Overview
Since the discovery of Helicobacter pylori, the urease activity of this bacterial
pathogen has been identified as the key factor in infection and acid acclimation in
the human stomach. Ureolytic activity plays a key role in the pathogenesis of several
bacteria, and urease has also been described as an emerging pathogenic factor during
fungal infection. However, urease produced by the oral bacteria community has been
shown to counteract caries, and caries-free subjects have high levels of urease activity
in plaque samples. Some lactic acid bacteria with documented probiotic behavior are
urease-positive. Likewise, other lactic acid bacterial species that are widely used
in yogurt production and other fermented dairy products use urease activity to counteract
acid stress and to feed several biosynthetic pathways with carbon dioxide and ammonia
derived from urea hydrolysis. Urease is also diffused in several species belonging
to the human gut microbiota, and it is estimated that this complex microbial community
is able to hydrolyze 15%–30% of the urea synthesized in normal subjects. In this context,
urease was proposed to serve as a microbial biomarker to distinguish microbiomes based
on age and geography, thus highlighting the crucial involvement of this enzymatic
activity in nitrogen recycling when dietary nitrogen is limiting. In light of these
considerations, the designation of urease as a microbial virulence factor would be
misleading, and the proposed use of urease as a therapeutic target to counteract microbial
infections should be carefully evaluated.
Urease: Multifunctional Roles in Microbial Physiology
Urease and its substrate urea represent historically important milestones in early
scientific investigation. Urea was the first organic molecule synthesized, and urease
from jack bean was the first enzyme crystallized, in addition to being the first enzyme
shown to contain nickel [1]–[4]. The scientific interest in microbial urease is largely
related to the relevance of this enzymatic activity in infection. This interest has
been strongly stimulated since the discovery of the association of H. pylori with
gastritis and stomach cancer [5]. Moreover, urease has served as a paradigm for understanding
the activation mechanisms of many metalloenzymes that require accessory proteins for
their catalytic activity [3], [4]. Urease is a urea amidohydrolase (EC 3.5.1.5) that
catalyzes the hydrolysis of urea to yield ammonia and carbamate, which spontaneously
decomposes to yield a second molecule of ammonia and carbonic acid. The released carbonic
acid and the two molecules of ammonia are in equilibrium with their deprotonated and
protonated forms, respectively, and the net effect of these reactions is an increase
in the pH of the environment that surrounds the urease-positive microorganisms (Fig.
1). For this reason, urease is considered a stress response that was developed by
several bacteria to counteract a low environmental pH [6]. In Streptococcus thermophilus,
urease is metabolically related to the biosynthetic pathways involved in aspartate,
glutamine, arginine, and carbon dioxide metabolism [7], [8]. Notably, urea hydrolysis
increases the catabolic efficiency of S. thermophilus by modulating the intracellular
pH and increasing the activity of β-galactosidase, glycolytic enzymes, and lactate
dehydrogenase [9]. Urea hydrolysis results in increases in both the pHin and the pHout
due to the rapid diffusion of ammonia outside of the cell. Consequently, in the presence
of urea and a urease-positive microorganism, urease-negative microorganisms share
the environmental benefit derived from the transient local pH increase [9].
10.1371/journal.ppat.1004472.g001
Figure 1
Schematic representation of the reaction catalyzed by microbial urease and the involvement
of these enzymes in microbial physiology, human health, and disease.
Beyond that, the role of urease in most of the microorganisms that show this enzymatic
activity is primarily linked to the recycling of nitrogenous wastes and nitrogen assimilation.
Human Microbiota Urease As a “Health-Associated Factor”
Urea is the major nitrogenous waste product of most terrestrial animals. Ammonium,
released from the urea present in the secretions of major and minor exocrine glands,
provides a nitrogen source for bacteria that colonize the human body. It was estimated
that 15%–30% of the urea synthesized in healthy subjects is continually hydrolyzed
by microbial ureases [10]. Several microbial species belonging to the human microbiota
produce active urease, and these species take advantage of urea hydrolysis, as has
been demonstrated for the oral bacteria Streptococcus salivarius
[11] and Actinomyces naeslundii
[12] and hypothesized for other species of the gut microbiota [13], [14]. Urea is
also present in human milk. Human milk contains only approximately 15% of its nitrogen
in the form of urea. It is therefore believed that the total nitrogen in the infant
lower gastrointestinal tract (GIT) may be present at suboptimal levels [15]. An increase
in postnatal nitrogen levels is likely necessary to satisfy the growth and metabolism
requirements of the infant and the GIT microbiota. In human infants, it has been determined
that the amino acids in plasma can be derived from urea after hydrolysis and utilization
of nitrogen by the intestinal microbiota [16]. It is therefore not surprising that
an early colonizer of the GIT of humans, the Bifidobacterium longum subsp. Infantis,
produced an active urease [17]. By contrast, in subjects with acute liver failure,
the major clinical problem is the development of hepatic encephalopathy (HE) that
is associated with high level of gut-derived ammonia. In this scenario, the microbial
gut community, especially urease-positive species such as Klebsiella spp. and Proteus
spp., is an important source of ammonia in humans in the pathogenesis of HE [18].
Several clinical cases have suggested that the severity of HE can be reduced by modulating
the microbial gut community using agents that lead to a normalization of gut microbiota,
such as rifaximin, lactulose, prebiotics, and probiotics. However, even probiotics
can be urease-positive. Interestingly, a recent study performed using a murine model
reported that the administration of the probiotic urease-positive Lactobacillus reuteri
reduced the amount of urease activity in the murine gut, presumably due to the suppression
of fecal bacteria [19]. Another bacterial species that is currently used as a probiotic
for the oropharyngeal tract is the urease-positive S. salivarius strain K12 [20].
Following oral administration, strain K12 can colonize the oral mucosae of infants
and adults and down-regulate the innate immune responses of human epithelial cells.
It is also active against S. pyogenes and safe and well tolerated by the human host
[21]. In a more general food context, it is worth mentioning that yogurt consumption
is commonly associated with a health benefit by the consumers [21], and one of the
two species of the yogurt consortium, S. thermophilus, is urease-positive [22]. S.
thermophilus is widely used in the manufacturing of dairy products, yogurt, fermented
milk, and cheeses, and as a consequence, over 1021 living cells carrying active urease
molecules are ingested annually by the human population.
A recent study [14] focused on the characterization of gut microbial communities in
two human populations revealed that urease gene frequency was significantly higher
in Malawian and Amerindian infant microbiomes and that it decreased with age in these
two populations, unlike in the United States, where it remains low from infancy to
adulthood. Considering that urease has a crucial involvement in nitrogen recycling,
particularly when diets are deficient in protein, the ability of the microbiome to
use urea would presumably be advantageous to both microbes and host.
Urea is secreted into all parts of the digestive tract starting from the oral cavity.
In saliva, urea is present at a concentration of 3–10 mM, and it represents a relevant
nitrogen source for several species belonging to the oral microbiota, including S.
salivarius, S. vestibularis and Actinomyces naeslundii. A substantial body of evidence
is beginning to accumulate that indicates a direct contribution of alkali generation
in dental biofilms to the inhibition of dental caries [23]. The development of dental
caries is favored by tooth demineralization that happens as a consequence of the frequent
acidification of dental biofilms and the subsequent emergence of acidogenic and acid-tolerant
microorganisms, including mutans streptococci and Lactobacillus spp., which ferment
dietary carbohydrates rapidly and lower the pH. The increasing number of acid-tolerant
microorganisms results in a simultaneous decrease in the less acid-tolerant species
that are often associated with dental health [24]. Notably, bacteria associated with
dental health are able to use urea and/or arginine to generate ammonia. Alkali production
by these microorganisms positively affects the balance between the remineralization
and demineralization of the tooth and may help prevent the emergence of cariogenic
microorganisms [25]. The real scenario is actually more complex than it might appear.
In fact, while the urease activity associated with plaque seems to correlate with
a decrease in the incidence of caries, the urease activity associated with the saliva
had a significant effect on the risk of developing caries, and this effect was not
protective but instead promoted the development of caries [26]. It therefore appears
that the oral localization of urease activity is fundamental in preventing caries.
Interestingly, in mice, the carcinogenicity of the plaque bacterium S. mutans (naturally
urease-negative) was dramatically reduced in a derivative recombinant strain of S.
mutans that was able to produce an active urease, thus suggesting that recombinant
ureolytic bacteria may be useful in promoting dental health [27].
Microbial Urease As a General “Virulence Factor”
In addition to the positive aspects of microbial ureases in human health, a consistent
body of evidence has identified urease as a virulence factor for several microbial
pathogens (Fig. 1). In fact, ureolytic activity has a key role in the pathogenesis
of bacteria such as Clostridium perfringens, Helicobacter pylori, Klebsiella pneumoniae,
Proteus mirabilis, Salmonella spp., Staphylococcus saprophyticus, Ureoplasma urealyticum,
and Yersinia enterocolitica, and such activity has been reported in diseases such
as urolithiasis, pyelonephritis, ammonia encephalopathy, HE, hepatic coma, and gastroduodenal
infections [4], [28]. The role of urease in microbial infection has been well established
in H. pylori. Hydrolysis of urea in the human stomach provides NH3 that is essential
for acid neutralization, enabling H. pylori to raise the pH in its microenvironment
and periplasm, thus maintaining the proton motive force [28]. Moreover, the urea-dependent
ammonia production appears to be partially responsible for the gastric mucosal injury
found in association with H. pylori infection [29]. A proton-gated channel, UreI,
which regulates the uptake of urea [4], is only active at acidic pH and therefore
does not allow for the transport of urea into the bacterial cell at neutral pH, thus
preventing lethal alkalinization of the cytoplasm [4]. Without this mechanism, H.
pylori is unable to develop the infection process in the stomach [30], [31]. Similarly,
the urease activity allows the survival to the gastric transit of Y. enterocolitica
[32].
The role of urease activity in urinary tract infections and struvite and carbonate
apatite stones formation was described for P. mirabilis and Sta. saprophyticus. The
urease-dependent invasive property of P. mirabilis was supported by in vitro observation
and by the use of urease-negative mutants. P. mirabilis defective in urease exhibited
in a mouse model an ID50 more than 1,000-fold higher than the wild-type strain, and
only the wild-type strain was able to persist significantly [33]. Likewise, the contribution
of urease to the cytopathogenicity of Sta. saprophyticus has been demonstrated in
a rat model using a chemically mutagenized urease-deficient strain, and by the heterologous
expression of an active urease in the nonureolytic Staphylococcus carnosus strain
[34], [35].
More recently [36], the role of urease as a general microbial virulence factor was
proposed, highlighting the emerging pathogenic roles of urease during infection of
the fungal species Cryptococcus neoformans (a basidiomycete) and Coccidioides posadasii
(an ascomycete). During fungal lung infection, the urea present in the epithelial
lining fluid of the lungs is hydrolyzed by fungal urease, and the generated ammonia
inhibits immune function and contributes to lung tissue damage [36].
Other human pathogens are urease-positive, and in many cases, urea hydrolysis is thought
to have a role in the infectivity or persistence of the microorganisms. In this context,
although largely unexplored, the positive role of urease in microbial physiology (Fig.
1) can be an advantage for a pathogen during the various stages of the infection process
in terms of competition with commensal microorganisms associated with the human body.
Perspectives
Because of the facts that the human genome does not contain urease-encoding genes
and that no human nickel-containing enzymes are known, urease was proposed as a potential
therapeutic target [36] without taking into consideration all the positive aspects
linked to the microbial ureases of the human microbiota. In this context, the use
of the term “virulence factor” for microbial ureases should be carefully evaluated.
Microbiologists working on infectious organisms routinely define any gene product
that contribute to the virulence potential of a pathogen as a “virulence factor.”
Recently, the increasing interest in the human microbiota raises questions about the
terminology we use to describe the molecular and metabolic strategies that pathogenic
microbes use to compete in these complex biological systems [37]. In the GIT, many
pathogens and commensals use similar strategies to overcome the challenges associated
with this particular environment. It would therefore be misleading to describe the
same strategies and structures found in harmless or beneficial commensals as “virulence
factors” simply because they were acquired or evolved to survive in the GIT. The term
“niche factors” was therefore proposed [37] to describe the molecular and metabolic
strategies evolved by beneficial gut microbes to colonize this complex environment.