Characterization of Burkholderia pseudomallei protein BPSL1375 validates the Putative hemolytic activity of the COG3176 N-Acyltransferase family
© Ahmad et al. 2015
Received: 13 April 2015
Accepted: 13 November 2015
Published: 23 November 2015
There are still numerous protein subfamilies within families and superfamilies that do not yet have conclusive empirical experimental evidence providing a specific function. These proteins persist in databases with the annotation of a specific ‘putative’ function made by association with discernible features in the protein sequence.
Here, we report the characterization of one such protein produced by the pathogenic soil bacterium Burkholderia pseudomallei, BPSL1375, which provided evidence for putative hemolysins in the COG3176 family to have experimentally validated hemolytic activity. BPSL1375 can be classified into the N-acyltransferase superfamily, specifically to members of the COG3176 family. Sequence alignments identified seven highly conserved residues (Arg54, Phe58, Asp75, Asp78, Arg99, Glu132 and Arg135), of which several have been implicated with N-acyltransferase activity in previously characterized examples. Using the 3D model of an N-acyltransferase example as a reference, an acyl homoserine lactone synthase, we generated 3D structure models for mutants of six of the seven N-acyltransferase conserved residues (R54, D75, D78, R99, E132 and R135). Both the R99 and R135 mutants resulted in a loss of hemolytic activity while mutations at the other five positions resulted in either reduction or increment in hemolytic activity.
The implication of residues previously characterized to be important for N-acyltransferase activity to hemolytic activity for the COG3176 family members of the N-acyltransferase provides validation of the correct placement of the hemolytic capability annotation within the N-acyltransferase superfamily.
Burkholderia pseudomallei is a soil bacterium that causes melioidosis, a deadly disease endemic to South East Asia and Northern Australia . This bacterium is recognized as a potential category B biothreat agent due to its aerosol infectivity and severe course of infection . Pathogenesis of B. pseudomallei remains poorly understood despite a number of previous studies having described the expression of several virulence factors, particularly extracellular products with proteolytic, lipolytic and hemolytic activity [3, 4], the Type VI secretion system  and the capsule . A toxin that deamidates and subsequently inactivates helicase activity of the translation initiation factor (eIF4a) has been reported . Other aspects of this bacterium’s genome has also been explored including the existence of small RNAs  and methylation of genomic DNA by restriction modification systems . Nevertheless, much of the genome remains annotated as genes encoding hypothetical proteins.
Most bacterial pathogens have the capacity to acquire nutrients and minerals from the infected hosts . Free iron may generate damaging free radicals and therefore is typically maintained at low concentrations within the host. Bacterial pathogens are known to secrete extracellular proteins that lyse erythrocytes in order to acquire the free iron released from heme . Hemolysins, one such class of secreted proteins, are exotoxins that have lytic activity against erythrocytes and are often implicated as virulence factors. These proteins also allow bacteria to evade the immune system by escaping from phagosomes . Many hemolysins act by forming pores on the target cell’s membrane while others act through surfactant-like or enzymatic mechanisms .
Hemolysins have been implicated in a variety of complications arising from bacterial infections, ranging from intestinal tract disease to septicemia. Hemolysins produced by Escherichia coli have been associated with enterotoxicity and septicemia , whilst those secreted by Vibrio vulnificus , Aeromonas hydrophila  and Yersinia ruckeri  have been detected in high concentrations during fatal infections. Radke et al.  reported that Listeria monocytogenes listeriolysin disrupts lysosomal and phagosomal membranes to allow for bacterial escape into the host cytoplasm to initiate bacterial replication. Hemolysins have also been previously reported for members of the B. cepacia complex [19, 20] and it is known that different strains produced different hemolysins with differing functions. The Japanese clinical strain had been reported to produce a hemolysin called cepalysin that is capable of forming pores up to 30 nm in diameter . Two separate hemolysins have been described for U.S clinical isolates with a size of 72 kDa and 22 kDa . The hemolysins of British isolates are capable of inducing apoptosis and degranulation of mammalian phagocytes  and thus quite distinct from both the Japanese and the U.S strains due to its lack of pore forming capacity.
A number of studies have reported the expression of several B. pseudomallei heat-stable and heat-labile toxins that exhibited cytolytic and hemolytic activity [23–25]. Elevated levels of either cytotoxicity or hemolytic activity have also been detected in supernatant extracts of stationary growth phase cultures . However, it is still unclear if this hemolytic phenotype is the result of a single enzyme or a result of the combined outcomes from the reactions of different proteins.
Open reading frames (ORFs) annotated as hypothetical proteins make up almost 25 % of the B. pseudomallei genome . Cruz-Migoni et al.  reported a genome-wide project to characterize B. pseudomallei hypothetical proteins in an attempt to discover proteins with novel functions, which may include putative virulence factors. In this paper, we report the characterization of the protein encoded by the coding region bpsl1375 and provide definitive evidence of its ability to lyse erythrocytes. In silico analysis revealed sequence level similarities to members of the N-acyltransferase superfamily (Conserved Domains Database ID: cl00357) from the COG3176 group of the Clusters of Orthologous Groups (COGs) database. Members of COG3176 have previously been annotated as putative hemolysins.
BPSL1375 is a member of the N-acyltransferase superfamily
A systematic approach to characterize the functions of coding regions annotated as hypothetical proteins by Holden et al.  resulted in the extraction of all such sequences followed by resubmission for sequence alignment based analysis. This included targeted searches for previously reported virulence and pathogenicity factors such as various hemolysins from other Gram negative bacteria. One of these searches identified motifs previously observed in the hemolysin of Ralstonia eutropha within the coding region of an ORF originally annotated as encoding the hypothetical protein BPSL1375 by Holden et al. . A database search of the BPSL1375 sequence in GenBank revealed that the sequence contained domains that could be remotely associated with the N-acyltransferase (NAT) superfamily and shared sequence similarity to other proteins that were also annotated as hypothetical proteins. The NAT superfamily (Conserved Domains Database ID: cl00357) consists of various enzymes that characteristically catalyze the transfer of an acyl group onto its substrate. Among the known members of this superfamily are proteins that have been annotated as putative hemolysins (COG3176). The PATRIC database’s  entry for BPSL1375 has been updated to take into account this known homology to hemolysin and thus carries a revised annotation for BPSL1375 as ‘putative hemolysin’.
This homology to the NAT superfamily enabled us to refine our BLASTP database searches using the higher quality curated datasets in SWISSPROT/UniProt  and the Protein Data Bank (PDB) . The UniProt search yielded 13 hits, all with scores lower than 40, E values of 0.07 or higher and sequence identities of 25 % and lower. Two of the hits were for acyl-homoserine-lactone (AHL) synthase, a well characterized member of the NAT superfamily. The BLASTP search against the PDB also yielded hits with scores lower than 30, E values of 1.7 or higher and sequence identities of 37 % or lower. Again, we were able to observe a hit for an acyl-homoserine-lactone synthase, specifically, the sequence for the crystal structure of Pseudomonas aeruginosa LasI  with the PDB code 1RO5.
BPSL1375 and its orthologs are conserved in Burkholderia spp.
Scanning electron microscopy reveals evidence of hemolysis by BPSL1375
Modeling and structural analysis
The importance of the seven conserved residues identified by multiple sequence alignment of BPSL1375 with other available AHL synthases was initially investigated using the crystal structure of P. aeruginosa LasI (PDB: 1RO5) as reference. The corresponding conserved residues of BPSL1375 in LasI are R54/R23, F58/F27, D75/D44, D78/D47, R99/R70, E132/E101 and R135/R104 (Fig. 1b). The structural study of 1RO5 indicated that these residues were involved in the formation of the substrate binding pocket in an electrostatic cluster of the molecule , thus suggesting that these residues play key roles in supporting the proper structure and formation of the binding site.
Characterization of BPSL1375 and site directed mutants
Primers used for PCR amplification of bpsl1375 and site directed mutagenesis of bpsl1375
Primer Sequence (5’-3’)
The E. coli clones expressing recombinant BPSL1375 produced clear hemolytic zones around the colonies on trypticase soy agar containing 5 % sheep’s blood (Fig. 3d). Quantification of BPSL1375 hemolytic activity on 3 % sheep blood suspension for 1 h at 37 °C revealed up to 26.9 % hemolysis. To investigate the contribution of the conserved amino acids towards the observed BPSL1375 hemolytic activity, site directed mutagenesis was performed on R54, F58, D75, D78, R99, E132 and R135. Of the seven conserved positions, six residues were successfully substituted: R54K, D75E, D78E, R99K, E132D and R135K. All six successful site directed substitutions were then confirmed by Sanger sequencing.
Quantitation of hemolytic activity of the recombinant wild type and mutant BPSL1375 proteins
Protein / Control
Hemolysis Activity (%)a
B. pseudomallei D286 lysate
Wild type recombinant BPSL1375
Hemolysis induced by BPSL1375 wild type and mutant proteins are temperature dependent
BPSL1375 is inhibited by high erythrocyte concentrations
The amount of hemolysin needed to lyse a single erythrocyte molecule was determined by incubating a fixed quantity of B. pseudomallei lysate, BPSL1375 and the mutant proteins with different concentrations (0.2 to 2.5 %) of erythrocytes. According to Oberley et al. , one molecule of hemolytic protein is sufficient for lysis and therefore the amount of hemoglobin released by a constant amount of protein is proportional to the erythrocyte concentration until a plateau is reached. Therefore, if more than one molecule of protein is required for lysis, the amount of hemoglobin released should increase to a peak and then decrease. After 1 h at 37 °C, the mixtures were centrifuged and the amount of hemoglobin released into the supernatant was read at 545 nm. The B. pseudomallei lysate lysed erythrocytes at all concentrations of sheep blood while for BPSL1375 and its mutants, the percentage of hemolysis decreased as erythrocyte concentrations increased (Fig. 5b).
These results suggest that BPSL1375 employs a multi-hit hemolytic mechanism that is similar to the hemolysins of other pathogenic bacteria such as B. cereus , Gardenella vaginalis , Streptococcus suis and Synechoctus sp. . This mechanism is explained by the ability of a single toxin to bind only to a limited quantity of cells to induce hemolysis and is inhibited in the presence of a high ratio of erythrocytes to toxin molecule . Therefore, keeping the hemolysin concentration constant and increasing the number of erythrocytes suggests that the optimal ratio is not achieved due to the presence of excess erythrocytes. For instance, Inoue et al.  described that two to five molecules of streptolysin O and Clostridium perfringens delta toxin are required per erythrocyte to induce hemolysis. This characteristic was maintained in BPSL1375 and all of the mutants where similar reduction in hemolytic activity was observed at higher erythrocyte concentrations.
Our characterization of the B. pseudomallei protein BPSL1375 that was originally annotated as a hypothetical protein by Holden et al.  but subsequently revised to ‘putative hemolysin’ in the PATRIC database, has enabled this protein to be classified as a member of the N-acyltransferase (NAT) superfamily that includes several putative hemolysins, other hypothetical proteins and AHL synthases - namely LasI of Pseudomonas aeruginosa. The BPSL1375 sequence can be further grouped into the COG3176 cluster with members that contain hemolysin-like domains. This hemolytic potential was validated by hemolysis assays and site directed mutants. The hemolytic activity demonstrated by the expression of BPSL1375 (Fig. 3d) is evidence that the protein is the actual structural hemolysin and that BPSL1375 is not merely a regulator of hemolytic activity. To our knowledge, this is the first member of the NAT superfamily with experimental evidence of hemolytic activity.
Multiple sequence analysis to discover BPSL1375 amino acids that were shared with its homologs revealed seven conserved residues (R54, F58, D75, D78, R99, E132 and R135) that are likely important in the protein’s function. Cross-referencing of these residues with the observations reported by Gould et al.  for the LasI crystal structure revealed that these residues are known to be crucial for the functionality of LasI. These equivalent residues in LasI were deemed to be essential for AHL synthesis and contribute towards the stability of intra-structural interactions with possible catalytic functions. Additionally, the equivalent residue for E101 (corresponding to E132 in BPSL1375) is bound to two commonly conserved water molecules in AHL synthase that is known to partake in the enzyme’s catalytic mechanism .
Based on our modeling of the LasI structure (PDB ID: 1RO5), the most suitable amino acid substitutions that preserved the function of the conserved residues were R54K, F58W, D75E, D78E, R99K, E132D and R135K. These substitute amino acids were selected because they can maintain a similar 3D space and amino acid sequence characteristic to that of the wild type protein. The substitution of R with K and D with E were carried out to minimize the disturbance of protein fold and structural characteristics, either locally or globally. In this study, the substituted residues were visually scrutinized using Chimera to show the reduction and addition of hydrogen bonds that resulted from the changes, suggesting that the resulting mutants of these residues may show different levels of hemolytic activity.
Consequently, site directed mutagenesis of these seven conserved residues resulted in six successful substitutions and each mutant displayed different degrees of hemolytic capacity. The mutants R99K and R135K showed a complete loss of hemolytic activity and interestingly the D78E mutant showed a significant 2.3 fold increase in hemolytic activity. Although the exact molecular mechanisms for the hemolysis we observed is not yet known, the differences between the degree of hemolysis for each mutant can provide important clues with regards to the contribution of these residues in molecular interactions that result in hemolytic activity. The complete loss of hemolytic activity for mutants of R99 and R135 highlighted that these residues must play a crucial role in the activity of BPSL1375.
Our investigations into the hemolytic activity of BPSL1375 and the six mutants on different erythrocyte concentrations revealed that hemolytic activity decreased with an increase in the ratio between the protein molecule and erythrocyte thus implying that one erythrocyte required a certain number of ‘hits’ by the protein to induce lysis, a mechanism known as ‘multi-hits’. This mechanism has also been observed in Synechocystic sp. hemolysin, a cyanobacterial toxin .
In this study, a B. pseudomallei open reading frame identified using the BpK96243 genome sequence  that was initially annotated to encode a hypothetical protein, was characterized as an N-acyltransferase that exhibits hemolytic activity. Genetic manipulation of the residues associated with N-acyltransferase activity resulted in marked detectable variations of hemolytic activity. Although the exact mechanism that resulted in the differences in the exhibited hemolytic activity could not be pinpointed, this study demonstrated the highly useful utility that modeling of amino acid substitutions of available well characterized high resolution structures in the PDB can be reliably transferred to a homolog with very low sequence similarity and thus enabling functional characterization of the remote homolog. Based on our results, R99 and R135 are most likely crucial for function due to the complete loss of activity resulting from their substitution and therefore likely to be among the most highly conserved residues for this family of hemolytic N-acyltansferase (COG3176 family). Our work also demonstrated that BPSL1375 and the mutants possess a multi-hit hemolytic activity towards erythrocytes and that this N-acyltransferase enzyme is not heat stable.
Sequence homologs of BPSL1375 were retrieved using BLASTP searches (http://blast.ncbi.nlm.nih.gov) against the following databases: GenBank refseq, SWISSPROT/UniProt , Protein Data Bank (PDB)  and the Burkholderia Genome Database . Multiple sequence alignments were performed with ClustalW .
Protein structure modeling and analysis
Models were visualized and analyzed using Chimera . Modeling of mutations, structure comparisons and model refinements were also carried out using Chimera
Strains, vectors, enzymes and chemicals
B. pseudomallei strain D286 was previously isolated from a fatal melioidosis case at Kuala Lumpur Hospital . A stock culture of this strain was obtained from the Pathogen Laboratory, Faculty of Science and Technology, Universiti Kebangsaan Malaysia. E. coli BL21(DE3) and pET200/D-TOPO were obtained from Invitrogen, USA and other kits as well as reagents were obtained from standard commercial resources.
The ORFs for B. pseudomallei K96243 were generated using ARTEMIS 13.0. Corresponding primers were then designed for the ORF identified as bpsl1375 (Table 1). An additional CACC sequence (5’-CACCATGCGAGAACTGCCGA-3’) was added to the 5’- end to allow direct cloning into the expression vector. The primers (Table 1) for Quik-Change site directed mutagenesis were designed using a program provided by Stratagene.
Genomic DNA preparation
B. pseudomallei D286 was plated on Ashdown selective agar plates. A single colony was inoculated into Brain Heart Infusion broth (Pronadisa, Spain) and grown overnight at 37 °C; the bacterial cells were harvested and the cell pellet was dissolved in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5). The pellet was treated with Proteinase K (20 mg/ml) and incubated at 37 °C for 1 h. CTAB-NaCl (5 M NaCl) (Sigma, USA) was added and the incubation was continued at 65 °C for 20 min. B. pseudomallei genomic DNA extraction was carried out according to standard protocols as described by Richards et al. . Purification of genomic DNA was carried out according to the standard phenol chloroform method and precipitated using 0.6 volume of isopropanol.
Amplification and cloning of bpsl1375
DNA amplification by polymerase chain reaction (PCR) was carried out in a final volume of 50 μl containing 10 X Expand High Fidelity buffer with 15 mM MgCl2 (Sigma Chemical, USA), 10 mM dNTP (Sigma Chemical, USA), 200 ng/μl B. pseudomallei genome, 25 μM forward and reverse primers, 3.5 U/μl High Fidelity Taq polymerase (Roche, German) and ddH2O. The amplification was performed over 30 cycles of denaturation at 94 °C for 1 min, annealing at 67 °C for 1 min and elongation at 72 °C for 1 min. A final extension step was carried out for 10 min at 72 °C. The amplified products were cloned into pET200/D-TOPO according to the manufacturer’s recommendation (Invitrogen, USA). The gene orientation and sequence fidelity were verified by Sanger DNA sequencing.
Construction of mutants
Single residue mutations were produced using the Stratagene QuikChange site-directed mutagenesis kit. The wild type pET200-bpsl1375 construct was used as a DNA template. The mutations were confirmed by automated DNA sequencing. Six mutants were produced: R54K, D75E, D78E, R99K, E132D and R135K.
Expression and purification of BPSL1375 and its related mutants
Recombinant bpsl1375 and the corresponding mutants were transformed into E. coli BL21 Star (DE3) according to the kit manufacturer’s recommendations (Invitrogen, USA). The bacterial cells were grown on LB (Pronadisa, Spain)-Kanamycin (30 μg/ml) agar plates for 16 h at 37 °C. A single colony was inoculated into 5 ml LB-Kanamycin (30 μg/ml) and incubated at 30 °C overnight with shaking at 250 rpm. The overnight culture was subcultured into 50 ml of LB-Kanamycin (30 μg/ml) and grown at 30 °C with shaking at 250 rpm. At OD600 = 0.5, expression was induced with 1 mM IPTG (Promega, USA) for 6 h at 30 °C. Cells were harvested and protein extraction procedures were performed at 4 °C. The cell pellet was resuspended in 1 mL PBS buffer, sonicated for 10 s at 50 Hz and then centrifuged at 3200 g for 30 min. The supernatant containing soluble protein was collected and stored at −20 °C. His-tagged protein purification was performed using a HisTrap TM HP column (GE Healthcare). The column was washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, pH 7.4) and eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH 7.4). The eluted protein fractions were concentrated using Vivaspin 6 (MW 10 kDa) (Vivascience, Germany). Protein concentration was determined using the bicinchoninic acid method (Sigma Chemical, USA) and analyzed by 12 % SDS-PAGE electrophoresis followed by Western immunoblotting with anti-mouse IgG conjugated with HRP (Invitrogen, USA). C
Ethics statement and consent
Blood samples from rabbit and sheep were generously provided by the Animal House Facility, Universiti Kebangsaan Malaysia (UKM). Blood was drawn in accordance with the UKM animal ethics guideline formulated based on the guidelines of the National Health and Medical Research Council of Australia. The experiments were approved by the UKM Animal Ethics Committee (UKMAEC) under approval number FST/SBB/2010/SHEILA/24-AUGUST/320. All animals were maintained in a positive pressure environment at 20–25 °C, subjected to a 12 h light/dark cycle and fed with a protein-enriched diet and water ad libitum. No human subjects were used in the experiments reported in this paper.
Preparation of rabbit erythrocyte suspensions
Rabbit whole blood in anticoagulant citrate dextrose was centrifuged at 750 g for 10 min. Plasma and white blood cells were discarded by removing the supernatant. Erythrocytes were washed with PBS three times following centrifugation at 750 g for 10 min.
Measurement of hemolytic activity
Rabbit erythrocyte suspensions were adjusted to 4 % with PBS. The purified proteins and B. pseudomallei lysates were adjusted to 10 μg/ml. Then, 0.1 ml of this solution was incubated with 0.1 ml of 4 % erythrocyte suspension at 37 °C for 1 h. The samples were centrifuged at 750 g for 5 min to remove undamaged erythrocytes. The concentration of released hemoglobin was estimated by reading the absorbance at 545 nm against the control background lysis solution (erythrocyte suspension with PBS) using the Ultraspec 2100 pro (Amersham Biosciences). The percentage of hemolysis was calculated by normalizing against the absorbance of a mixture of erythrocytes and distilled water at the same ratio, which showed complete hemolysis.
To determine the effect of hemolytic activity on different amounts of red blood cells, rabbit erythrocyte suspensions at different concentrations (0.2 %, 0.4 %, 0.6 %, 0.8 %, 1.0 %, 1.5 %, 2.0 % and 2.5 %) in PBS were used. These diluted cell suspensions were incubated with an equal volume (1.0 μg) of B. pseudomallei lysate, purified BPSL1375 or the mutant protein for 1 h at 37 °C and then centrifuged at 750 g for 5 min to determine the hemolytic activity. For temperature-dependent studies, equal volumes (1.0 μg) of B. pseudomallei lysate, purified BPSL1375 and the mutant proteins were incubated with 1 % rabbit erythrocytes at 4 °C, 20 °C and 37 °C for 1 h and 60 °C for 15 min. The mixtures were centrifuged at 750 g for 5 min and the residual hemolytic activity of the supernatant was determined.
Scanning electron microscopy
Sheep erythrocytes (5 %) incubated with BPSL1375 were fixed in 2 % (v/v) glutaraldehyde, 0.1 M PBS (pH 7.4) overnight. Prior to dehydration in ethanol, samples were washed in PBS three times for 10 min. Critical point drying was performed with acetone and sputter-coated with gold-palladium. The samples were observed with a Philips XL30 scanning electron microscope at the Universiti Kebangsaan Malaysia Electron Microscopy Facility.
The service provided by the Electron Microscopy Facility, Universiti Kebangsaan Malaysia is gratefully acknowledged. The authors thank Prof. David Rice and the late Prof. Peter J. Artymiuk (the University of Sheffield) for useful discussions and comments with regard to this work and an earlier draft manuscript.
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