Critical ReviewsTM in Eukaryotic Gene Expression, 21(4):337-346 (2011)

New Insights into the Pathogenesis of Tuberculosis

Revealed by Mycobacterium marinum:
The Zebrafish Model from the Systems
Biology Perspective
Wanyan Denga, Xiemei Tangb, Manmei Houb, Chunmei Lib & Jianping Xiea,b,*

Institute of Modern Biopharmaceuticals, State Key Laboratory Breeding Base of Eco-Enviroment and Bio-Resource
a,b

of the Three Gorges Area, School of Life Sciences, Southwest University, Beibei, Chongqing, China

*Address all correspondence to: Jianping Xie; Tel./Fax: 862368367108; Email: jianpingxie@vip.sina.com; jianpingxie@swu.edu.cn; georgex@swu.edu.cn

ABSTRACT: Tuberculosis remains a worldwide health concern, largely due to the emergence of multi-drug-
resistant (MDR) and extensive-drug-resistant (XDR) Mycobacterium tuberculosis co-infection with HIV. The
exact mechanism of Mycobacterium virulence, pathogenesis, and persistence is not fully understood. The hall-
mark of tuberculosis, granulomas are promoted by Mycobacterium virulence factors, and they have long
been considered a structural advantage to the host. However, this traditional view has been challenged re-
cently, largely due to the evidence originating from the M. marinum–zebrafish model. As a genetically trac-
table model, zebrafish provide unprecedented opportunities to address the pathogenesis of tuberculosis from a
systems biology perspective. The latest data from this model are summarized in this review, special attention is
given to the shared pathway and network between zebrafish and humans. This research serves to deepen our un-
derstanding of this complex process and to promote the discovery of better countermeasures against tuberculosis.

KEY WORDS: Tuberculosis, pathogenesis, M. marinum–zebrafish model, systems biology.

I. INTRODUCTION drawbacks of the poorly organized macrophage

Mycobacterium tuberculosis is the causative agent
of tuberculosis (TB), a leading infectious disease.
Approximately one-third of the global population
is infected by this pathogen. Currently, the sole
available vaccine and medications are problematic.1
Novel measures to combat TB are urgently needed.
Insight into the biology of M. tuberculosis is a pre-
requisite for future rational interventions. The read-
ily manipulated M. marinum, highly similar to M.
tuberculosis in sequence, is a unique and beneficial
surrogate of M. tuberculosis that serves to accelerate
our research efforts.
Animal models are crucial to discovering the
pathogenesis of Mycobacterium, and zebrafish pro-
vide an ideal model for this purpose. As the native
host of M. marinum, zebrafish can circumvent the
aggregates that do not undergo necrosis seen in
mouse models,2 the shortage of available immuno-
logical and molecular reagents seen in guinea pig
and rabbit models,3–4 and the ethical and cost issues
related to nonhuman primate models.3 The zebraf-
ish model is more relevant to humans than that of
amoeba (Dictyostelium discoideum) and fruit flies
(Drosophila melanogaster) for the pathogenesis of
TB.4
The typical feature of TB is the formation of
granuloma necroses, which are clusters of infect-
ed macrophages surrounded by additional macro-
phages, neutrophils, and lymphocytes.5 As the main
battlefield of confrontation between pathogen and
host, the traditional role of granulomas is being
challenged.6 Once regarded as beneficial to the host,
recent data cast some doubt on this paradigm. These

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338
new data are summarized here.
II. THE ADVANTAGES OF THE
M. MARINUM–ZEBRAFISH MODEL
The M. marinum–zebrafish model has been well
established as a useful model to address the
pathogenesis of Mycobacterium infections. The
relatively short generation time of 4 h of M. mari-
num in contrast with the >20-h generation time of
M. tuberculosis is a great advantage.7 The natu-
ral hosts of M. marinum are some poikilothermic
animals, including leopard frog (Rana pipiens),
goldfish (Carassius auratus), and zebrafish (Da-
nio rerio). Mycobacterium marinum are less like-
ly to infect humans because the optimum growth
temperature of M. marinum is comparatively
lower.8 The successful zebrafish developmental
model has been expanded for use as a model for
the analysis of host–pathogen interactions dur-
ing infectious disease.9 Zebrafish larvae are op-
tically transparent, allowing real-time monitor-
ing of the early steps of M. marinum infection
in live animals.10 The genome of M. marinum is
extremely conserved in this model, with an 85%
resemblance to human amino acid sequence; in
addition, the immune system of the zebrafish
bears remarkable similarities to its mammalian
counterparts.11 Zebrafish embryos have not yet
developed an adaptive immune system, which
enables the investigation of the role of the innate
immune response during Mycobacterium disease
separately from the role of the adaptive system.12
Importantly, zebrafish produce caseating granu-
lomas resembling human granulomas during M.
marinum infection.13 The apparent advantages of
the M. marinum–zebrafish model over two other
models (i.e., M. marinum–frog and M. marinum–
goldfish) are the availability of genetic research
tools and real-time monitoring of the infection.10
The ever-increasing role of innate immunity in
vertebrates against infection can be better ad-
dressed using the M. marinum–zebrafish model. A
high-throughput screening system can be directly
executed in the M. marinum–zebrafish infection
model, especially for screening compounds.14
Deng et al.
III. NEW INSIGHTS INTO THE
FUNCTIONS OF GRANULOMAS
Granulomas, aggregates of immune cells including
macrophages and lymphocytes, are the hallmark of
TB.15–17 Granulomas have long been viewed as pro-
tective structures that physically “wall off” persist-
ing bacteria to maintain the function of neighboring
tissue and to effectively prevent the replication and
transmission of the pathogen.17 Granulomas have
been viewed as an adaptive structure that forms af-
ter the initiation of adaptive immunity.17 The bacteria
load increases rapidly following the first 2 weeks
of infection, and the plateau coincides with the de-
velopment of adaptive immunity in animal models
of TB.13 Mature granulomas represent a stalemate
or equilibrium between host and pathogen, demon-
strated by the arrest of progressive infection and en-
capsulation of some live bacteria.18 However, recent
studies show that the role of granulomas is stage de-
pendent. The early granulomas facilitate Mycobac-
terium transmission before the formation of adap-
tive immunity,19 but they check the Mycobacterium
growth during later stages.20 Recent studies prefer the
view that granulomas serve as a place for bacterial
growth and spread.7,21 First, granuloma formation co-
incides with accelerated bacterial expansion; it does
not precede the expansion.22 Second, Mm lacking
the ESX-1/RD1 secretion system locus (DRD1 Mm)
is attenuated and is associated with poor granuloma
formation.23 Finally, epithelioid granulomas develop
within a few days of infection, prior to the presence
of adaptive immunity.10 Real-time 3D-DIC (long-
term three-dimensional differential interference con-
trast) and fluorescence in vivo microscopy techniques
used at the whole-animal level have revealed that the
region of difference 1 (RD1) might be responsible
for this subversive discovery.20 Mycobacterium RD1
can both enhance the recruitment of macrophages to
the nascent granulomas and accelerate the pathogen-
esis and transmission of Mycobacterium by invad-
ing the early granulomas.20 In fact, the RD1 region
can initiate granuloma formation.19 ESAT-6 (protein
6-kD early secreted antigenic target), encoded by the
RD1 region, can induce matrix metalloproteinase-9
(MMP9) in epithelial cells neighboring infected mac-
Critical ReviewsTM in Eukaryotic Gene Expression
New insight into Mycobacterium marinum- zebrafish model
rophages. MMP9 has been shown to enhance the re-
cruitment of macrophages. The disruption of MMP9
can attenuate granuloma formation and bacterial
growth. Therefore, interception of epithelial MMP9
production might be promising for host-targeting TB
therapeutics. Whether this scenario exactly mirrors
the M. tuberculosis infection in humans, however,
remains to be determined.
IV. NEW GENES AND FUNCTIONS
INVOLVED IN MYCOBACTERIUM
INFECTION
Many well-characterized and new virulence fac-
tors crucial for Mycobacterium pathogenesis
have been discovered based on research using
the M. marinum–zebrafish model,6 such as those
located in Esx-1 cluster EspA (MMAR_4166,
Rv3616c), EspB (MMAR_5457, Rv3881c),
EsxB (MMAR_0187, Rv3874), and ESAT-6
(MMAR_0188, Rv3875); secreted repeat proteins
Erp (MMAR_5374, Rv3810); phagosome matura-
tion inhibiting protein PmiA (MMAR_3386); Mel1,
Mel2 gene locus protein MelA (MMAR_2864,
Rv2560), MelB (MMAR_2165, Rv2566), MelC
(MMAR_2164, Rv2567), MelD (MMAR_2163,
Rv2568c), MelE (MMAR_2162, Rv2569c), MelF
(MMAR_2864, Rv1936), MelG (MMAR_2865,
Rv1937), Mel (MMAR_2866, EphB (Rv1938)),
MelI (MMAR_2867, Rv1939), MelJ (MMAR_2868,
Rv1940), MelK (MMAR_2869, Rv1941); and
methadone acyl-b-ketoacyl-acyl carrier protein
synthase BKasB (MMAR_3339, Rv2246). In this
section we highlight some specific findings from
both the host and the Mycobacterium.
A. Genes with Enzymatic Activity
Vitamins (i.e., vitamin B7 or H) often serve as
cofactor of enzymatic carboxylation reactions in-
volved in the carboxylation of fatty acid synthesis,
amino acid metabolism, and glycol-metabolism
necessary to all living organisms.24 Enzymes in-
volved in biotin biosynthesis are potential targets
for the development of antibiotics; biotin biosyn-
thesis is unique to plants and microorganisms. The
Volume 21, Number 4, 2011
339
M. marinum MMAR_2770 mutant is a biotin auxo-
troph and is defective in growth in macrophages
and zebrafish. In reconstructing MMAR_2770 or
its M. tuberculosis homolog Rv1882c (82% iden-
tity in amino acid sequence) to the MMAR_2770
mutant, the phenotype was restored,25 suggesting
that MMAR_2770 and its M. tuberculosis homo-
log Rv1882c serve as new enzymes involved in
biotin biosynthesis and as potential targets for the
development of antibiotics against Mycobacte-
rium. An attenuated mutant TesA was found from
a M. marinum transposon mutant library using a
Dictyostelium discoideum assay.26 TesA is required
for the synthesis of phthioglycol diphthioceranates
and phenolic glycolipids because both lipids were
absent in TesA mutants. This mutant was found
to be highly attenuated in zebrafish embryos, but
virulence was retained when bacteria were injected
into the notochord,26 suggesting the high suscep-
tibility of notochord to Mycobacterium infection.
B. Host Genes with New Functions
Lta4h encode leukotriene A4 hydrolase, which
catalyzes the final step in the synthesis of leukot-
riene B4 (LTB4), a potent chemoattractant and
proinflammatory eicosanoid. A zebrafish lta4h mu-
tant larvae showed innate susceptibility to M. mari-
num in a forward genetic screen. Lta4h mutations
confer hypersusceptibility independent of LTB4
reduction by redirecting eicosanoid substrates to
anti-inflammatory lipoxins A4 (LXA4).27 In addi-
tion, both TB and multibacillary leprosy protec-
tion are associated with heterozygosity for LTA4H
polymorphisms correlated with differential LTB4
production. Six LTA4H SNPs in 692 cases and 759
controls followed the Hardy-Weinberg equilibrium
(HWE) in the control series, but significantly de-
viated from HWE in the case series. At each site
among the cases, fewer heterozygotes were present
than expected by the HWE prediction, suggesting
that heterozygosity at this locus might be protec-
tive against TB.27 However, analyses of six LTA4H
gene polymorphisms in samples collected from
Russia yielded contrasting results, suggesting that
common polymorphisms in the LTA4H gene might
340
not play a major role in susceptibility to clinical
pulmonary TB.28 The Russian sample is European
in origin and is distinct from the Vietnamese and
Nepali samples associated with TB and leprosy.27
The exact roles of LTA4H SNPs in TB susceptibil-
ity remain to be validated in more samples from
diverse populations.
C. Genes Affect the Transmission
of M. marinum
Defects in the espL, located in ESX-1 gene clus-
ter, can reduce early granuloma formation.29 The
ESX-1 secretion system encoding genes include
ESAT-6, CFP-10, EspA, and EspB (Mh3881c).30–32
ESAT-6 may cause membrane pore formation in
the MCV (Mycobacterium-containing vacuole),
facilitating M. marinum escape from the vacuole
into the host-cell cytosol and to disseminate.33 The
M. marinum locus of two genes (iipA and iipB) is
required for both invasion and intracellular sur-
vival within macrophages. Mycobacterium tuber-
culosis Rv1477 and Rv1478 are homologs of the
iipA and iipB gene, Rv1477, fully complementing
the Iip mutant for infectivity in vivo, as well as for
invasion and intracellular persistence in macro-
phages. Rv1478 only partially complemented the
Iip mutant in vivo and restored invasion but not
intracellular growth in macrophages.34 While IipA
and IipB differ at their N termini, they are highly
similar throughout their C-terminal NLPC_p60
domains. The p60 domain of Rv1478 is fully func-
tional to replace that of Rv1477, suggesting that the
N-terminal sequence of Rv1477 is required for full
virulence in vivo and in macrophages. Further mu-
tations have demonstrated that both Arg-Gly-Asp
(RGD) and Asp-Cys-Ser-Gly (DCSG) sequences
in the p60 domain are required for function.
V. NEW MYCOBACTERIUM
EFFECTORS AND HOST TARGETS
Host and high-risk environmental factors are cru-
cial for the transmission of M. tuberculosiss.35 The
virulence factor may affect the dissemination of
Mycobacterium36–38 by promoting the formation
Deng et al.
of granulomas.13 The most well-studied genetic
locus involved in Mycobacterium virulence and
granuloma formation is probably RD1, which in-
cludes nine genes and is deleted in all attenuated
M. bovis bacille Calmette-Guérin (BCG) vaccine
strains.39 The nine genes of the RD1 region are part
of the ESX-1 locus, which encodes a specialized
system responsible for the secretion of a number
of proteins, including the important virulence fac-
tors ESAT-6 and CFP-10.40 It has been reported
that ESAT-6 is involved in the disruption of mem-
branes; consequently, the protein has been asso-
ciated with the translocation of Mycobacterium
from the phagosome into the cytosol and subse-
quent intercellular spread.33,41 Reintroduction of
an intact version of RD1 into M. bovis BCG re-
sulted in increased bacterial growth and formation
of granuloma-like structures in immunodeficient
mice,42 whereas deletion of RD1 from M. tuber-
culosis led to attenuation of both bacterial replica-
tion and granuloma formation in a mouse infection
model.43 Mycobacterium marinum shows delayed
granuloma formation in zebrafish when RD1 is
absent.13,22 The disruption of host MMP9 (Matrix
metalloproteinase-9) attenuated granuloma forma-
tion and bacterial growth. Thus, host MMP9 does
not inhibit but rather promotes the transmission of
bacterium and the progress of infection by affect-
ing the formation of early granulomas.19,20 Recent
research has shown that three new Mycobacteri-
um factors (i.e., FAM53 (fadE33), FAM58 (espL
(mmar_5456), espB(mmar_5457)) and FAM6
7(mmar_5425 and mmar_5426)) involved in the
initial stages of granuloma are related to bacterial
virulence.44 These new identified Mycobacterium
factors may promote the formation of granulomas
structure, resulting in a reversal of the classical
view that granulomas are protective structures that
can defend against mycobacterial infection.
VI. SYSTEMS BIOLOGY INSIGHTS INTO THE
MYCOBACTERIUM INFECTION BASED ON
TRANSCRIPTOME
Digital gene expression profiling (DGE) combines
high-throughput sequencing with a high-perfor-
Critical ReviewsTM in Eukaryotic Gene Expression
New insight into Mycobacterium marinum- zebrafish model
FIGURE 1: Novel genes found to regulate metabolism and signaling crosslinking pathway.
mance computer analysis technique that can there-
by detect the tissue-specific gene expression.45,46
Using this technique, 159 genes were found to
be robustly regulated by Mycobacterium infec-
tion. Up-regulated genes include many known
components of inflammatory signaling, such as
Mycobacterium-infection-related genes and differ-
ent marker genes of the myeloid lineage that in ze-
brafish characterize response to infection.47 Novel
high-throughput, deep-sequencing technology has
dramatically changed the way that the functional
complexity of transcriptomes can be studied. My-
cobacterium-induced transcriptome changes in the
zebrafish model were investigated using Solexa/Il-
lumina’s DGE system.48
Up-regulated zebrafish genes have been
shown to involve immune-response-related tran-
scription factors, proinflammatory cytokines,
and MHC class II proteins. Gene groups encod-
ing proteolytic enzymes and lysosomal proton-
transporting ATPases were also enriched among
the up-regulated transcripts.47,48 Down-regulated
genes included genes involved in gene groups
Volume 21, Number 4, 2011
341
that encode enzymes involved in carbohydrate,
alcohol, steroid, amino acid, and lipid metabo-
lism. They also included cytoskeletal protein
coding genes, tight junction proteins, solute car-
riers, and fatty acid binding proteins.47,48 These
results suggest that deep-sequencing analysis
revealed the complexity of the host’s transcrip-
tional response to Mycobacterium infection.
Novel host genes regulated by Mycobacteri-
um were identified by comparing data from DGE
analysis with a previous multi-platform microar-
ray (i.e., UniGene, RefSeq, and Ensembl). These
genes include csf2rb (CSF2RB, colony stimulat-
ing factor 2 receptor), cmklr1 (CMKLR1), sart3
(SART3, squamous cell carcinoma antigen rec-
ognized by T cells 3), hspb8 (HspB8, heat shock
protein, alpha-crystallin-related, B8), LOC565410
(CAPG, capping protein), rhoae (RHOA),
zgc:171802 (GPR84, G protein-coupled recep-
tor 84), akap13 (AKAP13,A kinase anchor pro-
tein 13), ltb4dh (LTB4DH,leukotriene B4 12-hy-
droxydehydrogenase), LOC368614 (HLA-DPA1,
major histocompatibility complex, class II, DP
342 Deng et al.

TABLE 3. The new identified functions of partial human homologs regulated by

Mycobacterium from DGE.

Zebrafish gene Human homology DGE Function Reference

Component of the MHC class II protein com-

LOC368614 HLA-DPA1 up [52]
plex involved in antigen presentation
Regulate cell proliferation and cytokine pro-
zgc: 112143 TNFAIP9 up [53]
duction
LTF may serve as a negative effector in-
zgc: 112154 LTF up [54]
volved in participate in NPC carcinogenesis
Functions as a chaperone in association with
hspb8 HspB8 down macroautophagy stimulator Bag3 in protein [55-56]
aggregation diseases
Contribute to negative regulation of immune
rhogb RHOG up responses Immunological function. Promote [49,57]
cell survival by the activation of PI3K and Akt
CFB associated with aHUS (atypical hemo-
LOC792364 CFB up [58]
lytic uremic syndrome)

alpha 1), LOC557797 (CD18, ITGB2-integrin,
beta 2), zgc:86681, capg (CAPG, capping pro-
tein), LOC563727 (MAP3K8, mitogen-activated
protein kinase kinase kinase 8), LOC794777
(TNFAIP2, tumor necrosis factor, alpha-induced
protein 2), tnip1 (TNIP1, TNFAIP3 interacting
protein 1), cul5 (CUL5, cullin 5), irgq2 (IRGM),
novel/rgl2 (RGL2, ral guanine nucleotide dissoci-
ation stimulator-like 2), LOC564859 (STK40, ser-
ine/threonine kinase 40), zgc:112143 (TNFAIP9,
tumor necrosis factor alpha-induced protein 9),
il11a (IL11), LOC799527 (CXCR3), ptger2l (PT-
GER2, prostaglandin E receptor 2), tfa (LTF, lac-
totransferrin), rhogb (RHOG, ras homolog gene
family, member G), LOC792364 (CFB, comple-
ment factor B), mxi1 (MXI1, MXI1-MAX interac-
tor 1 isoform a), bcor (BCOR, BCL-6 interacting
corepressor isoform c), and LOC555512 (IFI44,
interferon-induced protein 44).
Some new molecules may play important roles
in the crosslinking response of three key metabo-
lism processes (i.e., glycolysis, protein synthesis
and degradation, and fatty acid oxidation) and in
the important signaling cascade pathways (i.e., NF-
κB, ERK, AKt, JNKK, and Ca2+ pathway) (Figure
1). Another new regulated human homology that
may be associated with human genetic disorders
was modulated by Mycobacterium (Table 1). Im-
portantly, few host genes identified from DGE di-
rectly related to TB caused by M. tuberculosis. For
example, human RhoG can activate both apoptotic
and anti-apoptotic pathways through JNK and NF-
kappaB-independent PI3K/Akt pathways, respec-
tively.49 Recent evidence has shown that the single
nucleotide polymorphism (SNPs) of the IRGM
promoter region (1208A/G) is associated with sus-
ceptibility to TB.50 In addition, IRGM has been
shown to eliminate intracellular mycobacteria by
inducing autophagy and reducing the intracellular
bacillary load.51 These results may suggest that
new molecules, identified by DGE based on the M.
marinum–zebrafish model, might have a function
in immune defense and might facilitate the Myco-
bacterium evasion of host immunity.
VII. CONCLUSION
The M. marinum–zebrafish model provides tre-

Critical ReviewsTM in Eukaryotic Gene Expression

New insight into Mycobacterium marinum- zebrafish model
mendously fresh vision for the development of
forthcoming therapies for TB. This model may
have transformed our traditional understanding of
the roles of granulomas. With it, multiple candidate
genes can be scrutinized. This model has helped to
identify the virulence factor with unknown func-
tion. Although seemingly independent, these pro-
teins may interplay during the pathogenesis of My-
cobacterium. The M. marinum–zebrafish model,
together with other high-throughput and high-con-
tent screening methodologies, are sure to expedite
the discovery of novel antibiotics against TB.
ACKNOWLEDGEMENT
This work was supported by the National Mega-
project for Key Infectious Disease (Grant No.
2012ZX10003-003), the Fundamental Research
Funds for the Central Universities (Grant No. XD-
JK2009A003), the National Natural Science Foun-
dation (Grant No. 81071316), and the Excellent
PhD Thesis Fellowship of Southwest University
(Grant Nos. kb2009010 and ky2009009).
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