact function is not clear.
Over the last years, different roles for PE and PPE proteins have been proposed.
Several PE and PPE proteins were found to be located on the cell surface of mycobacteria,
suggesting interaction with macrophages or other host cells [11-13]. Other studies showed
that individual PE and PPE proteins are involved in immune modulation of macrophages
or virulence in vivo [14-17]. In order to study the effects of a large group of PE and PPE
proteins simultaneously, mycobacterial pathogens disturbed in ESX-5- mediated protein
secretion can be used. With this approach, ESX-5 of M. marinum was found to be involved
in the induction of cell death and modulation of the cytokine response of macrophages
[18]. However, in vivo experiments with ESX-5-deficient mycobacteria that can provide
more insight into the role of multiple PE and PPE proteins during infection have not been
described yet. Therefore, we made use of the zebrafish infection model to study the effect
of all ESX-5 effector proteins during M. marinum infection of a natural host. Surprisingly,
our results show that loss of protein secretion by ESX-5-deficiency leads to hypervirulence
in adult zebrafish. In contrast, disruption of ESX-5 does not affect virulence in macrophages
and even leads to attenuation in zebrafish embryos. Our data shows that mycobacterial
ESX-5-mediated protein secretion plays an important role during infection in vivo.
49
RESULTS
Replication of ESX-5-deficient M. marinum is normal in human macrophages
Previously, disruption of M. marinum ESX-5 has been shown to affect cell death and
cytokine release of macrophages [18]. These results suggest that secreted PE and
PPE proteins play an important role during the initial steps of infection. To determine
whether the ESX-5 secretion system is also important for intracellular replication and
spread, THP-1 macrophages were infected with M. marinum wild-type and ESX-5
mutant strains and bacterial growth was assessed. Intracellular CFU counts up to
3 days after infection did not show a significant difference between both strains,
indicating that ESX-5 substrates have no significant effect on intracellular replication
of M. marinum (Fig. 1A). Fluorescence microscopy confirmed that also cell to cell
spread was not affected, since a comparable increase in the percentage of infected
macrophages throughout the course of infection with both strains was observed
(Fig. 1B). These results seem to contradict a report published previously, in which
ESX-5 was suggested to be involved in macrophage escape and spreading to other
cells [7]. However, those earlier results were obtained with a mutant that turned out to
be defective in both ESX-5 and ESX-1 [6], indicating that the observed effects could
be due to the absence of ESX-1 or a combination of the two mutations.
ESX-5 is involved in bacterial growth at the early stage of zebrafish
embryo infection
Although disruption of ESX-5 did not have an effect on intracellular replication of
M. marinum, it induces the production of pro-inflammatory cytokines such as TNF-α,
IL-12p40 and IL-6 in human macrophages within 24 hours [18] (Fig. S1). This increase
in pro-inflammatory cytokine levels may lead to enhanced attraction of other immune
cells, resulting in improved clearance of the bacteria in vivo. Alternatively, it could
facilitate bacterial spread to neighbouring cells. In order to investigate whether ESX-5
affects the outcome of infection in vivo, we infected zebrafish embryos with M. marinum
wild-type and ESX-5 mutant strains and determined bacterial growth by whole embryo
plating. Up to 5 days after infection, bacterial counts in zebrafish embryos infected
with the ESX-5 mutant were significantly reduced when compared with the M. marinum
wild-type strain (Fig. 2A). This effect could be partly complemented by reintroduction
of the disrupted gene (Mmar_2676) in the ESX-5 mutant strain. At later time points
however, equal amounts of bacteria were found in all groups. These results indicate
that the effect of inactivation of ESX-5-mediated protein secretion on the immune
response of macrophages may lead to increased bacterial clearance during the first
few days of infection in zebrafish embryos.
Since zebrafish embryos are transparent, clustering of cells infected with mCherry
expressing M. marinum strains –a process called early granuloma formation [19]– can
be visualized and followed in time. It is known that M. marinum strains deficient for
ESX-1-mediated protein secretion are impaired in the induction of this process as
well as growth in zebrafish embryos [20,21]. Since ESX-5-deficient M. marinum is also
50
A
10 8
Wild-type
Tn::ESX-5
CFU
10 7
3
10 6
10 4
B
Percentage infected cells
100
80
0
1
2
Days after infection
3
Wild-type
Tn::ESX-5
60
40
20
0
2
Days after infection
HYPERVIRULENCE OF ESX-5-DEFICIENT M. MARINUM
10 5
5
Figure 1. ESX-5 is not required for bacterial replication and spread in macrophages. A. PMAstimulated THP-1 cells were infected with wild-type and ESX-5-deficient M. marinum at an MOI
of 0.5 and at several time points intracellular bacterial growth was determined by CFU analysis.
Values represent mean ± standard error of four different experiments. B. THP-1 cells were
infected with wild-type and ESX-5-deficient M. marinum at an MOI of 10 and after 2 and 5 days
the percentage of infected cells was determined by fluorescence microscopy. Values represent
mean ± standard error of four different experiments.
attenuated in growth during the first few days of infection, we examined whether ESX-5
plays a role in early granuloma formation. Using fluorescence microscopy, we found that
ESX-5-deficient M. marinum was still able to elicit early granuloma formation, although
the amount of granulomas was considerably reduced as compared to the wild-type
strain (Fig. 2B and C). No differences in the size of early granulomas were observed
(Fig. 2D). Our results indicate that ESX-5 mediated protein secretion by M. marinum
plays a moderate role in the early phase of infection of the zebrafish embryo.
ESX-5 deficiency leads to increased bacterial growth in adult zebrafish
which is independent of the adaptive immune system
Zebrafish embryos are well-suited to study the early steps of infection with M. marinum
in a context of innate immunity. However, chronic mycobacterial infection is not
51
C
10 5
Wild-type
Tn::ESX-5
Tn::ESX-5c
CFU per fish
10 4
Fluoresence intensity (percentage)
A
10 3
10 2
10 1
0
5
10
Days after infection
B
15
D
*
120
Wild-type
100
80
60
40
20
0
Wild-type
Tn::ESX-5
Tn::ESX-5
Wild-type
Tn::ESX-5
Figure 2. ESX-5 is important at the initial stage of infection of zebrafish embryos. A. 28 Hpf
zebrafish embryos were infected with 102 CFU of wild-type, ESX-5-deficient, or complemented
ESX-5-deficient M. marinum and bacterial growth was determined by CFU analysis after whole
embryo plating. Values represent mean ± standard error of CFU from at least 10 fish per time
point. B. Zebrafish embryos were infected as indicated and after 5 days clustering of infected
macrophages (early granuloma formation) was visualized by fluorescence microscopy. Scale
bars are 500 μm. C. Early granuloma formation in zebrafish embryos infected with wild-type or
ESX-5-deficient M. marinum was quantified [20]. Values represent mean ± standard error of four
different experiments. D. High magnification of a cluster of infected macrophages from zebrafish
embryos infected for 5 days with wild-type or ESX-5-deficient M. marinum. Scale bars are 10 μm.
established in embryos due to the absence of full immunity. Therefore, we infected
one-year old adult zebrafish with wild-type and ESX-5-deficient M. marinum strains
and followed these fish in time. Normally, zebrafish infected with the M. marinum
wild-type strain used in this study (E11) do not show clear symptoms of disease
until 6-8 weeks of infection [22]. To our surprise, zebrafish infected with the isogenic
M. marinum ESX-5 mutant became moribund within 2-3 weeks of infection (Fig. 3A),
and had to be euthanized in accordance with the rules of our ethical committee.
In contrast, adult zebrafish that were infected with the wild-type strain appeared
completely healthy at this time. In a second experiment, we again observed increased
virulence, although this time the ESX-5 mutant induced terminal illness after 4 weeks
of infection. Determination of bacterial loads in the organs of infected fish revealed
that bacterial numbers of both wild-type and ESX-5-deficient M. marinum were similar
after 6 days of infection. However, at 11 days post infection the ESX-5 mutant showed
significantly increased bacterial numbers (Fig. 3B and C). In spleens and livers of fish
infected with the ESX-5 mutant, bacterial loads continued to increase up to 4 weeks
of infection, whereas CFU levels of the wild-type strain remained relatively constant
during this period. With the exception of the latest time point in the spleen, infection
with the complemented mutant strain resulted in CFU levels comparable to wild-type
52
3
HYPERVIRULENCE OF ESX-5-DEFICIENT M. MARINUM
infection indicating that the observed increase in bacterial numbers was due to the
absence of a functional ESX-5 secretion system.
Our experiments show that ESX-5-deficient M. marinum becomes hypervirulent
in adult zebrafish, but not in macrophages or embryonic zebrafish. Since embryos rely
solely on innate immunity for their defence against pathogens, we reasoned that the
adaptive immune system might mediate the increased virulence observed after one
week of infection in adult zebrafish. Therefore, we infected adult ragI-/- zebrafish, which
lack functional T and B lymphocytes but possess macrophages and granuloctyes [23].
Determination of bacterial loads in livers and spleens two weeks post infection revealed
that in ragI-/- zebrafish, ESX-5-deficient M. marinum still had a growth advantage over the
wild-type strain, indicating that its hypervirulence is not mediated by the adaptive immune
system of the host (Fig. 3D). We found decreased bacterial numbers in ragI-/- zebrafish
as compared to wild-type zebrafish, which contradicts with a previous report showing
hypersusceptibility of ragI-/- zebrafish to M. marinum M-strain infection within one week
post infection [24]. Possibly, the increased numbers of granulocytes that are present in
ragI-/- zebrafish [23] may lead to enhanced innate immunity and improved clearance of
the M. marinum wild-type strain E11 that is used in this study. This isolate induces chronic
progressive disease in zebrafish whereas the M-strain, that is used in most other studies,
causes acute disease [22]. In addition, studies in ragI-deficient mice show that increased
susceptibility to mycobacterial infection is not necessarily reflected in the bacterial burden
[25-27]. Overall, our results show that ESX-5-deficient M. marinum is hypervirulent in adult
zebrafish due to mechanisms that do not involve the adaptive immune system.
Absence of the ESX-5-mediated protein secretion leads to rapid
and increased granuloma formation in adult zebrafish
Histological analysis revealed a high amount of solid granulomas in the spleens and
pancreata of zebrafish infected for 11 days with the M. marinum ESX-5 mutant, whereas only
few granulomas were present in fish infected with the wild-type strain or complemented
mutant (Fig. 4A and B). Four weeks after infection, the high number of granulomas elicited
by the ESX-5 mutant largely destroyed the cellular organisation of the pancreas. About half
of these lesions contained a caseous necrotic center and some of them were multifocal. At
this time point of four weeks after infection, granulomas in the pancreata of fish infected
with the wild-type strain had developed to an extent comparable to the 11-day stage in
ESX-5 mutant infected fish (Fig. 4A and B) and were all non-necrotic (Fig. 4B and C). Very
few granulomas were present in the spleens. These data suggest that ESX-5 has an effect
on the timing of granuloma formation but not on the process itself.
Granuloma formation in zebrafish infected with ESX-5-deficient
M. marinum is accompanied by up-regulation of pro-inflammatory genes
In macrophage infection experiments, ESX-5-deficient M. marinum was shown to
specifically induce the production of pro-inflammatory cytokines [18]. In order to get
more insight in the immunological processes that occur during infection in vivo and
which may underlie the observed hypervirulence, we measured mRNA levels of several
53
B
100
*
CFU per liver
80
60
40
20
0
CFU per spleen
C
0
10
20
30
40
Days after infection
10 3
*
CFU per liver
***
** ** **
Wt
Tn::ESX-5
Tn::ESX-5c
Wt+Tn::ESX-5
D
*
Wt+Tn::ESX-5
10 1
6
11
Days after infection
***
ion
54
***
Wt
Tn::ESX-5
Tn::ESX-5c
Wt +Tn::ESX-5
10 2
6
11
Days after infection
28
10 5
Wt ragI+/+
Tn::ESX-5 ragI+/+
10 4
Wt ragI-/Tn::ESX-5 ragI-/-
10 3
10 2
10 1
Liver
Spleen
D
6
11
Days after infec
10 5
10 4
10 3
10 2
10 1
10 0
28
10 3
10 0
28
Tn::ESX-5
10 2
10 1
CFU per fish 14 dpi
tion
Wt
Tn::ESX-5c
10 4
50
10 2
10 1
50
***
** ** **
*
40
10 3
Wild-type
Tn::ESX-5
10 4
10 0
B
10 4
CFU per fish 14 dpi
Percent survival
A
Liver
◀
immunity-related genes in livers and spleens of adult zebrafish during infection with
M. marinum wild-type and ESX-5-deficient strains and compared them to uninfected
fish. After one week of infection with either of the strains, we did not find clear upor down-regulation for most of the genes tested (Fig. 5A), except for an infectiondependent increase in expression levels of the matrix metalloproteinase gene mmp13.
Two weeks after infection, expression levels of this tissue remodelling gene, which is
suggested be involved in granuloma formation [28], were still increased in organs of
fish infected with the ESX-5 mutant but not in those infected with the wild-type strain
(Fig. 5B). At this timepoint we also observed a specific up-regulation of transcript
levels of the pro-inflammatory cytokines tnf-α, ifn-γ and il-1β in organs of zebrafish
infected with ESX-5-deficient M. marinum. These gene expression patterns correlate
with the increased bacterial load and infection-associated pathology. Therefore, our
results suggest that there are no major mycobacteria-induced modifications of the
inflammatory response that precede specific outgrowth of ESX-5-deficient M. marinum,
but that the up-regulation of pro-inflammatory cytokines at the infection site is a
marker of disease progression. When mRNA levels of whole fish were compared,
specific effects of ESX-5-deficiency on host gene transcription were, probably due to
a dilution of the observed tissue effects, not observed (results not shown), underlining
the importance of using tissues with high numbers of infected host cells.
3
HYPERVIRULENCE OF ESX-5-DEFICIENT M. MARINUM
Figure 3. In adult zebrafish bacterial growth of ESX-5-deficient M. marinum is highly increased.
A. Kaplan-Meier survival curve of adult zebrafish following infection with 2*104 CFU wild-type or
ESX-5-deficient M. marinum. Fish were terminated when signs of severe disease were displayed
(i.e. skin ulcers, lethargy). At least 10 zebrafish per condition were used. Corresponding bacterial
loads in organs of these fish after 16 days of infection can be found in Fig. S2. B - C. Adult
zebrafish were infected with 2*104 CFU wild-type, ESX-5-deficient, complemented ESX-5deficient or 1*104 CFU wild-type together with 1*104 CFU ESX-5-deficient M. marinum and after
6, 11, and 28 days the bacterial loads in liver (B) and spleen (C) were determined by CFU analysis
on 7H10 plates. Values represent mean ± standard error of CFU counts obtained from 6 zebrafish
per group. Statistical differences were determined by the Student’s t-test (* p<0.05, ** p<0.01,
*** p<0.001). D. Adult isogenic rag1–/– and rag1+/+ zebrafish siblings were infected with 2*104
CFU wild-type or ESX-5-deficient M. marinum and after 14 days bacterial loads in livers and
spleens were determined by CFU analysis on 7H10 plates. Values represent mean ± SE of CFU
counts obtained from at least 3 zebrafish per group.
M. marinum ESX-5 does not affect the hypoxic response in vitro
Since we found no indications for differential host gene expression in response to
infection or involvement of the adaptive immune system in the increased bacterial
growth, rapid induction of granulomas and early death of adult zebrafish infected with
ESX-5-deficient M. marinum, we examined the possibility that these effects were not
due to an inappropriate host response but instead a result of bacterial adaptations
to the environment created by the host. Studies on different clinical isolates of M.
tuberculosis have shown that strains of the recently evolved Beijing lineage display
increased virulence, probably as a result of enhanced and constitutive expression
of the DosR regulon. This regulon is a dormancy-associated transcriptional program
55
A
Uninfected
Wild-type
Tn::ESX-5
A
Pancreas
*
*
*
Spleen
** *
*
B
Nr. of granulomas per fish
C
80
60
Wild-type
Tn::ESX-5c
*
*
*
*
Tn::ESX-5
Wt - Pancreas
Wt - Spleen
Tn::ESX-5 - Pancreas
Tn::ESX-5 - Spleen
*
40
20
0
6
11
Days after infection
28
Figure 4. ESX-5-deficient M. marinum causes increased granuloma formation in adult zebrafish.
A. Adult zebrafish infected for 11 days with wild-type, ESX-5-deficient or complemented ESX5-deficient bacteria were analyzed for granuloma formation by HE staining of histological
sections. Stars indicate (solid) granulomas. Scale bars are 20 μm. B. Quantification of the amount
of granulomas in zebrafish infected with M. marinum wild-type or ESX-5 mutant strain. Values
represent mean ± standard error of one (6 dpi), two (28 dpi) or three (11 dpi) fish per group. C. HE
staining of a typical granuloma in the pancreas of wild-type or ESX-5 mutant infected zebrafish
(28 dpi). Centre of granuloma in zebrafish infected with the ESX-5 mutant shows extensive
necrosis. Star indicates solid granuloma. Scale bars are 50 μm.
that is essential for mycobacteria to survive in hypoxic conditions and for a rapid
resumption of growth upon transition to more favourable conditions [29,30]. Compared
to macrophages and zebrafish embryos, tissues of adult zebrafish are probably more
prone to hypoxic conditions. For this reason increased growth of the M. marinum ESX-5
mutant in adult zebrafish may be a result of an enhanced ability to adapt to anaerobic
conditions. To test this possibility we measured mRNA expression of the M. marinum
orthologues of M. tuberculosis dosR and the DosR-regulated gene hspX in wild-type
and ESX-5 mutant strains under standard growth conditions in vitro. However, we did
not find any differences in gene expression indicating normal DosR-mediated gene
regulation (Fig. 6A). In addition, bacterial growth in vitro under normal and hypoxic
conditions did not differ between the two strains (Fig. 6B and C), nor was there a
difference in antibiotic sensitivity (not shown). Furthermore, ESX-5-deficient and
wild-type M. marinum re-isolated from infected adult zebrafish showed similar growth
rates during re-infection of THP-1 cells, indicating that presence in the host does not
lead to enduring changes in ESX-5 related growth characteristics (data not shown).
56
A
1
3
0
-1
-2
B
Wt
Tn::ESX-5
TNF-a IFN-y
IL-1b
IL-10
IL-4
MMP-9 MMP-13
Log fold change 14 dpi
2
Wt
Tn::ESX-5
1
0
-1
-2
TNF-a IFN-y
IL-1b
IL-10
IL-4
HYPERVIRULENCE OF ESX-5-DEFICIENT M. MARINUM
Log fold change 7 dpi
2
MMP-9 MMP-13
Figure 5. Gene expression levels in spleens of adult zebrafish during infection with M. marinum.
RNA was extracted from spleens of infected or uninfected adult zebrafish after 7 (A) and 14 (B) days
of infection and mRNA levels of ifn-γ, tnf-α, il-1β, il-4, il-10, mmp-9 and mmp-13 were measured.
Gene expression levels of zebrafish infected with M. marinum were compared with those of mockinfected fish. Graphs represent relative expression levels in spleens of three fish per group.
Wild-type mycobacteria cannot suppress hypervirulence
of the ESX-5 mutant strain
Previous in vitro experiments showed that the M. marinum wild-type strain is able
to suppress the macrophage pro-inflammatory immune response that is elicited by
ESX-5-deficient M. marinum [18]. Therefore, we investigated whether the M. marinum
wild-type strain is also able to suppress the detrimental effects caused by the ESX-5
mutant strain in vivo. To this end we infected adult zebrafish simultaneously with
wild-type and ESX-5 mutant bacteria. Total bacterial growth in this mixed infection
was comparable to wild-type-only infection up to 11 days (Fig. 3B and C), suggesting
that wild-type bacteria were indeed able to suppress hypervirulence of the ESX-5
mutant strain. However, four weeks after infection, bacterial loads in livers and spleens
had increased to levels observed in the mutant-only infection. Analysis of resulting
colonies showed that at this time point more than 95% of all bacteria were ESX-5-
57
A
Copy nr. relative to SigA
10 0
Wild-type
Tn::ESX-5
10 -1
10 -2
B
Hspx_1
CFU per ml
10 9
DosR (2)
Wild-type
Tn::ESX-5
10 8
10 7
10 6
C
0
10 9
CFU per mL
DosR (1)
20
40
60
Time (hours)
80
100
80
100
Wild-type
Tn::ESX-5
10 8
10 7
10 6
0
20
40
60
Time (hours)
Figure 6. Hypervirulence of ESX-5 mutant strain cannot be explained with in vitro growth
characteristics. A. qRT-PCR analysis of M. marinum dosR (Mmar_3480 (1) and Mmar_1516 (2))
and hspX_1 (Mmar_3484) mRNA levels. Gene expression levels were normalized to sigA. Values
represent the average ± standard error of three experiments. B - C. Mycobacterial growth of
M. marinum wild-type and ESX-5 mutant strains in 7H9 liquid agar with normal (B) or low oxygen
levels (C) was determined by OD600 and CFU analysis at several time points. Values represent the
average ± standard error of three experiments.
deficient, whereas equal numbers of wild-type and mutant bacteria had been injected.
This indicates that the ESX-5 mutant had outgrown the M. marinum wild-type strain
during infection. In addition, these data suggest that the wild-type strain is not able
to suppress the detrimental effects caused by the ESX-5 mutant and moreover, that
wild-type bacteria were not able to profit from the increased granuloma formation and
58
putative immune modulation caused by the mutant. Together, our data indicates that
ESX-5-deficient M. marinum has a specific growth advantage in adult zebrafish.
DISCUSSION
3
HYPERVIRULENCE OF ESX-5-DEFICIENT M. MARINUM
In this report, we show that inactivation of the ESX-5 secretion system in M. marinum
leads to increased virulence in a natural host. Hypervirulence of this M. marinum ESX-5
mutant strain was characterized by highly increased bacterial loads and early death of
adult zebrafish. In addition, well-structured granulomas developed much more rapidly and
in higher numbers than in zebrafish infected by wild-type bacteria. Hypervirulence as a
consequence of gene deletion in mycobacteria has been described before. For example,
disruption of the mce1 operon in M. tuberculosis leads to increased bacterial growth, poorly
organized granulomas and a diminished pro-inflammatory cytokine response in mice [31].
Interestingly, overexpression of the mce1 operon by disrupting the mce1 repressor protein
mce1R had a similar effect on virulence, although in this case an accelerated immune
response and granuloma formation was observed [32]. The latter situation is reminiscent of
the results we obtained with the M. marinum ESX-5 mutant strain in zebrafish, opening the
interesting possibility of a link between the ESX-5 and Mce1.
It is remarkable that the inability to secrete a substantial amount of proteins, which
include all PE_PGRS proteins that are expressed in vitro and may add up to more than 100
proteins, results in such a large increase in virulence. Given the evolutionary expansion
of PE and PPE protein family members in pathogenic mycobacterial species [9], these
proteins would be expected to have an important function in virulence. One possible
explanation for hypervirulence of ESX-5-deficient M. marinum would be that ESX-5mediated PE and PPE protein secretion imposes a high metabolic burden on the bacteria
explaining the increased bacterial growth rate of the mutant. However, our experiments
showed that this is not the case during growth in culture medium or during infection of
macrophages and zebrafish embryos. In addition, the ESX-5 mutant strain of M. marinum
did not display an increased growth rate in vitro during hypoxia nor altered hspX/dosR
gene expression, which are indicative for increased virulence in the M. tuberculosis Beijing
family strains [29]. Therefore our data suggests that the hypervirulent characteristics of
ESX-5-deficient M. marinum are due to the interplay between bacteria and host.
Previous experiments showed that ESX-5 substrates suppress the release of proinflammatory cytokines by macrophages [18]. Elevated pro-inflammatory cytokine
levels may be the cause of the reduced bacterial numbers we found in zebrafish
embryos during the early phase of infection with the ESX-5 mutant. However, at later
stages of infection attenuation is not observed anymore in these embryos. In contrast,
in adult zebrafish infected with the M. marinum ESX-5 mutant bacterial loads were
already after 11 days of infection at least 1 log higher as compared to the wild-type
strain. One of the differences between embryonic and adult zebrafish is the presence
of a functional adaptive immune system. In embryos, it takes several weeks to achieve
immunocompetence [33]. Before this time point, bacterial infections are primarily
dealt with by the innate immune system. Adult zebrafish can also rely on the adaptive
59
immune system, which plays an important role in the defence against pathogens. This
was illustrated by a study using adult ragI-/- zebrafish, which showed the detrimental
effects of the absence of adaptive immunity already one week after mycobacterial
infection [24]. As hypervirulence of the M. marinum ESX-5 mutant was only observed
in the immunocompetent adult zebrafish after more than one week of infection, we
hypothesized that substrates of ESX-5 may interact with mediators of the adaptive
immune system leading to control of infection. However, infection of ragI-/- adult
zebrafish showed a growth advantage of ESX-5-deficient M. marinum despite the
absence of a functional adaptive immune system. Hypervirulence is therefore probably
mediated by other factors that differ between embryonic and adult zebrafish. We
showed that during infection of adult zebrafish with a mixture of both the M. marinum
wild-type and ESX-5 mutant strains, the mutant outcompeted the wild-type strain. The
fact that wild-type bacteria were not able to profit from the mutant’s hypervirulence,
suggests that the advantage of ESX-5-deficient M. marinum does not result from an
altered extracellular environment or general immune response but instead may be
due to more local and possibly intracellular effects.
Although many hematopoietic cell types are produced already at early stages
during embryonic development, these cells can be quite different from the ones that
are present in adult zebrafish. This is a result of several waves of hematopoiesis that
occur during development of the embryo [34]. Therefore, there may be specific markers
of adult macrophages that are not present on or in primitive cells that interact with
ESX-5 substrates resulting in control of infection. It is also possible that ESX-5-deficient
M. marinum is able to spread more easily to other cell types. Due to the absence of
cell-surface localized PE_PGRS and other ESX-5-dependent proteins, the cell wall of
ESX-5-deficient M. marinum is altered (J. Bestebroer, unpublished), which may have
an effect on interaction with different types of host cells. The structural changes of the
mycomembrane may also have an effect on the exposure of cell wall lipids, some of
which induce granuloma formation [35]. It is possible that altered exposure of these cell
surface lipids may result in dramatic differences in the outcome of infection.
The granulomas that are formed in zebrafish during infection with both ESX-5 mutant
and wild-type M. marinum are structurally well-organized, which is also characteristic
for human M. tuberculosis granulomas. As granuloma development progresses,
infected macrophages in the centre undergo necrosis, leading to the accumulation
of caseum in which mycobacteria can multiply [24,36]. Necrotizing granulomas are
usually formed at late stages of mycobacterial infection. In organs of zebrafish, necrotic
granulomas are present after eight weeks of infection with wild-type M. marinum [22].
However, zebrafish infected with ESX-5 mutant mycobacteria develop necrotizing
granulomas much more rapidly and in high amounts. The rapid necrosis, which
facilitates mycobacterial replication, may account for the increased CFU numbers
we find in these fish. Possibly, the type of cell death induced by the ESX-5 mutant
strain during infection is necrotic rather than apoptotic leading to specific outgrowth.
Recently, ESX-5 has been shown to play a role in host cell death [37]. As the induction
60
3
HYPERVIRULENCE OF ESX-5-DEFICIENT M. MARINUM
of apoptosis by PE proteins has also been reported [38,39], it may be possible that
the absence of these proteins may lead to a shift to a more necrotic type of cell death.
The rapid development of granulomas and eventual tissue destruction in zebrafish
infected with the ESX-5-deficient M. marinum correlates with the prolonged induction
of the tissue remodelling genes mmp9 and mmp13. These genes encode matrix
metalloproteinases that have been suggested to be involved in granuloma formation
during mycobacterial infection [28,40,41]. The large increase in transcription of ifn-γ,
tnf-α and il-1β after two weeks indicates high levels of inflammation induced by the
rapidly progressing infection, which probably leads to tissue damage rather than
effective bacterial clearance. At one week of infection, just before the induction of
granulomas, we did not observe significantly altered gene expression levels. Since an
up-regulation of pro-inflammatory cytokines has also been observed at the end-stage
of E11 infection (6-8 weeks) [28], these data suggest that the host’s immune response
to the M. marinum ESX-5 mutant strain is pushed forward in time but not significantly
different from the response to wild-type M. marinum infection.
In summary, we show here that the ESX-5 protein secretion system of M. marinum
plays an important role in progression of infection in a natural host. Our research
indicates that ESX-5-mediated protein secretion is important for the establishment
of a moderate and persistent infection. Consequently, PE and PPE proteins might be
very important for mycobacterial persistence. In this respect it is interesting to note
that recently many members of these protein families were found to be abundantly
expressed at the chronic state of M. tuberculosis infection in guinea pigs [42], which
would support this hypothesis.
EXPERIMENTAL PROCEDURES
Bacterial strains and growth conditions
The mCherry-expressing M. marinum wild-type strain E11 [22], its isogenic ESX-5deficient transposon mutant (Tn::ESX-5, transposon mutant of Mmar_2676) and the
complemented ESX-5 mutant (Tn::ESX-5c) [6] used in this study were grown at 30˚C on
Middlebrook 7H10 agar plates supplemented with 10% OADC (oleic acid–albumin–
dextrose- catalase, BD Biosciences) or in shaking cultures in Middlebrook 7H9 liquid
medium supplemented with 10% ADC (albumin–dextrose-catalase, BD Biosciences)
and 0.05% Tween 80. Kanamycin, hygromycin and chloramphenicol were added when
required at a concentration of 25, 50 and 30 μg/ml, respectively. Anaerobic conditions
were set up according to the Wayne model [43].
Animals
Male 1 year old Danio rerio wild-type and ragI-/- zebrafish were maintained at 28˚C
in 10 L tanks with aerated freshwater on a 14h light/10h dark cycle as described
previously [22]. Prior to infection experiments, wild-type and isogenic ragI-/- zebrafish
were genotyped to confirm mutation of the ragI gene.
61
Cell culture and macrophage infection
The human monocytic cell line THP-1 was cultured at 37˚C and 5% CO2 in RPMI-1640
with Glutamax-1 (Gibco) supplemented with 10% FBS, 100 μg/ml streptomycin and
100 U/ml penicillin. Monocytic differentiation into macrophage-like cells was induced
by overnight incubation with 20 ng/ml PMA (Sigma). For cell infection experiments,
THP-1 cells were seeded in 24-well plates (Costar) at a density of 4*105 cells per well.
After differentiation, cells were washed with infection medium (RPMI + 10% FBS)
and infected with mycobacteria either at an MOI of 1 for 2 hours (for mycobacterial
replication analysis) or at an MOI of 20 for one hour (for cytokine measurement) at
32˚C and 5% CO2. Subsequently, cells were washed three times with infection medium
and incubated at 32˚C and 5% CO2. To analyze intracellular mycobacterial replication,
cells were collected at several time points, lysed with 1% Triton X-100 and plated in
serial dilutions on 7H10 agar plates. For fluorescence microscopy, cells were seeded
on glass cover slips, infected as described above and fixed for 2 hours with 2%
paraformaldehyde and 0.2% glutaraldehyde.
TNF- α secretion
Cell culture supernatants of THP-1 cells were harvested 24 hours after infection and assayed
for TNF-α by ELISA kit (Biosource, Invitrogen) according to the manufacturer’s instructions.
Zebrafish infection
Mycobacteria were grown in 7H9 liquid medium to an OD600 of 1.0 and washed in
phosphate-buffered saline (PBS) prior to infection. Zebrafish embryos were infected 28
hours post fertilization with 100 colony-forming units (CFU) of M. marinum wild-type,
Tn::ESX-5, Tn::ESX-5c or 1 nl PBS by micro-injection in the caudal vein, as described
previously [20]. Adult zebrafish were anaesthetized in 0.02% MS-222 (Sigma) and injected
intraperitoneally with 2*104 CFU of M. marinum wild-type, Tn::ESX-5, Tn::ESX-5c or 1*104
CFU wild-type together with 1*104 CFU Tn::ESX-5. Uninfected control adult zebrafish were
injected with 10 μl PBS. By plating on 7H10 agar, the bacterial CFU in the injected inoculum
was confirmed. All adult fish infection experiments were performed double-blind.
Bacterial quantification
At several time points after infection, at least 10 zebrafish embryos per group were
homogenized in BBL Mycoprep (BD Diagnostics) and plated in 10-fold serial dilutions
on 7H10 agar plates. Adult zebrafish were subjected to terminal anesthesia in MS-222
at 6, 11, 14 and 28 days after infection. Subsequently, the livers and spleens of 6
(6, 11 and 28 dpi) or 3 (14 dpi) zebrafish per group were homogenized in BBL MycoPrep
and plated in 10-fold serial dilutions on 7H10 agar plates to determine bacterial CFU.
Histopathological analysis
At 6, 11 and 28 days after infection, 3 adult zebrafish fish per group were fixed in 4%
paraformaldehyde. Fixed animals were embedded in paraffin and cut frontally in serial
sections from ventral to dorsal. Sections were stained with hematoxylin and eosin (HE).
62
A Zeiss Axioskop light microscope equipped with a Leica DC500 camera was used for
imaging. Image J software was used to adjust brightness and contrast.
RNA extraction and quantitative RT-PCR
Statistical analysis
Adult zebrafish infection experiments were performed at three independent occasions
and similar differences between infection groups were found. Data shown here is
derived from one representative experiment. Infection of ragI-/- zebrafish was performed
twice. All statistical analyses were performed with GraphPad Prism software.
3
HYPERVIRULENCE OF ESX-5-DEFICIENT M. MARINUM
At 7 and 14 days after infection, livers and spleens of 3 adult zebrafish from each
group were snap frozen in liquid nitrogen and stored at -80˚C. Zebrafish organs were
homogenized in TRIzol (Invitrogen) using a drill and micropestles (Eppendorf). For
mycobacterial RNA isolation, pellets of 25 ml bacterial cultures at an OD600 of 1.0 were
resuspended in 1 ml TRIzol. Bacterial cells were disrupted by bead beating, followed by
incubation at 60˚C for 10 minutes. After centrifugation of homogenized zebrafish or lysed
mycobacteria, supernatants were extracted with chloroform and RNA was precipitated
with isopropanol. RNA pellets were washed with 75% ethanol and taken up in RNAse
free water. Contaminating DNA was removed with RNase-free DNase (Fermentas) prior
to RNA purification through a RNeasy MinElute Cleanup kit (Qiagen). SuperScript III
reverse transcriptase (Invitrogen) was used to generate cDNA, of which 1 μg zebrafish
RNA or 200 ng mycobacterial RNA was used in the quantitative real time PCR (qPCR)
reactions. qPCR was perfomed using the SYBR GreenER qPCR kit (Invitrogen) and the
LightCycler 480 (Roche). Ct values were normalized to values obtained for β-actin, a
constitutively expressed zebrafish gene, or the mycobacterial housekeeping gene sigA.
ACKNOWLEDGEMENTS
We thank Wim Schouten for fish care, Theo Verboom, Gunny van den Brink and Esther
Stoop for technical assistance, Ji-Ying Song for help with histopathological analysis of the
zebrafish sections and Peter Peters for providing the environment to perform microscopy
experiments. This work has received funding from the European Community’s Seventh
Framework Programme ([FP7/2007–2013]) under grant agreement n°201762.
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65
SUPPORTING INFORMATION
TNF- (pg/ml)
1500
1000
500
0
Medium
Wild-type
Tn::ESX-5
Tn::ESX-5c
Figure S1. THP-1 cells were infected with wild-type, ESX-5-deficient or complemented ESX-5deficient M. marinum at an MOI of 20 and after 24 hours cell culture supernatant was assayed
for TNF-α levels by ELISA.
10 5
**
*
Liver
Spleen
CFU 16 dpi
10 4
Wt
Tn::ESX-5
10 3
10 2
10 1
10 0
Figure S2. Bacterial loads (CFU) in organs of fish infected with the M. marinum ESX-5 mutant
and wild-type strain after 16 days after infection. Statistical differences were determined by the
Student’s t-test (* p<0.05, ** p<0.01).
66