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International Journal of
Molecular Sciences
Review
The Mechanism of Facultative Intracellular Parasitism
of Brucella
Hanwei Jiao 1,2,3,*, Zhixiong Zhou 2, Bowen Li 2, Yu Xiao 2, Mengjuan Li 2, Hui Zeng 2, Xiaoyi Guo 2
and Guojing Gu 2
Citation: Jiao, H.; Zhou, Z.; Li, B.;
Xiao, Y.; Li, M.; Zeng, H.; Guo, X.; Gu,
G. The Mechanism of Facultative
Intracellular Parasitism of Brucella.
Int. J. Mol. Sci. 2021,22, 3673.
https://doi.org/10.3390/ijms22073673
Academic Editor: Rustam I. Aminov
Received: 24 February 2021
Accepted: 30 March 2021
Published: 1 April 2021
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1Immunology Research Center, Medical Research Institute, Southwest University, Chongqing 402460, China
2College of Veterinary Medicine, Southwest University, Chongqing 402460, China;
zzx449090405@email.swu.edu.cn (Z.Z.); libowenswu@email.swu.edu.cn (B.L.);
xiaoyulike@email.swu.edu.cn (Y.X.); lmj123@email.swu.edu.cn (M.L.); zenghui1234@email.swu.edu.cn (H.Z.);
greataguo4@email.swu.edu.cn (X.G.); ggj19970819@email.swu.edu.cn (G.G.)
3Veterinary Scientific Engineering Research Center, Chongqing 402460, China
*Correspondence: jiaohanwei@swu.edu.cn
Abstract:
Brucellosis is a highly prevalent zoonotic disease characterized by abortion and repro-
ductive dysfunction in pregnant animals. Although the mortality rate of Brucellosis is low, it is
harmful to human health, and also seriously affects the development of animal husbandry, tourism
and international trade. Brucellosis is caused by Brucella, which is a facultative intracellular parasitic
bacteria. It mainly forms Brucella-containing vacuoles (BCV) in the host cell to avoid the combination
with lysosome (Lys), so as to avoid the elimination of it by the host immune system. Brucella not
only has the ability to resist the phagocytic bactericidal effect, but also can make the host cells form a
microenvironment which is conducive to its survival, reproduction and replication, and survive in
the host cells for a long time, which eventually leads to the formation of chronic persistent infection.
Brucella can proliferate and replicate in cells, evade host immune response and induce persistent
infection, which are difficult problems in the treatment and prevention of Brucellosis. Therefore, the
paper provides a preliminary overview of the facultative intracellular parasitic and immune escape
mechanisms of Brucella, which provides a theoretical basis for the later study on the pathogenesis
of Brucella.
Keywords:
Brucella; facultative intracellular parasitism; immune escape; persistent infection; re-
search progress
1. Introduction
Brucellosis is a zoonotic systemic chronic infectious disease caused by Brucella [
1
]. Bru-
cellosis is often called “Mediterranean fever” or “Malta fever”. The main clinical features
of the disease are high fever, enlargement of liver and spleen, joint pain, inflammation
of reproductive organs and fetal membranes, infertility and localized lesions of various
tissues [
2
,
3
]. Brucellosis is widely distributed around the world; only a few countries
in Northern Europe and Central Europe, as well as Canada, Japan, Australia and New
Zealand, have eliminated Brucellosis. The Mediterranean area, Asia and central and South
America are the high incidence areas of Brucellosis. Indeed, Brucellosis has been described
as being the most common zoonotic disease worldwide with more than 500,000 new human
cases annually [1].
Brucella is a Gram-negative facultative intracellular parasitic bacteria—it has no spores,
flagella and capsules [
4
]. In 1886, David Bruce first identified and isolated Brucella from
the spleens of soldiers who died of “Maltese fever” [
5
]. In 1985, the WHO Brucellosis
expert committee divided Brucella spp. into six species according to the differences of
infected animals and antigenicity. Brucella melitensis (B.melitensis) is the most common
and virulent bacteria, followed by Brucella abortus (B.abortus). Brucella mainly infects
Int. J. Mol. Sci. 2021,22, 3673. https://doi.org/10.3390/ijms22073673 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2021,22, 3673 2 of 13
ruminants and causes abortion and infertility in pregnant animals [
6
]. The Brucella cell
wall consists of an inner and an outer membrane. The outer membrane of Brucella consists
of lipopolysaccharide (LPS), outer membrane protein (OMP) and phospholipid layer.
Lipid A, core polysaccharide and O antigen constitute the LPS of Brucella, which is an
important virulence factor of Brucella and an important antigen that mediates the immune
production of the animal body [
7
]. Brucella mainly invades macrophages and trophoblast
cells, parasitizes in host cells through specific molecular mechanism, affects the apoptosis
of host cells, thus mediates the autophagy of host cells, and creates favorable conditions
for its survival and reproduction in the host cells. After Brucella invades the host cells, it
mainly causes chronic infection by avoiding the host immune-reaction system [
8
], but the
molecular mechanism of Brucella facultative intracellular parasitism has not been clear.
Therefore, through an overview of the mechanism of Brucella facultative intracellular
parasitism, this article provides a theoretical basis for the later excavation of the pathogenic
mechanism of Brucella and related research.
2. Intracellular Life Cycle of Brucella
Brucella has been initially described as a facultative intracellular parasitic bacteria
able to replicate in professional phagocytes such as macrophages, dendritic cells (DC) and
granulocytes as well as nonprofessional phagocytes, including epithelial, fibroblastic and
trophoblastic cells [
9
]. Brucella interacts with the cell membrane of macrophages through
lipid rafts and enters the host cells to form Brucella-containing vacuoles (BCV) surrounded
by phagocytic vesicles [
10
]. From 8 to 12 h after Brucella invades the cell, BCV obtains some
host marker molecules through interaction with lysosome (Lys) and endosomes, matures
the endosomes in the membrane bound vacuoles, and forms acidified endosomes. At
this time, BCV is called endosomal Brucella containing vacuole (eBCV). As BCV develops
and matures, the Type IV secretory system (T4SS) mediates the interaction between the
effector protein and the endoplasmic reticulum (ER) exit site, and obtains ER and Golgi
apparatus-derived membranes. After losing the early host marker molecules, the eBCV
obtained Lys marker molecules (such as Rab7, LAMP-1, etc.) [
11
]. The BCV escaping
Lys degradation will reach the ER and fuse with the ER in a Sar1 and Rab2 dependent
manner [
12
]. At this time, the BCV is called repetitive Brucella containing vacuole (rBCV).
At the later stage of infection, rBCV will be transformed into autophagic Brucella containing
vacuole (aBCV) (Figure 1). At this time, the aBCV will not continue to mature and kill cells.
Thus far, Brucella completes the intracellular circulation, and the organism finally releases
pathogens through lysis and nonlysis mechanisms [13].
Brucella interacts with lipid rafts on the plasma membrane, which promotes Brucella to
contact with the host cells and mediates its internalization into phagocytes. The lipid rafts
contain glycosphingolipids and cholesterol, which can promote the membrane-related bio-
logical processes, such as the formation of polybasic membrane complexes, transmembrane
signaling and membrane fusion [
14
]. LPS is a key molecule in the interaction between
Brucella and host cell lipid rafts, and can prevent complement mediated bacterial lysis and
host cell apoptosis [
15
]. It has been shown that class A scavenger receptor (SR-A) and
prion protein (Pr Pc) are involved in the process of Brucella invading cells through lipid
rafts [
16
,
17
]. Prion protein and SR-A, as receptor proteins of heat shock protein 60 (HSP60)
and LPS, exist in specific lipid rafts. The destruction of lipid rafts can effectively reduce
the early survival of Brucella in macrophages, indicating that the introduction of lipid rafts
is a necessary condition for the early survival of bacteria [
18
]. Brucella enter the cell to
form phagosome and participate in the endocytosis pathway, but Brucella can be quickly
separated from the phagosome, indicating that the early survival of Brucella is related to
the lipid raft mediated signaling pathway [
19
]. In the process of Brucella’s intracellular
circulation, it needs aBCV to complete the intracellular life cycle and cell–cell diffusion [
20
].
The host protein (Yip1A) plays an important role in the formation of rBCV and aBCV. In
Yip1A knockout cells, Brucella could not form rBCV, so it could only remain in the Lys body.
The formation of aBCV depends on the small GTP enzyme Rab9 [
13
]. When the ER Beclin1
Int. J. Mol. Sci. 2021,22, 3673 3 of 13
and PI3K form a complex, rBCV begins to transform into aBCV, but with the consumption
of ATG14L, the formation of aBCV decreases gradually. By interacting with the conserved
oligomeric Golgi (COG), the effector protein BspB regulates the COG dependent transport,
reorients the golgi body derived vesicles to BCV, promotes the formation of rBCV, and
promotes the intracellular proliferation of Brucella.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 14
Figure 1. Brucella interacts with lipid rafts on the surface of cell membrane to enter macrophages
and form Brucella-containing vacuoles (BCV). The early BCV in macrophages is called eBCV, it ac-
quires some host marker molecules. With the maturation of eBCV, eBCV loses the marker of early
endosome, and obtains the marker molecules of late endosome and lysosome recognition, so as to
promote the fusion of eBCV and lysosome. Part of the eBCV escaped lysosome degradation and
reached the ER, and then fused with the ER by Sar1 and Rab2 to form rBCV. Brucella proliferated in
rBCV. At the late stage of infection, the rBCV containing a large number of Brucella transformed into
aBCV. The aBCV released pathogens through cleavage and noncleavage mechanisms, and the in-
tracellular circulation of Brucella ended.
Brucella interacts with lipid rafts on the plasma membrane, which promotes Brucella
to contact with the host cells and mediates its internalization into phagocytes. The lipid
rafts contain glycosphingolipids and cholesterol, which can promote the membrane-re-
lated biological processes, such as the formation of polybasic membrane complexes, trans-
membrane signaling and membrane fusion [14]. LPS is a key molecule in the interaction
between Brucella and host cell lipid rafts, and can prevent complement mediated bacterial
lysis and host cell apoptosis [15]. It has been shown that class A scavenger receptor (SR-
A) and prion protein (Pr Pc) are involved in the process of Brucella invading cells through
lipid rafts [16,17]. Prion protein and SR-A, as receptor proteins of heat shock protein 60
(HSP60) and LPS, exist in specific lipid rafts. The destruction of lipid rafts can effectively
reduce the early survival of Brucella in macrophages, indicating that the introduction of
lipid rafts is a necessary condition for the early survival of bacteria [18]. Brucella enter the
cell to form phagosome and participate in the endocytosis pathway, but Brucella can be
quickly separated from the phagosome, indicating that the early survival of Brucella is
related to the lipid raft mediated signaling pathway [19]. In the process of Brucella’s intra-
cellular circulation, it needs aBCV to complete the intracellular life cycle and cell–cell dif-
fusion [20]. The host protein (Yip1A) plays an important role in the formation of rBCV
and aBCV. In Yip1A knockout cells, Brucella could not form rBCV, so it could only remain
in the Lys body. The formation of aBCV depends on the small GTP enzyme Rab9 [13].
When the ER Beclin1 and PI3K form a complex, rBCV begins to transform into aBCV, but
with the consumption of ATG14L, the formation of aBCV decreases gradually. By inter-
acting with the conserved oligomeric Golgi (COG), the effector protein BspB regulates the
COG dependent transport, reorients the golgi body derived vesicles to BCV, promotes the
formation of rBCV, and promotes the intracellular proliferation of Brucella.
Figure 1.
Brucella interacts with lipid rafts on the surface of cell membrane to enter macrophages and
form Brucella-containing vacuoles (BCV). The early BCV in macrophages is called eBCV, it acquires
some host marker molecules. With the maturation of eBCV, eBCV loses the marker of early endosome,
and obtains the marker molecules of late endosome and lysosome recognition, so as to promote the
fusion of eBCV and lysosome. Part of the eBCV escaped lysosome degradation and reached the ER,
and then fused with the ER by Sar1 and Rab2 to form rBCV. Brucella proliferated in rBCV. At the late
stage of infection, the rBCV containing a large number of Brucella transformed into aBCV. The aBCV
released pathogens through cleavage and noncleavage mechanisms, and the intracellular circulation
of Brucella ended.
3. The Survival and Replication of Brucella in Cells
After Brucella invades the host cell, it circulates in the cell [
21
]. The rBCV stage is
tightly associated with bacterial proliferation (between 12 and 48 h post infection). At the
same time, rBCV obtains a large number of ER molecular markers, such as calmodulin,
calreticulin and ER protein Sec61. After Brucella infection, unfolded protein response (UPR)
is induced by secreting a variety of effector proteins. IRE1 and Yip1A mediate UPR to form a
complex at endoplasmic reticulum export sites (ERES) to selfphosphorylate [
22
]. Activated
IRE1 can promote the formation of ER derived vesicles. The ER derived vesicles fuse
with the Lys vesicles containing Brucella to form rBCV, which promote the proliferation of
Brucella by continuously fusing with the secretory ER derived vesicles. Brucella can survive
and propagate in the host cell mainly by the action of LPS, OMP, T4SS, two-component
regulatory system and other virulence factors [
23
,
24
]. These factors are necessary for
Brucella to invade host cells and survive and replicate in cells.
As the virulence factor of Brucella, the T4SS encoded by VirB can affect the survival
and replication of Brucella (Figure 2). VirB is a virulence gene of Brucella. Its expression in
host cells can affect the intracellular survival and replication of Brucella. The functions of
the 12 genes of VirB operon are different. At present, the effects of the 12 VirB genes on the
virulence of Brucella are mainly focused on Brucella. abortus,Brucella. melitensis and Brucella.
ovis (Table 1).
Int. J. Mol. Sci. 2021,22, 3673 4 of 13
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 14
3. The Survival and Replication of Brucella in Cells
After Brucella invades the host cell, it circulates in the cell [21]. The rBCV stage is
tightly associated with bacterial proliferation (between 12 and 48 h post infection). At the
same time, rBCV obtains a large number of ER molecular markers, such as calmodulin,
calreticulin and ER protein Sec61. After Brucella infection, unfolded protein response (UPR)
is induced by secreting a variety of effector proteins. IRE1 and Yip1A mediate UPR to
form a complex at endoplasmic reticulum export sites (ERES) to selfphosphorylate [22].
Activated IRE1 can promote the formation of ER derived vesicles. The ER derived vesicles
fuse with the Lys vesicles containing Brucella to form rBCV, which promote the prolifera-
tion of Brucella by continuously fusing with the secretory ER derived vesicles. Brucella can
survive and propagate in the host cell mainly by the action of LPS, OMP, T4SS, two-com-
ponent regulatory system and other virulence factors [23,24]. These factors are necessary
for Brucella to invade host cells and survive and replicate in cells.
As the virulence factor of Brucella, the T4SS encoded by VirB can affect the survival
and replication of Brucella (Figure 2). VirB is a virulence gene of Brucella. Its expression in
host cells can affect the intracellular survival and replication of Brucella. The functions of
the 12 genes of VirB operon are different. At present, the effects of the 12 VirB genes on
the virulence of Brucella are mainly focused on Brucella. abortus, Brucella. melitensis and
Brucella. ovis (Table 1).
Figure 2. T4SS of Brucella is a multiprotein complex encoded by VirB operon, which participates in
the intracellular activities of Brucella. The T4SS is mainly divided into the following five parts: the
elongation region is composed of VirB2; the central and outer membrane regions were composed
of VirB7, VirB9 and VirB10; the junction region is composed of VirB5 and VirB10; the intimal re-
gion was composed of VirB3, VirB4, VirB6, VirB8 and VirB10; ATP energy region composed of
VirB4 and VirB11.
Figure 2.
T4SS of Brucella is a multiprotein complex encoded by VirB operon, which participates in
the intracellular activities of Brucella. The T4SS is mainly divided into the following five parts: the
elongation region is composed of VirB2; the central and outer membrane regions were composed
of VirB7, VirB9 and VirB10; the junction region is composed of VirB5 and VirB10; the intimal region
was composed of VirB3, VirB4, VirB6, VirB8 and VirB10; ATP energy region composed of VirB4
and VirB11.
Table 1.
The 12 genes of VirB operon and their respective function Brucella. abortus,Brucella.melitensis and Brucella.
Ovis [25–29].
Operon
Whether or Not the Virulence Decreases
after Deletion Function
Brucella.
abortus
Brucella.
melitensis Brucella. ovis
VirB1 decrease unknown unknown Dissolves glycosyltransferase, which makes T4SS
easier to assemble and assemble.
VirB2 decrease decrease decrease Participate in the immune protection of the body and
affect the production of immune protection.
VirB3, VirB6,
VirB7, VirB10 decrease unknown unknown Signal transmission of bacterial transmembrane
proteins.
VirB4 decrease decrease unknown Transport substances and effectively prevent BCV and
dissolution Enzyme fusion.
VirB5, VirB8 decrease unknown unknown Regulation of intracellular transport in Brucella.
VirB9 decrease decrease unknown
As a part of the outer membrane of Brucella type IV
secretion system, it can stimulate the body to produce
immune responsean.
VirB11 decrease unknown unknown It provides energy for the secretion process of Brucella.
VirB12 unchanged unchanged unchanged Immune antigen.
Int. J. Mol. Sci. 2021,22, 3673 5 of 13
In the process of Brucella cell replication, T4SS plays an important role in a series
of processes [
30
], such as late intracellular and lysozyme markers, the recognition of
ER markers and acting on the secretion pathway, the recognition of autophagy markers,
resistance to the harsh intracellular environment and the regulation of the activation of
the immune pathway [
30
]. With the help of T4SS, Brucella transfers to ER and produces
acidic BCV under the induction of expression of VirB operon encoded by T4SS. The acidic
environment of BCV is conducive to the survival of Brucella [
31
]. BCV acidification is
an important step in the maturation of BCV, and acidic environment contributes to the
expression of VirB operon in Brucella [
32
]. Brucella induces the expression of VirB operon
in acidic environment and controls the expression of genes related to T4SS. Brucella uses
T4SS to transport effector from the membrane space to the host cell cytoplasm, so as to
regulate the signal transduction of the host cell to facilitate its survival in the host cell. In
addition to T4SS, the two-component regulatory system (BvrS/BvrR), cyclic
β
-glucan, Lux
R-like transcriptional regulator (VjbR), LPS, flagellumlike structure, and transporter-like
protein (Baca) and phosphatidylcholine (PC) are necessary components for Brucella to
invade cells and live in cells [
33
]. Cd98hc transmembrane protein also plays an important
role in intracellular proliferation and signal pathway regulation [34].
Brucella is similar to other intracellular bacteria, it infection of the host cells includes
adhesion, invasion, establishment of infection and diffusion. The interaction between host
and LPS may play an important role in Brucella’s cell viability. The essential gene (manB,
wboA) for the O-side chain synthesis of LPS is a necessary factor for Brucella to establish
the replication region in the cells [
35
]. Smooth Brucella glabrata inhibits host cell apoptosis
and promotes its survival and replication in host cells through the interaction of the o-chain
and TNF-
α
[
36
]. Thus, dead cells do not release specific factors, therefore they do not
activate the immune system and Brucella are able to avoid host immune surveillance [
37
].
In this way, Brucella enters its replication niche and proliferates in the host cells. In addition,
LPS is considered to be the main determinant of virulence, and the survival rate of the
LPS-deficient strains in the host cells is reduced. Brucella can not only escape from immune
surveillance, but also adapt to the internal environment of phagocytes and nonphagocytes
and survive in the cell. Based on the inhibition of the phagosomal–lysosomal maturation
pathway and the maladjustment of the intracellular transport, Brucella enters the cell and
then reaches the ER-derived replication niche [38].
The propagation of Brucella in cells includes two stages: the stable stage and the
exponential stage. The physiological state of the stable stage is favorable for Brucella to
adapt to the harsh living conditions in the phagocytosis body, while the exponential stage
is used to replicate under the appropriate environmental conditions, and this adaptive
regulation is completed by molecular mechanism. It has been found that the expression
of VirB operon is high in the exponential growth stage, but it is inhibited after entering
the stable stage [
39
]. Like other intracellular bacteria, Brucella have adapted to their
intracellular lifestyle and no longer need to accumulate energy storage molecules [
40
].
Brucella can survive opsonin mediated phagocytosis and replicate in cells [
41
]. The presence
of cytochromes (such as cytochrome bc1 complex or hydroquinone) with high oxygen
affinity play an important role in Brucella’s adaptation to intracellular survival. It has
been confirmed that Brucella uses heme iron polypeptide as an iron source in vitro. When
Brucella replicates in trophoblast cells, heme iron polypeptide is needed to participate.
Macrophages are the preferred host cells of Brucella, which are very important for the
heme iron polypeptide cycle in mammals [
41
]. The mutations of iron regulatory genes can
weaken the virulence of Brucella in mammalian host cells and make it more sensitive to
oxidative damage, which indicates that these mutations are involved in the growth and
intracellular survival of Brucella [42].
Int. J. Mol. Sci. 2021,22, 3673 6 of 13
4. Brucella Evades Killing of Host Immune System
Innate immunity and adaptive immunity play an important role in host immunity
against Brucella. Innate cellular immunity antiinfection effect is not good; the host immune
response caused by Brucella invasion is mainly adaptive cellular immunity [38].
4.1. Brucella Interferes with Innate Immune Recognition and Response of Host
As the first immune defense line, innate immune response plays a very important
role in the process of protecting the body from pathogens [
43
]. Brucella has been living in
cells for a long time and Brucella has evolved many mechanisms that interfere with innate
immune recognition and response in its interaction with the body (Figure 3).
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 14
maturation pathway and the maladjustment of the intracellular transport, Brucella enters
the cell and then reaches the ER-derived replication niche [38].
The propagation of Brucella in cells includes two stages: the stable stage and the ex-
ponential stage. The physiological state of the stable stage is favorable for Brucella to adapt
to the harsh living conditions in the phagocytosis body, while the exponential stage is
used to replicate under the appropriate environmental conditions, and this adaptive reg-
ulation is completed by molecular mechanism. It has been found that the expression of
VirB operon is high in the exponential growth stage, but it is inhibited after entering the
stable stage [39]. Like other intracellular bacteria, Brucella have adapted to their intracel-
lular lifestyle and no longer need to accumulate energy storage molecules [40]. Brucella
can survive opsonin mediated phagocytosis and replicate in cells [41]. The presence of
cytochromes (such as cytochrome bc1 complex or hydroquinone) with high oxygen affin-
ity play an important role in Brucella’s adaptation to intracellular survival. It has been
confirmed that Brucella uses heme iron polypeptide as an iron source in vitro. When Bru-
cella replicates in trophoblast cells, heme iron polypeptide is needed to participate. Mac-
rophages are the preferred host cells of Brucella, which are very important for the heme
iron polypeptide cycle in mammals [41]. The mutations of iron regulatory genes can
weaken the virulence of Brucella in mammalian host cells and make it more sensitive to
oxidative damage, which indicates that these mutations are involved in the growth and
intracellular survival of Brucella [42].
4. Brucella Evades Killing of Host Immune System
Innate immunity and adaptive immunity play an important role in host immunity
against Brucella. Innate cellular immunity antiinfection effect is not good; the host immune
response caused by Brucella invasion is mainly adaptive cellular immunity [38].
4.1. Brucella Interferes with Innate Immune Recognition and Response of Host
As the first immune defense line, innate immune response plays a very important
role in the process of protecting the body from pathogens [43]. Brucella has been living in
cells for a long time and Brucella has evolved many mechanisms that interfere with innate
immune recognition and response in its interaction with the body (Figure 3).
Figure 3. Brucella can inhibit the secretion of IL-2 by antigen presenting cells, and then prevent natural killer cells (NK)
from secreting inflammatory factors such as IFN-γ and TNF-α. Brucella can also inhibit IFN-γ mediated phagocytosis to
Figure 3.
Brucella can inhibit the secretion of IL-2 by antigen presenting cells, and then prevent natural killer cells (NK) from
secreting inflammatory factors such as IFN-γand TNF-α.Brucella can also inhibit IFN-γmediated phagocytosis to escape
the killing of immune system. Brucella affects the maturation of DC by blocking TLR2 receptor pathway, and interferes with
the establishment of Th1 immune response by inhibiting macrophages to reduce IL-12 secretion and preventing DC from
activating T-lymphocytes.
The innate immune system recognizes microbes by characteristic molecules like the
Gram-negative LPS Lipid A (the LPS bioactive moiety) signals through Toll-like receptors
(TLRs) to induce proinflammatory molecules and small GTPases of the p47 family involved
in intracellular pathogen control. Brucella LPS exhibits a low toxicity and its atypical
structure was postulated to delay the host immune response, favouring the establishment
of chronic disease. Brucella Lipid A is a 2,3-diaminoglucose disaccharide substituted with
C16, C18, C28 and other very long acyl chains. This peculiar structure is a poor agonist of
TLR4/myeloid differentiation-2 (MD-2) and therefore a paradigm has emerged proposing
Brucella LPS as a crucial virulence factor that hampers recognition by pattern recognition
receptors (PRR) and plays essential roles during infection. Research shows that Brucella
LPS did not induce inflammatory responses in macrophages and DCs, two of the most
important sentinels of the immune system.This was attributed to its poor recognition by
TLR4/MD-2, which is widely considered to be the major receptor complex for LPS binding
and signalling [44].
Host cells recognize the harmful substances by the interaction of PRR and pathogen
associated molecular pattern (PAMPs), so as to stimulate the body to produce related
immune response to kill and eliminate pathogens. However, there is increasing evidence
that Brucella display altered PAMPs in key molecules, suggesting that to escape detection by
innate immunity is a survival strategy. One of the best examples of a structure with altered
PAMPs is the LPS of Brucella.Brucella LPS bears a noncanonical lipid A and, although it
Int. J. Mol. Sci. 2021,22, 3673 7 of 13
signals TLR4, it is active only at very high concentrations [
45
]. The flagellin of Brucella lacks
the specific domain recognized by TLR5 and plays an important role in immune escape [
46
].
Moreover, Brucella LPS confers a highly resistant phenotype to cationic bactericidal peptides
and makes Brucella a poor activator of the complement system [
47
]. The Lipid A of Brucella
contains a longer fatty acid chain (C28), which greatly reduces its endotoxin properties [
23
].
There are free hydroxyl residues in the O-chain of bacterial LPS, which is favorable for
binding with C3. However, the O-chain of Brucella lacks free hydroxyl. Complement C3 can
inhibit the production of C3a and C5a by contacting with the specific O-chain of Brucella
LPS, thus avoiding the capture of the host immune system. At the same time, LPS on the
surface of the cell wall has many long side chains, which prevent the membrane attacking
complex from contacting the cell membrane (Figure 4).
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 14
escape the killing of immune system. Brucella affects the maturation of DC by blocking TLR2 receptor pathway, and inter-
feres with the establishment of Th1 immune response by inhibiting macrophages to reduce IL-12 secretion and preventing
DC from activating T-lymphocytes.
The innate immune system recognizes microbes by characteristic molecules like the
Gram-negative LPS Lipid A (the LPS bioactive moiety) signals through Toll-like receptors
(TLRs) to induce proinflammatory molecules and small GTPases of the p47 family in-
volved in intracellular pathogen control. Brucella LPS exhibits a low toxicity and its atyp-
ical structure was postulated to delay the host immune response, favouring the establish-
ment of chronic disease. Brucella Lipid A is a 2,3-diaminoglucose disaccharide substituted
with C16, C18, C28 and other very long acyl chains. This peculiar structure is a poor ago-
nist of TLR4/myeloid differentiation-2 (MD-2) and therefore a paradigm has emerged pro-
posing Brucella LPS as a crucial virulence factor that hampers recognition by pattern
recognition receptors (PRR) and plays essential roles during infection. Research shows
that Brucella LPS did not induce inflammatory responses in macrophages and DCs, two
of the most important sentinels of the immune system.This was attributed to its poor
recognition by TLR4/MD-2, which is widely considered to be the major receptor complex
for LPS binding and signalling [44].
Host cells recognize the harmful substances by the interaction of PRR and pathogen
associated molecular pattern (PAMPs), so as to stimulate the body to produce related im-
mune response to kill and eliminate pathogens. However, there is increasing evidence
that Brucella display altered PAMPs in key molecules, suggesting that to escape detection
by innate immunity is a survival strategy. One of the best examples of a structure with
altered PAMPs is the LPS of Brucella. Brucella LPS bears a noncanonical lipid A and, alt-
hough it signals TLR4, it is active only at very high concentrations [45]. The flagellin of
Brucella lacks the specific domain recognized by TLR5 and plays an important role in im-
mune escape [46]. Moreover, Brucella LPS confers a highly resistant phenotype to cationic
bactericidal peptides and makes Brucella a poor activator of the complement system [47].
The Lipid A of Brucella contains a longer fatty acid chain (C28), which greatly reduces its
endotoxin properties [23]. There are free hydroxyl residues in the O-chain of bacterial LPS,
which is favorable for binding with C3. However, the O-chain of Brucella lacks free hy-
droxyl. Complement C3 can inhibit the production of C3a and C5a by contacting with the
specific O-chain of Brucella LPS, thus avoiding the capture of the host immune system. At
the same time, LPS on the surface of the cell wall has many long side chains, which prevent
the membrane attacking complex from contacting the cell membrane (Figure 4).
Figure 4. As an important virulence factor of Brucella, LPS plays an important role in escaping from the host immune
system. The acetyl side chain (C28) of Brucella LPS reduces the nature of endotoxin to avoid the recognition of Toll-like
receptor 4 (TLR4), thus avoiding the monitoring of host immune system. The specific O-chain contained in Brucella LPS
can inhibit the production of C3a and C5a by complement C3, and then inhibit the degranulation of neutrophils, so as to
Figure 4.
As an important virulence factor of Brucella, LPS plays an important role in escaping from the host immune
system. The acetyl side chain (C28) of Brucella LPS reduces the nature of endotoxin to avoid the recognition of Toll-like
receptor 4 (TLR4), thus avoiding the monitoring of host immune system. The specific O-chain contained in Brucella LPS
can inhibit the production of C3a and C5a by complement C3, and then inhibit the degranulation of neutrophils, so as to
prevent the release of myeloperoxidase (MPO) and other lysosomal substances, and prevent them from being captured by
the host immune system.
It was found that the removal of polymorphonuclear before the start of adaptive
immunity was helpful to the elimination of bacteria in mice, indicating that neutrophils
inhibited the immune response of the body to Brucella [
48
,
49
]. Brucella also has different re-
sistance mechanisms to phospholipase A2, catheteridin, Lys and defensins, so as to ensure
its transport in lymphoid tissue. Brucella induces antigen-presenting cells to secrete IL-2
and activate NK cells. NK cells secrete IFN-
γ
, TNF-
α
, GM-CSF and other cytokines, which
play an important role in Th1 and Tc1 reactions [
50
]. Brucella evades the host immune
response by affecting macrophage function. Brucella can inhibit IFN-
γ
mediated phago-
cytosis and TNF-
α
expression in macrophages [
51
]. Infected macrophages can produce
proinflammatory factors (TNF-
α
, IL-6, IL-12) and inflammatory chemokines (GRO-
α
, IL-8,
MCP-1). TNF-
α
can significantly enhance the bactericidal ability of macrophages, IL-12
can induce Th1 immune response and produce IFN-
γ
.Brucella regulates the expression of
MHC-I and MHC-II by regulating IFN-
γ
secretion. IFN-
γ
mediated Th1 immune response
is essential for Brucella clearance [
52
]. Brucella can affect the maturation of DC by blocking
the TLR2 receptor pathway, and interfere with the establishment of type Th1 immune
response by reducing the secretion of IL-12 and preventing the activation of T-lymphocyte
by DC [53].
4.2. Transmission Mechanism of Brucella Interfering Host Adaptive Immune Response
The adaptive immune response of the organism mainly includes the humoral immune
response and the cellular immune response [
54
]. In the long-term evolution, Brucella has
Int. J. Mol. Sci. 2021,22, 3673 8 of 13
produced the transmission mechanism of interfering information from innate immunity to
acquired immunity, so as to establish chronic infection [49].
The decrease in macrophage and DCs recruitment after Brucella infection leads to the
decrease of CD8+ T lymphocyte activation, which leads to the formation of immunosup-
pression, which is conducive to Brucella replication and chronic infection [
55
]. Brucella
lumazine synthase (BLS) transmits signals through TLR4 and induces DC maturation
and CD8+ T-lymphocytel toxicity, so as to inhibit tumor growth and regulate innate and
adaptive immune responses. As one of several virulence factors necessary for Brucella to
establish chronic infection, prpA can interact with macrophages to promote B-cell pro-
liferation [
56
]. PrpA can regulate IFN-
γ
, TNF-
α
, IL-10 and TGF-
β
1 in the early stage of
infection [
57
]. The results showed that prpA, Btp1/TcpB and LPS, as immunomodulators,
had the ability to inhibit IFN-
γ
secretion and promote IL-10 secretion, thus affecting Th1
immune response [58].
Brucella effectors can control the TLR signaling pathway involved in DC maturation,
and have a significant effect on T lymphocyte activation and antigen presentation. The
sequence of Brucella TIR protein 1 (btp1) is similar to toll/IL-1 domain family, because
of the significance of the TIR domain in TLR signal [
23
,
59
]. BTP 1 not only inhibited
the production of proinflammatory cytokines, but also inhibited the maturation of DC,
resulting in the inhibition of TLR2 and TLR4 signals [60].
Brucella inhibits immune signal transduction by expressing secreted proteins contain-
ing the TIR domain Btp1/TcpB [
61
]. The detailed mechanism of this protein is still not fully
understood, but there is evidence that when it binds to the TIR domain-containing adaptor
protein (TIRAP/Mal), it competes with myeloid differentiation response gene 88 (MyD88),
which not only promotes TIRAP/Mal ubiquitination degradation but also inhibits TLR4
and TLR2 signal transduction [
62
]. In this way, Brucella inhibits DC maturation and the
production of proinflammatory cytokines IL-12 and TNF-
α
. In addition, this protein also
inhibits the killing effect of CD8+ T-lymphocyte on Brucella target cells [63].
5. Chronic Infection Caused by Brucella
After Brucella invade the macrophage, with the proliferation of BCV in ER, some
components of Brucella can interact with host cells, which can inhibit the synthesis of
bactericides, affect the activation of signal transduction pathway, induce super allergic
reaction, and finally cause systemic persistent infection [
64
]. When the T4SS encoded by
VirB stimulates the body, the immune response produced by the host can inhibit the growth
and reproduction of Brucella, but it cannot kill Brucella, resulting in a confrontation between
Brucella and the host, causing the body to develop persistent infection [65].
The interaction between Brucella and host immune system is very important in caus-
ing persistent infection, but immune evasion is not the only mechanism. Studies have
shown that the genes needed for Brucella persistence are related to changes in bacterial
metabolism and the ability of pathogens to utilize specific nutrients [
66
]. The macrophage
is the main target cell of Brucella persistence, which provides a place for Brucella replication
and survival [
67
]. The key to understanding Brucella’s cell viability and chronic infection
is to understand the interaction between Brucella and different macrophage subsets [
68
].
Several factors required for the long-term existence of Brucella in the host cannot medi-
ate the replication of Brucella in
in vitro
cultured macrophages, indicating that different
macrophage populations and their metabolic status may be the determinants of chronic
diseases [66,69].
Brucella not only has the ability to resist phagocyte sterilization, but also can prevent
antigen-specific T-lymphocyte from recognizing themselves, thus forming a microenvi-
ronment conducive to their survival and reproduction, leading to chronic persistent infec-
tion [
28
]. The proliferation and replication of Brucella weakens the phagocytic function of
macrophages, which results in the loss of the cell killing and antigen presenting functions of
macrophages, thus avoiding the host’s immune defense mechanism and causing persistent
infection [
70
]. Persistent infection is determined not only by Brucella’s ability to evade host
Int. J. Mol. Sci. 2021,22, 3673 9 of 13
immune response, but also by its ability to utilize available nutrients in the chronic phase
of infection. Macrophages play an important role in host physiology and metabolism [
71
],
which are the key to the survival of Brucella.Brucella not only has the ability to resist the
phagocytic bactericidal effect, but also makes the host cells form a microenvironment which
is conducive to its survival and reproduction. This microenvironment can make Brucella
live in the host cell for a long time, and eventually lead to chronic persistent infection.
The macrophage is the main target cell of Brucella.Brucella can inhibit the apoptosis
of the macrophage by inhibiting the secretion of TNF-
α
so that it can survive in the cell
and multiply in large numbers, weaken the phagocytic function of macrophage, lose the
killing effect and antigenic presenting function of macrophage, and finally escape the
immune surveillance of host [
72
]. On the other hand, macrophages have a weak ability to
activate the initial T-lymphocyte and do not express CD1 molecules with antigen presenting
function, so they cannot effectively present lipid antigens to NK cells, which may be the
reason for the formation of Brucella persistent infection. Brucella regulates the immune
response by inducing regulatory cytokines (such as IL-10), which indicates that the IL-10
pathway plays an important role in the chronic infection caused by Brucella [73–75].
Brucella evades the host’s initial natural immunity through Toll-like receptor (TLR),
and causes a slight inflammatory reaction by modifying virulent factors such as LPS and
flagellin [
51
], which causes Brucella to develop persistent infection in the body [
23
]. In
addition, Brucella can regulate immune response by inducing regulatory cytokines (such
as IL-10), indicates that IL-10 pathway can play an important role in bacterial persistent
infection [
73
–
75
]. It is worth noting that Brucella IL-10 inhibits the bactericidal ability
of macrophages activated by IFN-
γ
and the production of proinflammatory cytokine
in vitro [74,75].
Brucella can induce CD4(+) CD25(+) T-lymphocyte to produce anti-inflammatory cy-
tokine IL-10, thus inhibiting the immune activation of macrophages. The early production
of IL-10 by CD25 (+) CD4 (+) T-lymphocyte can regulate the function of macrophages
and help to promote the initial balance between proinflammatory cytokines and anti-
inflammatory cytokines beneficial to pathogens, so as to promote the survival and persis-
tent infection of bacteria [
76
]. Proline racemase of Brucella acts as mitogen of B lymphocytes
and induces spleen cells to secrete IL-10, which may be the basis of its persistent infec-
tion of mononuclear phagocyte system in mice [
77
]. In conclusion, these data indicate
that IL-10 plays an important role in regulating the initial immune response. Brucella
infection can increase the survival and persistent infection of pathogens by regulating
macrophage function.
Trophoblast cells are also the key target cells for the survival and replication of Brucella.
The replication ability of Brucella in host cells is the core of its pathogenicity. It was
found that the accumulation of Brucella in trophoblast cells was higher than that in other
organs [
78
]. In the extravillous trophoblast (EVTs), Brucella abortus forms an acidified
lysosomal vesicle in which it proliferates and replicates. These vesicles play an important
role in the chronic infection caused by Brucella [
79
]. It was found that the invasion of
Brucella to trophoblast cells mainly caused cell necrosis and released a large number of
Brucella. The necrotic foci of Brucella spread continuously through the capillaries and
became the main source of chronic infection of the placenta [80].
It has been found that the proliferation of Brucella in trophoblast cells is related to
the ER network, and Brucella establishes a proliferative living environment through the
interaction with the endoplasmic network [
81
]. Brucella abortus invades and replicates in the
human trophoblastic cell line Swan-71 and that the intracellular survival of the bacterium
depends on a functional VirB operon. After Brucella infection, the trophoblast cells of
ruminants can produce a lot of erythritol, which can promote the growth and reproduction
of bacteria. At present, little is known about the changes of the trophoblast environment
after Brucella infection, which need further research and discovery.
Int. J. Mol. Sci. 2021,22, 3673 10 of 13
6. Conclusions
The relationship between intracellular bacteria and host is very complex, involving a
variety of biological factors and signal pathways. As a facultative intracellular bacteria,
Brucella can colonize, grow and reproduce for a long time after invading the host cell, which
is completely dependent on its survival cycle in the host cell and the function of escaping
from the host immune system. Although the research on the facultative intracellular
parasitism mechanism of Brucella is still in progress, there are still many problems to be
further explained.
Author Contributions:
Conceptualization, H.J. and Z.Z.; methodology, Y.X.; software, B.L.; valida-
tion, H.J., Z.Z. and B.L.; formal analysis, M.L.; investigation, H.Z.; resources, X.G.; data curation,
G.G.; writing—original draft preparation, Z.Z.; writing—review and editing, H.J.; visualization, B.L.;
supervision, Y.X.; project administration, H.J.; funding acquisition, H.J. All authors have read and
agreed to the published version of the manuscript.
Funding:
This work was financially supported by the National Science Foundation for Young Scien-
tists of China (No. 31802215), the Natural Science Foundation of Chongqing (No. cstc2018jcyjA0807,
cstc2020jcyj-msxm0522), and the Fundamental Research Funds for the Central Universities (Nos.
XDJK2020C022, XDJK2019C024).
Acknowledgments:
We are very grateful to Jianjun Wen from University of Texas Medical Branch
for helpful discussions during the preparation of this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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