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Microbial Urease in Health and Disease

Microbial Urease in Health and Disease Pearls Diego Mora*, Stefania Arioli Department of Food Environmental and Nutritional Sciences, University of Milan, Milan, Italy the intracellular pH and increasing the activity of b-galactosidase, Overview glycolytic enzymes, and lactate dehydrogenase [9]. Urea hydro- Since the discovery of Helicobacter pylori, the urease activity of lysis results in increases in both the pH and the pH due to the in out this bacterial pathogen has been identified as the key factor in rapid diffusion of ammonia outside of the cell. Consequently, in infection and acid acclimation in the human stomach. Ureolytic the presence of urea and a urease-positive microorganism, urease- activity plays a key role in the pathogenesis of several bacteria, and negative microorganisms share the environmental benefit derived urease has also been described as an emerging pathogenic factor from the transient local pH increase [9]. during fungal infection. However, urease produced by the oral Beyond that, the role of urease in most of the microorganisms bacteria community has been shown to counteract caries, and that show this enzymatic activity is primarily linked to the caries-free subjects have high levels of urease activity in plaque recycling of nitrogenous wastes and nitrogen assimilation. samples. Some lactic acid bacteria with documented probiotic behavior are urease-positive. Likewise, other lactic acid bacterial Human Microbiota Urease As a ‘‘Health- species that are widely used in yogurt production and other Associated Factor’’ fermented dairy products use urease activity to counteract acid stress and to feed several biosynthetic pathways with carbon Urea is the major nitrogenous waste product of most dioxide and ammonia derived from urea hydrolysis. Urease is also terrestrial animals. Ammonium, released from the urea present diffused in several species belonging to the human gut microbiota, in the secretions of major and minor exocrine glands, provides a and it is estimated that this complex microbial community is able nitrogen source for bacteria that colonize the human body. It to hydrolyze 15%–30% of the urea synthesized in normal subjects. was estimated that 15%–30% of the urea synthesized in healthy In this context, urease was proposed to serve as a microbial subjects is continually hydrolyzed by microbial ureases [10]. biomarker to distinguish microbiomes based on age and geogra- Several microbial species belonging to the human microbiota phy, thus highlighting the crucial involvement of this enzymatic produce active urease, and these species take advantage of urea activity in nitrogen recycling when dietary nitrogen is limiting. In hydrolysis, as has been demonstrated for the oral bacteria light of these considerations, the designation of urease as a Streptococcus salivarius [11] and Actinomyces naeslundii [12] microbial virulence factor would be misleading, and the proposed and hypothesized for other species of the gut microbiota use of urease as a therapeutic target to counteract microbial [13,14]. Urea is also present in human milk. Human milk infections should be carefully evaluated. contains only approximately 15% of its nitrogen in the form of urea. It is therefore believed that the total nitrogen in the infant Urease: Multifunctional Roles in Microbial lower gastrointestinal tract (GIT) may be present at suboptimal levels [15]. An increase in postnatal nitrogen levels is likely Physiology necessary to satisfy the growth and metabolism requirements of Urease and its substrate urea represent historically important the infant and the GIT microbiota. In human infants, it has milestones in early scientific investigation. Urea was the first been determined that the amino acids in plasma can be derived organic molecule synthesized, and urease from jack bean was the from urea after hydrolysis and utilization of nitrogen by the first enzyme crystallized, in addition to being the first enzyme intestinal microbiota [16]. It is therefore not surprising that an shown to contain nickel [1–4]. The scientific interest in microbial early colonizer of the GIT of humans, the Bifidobacterium urease is largely related to the relevance of this enzymatic activity longum subsp. Infantis, produced an active urease [17]. By in infection. This interest has been strongly stimulated since the contrast, in subjects with acute liver failure, the major clinical discovery of the association of H. pylori with gastritis and stomach problem is the development of hepatic encephalopathy (HE) cancer [5]. Moreover, urease has served as a paradigm for that is associated with high level of gut-derived ammonia. In this understanding the activation mechanisms of many metalloen- scenario, the microbial gut community, especially urease- zymes that require accessory proteins for their catalytic activity [3,4]. Urease is a urea amidohydrolase (EC 3.5.1.5) that catalyzes Citation: Mora D, Arioli S (2014) Microbial Urease in Health and Disease. PLoS the hydrolysis of urea to yield ammonia and carbamate, which Pathog 10(12): e1004472. doi:10.1371/journal.ppat.1004472 spontaneously decomposes to yield a second molecule of ammonia Editor: Virginia Miller, University of North Carolina at Chapel Hill School of and carbonic acid. The released carbonic acid and the two Medicine, United States of America molecules of ammonia are in equilibrium with their deprotonated and protonated forms, respectively, and the net effect of these Published December 11, 2014 reactions is an increase in the pH of the environment that Copyright:  2014 Mora, Arioli. This is an open-access article distributed under surrounds the urease-positive microorganisms (Fig. 1). For this the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the reason, urease is considered a stress response that was developed original author and source are credited. by several bacteria to counteract a low environmental pH [6]. In Funding: The authors received no specific funding for this study. Streptococcus thermophilus, urease is metabolically related to the biosynthetic pathways involved in aspartate, glutamine, arginine, Competing Interests: The authors have declared that no competing interests exist. and carbon dioxide metabolism [7,8]. Notably, urea hydrolysis increases the catabolic efficiency of S. thermophilus by modulating * Email: diego.mora@unimi.it PLOS Pathogens | www.plospathogens.org 1 December 2014 | Volume 10 | Issue 12 | e1004472 Figure 1. Schematic representation of the reaction catalyzed by microbial urease and the involvement of these enzymes in micro- bial physiology, human health, and disease. doi:10.1371/journal.ppat.1004472.g001 positive species such as Klebsiella spp. and Proteus spp., is an carrying active urease molecules are ingested annually by the important source of ammonia in humans in the pathogenesis of human population. HE [18]. Several clinical cases have suggested that the severity A recent study [14] focused on the characterization of gut of HE can be reduced by modulating the microbial gut microbial communities in two human populations revealed that community using agents that lead to a normalization of gut urease gene frequency was significantly higher in Malawian and microbiota, such as rifaximin, lactulose, prebiotics, and Amerindian infant microbiomes and that it decreased with age in probiotics. However, even probiotics can be urease-positive. these two populations, unlike in the United States, where it Interestingly, a recent study performed using a murine model remains low from infancy to adulthood. Considering that urease reported that the administration of the probiotic urease-positive has a crucial involvement in nitrogen recycling, particularly when Lactobacillus reuteri reduced the amount of urease activity in diets are deficient in protein, the ability of the microbiome to use the murine gut, presumably due to the suppression of fecal urea would presumably be advantageous to both microbes and bacteria [19]. Another bacterial species that is currently used as host. a probiotic for the oropharyngeal tract is the urease-positive S. Urea is secreted into all parts of the digestive tract starting from salivarius strain K12 [20]. Following oral administration, strain the oral cavity. In saliva, urea is present at a concentration of 3– K12 can colonize the oral mucosae of infants and adults and 10 mM, and it represents a relevant nitrogen source for several down-regulate the innate immune responses of human epithelial species belonging to the oral microbiota, including S. salivarius, S. cells. It is also active against S. pyogenes and safe and well vestibularis and Actinomyces naeslundii. A substantial body of tolerated by the human host [21]. In a more general food evidence is beginning to accumulate that indicates a direct context, it is worth mentioning that yogurt consumption is contribution of alkali generation in dental biofilms to the inhibition commonly associated with a health benefit by the consumers of dental caries [23]. The development of dental caries is favored [21], and one of the two species of the yogurt consortium, S. by tooth demineralization that happens as a consequence of the thermophilus, is urease-positive [22]. S. thermophilus is widely frequent acidification of dental biofilms and the subsequent used in the manufacturing of dairy products, yogurt, fermented emergence of acidogenic and acid-tolerant microorganisms, milk, and cheeses, and as a consequence, over 10 living cells including mutans streptococci and Lactobacillus spp., which PLOS Pathogens | www.plospathogens.org 2 December 2014 | Volume 10 | Issue 12 | e1004472 ferment dietary carbohydrates rapidly and lower the pH. The property of P. mirabilis was supported by in vitro observation and increasing number of acid-tolerant microorganisms results in a by the use of urease-negative mutants. P. mirabilis defective in simultaneous decrease in the less acid-tolerant species that are urease exhibited in a mouse model an ID50 more than 1,000-fold often associated with dental health [24]. Notably, bacteria higher than the wild-type strain, and only the wild-type strain was associated with dental health are able to use urea and/or arginine able to persist significantly [33]. Likewise, the contribution of to generate ammonia. Alkali production by these microorganisms urease to the cytopathogenicity of Sta. saprophyticus has been positively affects the balance between the remineralization and demonstrated in a rat model using a chemically mutagenized demineralization of the tooth and may help prevent the emergence urease-deficient strain, and by the heterologous expression of an of cariogenic microorganisms [25]. The real scenario is actually active urease in the nonureolytic Staphylococcus carnosus strain more complex than it might appear. In fact, while the urease [34,35]. activity associated with plaque seems to correlate with a decrease More recently [36], the role of urease as a general microbial in the incidence of caries, the urease activity associated with the virulence factor was proposed, highlighting the emerging patho- saliva had a significant effect on the risk of developing caries, and genic roles of urease during infection of the fungal species this effect was not protective but instead promoted the develop- Cryptococcus neoformans (a basidiomycete) and Coccidioides ment of caries [26]. It therefore appears that the oral localization posadasii (an ascomycete). During fungal lung infection, the urea of urease activity is fundamental in preventing caries. Interestingly, present in the epithelial lining fluid of the lungs is hydrolyzed by in mice, the carcinogenicity of the plaque bacterium S. mutans fungal urease, and the generated ammonia inhibits immune (naturally urease-negative) was dramatically reduced in a deriva- function and contributes to lung tissue damage [36]. tive recombinant strain of S. mutans that was able to produce an Other human pathogens are urease-positive, and in many cases, active urease, thus suggesting that recombinant ureolytic bacteria urea hydrolysis is thought to have a role in the infectivity or may be useful in promoting dental health [27]. persistence of the microorganisms. In this context, although largely unexplored, the positive role of urease in microbial physiology Microbial Urease As a General ‘‘Virulence Factor’’ (Fig. 1) can be an advantage for a pathogen during the various stages of the infection process in terms of competition with In addition to the positive aspects of microbial ureases in human commensal microorganisms associated with the human body. 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 Perspectives as Clostridium perfringens, Helicobacter pylori, Klebsiella pneu- Because of the facts that the human genome does not contain moniae, Proteus mirabilis, Salmonella spp., Staphylococcus sapro- urease-encoding genes and that no human nickel-containing phyticus, Ureoplasma urealyticum, and Yersinia enterocolitica, and enzymes are known, urease was proposed as a potential such activity has been reported in diseases such as urolithiasis, therapeutic target [36] without taking into consideration all the pyelonephritis, ammonia encephalopathy, HE, hepatic coma, and positive aspects linked to the microbial ureases of the human gastroduodenal infections [4,28]. The role of urease in microbial microbiota. In this context, the use of the term ‘‘virulence factor’’ infection has been well established in H. pylori. Hydrolysis of urea for microbial ureases should be carefully evaluated. Microbiolo- in the human stomach provides NH that is essential for acid gists working on infectious organisms routinely define any gene neutralization, enabling H. pylori to raise the pH in its product that contribute to the virulence potential of a pathogen as microenvironment and periplasm, thus maintaining the proton a ‘‘virulence factor.’’ Recently, the increasing interest in the motive force [28]. Moreover, the urea-dependent ammonia human microbiota raises questions about the terminology we use production appears to be partially responsible for the gastric to describe the molecular and metabolic strategies that pathogenic mucosal injury found in association with H. pylori infection [29]. A microbes use to compete in these complex biological systems [37]. proton-gated channel, UreI, which regulates the uptake of urea In the GIT, many pathogens and commensals use similar [4], is only active at acidic pH and therefore does not allow for the strategies to overcome the challenges associated with this transport of urea into the bacterial cell at neutral pH, thus particular environment. It would therefore be misleading to preventing lethal alkalinization of the cytoplasm [4]. Without this describe the same strategies and structures found in harmless or mechanism, H. pylori is unable to develop the infection process in beneficial commensals as ‘‘virulence factors’’ simply because they the stomach [30,31]. Similarly, the urease activity allows the were acquired or evolved to survive in the GIT. The term ‘‘niche survival to the gastric transit of Y. enterocolitica [32]. factors’’ was therefore proposed [37] to describe the molecular and The role of urease activity in urinary tract infections and struvite and carbonate apatite stones formation was described for P. metabolic strategies evolved by beneficial gut microbes to colonize mirabilis and Sta. saprophyticus. The urease-dependent invasive this complex environment. References 1. Sumner JB (1926) The isolation and crystallization of the enzyme urease. J Biol 7. Arioli S, Monnet C, Guglielmetti S, Parini C, De Noni, et al. (2007) Aspartate Chem 69: 435–441. biosynthesis is essential for the growth of Streptococcus thermophilus in milk, and 2. Dixon NE, Gazzola C, Blakeley RL, Zerner B (1975) Jack bean urease (EC aspartate availability modulates the level of urease activity. Appl Environ 3.5.1.5). A metalloenzyme. A simple biological role for nickel? J American Chem Microbiol 73: 5789–5796. Society 97: 4131–4133. 8. Arioli S, Roncada P, Salzano AM, Deriu F, Corona S, et al. (2009) The 3. Andrews RK, Blakeley RL, Zerner B (1984) Urea and urease. Adv Inorg relevance of carbon dioxide metabolism in Streptococcus thermophilus. Microbiol Biochem 6: 245–283. 155: 1953–1965. 4. Weeks Dl, Eskandari S, Scott DR, Sachs G (2000) A H+ - gated urea channel: the link 9. Arioli S, Ragg EM, Scaglioni L, Fessas D, Signorelli M, et al. (2010) Alkalizing between Helicobacter pylori urease and gastric colonization. Science 287: reactions streamline cellular metabolism in acidogenic microorganisms. PLoS ONE 5: e15520 482–485. 5. Mobley HLT, Island MD, Hausinger RP (1995) Molecular biology of microbial 10. Walser M, Bodenlos L (1959) Urea metabolism in man. J Clin Invest 38: 1617–1626. ureases. Microbiol Rev 59: 451–480. 11. Chen YM, Burne RA (2003) Identification and characterization of the nickel transport system for urease biogenesis in Streptococcus salivaius 57.1. J Bacteriol 6. Cotter PD, Hill C (2003) Surviving the acid test: responses of Gram-positive bacteria to low pH. Microbiol. Mol Biol Rev 67: 429–453. 185: 6773–6779. PLOS Pathogens | www.plospathogens.org 3 December 2014 | Volume 10 | Issue 12 | e1004472 12. Morou-Bermudez E, Burne RA (2000) Genetic and physiologic 25. Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, et al. (2002) characterization of urease of Actinomyces naeslundii. Infect Immun 67: 504–512. Molecular analysis of bacterial species associated with childhood caries. J Clin 13. Wegmann U, Louis P, Goesmann A, Henrissat B, Duncan SH, et al. (2013) Microbiol 40: 1001–1009. Complete genome of a new Firmicutes species belonging to the dominant 26. Morou-Bermudez E, Elias-Boneta A, Billings RJ, Burne RA, Garcia-Rivas V, et human colonic microbiota (‘Ruminococcus bicirculans’) reveals two al. (2011) Urease activity as a risk factor for caries development in children chromosomes and a selective capacity to utilize plant glucans. Environ Microbiol during a three-year study period: A survival analysis approach. Archives Oral 16: 2879–2890. doi:10.1111/1462-2920.12217 Microbiol 56: 1560–1568. 14. Yatsunenko T, Rey FE, Manary M, Trehan I, Dominguez-Bello MG, et al. 27. Clancy KA, Pearson S, Bowen WH, Burne RA (2000) Characterization of (2012) Human gut microbiome viewed across age and geography. Nature 486: recombinant, ureolytic Streptococcus mutans demonstrates an inverse relation- 222–227. ship between dental plaque ureolytic capacity and cariogenicity. Infect Immun 15. Fuller MF, Reeds PJ 1998. Nitrogen cycling in the gut. Annu Rev Nutr 18: 385– 68: 2621–2629. 28. Burne RA, Chen YM (2000) Bacterial ureases in infectious diseases. Microb 16. Millward DJ, Forrester T, Ah-Sing E, Yeboah N, Gibson N, et al. (2000) The Infect 2: 533–542. transfer of 15N from urea to lysine in the human infant. British J Nut 83: 505– 29. Smoot DT, Mobley HL, Chippendale GR, Lewison JF, Resau JH (1990) Helicobacter pylori urease activity is toxic to human gastric epithelial cells. Infect 17. LoCascio RG, Desai P, Sela DA, Weimer B, Mills DA (2010) Broad Immun 58: 1992–1994. conservation of milk utilization genes in Bifidobacterium longum subsp. infantis 30. Mollenhauer-Rektorschek M, Hanauer G, Sacks G, Melchers K (2002) as revealed by comparative genomic hybridization. Appl Environ Microbiol 76: Expression of UreI is required for intragastric transit and colonization of gerbil 7373–7381. gastric mucosa by Helicobacter pylori. Res Microbiol 153: 659–666. 18. Riordan SM, Williams R (2010) Gut flora and hepatic encephalopathy in 31. Skouloubris S, Thiberge JM, Labigne A, De Reuse H (1998) The helicobacter patients with cirrhosis. N Engl J Med 362: 1140–1142. pylori UreI protein is not involved in urease activity but is essential for bacterial 19. Wilson CM, Loach D, Lawley B, Bell T, Sims I, et al. (2014) Lactobacillus reuteri survival in vivo. Infect Immun 66: 4517–4521. 100-23 modulates urea hydrolysis in the murine stomach. Appl Environ 32. De Koning-Ward TF, Robins-Browne RM (1995) Contribution of urease to acid Microbiol 80: 6104–6113. doi:10.1128/AEM.01876-14. tolerance in Yersinia enterocolitica. Infect Immun 63: 3790–3795. 20. Power DA, Burton JP, Chilcott CN, Dawes PJ, Tagg JR (2008). Preliminary 33. Johnson DE, Russel RG, Lockatell CV, Zulty JC, Warren JW, et al. (1993) investigations of the colonization of upper respiratory tract tissues of infants Contribution of Proteus mirabilis urease to persistence, urolithiasis, and acute using a pediatric formulation of the oral probiotic Streptococcus salivarius K12. pyelonephritis in a mouse model of ascending urinary tract infection. Infect Eur J Clin Microbiol Infect Dis 27: 1261–1263. Immun 61: 2748–2754. 21. Burton JP, Cowley S, Simon RR, McKinney J, Wescombe PA, et al. (2011) 34. Gatermann S, John J, Marre R (1989) Staphylococcus saprophyticus urease: Evaluation of safety and human tolerance of the oral probiotic Streptococcus characterization and contribution to uropathogenicity in unobstructed urinary salivarius K12: A randomized, placebo-controlled, double-blind study. Food Chem Toxicol 49: 2356–2364. tract infection of rats. Infect Immun 57: 110–116. 22. Mora D, Maguin E, Masiero M, Ricci G, Parini C, et al. (2004) Characterization 35. Gatermann S, Marre R (1989) Cloning and expression of Staphylococcus of urease genes cluster of Streptococcus thermophilus. J Appl Microbiol 96: 209– saprophyticus urease gene sequences in Staphylococcus carnosus and contribution of the enzyme to virulence. Infect Immun 57: 2998–3002. 23. Zhu Y, Wang H, Hollis JH, Jacques PF (2014) The association between yogurt 36. Rutherford JC (2014) The emerging role of urease as a general microbial consumption, diet quality, and metabolic profiles in children in the USA. Eur J virulence factor. PLoS Pathog 10: e1004062. Nutr. E-pub ahead of print. doi:10.1007/s00394-014-0735-7 37. Hill C (2012) Virulence or niche factors: what’s in a name? J Bacteriol 194: 24. Liu Y-L, Nascimento M, Burne R (2012) Progress toward understanding the 5725–5727. contribution of alkali generation in dental biofilms to inhibition of dental caries. Int J Oral Sci 4: 135–140. PLOS Pathogens | www.plospathogens.org 4 December 2014 | Volume 10 | Issue 12 | e1004472 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS Pathogens Public Library of Science (PLoS) Journal

Microbial Urease in Health and Disease

PLoS Pathogens , Volume 10 (12) – Dec 11, 2014

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Copyright: © 2014 Mora, Arioli. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors received no specific funding for this study. Competing interests: The authors have declared that no competing interests exist.
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10.1371/journal.ppat.1004472
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Abstract

Pearls Diego Mora*, Stefania Arioli Department of Food Environmental and Nutritional Sciences, University of Milan, Milan, Italy the intracellular pH and increasing the activity of b-galactosidase, Overview glycolytic enzymes, and lactate dehydrogenase [9]. Urea hydro- Since the discovery of Helicobacter pylori, the urease activity of lysis results in increases in both the pH and the pH due to the in out this bacterial pathogen has been identified as the key factor in rapid diffusion of ammonia outside of the cell. Consequently, in infection and acid acclimation in the human stomach. Ureolytic the presence of urea and a urease-positive microorganism, urease- activity plays a key role in the pathogenesis of several bacteria, and negative microorganisms share the environmental benefit derived urease has also been described as an emerging pathogenic factor from the transient local pH increase [9]. during fungal infection. However, urease produced by the oral Beyond that, the role of urease in most of the microorganisms bacteria community has been shown to counteract caries, and that show this enzymatic activity is primarily linked to the caries-free subjects have high levels of urease activity in plaque recycling of nitrogenous wastes and nitrogen assimilation. samples. Some lactic acid bacteria with documented probiotic behavior are urease-positive. Likewise, other lactic acid bacterial Human Microbiota Urease As a ‘‘Health- species that are widely used in yogurt production and other Associated Factor’’ fermented dairy products use urease activity to counteract acid stress and to feed several biosynthetic pathways with carbon Urea is the major nitrogenous waste product of most dioxide and ammonia derived from urea hydrolysis. Urease is also terrestrial animals. Ammonium, released from the urea present diffused in several species belonging to the human gut microbiota, in the secretions of major and minor exocrine glands, provides a and it is estimated that this complex microbial community is able nitrogen source for bacteria that colonize the human body. It to hydrolyze 15%–30% of the urea synthesized in normal subjects. was estimated that 15%–30% of the urea synthesized in healthy In this context, urease was proposed to serve as a microbial subjects is continually hydrolyzed by microbial ureases [10]. biomarker to distinguish microbiomes based on age and geogra- Several microbial species belonging to the human microbiota phy, thus highlighting the crucial involvement of this enzymatic produce active urease, and these species take advantage of urea activity in nitrogen recycling when dietary nitrogen is limiting. In hydrolysis, as has been demonstrated for the oral bacteria light of these considerations, the designation of urease as a Streptococcus salivarius [11] and Actinomyces naeslundii [12] microbial virulence factor would be misleading, and the proposed and hypothesized for other species of the gut microbiota use of urease as a therapeutic target to counteract microbial [13,14]. Urea is also present in human milk. Human milk infections should be carefully evaluated. contains only approximately 15% of its nitrogen in the form of urea. It is therefore believed that the total nitrogen in the infant Urease: Multifunctional Roles in Microbial lower gastrointestinal tract (GIT) may be present at suboptimal levels [15]. An increase in postnatal nitrogen levels is likely Physiology necessary to satisfy the growth and metabolism requirements of Urease and its substrate urea represent historically important the infant and the GIT microbiota. In human infants, it has milestones in early scientific investigation. Urea was the first been determined that the amino acids in plasma can be derived organic molecule synthesized, and urease from jack bean was the from urea after hydrolysis and utilization of nitrogen by the first enzyme crystallized, in addition to being the first enzyme intestinal microbiota [16]. It is therefore not surprising that an shown to contain nickel [1–4]. The scientific interest in microbial early colonizer of the GIT of humans, the Bifidobacterium urease is largely related to the relevance of this enzymatic activity longum subsp. Infantis, produced an active urease [17]. By in infection. This interest has been strongly stimulated since the contrast, in subjects with acute liver failure, the major clinical discovery of the association of H. pylori with gastritis and stomach problem is the development of hepatic encephalopathy (HE) cancer [5]. Moreover, urease has served as a paradigm for that is associated with high level of gut-derived ammonia. In this understanding the activation mechanisms of many metalloen- scenario, the microbial gut community, especially urease- zymes that require accessory proteins for their catalytic activity [3,4]. Urease is a urea amidohydrolase (EC 3.5.1.5) that catalyzes Citation: Mora D, Arioli S (2014) Microbial Urease in Health and Disease. PLoS the hydrolysis of urea to yield ammonia and carbamate, which Pathog 10(12): e1004472. doi:10.1371/journal.ppat.1004472 spontaneously decomposes to yield a second molecule of ammonia Editor: Virginia Miller, University of North Carolina at Chapel Hill School of and carbonic acid. The released carbonic acid and the two Medicine, United States of America molecules of ammonia are in equilibrium with their deprotonated and protonated forms, respectively, and the net effect of these Published December 11, 2014 reactions is an increase in the pH of the environment that Copyright:  2014 Mora, Arioli. This is an open-access article distributed under surrounds the urease-positive microorganisms (Fig. 1). For this the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the reason, urease is considered a stress response that was developed original author and source are credited. by several bacteria to counteract a low environmental pH [6]. In Funding: The authors received no specific funding for this study. Streptococcus thermophilus, urease is metabolically related to the biosynthetic pathways involved in aspartate, glutamine, arginine, Competing Interests: The authors have declared that no competing interests exist. and carbon dioxide metabolism [7,8]. Notably, urea hydrolysis increases the catabolic efficiency of S. thermophilus by modulating * Email: diego.mora@unimi.it PLOS Pathogens | www.plospathogens.org 1 December 2014 | Volume 10 | Issue 12 | e1004472 Figure 1. Schematic representation of the reaction catalyzed by microbial urease and the involvement of these enzymes in micro- bial physiology, human health, and disease. doi:10.1371/journal.ppat.1004472.g001 positive species such as Klebsiella spp. and Proteus spp., is an carrying active urease molecules are ingested annually by the important source of ammonia in humans in the pathogenesis of human population. HE [18]. Several clinical cases have suggested that the severity A recent study [14] focused on the characterization of gut of HE can be reduced by modulating the microbial gut microbial communities in two human populations revealed that community using agents that lead to a normalization of gut urease gene frequency was significantly higher in Malawian and microbiota, such as rifaximin, lactulose, prebiotics, and Amerindian infant microbiomes and that it decreased with age in probiotics. However, even probiotics can be urease-positive. these two populations, unlike in the United States, where it Interestingly, a recent study performed using a murine model remains low from infancy to adulthood. Considering that urease reported that the administration of the probiotic urease-positive has a crucial involvement in nitrogen recycling, particularly when Lactobacillus reuteri reduced the amount of urease activity in diets are deficient in protein, the ability of the microbiome to use the murine gut, presumably due to the suppression of fecal urea would presumably be advantageous to both microbes and bacteria [19]. Another bacterial species that is currently used as host. a probiotic for the oropharyngeal tract is the urease-positive S. Urea is secreted into all parts of the digestive tract starting from salivarius strain K12 [20]. Following oral administration, strain the oral cavity. In saliva, urea is present at a concentration of 3– K12 can colonize the oral mucosae of infants and adults and 10 mM, and it represents a relevant nitrogen source for several down-regulate the innate immune responses of human epithelial species belonging to the oral microbiota, including S. salivarius, S. cells. It is also active against S. pyogenes and safe and well vestibularis and Actinomyces naeslundii. A substantial body of tolerated by the human host [21]. In a more general food evidence is beginning to accumulate that indicates a direct context, it is worth mentioning that yogurt consumption is contribution of alkali generation in dental biofilms to the inhibition commonly associated with a health benefit by the consumers of dental caries [23]. The development of dental caries is favored [21], and one of the two species of the yogurt consortium, S. by tooth demineralization that happens as a consequence of the thermophilus, is urease-positive [22]. S. thermophilus is widely frequent acidification of dental biofilms and the subsequent used in the manufacturing of dairy products, yogurt, fermented emergence of acidogenic and acid-tolerant microorganisms, milk, and cheeses, and as a consequence, over 10 living cells including mutans streptococci and Lactobacillus spp., which PLOS Pathogens | www.plospathogens.org 2 December 2014 | Volume 10 | Issue 12 | e1004472 ferment dietary carbohydrates rapidly and lower the pH. The property of P. mirabilis was supported by in vitro observation and increasing number of acid-tolerant microorganisms results in a by the use of urease-negative mutants. P. mirabilis defective in simultaneous decrease in the less acid-tolerant species that are urease exhibited in a mouse model an ID50 more than 1,000-fold often associated with dental health [24]. Notably, bacteria higher than the wild-type strain, and only the wild-type strain was associated with dental health are able to use urea and/or arginine able to persist significantly [33]. Likewise, the contribution of to generate ammonia. Alkali production by these microorganisms urease to the cytopathogenicity of Sta. saprophyticus has been positively affects the balance between the remineralization and demonstrated in a rat model using a chemically mutagenized demineralization of the tooth and may help prevent the emergence urease-deficient strain, and by the heterologous expression of an of cariogenic microorganisms [25]. The real scenario is actually active urease in the nonureolytic Staphylococcus carnosus strain more complex than it might appear. In fact, while the urease [34,35]. activity associated with plaque seems to correlate with a decrease More recently [36], the role of urease as a general microbial in the incidence of caries, the urease activity associated with the virulence factor was proposed, highlighting the emerging patho- saliva had a significant effect on the risk of developing caries, and genic roles of urease during infection of the fungal species this effect was not protective but instead promoted the develop- Cryptococcus neoformans (a basidiomycete) and Coccidioides ment of caries [26]. It therefore appears that the oral localization posadasii (an ascomycete). During fungal lung infection, the urea of urease activity is fundamental in preventing caries. Interestingly, present in the epithelial lining fluid of the lungs is hydrolyzed by in mice, the carcinogenicity of the plaque bacterium S. mutans fungal urease, and the generated ammonia inhibits immune (naturally urease-negative) was dramatically reduced in a deriva- function and contributes to lung tissue damage [36]. tive recombinant strain of S. mutans that was able to produce an Other human pathogens are urease-positive, and in many cases, active urease, thus suggesting that recombinant ureolytic bacteria urea hydrolysis is thought to have a role in the infectivity or may be useful in promoting dental health [27]. persistence of the microorganisms. In this context, although largely unexplored, the positive role of urease in microbial physiology Microbial Urease As a General ‘‘Virulence Factor’’ (Fig. 1) can be an advantage for a pathogen during the various stages of the infection process in terms of competition with In addition to the positive aspects of microbial ureases in human commensal microorganisms associated with the human body. 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 Perspectives as Clostridium perfringens, Helicobacter pylori, Klebsiella pneu- Because of the facts that the human genome does not contain moniae, Proteus mirabilis, Salmonella spp., Staphylococcus sapro- urease-encoding genes and that no human nickel-containing phyticus, Ureoplasma urealyticum, and Yersinia enterocolitica, and enzymes are known, urease was proposed as a potential such activity has been reported in diseases such as urolithiasis, therapeutic target [36] without taking into consideration all the pyelonephritis, ammonia encephalopathy, HE, hepatic coma, and positive aspects linked to the microbial ureases of the human gastroduodenal infections [4,28]. The role of urease in microbial microbiota. In this context, the use of the term ‘‘virulence factor’’ infection has been well established in H. pylori. Hydrolysis of urea for microbial ureases should be carefully evaluated. Microbiolo- in the human stomach provides NH that is essential for acid gists working on infectious organisms routinely define any gene neutralization, enabling H. pylori to raise the pH in its product that contribute to the virulence potential of a pathogen as microenvironment and periplasm, thus maintaining the proton a ‘‘virulence factor.’’ Recently, the increasing interest in the motive force [28]. Moreover, the urea-dependent ammonia human microbiota raises questions about the terminology we use production appears to be partially responsible for the gastric to describe the molecular and metabolic strategies that pathogenic mucosal injury found in association with H. pylori infection [29]. A microbes use to compete in these complex biological systems [37]. proton-gated channel, UreI, which regulates the uptake of urea In the GIT, many pathogens and commensals use similar [4], is only active at acidic pH and therefore does not allow for the strategies to overcome the challenges associated with this transport of urea into the bacterial cell at neutral pH, thus particular environment. It would therefore be misleading to preventing lethal alkalinization of the cytoplasm [4]. Without this describe the same strategies and structures found in harmless or mechanism, H. pylori is unable to develop the infection process in beneficial commensals as ‘‘virulence factors’’ simply because they the stomach [30,31]. Similarly, the urease activity allows the were acquired or evolved to survive in the GIT. The term ‘‘niche survival to the gastric transit of Y. enterocolitica [32]. factors’’ was therefore proposed [37] to describe the molecular and The role of urease activity in urinary tract infections and struvite and carbonate apatite stones formation was described for P. metabolic strategies evolved by beneficial gut microbes to colonize mirabilis and Sta. saprophyticus. The urease-dependent invasive this complex environment. References 1. Sumner JB (1926) The isolation and crystallization of the enzyme urease. J Biol 7. Arioli S, Monnet C, Guglielmetti S, Parini C, De Noni, et al. (2007) Aspartate Chem 69: 435–441. biosynthesis is essential for the growth of Streptococcus thermophilus in milk, and 2. Dixon NE, Gazzola C, Blakeley RL, Zerner B (1975) Jack bean urease (EC aspartate availability modulates the level of urease activity. Appl Environ 3.5.1.5). A metalloenzyme. A simple biological role for nickel? J American Chem Microbiol 73: 5789–5796. Society 97: 4131–4133. 8. 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Eur J virulence factor. PLoS Pathog 10: e1004062. Nutr. E-pub ahead of print. doi:10.1007/s00394-014-0735-7 37. Hill C (2012) Virulence or niche factors: what’s in a name? J Bacteriol 194: 24. Liu Y-L, Nascimento M, Burne R (2012) Progress toward understanding the 5725–5727. contribution of alkali generation in dental biofilms to inhibition of dental caries. Int J Oral Sci 4: 135–140. PLOS Pathogens | www.plospathogens.org 4 December 2014 | Volume 10 | Issue 12 | e1004472

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