203 

ISJ 15: 203-209, 2018                                             ISSN 1824-307X 

 
 

RESEARCH REPORT 

 
Myeloid leukemia factor participates in the antibacterial immune reaction of kuruma 
shrimp Marsupenaeus japonicus  

 
X-W Feng, L-J Huo, X-Z Shi* 

 
Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Sciences, 
Shandong University, Jinan, Shandong, 250100, China 

 
 

   Accepted June 12, 2018 

 
Abstract 

Myeloid leukemia factor (MLF) plays an important role in the development of Drosophila. It is 
reported that MLF could inhibit white spot syndrome virus (WSSV) replication in shrimp. However, the 
function and mechanism of MLF in shrimp antibacterial immunity are still elusive. In this study, we 
found MjMLF could be upregulated by Vibro anguillarum challenge in hemocytes, gills, and intestine of 
kuruma shrimp. RNAi assay showed that MjMLF could facilitate bacterial clearance in shrimp, and it 
was beneficial for shrimp survival post V. anguillarum infection. Pull down assay showed that rMjMLF 
could bind to rMj14-3-3 in vitro. Some antimicrobial peptide genes could be regulated by MjMLF. The 
results indicated that MjMLF might participate in the anti-bacterial immune reaction of shrimp. 

 
Key Words: shrimp; MjMLF; V. anguillarum; AMPs; Mj14-3-3 

 

 
Introduction 

 
When organisms were challenged by invading 

pathogens, immune reaction could be initiated. 
Immune response contains humoral reaction and 
cellular reaction (Iwanaga et al., 2005; Li et al., 
2013). Humoral reaction includes prophenoloxidase, 
clotting cascade, and the production of some 
antimicrobial peptides (AMPs), while cellular 
reaction consists of apoptosis, encapsulation, 
nodule formation, and phagocytosis (Iwanaga et al., 
2005; Li et al., 2013).  

It is reported production of AMPs is one 
hallmark in host defense of Drosophila, as well as 
some other holometabolous insects (Lemaitre et al., 
2007). When microorganism invades, the synthesis 
and secretion of potent AMPs are induced and 
accumulated in hemolymph. Microarray studies 
indicate the gene expression program in Drosophila 
go through intensive change due to the microbial 
infection. AMP genes and some other immune 
responsive genes could be regulated by Toll/IMD 
cascades pathway in Drosophila (De Gregorio et al., 
2002). In Drosophila, the infection of some viruses 
(such as HIV-1 and cytomegalovirus) could also 
activate Toll/IMD pathway and induce the 
expression of some immune related genes. 
___________________________________________________________________________ 

 
Corresponding author: 

Xiu-Zhen Shi 
School of Life Sciences 
Shandong University 

Jinan, Shandong 250100, China 
E-mail: shixiuzhen@sdu.edu.cn 

 

 
 
Myeloid leukemia factor (MLF) was discovered 

in acute myeloid leukemia (AML) caused by 
chromosomal rearrangement. MLF protein is a small 
nucleo-cytoplasmic shuttling protein, and contains a 
consensus 14-3-3 binding motif (Gobert, et al., 2012). 
Some MLF interaction proteins, such as 
MLF-interaction protein (MLFIP) and MLF adaptor 
molecule (MADM), were identified through yeast two 
hybrid screens (Lim, et al., 2002; Hanissian, et al., 
2004). MLF is able to decrease the occurrence of 
Trib1-driven acute myeloid leukemia, as tumor 
suppressor CCAT/enhancer binding protein 

(C/EBP) could be stabilized by MLF (Nakamae, et 
al., 2017; Koschmieder et al., 2009). MLF plays an 
important role in regulating mechanosensitive 
signaling pathway, which is responsible for typical 
characteristics in neonatal rat cardiomyocytes 
(NRVCMs). The overexpression of MLF in NRVCMs 
could inhibit cell proliferation and increase apoptosis, 
while knockdown of MLF causes opposite results by 
protecting cell from apoptosis and increasing 
proliferation. Besides, knockdown of MLF could 
upregulate D cyclins expression, which indicates 
MLF functions in regulating cell proliferation 
(Yoneda-Kato et al., 2005; Rangrez et al., 2017). In 
addition, MLF is reported to function as proapoptotic 
regulator to modulate cell survival by associating 
with HOP complex molecule (HAX-1 (HS1 associated 
protein X-1)/Htra2-OMI/PARL (presenilins-associated 
rhomboid-like)) (Sun et al., 2017b). MLF is able to 
interact with HAX1 and Htra2 physically, and prevent 
Htra2 from cleavage and activation. 
 

mailto:shixiuzhen@sdu.edu.cn


204 

 
 
Fig. 1 The temporal course of MjMLF in hemocytes, gills, and intestine after V. anguillarum injection at 0, 6, 12, 24, 

48 h by qRT-PCR at transcriptional level. -actin was used as the control. The significance was detected by t-test 
between PBS group and infected group (P < 0.05, P < 0.001) 
 
 
 
 
 

It is reported that MLF1 could associate with 
14-3-3 via a classic RSXSXP sequence in murine 
and human (Lim et al., 2002). MLF1 could be 
redirected from cytoplasm into nucleus by 
MLF1-associated nuclear protein (Manp), and also 
be regulated by 14-3-3 via binding to the 14-3-3 
binding motif on MLF1 N terminus (Winteringham et 
al., 2006). And in shrimp, 14-3-3 could inhibit the 
expression of some AMPs and negatively influence 
the Akirin-Relish function (Liu et al., 2016). 

In shrimp, MLF functions importantly by 
inhibiting WSSV replication and inducing hemocytes 
apoptosis (Feng et al., 2017). In this study, the 
function of MLF in shrimp innate immunity against 
bacteria was studied. And results showed that 
MjMLF could enhance the exogenous Vibrio 
anguillarum clearance, regulate the expression of 
some AMPs, interact with 14-3-3, and affect shrimp 
survival. MjMLF participated in the innate immune 
reaction of kuruma shrimp against V. anguillarum. 
 
Materials and methods 
 
Animal and tissue collection 

Healthy kuruma shrimp Marsupenaeus 

japonicus (6-8 g/individual) were purchased from 
an aquaculture market in Jinan, Shandong Province, 
China and cultured in artificial aquarium with 
pumped air (Sun et al., 2017a). Three shrimp were 
randomly selected to extract total RNAs from 
hemocytes, gills, and intestine with TRIpure Reagent 
(Bioteke Corporation, Beijing, China) following the 

manufactures instruction. 
 
Expression profiles analysis after V. anguillarum 
challenge 

30 L (1×106 CFU) V. anguillarum was injected 
into the last second segment of shrimp. Meanwhile, 
the same volume of phosphate-buffered saline (PBS) 
(0.14 M NaCl, 1.8 mM KH2PO4, 2.7 mM KCl, and 10 
mM Na2HPO4) was injected in the other shrimp 
group, which were used as the control. Tissues 
including hemocytes, gills, and intestine from V. 
anguillarum injected shrimp and PBS injected 
shrimp were collected at 6, 12, 24, and 48 h post 
injection for total RNA extraction. Total RNAs were 

obtained from at least three shrimp, and reversely 
transcribed into the first stand cDNAs with primer 
oligo-anchorR (Feng et al., 2017). The obtained 
cDNAs were diluted 20-fold as the templates for 
quantitative real time Polymerase Chain Reaction 
(qRT-PCR). Primer MjMLF-RTF1 and 
MjMLF-RTR1 were used as the previous report 
(Feng et al., 2017). 
 
DsRNA preparation 

A partial cDNA fragment of MjMLF was 
amplified by PCR with primer MjMLF-Fi and 
MjMLF-Ri and used as template for dsRNA 
synthesis (Feng et al., 2017). The control dsGFP 
was produced with the same method (Yang et al., 
2015). The synthesized dsRNAs were purified by 
hydroxybenzene and chloroform extraction. And the 
final dsRNAs were dissolved in RNase-free water, 

and the concentration was adjusted to 1 g/l. 
RNA interference (RNAi) was performed as 

previously reported (Shi et al., 2015). At 24 h post 
the second dsRNA injection, shrimp hemocytes 
were collected for total RNAs extraction. To test the 
RNAi efficiency, the first strand cDNAs were 
reversely transcribed and diluted 20-fold as the 
qRT-PCR templates with primer MjMLF-RTF2 and 
MjMLF-RTR2 (Feng et al., 2017). 
 
Bacterial clearance assay 

For the bacterial clearance, shrimp was divided 
into two groups, dsMjMLF group and dsGFP group. 
After RNAi, 1×106 CFU V. anguillarum were injected 
into the shrimp abdominal segment. At 30 min after 
V. anguillarum injection, hemolymph was extracted 
from at least three shrimp. The hemolymph was 
gradiently diluted and spread onto the solid medium 
plates (1% Tryptone, 0.5% Yeast extraction, 0.1% 
FeCl3, 3% NaCl, 1.5% Agar) (Zhang et al., 2014). 

After the plates were cultured at 37 C overnight, the 
number of bacteria colonies was counted. Results 
were analyzed by GraphPad Prism 5.0 software with 

Students paired t test. 
 
Survival rate assay 

For the survival assay, shrimp were divided into 
three groups, PBS group, dsGFP group, and 



205 

dsMjMLF group (each group containing 30 shrimp). 
RNAi assay was performed as the above mentioned. 
And also, the same volume of PBS was injected into 
shrimp as the PBS group. After RNAi, 1×106 CFU V. 
anguillarum were injected into shrimp of the three 
groups. The dead shrimp were numbered at 6, 12, 
24, 48, 72, 96, 120, 144 h post V. anguillarum 
injection. The dsGFP-injected shrimp and 
PBS-injected shrimp were used as the controls. The 
P value was detected by GraphPad Prism 5.0 
software with log–rank (mantel-cox) test. 
 
The expression detection of some AMP genes 

V. anguillarum was incubated in liquid medium 
(1% Tryptone, 0.5% Yeast extraction, 0.1% FeCl3, 
3% NaCl) overnight. The bacteria were fully washed 
by sterile PBS twice. After RNAi, 1×106 CFU V. 
anguillarum were injected. The relative expression of 
some AMP genes including MjALFE1, MjALFE2, 
MjcrustinI-2, and MjcrustinI-4 at 6 h post bacteria 
injection was detected by qRT-PCR in shrimp 

hemocytes. -actin was the internal control. The 
primers could be found in the previous report (Bi et 
al., 2015; Yang et al., 2017). P value was analyzed 

and accumulated by Students t test. 
 
Pull-down assay 

To detect the interaction of MjMLF with 
Mj14-3-3, GST pull-down assay was performed. 200 

g purified recombinant Mj14-3-3 protein (rMj14-3-3, 
with GST tag) or GST tag protein (as the control) 
was incubated with the proteinlso GST resin 
(TransGen Biotech, Beijing, China) respectively for 
20 min at room temperature. The resins saturated 
with rMj14-3-3 or GST protein were sufficiently 
washed by PBS, till no protein could be detected in 

the wash solution. 200 g rMjMLF (with His-tag) 
were added into the resins separately, and incubated 

 

with gentle shaking for 2 h at room temperature. 
Then the resins was adequately washed by PBS for 
at least six times to clean up the weak binding 
protein. The washing solution of the last time was 
collected as the wash fraction. And finally, the 
elution buffer (10 mM Glutathione) was added to 
elute the protein on resin. The eluted fraction and 
the wash fraction were separated by 15% 
SDS-PAGE. SDS-PAGE gel was stained by 
Coomassie brilliant blue G-250 (Sangon Biotech, 
Shanghai, China). The purified rMj14-3-3, rMjMLF, 
and GST tag protein were also run on SDS-PAGE 
as the control. 

As the predicted molecular mass of rMj14-3-3 
was 53.92 kDa and that of rMjMLF was 52.62 kDa, it 
was hard to separate the two bands on normal 
SDS-PAGE. So SDS-PAGE was run at 100 V for 
about 6 h to separate the two bands on the same 
lane. GST tag protein was used as the negative 
control. 

 
Results 
 
MjMLF was upregulated by V. anguillarum 

To study the function of MjMLF in antibacterial 
immune response, qRT-PCR was carried out to 
analyze the temporal expression of MjMLF in 
hemocytes, gills, and intestine of shrimp after V. 
anguillarum challenge. The results showed that 
MjMLF could be upregulated in hemocytes, gills, and 
intestine by V. anguillarum challenge (Fig. 1). The 
expression level of MjMLF in hemocytes and gills 
reached the peak at 24 h after V. anguillarum 
infection (Fig. 1A and 1B), while in intestine it 
exhibited the highest expression level at 12 h post 
challenge (Fig. 1C). These results indicated that 
MjMLF might be involved in the immune defense 
against V. anguillarum. 

 
 
 
 

 
 
Fig. 2 Bacterial clearance assay and survival assay in shrimp. The dsGFP was injected to use as control. (A) The 
interference efficiency of MjMLF in hemocytes was analyzed by qRT-PCR; (B) The bacterial clearance was 
analyzed by calculating the V. anguillarum number per milliliter hemolymph. The significant difference was 

calculated between dsMjMLF group and dsGFP group. The significances were calculated by t-test (P < 0.05,  P 
< 0.01). (C) The shrimp survival rate was analyzed by RNAi and V. anguillarum challenge. The shrimp mortality 
was detected for calculation of the shrimp survival rate. The dsGFP group and PBS group were used as the control. 
The survival rate was analyzed with the Kaplan-Meier method. The significant differences were detected by 

log-rank test (P < 0.05) 



206 

 
 
Fig. 3 The expression analysis of AMPs (MjALFE1, MjALFE2, MjcrustinI-2, and MjcrustinI-4) in shrimp hemocytes. 
(A) The knocking down efficiency of dsMjMLF in hemocytes was detected after the dsRNA injection. The relative 
expression of MjALFE2 (B), MjcrustinI-2 (C), MjcrustinI-4 (D), and MjALFE1 (E) was analyzed by qRT-PCR at 6 h 

post the V. anguillarum challenge. The significant differences were analyzed by t-test (P < 0.05,  P < 0.01) 
 
 
 
 
 
 
 
V. anguillarum clearance rate decreased in 
MjMLF-silenced shrimp 

To determine the function of MjMLF in immune 
defense against V. anguillarum, RNAi and bacterial 
clearance assay were performed. After knocking 
down the expression of MjMLF (Fig. 2A), V. 
anguillarum were injected into shrimp. Compared to 
the dsGFP-injected shrimp, the V. anguillarum 
number in dsMjMLF-injected shrimp was much 
higher, which indicated the clearance rate in the 
dsMjMLF-injected shrimp was much slower (Fig. 2B). 
MjMLF could accelerate the bacterial clearance in 
shrimp. The result implied that MjMLF might have an 
important function in the immune defense against V. 
anguillarum in shrimp. 

The expression of some AMPs was downregulated 
in dsMjMLF-injected shrimp 

To test whether some effector molecules could 
be regulated by MjMLF, the expression of some 
AMPs (MjALFE1, MjALFE2, MjcrustinI-2, and 
MjcrustinI-4) was analyzed after RNAi by qRT-PCR. 
After knocking down MjMLF (Fig. 3A), the 
expression of MjALFE2, MjcrustinI-2, and 
MjcrustinI-4 decreased significantly in 
dsMjMLF-injected shrimp compared with the 
dsGFP-injected shrimp (Fig. 3B, C, D), while there 
was no significant change for the expression of 
MjALFE1 (Fig. 3E). The results indicated that MjMLF 
might protect shrimp from V. anguillarum infection by 
regulating some specific AMPs expression. 



207 

 
 
Fig. 4 Pull down assay was used to check the interaction of rMjMLF and rMj14-3-3. (A) Purified recombinant 
rMj14-3-3 was bound to GST resin, and then rMjMLF was added. After sufficient washing, the eluted fraction was 
collected. rMj14-3-3, the wash fraction, the elute fraction, and rMjMLF were separated by 15% SDS-PAGE for 6 h 
at 100 V. (B) Purified GST tag protein was used as the control and separated on 15% SDS-PAGE at 100 V for 2 h. 
The gels were stained by Coomassie brilliant blue 
 
 
 
 
 
rMjMLF interacts with rMj14-3-3 in vitro 

It is reported that MLF could enhance apoptosis, 
and bind to 14-3-3. And MLF is negatively regulated 
by 14-3-3 (Sun et al., 2015). In shrimp, Mj14-3-3 is 
involved in regulating AMPs expression (Liu et al., 
2016). To understand the mechanism of MjMLF in 
antibacterial immunity of shrimp, GST-pulldown 
assay was performed to investigate the interaction of 
rMjMLF with rMj14-3-3. The results showed that 
rMjMLF could interact with rMj14-3-3 in vitro (Fig. 
4A), while no interaction was detected between 
rMjMLF and GST protein (Fig. 4B). It demonstrated 
that rMjMLF had the binding activity to rMj14-3-3, but 
not the GST tag. And it has been reported His tag 
protein could not bind to GST tag protein (Xu et al., 
2017). The results suggested that rMjMLF could bind 
to rMj14-3-3 directly in vitro. 
 
Discussion 

 
In this study, the role of MjMLF in antibacterial 

immune reaction was researched. As far as we know, 
it is the first report about the function of MLF in 
antibacterial immunity of kuruma shrimp M. 
japonicus. 

As reported previously, the expression of 
MjMLF was upregulated in hemocytes and 
hepatopancreas by WSSV challenge. And MjMLF 
inhibits WSSV replication by influencing hemocytes 
apoptosis. MjMLF could increase the survival rate 
and play an important function in antivirus 
immunology (Feng et al., 2017). The expression 
pattern assay showed that expression of MjMLF was 
upregulated in hemocytes, gills, and intestine of 
shrimp when shrimp were challenged by V. 
anguillarum. The result suggested that the potent 

role of MjMLF in shrimp antibacterial immunity. To 
research the function of MjMLF in antibacterial 
immune reaction, RNAi assay and exogenous 
bacterial clearance assay were performed. The V. 
anguillarum clearance rate was much slower in 
dsMjMLF-injected shrimp, which indicated the critical 
role of MjMLF in antibacterial immune reaction. 
Besides, the shrimp survival rate in 
dsMjMLF-injected shrimp was lower than the control 
groups. MjMLF could be involved in shrimp 
antibacterial immune reaction. 

In Drosophila, MLF could promote the 
expression of JNK (c-Jun N-terninal kinase) (Yanai 
et al., 2014). Further research indicates that JNK 
pathway is a critical signaling pathway that initiating 
some relevant effector molecular (Boutros et al., 
2002; Park et al., 2004; Kallio et al., 2005; Delaney 
et al., 2006). It is reported that dMLF could associate 
with DREF (DNA replication-related element-binding 
factor) and initiate bsk promoter. The interaction 
could further activate the JNK signaling pathway 
(Yanai et al., 2014). In Drosophila S2 cells, the JNK 
signaling pathway plays an essential role in 
regulating normal AMPs release (Kallio et al., 2005). 
The production of AMPs could regard as local 
immune response relating to external environment 
(Lemaitre et al., 2007). And in Drosophila and other 
holometabolous insects, the synthesis and secretion 
of the potent AMPs could be induced and 
accumulated in hemolymph when invading 
microorganisms challenge, and it was the critical 
hallmark of host defense (Lemaitre et al., 2007). In 
Drosophila, most of the immune responsive genes 
are believed to be regulated by immune signaling 
pathway, such as Toll and IMD pathway (De 
Gregorio et al., 2002). 



208 

In our study, we found MjMLF could positively 
regulate the expression of MjALFE2, MjcrustinI-2, 
and MjcrustinI-4. Additionally, it is reported that MLF 
is able to interact with 14-3-3 with phosphorylated 
motif (Lim et al., 2002). In shrimp, we detected their 
direct interaction in vitro by pull down assay. 
Mj14-3-3 was able to regulate the expression of 
some AMPs (Liu et al., 2016). And Mj14-3-3 could 
interact with Akirin, which suppressed the 
expression of some AMPs. Akirin participates in 
IMD-Relish pathway via associating with Relish and 
activate Relish-regulated AMP genes. The 
interaction of rMjMLF and rMj14-3-3 indicated that 
MjMLF might suppress the regulation of Mj14-3-3 on 
AMPs expression by binding to Mj14-3-3. So MjMLF 
might participate in shrimp antibacterial innate 
immunity by regulating the expression of some 
AMPs. Further research such the sublocation of 
Mj14-3-3 in hemocytes after knocking down MjMLF 
or co-expression of Mj14-3-3 and MjMLF in insect 
cell lines would help us fully understand the 
mechanism of MjMLF in shrimp antibacterial 
immunity. 

In conclusion, MjMLF could protect shrimp 
from bacterial infection by regulating some AMPs 
expression, enhancing bacterial clearance, and 
affecting the shrimp survival rate. MjMLF might play 
a critical function in antibacterial immunity of 
shrimp. 
 
Acknowledgement 

This work was supported by grant from National 
Natural Science Foundation of China 
(No.31301879). 
 
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