Characterization and regulation of the trehalose synthesis pathway and its importance in the pathogenicity of Cryptococcus neoformans

The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of Cryptococcus neoformans identified the presence of a functioning trehalose pathway during infection and suggested its importance for C. neoformans survival in the host. Therefore, in C. neoformans we created null mutants of the trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-6-phophate phosphatase (TPS2), and neutral trehalase (NTH1) genes. We found that both TPS1 and TPS2 are required for high-temperature (37 degrees C) growth and glycolysis but that the block at TPS2 results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the tps1 and tps2 mutants at 37 degrees C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in C. neoformans. The essential nature of this pathway for disease was confirmed when a tps1 mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate C. elegans, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the tps1 mutant, and this supports the hypothesis that the trehalose pathway in C. neoformans is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in C. neoformans and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.     

Trehalose hydrolysis is not required for human serum-induced dimorphic transition in Candida albicans: evidence from a tps1/tps1 mutant deficient in trehalose synthesis

Exponential yeast-like cells of a Candida albicans wild-type strain exhibited strong capacity for germ tube formation in a glucose-containing medium (YPD) after induction with human serum at 37 degrees C, whereas the isogenic double disruptant tps1/tps1 mutant, which is deficient in trehalose synthesis, failed to produce germ tubes. In a medium without glucose (YP), the morphological transition fraction was roughly equivalent in both strains. Substitution of glucose by galactose or glycerol increased the number of wild-type proliferating cells able to enter the dimorphic program with no noticeable change in their trehalose content, while stationary cells, which accumulate a large amount of trehalose, did not form germ tubes. When fresh medium was added, a high proportion of these resting cells recovered their ability to carry out dimorphic transition. The tps1/tps1 mutant followed the same pattern of hyphae formation, despite the fact that it was unable to accumulate trehalose either during dimorphism induction or after several stress challenges. Furthermore, trehalose-6-phosphate synthase activity was barely detectable in the mutant. These results strongly suggest that serum-induced dimorphic transition does not require trehalose mobilization; they also support the idea that TPS1 is the only activity involved in trehalose biosynthesis in C. albicans.     

Role of trehalose in resistance to macrophage killing: study with a tps1/tps1 trehalose-deficient mutant of Candida albicans

Accumulation of trehalose by yeast is an important protective mechanism against different stress conditions. This study examined the effect of trehalose on several growth features, as well as its association with the intracellular survival of yeasts exposed to macrophages. A tps1/tps1 mutant and its parental counterpart, CAI4, exhibited similar growth rates and preserved their dimorphic conversion and agglutination ability. However, electron-microscopy of cell-wall architecture showed a partial loss of material from the outer cell-wall layer in the tps1/tps1 mutant. Flow-cytometry revealed that the mutant had lower auto-fluorescence levels and a higher fluorescein isothiocynate staining efficiency. When co-cultured with macrophages, a slight reduction in binding to macrophages and slower ingestion kinetics were revealed for the tps1/tps1 mutant, but these did not interfere significantly with the amount of yeast ingested by macrophages after co-incubation for 2 h. Under the same conditions, CAI4 cells were more resistant to macrophage killing than was the tps1 null mutant, provided that the macrophages had been stimulated previously with interferon-gamma. Measurement of trehalose content and the anti-oxidant activities of yeast cells recovered after phagocytosis revealed that the trehalose content and the glutathione reductase activity were increased only in CAI4 cells, whereas levels of catalase activity were increased similarly in both strains. These results suggest that the presence of trehalose in Candida albicans is a contributory factor that protects the cell from injury caused by macrophages.     

Disruption of the Candida albicans TPS2 gene encoding trehalose-6-phosphate phosphatase decreases infectivity without affecting hypha formation

Deletion of trehalose-6-phosphate phosphatase, encoded by TPS2, in Saccharomyces cerevisiae results in accumulation of trehalose-6-phosphate (Tre6P) instead of trehalose under stress conditions. Since trehalose is an important stress protectant and Tre6P accumulation is toxic, we have investigated whether Tre6P phosphatase could be a useful target for antifungals in Candida albicans. We have cloned the C. albicans TPS2 (CaTPS2) gene and constructed heterozygous and homozygous deletion strains. As in S. cerevisiae, complete inactivation of Tre6P phosphatase in C. albicans results in 50-fold hyperaccumulation of Tre6P, thermosensitivity, and rapid death of the cells after a few hours at 44 degrees C. As opposed to inactivation of Tre6P synthase by deletion of CaTPS1, deletion of CaTPS2 does not affect hypha formation on a solid glucose-containing medium. In spite of this, virulence of the homozygous deletion mutant is strongly reduced in a mouse model of systemic infection. The pathogenicity of the heterozygous deletion mutant is similar to that of the wild-type strain. CaTPS2 is a new example of a gene not required for growth under standard conditions but required for pathogenicity in a host. Our results suggest that Tre6P phosphatase may serve as a potential target for antifungal drugs. Neither Tre6P phosphatase nor its substrate is present in mammals, and assay of the enzymes is simple and easily automated for high-throughput screening.     

Pseudohyphal differentiation defect due to mutations in GPCR and ammonium signaling is suppressed by low glucose concentration: a possible integrated role for carbon and nitrogen limitation

In response to carbon and/or nitrogen limitation, diploid cells of Saccharomyces cerevisiae either sporulate or develop pseudohyphae. Although the signal transduction pathways leading to these developmental changes have been extensively studied, how nutritional signals are integrated is not clearly understood. Results of this study indicate that reducing glucose concentration from 2% (SLAD) to 0.05% (SLALD) causes an increase in the magnitude of filamentation as well as a discernible reduction in the time required for pseudohyphal development. Further, the pseudohyphal defect of gpa2, gpr1and gpa2gpr1 but not the mep2 mutant strain is overcome on SLALD. Low glucose also induced pseudohyphae in mep2gpr1 but not mep2gpa2 strain suggesting that GPR1 inhibits pseudohyphae by inhibiting GPA2 function. Accordingly, deleting GPA2 in mep2gpr1 mutant abrogated pseudohyphae formation in SLALD. Further, replenishment of glucose suppressed pseudohyphal differentiation in wild-type cells grown in SLAD medium. However, in SLALD, glucose replenishment suppressed the filamentation response of gpa2 mutants but not that of strains carrying the wild-type GPA2. Increased trehalose levels correlated with decreased pseudohyphae formation. Results of this study demonstrate that filamentation in response to nitrogen limitation occurs as glucose becomes limiting.     

Titan Cell Production Enhances the Virulence of Cryptococcus neoformans

Infection with Cryptococcus neoformans begins when desiccated yeast cells or spores are inhaled and lodge in the alveoli of the lungs. A subset of cryptococcal cells in the lungs differentiate into enlarged cells, referred to as titan cells. Titan cells can be as large as 50 to 100 μm in diameter and exhibit a number of features that may affect interactions with host immune defenses. To characterize the effect of titan cell formation on the host-pathogen interaction, we utilized a previously described C. neoformans mutant, the gpr4Δ gpr5Δ mutant, which has minimal titan cell production in vivo. The gpr4Δ gpr5Δ mutant strain had attenuated virulence, a lower CFU, and reduced dissemination compared to the wild-type strain. Titan cell production by the wild-type strain also resulted in increased eosinophil accumulation and decreased phagocytosis in the lungs compared to those with the gpr4Δ gpr5Δ mutant strain. Phagocytosed cryptococcal cells exhibited less viability than nonphagocytosed cells, which potentially explains the reduced cell survival and overall attenuation of virulence in the absence of titan cells. These data show that titan cell formation is a novel virulence factor in C. neoformans that promotes establishment of the initial pulmonary infection and plays a key role in disease progression.     

Role of trehalose biosynthesis in environmental survival and virulence of Salmonella enterica serovar typhimurium

The otsA and otsB genes, encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase respectively, have been isolated from Salmonella enterica serovar typhimurium and nucleotide-sequenced. Induction of trehalose biosynthesis by exposure of bacteria to high osmotic strength resulted in the intracellular accumulation of trehalose. An otsA mutant of S. enterica serovar typhimurium was more susceptible to killing by heat, and grew poorly under conditions of high osmolarity. The wild-type and otsA mutant strains showed similar abilities to colonise spleen tissues after oral dosing of mice. These findings suggest that the otsBA gene products play a role in environmental survival, but not in virulence, of S. enterica serovar typhimurium.     

Role of Trehalose Biosynthesis in Aspergillus fumigatus Development,Stress Response,and Virulence

Aspergillus fumigatus is a pathogenic mold which causes invasive, often fatal, pulmonary disease in immunocompromised individuals. Recently, proteins involved in the biosynthesis of trehalose have been linked with virulence in other pathogenic fungi. We found that the trehalose content increased during the developmental life cycle of A. fumigatus, throughout which putative trehalose synthase genes tpsA and tpsB were significantly expressed. The trehalose content of A. fumigatus hyphae also increased after heat shock but not in response to other stressors. This increase in trehalose directly correlated with an increase in expression of tpsB but not tpsA. However, deletion of both tpsA and tpsB was required to block trehalose accumulation during development and heat shock. The ΔtpsAB double mutant had delayed germination at 37°C, suggesting a developmental defect. At 50°C, the majority of ΔtpsAB spores were found to be nonviable, and those that were viable had severely delayed germination, growth, and subsequent sporulation. ΔtpsAB spores were also susceptible to oxidative stress. Surprisingly, the ΔtpsAB double mutant was hypervirulent in a murine model of invasive aspergillosis, and this increased virulence was associated with alterations in the cell wall and resistance to macrophage phagocytosis. Thus, while trehalose biosynthesis is required for a number of biological processes that both promote and inhibit virulence, in A. fumigatus the predominant effect is a reduction in pathogenicity. This finding contrasts sharply with those for other fungi, in which trehalose biosynthesis acts to enhance virulence.Aspergillus fumigatus is a ubiquitous thermotolerant mold which plays an important role in the recycling of environmental carbon and nitrogen (32, 38). A. fumigatus is also an important opportunistic human pathogen that invades the lungs of immunocompromised patients, causing a progressive pneumonia that can disseminate to the heart, brain, and other organs (21). Invasive aspergillosis is a leading cause of death in transplant and leukemic patients, with a mortality rate exceeding 50% despite the best available antifungal therapy (19). This poor response to existing antifungal therapy has led to a great interest in novel therapeutic approaches directed against this organism.In both the environment and the host, A. fumigatus is exposed to a variety of stressors. While growing in compost, A. fumigatus is commonly found at temperatures exceeding 50°C (5). Similarly, in human tissues, hyphae are subjected to nutrient deprivation and oxidative stress from host immune cells (24, 31). Little is known about the mechanisms by which A. fumigatus survives under these conditions. One possible mechanism by which A. fumigatus adapts to environmental stress is via the biosynthesis of the carbohydrate trehalose, which is involved in mediating the stress response and virulence of other pathogenic fungi such as Candida albicans and Cryptococcus neoformans (1, 22, 30, 48). The trehalose content of these fungal cells may increase up to 50-fold in response to oxidative stress, osmotic stress, and heat shock (27).Trehalose is a nonreducing disaccharide of glucose that is synthesized by bacteria, plants, insects, and fungi. In fungi, trehalose functions both as a reserve carbohydrate and as a stress metabolite (37, 39, 40). As a reserve carbohydrate, trehalose is found in vegetative resting cells and spores, where it can constitute up to 15% of the dry weight of these structures (46). It is an important source of energy in fungal development, as it is utilized in cell processes such as glycolysis, sporulation, and germination. In addition, trehalose helps the cell withstand environmental stress and nutrient limitation. Trehalose molecules protect the cell by preventing aggregation of denatured proteins and scavenging free radicals (37). Interestingly, trehalose is absent from mammalian cells, and therefore enzymes involved in trehalose biosynthesis have been considered as potential targets for antifungal therapy.In medically relevant fungi, trehalose is synthesized from glucose. First, the enzyme hexokinase converts a molecule of glucose into glucose-6-phosphate. Then, trehalose-6-phosphate synthase (Tps) catalyzes the production of trehalose-6-phosphate (T6P) from glucose-6-phosphate and a molecule of UDP-glucose. Finally, a phosphate group is removed from trehalose-6-phosphate by trehalose-6-phosphate phosphatase (Tpp) to yield trehalose (29, 42). When trehalose is required for energy, it can be recycled back to glucose through the universal enzyme trehalase (20). The enzymes involved in fungal trehalose biosynthesis have been well characterized in Saccharomyces cerevisiae. These proteins are encoded by four genes: TPS1, which encodes trehalose-6-phosphate synthase; TPS2, which encodes trehalose-6-phosphate phosphatase; and TPS3 and TSL1, redundant genes which encode a large regulatory subunit of the synthase complex (6, 15, 28, 29, 42, 44). All four proteins are tightly regulated both at the genetic level and at the protein level, and they form a protein complex to catalyze the synthesis of trehalose (6, 45).In other fungi, trehalose biosynthesis appears to play similar but not identical roles in governing growth, stress response, and virulence. In the pathogenic yeasts C. albicans and C. neoformans, the single-copy trehalose-6-phosphate synthase Tps1 is required for normal oxidative stress response and hyphal development and has a role in mediating virulence (1, 22, 30, 48). In Aspergillus nidulans, the trehalose-6-phosphate synthase TpsA is necessary for normal fungal development, thermosensitive growth and response to sublethal exposure to oxidative stress (16). In Aspergillus niger, two putative trehalose-6-phosphate synthases, TpsA and TpsB, have been identified. Inactivation of tpsA resulted in a reduction in T6P activity; however, the role of TpsA and TpsB in trehalose metabolism has not been examined (47). Importantly, trehalose biosynthesis has not been studied in A. fumigatus, which is the most important filamentous fungus causing human disease.We undertook the study of the role of trehalose biosynthesis in A. fumigatus development, stress response, and virulence. We found that the trehalose content increased in A. fumigatus hyphae throughout development and when actively growing hyphae were exposed to heat shock, but not when they were exposed to other environmental stresses. Increases in trehalose content directly correlated with elevated expression levels of two putative trehalose-6-phosphate synthase genes, tpsA and tpsB. Disruption of both tpsA and tpsB was required to prevent trehalose biosynthesis. Abolition of trehalose biosynthesis via disruption of both tpsA and tpsB affected conidial germination, thermotolerance, and high-level oxidative stress response in vitro. Surprisingly, disruption of tpsA and tpsB resulted in a strain that was hypervirulent by several measures in a murine model of invasive aspergillosis.     

Defective Hyphal induction of a Candida albicans phosphatidylinositol 3-phosphate 5-kinase null mutant on solid media does not lead to decreased virulence

A phosphatidylinositol 3-phosphate [PI(3)P] 5-kinase gene (CaFAB1) of the most important human pathogenic yeast, Candida albicans, was cloned and sequenced. An open reading frame was detected which encodes a 2,369-amino-acid protein with a calculated molecular mass of 268 kDa and a relative isoelectric point of 6.76. This protein exhibits 38% overall amino acid sequence identity with Saccharomyces cerevisiae Fab1p. We localized the CaFAB1 gene on chromosome R. To determine the influence of the PI(3)P 5-kinase CaFab1p on processes involved in C. albicans morphogenesis and pathogenicity, we sequentially disrupted both copies of the gene. Homozygous deletion of C. albicans CaFAB1 resulted in a mutant strain which exhibited defects in morphogenesis. A Cafab1 null mutant had enlarged vacuoles, an acidification defect, and increased generation times and was unable to form hyphae on different solid media. The sensitivities to hyperosmotic and high-temperature stresses, adherence, and virulence compared to those of wild-type strain SC5314 were not affected.     

Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis

The GGS1/TPS1 gene of the yeast Saccharomyces cerevisiae encodes the trehalose-6-phosphate synthase subunit of the trehalose synthase complex. Mutants defective in GGS1/TPS1 have been isolated repeatedly and they showed variable pleiotropic phenotypes, in particular with respect to trehalose content, ability to grow on fermentable sugars, glucose-induced signaling and sporulation capacity. We have introduced the fdp1, cif1, byp1 and glc6 alleles and the ggs1/tps1 deletion into three different wild-type strains, M5, SP1 and W303-1A. This set of strains will aid further studies on the molecular basis of the complex pleiotropic phenotypes of ggs1/tps1 mutants. The phenotypes conferred by specific alleles were clearly dependent on the genetic background and also differed for some of the alleles. Our results show that the lethality caused by single gene deletion in one genetic background can become undetectable in another background. The sporulation defect of ggs1/tps1 diploids was neither due to a deficiency in G₁ arrest, nor to the inability to accumulate trehalose. Ggs1/tps1 mutants were very sensitive to glucose and fructose, even in the presence of a 100-fold higher galactose concentration. Fifty-percent inhibition occurred at concentrations similar to the K_m values of glucose and fructose transport. The inhibitory effect of glucose in the presence of a large excess of galactose argues against an overactive glycolytic flux as the cause of the growth defect. Deletion of genes of the glucose carrier family shifted the 50% growth inhibition to higher sugar concentrations. This finding allows for a novel approach to estimate the relevance of the many putative glucose carrier genes in S. cerevisiae. We also show that the GGS1/TPS1 gene product is not only required for the transition from respirative to fermentative metabolism but continuously during logarithmic growth on glucose, in spite of the absence of trehalose under such conditions.     

The stringent response is essential for Pseudomonas aeruginosa virulence in the rat lung agar bead and Drosophila melanogaster feeding models of infection

The stringent response is a regulatory system that allows bacteria to sense and adapt to nutrient-poor environments. The central mediator of the stringent response is the molecule guanosine 3',5'-bispyrophosphate (ppGpp), which is synthesized by the enzymes RelA and SpoT and which is also degraded by SpoT. Our laboratory previously demonstrated that a relA mutant of Pseudomonas aeruginosa, the principal cause of lung infections in cystic fibrosis patients, was attenuated in virulence in a Drosophila melanogaster feeding model of infection. In this study, we examined the role of spoT in P. aeruginosa virulence. We generated an insertion mutation in spoT within the previously constructed relA mutant, thereby producing a ppGpp-devoid strain. The relA spoT double mutant was unable to establish a chronic infection in D. melanogaster and was also avirulent in the rat lung agar bead model of infection, a model in which the relA mutant is fully virulent. Synthesis of the virulence determinants pyocyanin, elastase, protease, and siderophores was impaired in the relA spoT double mutant. This mutant was also defective in swarming and twitching, but not in swimming motility. The relA spoT mutant and, to a lesser extent, the relA mutant were less able to withstand stresses such as heat shock and oxidative stress than the wild-type strain PAO1, which may partially account for the inability of the relA spoT mutant to successfully colonize the rat lung. Our results indicate that the stringent response, and SpoT in particular, is a crucial regulator of virulence processes in P. aeruginosa.     

Role of sigma E-regulated genes in Escherichia coli uropathogenesis

The sigma E regulon encodes proteins for maintenance and repair of the Escherichia coli cell envelope. Previously, we observed that an antirepressor of sigma E, DegS, is essential for uropathogenic E. coli virulence. Here we use a mouse urinary tract infection model to assay the virulence of mutants of E. coli genes described as sigma E dependent. Deletion mutants of candidate genes were made in the uropathogenic E. coli strain CFT073. Swiss Webster female mice were inoculated with a mixture of mutant and wild-type strains. Bladder and kidney homogenates were cultured 2 days after infection, and CFU of the wild type and mutant were compared. Eleven mutants were assayed, and two, CFT073 degP and CFT073 skp, showed significantly diminished survival compared to wild type. DegP is a chaperone and degradase active in the periplasm. Skp is also a periplasmic chaperone. The virulence of the skp deletion mutant could not be restored by complementation with skp. The virulence of the degP deletion mutant, in contrast, could be restored. However, complementation with a degP allele encoding a serine-to-alanine (S210A) mutation at the protease active site fails to restore virulence. Unlike degP mutants in other bacteria, the E. coli degP mutant is tolerant of oxidative stress. It disappears abruptly from bladder and kidney cultures between 6 and 12 hours after inoculation. A mutant of degQ, a close homolog of degP, was not attenuated in mice. This is the first report that the DegP degradase is an E. coli virulence factor in an animal infection model.     

Attenuation of Virulence and Changes in Morphology in Candida albicans by Disruption of the N-Acetylglucosamine Catabolic Pathway

A Candida albicans mutant with mutations in the N-acetylglucosamine (GlcNAc) catabolic pathway gene cluster, including the GlcNAc-6-phosphate deacetylase (DAC1), glucosamine-6-phosphate deaminase (NAG1), and GlcNAc kinase (HXK1) genes, was not able to grow on amino sugars, exhibited highly attenuated virulence in a murine systemic candidiasis model, and was less adherent to human buccal epithelial cells in vitro. No germ tubes were formed by the mutant after induction with GlcNAc, but the mutant exhibited hyperfilamentation under stress-induced filamentation conditions. In addition, the GlcNAc catabolic pathway played a vital role in determining the colony phenotype. Our results imply that this pathway is very important because of its diverse links with pathways involved in virulence and morphogenesis of the organism.     

Relative contributions of hyaluronic acid capsule and M protein to virulence in a mucoid strain of the group A Streptococcus.

The antiphagocytic effect of M protein has been considered a critical element in virulence of the group A streptococcus. The hyaluronic acid capsule also appears to play an important role: studies of an acapsular mutant derived from the mucoid or highly encapsulated M protein type 18 group A streptococcal strain 282 indicated that loss of capsule expression was associated with decreased resistance to phagocytic killing and with reduced virulence in mice. To study directly the relative contributions to virulence of M protein and the hyaluronic acid capsule in strain 282, we inactivated the gene encoding the M protein (emm18) both in wild-type strain 282 and in its acapsular mutant, strain TX72. Inactivation of emm18 was accomplished by integrational plasmid mutagenesis, using the temperature-sensitive shuttle vector pJRS233 harboring a 5' DNA segment of emm18. As reported previously, wild-type strain 282 was resistant to phagocytic killing in vitro, both in whole human blood and in 10% serum. The capsule mutant TX72 was highly susceptible to phagocytic killing in 10% serum and moderately sensitive in whole blood. The M protein mutant 282KZ was highly susceptible to phagocytic killing in blood but only moderately sensitive in 10% serum. The double mutant TX74 was sensitive to killing in both conditions. In a mouse infection model, the 50% lethal dose was increased by 60- and 80-fold for the capsule and double mutants, respectively, compared with that of strain 282, but only by 6-fold for the M protein mutant. Integration of the strain 282 capsule genes into the chromosome of a nonmucoid M1 strain resulted in high-level capsule production and rendered the transformed strain resistant to phagocytic killing in 10% serum. These results provide further evidence that the hyaluronic acid capsule confers resistance to phagocytosis and enhances group A streptococcal virulence. The results suggest also that assessment of in vitro resistance to phagocytosis in 10% serum rather than in whole blood may be a more accurate reflection of virulence in vivo of group A streptococci.     

Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection

Cell-to-cell signaling controls many virulence genes in Pseudomonas aeruginosa. We tested the virulence of las and rhl quorum-sensing mutants in neonatal mice. A lasI rhlI double mutant was nearly avirulent, and the respective single mutant strains were reduced in virulence compared with the wild-type strain. Quorum sensing plays a role in P. aeruginosa pneumonia in neonatal mice.     

Mutations in the cueA gene encoding a copper homeostasis P-type ATPase reduce the pathogenicity of Pseudomonas aeruginosa in mice

A sequencing project identified a putative copper homeostasis gene, cueA, in Pseudomonas aeruginosa strain PAO1. Strains with mutations of the cueA gene, encoding a P-type ATPase linked to copper homeostasis in P. putida, displayed greater sensitivity to copper compared to wild-type bacteria using MIC determinations and in vitro passage in growth media with different concentrations of copper added. An LD50 assay showed a cueA deletion mutant was 50-fold more attenuated than wild-type strain PAO1 bacteria. Complementation of the cueA mutation restored in vitro tolerance to copper and virulence in a systemic model of infection to near wild-type levels. Competition assays between cueA mutants and wild-type P. aeruginosa strains demonstrated 20-fold attenuation by the cueA mutants within spleens of mice. This data suggests the P. aeruginosa CueA protein may be important in maintaining copper homeostasis both in vitro and in vivo.