Preface
Acid is fundamental to the immune mechanisms of eukaryotes. Therefore pathogens have
evolved a myriad of strategies to evade, suppress, or exploit biological acids to
gain access to host resources. Here, we describe several intracellular and extracellular
pathogens to illustrate our current understanding of how acid plays central roles
in animal and plant immunity, and also how it can be produced and exploited by microbes
for pathogenic success.
The Importance of Acid
The pH of an aqueous solution constitutes one of its most fundamental properties.
Cells and organisms are largely aqueous entities and have evolved sophisticated strategies
for sensing, exploiting, and modifying the pH of their surrounding environments for
assorted biological processes, including nutrient acquisition, intercellular communication,
virulence, and defense against invading pathogens. At the same time, abnormal intracellular
or extracellular pH values are found in several human and plant diseases, including
cancer [1] and tumorigenesis in plants. The pH of the cytosol of most eukaryotic cells
is slightly alkaline, at 7.2–7.4 [2]. However, cells generate and exploit biological
acids for essential functions, including the denaturation of proteins destined for
degradation and the activation of acid hydrolases that mediate this process [3], and
as potent activators of intracellular signaling events [4]–[7]. Here, we focus on
how biological acids are generated, exploited, and manipulated by hosts and pathogens
during infection and disease progression. Our analysis features a comparative approach.
We consider the function and measurement of acid in several host-pathogen systems.
We provide examples of how acid is used in innate and adaptive immunity, inside and
outside of cells, by pathogens of medical, agricultural, and economic importance,
from bacterial and fungal Kingdoms, parasitizing both plants and animals (Figure 1).
We conclude by comparing broad themes that bridge or separate the mechanisms by which
evolutionarily divergent pathogens and hosts evade, subvert, or exploit biological
acids.
10.1371/journal.ppat.1003402.g001
Figure 1
Acid is found in pathogenesis and defense in diverse symbiotic relationships.
Cellular schematic shows the use of acid in innate and adaptive immunity of plant
and animal cells (top). Subversion strategies of five model pathogens discussed in
detail are shown in lower insets. Acid is denoted by red or H+. PAMP, pathogen-associated
molecular patterns; PRR, pattern recognition receptors; R, (plant) resistance genes;
MHC, major histocompatibility complex; SA, salicylic acid; JA, jasmonic acid; OA,
oxalic acid.
Intracellular Acids and Their Measurement
The endocytic and phagocytic pathways of eukaryotic cells contain acidic intracellular
compartments that carry out diverse functions, including nutrient acquisition [8],
[9], immunological information processing [10], protein and membrane degradation [3],
apoptosis [11], cellular repair and autophagy [12], and host defense [13]. Specialized
functions for these pathways have often been described. In plants, for example, endocytic
physiology influences gravitropism, guard cell movement, and plant hormone transport
(for review, see [14]). Low pH is a critical requirement in several of these processes.
The pH of early endosomes, late endosomes, and lysosomes/vacuoles are approximately
6, 5.5, and below 5, respectively, although their pH varies with cell type, cultivation
conditions, and biological context [15]. These acidic compartments communicate with
one another through the vectoral exchange of membrane and protein along evolutionarily
conserved trafficking pathways [10], [16]. In the endocytic pathway, extracellular
proteins that are destined for internalization and degradation are captured by cell
surface receptors or fluid phase engulfment. The internalized materials are sequentially
trafficked from early to late endocytic compartments for delivery to acidic terminal
compartments, which are designated lysosomes or terminal vesicles/vacuoles in animal
and plant systems, respectively. During this process, the acidity of endocytic organelles
increases. Newly synthesized proteins can also be directly delivered to endocytic
compartments from the trans-Golgi network, or transported to the plasma membrane and
then subsequently endocytosed. The mannose-6-phosphate receptor pathway mediates the
former trafficking event [17]. Finally, contents can be delivered to acidic organelles
via the autophagy pathway. In this process, membranes engulf cytoplasmic contents
and/or subcellular organelles for eventual maturation and delivery to lysosomes/vacuoles,
where these materials are degraded [18]. The autophagy pathway has been shown to be
critical for diverse biological processes in plants, animals, and fungi, including
survival during periods of nutrient limitation, (embryonic) development, apoptosis,
abiotic stress, and host defense [19].
Considerable research, using yeast, insect, worm, plant, and mammalian model systems,
has been performed to elucidate the mechanisms by which acidic pH is achieved within
the organelles of eukaryotic cells. The acidification of organelles in the endocytic
pathway has been shown to be mediated by the activity of vacuolar-type ATPase (V-ATPase)
enzymes [20]. These proteins exploit ATP to drive protons into the lumen of endocytic
membranes [21], [22]. In parallel with this movement of protons is the pumping of
cations out of the organelle, to dissipate the development of a restrictive electrochemical
gradient across the vesicular membrane [23]. Mechanisms mediating the biogenesis of
acidic compartments have also been extensively investigated. In fact, the biogenesis
of lysosomes and vacuoles constitutes an important subfield of cell biology [24],
and several excellent reviews on this subject have recently been published [14], [25]–[27].
The task of measuring the pH of intracellular organelles presents several challenges.
First, the subcellular organelles of eukaryotic cells are small (from 0.1 µm in fungi
to 50 µm in plants) and provide no direct access to the outside environment once formed
[28], [29]. Therefore, biochemical analysis of the lumen of these compartments requires
their fractionation and isolation. Moreover, intracellular organelles are highly dynamic
entities that change composition as they mature or travel along defined membrane trafficking
pathways. This fact poses challenges to the analysis of heterogenous, often nonsynchronous
populations of subcellular compartments. Hence, light microscopy approaches are favored
for tracking organellar pH in living cells, and a variety of tools have been developed
for this purpose.
Early estimates of the pH of intracellular organelles of plants, animals, and microbes
were based upon the use of acidotropic dyes [30]–[33]. These molecules traverse biological
membranes and accumulate in acidic intracellular organelles, thereby providing an
indirect, low cost, and qualitative estimate of organellar pH. More recently, a variety
of sophisticated technologies have been used to estimate intracellular pH values,
including the pH of endocytic and other intracellular organelles. These technologies
include pH-responsive microelectrodes [34], NMR [35], and absorbance spectroscopy
[36]. However, fluorescence microscopy provides the most compelling technology for
analyzing (with high sensitivity and resolution) the spatial and temporal dynamics
of pH changes inside living cells. For example, probes that emit fluorescence in a
manner that is dictated by their state of protonation can be used to estimate organellar
pH [37]. These reagents, which include Oregon Green and engineered green fluorescent
protein variants [38], also support ratiometric measurements, which can then be converted
to absolute pH levels by comparison to calibration curves [39]–[41]. These ratiometric
methods are also insensitive to changes in fluorescence introduced by parameters other
than pH, including focal plane or photobleaching. Therefore, approaches that exploit
ratiometric indicators provide a more precise, specific, and robust measure of organellar
pH than their acidotropic fluorophore counterparts, like LysoTracker [42]. Finally,
fluorescent ratiometric analyses of intracellular pH can be extended to investigate
interactions between intracellular pathogens and host cells by labeling live pathogens
with pH-sensitive dyes (carboxyfluorescein, fluorescein, or Oregon Green) so that
viability is not compromised, infecting host cells with the labeled pathogens, and
then performing coincident analysis of pathogen intracellular trafficking and vacuolar
pH [43].
Acids in Immune Systems
Biological acids play central roles in both innate and adaptive immune systems that
dictate host-pathogen interactions. All living things possess innate immune mechanisms,
while vertebrates benefit from the classically defined, lymphocyte-mediated adaptive
immunity as well.
Acid in Innate Immunity
In animal innate immunity, acid functions extracellularly and intracellularly. Low
pH plays an important role extracellularly in controlling the microbial flora at mucosal
sites, particularly in tetrapod vertebrates. The gastric acid of the stomach limits
the range of prokaryotes that can continue down the alimentary canal to join intestinal
populations [44]. The vaginal microbiome maintains an acidic environment that limits
protozoan, fungal, and bacterial infections [45]. Intracellularly, biological acids
and acid hydrolase enzymes mediate the killing of non–acid-adapted organisms that
are phagocytosed into progressively acidifying vesicles epitomized by the lysosome.
This process still provides predatory feeding and phagotrophic nutrition for some
protists [46]. Pattern recognition receptors (PRR) such as the Toll-like receptors
(TLR) bind perceived threats by recognition of pathogen-associated molecular patterns
(PAMPs). In addition to signaling the innate immune system, PRR recognition of PAMPs
in triploblastic animals can doom PAMP-bearing microbes to phagosomal degradation
in acidic vesicles [47]. Moreover, several PRR such as TLR-3, TLR-7, and TLR-9 have
evolved to sense nucleic acid PAMP from acidic endosomes, rather than the cell surface,
taking advantage of the acidic degradation of virus and virus-infected cells by the
low-pH vesicle to sense pathogen-indicative double-stranded RNA, single-stranded RNA,
or DNA with unmethylated CpG dinucleotides [48]. Thus, acid is used both to selectively
control microbial populations on animal surfaces by filtering them based upon acid
tolerance, and to identify and eradicate those that are engulfed by patrolling leukocytes.
Plants rely on the innate immune system for defense by sensing pathogen-derived molecules
for nonself recognition [49]. Plant immune receptors are known as PRR that recognize
PAMPs, such as bacterial flagellin and fungal chitin as well as plant-derived signals
that arise from damage caused by pathogen challenge, known as damage-associated molecular
patterns (DAMPs). PAMP and DAMP recognition is ancient and shared by plants and animals.
PRR binding activates broadly but moderately effective PAMP-triggered immunity (PTI).
Plant pathogens have evolved mechanisms to breach this line of defense by acquisition
of effector molecules that are secreted into the plant cell and perturb host immune
responses by either avoiding detection or suppressing PTI signal transduction. In
the continuing arms race, plants have developed a second tier of defense in which
resistance gene products (R proteins) [49] mediate recognition of specific pathogen
effectors and trigger effector-triggered immunity (ETI), which generally culminates
in host programmed cell death. R gene products contain leucine-rich repeats (LRR)
and nucleotide binding sites and belong to the CATERPILLAR/NOD/NLR family of proteins
that mediate cytosolic PAMP surveillance in animals as well [50], thus exhibiting
trans-kingdom conservation.
Organic acids are crucial messengers of effector-triggered immunity activated by R
gene products of plants. These include the hormone salicylic acid that mediates endogenous
defense signals and transmits systemic responses that orchestrate systemic acquired
resistance [51]. This response is thought to be one of an antagonistic triumvirate
of acid-regulated stress responses in plants, including jasmonic acid for wound healing
and insect protection and abscisic acid for environmental stresses [52]. Ethylene
signaling has somewhat redundant overlap with these latter two. Acids are also used
in the pathogenic counterattack, as several necrotrophic (requiring dead tissue/cells
for growth and reproduction) plant pathogens produce oxalic acid to control host cell
death programs, as discussed in more detail below.
Acid in Adaptive Immunity
The adaptive immune system shared by jawed vertebrates is mediated by lymphocytes
and has hallmark characteristics of specificity and memory. The adaptive system enables
the preemptive engineering of our lymphocyte repertoires through immunization, one
of the most powerful tools for global public health in the face of infectious disease.
Like the plant ETI, the adaptive system is a second line of defense behind the innate
system. While slower in initial response in comparison to the innate, the adaptive
system is plastic in its mitotic expansion and contraction capability, surgical in
the fine molecular specificity of its targeting, and diverse in its effector mechanisms
tailored to classes and locales of pathogen.
The vertebrate adaptive immune system works in conjunction with the more ancient innate
system. The adaptive system may have originally evolved to manage the vast populations
of commensal (better termed mutualistic) bacteria at mucosal surfaces where low pH
was already in use [53]. When the relationship between host animal and symbiont turns
parasitic, immune mechanisms are used to eliminate or contain the microbe and limit
pathology, akin to the “hypersensitive response” of plants.
Most adaptive immune responses require activation of specific helper T lymphocytes,
and the low-pH phagolysosomal system is important for this activation. Unlike B cells
that recognize free antigen, T cells recognize peptide antigen in the context of major
histocompatibility complex (MHC) molecules. Two different classes of MHC molecules
present peptide to two different classes of T cells [54]. Helper T cells are restricted
to being activated only by MHC class II molecules on antigen-presenting cells (APC)
that present peptide antigen. Dendritic cells are the best APC, but macrophages and
B cells are also very competent in T cell activation. This APC hurdle is a major checkpoint
to initiating an adaptive immune response (Figure 2). For example, macrophages harboring
wily intracellular stowaways typically need “help” via cytokine and co-stimulatory
signals from an activated helper T cell to in turn become activated and execute effector
functions. Helper T cells can only be activated by presentation of peptide antigens
specific for their somatically recombined T cell receptor gene products in the context
of self-MHC class II molecules. This MHC class II–presented antigen must arise from
an APC that has itself been activated by ligation of its innate PRR (such as TLR).
The generation of the antigenic peptides presented by APCs is acid dependent.
10.1371/journal.ppat.1003402.g002
Figure 2
Acid's role in initiating adaptive immunity.
An antigen-presenting cell activated by innate PRR will present peptide antigen generated
in acidic vesicles to a helper T cell via MHC class II. Activated by this presentation
of specific antigen, the helper T cell can then mediate many different immune effector
functions, depending on the subtype of helper CD4+ T cell, context, and signals from
the APC. Five such major immune effector pathways are suggested here.
The low pH of compartments of the phagolysosomal pathway is co-opted by the adaptive
as well as the innate immune system for defensive purposes. We suggest that the cellular
physiology of this pathway has evolved from functioning in recycling of cellular debris
and tissue remodeling in early eukaryotes [46], to functioning primarily for nutritional
purposes in heterotrophic protozoa, on to the killing of pathogens in innate immunity
[55], and finally to elegant regulation of adaptive immunity via antigen processing
[56] (Figure 3). Jawed vertebrates use a sophisticated antigen processing pathway
to load MHC class II with peptide antigen from the acidified phagolysosome. Newly
synthesized class II α and β chains assemble in the pH-neutral endoplasmic reticulum
[57] together with a glycoprotein called the invariant chain [58], [59]. An important
role of the invariant chain is to keep MHC class II from being loaded prematurely
with peptide from the endoplasmic reticulum or Golgi, retaining that place for products
of the acidified phagolysosomal system.
10.1371/journal.ppat.1003402.g003
Figure 3
Phylogeny of acidic phagolysosome use in immunity.
Simplified phylogeny of life, marking major hypothesized steps supported by current
comparative biology in the co-opting of the acidic phagolysosome system in innate
and adaptive immunity (blue). Sister taxon names are illustrative and not necessarily
of same phylogenetic rank, and genetic distances are not to scale. All life has innate
immunity, but only vertebrates have adaptive immunity (red). Origins of key proteins
that regulate the system are shown in green.
In the trans-Golgi, the MHC class II/invariant chain complex is diverted from the
secretory pathway to the endocytic pathway (Figure 4). In the low pH of the MHC class
II compartment, cathepsin proteases are activated to further cleave the invariant
chain, leaving only CLIP (the class II–associated invariant chain peptide) in the
peptide binding cleft [60]. HLA-DM can then bind class II and catalyze the release
of CLIP, facilitating its exchange for phagolysosomal antigenic peptides before MHC
transport to the cell surface. Pathogen blockade (e.g., by the human immunodeficiency
virus) of the progressive cleavage of the invariant chain results in the accumulation
of invariant chain intermediates, constipation of the antigen presentation pathway,
and immunoevasive reduced surface expression of MHC class II [61].
10.1371/journal.ppat.1003402.g004
Figure 4
Acid-active cathepsins cleave phagolysosomal antigens in the MHC class II pathway.
Phagocytosed antigens are degraded to peptides (grey) by acids and acid-active cathepsin
proteases as the endosomal pH decreases due to fusion with lysosomes (1). During their
trafficking from the ER to the cell surface, MHC class II molecules (light green)
pass through these acidified vesicles (2). Invariant chain (red) chaperones MHC class
II from the ER to an acidified endosome, all the while protecting the peptide binding
groove of MHC class II from premature loading (3). Invariant chain is cleaved by cathepsins
but leaves the CLIP portion (red triangle) in the MHC peptide binding site (4). In
a specialized late endosome, the MHC homolog HLA-DM finally binds to the MHC class
II/CLIP complex and releases CLIP (5), allowing other peptides to bind before the
MHC class II travels to the cell surface (6). There it can present antigen to T cells
(7).
Cathepsins, related to papain, are the crucial acid-activated proteases that cleave
the invariant chain and also degrade lysosomal contents to peptides appropriate for
antigen to be loaded in MHC class II [62]. The cathepsins primarily involved in antigen
processing are L and S. Although invariant chain and cathepsins of adaptive immunity
likely evolved with the jawed cartilaginous fishes (gnathostomes, see Figure 3) [63],
comparative phagosome proteomics show acid-active cathepsins to be an original and
fundamental component of the ancestral eukaryotic phagolysosomal system [64].
Thus, the acidic phagolysosomal system prepares peptide antigens for the initiation
of most cellular adaptive immune responses, mediated by the MHC class II system of
presentation to helper T cells.
Acids in Host-Pathogen Interactions
Given the role that acid plays in host defense and antigen presentation in animal
pathosystems, it is perhaps not surprising that both plant and animal pathogens have
evolved sophisticated systems for adapting to, avoiding, or subverting the threats
that acidic environments and acid-mediated defense processes pose. We illustrate this
point using two intracellular bacterial pathogens—Coxiella burnetii and Brucella melitensis—which
have evolved disparate strategies for adapting to life in acidic environments and
avoiding killing by acidic organelles. Two extracellular mucosal pathogens—Helicobacter
pylori and Escherichia coli—exemplify tactics used to colonize the extreme pH of the
alimentary canal. In addition, we describe the way in which the plant pathogens Agrobacterium
tumefaciens and Sclerotinia sclerotium exploit and produce acidic environments, respectively,
to promote their pathogenic programs. These host-pathogen systems have been chosen
both for their position as major models in which acid defense mechanisms have been
elucidated as well as their importance in human, animal, and plant pathology.
Intracellular Acid
Many organisms generate and use acidic environments to thwart infection by pathogens.
However, some pathogens have evolved elegant strategies to defeat these acidic defense
mechanisms, including the ability to withstand or thrive in highly acidic environments.
The intracellular pathogen Coxiella burnetii provides a compelling example of such
an adaptation. The Gram-negative bacterium C. burnetii is the causative agent of Q
fever. Its natural reservoir in the United States consists mainly of dairy cattle,
sheep, and goats [65], and its extreme infectivity (single bacterium [66]) led to
its weaponization before the United States' biological warfare program was terminated
in 1969 [67].
C. burnetii replicates within phagolysosome-like Coxiella-containing vacuoles (CCVs)
and is dependent on the low pH of this compartment to activate a developmental process
that turns metabolically quiescent small cell variants (SCV) into their metabolically
active, replicating large cell counterparts [68] (Figure 5A). C. burnetii is taken
up via complement receptor 3 and αvβ3 integrin-mediated mechanisms into macrophages
[69]. Actin-dependent phagocytosis leads to trafficking through an early endosome,
progressing to late endosome and the phagolysosome-like terminal CCV [70], [71]. The
trafficking appears slightly delayed compared with latex bead uptake [72], and membrane
markers for Rab5, Rab7, Rab24, microtubule-associated protein-1 light chain alpha
3 (LC3), lysosomal-associated membrane proteins (LAMP)-1, LAMP-2, and LAMP-3, and
flotillin 1 and 2 progressively decorate the CCV [73]–[75]. Connection to the autophagosome
compartment appears essential to support the biogenesis of a compartment that can
support productive replication of the pathogen [74], [76]. In addition, CCV membrane
development to a spacious vacuole requires access to continual host-derived cholesterol
biosynthesis [77], [78] and connection to a canonical secretory pathway [79].
10.1371/journal.ppat.1003402.g005
Figure 5
Coxiella and Brucella use distinct mechanisms for intracellular pathogenesis.
A.
C. burnetti thrives in the acidic phagolysosome system, requiring low pH for the transition
from quiescent small cell variants (SCV) to metabolically active large cell variants
(LCV). Several of the transmembrane proteins that mark the Coxiella-containing vacuole
(CCV) through this transition are shown. The Dot/Icm type IV secretion system is used
by C. burnetti to deliver proteins into the host cytosol [138], and renovate the lysosome
into a CCV [139]. Cb, Coxiella burnetti; Atg, autophagosome; Lys, lysosome. B. Working
model of Brucella intracellular parasitism. Brucella-containing vacuoles avoid fusion
with acidic lysosomes, and instead traffic to a compartment that is decorated with
ER markers for replication. The Type IV secretion system (T4SS) of the pathogen is
critical for appropriate trafficking, and mutants that harbor mutations in the T4SS
traffic to the lysosome where they are killed. Several T4SS secretion substrates have
been identified, and it has been postulated that these molecules contribute to supporting
the intracellular lifestyle of the pathogen. Replicative Brucella can exit cells by
trafficking along a pathway that involves selective interactions with components of
the host cell autophagy biogenesis machinery. Approximate vesicular/vacuolar pH is
indicated by color, and the Golgi is generally more acidic than the ER [57, 140–142].
Several lines of investigation support the hypothesis that maintenance of the acidic
pH of the spacious vacuole is essential for C. burnetii replication. First, studies
using ratiometric, pH-sensitive probes have demonstrated that C. burnetii replicates
in vacuoles that possess an acidic pH [80]. Inhibition of host vacuolar ATPase activities
that maintain the acidic pH of late endocytic and lysosomal compartments by treatment
with bafilomycin significantly impairs the intracellular replication of the pathogen.
Similarly, neutralization of vacuolar pH by treatment with the chaotropic agent chloroquine
inhibits intracellular replication [81]. Taken together, these data indicate that
the C. burnetii replicative niche has an acidic pH, and raise many questions about
the mechanisms by which Coxiella and other acid-adapted intracellular pathogens survive
in the highly acidic environment of the CCV terminal compartment [82], [83].
Several strategies have been proposed to contribute to acid tolerance of C. burnetii,
and the development of axenic growth conditions and suicide plasmid-based targeted
gene deletion methods is allowing identification of virulence mechanisms and genes
in this model [84]. First, the intracellular trafficking of the pathogen along the
endolysosomal pathway pauses immediately after entry [72]. This pause in phagolysosomal
maturation has been hypothesized to play a critical role in acid adaptation by providing
the pathogen with sufficient time to prepare (through the expression of acid tolerance
factors) for the onslaught of acid that follows. Second, stress-response and vacuole-detoxification
genes are dramatically upregulated when the pathogen invades host cells, thereby supporting
its adaptation to the harsh vacuolar environment [85]. Finally, C. burnetii encodes
an unusually high number of basic proteins. The average pI value for all predicted
proteins in the genome of the Nine Mile reference strain is 8.25 [86], [87]; 60% of
the proteome is acidic [88]. Moreover, approximately 45% of C. burnetii proteins were
found to have a pI value of ≥9, which is higher than the sequenced genomes of other
intracellular bacterial pathogens [89]. Orthologous products of the RpoS genes of
E. coli (pI 4.6) and C. burnetti (pH 9.6) serve as striking examples of the extreme
acid adaptation of a protein while maintaining a conserved function. It is hypothesized
that Coxiella's skew toward production of basic proteins provides a proton sink to
buffer those protons that enter the cytoplasm [88], [90]. While the low pH of the
phagolysosome is a crucial parameter, Coxiella tolerates cathepsin and other acid
protease action while thriving in this environment. Coxiella therefore illustrates
how pathogens can adapt to acidic compartments.
In an alternative strategy, intracellular bacterial pathogens can address the threat
that acidic intracellular compartments pose by minimizing their interactions with
them. The intracellular bacterial pathogen Brucella spp. provide excellent models
to analyze this strategy for addressing the threat that intracellular acidic vacuoles
pose to invading pathogens. Brucella spp. are causative agents of brucellosis, a zoonosis
of global importance [91]. In humans, the disease causes severe, debilitating, and
protracted symptoms, and affects practically every organ system of the body. Brucellosis
often presents in the clinic as an undulant fever [92]. However, chronic infections
are frequently associated with osteoarticular disease with neurological complications.
The reproductive system is also a common site of infection, and infection during pregnancy
may increase the risk of spontaneous abortion. Consumption of unpasteurized milk products
from infected animals is the most common route to human infection [92]. However, Brucella
is highly infectious and can be readily transmitted in aerosolized form [93]. Brucellosis
has eluded systematic attempts at eradication, even in most developed countries, and
no human vaccine is available [94]. These features contribute to the classification
of Brucella as a potential bioterror agent, and to the interest the biosecurity and
world health communities have expressed in this organism.
During their intracellular trafficking within host cells, Brucella-containing vacuoles
(BCVs) interact but avoid fusion with the host lysosome [95], [96] (Figure 5B). Instead,
replicative BCVs become decorated with markers for ER [97]. Mutant strains harboring
defects in the Type IV secretion system do not avoid fusion with acidic lysosomal
compartments, and instead are rapidly killed after fusion with this organelle [98].
In mouse models of brucellosis, the organism persists for months in the lymph nodes
and spleen [99]. However, colonization by even the most virulent strains becomes undetectable
with time [100]. The persistence of this organism in the ER of host APCs may be crucial
for chronic infection. Thus, avoiding the harsh environment of the host cell's acidic
degradative organelles is critical to the survival and replication of this intracellular
bacterial pathogen.
Extracellular Acid in Animals
Several notable microbial pathogens, including those that infect the gastrointestinal
or urogenital tracts of humans and animals, exploit or create extracellular acidic
environments to promote their pathogenic programs. For example, pathogenic E. coli
(strain 0157:H7), Vibrio cholerae, Vibrio vulnifus, Shigella flexneri, and Salmonella
typhimurium are found in neutral pH environments (on food products or in water). However,
after ingestion by their mammalian hosts, these pathogens encounter the severe acidic
environment of the stomach (pH = 2 or lower) or urogenital systems, which normally
constitute important barriers to infection by non–acid-adapted organisms. These parasites,
however, have evolved sophisticated and divergent strategies to tolerate these harsh
acidic environments.
A significant human pathogen that provides an understanding of an alternative strategy
by which an extracellular acidic niche can be occupied is Helicobacter pylori, the
agent whose colonization is associated with chronic gastrointestinal diseases ranging
from dyspepsia to gastric and duodenal ulcers to gastric carcinoma [101], [102]. This
organism chronically infects the stomach, surviving the very low pH of the lumen,
burrowing into the mucus with flagella to attach to and occasionally invade epithelial
cells. Survival in this environment is dependent on expression of copious amounts
of urease, converting urea into buffering ammonia plus carbon dioxide [102]. In this
strategy, H. pylori maintains a periplasmic pH at ∼6.1, while the extracellular environment
can be as low as 2.0. The reaction products of urease apoenzyme (ureA and ureB) are
driven specifically to the periplasmic compartment by a protein (UreI) encoded in
a gene cluster with urease. UreI is a pH-gated inner membrane urea channel allowing
for efficient transit to the cytoplasm of substrate (urea) and periplasmic release
of reactant NH3, which is rapidly converted to NH4 by membrane-bound α-carbonic anhydrase.
The transcriptional expression control, post-transcriptional recruitment, and enzymatic
activity of H. pylori urease is optimized to function in a microenvironment of pH
3.5–∼6.0 [103].
H. pylori is specialized for the extreme pH of the stomach, but enteric species survive
passage through the stomach for colonization of the lower gut. Many E. coli efficiently
colonize the intestine, and provide a complementary window for understanding how pathogens
address the challenge of acidic environments. The acid stress response systems of
Gram-negative enteric pathogens are both enzyme and chaperone based, and include resistance
pathways that exploit glutamate-, arginine-, and lysine-decarboxylase enzymes [104].
E. coli contains five acid resistance pathways (AR1–5), which work in concert to resist
the highly acidic environment that the pathogen encounters in the gut [105]. In the
AR2 system of E. coli, for example, the pyridoxal 5′ phosphate (PLP)-dependent GadA
and GadB decarboxylases convert glutamate to gamma-amino butyric acid (GABA) and carbon
dioxide (CO2) in a reaction that consumes a cytoplasmic proton [106]. The inner membrane
antiporter GadC then transports GABA out of the cell in exchange for additional glutamate
[106]. Thus, the pathogen mitigates acid stress by promoting the net export of protons
outside of the cell at the expense of intracellular glutamate. Analogous systems that
exploit arginine and lysine also contribute to maintaining the pH homeostasis of the
cytoplasm.
Thus gut pathogens use a variety of active biochemical systems to maintain periplasmic
and cytoplasmic pH at tolerable levels amidst the low luminal pH of the gastrointestinal
tract.
Extracellular Acid in Plants
Analysis of interactions between plant pathogens and their host plants provides additional
insights into the role of acid in host-pathogen interactions that cannot be fully
appreciated by the exclusive analysis of animal systems. The bacterial pathogens Agrobacterium
tumefaciens and Erwinia amylovora, the causative agents of crown gall disease in diverse
dicotyledenous plants and fire blight in apples, pears, and Rosaceous crops, respectively,
induce disparate and nonoverlapping disease symptoms in plants. Nevertheless, these
pathogens respond to environmental acids by modulating their virulence programs, and
thus provide ideal illustrations of an important mechanism by which pathogens of both
plants and animals respond to acidic extracellular environments in the context of
the host-pathogen interaction.
In the Agrobacterium system, environmental acids drive the induction of the pathogen's
virulence program. Specifically, the acidic environment created by wounded plant tissues,
as well as plant-derived phenolic compounds present there, activate the expression
of bacterial virulence genes (vir regulon) on the tumor-inducing (Ti) plasmid [107].
This activity, in turn, drives the assembly and transfer of the Agrobacterium-derived
T-DNA from the bacterial pathogen to host cells. The T-DNA integrates into the host
plant genome, triggering factors that mediate the generation of a tumor (gall) in
plants. Interestingly, the acidic conditions that initiate the T-DNA virulence program
of Agrobacterium elicit two additional and distinct responses in the pathogen—a conserved
response associated with the adaptation of the pathogen to environmental acidification,
and the intricate response that regulates the establishment of a stable, long-term
plant-pathogen interaction [108]. Genes induced by the former response, such as the
motility gene flaA and the heat-shock protein ibpA, are highly conserved and play
corresponding roles in acid adaptation in other microbial systems, including in the
bacterial pathogens of animals (e.g., E. coli, Salmonella spp.) [108]. An important
component of the latter response is regulated by the activities of acidic plant hormone
signaling molecules, including salicylic acid (SA), indole-3-acetic acid (IAA), and
gamma-amino butyric acid (GABA) [109] (more on plant hormones in the following section).
These plant acids generally signal through biochemically distinct and independent
bacterial pathways to function additively to shut off the Agrobacterium virulence
program and activate the quorum-quenching machinery, which promotes the establishment
of a stable host-pathogen interaction. However, signal input from one pathway (an
environmental stress response signaled through abscisic acid during a drought, for
example) can inhibit the activation of another (such as a jasmonic acid signal for
wound repair stimulated by herbivory). The activation of quorum-sensing machinery
as part of the process of establishing a stable host-pathogen interaction represents
a conserved theme in the virulence programs of many bacterial pathogens [110].
Extracellular acidic environments can also influence the virulence of fungal pathogens
of plants, including Ustilago maydis
[111], Fusarium oxysporum
[112], and Sclerotinia sclerotiorum
[113], the causative agents of tumorigenic corn smut, Fusarium wilt, and white mold
diseases of all broadleaf plants, respectively.
Plant Hormones
Analogous to animal hormones, plant hormones play key roles in the control of development,
growth, reproduction, and, of relevance for this discussion, the regulation of immune
responses to microbial pathogens. Of the principal plant hormones, five are acids,
including: salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), indole acetic
acid (IAA), and gibberellic acid (GA) [114]. SA and JA and their derivatives are structurally
and functionally akin to aspirin and prostaglandins, respectively. SA and JA are recognized
as major defense hormones with the classical view that SA is effective against biotrophic
pathogens and JA against necrotrophs, although there are exceptions [52]. Moreover,
crosstalk occurs between these hormones that is often antagonistic; elevated biotroph
resistance (SA) results in elevated necrotroph susceptibility and vice versa.
Systemic acquired resistance (SAR) is induced in the plant by SA following pathogen
challenge. When established, SAR affords long-lasting and broad-spectrum resistance
including uninfected tissue. SA levels increase following initial pathogen attack
and are strongly correlated with establishment of SAR [115]. This observation is strengthened
by the fact that plant treatment with exogenous SA or biologically active chemical
analogs leads to SAR. Moreover, blocking SA synthesis inhibits SAR [116]. Considerable
effort has been undertaken to identify the regulatory pathways mediating SAR. Of note,
mutants of the npr1 locus were found to prevent SA signaling [117]. In the uninduced
state, cytosolic NPR1 is present as an oligomer via intermolecular disulfide bridges.
Following SA-mediated SAR induction, alterations in cellular redox result in a reduced
state leading to disassociation of the complex and release of monomeric species. Monomeric
NPR1 translocates to the nucleus, interacts with the leucine zipper transcription
factor TGA1 that binds to promoters of SA-responsive genes, and activates defense
gene expression [118].
The jasmonate family comprises lipid-derived metabolites synthesized via the oxylipin
pathway [119]. Upon synthesis, JA can be metabolized to methyl jasmonate or conjugated
to amino acids [120]. Most JA responses are mediated by the F-box protein coronatine
insentive 1 (COI1). Coi1 mutants are more resistant to bacterial pathogens and show
elevated SA levels [121]. In accordance with SA-JA antagonism, coi1 plants are more
susceptible to several, but not all, necrotrophic fungi. Exogenous application of
JA induces broad changes in transcription patterns—in particular, of genes regulated
by MYC2, a basic helix loop helix transcription factor [122]. Genetic studies revealed
a family of 12 jasmonate ZIM-domain–containing (JAZ) proteins that repress JA signaling.
JAZ proteins can homo- and heterodimerize in vitro, suggesting a possible mechanism
for fine-tuning signaling responses [123]. Binding of conjugated JA or coronatine
to SCFCOI1 promotes ubiquitination of JAZs leading to proteasome degradation, relieving
repression of MYC2, and facilitating activation of JA-responsive genes [124]. Alternative
splicing of the c-terminal JAS domain of JAZ proteins results in reduced ubiquitination
and thus reduced degradation.
Although JA is central to modulating defense against necrotrophic pathogens, it is
increasingly important in other aspects of plant-pathogen interactions including SAR
[125]. Once the JA pathway is activated (e.g., after wounding), a similar JA response
can be triggered in distal undamaged parts of the plant. The antagonism between SA
and JA signaling pathways in plants show a dramatic resemblance to the effect of the
anti-inflammatory drug aspirin, which is an acetylated form of SA, on prostaglandins.
Prostaglandins are structurally related to JA, and these hormones function at sites
of infection or injury. SA/JA crosstalk shows similarities with the inhibitory effect
of aspirin to prostaglandins in mammalian cells; however, the molecular bases of these
interactions are not identical [126].
The phytopathogenic fungus Sclerotinia sclerotiorum provides an informative model
to illustrate acid-dependent pathogenic development. This fungus produces copious
amounts of the dicarboxylic acid oxalic acid (OA) as part of its virulence arsenal,
but also uses OA to reprogram host signaling and modulate programmed cell death to
coordinate effective pathogenesis [113].
Sclerotinia is an economically important necrotrophic fungal pathogen of plants with
an extremely broad host range (all broadleaf dicot plants). The primary component
for pathogenic success is the production and secretion of oxalic acid by the fungus
[127]. Oxalic acid is found in plants, animals, and humans and is the endpoint of
several metabolic processes such as the breakdown of glyoxylic acid or ascorbic acid
[128], [129]. In mammals, oxalic acid plays an important role in kidney stone formation
as a counter ion of calcium-forming oxalate crystals [130].
In fungi, this “simple” organic acid is remarkably multifunctional and contributes
to numerous physiological and pathogenic processes [131] (Figure 6). Oxalate-deficient
mutants of S. sclerotiorum are nonpathogenic on all host plants tested and are also
unable to develop sclerotia—highly melanized, durable overwintering structures. Oxalate
secretion might enhance Sclerotinia virulence in several ways. Many fungal enzymes
secreted during invasion of plant tissues (e.g., pectinases) have maximal activities
at acidic pH and have been shown to be activated by OA. A Sclerotinia MAP kinase has
also been identified and characterized [132], and has been shown to be required for
sclerotia formation in S. sclerotiorum. Gene expression of this Erk-like MAPK (SMK1)
is triggered by acidic pH mediated by OA; if acidification does not occur, pathogenic
development is blocked [133], [134].
10.1371/journal.ppat.1003402.g006
Figure 6
Stages of Sclerotinia pathogenesis.
Early steps of infection create a reducing environment that dampens host defense responses
and inhibits reactive oxygen species (ROS). This allows the fungal pathogen to establish
and damage host tissues with cell wall degradative enzymes (CWDE). When eventual apoptotic
cascades are induced, recognition occurs but too late for the host plant to prevail.
(adapted from [143]).
OA has several other deleterious effects upon the plant. It can degrade or weaken
the plant cell wall via acidity and/or chelation of cell wall Ca2+. Oxalic acid crystals
are sequestered in vacuoles, and when decompartmentalized during infection, these
crystals can plug the vascular system. Oxalic acid in and of itself is directly toxic,
functioning as a non–host-specific phytotoxin. While these features are correlated
with fungal disease development, they do not account for wild-type pathogenesis [135].
OA can function as an elicitor of apoptotic-like plant programmed cell death, involving
the modulation of the host redox environment [136]. The induction of apoptosis and
disease requires generation of reactive oxygen species (ROS) in the host, a process
triggered by fungal-secreted OA. Curiously, the programmed cell death process mediated
by OA is independent of acidification; a direct correlation between DNA laddering,
ROS induction, and cell death were all observed at neutral pH. When acidification
via OA occurs, cells also die but in a mechanistically different way: necrotically
without hallmarks of apoptosis such as plasma membrane blebbing and chromosomal DNA
fragmentation. The specificity of this interaction is further supported by the observation
that DNA fragmentation is specific to OA, as other acids such as citric acid, succinic
acid, and hydrochloric acid do not induce DNA ladders [137]. DNA cleavage also was
independent of oxalate formulation because OA, potassium oxalate, and sodium oxalate
all caused DNA laddering, thus suggesting that programmed cell death induction is
not due to the acidic nature of oxalate but rather to a property of OA itself.
Conversely, during the initial stages of infection, OA also dampens the plant oxidative
burst—an early host response generally associated with plant defense. Experiments
using a transgenic redox-regulated GFP reporter show that, initially, Sclerotinia
(via OA) generates a reducing environment in host cells that suppresses host defense
responses including the oxidative burst. Once infection is established, however, this
necrotroph induces the generation of plant ROS leading to PCD of host tissue, the
result of which is of direct and sole benefit to the pathogen. In contrast, a nonpathogenic,
OA-deficient mutant failed to alter host redox status and induced autophagy and restricted
growth. These results indicate active recognition of the mutant by the plant and further
point to the importance of cell death control in mediating host-pathogen interactions.
Taken together, these data suggest that Sclerotinia establishes reductive conditions
that dampen the host oxidative burst and suppress defense responses [135]. This scenario
buys precious time, allowing for unimpeded fungal growth and establishment. When the
plant eventually senses the presence of nonself, it is too late; the fungus is already
inducing the programmed cell death of host cells.
Conclusions and Perspective
Just as pH is a fundamental property of living systems, acid is employed by both host
and pathogen in their ever-escalating arms race. On the side of the host, acid is
used in a variety of mechanisms, including extracellular innate immunity at mucosal
surfaces and extreme pH in the case of the mammalian stomach. Yet some pathogens have
evolved mechanisms to defeat the natural defense that the acidic environment provides,
exemplified by Helicobacter's urease system that keeps its periplasm close to neutral
and E. coli's glutamate decarboxylase pathway that removes cytoplasmic protons as
a component of its acid response. Acid is important in plant extracellular immunity
as well and is a characteristic of wounding, but Agrobacterium senses this drop in
pH and counters with acid-based signaling programs to ramp up and attenuate virulence
effectors. Fungi such as Sclerotinia use OA to subvert host defense and co-opt host
signaling pathways, triggering cell death via apoptosis of host cells. Thus fungal
(OA) induced metabolic reprogramming of the host results in apoptosis, providing nutrients
exclusively for the benefit of the necrotrophic organism.
The adaptive immune system of vertebrates pirated the acidic phagolysosomal system
for not only pathogen killing but also for antigen presentation to T cells. Vesicular
acid and acid-activated proteases such as cathepsins are critical to antigen processing
for MHC loading. But some microbes have evolved mechanisms to cope with the low pH
of vacuoles, such as Coxiella, while others have managed to reprogram the vesicle
trafficking to mitigate interactions with harmful acidic organelles (e.g., lysosomes),
as does Brucella. Ratiometric dyes have been crucial in facilitating the analysis
of these intracellular organelles and the pathogenesis that occurs there. Table 1
lists additional pathogens that exploit or evade acid beyond the scope of this review.
10.1371/journal.ppat.1003402.t001
Table 1
Notable pathogens evolved for acid management or evasion.
Mechanism
Species
Phagolysosome adaptation
Coxiella burnetii
Phagolysosome evasion
Brucella melitensis
Mycobacterium tuberculosis
Listeria monocytogenes
Toxoplasma gondii
Chlamydia trachomatis
Legionella pneumophila
Adaptation to extracellular acidic mucosa
Helicobacter pylori
Escherichia coli
Vibrio cholerae
Trichomonas vaginalis
Shigella flexneri
Campylobacter jejuni
Clostridium perfringens
Salmonella enterica
Shigella dydenteriae
Neisseria gonorrhoeae
Treponema palladum
Yersinia enterocolitica
Clostridium difficile
Staphylococcus aureus
Adaptation to plant acidic environments
Agrobacterium tumefaciens
Sclerotinia sclerotium
Erwinia amylovora
From the perspective of natural history, we see the mechanisms by which organisms
exploit or subvert biological acids as central to their evolutionary success. Thus
acid is one of a small, select number of fundamental biological arenas in which organic
life has long fought. For example, membranes create a defined space for orderly biochemistry
where metabolism and other necessities can occur protected from the chaos beyond the
phospholipid bilayer. Naturally, immune systems evolve enzymes such as lysozyme, perforin,
and the membrane attack complex of the complement cascade to disrupt the membranes,
but pathogenic microbes evolve complex cell walls and capsules to protect and then
elaborate secretion systems to breach these barriers. These are but a few of the many
mechanisms and counter-mechanisms that operate at the theater of war that is the plasma
membrane. Similarly, genomic integrity is also an important battleground; therefore,
the simplest of organisms have elegant DNA repair systems, high-fidelity replication
enzymes, and restriction endonucleases. Yet viruses still integrate, and even counter
by exploiting the rapid evolution afforded by replication errors. In turn, the adaptive
immune system employs somatic hypermutation of antibody genes in a Darwinian germinal
center reaction to hone high-affinity receptors against ever-shifting pathogen antigens.
Just as the plasma membrane and the genome are core components of life to be protected,
exploited, and used in defense and offense, so is the pH of spaces large (stomach)
and small (phagolysosome). It should come as no surprise that acids are used to great
effect in signaling, killing, protecting, hiding, sampling, and evading by both host
and microbe, and we should look to the trenches of protons and hydroxides for continued
exploitation by both immune and pathogen systems.