Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.
Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.
Chronic lung infections with Pseudomonas aeruginosa are a major cause of morbidity and mortality in patients with cystic fibrosis (CF). During early childhood, patients are colonized by multiple bacterial pathogens including nonmucoid isolates of P. aeruginosa1,2. Emergence of the small colony variant (SCV) isolates as well as mucoid isolates is a marker for the onset to chronic infections. SCV isolates are highly drug resistant3 due to their slow growth rates4, which renders them a severe deterrent in the treatment regiments and other chronic infections5 by P. aeruginosa. Work by Al Ahmar et al.6 showed a link between SCV and mucoidy linked by de novo pyrimidine biosynthesis. Pyrimidine starvation, due to mutations in genes involved with pyrimidine production, resulted in SCV phenotype in the nonmucoid reference strain PAO1 and the mucoid derivative, PAO581 (PAO1mucA25).
Even though alginate overproduction is an important disease marker for chronic lung infections in CF, it is not clear whether there is a direct correlation between the amount of alginate and lung pathology, and it is unclear if alginate can be used as a prognosis marker for treatment7. Alginate production is mainly regulated by two operons, a regulatory operon (algUmucABCD)8,9 and the biosynthetic operon (algD operon)10,11. Alginate production is tightly regulated by the sigma factor AlgU9,12 (also known as AlgT) and the degradation of the anti-sigma factor MucA13. The ability to monitor the production of alginate in situ from the patients' sputum specimens can aid in the development of novel therapeutic options.
Here, we describe a growth condition that detects the presence of SCV caused by mutants that cannot synthesize the pyrimidine de novo. Supplementation of uracil and/or cytosine, the nitrogenous base of pyrimidine nucleotide, to the medium activates the salvage pathway, thus restoring the normal growth in mutants. This growth method for these specific SCV mutants may be used as a screening method to identify pyrimidine mutations in patient samples. In addition, we discuss two methods for detection and measurement of alginate produced and secreted by P. aeruginosa. The first is the traditional method14,15,16 of degrading the polysaccharide using a high concentration of acid and then adding a colorimetric indicator to quantitate the concentration in the sample. The second method, developed in our laboratory, utilizes the Enzyme-Linked Immunosorbent Assay (ELISA) using an anti-alginate monoclonal antibody (mAb) developed by QED Biosciences. The ELISA method proves to be more specific and sensitive than the uronic acid assay and allows for safer use due to the avoidance of the highly concentrated sulfuric acid. With the ability of the ELISA to be used directly on patient sputum samples to measure alginate, it can be developed as a monitoring diagnostic tool to follow the amount of alginate present in the lungs at different periods of the infection.
1. SCV Growth Conditions and Physiological Activation of the Salvage Pathway
2. Uronic Acid Carbazole Assay
3. ELISA for Alginate Quantitation
4. Statistical Analysis of Alginate Measurement
Figure 1 shows plates of PAO1 and PAO581 with or without in-frame deletion in the pyrD gene (a gene in the pyrimidine biosynthesis pathway) that results in SCV6. The PAO1 SCV mutant was restored to normal growth in response to uracil supplementation (Figure 1A,B). Furthermore, the PAO581ΔpyrDSCV mutant was returned to mucoidy with the same uracil treatment, because the parent strain PAO581 has an additional mucA25 mutation (Figure 1C,D). The results for the uronic acid carbazole assay are shown in Figure 26. The data represents samples of PAO581 and PAO581 with mutations in genes regulating pyrimidine de novo biosynthesis grown on PIA and PIA supplemented with 0.1 mM of uracil (Figure 2A). The data shows that the presence of uracil in the media results in the conversion of the mutant strain back to mucoidy (as seen by the increase/restored alginate production) (Figure 2B). The results for the anti-alginate monoclonal antibody based ELISA are represented in Figure 3. The data shows PAO1, PAO1 with an in-frame deletion of the algD gene encoding the key alginate biosynthetic enzyme GDP-mannose dehydrogenase, and PAO1 carrying an expression plasmid pHERD20T with the main alginate-specific sigma factor algU when grown on PIA plates with arabinose for the induction the pBAD promoter in pHERD20T. This data shows the non-mucoid levels of alginate measured for PAO1, and PAO1ΔalgD versus the mucoid levels of alginate measured for PAO1+pHERD20T-algU. Figure 4 compares the two methods of alginate measurements together. The results were not statistically significant when compared to each other using a two-way ANOVA with p < 0.01. Figure 5A compares the cross reactivity of the anti-alginate mAb against other polysaccharides including amylopectin, amylose, collagen, and glycogen. Figure 5B shows the comparison in specificity and sensitivity of the uronic acid carbazole assay to the newly developed anti-alginate monoclonal antibody-based ELISA with the control utilizing the highly purified seaweed alginate (Table of Materials). Figure 6 shows direct testing of the ELISA on patient sputum samples that were positive for mucoid P. aeruginosa and patients that did not contain mucoid P. aeruginosa.
Figure 1: Representative images of the de novo pyrimidine biosynthesis mutations that result in SCV phenotype in PAO1 and PAO581. Image shows PAO1 (left) and PAO1ΔpyrD (right) on PIA plates (A) and PIA plates supplemented with uracil (B) grown at 37 °C for 48 h. Image shows PAO581 (left) and PAO581ΔpyrD (right) on PIA plates (C) and PIA plates supplemented with uracil (D) grown at 37 °C for 48 h. This figure has been modified from work done by Al Ahmar et al.6. Please click here to view a larger version of this figure.
Figure 2: Representative graph of uronic acid carbazole assay. (A) The image of mucoid P. aeruginosa strain PAO581(PAO1mucA25) grown at 37 °C for 24 h on PIA plates (left) and PIA plates with uracil (right). (B) Alginate production for PAO581, PAO581carA, PAO581carB, and PAO581pyrD when grown on PIA plates with and without uracil at 37 °C for 24 h. Alginate was collected and measured using the standard carbazole assay. Values shown are mean alginate ± standard deviation of triplicate reads. (**** = p < 0.0001). This figure has been modified from work done by Al Ahmar et al.6. Please click here to view a larger version of this figure.
Figure 3: Representative graph of anti-alginate mAb-based ELISA. Alginate production for PAO1, PAO1ΔalgD and PAO1 carrying the expression vector pHERD20T-algU grown on PIA with 0.1% arabinose at 37 °C for 24 h. Alginate was collected and measured using the anti-alginate ELISA with the mouse anti-alginate monoclonal antibody. Values shown are mean alginate ± standard deviation of triplicate reads. ****p < 0.0001. Please click here to view a larger version of this figure.
Figure 4: Comparison between the results obtained from uronic acid assay and anti-alginate mAb-based ELISA. Alginate production from five different mucoid P. aeruginosa proprietary strains grown on PIA plates at 37 °C for 24 h. Alginate was collected and measured by the uronic acid assay and anti-alginate ELISA. Values shown are mean alginate ± standard deviation of triplicate reads. Please click here to view a larger version of this figure.
Figure 5: Specificity and sensitivity of the anti-alginate mAb based ELISA in comparison to the uronic acid carbazole assay. (A) ELISA was run with high (800 µg/mL) and low (100 µg/mL) internal assay controls of the seaweed alginate. This alginate was also used as a standard for the ELISA. Other polysaccharides tested that may cross react with anti-alginate mAb were amylopectin, amylose, collagen, and glycogen (500 µg/mL each). (B) Uronic acid carbazole assay and ELISA were run using the same range of standard concentrations with seaweed alginate: 50 µg/mL, 5 µg/mL, 1 µg/mL, 0.5 µg/mL, 0.1 µg/mL, and 0.05 µg/mL. Values shown are mean alginate ± standard deviation of triplicate reads. Please click here to view a larger version of this figure.
Figure 6: Direct patient sample testing. Anti-alginate ELISA was tested on patients' sputum samples without prior growth on plates. Three CF sputum samples that had growth of mucoid P. aeruginosa were used as well as two patient sputum samples that contained either non-mucoid P. aeruginosa (Neg 1) or no P. aeruginosa growth (Neg 2). Values shown are mean alginate ± standard deviation of triplicate reads. Please click here to view a larger version of this figure.
Both SCV and alginate are important disease markers implicated in several chronic infections. Therefore, the ability to grow SCV as well as study the regulation and production of alginate by P. aeruginosa is integral to the discovery of novel treatments for these chronic illnesses.
SCV strains are notoriously difficult to grow due to their slow growth rate4 as compared to other P. aeruginosa strains, which aids in their antimicrobial resistance3. Our work identifies a specific form of P. aeruginosa SCV that are a result of mutations in the de novo pyrimidine biosynthesis. Here, we discuss a growth condition for such SCV to revert to a normal growth phenotype when nucleoside uracil is supplied. However, when UMP/UTP was added to the medium, the defective growth was not restored (data not shown). The porin(s) responsible for this selectivity needs to be further studied. Supplementation of the growth media with uracil aided in relieving the stress induced from pyrimidine starvation (Figure 1). Similarly, addition of free cytosine to the media in the same method of addition of uracil, aided in the relief of pyrimidine starvation (data not shown). Both free uracil and free cytosine in the media enter the cells and help return the normal levels of uridine monophosphate (UMP) and uridine tri-phosphate (UTP)6 in the cell, which is how the SCV isolates reverted back to normal growth.
Several critical steps for the alginate measurements exist that might aid in reproducibility of the results. Initially the samples, after being scrapped from the plates, must remain on ice to help block the degradation of alginate in the sample and result in lower measured concentrations. In addition, prior to OD600 measurements, it is important to thoroughly mix the sample to obtain a homogeneous solution as clumps do form in samples that have been sitting for a while and that might interfere with the OD measurement and thereby with the final concentration calculations. When using the ELISA method, samples from growth plates may need to be diluted especially when using strains that produce a large amount of alginate since results might be outside of the standard curve range. When using the uronic acid, thoroughly vortex the culture tubes after addition of the carbazole and after the incubation to ensure a homogeneous sample. When working with the uronic acid assay, it is important to be cautious when handling the acid mixture and to dispose of the samples properly.
The traditional method of measurement requiring acid hydrolysis of alginate described above has shown great potential and has been utilized by many researchers for several years. In this work, we show the procedure for the traditional assay along with an in-house developed ELISA using an anti-alginate monoclonal antibody. These antibodies were developed in mice and can recognize alginate at a yet to be identified epitope. The specificity of the ELISA method was thoroughly tested against alginate from P. aeruginosa as well as from seaweeds. Moreover, the ELISA was comparable to the uronic acid assay in quantifying alginate in bacterial samples tested (Figure 3). In addition, it was tested against other polysaccharides that might result in false positive results. Our work showed a high specificity of the antibody to alginate and its ability to distinguish between alginate and the other tested polysaccharides (Figure 5A). The ELISA protocol has higher sensitivity as compared to the uronic acid assay (Figure 5B) since we were able to detect trace levels of alginate using ELISA that were not detected using the uronic acid assay when testing patient sputum samples (Figure 6). The ELISA method can be adapted for in vivo measurement of alginate from the patients' sputum (Figure 6). Both the growth conditions using uracil supplementation as well as the newly developed ELISA would be powerful tools in better understanding P. aeruginosa pathogenesis in CF.
The authors have nothing to disclose.
This work was supported by the National Institutes of Health (NIH) grants R44GM113545 and P20GM103434.
1-Step Ultra TMB-ELISA | Thermo Scientific | 34028 | via Fisher Scientific |
Absolute Ethanol (200 Proof) | Fisher Scientific | BP2818-4 | Molecular Bio-grade |
Accu Block Digital Dry Bath | Labnet | NC0205808 | via Fisher Scientific |
Assay Plates 96-well | CoStar | 2021-12-20 | |
Bench Top Vortex-Genie 2 | Scientific Industries | G560 | |
Boric Acid | Research Products International Corp. | 10043-35-3 | |
Cabinet Incubator | VWR | 1540 | |
Carbazole | Sigma | C-5132 | |
Carbonate-Bicarbonate Buffer | Sigma | C3041 | |
Centrifuge Tubes (50 ml) | Fisher Scientific | 05-539-13 | via Fisher Scientific |
Culture Test Tubes | Fisher Scientific | 14-956-6D | via Fisher Scientific |
Cuvette Polystyrene (1.5 ml) | Fisher Scientific | 14955127 | via Fisher Scientific |
Cytosine | Acros Organics | 71-30-7 | |
Diposable Inoculation Loops | Fisher Scientific | 22-363-597 | |
D-Mannuronic Acid Sodium | Sigma Aldrich | SMB00280 | |
FMC Alginate | FMC | 2133 | |
Glycerol | Fisher Scientific | BP906-5 | For Molecular Biology |
Mouse Anti-Alginate Monoclonal Antibody | QED Biosciences | N/A | Lot # :15725/15726 |
Phosphate Buffered Saline Powder (PBS) | Sigma | P3813 | |
Pierce Goat Anti-Mouse Poly-HRP Antibody | Thermo Scientific | 32230 | via Fisher Scientific |
Potassium Hydroxide | Fisher Scientific | 1310-58-3 | via Fisher Scientific |
Prism 7 | GraphPad | ||
Pseudomonas Isolation Agar (PIA) | Difco | 292710 | via Fisher Scientific |
Pseudomonas Isolation Broth (PIB) | Alpha Biosciences | P16-115 | via Fisher Scientific |
Round Toothpicks | Diamond | Any brand | |
Seaweed alginate (Protanal CR 8133) | FMC Corporation | ||
Skim Milk | Difco | 232100 | via Fisher Scientific |
SmartSpec Plus Spectrophotometer | BioRad | 170-2525 | or preferred vendor |
Sodium Chloride (NaCl) | Sigma | S-5886 | |
SpectraMax i3x Multi-mode MicroPlate Reader | Molecular Devices | i3x | or preferred vendor |
Sterile Petri Dish 100mm x 15mm | Fisher Scientific | FB0875713 | via Fisher Scientific |
Sulfuric Acid | Fisher Scientific | A298-212 | Technical Grade |
Sulfuric Acid (2 Normal -Stop Solution) | R&D Systems | DY994 | |
Tween 20 | Sigma | P2287 | |
Uracil | Acros Organics | 66-22-8 |