Microbiological Research 193 (2016) 30–38

Contents lists available at ScienceDirect

Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Acanthamoeba spp. as a universal host for pathogenic

microorganisms: One bridge from environment to host virulence
Allan J. Guimaraes a,∗,1 , Kamilla Xavier Gomes a,1 , Juliana Reis Cortines b ,
José Mauro Peralta c,∗∗ , Regina H.Saramago Peralta d
a
Departamento de Microbiologia e Parasitologia, Instituto Biomédico, Universidade Federal Fluminense, Brazil
b
Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Brazil
c
Departamento de Imunologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Brazil
d
Departamento de Patologia, Faculdade de Medicina, Universidade Federal Fluminense, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Free-living amoebas (FLA) are ubiquitous environmental protists that have enormously contributed to the
Received 2 March 2016 microbiological contamination of water sources. FLAs have displayed resistance to environmental adver-
Received in revised form 2 June 2016 sities and germicides and have played important roles in the population control of microbial communities
Accepted 1 August 2016
due to its predatory behavior and microbicidal activity. However, some organisms have developed resis-
Available online 2 August 2016
tance to the intracellular milieu of amoebas, as in the case of Acanthamoebas, which in turn, have been
functioning as excellent reservoirs for amoeba-resistant microorganisms (ARMs), such as bacteria, viruses
Keywords:
and fungi. Little is known about these relationships and interaction mechanisms, but it is speculated that
Acanthamoeba castellanii
Environment
the FLAs need a very broad repertoire or universal class of receptors to bind and recognize these diverse
Host species of microorganisms. By harboring these organisms as a “Trojan Horse”, the Achantamoeba has been
Virulence working as an excellent vector for pathogens. Moreover, studies have demonstrated that the interaction
Pathogens of pathogens with Acanthamoeba results in environmental selective pressure responsible for induction
and maintenance of virulence factors and increase in microbial pathogenicity. This phenomenon is cor-
related to the observation of higher gene number and DNA content of ARMs, when compared to their
relatives which are adapted to other hosts, due to allopatric or sympatric gene transfer and acquisition,
contradicting the overall genome reduction theory for intracellularly adapted pathogens. Thus, adapta-
tion to FLAs indirectly provided a “learning” environment for pathogens to resist later to macrophages;
besides the evolutionary distance, these phagocytes share similar predatory mechanisms, such as phago-
cytosis and phagolysossomal degradation. In this mini-review, we cover the most important aspects of
Acanthamoeba biology and their interactions with endemically important human pathogens.
© 2016 Elsevier GmbH. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2. Free-living amoebae as environmental hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3. Amoeba-resistant microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.1. Legionella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2. Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3. Pathogenic and non-pathogenic fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.4. Giant viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

∗ Corresponding author at: Instituto Biomédico, Dept. de Microbiologia e Parasitologia – MIP, Universidade Federal Fluminense, Rua Prof. Hernani Pires de Melo 101, São
Domingos, Niterói, 24210-130, Brazil.
∗∗ Corresponding author at: Instituto de Microbiologia Prof. Paulo de Góes CCS – Centro de Ciências da Saúde – Bloco I- 2 andar, sala 046, Avenida Carlos Chagas Filho, 373,
Cidade Universitária, RJ, CEP. 21941-902, Brazil.
E-mail addresses: allanguimaraes@id.uff.br (A.J. Guimaraes), peralta@micro.ufrj.br (J.M. Peralta).
1
Authors contributed equally to this work.

http://dx.doi.org/10.1016/j.micres.2016.08.001
0944-5013/© 2016 Elsevier GmbH. All rights reserved.
 A.J. Guimaraes et al. / Microbiological Research 193 (2016) 30–38 31

4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

1. Introduction Hogan et al., 1996). Soil phagocytic microorganisms such as A.

castellanii, are nourished mainly with fungi and bacteria; in turn,
Pathogenic microorganisms belong to two main categories, survival of these engulfed microorganisms within the host depends
depending on whether they are acquired from the environment on the expression of hydrolytic enzymes such as proteases, phos-
or from hosts, such as human or other animals. Environmentally pholipases, lipases and DNases for nutrition. Due to the enormous
acquired microorganisms are usually able to survive in their niche genetic diversity of the community of soil microorganisms, each has
and also in a wide variety of hosts they interact with (Casadevall, a unique combination of features and distinctive attributes to pre-
2008). For example, endemic pathogenic fungi such as Histoplasma vent the action of phagocytic predators, such as chemical defenses
capsulatum, Cryptococcus neoformans and Coccidioides spp. are including the production of anti-phagocytic capsule and host dam-
found in soil and apparently do not require specific hosts; therefore, aging toxins, the expression of several mechanisms for host iron
these organisms can infect many hosts and are commonly consid- sequestration and acquisition, expression of catalases and super-
ered nonspecific pathogens. In contrast, microorganisms acquired oxide dismutase and biofilm formation, among others (Casadevall,
by contact between individuals often have complex interactions 2008; Casadevall, 2006; Casadevall and Pirofski, 2001; Hogan et al.,
with their host of origin, in which they rely on for its survival and 1996).
replication. Within this group, we can find fungi such as the der- As for the promiscuous nature of amoebae, several pathogens
matophytes, which can be acquired from hosts such as human and are able to grow within these organisms, including those without
animals, but also can independently live in the environment (White classically known reservoirs. Many features necessary for survival
et al., 2014). On the other extreme of this group, viruses entirely of microorganisms during interaction with environmental hosts
depend on their hosts for replication and have co-evolved with have been also involved in pathogenicity to mammalian hosts. For
them. Between these two distinct poles, microorganisms such as instance, microbial phospholipases are important for eicosanoid
Legionella pneumophilla, found in soils and contaminated waters productions within mammalian cells and phospholipid degrada-
(Thomas and Ashbolt, 2011; Walser et al., 2014), are able to adapt tion, and also play important nutritional roles in the environment
quickly and replicate within mammalian hosts (Escoll et al., 2013). such as phospholipids digestion and fatty acid acquisition. Melanins
Host-restricted microorganisms are more limited regarding function for iron acquisition and as antioxidant, either upon mam-
their survival strategies, infecting only those displaying similar malian infection or in the environment. These suggest that the
characteristics. For host-restricted organisms, virulence is best virulence may have evolved by interaction with soil predators
defined by the ability to cause damage to their host, and new and can be maintained as a defense mechanism against amoeboid,
infections become critical for pathogen survival and maintenance nematodes and other small invertebrate hosts that feed on microor-
(Casadevall, 2006; Casadevall and Pirofski, 2001). On the other ganisms as a stochastic process, and these virulence factors might
hand, free-living pathogens do not require a specific host for sur- also have similar functions in mammalian virulence (Casadevall,
vival, and infection to superior organisms may result in colonization 2008; Casadevall, 2006; Casadevall, 2012).
and subsequent damage due to stochastic processes. Therefore, Little is known about the mechanisms and dynamics of inter-
free-living microorganisms inhabiting the soil are more likely to action between pathogens and FLAs. This propels the need to
have a more diverse set of molecular defenses against preda- understand the molecular mechanisms enabling FLAs to interact
tors than host-restricted microorganisms, as there is no selective with a broad variety of pathogens. They may express universal
pressure for possible host damage. Upon association between free- receptors allowing the interaction with various microorganisms,
living soil microorganisms and their hosts, possible outcomes are such as fungi, bacteria and viruses (Fig. 1). Interestingly, from the
death of the microorganism, death of the host or persistence of host point-of-view, macrophages and amoebae display similari-
the microorganism within the host (Mylonakis et al., 2007); after ties on their interactions, which can be important for infections
death of the host, free-living soil microorganisms usually return to in mammalians. Both cells ingest particles into phagosomes and
the environment (Casadevall, 2008; Casadevall and Pirofski, 2001). have lysosomal enzymes responsible for the digestion and nutrient
The lack of host-requirement raises fundamental questions acquisition (German et al., 2013; Siddiqui and Khan, 2012; Bowers
regarding the origin of virulence and pathogenicity of free-living and Olszewski, 1983). These features would make these cells inhos-
soil microorganisms (Mylonakis et al., 2007; Casadevall, 2012; pitable to infection by pathogens. However, several pathogens
Casadevall and Pirofski, 2007). Recent studies focusing on the inter- have developed mechanisms to subvert the phagocytic function
action between soil microorganisms, such as fungi, bacteria or of macrophages and amoebae. Another importance to the parallel
virus with free-living amoebae (FLA), suggest distinct mechanisms established between FLAs and macrophages is the fact that escape
whereby virulence can emerge or be selected upon interaction with mechanisms and survival of fungi in these amoebas were shown to
such “accidental” host, as most of these are non-obligate intracellu- be common to those used by these pathogens within macrophages,
lar pathogens (Casadevall, 2008; Casadevall, 2006; Casadevall and which may explain why these cells have evolved from infection of
Pirofski, 2001; Marciano-Cabral and Cabral, 2003). simple eukaryotes to more complex hosts, such as mammals (Greub
In the soil environment, microorganisms compete for nutri- and Raoult, 2004).
ents and are subjected to extreme conditions of moisture, oxygen, Therefore, as the innate immunity pathways are widely
temperature, radiation, ionic strength and pH (Casadevall, 2006). conserved in nature, interaction studies between amoeboid or
The presence of a large variety of potential hosts in the soil, invertebrate hosts with pathogenic microorganisms consist of
including several predators with a wide arsenal of receptors and important models for understanding the mechanisms of micro-
antimicrobial defenses, provides a hostile environment for many bial virulence and host immunity. These easy-to-use models
microorganisms, suggesting that only the expression of special complement the use of mammalian models, represented mainly
features enables their survival after the contact with their hosts by macrophages at the cellular level to complex organisms
(Casadevall, 2008; Casadevall, 2006; Casadevall and Pirofski, 2001; (Steenbergen et al., 2001; Sandstrom et al., 2011). Most impor-
32 A.J. Guimaraes et al. / Microbiological Research 193 (2016) 30–38

Fig. 1. Acanthamoeba castellanii interaction with bacteria, giant viruses and fungi and their host intracellular fate. Legionella spp. (1) attachment of Legionella to A. castellanii by
mannose or Gal/GalNAc receptor; (2) internalization via “coiling” phagocytosis; (3) Avoidance of lysosome fusion and high resistance to biocidal activity; (4) remodelation of
the Legionella containing vacuole (LCV) distributed throughout the cytoplasmic space; (5) Legionella intracellular replication within LCVs. Mimiviridae (I) viral attachment to A.
castellanii surface via a mannose receptor, lectin or Fc; (II) internalization of capsid in phagosome; (III) liberation viral DNA; (IV) formation of viral factory for DNA replication
and production of newly synthesized particles. Fungi (A’) filamentous fungi and yeast attachment to A. castellanii surface via an unknown receptor; (B’) internalization
via “coiling” phagocytosis; (C’) fungi filled phagosome distributed throughout the cytoplasm; (D’) inhibition of phagolysosome formation and high resistance to biocidal
activity; (E’) Yeast can convert to filamentous form in the case of dimorphic fungi, which in turn replicate intracellularly; (F’) Filamentous phase can overgrow and disrupt
the Acanthamoeba surface. (B) The main observed characteristics of the pathogens covered in this review upon interaction with amoebas. Legionella sp., mimivirus and fungi
can somehow be changed by selective pressure from Acanthamoeba enabling higher environmental resistance and frequently virulence enhancement.
 A.J. Guimaraes et al. / Microbiological Research 193 (2016) 30–38
tantly, these alternatives usually have much lower costs and
avoid existing ethical issues with mammalian models of infection
(Mylonakis et al., 2007; Glavis-Bloom et al., 2012).
2. Free-living amoebae as environmental hosts
FLAs are ubiquitous protozoa in the biosphere, frequently found
in soil and natural or man-made aquatic environments (Thomas
and Ashbolt, 2011; Ashbolt, 2015; Trabelsi et al., 2012), such as
water treatment and cooling systems. In these sites, they feed
on microbial biofilms (Marciano-Cabral and Cabral, 2003) and are
important for the predatory control of microbial communities
(Molmeret et al., 2005).
FLAs can invade a host and occasionally, parasite host tis-
sues, thus also been called amphizoic amoebas, but rarely causing
devastating disease. Among many FLAs, only the four genera Acan-
thamoeba, Naegleria, Balamuthia and Sappinia, have been associated
to opportunistic and non opportunistic infections in humans and
other animals (Visvesvara et al., 2007). Successful treatment guide-
lines for these morbidities have not been established in literature,
although some lysophospatidylcholine derivatives, such as milte-
fosine show certain promise (Dunn et al., 2016; Linam et al., 2015;
Roy et al., 2015).
The genre Acanthamoeba spp. is primarily associated with
human diseases in immunocompromised patients, particularly
those undergoing chemotherapy for cancer, individuals who have
undergone organ transplants and HIV-positive individuals, with
clinical presentations ranging from cutaneous, nasopharyngeal,
pulmonary and kidney lesions, to a granulomatous encephalitis
(Trabelsi et al., 2012; Baig, 2015). In immunocompetent individ-
uals, infections are presented as amoebic keratitis (Robaei et al.,
2015), often associated with contamination of contact lenses due
to usage of contaminated cleaning solutions (Carnt and Stapleton,
2016; Lorenzo-Morales et al., 2013a). Other FLAs also display
epidemiological expression in some geographic areas or related
to patientı́s age (Trabelsi et al., 2012; Scheid, 2014; Scheid and
Schwarzenberger, 2012). The species Balamuthia mandrillaris, can
also cause cutaneous lesions, lung infections and granuloma-
tous encephalitis, mostly related to immunocompetent infants
(Lorenzo-Morales et al., 2013b; Visvesvara, 2013). Cases of ame-
biasis by Naegleria fowleri generally occurs in previously healthy
children and young adults, frequently related to recreational activ-
ities in freshwater lakes or other water reservoirs and manifest as
a primary amoebic meningoencephalitis, with usually fatal course
due to rapid hemorrhagic necrotizing infection of the central ner-
vous system (Visvesvara, 2013; Grace et al., 2015). Only few cases
of encephalitis by the FLA Sappinia diploidea have been described
(Visvesvara et al., 2007; Visvesvara, 2013; Grace et al., 2015;
Qvarnstrom et al., 2009; Walochnik et al., 2010).
From all FLAs related to human infections, Acanthamoeba spp.
has been the most abundant protozoan in the environment and
can be easily isolated from soil, dust, water (natural, irrigation and
bottled), medical devices, dialysis, dentures, contact lenses and as
culture contaminants of fungal, bacterial and mammalian cells. For
instance, one of the main species, Acanthamoeba castellanii, was
firstly isolated by Castellanii in 1930 from Cryptococcus pararoseus
cultures (Castellanii, 1930).
The Acanthamoeba sp. cycle comprises two phases: trophozoite
and cyst. The first is considered to be the biochemically active stage,
with cell dimensions between 25 and 40 ␮m; it divides by mitosis
and has major organelles and characteristics of eukaryotic cells. The
trophozoites exhibit vegetative growth and feed through phago-
cytosis of bacteria, yeasts and algae present in the environment,
where the digestion takes place within phagolysosomes (German
et al., 2013; Siddiqui and Khan, 2012; Bowers and Olszewski, 1983),
33
and through pinocytosis, with liquid nutrients absorption. The con-
version to the dormant phase is characterized by encystment, and
is triggered by food deprivation or adverse environmental con-
ditions such as low/high temperatures or pH variation. The cysts
range from 15 to 28 ␮m in size and have a double-layered wall; the
ectocyst is the outer layer, and the endocyst, usually formed after
the ectocyst, is a fibril layer composed of acid-insoluble proteins
and cellulose, which confers resistance to harsh conditions such
as germicides, 70% alcohol, and chlorinated derivatives (Greub and
Raoult, 2004; Trabelsi et al., 2012; Torvinen et al., 2004; Cursons
et al., 1980). Increasing resistance to most chemical agents are usu-
ally correlated with increasing content in alkali-insoluble residues
(cellulose) and variations among Acanthamoeba isolates regarding
resistance to closely related biocides correlated to variations on the
cyst wall composition of these isolates (Turner et al., 2000).
Recently, it has been observed that water contamination by FLAs
can be an important problem for emerging epidemics (Greub and
Raoult, 2004; Scheid, 2014). Not only because they are potential
human pathogens, but also they host and protect other microor-
ganisms. Thus, some pathogenic bacteria, fungi and viruses have
developed strategies to escape the killing/inactivation mechanisms
of Acanthamoeba, using the amoeba as a reservoir for their own
benefit; or as a “Trojan horse”, as in the case of co-infection of
amoeba harboring pathogenic microorganisms (Greub and Raoult,
2004; Carter, 2009). Once adapted to the intracellular milieu of
amoebae, these pathogens are relatively protected from biocides
and environmental conditions, making their survival and dissem-
ination more effective (Steenbergen et al., 2001). Because of this
extensive association with various pathogens, A. castellanii has been
appropriately used as an amoeboid model to study host-parasite
interactions (Sandstrom et al., 2011; Steenbergen and Casadevall,
2003).
3. Amoeba-resistant microorganisms
As most pathogens are in the soil, A. castellanii and other
phagocytic predators may feed on several microorganisms;
amoeba-resistant microorganisms (ARMs) probably arose due to
the predatory behavior of amoebas on microorganisms. About
22% of Acanthamoeba isolates have some endosymbiont, includ-
ing organisms from over 30 species of pathogenic bacteria, such as
members of the Legionella spp. family, Mycobacterium avium, Rick-
etsialis and Chlamydialis, yeasts such as Cryptococcus neoformans,
Histoplasma capsulatum and Blastomyces dermatitides and viruses
such as adenovirus and mimivirus (Steenbergen et al., 2001; Scheid,
2014; Scheid and Schwarzenberger, 2012; Campos et al., 2014;
Lorenzo-Morales et al., 2007; Mattana et al., 2006; Taylor et al.,
2003; Whan et al., 2006).
Many pathogens possess the ability to replicate inside FLAs,
such as Acanthamoeba spp., and escape the microbicidal activity
of these organisms using similar mechanisms to those used for
survival within macrophages (Fig. 1) (Greub and Raoult, 2004;
Sandstrom et al., 2011; Molmeret et al., 2005; Carter, 2009; Segal
and Shuman, 1999; Steenbergen et al., 2003; Steenbergen et al.,
2004). Despite the large phylogenetic diversity of ARMs, these
pathogens evolutionarily developed/adapted characteristics that
make them resistant to the intracellular microenvironment of these
protozoa.
Greub and Raoult (Greub and Raoult, 2004) reported the great
diversity of the main infectious agents previously found in amoe-
bae, such as L. pneumophila, Coxiella burnetii, Francisella tularensis,
Listeria monocytogenes, Helicobacter pylori, C. neoformans, members
of the Enterobacteriaceae family, Vibrionaceae, Mycobacteriaceae
and Pseudomonacea. In addition, herpes viruses, enteroviruses and
34 A.J. Guimaraes et al. / Microbiological Research 193 (2016) 30–38
mimivirus have also been isolated from samples of FLAs (Greub and
Raoult, 2004; Rumelt et al., 2000).
In this symbiotic process, the virulence of amoebae appears to be
increased, as endosymbiont-free Acanthamoeba has been reported
to have lower ability to produce cytopathic effects on fibroblast
cultures (Fritsche et al., 1998). This strengthens the hypothe-
sis of allopatric gene exchange between Acanthamoeba and their
intracellular parasites (Casadevall, 2012; Molmeret et al., 2005;
Steenbergen and Casadevall, 2003; Moliner et al., 2010; Bertelli and
Greub, 2012; Matz, 2011 Moliner et al., 2010; Bertelli and Greub,
2012; Matz, 2011).
From the pathogen point-of-view, upon interaction with a host,
two behaviors could be observed: (i) some pathogens have spe-
cialized and adapted to specific eukaryotic hosts, and undergone
loss of large amount of genes; (ii) in the case of ARMs, amoebas
as hosts exert selective pressure on several of these pathogens, as
specifically the ARMs continue to live and multiply within these
cells. These ARMS do not undergo gene loss, maintaining their
genome size, thus favoring the retention of virulence factors which
confers advantages during infection to higher hosts (Bertelli and
Greub, 2012). Additionally, several ARMs display larger genomes
than their relatives, which also supports the idea of a reverse
gene transfer from Acanthamoeba to their endosymbionts (Moliner
et al., 2010). These reports are consistent with the idea that certain
aspects of microbial pathogenesis are derived from mechanisms
used by pathogens to survive in the presence of environmental
amoeboid organisms.
3.1. Legionella
The Gram negative bacteria Legionella spp. is the causative
agent of legionellosis’ disease. It is transmitted through Legionella-
contaminated aerosols emanating from man-made water systems
such as cooling towers, air conditioning systems and showers
(Amato-Gauci et al., 2011); human-to-human transmission has
not been reported to the best of our knowledge. Up to 4% of
community-acquired pneumonia and nosocomial pneumonia are
due to Legionella, mainly L. pneumophila. After inhalation of con-
taminated aerosols the bacteria reaches the lungs, and upon
phagocytosis by pulmonary macrophages, it replicates within these
cells resulting in the onset of a pneumonia case (Escoll et al., 2013).
The Legionella’s virulence, growth and survival in the environ-
ment are enhanced by their ability to form a symbiotic relationship
with several hosts. Legionella can invade and replicate intracellu-
larly into at least 13 species of amoebae, including Acanthamoeba
and Naegleria, and some ciliates, producing vesicles that contain
hundreds of bacterial cells (Lee and West, 1991; Magnet et al.,
2015). The rapid re-colonization of Legionella from distinct water
systems after a disinfection program can be attributed to the pro-
tective shield to the bacteria provided by the amoeba. Furthermore,
there is convincing evidence that intracellular multiplication of L.
pneumophila in free-living amoebae is a prerequisite for human
infection (Molmeret et al., 2005), but interaction with other hosts
closer to higher eukaryotes cannot be excluded (Escoll et al., 2013;
Molmeret et al., 2005; Fritsche et al., 1998).
L. pneumophila is considered a facultative intracellular pathogen
and was the first characterized human pathogen able to inter-
act with and survive within macrophages and amoebae (Table 1).
Intracellular events after infection and invasion of amoebae and
macrophages by L. pneumophila are similar in several pathways
(Fig. 1A). Although the binding of Legionella spp. with the cell
is a specific process for each host, the molecular events that
allow binding and uptake of these bacteria by amoebas still have
some gaps. In amoebae, it seems that bacteria binding occurs
via an 170 kDa N-acetyllactosamine galactose-inhibitable lectin
(Gal/GalNAc) (Declerck et al., 2007); however in macrophages,
complement receptors CR1 (CD35) and CR3 (CD18/CD11b) mediate
these interactions (Clemens and Horwitz, 1992). For L. pneumophila
it has been reported that the phagocytosis pathways are not totally
conserved between amoebae and macrophages, but the endocytic
system evasion mechanisms used by Legionella, such as inhibition of
phagosome-lysosome fusion, are common for escaping from either
hosts. After uptake and avoidance of degradation in lysosomes, vac-
uoles are formed (Legionella containing vacuole – LCV) and bacteria
replication occurs (Berk et al., 1998). The egress from the host cell is
an unknown process probably involving lytic and non-lytic mech-
anisms, resulting respectively in death or survival of the host cell
(Escoll et al., 2013; Chen et al., 2004).
When grown in the presence of A. castellanii and compared
to bacteria cultured in vitro, L. pneumophila has a significantly
higher resistance to adverse environmental conditions, as well as
increased invasiveness towards epithelial cells and macrophages.
Phenotypic differences were observed for L. pneumophila after
invasive growth in A. castellanii, such as reduced size despite the
larger diameter, aggregates formation and the presence of vesi-
cles distributed throughout the cytoplasmic space. The growth in
A. castellanii also resulted in modification on the protein expres-
sion profile by L. pneumophila, favoring the entry into macrophages
via “coiling” phagocytosis mechanisms. A. castellanii is also capa-
ble of releasing vesicles populated by L. pneumophila, which are
highly resistant to biocidal activity (Fig. 1B). Thus, replication of L.
pneumophila within Acanthamoeba spp. may be essential for the
production of competent bacteria regarding infectiveness, viru-
lence and therefore, manifestation of pneumonia in humans (Escoll
et al., 2013).
3.2. Mycobacteria
These bacteria are a large group of microorganisms that inhabit
a diverse range of natural environments and are a frequent cause
of opportunistic infection in humans and livestock (Primm et al.,
2004; Vaerewijck et al., 2005). Main examples of environmental
mycobacteria are the Mycobacterium avium complex, M. gordonae,
M. malmoense, M. simiae, M. marinum, M. tusciae, and M. lenti-
flavum, which have been found in natural fresh waters (Torvinen
et al., 2004; Vaerewijck et al., 2005). Several experimental studies
have further demonstrated M. avium-FLA interactions, including
Acanthamoeba spp.(Ovrutsky et al., 2013) and Dictyostelium spp.
(Hagedorn et al., 2009; Unal and Steinert, 2006). M. avium and
M. intracellulare have also been grown in the ciliated, unicellu-
lar protist Tetrahymenapyriformis (Strahl et al., 2001). It has been
demonstrated that M. avium subsp. avium and M. avium subsp.
paratuberculosis are able to survive within FLAs (Whan et al.,
2006), which result in their increased virulence (Whan et al., 2006;
Danelishvili et al., 2007) and enable bacterial protection against
adverse situations including exposure to antibiotics (Cursons et al.,
1980; Ovrutsky et al., 2013). Mycobacterium and Acanthamoeba
interactions are to date poorly understood (Baig, 2015; Robaei et al.,
2015). In a recent study by Weenderburg et al. (Weerdenburg et al.,
2015) for the characterization of virulence of M. marinum, several
virulence determinants were described for the infection of Acan-
thamoeba, including a type VII protein secretion system ESX-1,
utilization of sterols and biosynthesis of polyketide lipids. How-
ever, this microorganism contains also a large set of host-specific
virulence determinants, such as enzymes involved in the modi-
fication of surface glycolipids and ESX-1 system proteins. These
findings suggest that although Acanthamoeba could function as a
selective agent for virulence conservation and expression of M. mar-
inum virulence factors, they do not capacitate these pathogens to
cross inter-specific barriers (Weerdenburg et al., 2015).
 A.J. Guimaraes et al. / Microbiological Research 193 (2016) 30–38
Table 1
Selection of microorganisms that interact with Acanthamoeba and their markers described in the literature.
Microorganisms Markers described in interaction with Acanthamoeba
Escherichia coli K1 Derivatives lacking genomic islands
Escherichia coli K1 Acquired type III secretion system
Escherichia coli K1 Capsule
Francisella tularensis REP34
Legionella Rab GTPases
Legionella longbeachae Icm/Dot substrate SidC
Legionella pneumophila Collagen-like protein
Legionella pneumophila Orphan sensor kinase LqsT
Legionella pneumophila F-box protein Lpp2082
Legionella pneumophila IcmS-LvgA protein complex
Legionella pneumophila Gal/GalNAc Receptor
Parachlamydia acanthamoebae Type III secretion system
Parachlamydia acanthamoebae Serine protease activity
Vibrio parahaemolyticus Acquired type III secretion system
3.3. Pathogenic and non-pathogenic fungi
Except for Candida albicans, which is a commensal microor-
ganism commonly present on human skin and mucosas, most
pathogenic fungi are free-living saprophytes organisms and do
not require hosts to spread (Casadevall, 2012; Hogan et al., 1996;
Steenbergen and Casadevall, 2003). Due to the absence of speci-
ficity for a particular host, they are capable of causing disease in
many animal species.
Pathogenic fungi are present with a wide variety of pheno-
types in the environment. For instance, the fungus C. neoformans
can cause pneumonia and meningoencephalitis in humans, being
the main risk factor for HIV-infected individuals (Kwon-Chung,
2014). It is presented as a yeast surrounded by a polysaccharide
capsule, considered its most important virulence factor (Doering,
2009; Ma and May, 2009; Zaragoza et al., 2009). Capsular growth
is induced during infection and contributes to the virulence by
multiple mechanisms such as inhibition of phagocytosis, dysreg-
ulation of immunoresponses with reduced leukocyte migration,
complement depletion, interference on antigen presentation, T-cell
suppression with subsequent inhibition of inflammatory cytokines
production (Zaragoza et al., 2009; Yauch et al., 2006) and pro-
tection against free radicals and antifungal drugs (Zaragoza et al.,
2009).
C. neoformans is considered a facultative intracellular parasite of
macrophages (Cursons et al., 1980; Turner et al., 2000), and capsu-
lar induction generally occurs upon entrance in these phagocytes
(Zaragoza et al., 2008; Feldmesser et al., 2001). The mechanisms
of interaction between C. neoformans and other host phagocytic
cells seem to be conserved in nature, including those observed with
“accidental” hosts in the soil (Casadevall, 2012; Steenbergen and
Casadevall, 2003), such as A. castellanii.
In fact, the interaction of C. neoformans with amoeboid hosts
by still unknown mechanisms (Fig. 1A), also results in increased
fungal virulence (Steenbergen and Casadevall, 2003), mainly due to
the induction of expression of capsular polysaccharides (Chrisman,
2011) (Fig. 1B). Particularly polar lipids from A. castellanii, such as
phosphatidylcholine, or their digestion by fungal phospholipases,
which results in the release of glycerophosphocholine, could induce
the formation of giant cells. These mechanisms illustrate how polar
lipids of amoeboid origin could mimic the presence of A. castellanii,
resulting in capsular induction by C. neoformans as a nonspecific
defense mechanism against potential predators.
As C. neoformans virulence factors, such as the aforementioned
capsule, melanin, lipases, mannitol production, among others
(Casadevall, 2006) display a “dual use” during Acanthamoeba infec-
tion and in mammalian models, it is believed they may have arisen
as a result of interaction with their soil hosts (Steenbergen et al.,
2001; Bunting et al., 1979) (Table 1; Fig. 1), and these same virulence
35
Citations
Yousuf et al. (2014)
Siddiqui et al. (2011)
Jung et al. (2007)
Feld et al. (2014)
Hilbi et al. (2014)
Dolinsky et al. (2014)
Abdel-Nour et al. (2014)
Kessler et al. (2013)
Lomma et al. (2010)
Vincent and Vogel (2006)
Declerck et al. (2007)
Croxatto et al. (2013; Strahl et al. (2001)
Hayashi et al. (2012)
Matz (2011)
factors could be used by the fungi during infection and pathogenesis
on superior hosts (Casadevall, 2006).
Recently, Derengowski et al. established a parallel for gene
expression of the fungus C. neoformans upon infection of Acan-
thamoeba and macrophages. This work was the first attempt to
delineate evolutionary pressures on the fungus in nature by pro-
tozoan predation, which could have promoted the selection of
strategies that allow intracellular survival in macrophages and
adaptation to mammalian hosts (Table 1). Several common fungal
genes were overexpressed by C. neoformans in both hosts, among
which one specific, PTP1 (polyol transporter protein 1), involved
in the transport of 5- and 6-carbon polyols, was highly expressed,
but had no involvement on cryptococcal virulence in in vivo models
(Derengowski Lda et al., 2013),
Dimorphic fungi, such as Blastomyces dermatitidis, Paracoccid-
ioides brasiliensis, Sporothryx schenckii and Histoplasma capsulatum
are capable of inter-converting the mycelium-to-yeast form
depending on the surrounding temperature. Usually upon mam-
malian infection, the filamentous form converts to yeast form when
the fungus adapts to their body temperature. However, either
in vitro or probably when the yeasts returns to the environment,
A. castellanii is able to phagocytose these yeasts and trigger the
conversion to filamentous form, which frequently results in death
of the amoeba due to hyphal apical elongation and fungal growth
(Steenbergen et al., 2004) (Fig. 1A and B). In the specific case of H.
capsulatum, exposure to and recovery of yeasts of avirulent strains
from A. castellanii culture was able to select or induce more viru-
lent phenotypes of H. capsulatum, characterized by higher number
of pulmonary inflammatory cells and persistent infection in murine
models (Steenbergen et al., 2004). These observations suggest that
A. castellanii, in particular, plays a fundamental role in the main-
tenance of these organisms in the environment and selection of
fungal virulence factors.
3.4. Giant viruses
Amoeba infecting viruses are not uncommon, and several
have been isolated from amoeba cultures, such as adenoviruses
(Lorenzo-Morales et al., 2007) and enteroviruses (Greub and Raoult,
2004; Mattana et al., 2006). As in the case for other microorganisms,
amoebas have also been recognized as biological “Trojan Horses”
for viruses and have been charged of increasing the virulence
and protecting human viruses against environmental harshness,
as mentioned above for other pathogens. For example, intracellu-
lar localization of adenovirus could be observed using fluorescence
microscopy (Scheid, 2014). Besides bearing intracellular viruses,
previous studies suggest also that in the case of enteroviruses,
in contrast, they are maintained viable in the environment due
to their attachment to amoebaı́s surface only, with no detectable
36 A.J. Guimaraes et al. / Microbiological Research 193 (2016) 30–38
particles internalization (Danes and Cerva, 1981). In any case, the
receptors and the internalization pathways for amoeba-virus inter-
action have not been described to date to the best of our knowledge
(Fig. 1A).
In the late 90s, a search for a mysterious human pneumonia
causative agents resulted in the discovery of a yet unknown bac-
teria, at the time thought to be related to Chlamydia (Birtles et al.,
1997). Amoeba cultures were used as a common protocol to isolate
these microorganisms and purify the agent. When examined closer,
about 10 years later, the previously identified “bacteria” has been
characterized as the first ever giant virus (La Scola, 2003). Since
then, many other members of the ever-growing giant virus fam-
ilies have been identified in A. castellanii cultures from the most
diverse environments (Scheid, 2014). These viruses were named as
to “mimic” bacteria due to its surface covered LPS-like molecules
and a size of ∼750 nm, which potentially signals to phagocytic path-
ways of amoeba, normally triggered by particles with >0.5 ␮m.
This observation was confirmed by Ghigo et al., who later showed
that productive mimivirus infection could only be supported by
professional phagocytic cells, such as macrophages or monocytes.
For these cells, virus surface interacts with either mannose, Fc or
lectin-like receptors and may initiate particles internalization via
a phagocytic pathway (Fig. 1A), which is dependent on mobiliza-
tion of cytoskeleton actin and activation of phosphatydil inositol
3-kinases. Upon entrance, the replication process could be carried
on successfully (Ghigo, 2008).
Since its discovery, viruses from the Mimiviridae family have
been associated as possible causative agents of pneumonia and
other respiratory tract infections in humans. Unfortunately, this
is still obscure, since available data to date are very contradictory
(Moliner et al., 2010). Due to their size, infection of human cells
might be restricted to macrophages. Therefore, deciphering the
cellular receptors involved in virus binding/recognition and thus
understanding the infection and replication processes in amoeba
could enable us to extrapolate a similar mechanism to determine
the role of host cells during giant viruses-caused diseases. Since
this is a relatively novel family of viruses, information on how viru-
lence factors are affected by this symbiotic interaction is still to be
clarified. However, phylogenetic studies suggest that mimivirus’
conserved genes have been acquired by horizontal gene trans-
fer (Moreira and Brochier-Armanet, 2008), i.e., virulence factors
on these unknown giants may have been inherited from other
FLAs-infecting microorganisms, such as Legionella spp. One of the
advantages of infection of Acanthamoeba for the mimivirus is the
increased resistance to harsh environment conditions, such as UV
radiation, temperature and pH variation (Boratto et al., 2014) and
thus allowing for a successful spread to their next host (Fig. 1B).
4. Conclusion
In general, as most of pathogens interact with amoebas when
in their environmental cycle, they might undergo an evolution-
ary pressure within these host cells. These interactions might have
been responsible for the virulence evolutionary cradles, depicted
by addition or deletion of genes that may influence virulence of
species, as it has been observed for Legionella spp.
As the FLA, A. castellanii is able to interact with a wide vari-
ety of microorganisms, understanding the molecular basis of the
interaction between A. castellanii and distinct pathogens, including
the characterization of the arsenal of receptors used by the amoe-
bas to engulf these pathogens, would drive the knowledge on how
pathogenicity could be enhanced. From the pathogen point of view,
evaluating also how gene expression is triggered inside A. castel-
lanii, and drawing a comparison to expression within mammalian
cells, might provide clues on how pathogens evolved and adapted
to distinct hosts. These two points all together, may be the key to
solve the role of amoeba upon the onset of diseases caused by fungi,
bacteria and virus that are distributed in the environment.
Acknowledgements
Allan J. Guimaraes, Juliana R. Cortines, Jose M. Peralta and Regina
H. S. Peralta are supported by grants from the Brazilian agencies
CNPq (National Counsel of Technological and Scientific Develop-
ment) and FAPERJ (Fundação Carlos Chagas Filho de Amparo à
Pesquisa do Estado do Rio de Janeiro).
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