BACKGROUND
burkholderia pseudomallei is a soil bacterium that causes melioidosis, a deadly disease endemic to south east asia and northern australia  <cit> .
this bacterium is recognized as a potential category b biothreat agent due to its aerosol infectivity and severe course of infection  <cit> .
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  <cit> , the type vi secretion system  <cit>  and the capsule  <cit> .
a toxin that deamidates and subsequently inactivates helicase activity of the translation initiation factor  has been reported  <cit> .
other aspects of this bacterium’s genome has also been explored including the existence of small rnas  <cit>  and methylation of genomic dna by restriction modification systems  <cit> .
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  <cit> .
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  <cit> .
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  <cit> .
many hemolysins act by forming pores on the target cell’s membrane while others act through surfactant-like or enzymatic mechanisms  <cit> .
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  <cit> , whilst those secreted by vibrio vulnificus  <cit> , aeromonas hydrophila  <cit>  and yersinia ruckeri  <cit>  have been detected in high concentrations during fatal infections.
radke et al.
<cit>  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  <cit>  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  <cit> .
two separate hemolysins have been described for u.s clinical isolates with a size of 72 kda and 22 kda  <cit> .
the hemolysins of british isolates are capable of inducing apoptosis and degranulation of mammalian phagocytes  <cit>  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 .
elevated levels of either cytotoxicity or hemolytic activity have also been detected in supernatant extracts of stationary growth phase cultures  <cit> .
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  annotated as hypothetical proteins make up almost 25 % of the b. pseudomallei genome  <cit> .
cruz-migoni et al.
<cit>  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 bpsl <dig> and provide definitive evidence of its ability to lyse erythrocytes.
in silico analysis revealed sequence level similarities to members of the n-acyltransferase superfamily  from the cog <dig> group of the clusters of orthologous groups  database.
members of cog <dig> have previously been annotated as putative hemolysins.
RESULTS
bpsl <dig> 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.
<cit>  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 bpsl <dig> by holden et al.
<cit> .
a database search of the bpsl <dig> sequence in genbank revealed that the sequence contained domains that could be remotely associated with the n-acyltransferase  superfamily and shared sequence similarity to other proteins that were also annotated as hypothetical proteins.
the nat superfamily  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 .
the patric database’s  <cit>  entry for bpsl <dig> has been updated to take into account this known homology to hemolysin and thus carries a revised annotation for bpsl <dig> 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  <cit>  and the protein data bank   <cit> .
the uniprot search yielded  <dig> hits, all with scores lower than  <dig>  e values of  <dig>  or higher and sequence identities of 25 % and lower.
two of the hits were for acyl-homoserine-lactone  synthase, a well characterized member of the nat superfamily.
the blastp search against the pdb also yielded hits with scores lower than  <dig>  e values of  <dig>  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  <cit>  with the pdb code 1ro <dig>
to identify highly conserved regions such as residues that may be potential active sites or binding motifs, we carried out multiple sequence alignment of the bpsl <dig> sequence against  <dig> other sequences from the cog <dig> family and representatives from the lysophospholipid acyltransferase  family to which ahl synthase belongs.
from the alignments, we identified  <dig> residue positions – arginine  <dig> , phenylalanine  <dig> , aspartic acid  <dig> , aspartic acid  <dig> , arginine  <dig> , glutamic acid  <dig>  and arginine  <dig>  that were conserved in all  <dig> sequences .
the equivalent residue numbers for the lasi sequence are provided in fig.
1b.
six of these residues  carried charged side chains while f <dig> is a hydrophobic amino acid.fig.
<dig> multiple sequence alignment of bpsl <dig> and corresponding residue numbers of bpsl <dig> and lasi/1ro <dig>  multiple sequence alignment has been done to identify highly conserved regions such as residues that may be potential active sites or binding motifs of bpsl <dig> sequence against  <dig> other sequences from the cog <dig> family and representatives from the lysophospholipid acyltransferase  family to which ahl synthase belongs.
a multiple sequence alignment of bpsl <dig> with representatives of the cog <dig> family and representatives of the lplat family including the lasi sequence  – each sequence is labeled as either genus.species_protein name_accesion number for uniprot sourced data or genus.species_accession_number for genbank sourced data; b corresponding residue numbers in bpsl <dig> and p. aeruginosa lasi/1ro <dig> for the seven residues conserved throughout the alignment
bpsl <dig> and its orthologs are conserved in burkholderia spp.
we then investigated whether bpsl <dig> is common to all burkholderia spp.
and twelve orthologs were discovered in other burkholderia species with available genome sequences.
these burkholderia spp.
orthologs were mainly annotated as either hemolysin-like proteins or ornithine-acyl n-acyltransferases.
we further explored this conservation by analyzing the presence and arrangement of the bpsl <dig> orthologs and the flanking coding regions in the other burkholderia species.
we were able to match twelve orthologous sequence regions within the burkholderia genus although seven of these appear to have possibly undergone various levels of rearrangement .fig.
<dig> conservation of bpsl <dig> and neighboring genes in other members of the burkholderia genus.
the conservation of bpsl <dig> matched twelve orthologous sequence regions within the burkholderia genus although seven of these appear to have possibly undergone various levels of rearrangement.
bp: b. pseudomallei; ba: b. ambifaria; bc: b. cenopacia; bt: b. thailandensis; bm: b. mallei; b. multi atcc: b. multivorans atcc17616; bphy: b. phymatum; bx: b. xenovorans
scanning electron microscopy reveals evidence of hemolysis by bpsl1375
the potential for bpsl <dig> to have hemolytic activity as suggested by the sequence alignment was investigated by incubating purified recombinant bpsl <dig>  with sheep erythrocytes.
the expression and purification of recombinant bpsl <dig> is discussed in more detail below.
scanning electron microscopy  revealed that sheep erythrocytes treated with the recombinant bpsl <dig> displayed crenated shapes with cytoplasmic blebbing on the membrane  compared to untreated erythrocytes that presented the usual biconcave discs appearance , nevertheless, blebbing was not as notable as the positive control for lysis .
recombinant e. coli carrying the bpsl <dig> gene  that were grown on blood agar plates also showed clear hemolytic zones .
confirmation of bpsl <dig> hemolytic activity then led us to perform modeling and generation of recombinant mutants to enable the determination of key residue required for hemolytic activity.fig.
<dig> a comparison between the electron micrographs of cytoplasmic blebbing on the membrane and recombinant e. coli carrying the bpsl <dig> gene grown on blood agar plates.
scanning electron microscopy revealed that sheep erythrocytes treated with the recombinant bpsl <dig> displayed crenated shapes with cytoplasmic blebbing on the membrane  compared to untreated erythrocytes that presented the usual biconcave discs appearance , nevertheless, blebbing was not as notable as the positive control for lysis .
recombinant e. coli carrying the bpsl <dig> gene  that were grown on blood agar plates also showed clear hemolytic zones .
a normal biconcave disc shape of untreated erythrocytes in pbs; b bpsl <dig> treated erythrocytes showing the presence of discrete blebs  on the erythrocyte; and c erythrocytes in sterile distilled water that also show the blebs  as a positive control for lysis.
d recombinant e. coli bpsl1375colonies grown on trypticase soy agar with 5 % sheep blood after  <dig>   <dig> and 72 h of incubation at 37 °c showing clear hemolytic zones
modeling and structural analysis
the importance of the seven conserved residues identified by multiple sequence alignment of bpsl <dig> with other available ahl synthases was initially investigated using the crystal structure of p. aeruginosa lasi  as reference.
the corresponding conserved residues of bpsl <dig> in lasi are r54/r <dig>  f58/f <dig>  d75/d <dig>  d78/d <dig>  r99/r <dig>  e132/e <dig> and r135/r <dig> .
the structural study of 1ro <dig> indicated that these residues were involved in the formation of the substrate binding pocket in an electrostatic cluster of the molecule  <cit> , thus suggesting that these residues play key roles in supporting the proper structure and formation of the binding site.
each of the seven conserved residues was substituted in silico with residues from the same group of amino acids such as r with k and d with e in order to maintain the protein’s general fold as well as the local conformations at the substitution sites  <cit> .
visual analysis using chimera molecular graphics  <cit>  revealed the consistency of the local conformation for the substituted residues compared to the original residues indicating the suitability of the substitution in conserving the position and orientation of the original residue in 3d space .
we further investigated changes in the number of hydrogen bonds with the neighboring residues that resulted from the substitution.
the substitution of r23k reduced the number of hydrogen bonds formed from  <dig> to  <dig> , d44e from  <dig> bonds to  <dig> , d47e from  <dig> bonds to  <dig> bonds , r70k from  <dig> bonds to  <dig> , e101d from  <dig> bonds to  <dig> bonds  and r104k with  <dig> bonds reduced to  <dig> bonds .
with these mutant models as a guide, we expect the changes in the number of hydrogen bonds to change the interactions between the residues involved, thus, possibly affecting the overall catalytic properties of the molecule.
in the next section, we demonstrate the effect of each substitution on the hemolytic activity of bpsl <dig> fig.
<dig> models of the amino acid substitutions generated using chimera.
substitutions of the different residues also resulted in changes in the number of hydrogen bonds with the neighboring residues.
a r23k, b d44e, c d47e, d r70k, e e101d and f r104k
characterization of bpsl <dig> and site directed mutants
in order to characterize the bpsl <dig> protein, the bpsl <dig> gene was cloned from a clinical isolate  available in our laboratory.
the primers used for the work reported here  were designed using the available b. pseudomallei k <dig> genome sequence.
the amplified 819 bp orf of bpsl <dig>  encoding  <dig> amino acids, was successfully ligated into the expression vector pet200/d-topo.
the 37 kda bpsl <dig> protein was expressed by e. coli bl <dig>  and purified by affinity chromatography.table  <dig> primers used for pcr amplification of bpsl <dig> and site directed mutagenesis of bpsl1375
the e. coli clones expressing recombinant bpsl <dig> produced clear hemolytic zones around the colonies on trypticase soy agar containing 5 % sheep’s blood .
quantification of bpsl <dig> hemolytic activity on 3 % sheep blood suspension for 1 h at 37 °c revealed up to  <dig>  % hemolysis.
to investigate the contribution of the conserved amino acids towards the observed bpsl <dig> hemolytic activity, site directed mutagenesis was performed on r <dig>  f <dig>  d <dig>  d <dig>  r <dig>  e <dig> and r <dig>  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.
the r135k and r99k substitutions resulted in a complete loss of hemolytic activity indicating that these residues may be critical for the hemolytic activity observed for bpsl <dig> .
the other three substitutions of e132d, d75e and r54k resulted in a non-significant decrease of hemolytic activity.
the hemolysis activity of e132d was recorded at 22 % whereas d75e and r54k hemolysis were at 18 % and 14 % respectively.
this implied that although these residues are conserved within the cog <dig> family members and most likely important for bpsl <dig> hemolytic activity, they may not be directly involved in the catalytic reaction or interactions associated with hemolysis and perhaps, have substrate binding roles associated to substrate specificity determination.
we also noted that the hemolytic activity of the d78e mutant increased up to  <dig>  fold higher than the wild-type .
for the work reported here, a single sampling to quantify hemolysis by bpsl <dig> was conducted at 1 h. future studies should investigate the hemolytic activity of bpsl <dig> and the site directed mutants over time.table  <dig> quantitation of hemolytic activity of the recombinant wild type and mutant bpsl <dig> proteins
aincubation with erythrocytes for 60 min at 37 °c
hemolysis induced by bpsl <dig> wild type and mutant proteins are temperature dependent
the bpsl <dig> recombinant wild type and mutant proteins were incubated individually in erythrocyte suspensions to observe hemolysis at four different temperatures; 4 °c, 20°, 37 °c and 60 °c .
incubation was fixed at 60 min for all reactions except for the 60 °c sample which was limited to 15 min.
the mixtures were centrifuged and the level of hemoglobin released was measured at 545 nm.
poor hemolysis was observed for all samples at 4 °c, including the positive control b. pseudomallei lysate.
however, hemolysis at 20 °c was the highest for b. pseudomallei lysate, bpsl <dig>  bpsl1375/d78e, bpsl1375/r99k and bpsl1375/r135k  whilst the highest hemolysis at 37 °c was noted for bpsl1375/e132d.
incubation at 60 °c abolished the activity of all samples except for b. pseudomallei lysate.
the presence of a heat labile hemolysin has been reported for b. pseudomallei although it has not been specifically identified  <cit> .
our results suggest that bpsl <dig> as wellas the mutants are heat-labile, and bpsl <dig> may therefore be the previously uncharacterized heat labile hemolysin of b. pseudomallei first reported by ashdown and koehler  <cit> .fig.
<dig> hemolytic activity of the wild type and mutant proteins.
the hemolytic activity was carried out on 1 % rabbit erythrocyte solution at different temperatures a and at different erythrocyte concentrations ranging from  <dig>  to  <dig>  % b after 60 min incubation at 37 °c.
‘b.
pseudomallei’ refers to b. pseudomallei lysate;‘bpsl175’ refers to the recombinant protein; mutants are denoted by the scheme “m_original residue_residue number_substituted residue”; pbs refers to phosphate buffered saline
bpsl <dig> 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, bpsl <dig> and the mutant proteins with different concentrations  of erythrocytes.
according to oberley et al.
<cit> , 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 bpsl <dig> and its mutants, the percentage of hemolysis decreased as erythrocyte concentrations increased .
these results suggest that bpsl <dig> employs a multi-hit hemolytic mechanism that is similar to the hemolysins of other pathogenic bacteria such as b. cereus  <cit> , gardenella vaginalis  <cit> , streptococcus suis and synechoctus sp.
<cit> .
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  <cit> .
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.
<cit>  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 bpsl <dig> and all of the mutants where similar reduction in hemolytic activity was observed at higher erythrocyte concentrations.
discussion
our characterization of the b. pseudomallei protein bpsl <dig> that was originally annotated as a hypothetical protein by holden et al.
<cit>  but subsequently revised to ‘putative hemolysin’ in the patric database, has enabled this protein to be classified as a member of the n-acyltransferase  superfamily that includes several putative hemolysins, other hypothetical proteins and ahl synthases - namely lasi of pseudomonas aeruginosa.
the bpsl <dig> sequence can be further grouped into the cog <dig> 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 bpsl <dig>  is evidence that the protein is the actual structural hemolysin and that bpsl <dig> 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 bpsl <dig> amino acids that were shared with its homologs revealed seven conserved residues  that are likely important in the protein’s function.
cross-referencing of these residues with the observations reported by gould et al.
<cit>  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 e <dig>  is bound to two commonly conserved water molecules in ahl synthase that is known to partake in the enzyme’s catalytic mechanism  <cit> .
based on our modeling of the lasi structure , 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  <dig>  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 r <dig> and r <dig> highlighted that these residues must play a crucial role in the activity of bpsl <dig>
our investigations into the hemolytic activity of bpsl <dig> 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  <cit> .
CONCLUSIONS
in this study, a b. pseudomallei open reading frame identified using the bpk <dig> genome sequence  <cit>  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, r <dig> and r <dig> 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 .
our work also demonstrated that bpsl <dig> and the mutants possess a multi-hit hemolytic activity towards erythrocytes and that this n-acyltransferase enzyme is not heat stable.

 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, the type vi secretion system and the capsule. a toxin that deamidates and subsequently inactivates helicase activity of the translation initiation factor 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 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. 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 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 bpsl and provide definitive evidence of its ability to lyse erythrocytes. in silico analysis revealed sequence level similarities to members of the n-acyltransferase superfamily from the cog group of the clusters of orthologous groups database. members of cog have previously been annotated as putative hemolysins.  bpsl 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 bpsl by holden et al.. a database search of the bpsl sequence in genbank revealed that the sequence contained domains that could be remotely associated with the n-acyltransferase superfamily and shared sequence similarity to other proteins that were also annotated as hypothetical proteins. the nat superfamily 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. the patric database’s entry for bpsl has been updated to take into account this known homology to hemolysin and thus carries a revised annotation for bpsl 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. the uniprot search yielded hits, all with scores lower than e values of or higher and sequence identities of 25 % and lower. two of the hits were for acyl-homoserine-lactone synthase, a well characterized member of the nat superfamily. the blastp search against the pdb also yielded hits with scores lower than e values of 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 1ro to identify highly conserved regions such as residues that may be potential active sites or binding motifs, we carried out multiple sequence alignment of the bpsl sequence against other sequences from the cog family and representatives from the lysophospholipid acyltransferase family to which ahl synthase belongs. from the alignments, we identified residue positions – arginine, phenylalanine, aspartic acid, aspartic acid, arginine, glutamic acid and arginine that were conserved in all sequences. the equivalent residue numbers for the lasi sequence are provided in fig. 1b. six of these residues carried charged side chains while f is a hydrophobic amino acid.fig. multiple sequence alignment of bpsl and corresponding residue numbers of bpsl and lasi/1ro multiple sequence alignment has been done to identify highly conserved regions such as residues that may be potential active sites or binding motifs of bpsl sequence against other sequences from the cog family and representatives from the lysophospholipid acyltransferase family to which ahl synthase belongs. a multiple sequence alignment of bpsl with representatives of the cog family and representatives of the lplat family including the lasi sequence – each sequence is labeled as either genus.species_protein name_accesion number for uniprot sourced data or genus.species_accession_number for genbank sourced data; b corresponding residue numbers in bpsl and p. aeruginosa lasi/1ro for the seven residues conserved throughout the alignment bpsl and its orthologs are conserved in burkholderia spp. we then investigated whether bpsl is common to all burkholderia spp. and twelve orthologs were discovered in other burkholderia species with available genome sequences. these burkholderia spp. orthologs were mainly annotated as either hemolysin-like proteins or ornithine-acyl n-acyltransferases. we further explored this conservation by analyzing the presence and arrangement of the bpsl orthologs and the flanking coding regions in the other burkholderia species. we were able to match twelve orthologous sequence regions within the burkholderia genus although seven of these appear to have possibly undergone various levels of rearrangement.fig. conservation of bpsl and neighboring genes in other members of the burkholderia genus. the conservation of bpsl matched twelve orthologous sequence regions within the burkholderia genus although seven of these appear to have possibly undergone various levels of rearrangement. bp: b. pseudomallei; ba: b. ambifaria; bc: b. cenopacia; bt: b. thailandensis; bm: b. mallei; b. multi atcc: b. multivorans atcc17616; bphy: b. phymatum; bx: b. xenovorans scanning electron microscopy reveals evidence of hemolysis by bpsl1375 the potential for bpsl to have hemolytic activity as suggested by the sequence alignment was investigated by incubating purified recombinant bpsl with sheep erythrocytes. the expression and purification of recombinant bpsl is discussed in more detail below. scanning electron microscopy revealed that sheep erythrocytes treated with the recombinant bpsl displayed crenated shapes with cytoplasmic blebbing on the membrane compared to untreated erythrocytes that presented the usual biconcave discs appearance, nevertheless, blebbing was not as notable as the positive control for lysis. recombinant e. coli carrying the bpsl gene that were grown on blood agar plates also showed clear hemolytic zones. confirmation of bpsl hemolytic activity then led us to perform modeling and generation of recombinant mutants to enable the determination of key residue required for hemolytic activity.fig. a comparison between the electron micrographs of cytoplasmic blebbing on the membrane and recombinant e. coli carrying the bpsl gene grown on blood agar plates. scanning electron microscopy revealed that sheep erythrocytes treated with the recombinant bpsl displayed crenated shapes with cytoplasmic blebbing on the membrane compared to untreated erythrocytes that presented the usual biconcave discs appearance, nevertheless, blebbing was not as notable as the positive control for lysis. recombinant e. coli carrying the bpsl gene that were grown on blood agar plates also showed clear hemolytic zones. a normal biconcave disc shape of untreated erythrocytes in pbs; b bpsl treated erythrocytes showing the presence of discrete blebs on the erythrocyte; and c erythrocytes in sterile distilled water that also show the blebs as a positive control for lysis. d recombinant e. coli bpsl1375colonies grown on trypticase soy agar with 5 % sheep blood after and 72 h of incubation at 37 °c showing clear hemolytic zones modeling and structural analysis the importance of the seven conserved residues identified by multiple sequence alignment of bpsl with other available ahl synthases was initially investigated using the crystal structure of p. aeruginosa lasi as reference. the corresponding conserved residues of bpsl in lasi are r54/r f58/f d75/d d78/d r99/r e132/e and r135/r. the structural study of 1ro 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. each of the seven conserved residues was substituted in silico with residues from the same group of amino acids such as r with k and d with e in order to maintain the protein’s general fold as well as the local conformations at the substitution sites. visual analysis using chimera molecular graphics revealed the consistency of the local conformation for the substituted residues compared to the original residues indicating the suitability of the substitution in conserving the position and orientation of the original residue in 3d space. we further investigated changes in the number of hydrogen bonds with the neighboring residues that resulted from the substitution. the substitution of r23k reduced the number of hydrogen bonds formed from to, d44e from bonds to, d47e from bonds to bonds, r70k from bonds to, e101d from bonds to bonds and r104k with bonds reduced to bonds. with these mutant models as a guide, we expect the changes in the number of hydrogen bonds to change the interactions between the residues involved, thus, possibly affecting the overall catalytic properties of the molecule. in the next section, we demonstrate the effect of each substitution on the hemolytic activity of bpsl fig. models of the amino acid substitutions generated using chimera. substitutions of the different residues also resulted in changes in the number of hydrogen bonds with the neighboring residues. a r23k, b d44e, c d47e, d r70k, e e101d and f r104k characterization of bpsl and site directed mutants in order to characterize the bpsl protein, the bpsl gene was cloned from a clinical isolate available in our laboratory. the primers used for the work reported here were designed using the available b. pseudomallei k genome sequence. the amplified 819 bp orf of bpsl encoding amino acids, was successfully ligated into the expression vector pet200/d-topo. the 37 kda bpsl protein was expressed by e. coli bl and purified by affinity chromatography.table primers used for pcr amplification of bpsl and site directed mutagenesis of bpsl1375 the e. coli clones expressing recombinant bpsl produced clear hemolytic zones around the colonies on trypticase soy agar containing 5 % sheep’s blood. quantification of bpsl hemolytic activity on 3 % sheep blood suspension for 1 h at 37 °c revealed up to  % hemolysis. to investigate the contribution of the conserved amino acids towards the observed bpsl hemolytic activity, site directed mutagenesis was performed on r f d d r e and r 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. the r135k and r99k substitutions resulted in a complete loss of hemolytic activity indicating that these residues may be critical for the hemolytic activity observed for bpsl. the other three substitutions of e132d, d75e and r54k resulted in a non-significant decrease of hemolytic activity. the hemolysis activity of e132d was recorded at 22 % whereas d75e and r54k hemolysis were at 18 % and 14 % respectively. this implied that although these residues are conserved within the cog family members and most likely important for bpsl hemolytic activity, they may not be directly involved in the catalytic reaction or interactions associated with hemolysis and perhaps, have substrate binding roles associated to substrate specificity determination. we also noted that the hemolytic activity of the d78e mutant increased up to fold higher than the wild-type. for the work reported here, a single sampling to quantify hemolysis by bpsl was conducted at 1 h. future studies should investigate the hemolytic activity of bpsl and the site directed mutants over time.table quantitation of hemolytic activity of the recombinant wild type and mutant bpsl proteins aincubation with erythrocytes for 60 min at 37 °c hemolysis induced by bpsl wild type and mutant proteins are temperature dependent the bpsl recombinant wild type and mutant proteins were incubated individually in erythrocyte suspensions to observe hemolysis at four different temperatures; 4 °c, 20°, 37 °c and 60 °c. incubation was fixed at 60 min for all reactions except for the 60 °c sample which was limited to 15 min. the mixtures were centrifuged and the level of hemoglobin released was measured at 545 nm. poor hemolysis was observed for all samples at 4 °c, including the positive control b. pseudomallei lysate. however, hemolysis at 20 °c was the highest for b. pseudomallei lysate, bpsl bpsl1375/d78e, bpsl1375/r99k and bpsl1375/r135k whilst the highest hemolysis at 37 °c was noted for bpsl1375/e132d. incubation at 60 °c abolished the activity of all samples except for b. pseudomallei lysate. the presence of a heat labile hemolysin has been reported for b. pseudomallei although it has not been specifically identified. our results suggest that bpsl as wellas the mutants are heat-labile, and bpsl may therefore be the previously uncharacterized heat labile hemolysin of b. pseudomallei first reported by ashdown and koehler.fig. hemolytic activity of the wild type and mutant proteins. the hemolytic activity was carried out on 1 % rabbit erythrocyte solution at different temperatures a and at different erythrocyte concentrations ranging from to  % b after 60 min incubation at 37 °c. ‘b. pseudomallei’ refers to b. pseudomallei lysate;‘bpsl175’ refers to the recombinant protein; mutants are denoted by the scheme “m_original residue_residue number_substituted residue”; pbs refers to phosphate buffered saline bpsl 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, bpsl and the mutant proteins with different concentrations 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 bpsl and its mutants, the percentage of hemolysis decreased as erythrocyte concentrations increased. these results suggest that bpsl 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 bpsl and all of the mutants where similar reduction in hemolytic activity was observed at higher erythrocyte concentrations. discussion our characterization of the b. pseudomallei protein bpsl 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 superfamily that includes several putative hemolysins, other hypothetical proteins and ahl synthases - namely lasi of pseudomonas aeruginosa. the bpsl sequence can be further grouped into the cog 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 bpsl is evidence that the protein is the actual structural hemolysin and that bpsl 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 bpsl amino acids that were shared with its homologs revealed seven conserved residues 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 e 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, 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 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 r and r highlighted that these residues must play a crucial role in the activity of bpsl our investigations into the hemolytic activity of bpsl 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 bpk 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, r and r 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. our work also demonstrated that bpsl and the mutants possess a multi-hit hemolytic activity towards erythrocytes and that this n-acyltransferase enzyme is not heat stable. 
there are still numerous protein subfamilies within families and superfamilies that do not yet have conclusive empirical experimental evidence providing a specific function. sequence alignments identified seven highly conserved residues, of which several have been implicated with n-acyltransferase activity in previously characterized examples. the implication of residues previously characterized to be important for n-acyltransferase activity to hemolytic activity for the cog family members of the n-acyltransferase provides validation of the correct placement of the hemolytic capability annotation within the n-acyltransferase superfamily. here, we report the characterization of one such protein produced by the pathogenic soil bacterium burkholderia pseudomallei, bpsl which provided evidence for putative hemolysins in the cog family to have experimentally validated hemolytic activity. both the r and r mutants resulted in a loss of hemolytic activity while mutations at the other five positions resulted in either reduction or increment in hemolytic activity.
 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, bpsl which provided evidence for putative hemolysins in the cog family to have experimentally validated hemolytic activity. bpsl can be classified into the n-acyltransferase superfamily, specifically to members of the cog family. sequence alignments identified seven highly conserved residues, 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. both the r and r 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 cog family members of the n-acyltransferase provides validation of the correct placement of the hemolytic capability annotation within the n-acyltransferase superfamily. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords burkholderia pseudomalleihemolysinbpsl1375n-acyltransferaseissue-copyright-statement© the author 2015 