BACKGROUND
numerous bacterial pathogens secrete virulence factors by a type v  pathway  <cit> .
crystallographic studies of three passenger domains secreted by a classical  autotransporter pathway revealed that each has a predominantly β-helical structure  <cit> , and it is predicted that nearly all autotransporter passenger domains share a β-helical fold  <cit> .
very little is known about the structural features that are responsible for the unique properties of individual autotransporter passenger domains.
the helicobacter pylori vaca toxin is one of the most extensively studied bacterial proteins secreted by a classical autotransporter pathway  <cit> .
vaca is classified as a pore-forming toxin, but unlike many other bacterial pore-forming toxins, vaca is internalized by cells and can cause cellular alterations by acting intracellularly  <cit> .
vaca causes a wide array of alterations in mammalian cells, including cell vacuolation, mitochondrial alterations, and plasma membrane permeabilization  <cit> , and targets a variety of cell types, including gastric epithelial cells  <cit> , t cells  <cit> , and mast cells  <cit> .
several lines of evidence suggest that vaca contributes to the development of h. pylori-associated peptic ulcer disease and gastric adenocarcinoma in humans  <cit> .
vaca is synthesized as a  <dig> kda precursor protein, which undergoes proteolytic processing to yield a 33-amino acid signal sequence, a mature  <dig> kda secreted toxin, a ~ <dig> kda secreted peptide, and a carboxy-terminal domain that remains associated with the bacteria  <cit> .
the mature  <dig> kda vaca passenger domain can be proteolytically processed into an amino-terminal  <dig> kda  fragment and a carboxy-terminal  <dig> kda  fragment  <cit> , which are considered to represent two domains or subunits of vaca  <cit>  .
when expressed intracellularly in eukaryotic cells, about  <dig> residues at the amino-terminus of vaca  are sufficient to cause cell vacuolation  <cit> .
previous studies have shown that the amino-terminal hydrophobic portion of the p <dig> domain has an important role in membrane channel formation  <cit> .
components of both the p <dig> domain and the p <dig> domain are required for vaca oligomerization  <cit> , and components of the p <dig> domain are required for vaca binding to host cells  <cit> .
recently the crystal structure of the p <dig> domain of a vaca protein was determined  <cit> .
the most striking feature of this domain is the presence of a right-handed parallel β-helical structure, composed of coiled, parallel β-sheet structures .
each coil of the parallel β-helix consists of three parallel β-strands connected by loops of different lengths.
the β-helical portion of the vaca p <dig> domain of h. pylori strain  <dig> consists of about  <dig> coils   <cit> .
substitution mutagenesis of single amino acids within the amino-terminal region of the p <dig> domain is sufficient to ablate multiple activities of vaca  <cit> , but in contrast, it has been difficult to identify small inactivating mutations within the p <dig> domain  <cit> .
the only known small inactivating mutation within the p <dig> domain is a deletion of two amino acids   <cit> , which results in defective oligomerization of vaca.
since it has been difficult to identify small inactivating mutations within the p <dig> domain  <cit> , we hypothesized that large portions of the p <dig> domain might be non-essential for vacuolating toxin activity.
to test this hypothesis, in the current study we generated a set of h. pylori mutant strains expressing vaca proteins in which individual coils of the p <dig> β-helix were deleted, and we then analyzed the secretion and activity of these mutant proteins.
we report that within the vaca β-helix, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or activity, as well as a carboxy-terminal region in which similar alterations result in impaired secretion and protein misfolding.
methods
h. pylori strains and growth conditions
h. pylori wild-type strain  <dig>  was the parent strain used for construction of all mutants in this study.
the sequence of the vaca protein encoded by this strain is deposited as genbank accession number q <dig>  throughout this study, we use an amino acid numbering system in which residue  <dig> refers to alanine  <dig> of the secreted  <dig> kda vaca protein, and the p <dig> domain corresponds to amino acids  <dig> to  <dig>  h. pylori strains were grown on trypticase soy agar plates containing 5% sheep blood at 37°c in ambient air containing 5% co <dig>  h. pylori liquid cultures were grown in sulfite-free brucella broth containing 10% fetal bovine serum .
introduction of deletion mutations into the chromosomal vaca gene of h. pylori
to introduce in-frame internal deletion mutations into a plasmid encoding vaca, we performed inverse pcr using pmm <dig>   <cit>  as template dna, 5'-phosphorylated primers, and pfu turbo polymerase .
the resulting pcr products were then ligated and transformed into e. coli dh5α.
each plasmid was analyzed by dna sequencing to verify that the desired deletion was present.
to introduce the mutations into the h. pylori chromosomal vaca gene  <cit> , h. pylori strains containing a sacb-kanamycin cassette within vaca  <cit>  were transformed with plasmids containing vaca deletion mutations.
three strains , each derived from h. pylori strain  <dig> and each containing the sacb-kanamycin cassette in a different site within vaca  <cit> , were used to facilitate construction of the desired mutants.
sucrose-resistant, kanamycin-sensitive transformants were selected by growth on brucella broth plates supplemented with 10% fbs and  <dig> % sucrose  <cit> .
full-length vaca sequences encoding the secreted p <dig> vaca protein were pcr-amplified from mutant strains, and the nucleotide sequences of pcr products were analyzed to confirm that the desired mutation had been introduced successfully into the chromosomal vaca gene.
immunoblot analysis of vaca
to detect vaca expression, proteins in individual samples were separated by sds-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and immunoblotted using a polyclonal rabbit anti-vaca antiserum  raised against the secreted  <dig> kda passenger domain  <cit> , followed by horseradish peroxidase-labeled rabbit igg.
peptide mapping experiments, using a set of overlapping 16-amino-acid peptides derived from vaca, indicate that the polyclonal anti-vaca antiserum # <dig> reacts with at least  <dig> different epitopes distributed throughout the secreted  <dig> kda vaca protein, including the amino-terminus  and the carboxy-terminus  .
to confirm similar loading of lysates from wild-type and mutant h. pylori strains, the lysates were immunoblotted with rabbit antiserum to hspb   <cit> .
the anti-hspb serum was also used to detect the potential release of hspb into culture supernatant by autolysis.
signals were generated by the enhanced chemiluminescence reaction and detected using x-ray film.
preparation of broth culture supernatants and normalization of vaca concentrations
h. pylori strains were grown in bb-fbs for  <dig> hours.
broth culture supernatants were concentrated 30-fold by ultrafiltration with a  <dig> kda cutoff membrane.
relative concentrations of vaca in different broth culture supernatant preparations were determined by antigen-detection elisa  <cit> .
broth culture supernatants were diluted in carbonate buffer  and allowed to adhere to an elisa plate overnight at room temperature.
after removal of unbound vaca proteins, wells were blocked with phosphate buffered saline  containing 3% bsa and  <dig> % tween  <dig>  vaca was detected with rabbit anti-vaca antiserum  and horseradish peroxidase-labeled rabbit igg followed by tmb substrate .
to permit normalization of vaca concentrations in different preparations, samples were diluted with appropriate quantities of culture supernatant from a vaca null mutant strain, based on the antigen-detection elisa results.
sonication of h. pylori
h. pylori grown on blood agar plates were suspended in sonication buffer  and sonicated on ice for three  <dig> second pulses.
the lysate was centrifuged at  <dig>  rpm and the supernatant collected.
susceptibility of vaca to proteolysis by trypsin
h. pylori grown on blood agar plates were suspended in phosphate buffered saline , and bacterial suspensions were treated with trypsin  for  <dig> min at 37°c.
after addition of a protease inhibitor cocktail, the bacteria were pelleted, and the pellet washed once with pbs containing protease inhibitor.
the pellet was then suspended in sds lysis buffer, boiled, and analyzed by immunoblot.
sonicated preparations of h. pylori were treated with trypsin and analyzed in the same manner.
analysis of vaca reactivity with a monoclonal antibody
concentrated culture supernatants containing different vaca mutant proteins were adjusted so that the vaca concentrations were normalized, and then were diluted in carbonate buffer and allowed to adhere to an elisa plate overnight at room temperature.
after removal of unbound vaca proteins, wells were blocked with phosphate buffered saline  containing 3% bsa and  <dig> % tween  <dig>  vaca was detected with mouse anti-vaca   <cit>  and horseradish peroxidase-labeled mouse igg followed by tmb substrate .
cell culture analysis of vaca proteins
hela cells were grown as described previously  <cit> .
az- <dig> cells  and rk <dig> cells  were grown in minimal essential medium supplemented with 10% fbs and  <dig> mm non-essential amino acids.
for vacuolating assays, cells were seeded at  <dig> ×  <dig> cells/well into 96-well plates  <dig> hours prior to each experiment.
the vaca content of different samples was normalized as described above.
serial dilutions of samples were added to serum-free tissue culture medium overlying cells  and incubated for 8- <dig> hours at 37°c.
an equivalent volume of a corresponding preparation from a vaca null mutant was used as a negative control.
after incubation, cell vacuolation was examined by inverted light microscopy and quantified by a neutral red uptake assay  <cit> .
neutral red uptake data are presented as a <dig> values .
background levels of neutral red uptake by cells treated with culture supernatant from a vaca null mutant were subtracted to yield net neutral red uptake values.
RESULTS
expression and secretion of mutant vaca proteins by h. pylori
the structure of the vaca p <dig> domain is dominated by β-helical coils  <cit> .
in previous studies, it has been difficult to identify specific amino acids within the p <dig> domain that are important for toxin activity  <cit> .
to determine whether specific β-helical elements within the vaca p <dig> domain are required for vaca activity, we introduced an ordered series of eight deletion mutations, each  <dig> to  <dig> amino acids in length, into a portion of the vaca gene that encodes the p <dig> domain.
these deletion mutations were designed so that each would result in the deletion of a single coil of the β-helix .
by designing the deletion mutations in this manner, it was predicted that the mutant proteins would exhibit reductions in the length of the β-helical region but would exhibit minimal changes in protein folding in comparison to the wild-type vaca protein.
all of the deletion mutations analyzed in this study are located outside of the vaca region  previously found to be required for cell vacuolation when vaca is expressed in transiently transfected cells  <cit> .
each of the mutations was introduced into the h. pylori chromosomal vaca gene by natural transformation and allelic exchange as described in methods.
each mutant h. pylori strain was tested by immunoblot analysis for the capacity to express vaca.
we first analyzed expression of the mutant strains grown on blood agar plates.
each mutant strain expressed a vaca protein with a mass of ~ <dig> kda , which indicated that in each case, the ~ <dig> kda vaca protoxin underwent proteolytic processing similar to wild-type vaca .
we next analyzed expression and secretion of vaca when the bacteria were grown in broth culture.
wild-type h. pylori and each of the mutant strains exhibited similar patterns of growth.
immunoblot analysis of the bacterial cell pellets indicated that, as expected, each of the mutant strains expressed an ~ <dig> kda vaca protein .
in comparison to wild-type vaca, several of the mutant vaca proteins were present in reduced amounts in the bacterial cell pellets .
immunoblot analysis of the broth culture supernatants indicated that each of the mutant strains secreted or released an ~ <dig> kda vaca protein.
in comparison to secretion of vaca by the wild-type strain, several of the mutant vaca proteins were secreted at moderately reduced levels, and three mutant proteins  were nearly undetectable in culture supernatant .
analysis of the culture supernatants by elisa yielded similar results .
thus, all eight of the mutant proteins were expressed and underwent proteolytic processing similar to that of wild-type vaca, but there was substantial variation among the mutant proteins in the levels of expression and secretion.
susceptibility of vaca mutant proteins to proteolytic cleavage by trypsin
previous studies have shown that the wild-type  <dig> kda vaca passenger domain is secreted and released into the extracellular space and that  <dig> kda proteins also remain localized on the surface of h. pylori  <cit> .
to investigate whether the mutant vaca proteins were able to localize on the bacterial surface similar to wild-type vaca, the wild-type and mutant h. pylori strains were harvested from blood agar plates and treated with trypsin as described in methods.
trypsin is expected to proteolytically cleave proteins on the surface of the bacteria, but not intracellular proteins  <cit> .
each of the ~ <dig> kda mutant proteins was cleaved by trypsin , which provided evidence that these mutant vaca proteins are transported across the inner and outer membranes and localize on the surface of the bacteria.
to analyze potential differences in folding properties of the vaca mutant proteins compared to wild-type vaca, we analyzed the susceptibility of these proteins to proteolytic cleavage.
lysates of h. pylori strains were generated by sonication, and the solubilized proteins were treated with trypsin as described in methods.
trypsin digestion of two of the mutant proteins  yielded proteolytic digest patterns that were identical to each other and similar to that of trypsin-digested wild-type vaca .
trypsin digestion of two other mutant proteins  yielded different digest patterns, but these mutant proteins were not completely degraded .
four mutant proteins  were completely degraded by trypsin .
in general, the four mutant proteins that exhibited relative resistance to trypsin digestion were secreted at relatively high levels compared to mutant proteins that were completely degraded by trypsin .
the observed variation among mutant vaca proteins in susceptibility to trypsin-mediated proteolysis suggested that the individual mutant proteins differed in folding properties.
the proteins that were highly susceptible to trypsin digestion and secreted at very low levels  were probably misfolded.
due to the very low concentrations of these four proteins in the broth culture supernatants, these mutant vaca proteins were not studied further.
to evaluate whether the four mutant proteins exhibiting relative resistance to trypsin-mediated proteolysis  shared other features with wild-type vaca, we analyzed the reactivity of these proteins with an anti-vaca monoclonal antibody  that recognizes a conformational epitope  <cit> .
each of the four mutant vaca proteins was recognized by the 5e <dig> antibody , which provided additional evidence that these mutant proteins were folded in a manner similar to that of wild-type vaca.
analysis of vacuolating activity of mutant vaca proteins
we next investigated whether the mutant vaca proteins retained vacuolating toxin activity.
as a first approach, we attempted to purify the mutant vaca proteins from h. pylori broth culture supernatants, using methods that are well-established for purification of water-soluble oligomeric forms of wild-type vaca or mutant vaca proteins that contain alterations in the p <dig> domain  <cit> .
we focused these purification efforts on the four mutant proteins that were secreted at the highest levels and that exhibited evidence of protein folding similar to that of wild-type vaca .
the yields of purified mutant proteins were markedly lower than yields of purified wild-type vaca, and several of the vaca mutant proteins were not successfully purified.
these results could be attributable to relative defects in oligomerization of mutant proteins compared to wild-type vaca, or could be attributable to other altered properties of the mutant proteins that resulted in aberrant behavior during the purification procedure.
since it was not possible to purify sufficient quantities of the mutant vaca proteins to permit analysis of vacuolating toxin activity, we used an alternative approach.
h. pylori culture supernatants containing wild-type vaca or mutant proteins were normalized by elisa so that the vaca concentrations were similar, as described in methods, and then were tested for vacuolating toxin activity.
using this approach, it was possible to test the activity of the four mutant proteins that were secreted at the highest levels and that exhibited evidence of protein folding similar to that of wild-type vaca , but analysis of the remaining vaca mutant proteins  was not possible due to prohibitively low concentrations of the secreted mutant proteins and inability to normalize the concentrations of these proteins.
the mutant proteins were initially tested for ability to induce vacuolation of hela cells, a cell line that is commonly used for the study of vaca activity.
each of the mutant proteins  induced vacuolation of hela cells , but one of the mutants, vaca Δ433- <dig>  exhibited reduced vacuolating activity compared to wild-type vaca.
the same preparations of mutant proteins were then tested for their ability to induce vacuolation of az- <dig> cells  and rk <dig> cells , two cells lines that have been used for analysis of vaca activity  <cit> .
vaca Δ484- <dig>  Δ511- <dig>  and Δ517- <dig> each caused vacuolation of rk <dig> and az- <dig> cells, but vaca Δ433- <dig> lacked detectable vacuolating activity for both rk <dig> and az- <dig> cells .
thus, three of mutant proteins caused vacuolation of all the tested cell lines in a manner similar to wild-type vaca, whereas vaca Δ433- <dig> caused reduced vacuolation of hela cells and did not cause detectable vacuolation of rk <dig> or az- <dig> cells.
these data suggest that residues within the region spanning amino acids  <dig> to  <dig> contribute to vaca activity, and indicate that the β-helical coils corresponding to amino acids 484- <dig>  511- <dig> and 517- <dig> are dispensable for vacuolating toxin activity.
discussion
in this study, we sought to identify regions of the p <dig> β-helix that are either essential or non-essential for vacuolating toxin activity.
all of the vaca mutant proteins analyzed in this study were designed in a manner that resulted in the deletion of a single coil of the β-helix, based on analysis of the crystal structure of the vaca p <dig> domain  <cit> .
we predicted that all of the mutant vaca proteins would retain a β-helical structure, and that this mutagenesis approach would result in minimal disruptions in protein folding.
as a first step, we analyzed the proteolytic processing and secretion of the mutant proteins.
all eight of the mutant vaca proteins were expressed by the corresponding h. pylori mutant strains and underwent proteolytic processing to yield ~ <dig> kda passenger domains.
we found that several individual coils within the p <dig> domain could be deleted without substantially altering the capacity of the proteins to undergo secretion by h. pylori.
in contrast, the deletion of other coils led to a marked defect in vaca secretion.
the mutant proteins that exhibited marked defects in secretion also exhibited increased susceptibility to proteolytic cleavage by trypsin, which suggested that these proteins were misfolded.
in addition to the mutant vaca proteins shown in figure  <dig>  we also generated h. pylori mutant strains expressing vaca proteins in which two coils  or four coils  of the β-helix were deleted.
these mutant strains expressed truncated vaca proteins of the expected size  at levels similar to the level of wild-type vaca expression, but these mutant proteins were poorly secreted .
these findings suggest that vaca proteins containing large deletions  within the β-helical region of the p <dig> domain are poorly secreted.
similarly, a previous study reported efforts to introduce large deletions into the region of the h. pylori chromosomal vaca gene that encodes the vaca p <dig> domain, and most of the resulting mutant proteins were neither expressed nor secreted by h. pylori  <cit> .
in the current study, the three mutant vaca proteins that exhibited the most striking defects in secretion  each contained deletions that are localized near the carboxy-terminus of the β-helix.
interestingly, a study of bordetella pertussis brka revealed that a β-helical region near the carboxy-terminus of the passenger domain is required for folding of this protein  <cit> .
the authors proposed that this domain acts as an intramolecular chaperone to promote folding of the passenger domain concurrent with or following translocation through the outer membrane.
similarly, studies of b. pertussis pertactin indicate that the carboxy-terminal β-helical region of this protein exhibits enhanced stability and can fold as a stable core structure  <cit> .
we speculate that vaca amino acids 559- <dig> have a similar functional role in promoting protein folding and secretion.
an important finding in the current study is that, within the vaca β-helix, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or toxin activity.
vaca Δ484- <dig>  Δ511- <dig>  and Δ517- <dig> mutant proteins each retained vacuolating activity similar to that of wild-type vaca, which indicates that the corresponding coils are dispensable for vacuolating toxin activity.
the retention of vacuolating activity despite the deletion of entire coils of the β-helix correlates well with results from a previous study, which reported that inactivating point mutations within the portion of vaca encoding the p <dig> domain could not be identified  <cit> .
one of the vaca mutant proteins analyzed in the current study  exhibited detectable vacuolating toxin activity on hela cells, but its activity on hela cells was reduced compared to that of wild-type vaca, and it lacked detectable activity on rk <dig> cells and az <dig> cells.
these data suggest that residues within this vaca region  have an important role in vaca activity.
further studies may lead to the identification of specific amino acids within this region that mediate interactions between vaca and host cells.
similar to most previous studies, the current study assessed the effects of vaca mutations on the ability of vaca to cause cell vacuolation.
future investigations may provide new insights into structural properties of vaca that are required for other actions of this multifunctional toxin.
CONCLUSIONS
vaca is a unique toxin that is considered to be an important determinant of h. pylori virulence, and therefore, it is important to have an in-depth understanding of vaca structure and function.
the vaca p <dig> structure is predominantly a right-handed parallel β-helix, which is a characteristic of autotransporter passenger domains.
previous studies suggested that the p <dig> domain contributes to vaca binding to cells, but it has been difficult to identify small inactivating mutations within the p <dig> domain.
therefore, we hypothesized that large segments of the p <dig> domain might be non-essential for vacuolating toxin activity.
to test this hypothesis, we constructed and analyzed a set of h. pylori mutant strains expressing vaca proteins in which individual coils of the beta-helix were deleted.
three mutant proteins containing deletions in the region spanning vaca amino acids 484- <dig> were efficiently secreted and induced vacuolation of mammalian cells, which indicates that these segments are dispensable for vacuolating toxin activity.
we also identified a region near the carboxy-terminal end of the β-helix , in which the introduction of similar deletion mutations resulted in marked defects in protein secretion and apparent defects in protein folding.
we propose that non-essential β-helical coils and a carboxy-terminal β-helical segment required for proper protein folding and secretion are features of numerous autotransporter passenger domains.
authors' contributions
conceived and designed the experiments: sei, msm, dbl, tlc.
performed the experiments: sei.
analyzed the data: sei, msm, hmsa, dbl, tlc.
wrote the paper: sei, tlc.
all authors read and approved the final manuscript.

 numerous bacterial pathogens secrete virulence factors by a type v pathway. crystallographic studies of three passenger domains secreted by a classical autotransporter pathway revealed that each has a predominantly β-helical structure, and it is predicted that nearly all autotransporter passenger domains share a β-helical fold. very little is known about the structural features that are responsible for the unique properties of individual autotransporter passenger domains. the helicobacter pylori vaca toxin is one of the most extensively studied bacterial proteins secreted by a classical autotransporter pathway. vaca is classified as a pore-forming toxin, but unlike many other bacterial pore-forming toxins, vaca is internalized by cells and can cause cellular alterations by acting intracellularly. vaca causes a wide array of alterations in mammalian cells, including cell vacuolation, mitochondrial alterations, and plasma membrane permeabilization, and targets a variety of cell types, including gastric epithelial cells, t cells, and mast cells. several lines of evidence suggest that vaca contributes to the development of h. pylori-associated peptic ulcer disease and gastric adenocarcinoma in humans. vaca is synthesized as a kda precursor protein, which undergoes proteolytic processing to yield a 33-amino acid signal sequence, a mature kda secreted toxin, a ~ kda secreted peptide, and a carboxy-terminal domain that remains associated with the bacteria. the mature kda vaca passenger domain can be proteolytically processed into an amino-terminal kda fragment and a carboxy-terminal kda fragment, which are considered to represent two domains or subunits of vaca. when expressed intracellularly in eukaryotic cells, about residues at the amino-terminus of vaca are sufficient to cause cell vacuolation. previous studies have shown that the amino-terminal hydrophobic portion of the p domain has an important role in membrane channel formation. components of both the p domain and the p domain are required for vaca oligomerization, and components of the p domain are required for vaca binding to host cells. recently the crystal structure of the p domain of a vaca protein was determined. the most striking feature of this domain is the presence of a right-handed parallel β-helical structure, composed of coiled, parallel β-sheet structures. each coil of the parallel β-helix consists of three parallel β-strands connected by loops of different lengths. the β-helical portion of the vaca p domain of h. pylori strain consists of about coils. substitution mutagenesis of single amino acids within the amino-terminal region of the p domain is sufficient to ablate multiple activities of vaca, but in contrast, it has been difficult to identify small inactivating mutations within the p domain. the only known small inactivating mutation within the p domain is a deletion of two amino acids, which results in defective oligomerization of vaca. since it has been difficult to identify small inactivating mutations within the p domain, we hypothesized that large portions of the p domain might be non-essential for vacuolating toxin activity. to test this hypothesis, in the current study we generated a set of h. pylori mutant strains expressing vaca proteins in which individual coils of the p β-helix were deleted, and we then analyzed the secretion and activity of these mutant proteins. we report that within the vaca β-helix, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or activity, as well as a carboxy-terminal region in which similar alterations result in impaired secretion and protein misfolding. methods h. pylori strains and growth conditions h. pylori wild-type strain was the parent strain used for construction of all mutants in this study. the sequence of the vaca protein encoded by this strain is deposited as genbank accession number q throughout this study, we use an amino acid numbering system in which residue refers to alanine of the secreted kda vaca protein, and the p domain corresponds to amino acids to h. pylori strains were grown on trypticase soy agar plates containing 5% sheep blood at 37°c in ambient air containing 5% co h. pylori liquid cultures were grown in sulfite-free brucella broth containing 10% fetal bovine serum. introduction of deletion mutations into the chromosomal vaca gene of h. pylori to introduce in-frame internal deletion mutations into a plasmid encoding vaca, we performed inverse pcr using pmm as template dna, 5'-phosphorylated primers, and pfu turbo polymerase. the resulting pcr products were then ligated and transformed into e. coli dh5α. each plasmid was analyzed by dna sequencing to verify that the desired deletion was present. to introduce the mutations into the h. pylori chromosomal vaca gene, h. pylori strains containing a sacb-kanamycin cassette within vaca were transformed with plasmids containing vaca deletion mutations. three strains, each derived from h. pylori strain and each containing the sacb-kanamycin cassette in a different site within vaca, were used to facilitate construction of the desired mutants. sucrose-resistant, kanamycin-sensitive transformants were selected by growth on brucella broth plates supplemented with 10% fbs and % sucrose. full-length vaca sequences encoding the secreted p vaca protein were pcr-amplified from mutant strains, and the nucleotide sequences of pcr products were analyzed to confirm that the desired mutation had been introduced successfully into the chromosomal vaca gene. immunoblot analysis of vaca to detect vaca expression, proteins in individual samples were separated by sds-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and immunoblotted using a polyclonal rabbit anti-vaca antiserum raised against the secreted kda passenger domain, followed by horseradish peroxidase-labeled rabbit igg. peptide mapping experiments, using a set of overlapping 16-amino-acid peptides derived from vaca, indicate that the polyclonal anti-vaca antiserum # reacts with at least different epitopes distributed throughout the secreted kda vaca protein, including the amino-terminus and the carboxy-terminus. to confirm similar loading of lysates from wild-type and mutant h. pylori strains, the lysates were immunoblotted with rabbit antiserum to hspb. the anti-hspb serum was also used to detect the potential release of hspb into culture supernatant by autolysis. signals were generated by the enhanced chemiluminescence reaction and detected using x-ray film. preparation of broth culture supernatants and normalization of vaca concentrations h. pylori strains were grown in bb-fbs for hours. broth culture supernatants were concentrated 30-fold by ultrafiltration with a kda cutoff membrane. relative concentrations of vaca in different broth culture supernatant preparations were determined by antigen-detection elisa. broth culture supernatants were diluted in carbonate buffer and allowed to adhere to an elisa plate overnight at room temperature. after removal of unbound vaca proteins, wells were blocked with phosphate buffered saline containing 3% bsa and % tween vaca was detected with rabbit anti-vaca antiserum and horseradish peroxidase-labeled rabbit igg followed by tmb substrate. to permit normalization of vaca concentrations in different preparations, samples were diluted with appropriate quantities of culture supernatant from a vaca null mutant strain, based on the antigen-detection elisa results. sonication of h. pylori h. pylori grown on blood agar plates were suspended in sonication buffer and sonicated on ice for three second pulses. the lysate was centrifuged at rpm and the supernatant collected. susceptibility of vaca to proteolysis by trypsin h. pylori grown on blood agar plates were suspended in phosphate buffered saline, and bacterial suspensions were treated with trypsin for min at 37°c. after addition of a protease inhibitor cocktail, the bacteria were pelleted, and the pellet washed once with pbs containing protease inhibitor. the pellet was then suspended in sds lysis buffer, boiled, and analyzed by immunoblot. sonicated preparations of h. pylori were treated with trypsin and analyzed in the same manner. analysis of vaca reactivity with a monoclonal antibody concentrated culture supernatants containing different vaca mutant proteins were adjusted so that the vaca concentrations were normalized, and then were diluted in carbonate buffer and allowed to adhere to an elisa plate overnight at room temperature. after removal of unbound vaca proteins, wells were blocked with phosphate buffered saline containing 3% bsa and % tween vaca was detected with mouse anti-vaca and horseradish peroxidase-labeled mouse igg followed by tmb substrate. cell culture analysis of vaca proteins hela cells were grown as described previously. az- cells and rk cells were grown in minimal essential medium supplemented with 10% fbs and mm non-essential amino acids. for vacuolating assays, cells were seeded at × cells/well into 96-well plates hours prior to each experiment. the vaca content of different samples was normalized as described above. serial dilutions of samples were added to serum-free tissue culture medium overlying cells and incubated for 8- hours at 37°c. an equivalent volume of a corresponding preparation from a vaca null mutant was used as a negative control. after incubation, cell vacuolation was examined by inverted light microscopy and quantified by a neutral red uptake assay. neutral red uptake data are presented as a values. background levels of neutral red uptake by cells treated with culture supernatant from a vaca null mutant were subtracted to yield net neutral red uptake values.  expression and secretion of mutant vaca proteins by h. pylori the structure of the vaca p domain is dominated by β-helical coils. in previous studies, it has been difficult to identify specific amino acids within the p domain that are important for toxin activity. to determine whether specific β-helical elements within the vaca p domain are required for vaca activity, we introduced an ordered series of eight deletion mutations, each to amino acids in length, into a portion of the vaca gene that encodes the p domain. these deletion mutations were designed so that each would result in the deletion of a single coil of the β-helix. by designing the deletion mutations in this manner, it was predicted that the mutant proteins would exhibit reductions in the length of the β-helical region but would exhibit minimal changes in protein folding in comparison to the wild-type vaca protein. all of the deletion mutations analyzed in this study are located outside of the vaca region previously found to be required for cell vacuolation when vaca is expressed in transiently transfected cells. each of the mutations was introduced into the h. pylori chromosomal vaca gene by natural transformation and allelic exchange as described in methods. each mutant h. pylori strain was tested by immunoblot analysis for the capacity to express vaca. we first analyzed expression of the mutant strains grown on blood agar plates. each mutant strain expressed a vaca protein with a mass of ~ kda, which indicated that in each case, the ~ kda vaca protoxin underwent proteolytic processing similar to wild-type vaca. we next analyzed expression and secretion of vaca when the bacteria were grown in broth culture. wild-type h. pylori and each of the mutant strains exhibited similar patterns of growth. immunoblot analysis of the bacterial cell pellets indicated that, as expected, each of the mutant strains expressed an ~ kda vaca protein. in comparison to wild-type vaca, several of the mutant vaca proteins were present in reduced amounts in the bacterial cell pellets. immunoblot analysis of the broth culture supernatants indicated that each of the mutant strains secreted or released an ~ kda vaca protein. in comparison to secretion of vaca by the wild-type strain, several of the mutant vaca proteins were secreted at moderately reduced levels, and three mutant proteins were nearly undetectable in culture supernatant. analysis of the culture supernatants by elisa yielded similar results. thus, all eight of the mutant proteins were expressed and underwent proteolytic processing similar to that of wild-type vaca, but there was substantial variation among the mutant proteins in the levels of expression and secretion. susceptibility of vaca mutant proteins to proteolytic cleavage by trypsin previous studies have shown that the wild-type kda vaca passenger domain is secreted and released into the extracellular space and that kda proteins also remain localized on the surface of h. pylori. to investigate whether the mutant vaca proteins were able to localize on the bacterial surface similar to wild-type vaca, the wild-type and mutant h. pylori strains were harvested from blood agar plates and treated with trypsin as described in methods. trypsin is expected to proteolytically cleave proteins on the surface of the bacteria, but not intracellular proteins. each of the ~ kda mutant proteins was cleaved by trypsin, which provided evidence that these mutant vaca proteins are transported across the inner and outer membranes and localize on the surface of the bacteria. to analyze potential differences in folding properties of the vaca mutant proteins compared to wild-type vaca, we analyzed the susceptibility of these proteins to proteolytic cleavage. lysates of h. pylori strains were generated by sonication, and the solubilized proteins were treated with trypsin as described in methods. trypsin digestion of two of the mutant proteins yielded proteolytic digest patterns that were identical to each other and similar to that of trypsin-digested wild-type vaca. trypsin digestion of two other mutant proteins yielded different digest patterns, but these mutant proteins were not completely degraded. four mutant proteins were completely degraded by trypsin. in general, the four mutant proteins that exhibited relative resistance to trypsin digestion were secreted at relatively high levels compared to mutant proteins that were completely degraded by trypsin. the observed variation among mutant vaca proteins in susceptibility to trypsin-mediated proteolysis suggested that the individual mutant proteins differed in folding properties. the proteins that were highly susceptible to trypsin digestion and secreted at very low levels were probably misfolded. due to the very low concentrations of these four proteins in the broth culture supernatants, these mutant vaca proteins were not studied further. to evaluate whether the four mutant proteins exhibiting relative resistance to trypsin-mediated proteolysis shared other features with wild-type vaca, we analyzed the reactivity of these proteins with an anti-vaca monoclonal antibody that recognizes a conformational epitope. each of the four mutant vaca proteins was recognized by the 5e antibody, which provided additional evidence that these mutant proteins were folded in a manner similar to that of wild-type vaca. analysis of vacuolating activity of mutant vaca proteins we next investigated whether the mutant vaca proteins retained vacuolating toxin activity. as a first approach, we attempted to purify the mutant vaca proteins from h. pylori broth culture supernatants, using methods that are well-established for purification of water-soluble oligomeric forms of wild-type vaca or mutant vaca proteins that contain alterations in the p domain. we focused these purification efforts on the four mutant proteins that were secreted at the highest levels and that exhibited evidence of protein folding similar to that of wild-type vaca. the yields of purified mutant proteins were markedly lower than yields of purified wild-type vaca, and several of the vaca mutant proteins were not successfully purified. these results could be attributable to relative defects in oligomerization of mutant proteins compared to wild-type vaca, or could be attributable to other altered properties of the mutant proteins that resulted in aberrant behavior during the purification procedure. since it was not possible to purify sufficient quantities of the mutant vaca proteins to permit analysis of vacuolating toxin activity, we used an alternative approach. h. pylori culture supernatants containing wild-type vaca or mutant proteins were normalized by elisa so that the vaca concentrations were similar, as described in methods, and then were tested for vacuolating toxin activity. using this approach, it was possible to test the activity of the four mutant proteins that were secreted at the highest levels and that exhibited evidence of protein folding similar to that of wild-type vaca, but analysis of the remaining vaca mutant proteins was not possible due to prohibitively low concentrations of the secreted mutant proteins and inability to normalize the concentrations of these proteins. the mutant proteins were initially tested for ability to induce vacuolation of hela cells, a cell line that is commonly used for the study of vaca activity. each of the mutant proteins induced vacuolation of hela cells, but one of the mutants, vaca Δ433- exhibited reduced vacuolating activity compared to wild-type vaca. the same preparations of mutant proteins were then tested for their ability to induce vacuolation of az- cells and rk cells, two cells lines that have been used for analysis of vaca activity. vaca Δ484- Δ511- and Δ517- each caused vacuolation of rk and az- cells, but vaca Δ433- lacked detectable vacuolating activity for both rk and az- cells. thus, three of mutant proteins caused vacuolation of all the tested cell lines in a manner similar to wild-type vaca, whereas vaca Δ433- caused reduced vacuolation of hela cells and did not cause detectable vacuolation of rk or az- cells. these data suggest that residues within the region spanning amino acids to contribute to vaca activity, and indicate that the β-helical coils corresponding to amino acids 484- 511- and 517- are dispensable for vacuolating toxin activity. discussion in this study, we sought to identify regions of the p β-helix that are either essential or non-essential for vacuolating toxin activity. all of the vaca mutant proteins analyzed in this study were designed in a manner that resulted in the deletion of a single coil of the β-helix, based on analysis of the crystal structure of the vaca p domain. we predicted that all of the mutant vaca proteins would retain a β-helical structure, and that this mutagenesis approach would result in minimal disruptions in protein folding. as a first step, we analyzed the proteolytic processing and secretion of the mutant proteins. all eight of the mutant vaca proteins were expressed by the corresponding h. pylori mutant strains and underwent proteolytic processing to yield ~ kda passenger domains. we found that several individual coils within the p domain could be deleted without substantially altering the capacity of the proteins to undergo secretion by h. pylori. in contrast, the deletion of other coils led to a marked defect in vaca secretion. the mutant proteins that exhibited marked defects in secretion also exhibited increased susceptibility to proteolytic cleavage by trypsin, which suggested that these proteins were misfolded. in addition to the mutant vaca proteins shown in figure we also generated h. pylori mutant strains expressing vaca proteins in which two coils or four coils of the β-helix were deleted. these mutant strains expressed truncated vaca proteins of the expected size at levels similar to the level of wild-type vaca expression, but these mutant proteins were poorly secreted. these findings suggest that vaca proteins containing large deletions within the β-helical region of the p domain are poorly secreted. similarly, a previous study reported efforts to introduce large deletions into the region of the h. pylori chromosomal vaca gene that encodes the vaca p domain, and most of the resulting mutant proteins were neither expressed nor secreted by h. pylori. in the current study, the three mutant vaca proteins that exhibited the most striking defects in secretion each contained deletions that are localized near the carboxy-terminus of the β-helix. interestingly, a study of bordetella pertussis brka revealed that a β-helical region near the carboxy-terminus of the passenger domain is required for folding of this protein. the authors proposed that this domain acts as an intramolecular chaperone to promote folding of the passenger domain concurrent with or following translocation through the outer membrane. similarly, studies of b. pertussis pertactin indicate that the carboxy-terminal β-helical region of this protein exhibits enhanced stability and can fold as a stable core structure. we speculate that vaca amino acids 559- have a similar functional role in promoting protein folding and secretion. an important finding in the current study is that, within the vaca β-helix, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or toxin activity. vaca Δ484- Δ511- and Δ517- mutant proteins each retained vacuolating activity similar to that of wild-type vaca, which indicates that the corresponding coils are dispensable for vacuolating toxin activity. the retention of vacuolating activity despite the deletion of entire coils of the β-helix correlates well with results from a previous study, which reported that inactivating point mutations within the portion of vaca encoding the p domain could not be identified. one of the vaca mutant proteins analyzed in the current study exhibited detectable vacuolating toxin activity on hela cells, but its activity on hela cells was reduced compared to that of wild-type vaca, and it lacked detectable activity on rk cells and az cells. these data suggest that residues within this vaca region have an important role in vaca activity. further studies may lead to the identification of specific amino acids within this region that mediate interactions between vaca and host cells. similar to most previous studies, the current study assessed the effects of vaca mutations on the ability of vaca to cause cell vacuolation. future investigations may provide new insights into structural properties of vaca that are required for other actions of this multifunctional toxin.  vaca is a unique toxin that is considered to be an important determinant of h. pylori virulence, and therefore, it is important to have an in-depth understanding of vaca structure and function. the vaca p structure is predominantly a right-handed parallel β-helix, which is a characteristic of autotransporter passenger domains. previous studies suggested that the p domain contributes to vaca binding to cells, but it has been difficult to identify small inactivating mutations within the p domain. therefore, we hypothesized that large segments of the p domain might be non-essential for vacuolating toxin activity. to test this hypothesis, we constructed and analyzed a set of h. pylori mutant strains expressing vaca proteins in which individual coils of the beta-helix were deleted. three mutant proteins containing deletions in the region spanning vaca amino acids 484- were efficiently secreted and induced vacuolation of mammalian cells, which indicates that these segments are dispensable for vacuolating toxin activity. we also identified a region near the carboxy-terminal end of the β-helix, in which the introduction of similar deletion mutations resulted in marked defects in protein secretion and apparent defects in protein folding. we propose that non-essential β-helical coils and a carboxy-terminal β-helical segment required for proper protein folding and secretion are features of numerous autotransporter passenger domains. authors' contributions conceived and designed the experiments: sei, msm, dbl, tlc. performed the experiments: sei. analyzed the data: sei, msm, hmsa, dbl, tlc. wrote the paper: sei, tlc. all authors read and approved the final manuscript. 
vaca, a toxin secreted by h. pylori, is comprised of two domains, designated p and p analysis of the crystal structure of the p domain indicated that its structure is predominantly a right-handed parallel β-helix, which is a characteristic of autotransporter passenger domains. we propose that non-essential β-helical coils and a carboxy-terminal β-helical segment required for proper protein folding and secretion are features shared by numerous autotransporter passenger domains. these results indicate that within the β-helical segment of the vaca p domain, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or activity, as well as a carboxy-terminal region in which similar alterations result in protein misfolding and impaired secretion. to test this hypothesis, we introduced eight deletion mutations into the h. pylori chromosomal vaca gene. therefore, we hypothesized that large portions of the p domain might be non-essential for vacuolating toxin activity. three mutant proteins were secreted and induced vacuolation of mammalian cells, which indicated that these β-helical coils were dispensable for vacuolating toxin activity. one mutant protein exhibited reduced vacuolating toxin activity compared to wild-type vaca. other mutant proteins, including those containing deletions near the carboxy-terminal end of the β-helical region, exhibited marked defects in secretion and increased susceptibility to proteolytic cleavage by trypsin, which suggested that these proteins were misfolded. all eight of the mutant vaca proteins were expressed by the corresponding h. pylori mutant strains and underwent proteolytic processing to yield ~ kda passenger domains. helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. substitution mutations of specific amino acids within the p domain abrogate vaca activity, but thus far, it has been difficult to identify small inactivating mutations within the p domain.
 helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. vaca, a toxin secreted by h. pylori, is comprised of two domains, designated p and p analysis of the crystal structure of the p domain indicated that its structure is predominantly a right-handed parallel β-helix, which is a characteristic of autotransporter passenger domains. substitution mutations of specific amino acids within the p domain abrogate vaca activity, but thus far, it has been difficult to identify small inactivating mutations within the p domain. therefore, we hypothesized that large portions of the p domain might be non-essential for vacuolating toxin activity. to test this hypothesis, we introduced eight deletion mutations into the h. pylori chromosomal vaca gene.  all eight of the mutant vaca proteins were expressed by the corresponding h. pylori mutant strains and underwent proteolytic processing to yield ~ kda passenger domains. three mutant proteins were secreted and induced vacuolation of mammalian cells, which indicated that these β-helical coils were dispensable for vacuolating toxin activity. one mutant protein exhibited reduced vacuolating toxin activity compared to wild-type vaca. other mutant proteins, including those containing deletions near the carboxy-terminal end of the β-helical region, exhibited marked defects in secretion and increased susceptibility to proteolytic cleavage by trypsin, which suggested that these proteins were misfolded.  these results indicate that within the β-helical segment of the vaca p domain, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or activity, as well as a carboxy-terminal region in which similar alterations result in protein misfolding and impaired secretion. we propose that non-essential β-helical coils and a carboxy-terminal β-helical segment required for proper protein folding and secretion are features shared by numerous autotransporter passenger domains. 