Brucella Spp. Virulence Factors: and Immunity

AV04CH09-Tsolis ARI 29 October 2015 12:45
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Brucella Spp. Virulence Factors

and Immunity
Mariana X. Byndloss and Renee M. Tsolis
Department of Medical Microbiology and Immunology, School of Medicine, University of
Annu. Rev. Anim. Biosci. 2016.4. Downloaded from www.annualreviews.org
California, Davis, California 95616; email: mxavier@ucdavis.edu, rmtsolis@ucdavis.edu

Access provided by Trent University on 12/24/15. For personal use only.
Annu. Rev. Anim. Biosci. 2016. 4:9.1–9.17 Keywords

The Annual Review of Animal Biosciences is online at brucellosis, immune response, reproduction, chronic disease
animal.annualreviews.org
This article’s doi: Abstract

10.1146/annurev-animal-021815-111326
Brucellosis, caused by bacteria of the genus Brucella, is an important zoonotic
Copyright c 2016 by Annual Reviews. infection that causes reproductive disease in domestic animals and chronic
All rights reserved
debilitating disease in humans. An intriguing aspect of Brucella infection is
the ability of these bacteria to evade the host immune response, leading
to pathogen persistence. Conversely, in the reproductive tract of infected
animals, this stealthy pathogen is able to cause an acute severe inflammatory
response. In this review, we discuss the different mechanisms used by Brucella
to cause disease, with emphasis on its virulence factors and the dichotomy
between chronic persistence and reproductive disease.
9.1
Changes may still occur before final publication online and in print
INTRODUCTION
Brucellosis is a zoonotic bacterial disease caused by bacteria of the genus Brucella, which are able to
establish long-term infections in their hosts (1, 2). Human brucellosis, caused most commonly by
Brucella melitensis and Brucella abortus, is considered one of the most important zoonotic diseases
worldwide. Although accurate epidemiological data are not available for many endemic areas, it
has been estimated that more than 500,000 new human cases occur annually (3). The disease
is characterized by a long incubation period that leads to a chronic, sometimes lifelong, debil-
itating infection with serious clinical manifestations, such as fever, arthritis, hepatomegaly, and
splenomegaly (2, 4).
In vivo, Brucella is found in association with phagocytic cells, most prominently macrophages,
in which a subset of bacteria is able to evade killing in phagolysosomes and replicate successively
with an endoplasmic reticulum–associated compartment and a modified autophagosome (5, 6).
The immunoevasive nature of this pathogen is further underscored by its ability to elude initial
innate immune recognition through Toll-like receptors (TLRs) (7) as well as through modifica-
tions of virulence factors such as lipopolysaccharide (LPS) (8) and flagellin (7), resulting in a mild
proinflammatory response that leads to bacterial persistence (2). Moreover, Brucella is able to mod-
ulate the immune response through induction of regulatory cytokines such as IL-10, suggesting
that the IL-10 pathway could play an important role in enabling bacterial persistence (9–11).
It is important to note that a key aspect of Brucella pathogenesis is its interaction with
macrophages. Indeed, research focusing on the in vitro Brucella/macrophage interaction has been
critical for the understanding of how B. abortus survives intracellularly (2, 12). However, several
factors required for chronic persistence in vivo do not appear to mediate intracellular replication
in cultured macrophages (13, 14), suggesting that the different macrophage populations, as well
as their metabolic state, may be determinant factors for chronic Brucella spp. persistence in vivo.
In the natural host, Brucella spp. cause an acute severe inflammatory response (15, 16), which can
lead to abortion and infertility (Table 1, Figure 1). The reproductive disease caused by Brucella
seems to be crucial for pathogen transmission and life cycle; however, the molecular pathogenesis
of animal brucellosis has been seldom explored, mainly owing to the lack of a model in which all
available tools can be used to decipher the disease.
BRUCELLA SPECIES: HOST SPECIFICITY AND CLINICAL

MANIFESTATIONS
Brucellosis in the Natural Host

Small ruminants are considered the preferential host for B. melitensis infection (16, 17), although
cattle and other ruminants may also become infected. This disease is considered endemic in several
parts of the world, including Mediterranean, Middle Eastern, and Latin American countries where
small ruminants are a major source of nutrition to the resident population (18, 19). In goats and
sheep, B. melitensis infection is characterized by orchitis in affected males and abortion and reduced
milk production in females. Therefore, in infected dams, the predominant clinical sign of acute
brucellosis is reproductive failure characterized by abortion in the late stages of gestation, stillbirth,
or birth of weak offspring (16). Interestingly, B. melitensis targets the udder in the majority of acute
natural infections in pregnant goats, which leads to intermittent shedding of the bacteria in the
milk during the subsequent lactation. Moreover, this pathogen can also cause a mild inflammation
of the mammary tissue, the most likely cause of reduced milk production in infected animals (20).
Importantly, the disease is transmitted by contact between susceptible animals and contaminated
secretions from the female genital tract (20).
9.2 Byndloss · Tsolis
Table 1 Host preference for Brucella species in domestic animals

Zoonotic
Species Natural host potential Clinical signs Transmission
Brucella Small High Female: abortion, weak offspring, reduced Oral: ingestion of contaminated
melitensis ruminants milk yield placenta, aborted fetus,
Male: infertility, orchitis, epididymitis (rare) contaminated milk
Brucella abortus Cattle Moderate Female: abortion, weak offspring, reduced Oral: ingestion of contaminated
milk yield placenta, aborted fetus,
Male: infertility, orchitis, epididymitis (rare) contaminated milk
Brucella suis Pig Moderate Female: abortion, weak offspring Oral: ingestion of contaminated
Male: infertility, orchitis, epididymitis, placenta, aborted fetus,
osteoarticular disorders contaminated milk
Venereal: breeding using
contaminated semen
Brucella canis Dog Mild Female: abortion at 45–55 days Oral: ingestion of contaminated
Male: infertility, orchitis, epididymitis placenta, aborted fetus,

Both genders: bacteremia contaminated milk
Venereal: breeding using
contaminated semen
Brucella ovis Sheep Absent Female: abortion, weak offspring (rare) Oral: close contact between
Male: infertility, orchitis, epididymitis rams
Venereal: use of infected rams
during mating season
Brucellosis in cattle is mainly caused by B. abortus, a pathogen that is also able to infect buffaloes,
camels, deer, dogs, horses, goats, and sheep (16). Similarly to what happens during B. melitensis
infection in goats, B. abortus infection in dairy herds causes abortion, stillbirth, postpartum metritis,
and inflammation of the mammary gland, leading to decreased milk production and shedding of
the organism in the milk of infected cows (15, 21). Additionally, B. abortus infection of the pregnant
uterus leads to a neutrophilic necrotizing placentitis, as well as fetal lesions, which are characterized
by fibrinous pleuritis and pericarditis, and interstitial pneumonia (15). Although last-trimester
abortion is the predominant clinical sign of bovine brucellosis, infected cows usually present with
only one episode of abortion, giving birth to weak or even healthy calves in subsequent gestations.
In fact, a significant percentage of infected cows will remain asymptomatic throughout their
lifetime, giving birth to healthy calves in all pregnancies (22), while still shedding the organism in
the placenta. In bulls, B. abortus is a common cause of temporary or permanent infertility, owing
to infection of both internal and external male sexual organs, leading to orchitis, epididymitis,
and seminal vesiculitis (reviewed in 16). Bovine brucellosis is transmitted by contact between
susceptible individuals and the contaminated fetus, fetal membranes, and uterine secretions from
an infected cow (22).
Swine brucellosis is considered an important reemerging disease of domestic and wild pigs in
Europe; North, South, and Central America; southern Asia; and the Pacific Islands (16, 23). The
etiologic agent, Brucella suis, is also able to infect other domestic animal species, such as cattle,
horses, rabbits, and dogs (23, 24). Interestingly, B. suis infection in pigs is often asymptomatic.
When present, clinical signs in sows are characterized by infertility, irregular estrus, and abortion
during any stage of gestation, as well as birth of weak piglets, usually associated with high rates of
neonatal mortality. In boars, B. suis infection results in reproductive failure owing to inflammation
of the testis and epididymis and is often accompanied by arthritis, osteomyelitis, spondylitis,
www.annualreviews.org • Brucella 9.3
Animal brucellosis Human brucellosis
Chronic debilitating with

Disease Reproductive symptoms systemic symptoms
Abortion Undulant fever

Weak offspring Osteoarticular pain
Clinical signs Infertility in males Splenomegaly
Arthritis, osteomyelitis, paralysis Hepatomegaly
(Brucella suis) Neurological signs
Females: acute neutrophilic

Pathology necrotizing placentitis Granuloma formation in
systemic organs
Males: orchitis/epididymitis
Transmission to Yes Rare

other hosts
Vaccine Yes (S19, RB51, REV1) No
Treatment No Yes
Figure 1
Comparison of features of animal and human infections with Brucella spp.
and paralysis. Additionally, the osteoarticular disorders can also be present in young pigs (25).
Therefore, swine brucellosis is considered a herd problem with major economic implications
owing to the fact that pigs of all ages may be affected, even though the disease is more common
in adult animals (16). In an infected herd, B. suis is transmitted via both venereal and oral routes,
through intermittent secretion of the bacteria for long periods in semen, urine, uterine discharges,
and milk (20). Moreover, asymptomatic pigs play an important role in the maintenance of the
disease in the herd, because B. suis shedding in bodily fluids can occur in the absence of evident
clinical signs (25).
Canine brucellosis, caused by Brucella canis, is a disease of domestic dogs and wild Canidae and
very rarely affects other domestic animals (26). The disease is more prevalent in Central and South
America and has been eradicated in most developed countries. However, in the United States it is
more prevalent in rural parts of the southeastern states, and recent outbreaks associated with the
dog trade have prompted a plan for an epidemiological survey by the Centers for Disease Control
(16, 26, 27). As described for animal infection with other Brucella species, dogs infected with B. canis
show symptoms of reproductive disease. In infected bitches, the predominant clinical sign is abor-
tion after 45–55 days of gestation, although early embryonic death and/or abortion 10–20 days after
mating may also occur (26, 28). Semen from infected dogs exhibits a large number of inflamma-
tory cells and abnormal sperm, as a consequence of the B. canis–induced epididymitis and orchitis,
which commonly leads to infertility (29). In addition to reproductive disease, B. canis infection in
dogs results in a long-lasting bacteremia, making blood culture a valuable approach for diagnosis
(28). In an affected kennel, B. canis is transmitted both via the venereal route and via breeding
using semen from an infected dog. Transmission via the oral route occurs through ingestion of
contaminated placental tissues, aborted fetuses, or vaginal secretions from infected bitches (28, 30).
Ovine brucellosis is considered to be a significant disease in most sheep-raising areas in the
world, such as Australia, New Zealand, North and South America, South Africa, and many
European countries (31). In contrast to other Brucella species, Brucella ovis affects mostly males,
causing primarily epididymitis and inflammation of sexual glands in adult rams, which leads to poor
semen quality characterized by decreased sperm concentration and presence of inflammatory cells
(32, 33). Importantly, in chronically infected rams, the B. ovis–induced lesion in the epididymis
is rarely noticed, and most infected rams never develop any clinical signs of the disease (31, 34).
Although usually asymptomatic, infected rams may shed B. ovis in the semen for long periods, and
therefore they are considered the most important reservoir of the pathogen in the herd (31, 34).
In ewes, B. ovis can occasionally cause abortion associated with placentitis at 30 days of gestation,
as well as stillbirth or birth of weak lambs (35). B. ovis transmission in sheep occurs primarily via
the venereal route, and occurs when ewes mate consecutively with an infected ram and then a
susceptible one during the same mating season. Additionally, the disease can be transmitted via
the oral/mucosal route by direct contact between rams kept in the same premises for prolonged
periods (36, 37).
Human Brucellosis
Human brucellosis is considered to be a geographically widespread bacterial zoonotic infection
of global importance owing to its significant morbidity in humans (2, 38). Although the lack of
accurate information can make it challenging to determine the real disease burden attributable to
Brucella infection in humans, a variety of studies have demonstrated that this disease is endemic
in a large number of developing countries, mainly those in the Mediterranean rim, the Middle
East, central Asia, sub-Saharan Africa, and Latin America (reviewed in 38). In countries such
as Iraq, Jordan, and Saudi Arabia, as well as Kyrgyzstan and Azerbaijan, the incidence of human
brucellosis is believed to be higher than 100 cases per 100,000 habitants each year (39–41), whereas
the incidence of the disease in African and Latin American countries is lower but still significant,
ranging from 12.8 to 35 cases per 100,000 inhabitants (42, 43).
Human brucellosis is caused by four species of Brucella, namely, B. melitensis, B. abortus, B. suis,
and B. canis (Table 1), with the majority of the disease in humans being attributed to B. melitensis,
although there is a possibility that human infection by the other three species is underappreciated
(44, 45). In humans, brucellosis is described as a chronic febrile debilitating disease with an
incubation period of 2 to 24 weeks, which leads to significant socioeconomic losses owing to
long-term treatment and inability of the affected individuals to provide for their families (2).
The most common clinical signs of Brucella infection are a chronic, low-grade relapsing fever,
followed by articular, muscular, and back pain resulting from pathogen-induced inflammation
in the joints and vertebrae. Brucella infection can also cause splenomegaly and hepatomegaly,
and less frequently can lead to endocarditis and neuropsychiatric manifestations (Figure 1)
(46). In the natural host, Brucella infection mainly causes a reproductive disease. In contrast,
clinical studies in humans have suggested that there is no significant correlation between a prior
brucellosis diagnosis and occurrence of abortion (47). However, high prevalence of abortion,
ranging from 14% to 43%, was observed in women who were diagnosed with either acute or
chronic brucellosis during pregnancy (48), raising the possibility that Brucella infection may be a
significant cause of prenatal complications in endemic regions.
In most cases, human infections occur through consumption of unpasteurized milk and dairy
products from infected animals. Veterinarians, farmers, and butchers are at higher risk of exposure
via mucosal contact with bodily fluids and tissues from infected animals and aborted fetuses (49).
Additionally, brucellosis is considered one of the most common laboratory-acquired infections
worldwide because Brucella can be easily transmitted via aerosol, a fact that has also led this
pathogen to be developed as a potential biological weapon in the past (16). Importantly, both
vertical and horizontal human-to-human transmission are rare (50), which leads to the conclusion
that infected humans are accidental hosts and should be considered a dead end as far as Brucella
species transmission is concerned.
At present, there is no available vaccine to prevent Brucella infection in humans. The treatment
of choice for human brucellosis consists of combination antibiotic therapy for long periods of at
least 6 weeks; however, significant treatment failures and relapses of infection have been reported
(38). Therefore, a better understanding of the disease pathogenesis in both animal and human
hosts is crucial for the development of more efficient tools for the prevention and treatment of
Brucella infection.
ROLE OF VIRULENCE FACTORS IN CHRONIC DISEASE
Brucella spp. Evasion of the Immune System

During infection, it is crucial for the host to be able to quickly detect invading pathogens, to induce
the initial inflammatory response necessary for control of the disease. Hence, the host has evolved
mechanisms to recognize the presence of bacteria in tissue through an innate immune surveillance
system, which is able to distinguish conserved pathogen-associated molecular patterns (PAMPs)
through pathogen recognition receptors. These receptors can be found in cell membranes (TLRs)
or in the cytosol (NOD-like receptors, NLRs) and have the ability to detect products considered
unique to bacteria, such as LPS, lipoteichoic acids, lipoproteins, and flagellin (51), leading to
activation of the initial proinflammatory response. As a chronic pathogen, Brucella has developed
passive and active mechanisms to evade detection by both TLRs and NLRs in order to persist and
cause long-lasting infection.
Multiple features of its LPS limit innate immune detection of Brucella spp. Modification of the
lipid A moiety of its LPS enables Brucella spp. to avoid detection by TLR4. Whereas most bacterial
pathogens, such as Enterobacteriaceae, have a lipid A moiety containing short fatty acid residues
(C12 –C16 ), Brucella lipid A contains a much longer one (C28 ), resulting in its greatly reduced
TLR4 agonist and endotoxic properties (8). An additional feature of B. abortus LPS that limits
recognition by TLR4 is the glycosylation pattern of its core oligosaccharide constituent, which
prevents binding to the TLR4 co-receptor MD-2 (PMID:22589715). The O-antigen moiety of
LPS is a target for complement deposition in many pathogens (52). However, an additional anti-
inflammatory feature of Brucella LPS is its resistance to deposition of complement component C3
(53, 54), which prevents generation of the anaphylatoxins C3a and C5a that synergize with TLRs
in the induction of proinflammatory cytokines (55, 56).
Interestingly, Brucella flagellin is able to avoid TLR5 detection, as it lacks a domain that is
essential for its recognition by this receptor (7). However, recent work has demonstrated that the
cytosolic receptor NLCR4 is able to detect Brucella flagellin and is important for pathogen control
in the mouse model of infection (57).
In addition to TLR4, TLR2 and TLR9 have also been implicated in sensing Brucella infection
(58–60). Therefore, as another strategy to avoid immune recognition, the Brucella genome en-
codes a protein that contains a Toll-interleukin-1 receptor (TIR) domain, named Btp1/BtpA in
B. abortus and TcpB in B. melitensis (61, 62). BtpA/TcpB acts by degrading the MyD88 adaptor-
like (MAL), which is required for both TLR2 and TLR4 but not TLR9 signaling (61, 63). As
a consequence, BtpA/TcpB is able to inhibit dendritic cell maturation and production of proin-
flammatory cytokines, contributing to long-term Brucella persistence. Recently, a second Brucella
TIR-containing effector protein has been described, named BtpB (64). BtpB, which appears to
have a stronger antagonist activity for TLR signaling than BtpA, is also believed to interfere with
Myd88-dependent signal transduction, although its role in modulating Brucella-induced inflam-
matory responses and bacterial persistence is not yet clear.
The Immune Response to Brucella Infection

The immune response to Brucella spp. has been characterized most extensively in the murine model.
During the initial stage of Brucella infection in mice, the host response developed resembles the
T helper 1 (Th1) type, with production of interferon γ (IFN-γ) by Th1 and natural killer cells, as
well as production of IL-12 and tumor necrosis factor α (TNF-α) by infected macrophages (60,
65–67). Moreover, in the murine model, both CD4+ and CD8+ T cells contribute to control of
the infection, and this is thought to occur via production of IFN-γ (10, 68). Indeed, induction of
a Th1 immune response during brucellosis seems to be critical, because mice deficient for IFN-γ
production or mice lacking interferon regulatory factor-1 are unable to control systemic bacterial
replication and succumb to an exacerbated B. abortus infection (69, 70). Additionally, IL-12 and
TNF-α depletion result in increased colony forming unit (CFU) counts in spleen from mice
infected with B. abortus; however, the phenotype observed is not as severe as the one described for
absence of IFN-γ (65, 66).
In humans, the initial immune response to Brucella spp. is also characterized by elevated levels
of proinflammatory cytokines linked to Th1 responses, such as IL-1β, IL-6, IL-12p40, TNF-α,
and IFN-γ (71, 72). Additionally, previous studies have demonstrated that mutations in genes
encoding the cytokines IFN-γ, IL-6, TNF-α, and IL-10 contribute to increased susceptibility to
human brucellosis (73, 74). However, during chronic human brucellosis, the initial Th1 response is
dampened and gains features of Th2 responses, such as an increase in IL-13-producing T cells (71).
Therefore, a plausible strategy used by Brucella to persist in the host for long periods would be to
induce the production of a cytokine, which is able to modulate the host proinflammatory response.
Indeed, in addition to an early proinflammatory Th1 response, B. abortus also induces the anti-
inflammatory cytokine IL-10 (11, 58, 75). Interestingly, anti-Brucella effector functions of IFN-γ-
activated macrophages, such as bactericidal capacity and production of proinflammatory cytokines,
were dampened by IL-10 during in vitro infection (10, 11). In vivo experiments demonstrated that
production of IL-10 by CD4+ CD25+ T cells was key for modulation of macrophage function
during early Brucella infection, because mice lacking IL-10 production by T cells or lacking the
presence of the IL-10R in macrophages exhibited decreased bacterial survival in spleen and liver, as
well as increased production of proinflammatory cytokines and pathology in affected organs (75).
Moreover, a B. abortus proline racemase, PrpA, was shown to act as a B cell mitogen and induce
IL-10 secretion by splenocytes, which may underlie its contribution to persistent infection of the
murine mononuclear phagocyte system (76). Taken together, these data suggest an important role
of IL-10 in modulating the initial immune response to Brucella infection through regulation of
macrophage function, resulting in increased pathogen survival and long-term persistence.
Type IV secretion system effectors and inflammation. In spite of their well-established im-
munoevasive behavior, Brucella spp. do rely on an important virulence factor for intracellular
survival, the type IV secretion system (T4SS) encoded by the genes virB1–virB12 (77–79). A func-
tional T4SS is required for survival of Brucella spp. within macrophages and epithelial cells in vitro,
as mutants lacking any of the virB genes fail to replicate intracellularly (77, 79). Moreover, the
Table 2 Brucella proteins requiring the type IV secretion system for translocation into host cells
Effector Function In vivo phenotype (reference)
VceA Unknown Unknown (88)
VceC Induction of the unfolded Yes—Brucella abortus vceC shows decreased induction of interleukin-6 in
protein response via IRE1α the mouse persistence model (90)
activation
BspA Inhibition of host protein Decreased B. abortus bspA mutant persistence in liver of infected mice (89)
secretion
BspB Inhibition of host protein Decreased B. abortus bspB mutant persistence in liver of infected mice (89)
secretion
BspC Unknown Unknown (89)
BspE Unknown Unknown (89)
BspF Inhibition of host protein Decreased B. abortus bspF mutant persistence in liver of infected mice (89)
secretion
RicA Interaction with small Unknown (111)

GTPase Rab2
Bab2_0123(BPE123) Localizes to the Unknown (87)
Brucella-containing vacuole
Bab1_2005(BPE005) Unknown Unknown (87)
SepA Early interaction with host Unknown (112)
cells
BtpB Inhibition of TLR2, TLR4 Suppression of inflammation in spleens of mice (64)
and TLR9 signaling
critical role of the Brucella T4SS during in vivo infection is demonstrated by the inability of T4SS-
deficient mutants to establish persistence in both the murine (13, 79, 80) and the caprine infection
models (81, 82). Interestingly, previous studies have demonstrated that the T4SS is required not
only for establishment of long-term infection but also for the induction of a Th1 immune response
in infected mice (83). This function was confirmed by the fact that a functional T4SS is necessary
for B cell maturation, activation of CD4+ T cells, and initial secretion of IL-12 and IFN-γ (67, 84).
Moreover, B. abortus detection by NLRs, leading to ASC-inflammasome-mediated production of
IL-1β and IL-18, was also shown to be dependent on the T4SS (85).
Virulence factors of pathogens provide important signals that allow the innate immune system
to differentiate between a harmless and a harmful insult (86). Among virulence factors, T4SS are
known to be crucial for induction of an immune response, because they are able to translocate
bacterial PAMPs, such as flagellin, peptidoglycan, nucleic acids, or effectors, into the host cell. In-
deed, multiple reports have identified Brucella proteins whose translocation into the host cytosol is
dependent on the T4SS (87–89) (Table 2). Although the function of the majority of these proteins
is still unknown, recent studies have shed light on the contribution of T4SS-secreted proteins for
Brucella infection (89, 90). Interestingly, to date VceC is the only T4SS effector shown to have a
clear effect in induction of inflammation by Brucella, because it affects the production of IL-6 by
macrophages in vitro and during in vivo infection. It is believed that VceC induces inflammation
by targeting the endoplasmic reticulum and activating the IRE-1α-dependent arm of the unfolded
protein response (90). Interestingly, the protein BtpB, one of the two TIR domain–containing
proteins of Brucella spp., was shown to be translocated into host cells in a T4SS-dependent
manner and has the opposite effect on inflammatory signaling (64). Other T4SS-secreted proteins,
namely BspA, BspB, and BspF, are necessary for B. abortus persistence in the mouse model and
were shown to affect the protein secretory pathway in infected cells. However, the effect of these
proteins on the immune response to Brucella infection remains to be investigated (89).
Target cell metabolism and Brucella persistence. The interactions of Brucella with the host
immune system during persistent infection have been studied extensively and contribute greatly to
the ability of this pathogen to cause chronic infection. However, evasion of the immune response is
not the only mechanism for pathogen persistence, because studies have shown that factors required
for establishment of chronic disease in vivo may not necessarily be dependent on the induction of
an immune response. Interestingly, a variety of genes required for Brucella persistence are related
to changes in bacterial metabolism and to the ability of this bacteria to use different nutrient
sources (13). This fact gives rise to the possibility that Brucella may have not only evolved to
take advantage of the different immune environment present during persistent infection but also
adapted to differences in intracellular nutrient availability during this period.
It is common knowledge that macrophages represent one of the main target cells for Bru-
cella persistence during infection (2). Therefore, interactions between Brucella and the different
macrophage subpopulations are key for understanding bacterial survival and disease progression.
Interestingly, in the mouse model, the macrophage subpopulations present in the spleen differ
significantly between acute and chronic stages of Brucella infection. During the acute stage of infec-
tion, a subpopulation of macrophages known as classically activated macrophages (CAM) predomi-
nates; these have a high proinflammatory and bactericidal capacity (91, 92). This fact correlates well
with higher IFN-γ levels as well as a decrease in B. abortus survival in spleen of infected mice during
acute infection (92). Conversely, during chronic infection, there is a shift in the macrophage popu-
lation structure, with predominance of the wound-healing, noninflammatory alternative activated
macrophage (AAM) subtypes, which correlates with persistent Brucella survival over time. Indeed,
AAM were more permissive for B. abortus survival and replication both in vitro and within the spleen
of infected mice, and the prevalence of AAM during chronic infection was dependent on activation
of the intracellular receptor peroxisome proliferator-activated receptor γ (PPARγ) (Figure 2).
The PPAR family member PPARγ is a nuclear receptor activated by fatty acids that has re-
cently been linked to the polarization of the macrophage phenotype (93). Therefore, even though
PPARγ is best known for its influence on adipocyte development and insulin resistance (94), it can
also have a global influence on macrophage biology (95, 96). Interestingly, studies using PPARγ–
deficient cells have demonstrated that, in the absence of PPARγ signaling, macrophages neither
appropriately suppress inflammatory cytokine production nor acquire an oxidative metabolic pro-
gram that is associated with the AAM macrophage phenotype (93, 94).
Importantly, one consequence of macrophage polarization is a shift in cellular metabolism,
which means that CAM and AAM use different sources of carbon and energy (97). This fact
raises the possibility that different nutrients are available intracellularly in different macrophage
subpopulations. Indeed, CAM rely on glycolysis for energy production and, therefore, consume
the majority of available intracellular glucose to meet their energetic needs. Conversely, AAM
obtain their ATP via degradation of fatty acids via the β-oxidation pathway in a PPAR-dependent
manner. Consequently, AAM exhibit an accumulation of glucose inside the cell, shown by higher
intracellular glucose levels in AAM when compared with CAM (92, 98). Interestingly, Brucella
makes use of this available glucose in AAM for long-term persistence, because a gluP mutant
defective in a transporter required for glucose uptake exhibits reduced intracellular persistence
within AAM in the mouse model. Additionally, this phenotype was dependent on PPARγ expres-
sion by macrophages, demonstrating that the PPARγ-dependent phenotypic switch to AAM is
important for maintenance of chronic B. abortus infection (13, 92).
Acute Brucella infection Chronic Brucella infection
IFN-γ IFN-γ
AAM CAM
AAM
CAM
IL-6
IL-6
IL-12
IL-12
TNF-α
PPARγ PPARγ TNF-α
Glucose Glucose
Figure 2
During the acute phase of Brucella abortus infection (left), interferon-γ (IFN-γ) is transiently produced,
resulting in a predominance of classically activated macrophages (CAM). In these cells, oxygen is consumed
by NADPH oxidase to generate superoxide radicals, and energy is produced by anaerobic glycolysis. Because
anaerobic glycolysis yields only 2ATP, the cell must consume more glucose to meet its energy needs. In
contrast, during the chronic infection phase (right), IFN-γ is absent, but IL-4 and IL-13 signal via STAT6
to induce the alternative activated macrophage (AAM) phenotype. Activation of STAT6 increases the
expression and activation of PPARγ, which in turn upregulates genes controlling β-oxidation, thereby
shifting cellular physiology toward oxidative pathways. As a result, less glucose is consumed for cellular
metabolism, and the intracellular glucose concentration increases. This glucose can be used by B. abortus for
its intracellular replication.
ROLE OF VIRULENCE FACTORS IN REPRODUCTIVE DISEASE

As previously discussed, in domestic animals, Brucella infection leads to reproductive disease in
which the main clinical sign is abortion and infertility secondary to a severe acute placentitis (2, 15).
Despite the importance of reproductive disease for Brucella survival and transmission, very little is
known about the immune response in the placenta of infected animals, as well as the interactions
of this pathogen with its target cell in the placenta, the trophoblast (99).
Studies in a bovine placental explant model have shown that Brucella initially downregulates
expression of proinflammatory cytokines on infection, and this suppression is dependent on the
T4SS and on the BtpB protein (100, 101). As infection progresses, Brucella induces the production
of proinflammatory cytokines and chemokines, such as CXCL-6 and IL-8, both in trophoblast
explants and in placental tissue from cows experimentally infected with B. abortus (100). These
studies are an important beginning toward understanding the pathogenesis of Brucella reproductive
disease. However, the mechanisms used by this pathogen to induce proinflammatory responses
and severe pathology in the placenta are far from understood.
For most Brucella species, transmission occurs primarily after abortion, via contact of a sus-
ceptible host with highly contaminated (up to 1013 CFU/g) placenta, fetus, fetal membranes, or
uterine secretions (99, 102). Moreover, Brucella clones can rapidly expand and transmit within
groups of animals, suggesting that selection of Brucella for higher transmissibility and replication
occurs through successive infections in hosts (103). Taken together, these facts stress the im-
portance of better understanding Brucella-induced placentitis, because placental infection is most
likely the niche used by this pathogen for its genetic evolution and long-term maintenance in the
host population.
Mouse Model of Brucella-Induced Placentitis: What Do We Know Thus Far?

Initially, the idea of using the natural host as a model to study animal brucellosis may sound very
appealing, because the research could be performed in the ideal environment for the pathogen.
However, the use of either small or large ruminants for experimental in vivo Brucella infections is
fraught with significant challenges, including high cost, appropriate biocontainment infrastruc-
ture, and biosafety measures. Therefore, to date, few virulence mechanisms identified in mice have
been tested for their role in placental infection of ruminants (81, 82, 104). To overcome these
barriers to progress in our understanding of Brucella transmission, there is a great need to develop
a safer and more cost-effective model to study the pathogenesis of the reproductive disease caused
by Brucella species.
The mouse has been used for decades as a model for persistent Brucella infection, because
Brucella is able to persist in systemic organs of infected mice, and these animals show clinical signs,
a Persistence model
Model for human disease
Acute phase Chronic phase
CFU Brucella (spleen)
0 3 9 15 21 30 45 60 Granuloma in systemic organs

Days post infection
b Pregnant model
Model for animal disease
CFU Brucella (placenta)
Infection
Neutrophilic necrotizing placentitis

0.0 3.0 6.0 9.0 13.5 18.0
Days of pregnancy
Figure 3
Comparison of bacterial growth kinetics and tissue pathology in (a) the murine model of persistent infection
and (b) the pregnant mouse model.
such as splenomegaly and hepatomegaly, that are compatible to those of the disease in humans
(105) (Figure 3). Although the course of murine brucellosis depends on bacterial strain virulence,
dose, and inoculation route, as well as breed, genetic background, age, sex, and physiological status
(105), the vast literature available has solidified the mouse as a valuable model for the study of
persistent brucellosis.
In the past decade, different groups have employed pregnant mice to study the pathogene-
sis of Brucella infection in the placenta (106–108) (Figure 3). Interestingly, Brucella was able to
colonize the placenta of infected mice to high numbers, despite the physiological and anatomi-
cal differences between the reproductive tracts of mice and ruminants. Moreover, these studies
demonstrated transmission of the bacteria to the fetus, as well as high rates of abortion (fetal
reabsorption) resulting from severe acute placentitis, which had pathological features similar to
the acute necrotizing placentitis seen in infected ruminants (106, 107). The studies went further
to demonstrate that the induction of abortion was dependent on a functional T4SS, because a
virB4 mutant was attenuated in the mouse placenta (107). Immunologically, transient induction
of IFN-γ was crucial for Brucella-induced reproductive disease in mice (107), which is the first
cause-and-effect set of data to shed some light on the possible immunological mechanisms of
Brucella-induced placentitis and abortion. These few studies suggest that the power of a defined
animal model, the mouse, may be useful in elucidating additional mechanisms of host-pathogen
interactions that contribute to placental inflammation and fetal demise during B. abortus infection.
CONCLUSION AND OPEN QUESTIONS

Recent advances in our understanding of host-pathogen interactions leading to clinical brucellosis
have focused on intracellular survival and on persistence in the mononuclear phagocyte system,
both of which are crucial for maintenance of Brucella species in their host populations between pe-
riods of transmission. These studies have revealed fascinating insights into how this organism uses
its virulence factors and stealthy design to prevent induction of an adequate antibacterial response
and subvert the immune system. However, our understanding of key parts of the infectious cycle
is incomplete. For example, although B. abortus is thought to traverse mucosal surfaces via M cells
(61, 109, 110), it not known whether specific virulence factors facilitate this transepithelial trans-
port. Perhaps most importantly, our understanding of the events leading to placental colonization
and transmission of Brucella spp. is still in its infancy. Considering that transmission is the driving
force behind selection of virulence attributes, a deeper understanding of this process is needed to
understand the function of positively selected virulence genes in eliciting the massive inflammatory
response that results in abortion and bacterial spread within the natural host populations.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
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Brucella Spp. Virulence Factors

California, Davis, California 95616; email: mxavier@ucdavis.edu, rmtsolis@ucdavis.edu

Annu. Rev. Anim. Biosci. 2016. 4:9.1–9.17 Keywords

This article’s doi: Abstract

BRUCELLA SPECIES: HOST SPECIFICITY AND CLINICAL

Brucellosis in the Natural Host

9.2 Byndloss · Tsolis

Table 1 Host preference for Brucella species in domestic animals

Male: infertility, orchitis, epididymitis placenta, aborted fetus,

www.annualreviews.org • Brucella 9.3

Animal brucellosis Human brucellosis

Chronic debilitating with

Abortion Undulant fever

Females: acute neutrophilic

Transmission to Yes Rare

Vaccine Yes (S19, RB51, REV1) No

9.4 Byndloss · Tsolis

www.annualreviews.org • Brucella 9.5

ROLE OF VIRULENCE FACTORS IN CHRONIC DISEASE

Brucella spp. Evasion of the Immune System

9.6 Byndloss · Tsolis

The Immune Response to Brucella Infection

www.annualreviews.org • Brucella 9.7

RicA Interaction with small Unknown (111)

9.8 Byndloss · Tsolis

www.annualreviews.org • Brucella 9.9

Acute Brucella infection Chronic Brucella infection

ROLE OF VIRULENCE FACTORS IN REPRODUCTIVE DISEASE

9.10 Byndloss · Tsolis

Mouse Model of Brucella-Induced Placentitis: What Do We Know Thus Far?

0 3 9 15 21 30 45 60 Granuloma in systemic organs

Neutrophilic necrotizing placentitis

www.annualreviews.org • Brucella 9.11

CONCLUSION AND OPEN QUESTIONS

9.12 Byndloss · Tsolis

www.annualreviews.org • Brucella 9.13

9.14 Byndloss · Tsolis

www.annualreviews.org • Brucella 9.15

9.16 Byndloss · Tsolis

www.annualreviews.org • Brucella 9.17

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