ABSTRACT
l-Serine is a nonessential amino acid and a key intermediate in several relevant metabolic pathways. In bacteria, the major source of l-serine is the phosphorylated pathway, which comprises three enzymes: d-3-phosphoglycerate dehydrogenase (PGDH; SerA), phosphoserine amino transferase (PSAT; SerC), and l-phosphoserine phosphatase (PSP; SerB). The Brucella abortus genome encodes two PGDHs (SerA-1 and SerA-2), involved in the first step in l-serine biosynthesis, and one PSAT and one PSP, responsible for the second and third steps, respectively. In this study, we demonstrate that the serA1 serA2 double mutant and the serC and serB single mutants are auxotrophic for l-serine. These auxotrophic mutants can be internalized but are unable to replicate in HeLa cells and in J774A.1 macrophage-like cells. Replication defects of auxotrophic mutants can be reverted by cell medium supplementation with l-serine at early times postinfection. In addition, the serB mutant is attenuated in the murine intraperitoneal infection model and has an altered lipid composition, since the lack of l-serine abrogates phosphatidylethanolamine synthesis in this strain. Taken together, these results reveal that limited availability of l-serine within the host cell impairs proliferation of the auxotrophic strains, highlighting the relevance of this biosynthetic pathway in Brucella pathogenicity.
INTRODUCTION
Bacteria of the genus Brucella are alpha-2-proteobacteria that cause brucellosis, a chronic infectious disease affecting diverse species of mammals and humans. Brucella infection causes sterility and abortion in animals and undulating fever and debilitating disorders in humans. Brucellosis remains endemic in many countries, resulting in serious public health problems and economic losses worldwide (1).
The ability to replicate in a wide range of mammalian cell types, including endothelial cells, fibroblasts, epithelial cells, and microglia, is essential to Brucella pathogenesis (2). The pathogen primarily infects and replicates inside phagocytic cells such as macrophages and dendritic cells before disseminating to placental trophoblasts, the reproductive tract, and the mononuclear phagocyte system, where it persists to establish a chronic infection in the host (3, 4).
After internalization into host cells, Brucella resides in a membrane-bound compartment known as the Brucella-containing vacuole (BCV). BCVs traffic along the endocytic and secretory pathways, allowing the bacterium to evade killing in phagolysosomes and to replicate in an endoplasmic reticulum (ER)-derived compartment (5, 6). Afterward, BCVs mature into compartments with autophagic features which are required for cell-to-cell spreading (7).
To date, efforts to characterize Brucella pathogenesis mechanisms have been focused on “classical” virulence determinants such as the type IV secretion system and its effectors (8–11), lipopolysaccharide (12), cyclic β-1,2-glucan (13, 14), the BvrS/BvrR two-component system (15), autotransporters and adhesins (16), and transcriptional regulators (17, 18). These virulence factors participate in key aspects of Brucella pathogenesis, such as host cell adhesion and internalization, intracellular replication, and innate immune evasion (19).
Recently, bacterial nutrition and metabolism during infection have emerged as new research topics in bacterial pathogenesis (20–22). A recent example is illustrated by Brucella abortus glutamate dehydrogenase (GdhZ), which plays an essential role during intracellular replication, since GdhZ constitutes an entry point into the tricarboxylic acid cycle (TCA) for several amino acids. These results emphasize the importance of amino acids as a main carbon source during the Brucella intracellular phase (23).
Even though it has been widely accepted that BCVs are nutritionally poor (24, 25), their exact nutrient composition is unknown. About 20 years ago, random-scale mutagenesis allowed identification of attenuated mutants in genes coding for transport and catabolism of carbohydrates, as well as genes encoding peptides and amino acid transporters, suggesting that they could be used by the bacterium as sources of carbon and energy during infection (25–31). Later, proteomics studies demonstrated reduced key metabolic pathways early after infection, such as TCA, pyruvate, and pentose phosphate shunt cycles, as well as sugar uptake systems (32). In contrast, enzymes involved in catabolism of amino acid and proteins are increased, suggesting that Brucella may obtain precursors for the TCA cycle from amino acids, such as glutamate, during early infection. Altogether, these studies emphasize the ability of Brucella spp. to adjust their metabolism to the intracellular conditions encountered at each stage of the infection process.
In contrast to what is described for many intracellular pathogenic bacteria, which have reduced their genomes after long periods of coevolution with their hosts (33), bacteria of the genus Brucella are prototrophic for all amino acids (34, 35). Amino acid biosynthesis has become relevant in Brucella virulence after identification of attenuated mutants in genes coding for enzymes involved in these pathways, suggesting that BCVs are poor in amino acids (27, 28, 36–38).
l-Serine biosynthesis is a major anabolic pathway in most organisms. Although commonly classified as a nonessential amino acid, l-serine plays essential roles as a precursor for glycine, cysteine, tryptophan, phosphatidyl l-serine, sphingolipids, porphyrins, purines, glyoxalate, and glycine (39). Also, as the precursor to glycine, l-serine is the major source of one-carbon units that serve as the donors in methylation reactions mediated by derivatives of tetrahydrofolate and S-adenosylmethionine (40). Conversion of l-serine to glycine is a reversible reaction catalyzed by serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) (41). Nonetheless, the major route for l-serine biosynthesis in bacteria is the phosphorylated pathway (Fig. 1A), which comprises three sequential steps catalyzed by 3-phosphoglycerate dehydrogenase (PGDH/SerA; EC 1.1.1.95), phosphoserine aminotransferase (PSAT/SerC; EC 2.6.1.52), and phosphoserine phosphatase (PSP/SerB; EC 3.1.3.3).
l-Serine auxotrophy in B. abortus. (A) Schematic representation of l-serine biosynthetic pathway in B. abortus. SerA-1/SerA-2, 3-phosphoglycerate dehydrogenase (EC 1.1.1.95); SerC, 3-phosphoserine aminotransferase (EC 2.6.1.52); SerB, phosphoserine phosphatase (EC 3.1.3.3). (B and D) Growth kinetics of B. abortus wild type (S2308) and the indicated mutant strains in GW medium. Overnight cultures of bacteria grown in TSB were pelleted, washed, and resuspended in fresh GW medium in the presence or absence of 10 mM l-serine. Growth was monitored by measuring the turbidity (OD600) at different times. (C and E) Growth kinetics of B. abortus wild type (S2308), auxotrophic mutants for l-serine, and complemented strains in GW medium without l-serine. Four independent experiments were performed in duplicates for each growth curve. The data are means ± the standard deviations (SD; error bars are within the size of the symbols) of a representative experiment performed in duplicate.
Given its central role in metabolism, we sought to characterize and determine the importance of the l-serine biosynthesis pathway in B. abortus. In the present study, three deletion mutants auxotrophic for l-serine were obtained and characterized in vitro and in vivo. All three auxotrophic mutants failed to replicate intracellularly, and one of them proved to be attenuated in mice in the acute and chronic phases of the infection. These results indicate that during intracellular stages, B. abortus depends on the biosynthesis of l-serine via the phosphorylated pathway to sustain its proliferation.
RESULTS
Generation and characterization of l-serine auxotrophic mutants.In the phosphorylated biosynthetic pathway, l-serine is derived from the glycolytic intermediate 3-phospho-d-glycerate (PGA) in a three-step reaction (Fig. 1A). The first enzyme is d-3-phosphoglycerate dehydrogenase (PGDH), which converts PGA to phosphohydroxypyruvate (PHP) with the concomitant reduction of NAD+ to NADH. The B. abortus genome encodes two isoforms of PGDH/SerA: SerA-1 (BAB1_1697) and SerA-2 (BAB2_0783). Both PGDHs are 38% identical and comprise the catalytic domain and a C-terminal allosteric substrate binding (ASB) domain in SerA-1 or an aspartate kinase–chorismate mutase–TyrA domain (ACT) at the C terminus of SerA-2.
In order to obtain l-serine auxotrophic mutants, genes coding for PGDHs were deleted to produce single (serA1 and serA2) and double (serA1 serA2) mutant strains. Unmarked gene deletion of serA1 or serA2 had no significant effect on vegetative growth in tryptic soy broth (TSB) (not shown) or in the gluconeogenic medium Gerhardt-Wilson (GW) without l-serine supplementation (Fig. 1B), demonstrating that the effect of the single mutations can be compensated by the presence of the second gene that encodes a functional PGDH. On the contrary, the serA1 serA2 double mutant required l-serine supplementation for growth in minimal medium (Fig. 1B), indicating that l-serine biosynthesis is abrogated in this strain. At this point, it is worth mentioning that 10 mM l-serine supplementation to the growth medium TSB or GW does not confer any advantage in vegetative growth to B. abortus wild-type strain S2308 (see Fig. S1A and B in the supplemental material). Genetic complementation of the double mutant with plasmids encoding SerA-1 and/or SerA-2 partially restored wild-type growth in minimal medium without l-serine (Fig. 1C), probably due to plasmid instabilities in these strains. These results suggest that SerA-1 and SerA-2 are involved in the first step of l-serine biosynthesis in B. abortus.
In the following steps, phosphoserine aminotransferase (henceforth SerC; BAB1_1699) converts PHP to l-phosphoserine (PS) with the concomitant conversion of glutamate to α-ketoglutarate, followed by the conversion of PS to l-serine by phosphoserine phosphatase (SerB; BAB1_1410) (Fig. 1A). Unmarked gene deletion of serC or serB had no significant effect on vegetative growth in TSB (data not shown). As expected, these mutants required exogenous l-serine (10 mM) for growth in GW medium (Fig. 1D). The growth defect of these auxotrophic mutants could be fully rescued by complementation with plasmids coding for the corresponding enzymes (Fig. 1E).
These results indicate that when grown in a gluconeogenic medium such as GW, where the only carbon and nitrogen sources are glycerol and glutamate, B. abortus depends on the biosynthesis of l-serine for growth. In addition, the ability to rescue the growth defect of the auxotrophs upon addition of l-serine to the media suggests that B. abortus can incorporate this amino acid through an transport system not yet identified.
l-Serine biosynthesis is necessary for B. abortus intracellular replication.Brucella can adhere, invade, and proliferate inside different cell types, including both professional and nonprofessional phagocytic cells. To assess the impact of l-serine auxotrophy on the intracellular lifestyle of B. abortus, we evaluated the intracellular replication of the wild-type S2308, as well as serA1 serA2, serC, and serB mutants, and the corresponding complemented strains in HeLa and J774A.1 cell lines, two widely used infection models of nonprofessional and professional phagocytic cells. Genetic complementation of the double mutant with only one of the genes did not fully restore intracellular replication levels in HeLa and J774A.1 cells (not shown). For this reason, the serA1 serA2 mutant was complemented with both genes in these experiments. As shown in Fig. 2, all mutant strains showed no differences compared to the wild type at 4 h postinfection (p.i.) but failed to replicate in both cell types, with 1- to 4-log decreases in CFU counts compared to S2308 at 24 or 48 h p.i. Genetic complementation of the mutants with plasmids encoding the corresponding enzymes restored intracellular replication of the mutants with the exception of the serC mutant at 24 h p.i. in both cell types (Fig. 2, green bars). Likewise, the addition of 10 mM l-serine to the cell medium RPMI 1640 at the beginning of the infection process rescued the intracellular growth defect of l-serine auxotrophic mutants (Fig. 2, blue bars) but had no effect on intracellular replication of wild-type B. abortus (Fig. S1C and D). These results demonstrate that B. abortus requires biosynthesis of l-serine to replicate intracellularly and that exogenous l-serine can be incorporated into the BCVs to rescue the replication defect of the auxotrophs. Since the three auxotrophic mutants exhibited similar phenotypes in cells, we decided to continue the characterization only with the serB mutant in the following experiments.
l-Serine biosynthesis is required for B. abortus intracellular replication. Intracellular replication of the indicated strains in HeLa cells (A, C, and E) and J774A.1 macrophage-like cells (B, D, and F) was assessed. CFU were enumerated at 4, 24, and 48 h p.i. Where indicated, the cell medium was supplemented with 10 mM l-serine at the beginning of the infection. The data are means ± the SD of a representative experiment performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
In order to further characterize l-serine requirements of intracellular B. abortus, the host cell medium was supplemented with the amino acid at 6, 10, and 24 h p.i. Interestingly, supplementation of cell medium with l-serine can only restore intracellular proliferation of the serB mutant in HeLa and J774A.1 macrophage-like cells if added before 24 h p.i. (Fig. 3). These results agree with our previous results demonstrating that B. abortus cannot use host cell l-serine to sustain the intracellular replication phase and therefore depends on de novo biosynthesis. The results also highlight the importance of l-serine availability during the first stages of infection in order to successfully proliferate within cells. The fact that supplementation of the cell culture medium with l-serine restores the intracellular growth of the mutant indicates that B. abortus can transport the amino acid through both the BCV and the bacterium membranes.
l-Serine supplementation rescues B. abortus serB intracellular replication during early infection phase. Intracellular replication of the indicated strains in HeLa cells (A) and in J774A.1 macrophages (B) was assessed. Where indicated, the medium was supplemented with 10 mM l-serine at the indicated times p.i. The data are means ± the SD of a representative experiment performed in triplicate. ***, P < 0.001.
Since intracellular levels of l-serine cannot support proliferation of auxotrophic mutants, we sought to determine the minimal concentration required to rescue the replication defect of the serB mutant by infecting HeLa cells and adding increasing concentrations of l-serine at the beginning of the in vitro infection assay. As shown in Fig. 4, there is a minimum threshold of l-serine concentration capable of rescuing the intracellular replication defect of the serB mutant. At least 1.6 mM l-serine needs to be added to the RPMI 1640 cell medium (containing 0.28 to 0.4 mM l-serine) to bypass the serB mutant proliferation defect. These results confirm that Brucella can incorporate l-serine through both the bacterial cell envelope and the BCV membrane to support intracellular replication.
Determination of the minimal concentration of l-serine necessary to rescue the B. abortus serB mutant intracellular replication defect. The intracellular replication of the indicated strains in HeLa cells was assessed. The cell medium was supplemented with the indicated concentrations of l-serine at the beginning of the infection. The data are means ± the SD of a representative experiment performed in triplicate. ***, P < 0.001.
Abrogation of l-serine biosynthesis impairs the biogenesis of replicative vacuoles in phagocytic cells.Once internalized, Brucella resides within a membrane-bound vacuole, the Brucella-containing vacuole (BCV), which undergoes remodeling from a compartment with endosomal/lysosomal features into an organelle derived from the host ER that supports bacterial replication (rBCV) (42–45). Given that B. abortus l-serine auxotrophic mutants displayed intracellular replication defects, we investigated the biogenesis of the rBCV in HeLa cells infected with the serB mutant. We first quantified acquisition and exclusion of the endosomal/lysosomal marker LAMP-1 at 4 and 24 h p.i., respectively. At 4 h p.i. 64.59% ± 6.06% of wild-type BCVs were positive for LAMP-1, compared to 67.23% ± 4.57% for serB mutant BCVs. At 24 h p.i., serB mutant BCVs excluded the lysosomal marker and only 31.20% ± 3.65% remained positive for LAMP-1, compared to 26.95% ± 5.16% of wild-type BCVs (Fig. S2A). The addition of 10 mM l-serine to the cell medium had no effect on LAMP-1 labeling of BCVs at 4 and 24 h p.i. To further characterize serB mutant intracellular traffic, we quantified acquisition of calnexin, an ER-resident protein. By 24 h p.i., 71.37% ± 5.13% of the wild-type BCVs and 69.95% ± 4.73% of the serB mutant BCVs were positive for calnexin (Fig. S2B).
Detailed inspection and enumeration of intracellular bacteria by immunofluorescence microscopy of infected HeLa cells at 24 h p.i. revealed that cells infected with wild-type B. abortus show foci of intracellular replication containing more than seven bacteria per cell, whereas no signs of intracellular replication were observed in cells infected with the serB mutant, with 98.53% of the cells with none or fewer than four bacteria (Fig. 5A). At 48 h p.i., the cells infected with the serB mutant remained free of intracellular replication foci, with 88.43% ± 1.31% of cells without bacteria and only 11.25% ± 1.47% with fewer than four bacteria (Fig. 5B). As expected, supplementation of the cell culture medium with 10 mM l-serine restored the replication defect of the auxotrophic mutant, since 2.34% ± 0.04% of cells presented intracellular foci with more than seven bacteria. These results are consistent with the intracellular replication curves (Fig. 2E) and are illustrated by representative confocal images of infected HeLa cells at 48 h p.i. (Fig. 5C). These images show that replicative foci are found in cells infected with the wild type but are barely detectable in those infected with the serB mutant. As expected, supplementation with 10 mM l-serine restored the intracellular proliferation of the mutant, as judged by the higher number of replicative foci (Fig. 5C). The images also illustrate calnexin association with BCVs in both strains. Together, these results indicate that B. abortus l-serine auxotrophs are competent to promote the biogenesis of rBCVs in HeLa cells but are unable to replicate within them.
The B. abortus serB mutant fails to establish intracellular replication foci. Intracellular bacteria in HeLa cells infected with the indicated strains at 24 h (A) and 48 h (B) p.i. were enumerated. Where indicated, the cell medium was supplemented with 10 mM l-serine at the beginning of the infection. The data are means ± the SD of a representative experiment performed in duplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Representative confocal micrographs of HeLa cells infected with the indicated strains at 48 h p.i. HeLa cells were labeled for calnexin (green) or Brucella (red) as described in Materials and Methods. Arrowheads indicate replication foci, and insets show BCVs colocalizing with calnexin (arrows).
To assess whether the same is true in professional phagocytic cells, LAMP-1 acquisition and exclusion were evaluated in infected J774A.1 macrophages. At 4 h p.i., 71.31% ± 4.84% of wild-type BCVs were decorated with LAMP-1, compared to 62.468% ± 3.49% for serB mutant BCVs. At 24 and 48 h p.i, wild-type BCVs were able to progressively exclude this lysosomal marker, with 30.27% ± 0.39% and 19.5% ± 3.5% of LAMP-1-positive vacuoles, respectively. In contrast, serB mutant BCVs remained positive for this marker at 24 and 48 h p.i., with 51.35% ± 5.63% and 55.50% ± 6.36% of LAMP-1-positive BCVs, respectively (Fig. 6A). Confocal microscopy images illustrate B. abortus wild-type and serB mutant strains inside J774A.1 macrophages at 48 h p.i. (Fig. 6B). In agreement with the intracellular replication curves (Fig. 2F), the serB mutant fails to replicate inside these cells and is decorated with LAMP-1. In addition, serB mutant lysosomal degradation inside these cells is evidenced by small puncta labeled with total anti-Brucella antibodies (Fig. 6B, smaller arrowheads). In contrast to what was observed in nonphagocytic HeLa cells, inside a competent phagocytic cell such as J774.A1 macrophages, the serB serine auxotroph could not complete the biogenesis of the rBCV, remaining mostly in LAMP-1-positive and calnexin-negative compartments, where it was eventually degraded.
serB is necessary for LAMP-1 exclusion from BCVs in phagocytic cells. (A) Quantification of LAMP-1 acquisition and exclusion by BCVs of wild-type and serB mutant strains at 4, 24, and 48 h postinfection in J774A.1 macrophages. Bars represent means ± the SD of a representative experiment performed in duplicate. **, P < 0.01; ***, P < 0.001. (B) Representative confocal micrographs of J774A.1 macrophages infected with the indicated strains at 48 h p.i. The cells were labeled for LAMP-1 (red) and Brucella (green) as described in Materials and Methods. Arrowheads indicate LAMP-1 and Brucella colocalization for the serB mutant, and smaller arrowheads show puncta/dots suggestive of bacterial lysosomal degradation.
l-Serine auxotrophy alters B. abortus membrane lipid composition.The Brucella cell envelope is composed of phosphatidylethanolamine (PE), phosphatidylcholine (PC), ornithine lipid (OL), cardiolipin (CL), and phosphatidylglycerol (PG) (46, 47). In B. abortus, PE is synthesized by the phosphatidylserine synthase pathway (Fig. 7A). The first reaction requires the condensation of l-serine with CDP-diacylglycerol catalyzed by the phosphatidylserine synthase (PssA) to produce phosphatidylserine, which is quickly decarboxylated by the phosphatidylserine decarboxylase (Psd) to produce PE (46). In order to assess whether l-serine auxotrophy affects the membrane lipid composition, B. abortus wild-type and serB mutant strains were grown in GW medium supplemented with choline, and the total lipids were extracted and analyzed by thin-layer chromatography and revealed by acid charring (total lipids) and ninhydrin staining for amino lipids. As expected, the spot corresponding to PE was absent when the mutant was grown in a defined medium without l-serine. This defect can be circumvented by supplementing the medium with 10 mM l-serine (Fig. 7B). The lack of PE in the cell envelope was compensated for by increasing the amount of OL. These findings indicate that impairment of l-serine biosynthesis abrogates PE formation, which impacts the lipid membrane composition, suggesting that B. abortus l-serine auxotrophs depend on exogenous l-serine to form PE, one of the major membrane phospholipids.
PE synthesis is abrogated in the B. abortus serB mutant. (A) Schematic representation of PE biosynthetic pathway in B. abortus. PssA, phosphatidylserine synthase; Psd, phosphatidylserine decarboxylase. B. abortus strains were grown in minimum medium with choline, and 10 mM l-serine was added to the medium where indicated. Total lipids were isolated and separated by thin-layer chromatography analysis. Lipid spots corresponding to phosphatidylglycerol (PG), ornithine lipid (OL), phosphatidylethanolamine (PE), cardiolipin (CL), and phosphatidylcholine (PC) are indicated. Total lipids were visualized by sulfuric acid charring (B) and amino lipids were visualized by ninhydrin staining (C).
Since the phospholipid composition in the bacterial cell envelope is critical for the interaction with the host, we analyzed whether the lack of PE in the serB mutant increased its susceptibility to acidic pH conditions or lysosome killing. First, we evaluated the sensitivity of the bacteria to low-pH conditions by incubating S2308 and serB and pssA mutant strains in phosphate-buffered saline (PBS; pH 4) for 4 h. No differences were detected in the number of viable bacteria recovered after 4 h of incubation in this acidic pH (Fig. S3A). The pssA mutant strain was included as a control, since it cannot synthesize PE, but the l-serine biosynthetic pathway remains intact (46). In addition, we evaluated whether the lack of PE synthesis increases serB mutant sensitivity to lysosomal proteolytic activity. To achieve this, microsomal fractions obtained from J774A.1 macrophages were incubated with S2308, the serB and pssA mutant strains, and the serB complemented mutant. After 8 h of incubation with the microsomal fractions, no differences in sensitivity to lysosomal proteolytic activity were detected among the strains (Fig. S3B). Therefore, the inability of the serB mutant to proliferate intracellularly, whether in professional or nonprofessional phagocytes, is not related to the impaired PE biosynthesis but to its failure to produce serine de novo and/or to extract it from the host cell.
l-Serine biosynthesis is required for full virulence in BALB/c mice.Since auxotrophic mutant strains fail to replicate intracellularly and to produce PE, we sought to evaluate the virulence of the serB mutant in the murine infection model. As shown in Fig. 8, the serB auxotrophic mutant exhibited significantly reduced levels of splenic colonization. At 7 days p.i., the serB mutant showed a 2-log reduction in CFU compared to the wild type. This difference became larger at 15 days p.i., with a 2.867-log reduction in CFU (Fig. 8). Although statistically significant, the difference in CFU between the wild type and the serB mutant diminished to 1.641-log CFU at 30 days p.i., indicating that this auxotrophic mutant was able not only to persist in the spleen but also to replicate. Genetic complementation with a plasmid encoding the fusion protein SerB_3×FLAG partly restored virulence in mice. These results can be explained by the instability of certain plasmids in the mouse infection model, where antibiotic selection cannot be achieved. The colonization defect of the serB mutant was concomitant with reduced splenomegaly and hepatomegaly, hallmarks of Brucella infection (data not shown). These results indicate that B. abortus requires l-serine biosynthesis to achieve an efficient infection in the mammalian host.
The B. abortus serB mutant is defective for mouse spleen colonization. BALB/c mice were inoculated intraperitoneally with the B. abortus wild type, the serB mutant, or the complemented strain, as indicated in Materials and Methods. Bacteria were recovered from spleens at 7, 15 and 30 days postinfection. Individual CFU values are plotted, and horizontal dashed lines represent the median bacterial loads for each treatment group. Statistical significance was determined by one-way ANOVA (**, P < 0.01; ***, P < 0.001).
DISCUSSION
l-Serine is a nonessential amino acid produced by a biosynthetic pathway in nearly all organisms (48). In this study, we show that the disruption of the genes coding for the enzymes involved in the anabolic pathway causes l-serine auxotrophy, which impairs the ability of B. abortus to proliferate inside the host cell and affects virulence in the mouse infection model of brucellosis.
The crucial role of l-serine biosynthesis in pathogenicity is supported by a previous study describing a transposon-insertion mutant in the B. suis gene coding for SerB, which displays an attenuated phenotype after 48 h of infection in THP-1 macrophages (25). To our knowledge, attenuated mutants in the genes coding for SerC or SerA have not been reported or identified in mutagenesis analyses. In the case of SerA, this can be explained by the presence of two PGDH isoforms encoded in the B. abortus genome. Despite the little identity and different domain architectures of SerA-1 and SerA-2, both enzymes proved to be functionally redundant. Single mutants were prototrophs for l-serine, and only simultaneous deletion of serA1 and serA2 produced a growth defect in minimal medium without l-serine. The B. melitensis SerA-2 crystallographic structure has been resolved (PDB 3K5P), and it closely resembles the Escherichia coli PGDH structure (T. E. Edwards, J. Abendroth, and E. R. Smith, unpublished data). The ACT domain in E. coli PGDH is allosterically and cooperatively inhibited by l-serine to achieve negative-feedback regulation of the biosynthetic pathway (49). Further studies are required in order to characterize the function of ACT and ASB domains of PGDH isoforms in the regulation of the biosynthetic pathway in B. abortus.
Apart from the phosphorylated pathway, l-serine can also be produced from glycine by SHMT (48). However, since this reaction depletes glycine levels, this is not a major route to producing l-serine. In fact, this pathway cannot compensate for the effect of the mutations responsible for l-serine auxotrophy. For these reasons, we postulate that B. abortus SHMT encoded by gene locus bab1_0787 functions in vivo not for serine biosynthesis but for glycine production.
Several amino acid auxotroph strains of bacterial pathogens are often attenuated for intracellular growth and infection. Mycobacterium tuberculosis proline and lysine auxotrophs (50–52) and Salmonella auxotrophs for histidine and methionine (53–55) are examples of attenuated mutants in vivo. In B. abortus, l-serine depletion in serA1 serA2, serC, and serB mutants was likely the cause of intracellular replication defects, which could be rescued by amino acid supplementation of the host cell medium. The fact that the mutants were able to transport and use extracellular l-serine from RPMI-supplemented medium suggests that wild-type B. abortus does not obtain enough l-serine from the host during infection and must synthesize its own. Delivery of extracellular l-serine into the B. abortus cytoplasm requires transport across three membranes: the host cell membrane, the phagosomal membrane, and ultimately the bacterial membranes. Amino acid supplementation and uptake by intracellular pathogens have already been described in Francisella (56, 57) and Legionella (58) spp. These bacteria are auxotrophic for some amino acids and must obtain them from the host in order to proliferate.
According to our results, l-serine availability contributes during the early infection phases to bacterial proliferation. A recent transposon mutagenesis study highlights and supports the importance of B. abortus amino acid biosynthetic pathways during infection (59). Mutant strains in the genes coding for the enzymes involved in the anabolism of histidine and isoleucine, leucine, and valine are attenuated at 24 h p.i. in RAW 264.7 macrophages. At 2 and 5 h p.i., the mutants resemble l-serine auxotrophs, showing no differences from the wild type in intracellular bacterial counts. Noticeably, although the serB auxotrophic mutant is attenuated at 24 and 48 h p.i., it is contained inside BCVs with replicative characteristics in HeLa cells, resembling wild-type B. abortus. In HeLa cells, similar kinetics of LAMP-1 exclusion and calnexin recruitment to the BCVs suggest a dissociation between traffic events and replication capability for the serB mutant. However, in cells with proficient phagocytic activity, such as J774A.1 macrophages, the biogenesis of the rBCV is impaired, and the serB mutant resides in intracellular compartments positive for the late endosome/lysosome marker LAMP-1 and negative for the ER chaperone calnexin, where it is subjected to lysosomal degradation. Further studies are needed to address and characterize these observations.
The virulence attenuation of B. abortus caused by l-serine auxotrophy might be merely based on the requirement of this amino acid for protein biosynthesis. In addition, the virulence attenuation might be due to the role of l-serine as a precursor in PE biosynthesis. Previous studies demonstrated that the absence of PE altered the Brucella cell surface properties, impairing intracellular survival and spleen colonization in mice (46). In the pssA mutant, the absence of PE was compensated for by increasing the relative amounts of the other lipids, in particular the ornithine lipid, as described here for the serB auxotrophic mutant. However, the absence of PE in the serB cell envelope does not affect its resistance to acidic pH or to lysosome killing, suggesting that the lack of l-serine is mainly responsible for the defect in intracellular replication.
In the mouse infection model, although attenuated in acute and chronic stages, the serB mutant manages to persist in the spleen. This finding suggests that host tissues contain enough l-serine to support the growth of this auxotrophic mutant. It is well established that macrophages can adopt two different immunological states: (i) classical activated macrophages (CAM) with inflammatory cytokine production and bactericidal activity and (ii) alternative activated macrophages (AAMs), which are less inflammatory and play relevant roles in homeostasis, allergic inflammation, wound healing, and tissue repair (60, 61). During in vivo infection, Brucella is found within CAMs at early infection times but survives and replicates preferentially in AAMs (62). In these cells, a metabolic shift to beta-oxidation of fatty acids induced by peroxisome proliferator-activated receptor γ (PPARγ) increases intracellular glucose availability, which promotes intracellular survival and persistence of Brucella in AAMs (62). The fact that the serB mutant is less attenuated in the late stages than in the early stages of mouse infection could be related in part to the availability of amino acids (in particular l-serine) in each subtype of infected macrophages. Therefore, a better nutritional characterization of the microenvironment surrounding B. abortus in infected tissues would be useful to understand how bacteria adapt their metabolism to the different conditions found during the course of infection.
In the intracellular pathogens Legionella pneumophila and Coxiella burnetii, the host-cell l-serine is transported into the phagosome, where the bacterial cell converts it into pyruvate to feed the TCA cycle, thus serving as the main source of carbon and energy (63). However, this seems not to be the case for Brucella spp., which mainly depend on glutamic acid as an entry point into the TCA cycle (23, 64). In agreement with this, preliminary results from our group indicate that the mutant in the gene coding for the l-serine deaminase, which is involved in l-serine deamination to yield pyruvate, is not attenuated intracellularly like the l-serine auxotrophic mutants.
In conclusion, although it is capable of incorporating l-serine from the extracellular milieu by an as-yet-unidentified transporter, B. abortus depends on the biosynthesis of l-serine to sustain intracellular proliferation inside phagocytic and nonphagocytic cells. These findings highlight the l-serine biosynthetic pathway as an interesting target for the development of new drugs and/or strategies to combat brucellosis.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions.Bacterial strains and plasmids used in this study are listed in Table S1 in the supplemental material. B. abortus strains were inoculated in tryptic soy agar (TSA; Difco/Becton Dickinson, Sparks, MD) or in TSB at 37°C on a rotary shaker for 16 to 20 h. When indicated, media were supplemented with 50 μg/ml kanamycin, 5 μg/ml nalidixic acid, and/or 50 μg/ml ampicillin. All work with live B. abortus was performed in a biosafety level 3 laboratory facility. E. coli strains were grown in Luria broth liquid or solid medium at 37°C overnight. Antibiotics, when required, were added at the following concentrations: 50 μg/ml kanamycin or 100 μg/ml ampicillin.
Construction of B. abortus mutants and genetic complementation.To obtain the mutant strains by unmarked gene deletion, the regions flanking each gene (serA1, bab1_1697; serA2, bab2_0783; serC, bab1_1699; and serB, bab1_1410) were amplified and ligated using recombinant PCR techniques (65). The primers used for PCR amplification of the 500 bp upstream and downstream regions are listed in Table S2. Both PCR fragments obtained were used in an overlapping PCR to obtain a 1,000-bp fragment. These fragments were ligated into pK18mob-sacB vector (66). The resulting plasmids were transformed in E. coli S17λpir and subsequently conjugated to B. abortus S2308 by biparental mating. Single recombinants were selected with kanamycin (Km) and replica plated in TSA supplemented with 10% sucrose (Suc). Kmr and Sucs colonies were grown overnight in TSB without antibiotics and plated on TSA with 10% Suc to counterselect the double recombinants. Double-recombination events (Kms Sucr) were selected, and gene deletion was confirmed by PCR. To obtain the serA1 serA2 double mutant, the plasmid pK18mob-sacB containing the 1,000-pb flanking region of serA2 was conjugated to the serA1 mutant. Selection of recombination events was performed like in single-mutant constructions.
Genetic complementation of the mutant strains was achieved by the expression of C-terminal 3×Flag-tagged versions of the proteins SerB, SerC, and SerA-2 from plasmid pBBR1-MCS4-3×Flag (pLF) (67). All genes were amplified by PCR from B. abortus S2308 genomic DNA using the primers listed in Table S3. PCR products were digested with BamHI and SpeI, and the resulting fragments were cloned into the same sites of pLF to generate in-frame fusions to the 3×FLAG epitope under lac promoter control. The resulting constructions were introduced in the corresponding B. abortus mutant strains by biparental mating. To complement the serA1 serA2 double mutant, the promoter regions and genes coding for SerA-1 or SerA-2 were amplified by PCR using primers listed in the supplemental material (Fw_SerA-1_pBBR2_SpeI/Rv_SerA-1_pBBR2_BamHI and Fw_SerA-2_pBBR2_SpeI/Rv_SerA-2_pBBR2_BamHI). The PCR products were digested with BamHI and SpeI, and the fragments were cloned into pBBR1-MCS2. The resulting plasmids were introduced in the B. abortus double-mutant strain by biparental mating. For genetic complementation with both genes, pLF_serA-2 and pBBR1 MCS2_serA-1 were introduced simultaneously in the double mutant.
Growth curve in TSB and minimal medium.Starter cultures were grown in TSB in a rotary shaker (200 rpm) overnight at 37°C and then diluted with the same medium to an optical density at 600 nm (OD600) of 0.1 (10-ml cultures in 50-ml flasks). Culture growth was monitored by measuring the absorbance at 600 nm every 4 h. When indicated, the medium was supplemented with 10 mM l-serine (Sigma-Aldrich). Bacterial growth in GW minimal medium (68) was measured as described above, except that the minimal medium was used as the diluting solution of starter cultures grown until exponential phase (0.6 to 0.8 OD600). Bacterial growth was monitored by measuring the OD600 every 24 h. When indicated, 10 mM l-serine (Sigma) was added to the growth medium. Four independent experiments were performed in duplicates.
Cell culture infection and replication assays.Log-phase bacteria grown in TSB were used to infect J774A.1 macrophage-like cells at a multiplicity of infection (MOI) of 50:1 or HeLa cells at an MOI of 1,000:1. Bacteria were centrifuged onto cells at 400 × g for 10 min to promote bacterium-cell contact. After 60 min, the wells were gently washed three times with PBS and then incubated for 120 min with fresh medium containing 50 μg ml−1 gentamicin and 100 μg ml−1 streptomycin to kill noninternalized bacteria. Then, the antibiotic concentrations were decreased to 10 μg ml−1 gentamicin and 20 μg ml−1 streptomycin. At the indicated times, infected cells were either washed three times with PBS and lysed with 500 μl of 0.1% Triton X-100 in PBS (Sigma-Aldrich) for CFU counts or processed for immunofluorescence staining as described below. Intracellular CFU counts were determined by plating serial dilutions on TSA with the appropriate antibiotic.
Immunofluorescence microscopy and antibodies.HeLa cells were seeded on 12-mm coverslips in 24-well plates at 5 × 104 cells per well. After 24 h, the cells were infected with the indicated B. abortus strains and, at different times postinfection, the cells were washed three times with PBS and fixed with 4% paraformaldehyde (pH 7.4) for 15 min at 37°C. Fixed cells were washed again twice, and coverslips were incubated for 30 min in blocking buffer (PBS with 10% horse serum and 0.1% saponin). Afterward, the coverslips were incubated for 60 min in blocking buffer containing primary antibodies. After two washes in 0.1% saponin-PBS, the coverslips were incubated for 60 min in blocking buffer containing secondary antibodies. Finally, the coverslips were washed three times in PBS and once in Milli-Q water and mounted on glass slides using Fluorsave (Calbiochem). The primary antibodies used were rabbit anti-Brucella antibody, monoclonal mouse anti-Brucella antibody (M84), monoclonal mouse anti-human LAMP-1 antibody (H4A3), monoclonal rat anti-mouse LAMP-1 antibody (1D4B; Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, University of Iowa), and anti-human calnexin antibody (Abcam). The secondary antibodies used were Alexa Fluor goat anti-mouse IgG, goat anti-rat IgG, and goat anti-rabbit IgG (Molecular Probes/Invitrogen). Confocal images were acquired using an IX-81 microscope attached to a FV-1000 confocal module, with a PLAN APO 60× NA 1.42 oil immersion objective (Olympus, Japan). The acquisition software used was FV 10-ASW 3.1. Images were treated using ImageJ 1.45s software (National Institutes of Health), and images (1,024 × 1,024 pixels) were then assembled using Adobe Photoshop CS. Bacterial enumeration in HeLa cells and quantification of LAMP-1 and calnexin colocalization with BCVs were performed on a Nikon microscope (Eclipse TE 2000) at a magnification of ×60 with a lens with a numerical aperture of 1.42. At least 300 bacteria in random fields were analyzed per sample.
Microsomal and acidic pH susceptibility assay.Microsomes derived from J774A.1 murine macrophages were obtained as described previously (69). The protease activity in the microsomal fractions was determined using casein-BODIPY-FL, whose fluorescence is quenched. Protease-catalyzed hydrolysis relieves this quenching, yielding bright green fluorescent peptides. The increase in fluorescence emission is proportional to casein digestion and protease activity. Fluorescence was measured with a fluorescence plate reader (FilterMaxF5; Molecular Devices). To determine the sensitivity of Brucella strains to lysosomal killing, 2.5 × 105 CFU was incubated with 20 μg of the purified microsomal fractions or buffer (negative control) for 8 h at 37°C. After incubation, serial dilutions were plated in TSA to determine the number of viable bacteria. Susceptibility to low pH was assessed by incubating log-phase bacteria grown in TSB in PBS (pH 4 or 7) at 37°C for 4 h. Serial dilutions were plated after incubation to determine the number of viable bacteria after the treatments.
Mouse infection.All experimental protocols of this study were approved by the Committee on the Ethics of Animal Experiments of the University of San Martín (CICUAE UNSAM) and were conducted in agreement with international ethical standards for animal experimentation (Guide for the Care and Use of Laboratory Animals [73]). Eight-week-old female BALB/c mice were intraperitoneally inoculated with 5 × 104 CFU of B. abortus strains in PBS (200 μl). At 7, 15, and 30 days postinoculation, the spleens from infected mice were removed, weighed, and homogenized in 3 ml of PBS. Spleen homogenates were serially diluted and plated in TSA for CFU enumeration. During the experimental protocol, mice were housed in an appropriate biosafety level 3 facility and handled according to international guidelines required for animal experiments.
Thin-layer chromatography lipid analysis.Log-phase bacteria were used to inoculate fresh GW medium supplemented with 100 μM of choline dihydrogen citrate (Sigma-Aldrich) at an initial OD600 of 0.2. Cultures were grown for 48 h until they reached an OD600 of ∼1, and bacteria were harvested by centrifugation. Lipids were extracted according to the method of Bligh and Dyer (70) and separated on silica gel plates (Kieselgel 60; Merck) by using chloroform-methanol-water (14:6:1 [vol/vol/vol]) as a running solvent. Amino lipids were revealed by spraying the plate with 0.2% ninhydrin in ethanol, followed by heating at 100°C for 3 min (71). Phospholipids were visualized by destructive treatment with a solution of 8% H3PO4–10% CuSO4 and charring (72).
Statistical analyses.Statistical analyses were performed with Prism 6 software (GraphPad) with one- or two-way analysis of variance (ANOVA) and a Bonferroni posttest for multiple comparisons to assess statistical differences between two experimental data sets. P values are denoted in the figures as follows: ns, not significant; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
ACKNOWLEDGMENTS
We thank Francisco Guaimas for help in confocal microscopy image acquisition and processing.
Individual author contributions were as follows: V.R., data acquisition, data analysis, data interpretation, and writing of the manuscript; M.I.M., data acquisition, data analysis, data interpretation, writing of the manuscript, and revising of the manuscript; and D.J.C., data analysis, data interpretation, writing of the manuscript, revising of the manuscript, and principal investigator.
This study was supported by grants PICT-2014-3359 and PICT-2016-1993 from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina, to D.J.C., and by grant PICT-2017-0784 from the ANPCyT to M.I.M. V.R. is a research fellow of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. D.J.C. and M.I.M. are career investigators of CONICET.
We declare that the research was conducted in the absence of any financial or commercial relationships that would be construed as a potential conflict of interest.
FOOTNOTES
- Received 4 November 2019.
- Accepted 11 November 2019.
- Accepted manuscript posted online 18 November 2019.
Supplemental material is available online only.
- Copyright © 2020 American Society for Microbiology.
