BACKGROUND
burkholderia pseudomallei is a gram-negative bacterium readily recovered from the water and wet soils of endemic areas bordering the equator, particularly southeast asia and northern australia  <cit> .
the organism is a motile, aerobic bacillus that can survive environmental extremes as well as the bactericidal activities of complement  <cit> , defensins  <cit> , and phagocytes  <cit> .
the genome of the b. pseudomallei isolate k <dig> has been published by the wellcome trust sanger institute and was shown to consist of two chromosomes of  <dig>  and  <dig>  mbp  <cit> .
burkholderia mallei is a non-motile, host-adapted clone of b. pseudomallei that does not persist outside of its equine host and is endemic to certain parts of asia, africa, the middle east and south america  <cit> .
the genomic sequence of the b. mallei strain atcc <dig> has been published by tigr  <cit>  and is smaller  than that of b. pseudomallei k <dig>  b. mallei atcc <dig> was found to specify a large number of mobile dna elements that have contributed to extensive deletions and rearrangements relative to the genome of b. pseudomallei k <dig>  despite these differences, the genes shared by the two isolates have an average identity of 99% at the nucleotide level  <cit> .
the genomic sequence of several b. pseudomallei and b. mallei isolates are also publicly available through the ncbi genomic blast service , which provides a wealth of resources to study these organisms.
b. pseudomallei causes the human disease melioidosis, which is notoriously difficult to diagnose.
clinical manifestations vary greatly and may present as flu-like symptoms, benign pneumonitis, acute and chronic pneumonia, or fulminating septicemia.
infection occurs via inhalation of contaminated aerosol particles or through skin abrasions, and the risk of contracting the disease is proportional to the concentration of b. pseudomallei in soil and water.
in endemic areas, heavy rainfalls result in a shift from percutaneous inoculation to inhalation as the primary mode of infection, which leads to a more severe illness.
melioidosis commonly affects the lungs and is characterized by the spread of bacteria to various internal organs including the spleen and liver.
many patients become bacteremic and the mortality rate is high  despite aggressive antimicrobial therapy  <cit> .
b. pseudomallei is refractory to most antibiotics and resistance mechanisms include efflux pumps and β-lactamases  <cit> .
the recommended treatment entails the use of ceftazidime, carbapenems, tmp-smz, chloramphenicol and/or augmentin for several weeks.
response to treatment is slow and eradication of b. pseudomallei is difficult to achieve, resulting in recrudescence  <cit> .
b. mallei causes the zoonosis glanders, which primarily affects solipeds  <cit> .
in humans, infection occurs by contact with infected animals via the cutaneous or respiratory route.
the clinical manifestations of the disease include febrile pneumonia associated with necrosis of the tracheobronchial tree or pustular skin lesions and the development of abscesses.
most patients become bacteremic and b. mallei disseminates to the liver and spleen where it rapidly causes necrosis.
even with antibiotic treatment, the mortality rate for human glanders is 50% and the basis for this high mortality rate is not understood, though b. mallei has been shown to be resistant to complement-mediated killing  <cit> , macrophages  <cit>  and antimicrobials  <cit> .
one key aspect of pathogenesis by b. mallei and b. pseudomallei is their ability to invade and multiply within a variety of eukaryotic cells, where bacteria are shielded from the host humoral immune response and antibiotics.
once internalized, b. mallei and b. pseudomallei escape from endocytic vacuoles and enter the cytoplasm of infected cells where they multiply.
the organisms subsequently spread to neighboring cells through a process involving the formation of actin tails and membrane protrusions that push bacteria from one cell to another.
this intracellular lifestyle is crucial to virulence and has been a focus of research efforts aimed at understanding pathogenesis by b. mallei and b. pseudomallei  <cit> .
several gene products, such as bima, type  <dig> secretion system effectors, and type  <dig> secretion proteins, have been shown to play key roles in this process.
by contrast, the mechanisms used by these organisms to adhere to eukaryotic cells are poorly defined.
adherence is an essential step of pathogenesis by most infectious agents because it is necessary for colonizing a new host  <cit> .
moreover, b. pseudomallei and b. mallei are facultative intracellular pathogens that gain access to the interior of target cells.
though not always a prerequisite for this process, bacterial adherence is a widespread strategy that precedes and promotes invasion  <cit> .
thus far, only the b. pseudomallei flagellum  <cit>  and type  <dig> pilus  <cit>  have been implicated in adherence and their exact roles remain to be elucidated.
the present study reports the identification of b. pseudomallei and b. mallei gene products that mediate adherence to epithelial cells derived from the human respiratory tract, thus relevant to the aerosol route of infection by these organisms.
RESULTS
identification of a gene shared by b. mallei and b. pseudomallei that encodes a potential autotransporter adhesin
analysis of the annotated genomic sequence of b. mallei atcc <dig> identified the orf locus tag number bmaa <dig> as resembling members of the oligomeric coiled-coil adhesin  family of autotransporter proteins  <cit> .
yersinia enterocolitica yada  <cit>  is the prototypical member of this group of adherence factors, which also includes haemophilus influenzae hia  <cit>  and moraxella catarrhalis hag  <cit> .
these oca proteins share structural features including a c-terminal outer membrane  anchor domain composed of  <dig> β-strands , a surface-exposed passenger domain often containing repeated amino acid  motifs, and a helical region of ~ <dig> residues that connects the om anchor to the surface-exposed passenger domain  <cit> .
as illustrated in fig 1a, bmaa <dig> is predicted to possess these features.
further sequence analysis of the b. mallei atcc <dig> gene product revealed that residues 208- <dig>  contain repeats with the consensus xxxavaigxxxax , which resemble motifs found in the n-terminus of y. enterocolitica yada   <cit>  and m. catarrhalis hag   <cit> .
in yada, these aig patterns have been shown to form a structure termed a β-roll and to specify adhesive properties.
the passenger domain of bmaa <dig> was also found to contain several serine-rich repeats beginning with residues slst .
additionally, searches using the pfam database indicated that aa 1456- <dig> of bmaa <dig> encode a yada-like c-terminal domain , which is present in most oca molecules and is described as important for oligomerization.
taken together, these observations suggest structural and functional similarities between bmaa <dig> and members of the oca family of autotransporters.
hence, we designated this orf of b. mallei atcc <dig> boaa .
table  <dig> lists characteristics of the boaa gene and its encoded product.
asequence analyses were performed using vector nti  and online tools available through the expasy proteomics server.
bthe putative signal sequence cleavage site was determined using the signalp  <dig>  server
nd = not determined
the published genome of b. pseudomallei k <dig> was also found to specify a boaa gene product  that is  <dig> % identical to that of b. mallei atcc <dig>  oligonucleotide primers were designed to amplify the entire boaa gene from the b. pseudomallei strain used in our laboratory, dd <dig>  and sequence analysis of this amplicon predicted a gene product that is  <dig> % and  <dig> % identical to boaa of b. mallei atcc <dig> and b. pseudomallei k <dig>  respectively.
database searches with the ncbi genomic blast service also identified boaa in several b. pseudomallei and b. mallei isolates.
all nine b. mallei and  <dig> b. pseudomallei strains for which sequences are available through this service were found to have the gene.
characteristics of some of these orfs are listed in tables  <dig> and  <dig>  overall, the boaa proteins are 82-94% identical and differ primarily in the number and/or arrangement of slst repeats in their predicted passenger domains .
based on these results, we conclude that boaa is a well-conserved gene product shared by b. mallei and b. pseudomallei.
bm = b. mallei
bp = b. pseudomallei
identification of a b. pseudomallei-specific gene encoding a putative autotransporter adhesin that resembles boaa
further analysis of the annotated genomic sequence of b. pseudomallei k <dig> identified the orf locus tag number bpsl <dig> as specifying a second oca-like protein that is ~60% identical to boaa.
the last  <dig> aa of bpsl <dig> and boaa are  <dig> % identical  and the very last  <dig> residues, which encompass the predicted c-terminal om-anchoring domain and α-helical region of the molecules, were found to be particularly well-conserved .
the bpsl <dig> orf is predicted to encode a protein of 148-kda which, as depicted in fig 1c, possesses many of the structural features observed in boaa including two sets of β-roll aig motifs with the consensus xxgaigxxxax and several slst repeats.
this high level of sequence and structural similarity between bpsl <dig> and boaa prompted us to designate this b. pseudomallei k <dig> gene product boab.
the boab gene was sequenced from b. pseudomallei dd <dig> and was predicted to encode a protein that is  <dig> % identical to boab of b. pseudomallei k <dig>  database searches using ncbi genomic blast revealed that the genomes of at least  <dig> more b. pseudomallei strains contain the gene.
overall, the boab proteins are highly-conserved  and characteristics of the orf from selected strains are shown in tables  <dig> and  <dig> and fig  <dig> for comparison purposes.
importantly, database searches also revealed that none of the b. mallei isolates available through the ncbi genomic blast service have a boab gene.
taken together, these results indicate that boab is a highly-conserved b. pseudomallei-specific molecule.
expression of the burkholderia boaa and boab proteins in e. coli
because of their sequence and structural similarities to known bacterial adhesins, we hypothesized that boaa and boab mediate adherence to human epithelial cells.
to test this hypothesis, the b. mallei atcc <dig> boaa and b. pseudomallei dd <dig> boab genes were cloned into the e. coli strain epi <dig>  this organism does not normally adhere well to human epithelial cells  <cit>  and therefore provides an appropriate heterologous genetic background for examining the adhesive properties of boaa and boab.
to verify gene expression, rna was prepared from e. coli harboring the plasmids pcc <dig>  , pslboaa  and pslboab , and analyzed by quantitative reverse-transcriptase pcr .
fig 3a demonstrates that the boaa and boab genes are expressed by recombinant bacteria and that the primers used in these experiments are specific for their corresponding genes.
sarkosyl-insoluble om proteins were also extracted from e. coli cells and analyzed by western blot to ensure production of the burkholderia proteins.
fig 3b shows that α-boaa antibodies  react with a band of 130-kda in the om of e. coli expressing boaa  whereas abs against boab bind to a 140-kda antigen in e. coli expressing boab .
these molecular weights  are consistent with the predicted masses of the gene products .
in addition to showing that boaa and boab are associated with the om by protein separation and western blot, we used immunofluorescent labeling of non-permeabilized e. coli cells to demonstrate their display on the bacterial surface.
as depicted in fig 3c, e. coli harboring pslboaa and pslboab are labeled by the α-boaa and α-boab abs, respectively, while recombinant bacteria carrying the control plasmid pcc <dig>  are not.
staining of nucleic acids with the fluorescent dye dapi verified that comparable numbers of bacterial cells were examined .
quantitative attachment assays revealed that e. coli expressing boab attach to hep <dig>  and a <dig>  epithelial cell lines at levels 18- and 68-fold greater than bacteria carrying pcc <dig> , respectively .
in addition, boab expression was found to increase adherence to differentiated primary cultures of normal human bronchial epithelium .
under the growth conditions used, nhbe cultures form a pseudostratified epithelium with tight junctions containing both ciliated and non-ciliated cells.
this epithelium exhibits transepithelial resistance, mucus secretion, mucociliary activity, and an apical surface not submerged in tissue culture medium, thus representing an environment that is similar to the airway lumen in vivo  <cit> .
expression of the b. mallei atcc <dig> boaa protein on the surface of e. coli also substantially increased adherence to monolayers of a <dig> and hep <dig> cells and to nhbe cultures.
taken together, these data demonstrate that boaa and boab are om proteins mediating adherence to epithelial cells of the human respiratory tract.
b. pseudomallei and b. mallei are facultative intracellular organisms that can invade, survive and replicate in a variety of eukaryotic cells.
moreover, autotransporter adhesins often specify additional biological functions such as invasion  <cit> , biofilm formation  <cit> , survival within host cells  <cit>  and intracellular motility  <cit> .
for these reasons, we measured the ability of e. coli expressing boaa and boab to invade epithelial cells as well as their ability to survive within murine macrophages.
we also measured the ability of these recombinant strains to form biofilms on the plastic support of tissue culture plates using a crystal violet-based assay.
the results of these experiments indicated that neither boaa nor boab substantially increase invasion of epithelial cells, phagocytosis of recombinant bacteria by j774a.
<dig> murine macrophages, survival inside these immune cells, or biofilm formation .
construction and characterization of burkholderia mutant strains
to study the functional properties of the boa gene products in the native burkholderia background, we constructed isogenic boaa mutants of b. pseudomallei dd <dig> and b. mallei atcc <dig> as well as an isogenic boab mutant of b. pseudomallei dd <dig>  a double mutant strain was also engineered in which inactivated versions of both boaa and boab were introduced in the genome of b. pseudomallei dd <dig>  whole cell lysates and sarkosyl-insoluble om proteins were prepared from these strains and analyzed by western blot to verify lack of boaa and boab expression in the mutants.
the α-boaa and α-boab abs, however, did not react with burkholderia protein preparations .
in order to determine whether the genes are expressed, total rna was isolated from b. pseudomallei dd <dig> and b. mallei atcc <dig> and the relative transcript levels of boaa and boab were assessed by qrt-pcr.
fig  <dig> shows that boaa and boab are expressed by b. pseudomallei while b. mallei only expresses boaa, which is in agreement with database searches revealing that b. mallei isolates do not contain a boab gene.
the qrt-pcr data also demonstrate that the genes are expressed at very low levels compared to burkholderia reca, which was used to normalize boaa and boab transcript levels.
these results are consistent with our inability to visualize the proteins by western blot.
other methods such as immunoprecipitation and immunofluorescence labeling also proved unsuccessful at detecting production of boaa and boab by burkholderia strains.
quantitative attachment assays with recombinant bacteria indicated that boaa or boab expression significantly increases the adherence of e. coli to monolayers of a <dig> and hep <dig> cells and to nhbe cultures .
we therefore compared the ability of burkholderia parent and boa mutant strains to attach to these respiratory cells.
as shown in fig 5a and 5d, inactivation of the boaa gene in b. mallei atcc <dig> and b. pseudomallei dd <dig> decreases adherence to a <dig> cells by  <dig> and 53%, respectively.
the boaa mutation also caused a 50% reduction in the binding of b. pseudomallei to hep <dig> monolayers , and reduced adherence of b. mallei to these laryngeal cells by 67% .
moreover, both boaa mutant strains displayed significant impairment in their abilities to attach to nhbe cultures .
the boab mutation in b. pseudomallei dd <dig> decreased attachment to a <dig> and hep <dig> cells by ~50%  and caused a 62% reduction in adherence to nhbe cultures .
as expected, the double mutant strain dd <dig> boaa.boab exhibited significantly lower attachment to epithelial cells compared to the parent strain dd <dig> .
the adherence levels of the double mutant, however, did not differ significantly from that of the single mutants in any of the cell types tested.
one possible explanation for this apparent lack of synergistic effect is that other adhesins expressed by the double mutant strain dd <dig> boaa.boab provide a high background level of adherence.
taken together, these results demonstrate that the boaa and boab gene products contribute to the adherence of b. mallei and b. pseudomallei to epithelial cells of the human respiratory tract.
as previously stated, autotransporter adhesins often specify additional biological functions including survival within host cells  <cit> .
in addition, b. pseudomallei and b. mallei are facultative intracellular pathogens that are particularly proficient at replicating inside professional phagocytic cells.
for these reasons, we measured the ability of our panel of burholderia mutant and parent strains to replicate within j774a.
<dig> murine macrophages.
in b. pseudomallei dd <dig>  inactivation of the boa genes had no effect on phagocytosis of the organism .
once inside macrophages, the boaa  and boab  single mutants replicated at rates equivalent to that of the progenitor strain dd <dig> .
however, when both boaa and boab genes were disrupted , intracellular growth was diminished by 60% .
to verify that this reduced intracellular fitness was not due to a global growth defect, we measured the growth of strains dd <dig> and dd <dig> boaa.boab in broth as well as in tissue culture medium.
we found that both strains grew at equivalent rates under both conditions .
interestingly, the double mutant did not exhibit a growth defect in epithelial cells .
these results suggest a role for the boaa and boab proteins in b. pseudomallei's ability to grow inside professional phagocytes.
no defect in uptake or intracellular growth was measured for the b. mallei atcc <dig> boaa mutant strain .
it should also be noted that none of the boa mutants showed decreased biofilm formation on the plastic support of tissue culture plates nor defects in resistance to the bactericidal activity of normal human serum , both biological functions that are also commonly associated with oca autotransporter adhesins  <cit> .
discussion
autotransporters are involved in various biological traits of gram-negative bacteria including invasion  <cit> , serum resistance  <cit> , phospholipolysis  <cit> , cytotoxicity  <cit> , adherence  <cit> , biofilm formation  <cit> , survival within eukaryotic cells  <cit>  and intracellular motility  <cit> .
these proteins share an n-terminal extracellular passenger domain that specifies the biological activity of the autotransporter and a c-terminus containing several β-strands, which tether the molecule to the om.
based on the structure of this membrane-anchoring domain, autotransporters can be classified as conventional  or trimeric   <cit> .
one of the best characterized trimeric autotransporters is the y. enterocolitica adhesin yada.
this protein, along with structurally-related adherence proteins such as m. catarrhalis hag and h. influenzae hia, are often referred to as oligomeric coiled-coil adhesins   <cit> .
tiyawisutsri and colleagues previously reported that the published genomic sequences of b. pseudomallei k <dig> and b. mallei atcc <dig> contain several orfs encoding putative trimeric autotransporters  <cit> .
of these, only bima  has been functionally characterized and shown to be required for actin-based motility of the organisms inside eukaryotic cells  <cit> .
in the present study, we identified the boaa orf based on similarities to the oca proteins y. enterocolitica yada and m. catarrhalis hag.
specifically, we searched the genome of b. mallei atcc <dig> for gene products specifying n-terminal aig β-roll motifs, a transporter module containing  <dig> β-strands, and a yada-like c-terminal domain .
we demonstrated that when expressed by e. coli, boaa increases adherence to the human epithelial cell lines hep <dig>  and a <dig>  grown as monolayers in submerged cultures.
though these cell types are relevant to the aerosol route of infection by b. mallei and b. pseudomallei, they lack important features of the airway mucosa such as cilia and mucociliary activity.
the ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving and contribute to preventing colonization by pathogens.
for these reasons, we also measured the adherence of e. coli expressing boaa to cultures of normal human bronchial epithelium  grown in an air-liquid interface system.
these cultures mimic the structure and function of the airway mucosa more accurately as they are fully differentiated, form a pseudostratified epithelium with tight junctions, contain ciliated and mucus-producing goblet cells, and exhibit mucociliary activity  <cit> .
quantitative attachment assays utilizing this culture system revealed that boaa expression increases adherence to nhbe cultures .
in addition to showing that boaa specifies adhesive properties when expressed in the heterologous genetic background of e. coli, we determined that disruption of the boaa gene in the genome of b. mallei atcc <dig> reduces adherence of the organism to monolayers of hep <dig> and a <dig> cells and to nhbe cultures, therefore substantiating the function of boaa as an adhesin.
database searches using the ncbi genomic blast service identified boaa in several b. pseudomallei and b. mallei isolates and we demonstrated that inactivation of boaa in the b. pseudomallei strain dd <dig> also decreases attachment to hep <dig> laryngeal cells, a <dig> pneumocytes, and nhbe cultures.
together, our data indicate that boaa is an adhesin common to b. mallei and b. pseudomallei and mediates adherence to host cells relevant to pathogenesis by the organisms.
these findings are consistent with the recent inclusion of boaa  in the virulome of b. mallei and b. pseudomallei, which consists of a set of  <dig> putative virulence genes that are shared by b. pseudomallei and b. mallei but are not present in five closely-related non-pathogenic burkholderia species  <cit> .
comparative genomic analyses revealed that several b. pseudomallei isolates possess a second oca-like gene product highly similar to boaa, which we termed boab.
the c-terminus of boab is strikingly similar to that of boaa  and the predicted passenger domains of the molecules contain numerous matching serine-rich slst motifs .
the proteins are also functionally related as they mediate adherence to the same types of host cells .
therefore, it is tempting to speculate that boaa and boab are the result of gene duplication.
this hypothesis would be consistent with the genomic organization of the genes.
in b. pseudomallei strains k <dig>  1710b,  <dig>   <dig> and mshr <dig>  the boab gene is located on chromosome  <dig> while boaa is on chromosome  <dig>  moreover, the boab gene in all these isolates is preceded by two orfs specifying an invertase and a transposase.
these genes may be the remnants of mobile genetic elements possibly involved in gene duplication.
database searches also revealed that b. mallei isolates do not possess a boab gene, which was likely lost during evolution of the organism into a host-adapted pathogen.
interestingly, the closely-related bacterium burkholderia thailandensis has been reported by others to bind poorly to epithelial cells  <cit> .
this organism exhibits high genomic similarities to b. pseudomallei and b. mallei and, like b. pseudomallei, is a natural inhabitant of the tropical soil environment.
however, b. thailandensis is not considered pathogenic to humans or higher animals  <cit> .
this difference in virulence can be attributed to the fact that b. thailandensis does not produce a capsule  <cit>  and lacks the  <dig> genes comprising the aforementioned virulome of b. mallei and b. pseudomallei.
analysis of the published genome of the b. thailandensis strain e <dig>  <cit>  indicated that it contains neither the boaa nor the boab gene.
b. pseudomallei dd <dig> and b. mallei atcc <dig> do not produce detectable amounts of the boaa and boab proteins under the conditions tested.
these results are consistent with qrt-pcr experiments demonstrating that the organisms express very low levels of the boa genes relative to the burkholderia reca control .
similar observations were made by druar and colleagues while studying expression of the burkholderia type  <dig> secretion system- <dig>  proteins bipb and bipd  <cit> .
these proteins were not detected in lysates of b. pseudomallei or b. mallei grown under different conditions, even though the antibodies used in their western blot experiments recognized recombinant forms of bipb and bipd.
the authors concluded that these two t3ss- <dig> molecules must be expressed in detectable amounts only under very specific in vitro conditions  <cit> .
using a gfp reporter strain, burtnick et al recently showed that the b. mallei type  <dig> secretion system- <dig>  gene tsse is not expressed at detectable levels when bacteria are grown in lslb or tissue culture medium, but is expressed upon phagocytosis of the organisms by murine macrophages  <cit> .
the protein preparations tested in our studies were obtained from bacteria cultured on lslb agar plates at 37°c, conditions which may not be optimal for expression of the boaa and boab proteins.
additionally, chantratita and colleagues reported that growth of b. pseudomallei under various conditions triggers a complex adaptive process altering the expression of surface molecules  <cit> .
this process, termed phenotypic plasticity, was correlated with changes in the morphology of b. pseudomallei colonies grown on agar plates and appears to modulate the environmental fitness, as well as virulence, of the organism.
given their surface location and likely role in virulence , it is possible that boaa and boab are subject to phenotypic plasticity and are expressed in detectable amounts only under very specific in vitro conditions.
in concordance, the reduced adherence phenotype of the boaa and boab mutant strains suggests increased level of expression of the genes when burkholderia is incubated with epithelial cells.
however, efforts to detect protein expression under these conditions  have been unsuccessful.
of further note, studies have shown that sera from horses infected with b. mallei and sera from melioidosis patients contain antibodies reacting with boaa   <cit>  and with boab  <cit> , respectively, which indicates expression of the autotransporters in vivo.
determining the conditions and mechanisms that modulate expression of the boa adhesins, and their influence on the binding of b. pseudomallei and b. mallei to host surfaces, represent key areas for future study.
disruption of boaa and boab in the b. pseudomallei double mutant strain dd <dig> boaa.boab was found to have a significant effect on the growth of the organism within murine macrophages .
at present, it is not clear whether boaa and boab play a direct role in intracellular replication.
it is possible that the absence of both boa proteins in the om of dd <dig> boaa.boab affects the proper surface display of another molecule involved in this phenotypic trait.
one candidate is lps, as this molecule was previously shown to play an important role in the ability of b. pseudomallei to grow inside host cells  <cit> .
b. pseudomallei produces multiple t3ss and t6ss that are involved in the intracellular lifestyle of the organism.
these specialized secretion apparatuses are used to inject bacterial effector proteins inside host cells where they exert cytopathic effects or manipulate signaling pathways.
one important step in this process is the proper docking of bacteria to the host cell to deliver the effectors.
given their roles in adherence, it is possible that the lack of expression of the boaa and boab gene products interferes with the delivery of t3ss and/or t6ss cell-altering effectors, which in turn reduces the intracellular fitness of the double mutant strain dd <dig> boaa.boab.
the yersinia pestis om adhesin ail was recently shown to affect delivery of yop effector proteins to hep <dig> cells and macrophages in such a manner  <cit> .
alternatively, the reduced intracellular growth of the double boaa boab mutant may be due to a greater sensitivity to immune effectors produced by the macrophages.
the molecular basis for this phenotype is currently being investigated.
CONCLUSIONS
the present study reports the identification of b. pseudomallei and b. mallei gene products mediating adherence to epithelial cells.
because of their classification as select agents, there is currently a shortage of tools for genetic studies in b. pseudomallei and b. mallei , which precluded us from complementing mutants.
our ability to express boaa and boab in e. coli, however, conclusively demonstrates that the proteins directly mediate binding to epithelial cells.
these results, along with our analyses of the mutant strains, clearly establish that these molecules participate in adherence by b. pseudomallei and b. mallei.
adherence is an essential step in pathogenesis by most infectious agents because it is necessary for colonization and precedes invasion of host cells by intracellular pathogens.
thus, continued investigation of boaa and boab will yield important information regarding the biology and virulence of these organisms.

 burkholderia pseudomallei is a gram-negative bacterium readily recovered from the water and wet soils of endemic areas bordering the equator, particularly southeast asia and northern australia. the organism is a motile, aerobic bacillus that can survive environmental extremes as well as the bactericidal activities of complement, defensins, and phagocytes. the genome of the b. pseudomallei isolate k has been published by the wellcome trust sanger institute and was shown to consist of two chromosomes of and mbp. burkholderia mallei is a non-motile, host-adapted clone of b. pseudomallei that does not persist outside of its equine host and is endemic to certain parts of asia, africa, the middle east and south america. the genomic sequence of the b. mallei strain atcc has been published by tigr and is smaller than that of b. pseudomallei k b. mallei atcc was found to specify a large number of mobile dna elements that have contributed to extensive deletions and rearrangements relative to the genome of b. pseudomallei k despite these differences, the genes shared by the two isolates have an average identity of 99% at the nucleotide level. the genomic sequence of several b. pseudomallei and b. mallei isolates are also publicly available through the ncbi genomic blast service, which provides a wealth of resources to study these organisms. b. pseudomallei causes the human disease melioidosis, which is notoriously difficult to diagnose. clinical manifestations vary greatly and may present as flu-like symptoms, benign pneumonitis, acute and chronic pneumonia, or fulminating septicemia. infection occurs via inhalation of contaminated aerosol particles or through skin abrasions, and the risk of contracting the disease is proportional to the concentration of b. pseudomallei in soil and water. in endemic areas, heavy rainfalls result in a shift from percutaneous inoculation to inhalation as the primary mode of infection, which leads to a more severe illness. melioidosis commonly affects the lungs and is characterized by the spread of bacteria to various internal organs including the spleen and liver. many patients become bacteremic and the mortality rate is high despite aggressive antimicrobial therapy. b. pseudomallei is refractory to most antibiotics and resistance mechanisms include efflux pumps and β-lactamases. the recommended treatment entails the use of ceftazidime, carbapenems, tmp-smz, chloramphenicol and/or augmentin for several weeks. response to treatment is slow and eradication of b. pseudomallei is difficult to achieve, resulting in recrudescence. b. mallei causes the zoonosis glanders, which primarily affects solipeds. in humans, infection occurs by contact with infected animals via the cutaneous or respiratory route. the clinical manifestations of the disease include febrile pneumonia associated with necrosis of the tracheobronchial tree or pustular skin lesions and the development of abscesses. most patients become bacteremic and b. mallei disseminates to the liver and spleen where it rapidly causes necrosis. even with antibiotic treatment, the mortality rate for human glanders is 50% and the basis for this high mortality rate is not understood, though b. mallei has been shown to be resistant to complement-mediated killing, macrophages and antimicrobials. one key aspect of pathogenesis by b. mallei and b. pseudomallei is their ability to invade and multiply within a variety of eukaryotic cells, where bacteria are shielded from the host humoral immune response and antibiotics. once internalized, b. mallei and b. pseudomallei escape from endocytic vacuoles and enter the cytoplasm of infected cells where they multiply. the organisms subsequently spread to neighboring cells through a process involving the formation of actin tails and membrane protrusions that push bacteria from one cell to another. this intracellular lifestyle is crucial to virulence and has been a focus of research efforts aimed at understanding pathogenesis by b. mallei and b. pseudomallei. several gene products, such as bima, type secretion system effectors, and type secretion proteins, have been shown to play key roles in this process. by contrast, the mechanisms used by these organisms to adhere to eukaryotic cells are poorly defined. adherence is an essential step of pathogenesis by most infectious agents because it is necessary for colonizing a new host. moreover, b. pseudomallei and b. mallei are facultative intracellular pathogens that gain access to the interior of target cells. though not always a prerequisite for this process, bacterial adherence is a widespread strategy that precedes and promotes invasion. thus far, only the b. pseudomallei flagellum and type pilus have been implicated in adherence and their exact roles remain to be elucidated. the present study reports the identification of b. pseudomallei and b. mallei gene products that mediate adherence to epithelial cells derived from the human respiratory tract, thus relevant to the aerosol route of infection by these organisms.  identification of a gene shared by b. mallei and b. pseudomallei that encodes a potential autotransporter adhesin analysis of the annotated genomic sequence of b. mallei atcc identified the orf locus tag number bmaa as resembling members of the oligomeric coiled-coil adhesin family of autotransporter proteins. yersinia enterocolitica yada is the prototypical member of this group of adherence factors, which also includes haemophilus influenzae hia and moraxella catarrhalis hag. these oca proteins share structural features including a c-terminal outer membrane anchor domain composed of β-strands, a surface-exposed passenger domain often containing repeated amino acid motifs, and a helical region of ~ residues that connects the om anchor to the surface-exposed passenger domain. as illustrated in fig 1a, bmaa is predicted to possess these features. further sequence analysis of the b. mallei atcc gene product revealed that residues 208- contain repeats with the consensus xxxavaigxxxax, which resemble motifs found in the n-terminus of y. enterocolitica yada and m. catarrhalis hag. in yada, these aig patterns have been shown to form a structure termed a β-roll and to specify adhesive properties. the passenger domain of bmaa was also found to contain several serine-rich repeats beginning with residues slst. additionally, searches using the pfam database indicated that aa 1456- of bmaa encode a yada-like c-terminal domain, which is present in most oca molecules and is described as important for oligomerization. taken together, these observations suggest structural and functional similarities between bmaa and members of the oca family of autotransporters. hence, we designated this orf of b. mallei atcc boaa. table lists characteristics of the boaa gene and its encoded product. asequence analyses were performed using vector nti and online tools available through the expasy proteomics server. bthe putative signal sequence cleavage site was determined using the signalp server nd = not determined the published genome of b. pseudomallei k was also found to specify a boaa gene product that is % identical to that of b. mallei atcc oligonucleotide primers were designed to amplify the entire boaa gene from the b. pseudomallei strain used in our laboratory, dd and sequence analysis of this amplicon predicted a gene product that is % and % identical to boaa of b. mallei atcc and b. pseudomallei k respectively. database searches with the ncbi genomic blast service also identified boaa in several b. pseudomallei and b. mallei isolates. all nine b. mallei and b. pseudomallei strains for which sequences are available through this service were found to have the gene. characteristics of some of these orfs are listed in tables and overall, the boaa proteins are 82-94% identical and differ primarily in the number and/or arrangement of slst repeats in their predicted passenger domains. based on these results, we conclude that boaa is a well-conserved gene product shared by b. mallei and b. pseudomallei. bm = b. mallei bp = b. pseudomallei identification of a b. pseudomallei-specific gene encoding a putative autotransporter adhesin that resembles boaa further analysis of the annotated genomic sequence of b. pseudomallei k identified the orf locus tag number bpsl as specifying a second oca-like protein that is ~60% identical to boaa. the last aa of bpsl and boaa are % identical and the very last residues, which encompass the predicted c-terminal om-anchoring domain and α-helical region of the molecules, were found to be particularly well-conserved. the bpsl orf is predicted to encode a protein of 148-kda which, as depicted in fig 1c, possesses many of the structural features observed in boaa including two sets of β-roll aig motifs with the consensus xxgaigxxxax and several slst repeats. this high level of sequence and structural similarity between bpsl and boaa prompted us to designate this b. pseudomallei k gene product boab. the boab gene was sequenced from b. pseudomallei dd and was predicted to encode a protein that is % identical to boab of b. pseudomallei k database searches using ncbi genomic blast revealed that the genomes of at least more b. pseudomallei strains contain the gene. overall, the boab proteins are highly-conserved and characteristics of the orf from selected strains are shown in tables and and fig for comparison purposes. importantly, database searches also revealed that none of the b. mallei isolates available through the ncbi genomic blast service have a boab gene. taken together, these results indicate that boab is a highly-conserved b. pseudomallei-specific molecule. expression of the burkholderia boaa and boab proteins in e. coli because of their sequence and structural similarities to known bacterial adhesins, we hypothesized that boaa and boab mediate adherence to human epithelial cells. to test this hypothesis, the b. mallei atcc boaa and b. pseudomallei dd boab genes were cloned into the e. coli strain epi this organism does not normally adhere well to human epithelial cells and therefore provides an appropriate heterologous genetic background for examining the adhesive properties of boaa and boab. to verify gene expression, rna was prepared from e. coli harboring the plasmids pcc, pslboaa and pslboab, and analyzed by quantitative reverse-transcriptase pcr. fig 3a demonstrates that the boaa and boab genes are expressed by recombinant bacteria and that the primers used in these experiments are specific for their corresponding genes. sarkosyl-insoluble om proteins were also extracted from e. coli cells and analyzed by western blot to ensure production of the burkholderia proteins. fig 3b shows that α-boaa antibodies react with a band of 130-kda in the om of e. coli expressing boaa whereas abs against boab bind to a 140-kda antigen in e. coli expressing boab. these molecular weights are consistent with the predicted masses of the gene products. in addition to showing that boaa and boab are associated with the om by protein separation and western blot, we used immunofluorescent labeling of non-permeabilized e. coli cells to demonstrate their display on the bacterial surface. as depicted in fig 3c, e. coli harboring pslboaa and pslboab are labeled by the α-boaa and α-boab abs, respectively, while recombinant bacteria carrying the control plasmid pcc are not. staining of nucleic acids with the fluorescent dye dapi verified that comparable numbers of bacterial cells were examined. quantitative attachment assays revealed that e. coli expressing boab attach to hep and a epithelial cell lines at levels 18- and 68-fold greater than bacteria carrying pcc, respectively. in addition, boab expression was found to increase adherence to differentiated primary cultures of normal human bronchial epithelium. under the growth conditions used, nhbe cultures form a pseudostratified epithelium with tight junctions containing both ciliated and non-ciliated cells. this epithelium exhibits transepithelial resistance, mucus secretion, mucociliary activity, and an apical surface not submerged in tissue culture medium, thus representing an environment that is similar to the airway lumen in vivo. expression of the b. mallei atcc boaa protein on the surface of e. coli also substantially increased adherence to monolayers of a and hep cells and to nhbe cultures. taken together, these data demonstrate that boaa and boab are om proteins mediating adherence to epithelial cells of the human respiratory tract. b. pseudomallei and b. mallei are facultative intracellular organisms that can invade, survive and replicate in a variety of eukaryotic cells. moreover, autotransporter adhesins often specify additional biological functions such as invasion, biofilm formation, survival within host cells and intracellular motility. for these reasons, we measured the ability of e. coli expressing boaa and boab to invade epithelial cells as well as their ability to survive within murine macrophages. we also measured the ability of these recombinant strains to form biofilms on the plastic support of tissue culture plates using a crystal violet-based assay. the results of these experiments indicated that neither boaa nor boab substantially increase invasion of epithelial cells, phagocytosis of recombinant bacteria by j774a. murine macrophages, survival inside these immune cells, or biofilm formation. construction and characterization of burkholderia mutant strains to study the functional properties of the boa gene products in the native burkholderia background, we constructed isogenic boaa mutants of b. pseudomallei dd and b. mallei atcc as well as an isogenic boab mutant of b. pseudomallei dd a double mutant strain was also engineered in which inactivated versions of both boaa and boab were introduced in the genome of b. pseudomallei dd whole cell lysates and sarkosyl-insoluble om proteins were prepared from these strains and analyzed by western blot to verify lack of boaa and boab expression in the mutants. the α-boaa and α-boab abs, however, did not react with burkholderia protein preparations. in order to determine whether the genes are expressed, total rna was isolated from b. pseudomallei dd and b. mallei atcc and the relative transcript levels of boaa and boab were assessed by qrt-pcr. fig shows that boaa and boab are expressed by b. pseudomallei while b. mallei only expresses boaa, which is in agreement with database searches revealing that b. mallei isolates do not contain a boab gene. the qrt-pcr data also demonstrate that the genes are expressed at very low levels compared to burkholderia reca, which was used to normalize boaa and boab transcript levels. these results are consistent with our inability to visualize the proteins by western blot. other methods such as immunoprecipitation and immunofluorescence labeling also proved unsuccessful at detecting production of boaa and boab by burkholderia strains. quantitative attachment assays with recombinant bacteria indicated that boaa or boab expression significantly increases the adherence of e. coli to monolayers of a and hep cells and to nhbe cultures. we therefore compared the ability of burkholderia parent and boa mutant strains to attach to these respiratory cells. as shown in fig 5a and 5d, inactivation of the boaa gene in b. mallei atcc and b. pseudomallei dd decreases adherence to a cells by and 53%, respectively. the boaa mutation also caused a 50% reduction in the binding of b. pseudomallei to hep monolayers, and reduced adherence of b. mallei to these laryngeal cells by 67%. moreover, both boaa mutant strains displayed significant impairment in their abilities to attach to nhbe cultures. the boab mutation in b. pseudomallei dd decreased attachment to a and hep cells by ~50% and caused a 62% reduction in adherence to nhbe cultures. as expected, the double mutant strain dd boaa.boab exhibited significantly lower attachment to epithelial cells compared to the parent strain dd. the adherence levels of the double mutant, however, did not differ significantly from that of the single mutants in any of the cell types tested. one possible explanation for this apparent lack of synergistic effect is that other adhesins expressed by the double mutant strain dd boaa.boab provide a high background level of adherence. taken together, these results demonstrate that the boaa and boab gene products contribute to the adherence of b. mallei and b. pseudomallei to epithelial cells of the human respiratory tract. as previously stated, autotransporter adhesins often specify additional biological functions including survival within host cells. in addition, b. pseudomallei and b. mallei are facultative intracellular pathogens that are particularly proficient at replicating inside professional phagocytic cells. for these reasons, we measured the ability of our panel of burholderia mutant and parent strains to replicate within j774a. murine macrophages. in b. pseudomallei dd inactivation of the boa genes had no effect on phagocytosis of the organism. once inside macrophages, the boaa and boab single mutants replicated at rates equivalent to that of the progenitor strain dd. however, when both boaa and boab genes were disrupted, intracellular growth was diminished by 60%. to verify that this reduced intracellular fitness was not due to a global growth defect, we measured the growth of strains dd and dd boaa.boab in broth as well as in tissue culture medium. we found that both strains grew at equivalent rates under both conditions. interestingly, the double mutant did not exhibit a growth defect in epithelial cells. these results suggest a role for the boaa and boab proteins in b. pseudomallei's ability to grow inside professional phagocytes. no defect in uptake or intracellular growth was measured for the b. mallei atcc boaa mutant strain. it should also be noted that none of the boa mutants showed decreased biofilm formation on the plastic support of tissue culture plates nor defects in resistance to the bactericidal activity of normal human serum, both biological functions that are also commonly associated with oca autotransporter adhesins. discussion autotransporters are involved in various biological traits of gram-negative bacteria including invasion, serum resistance, phospholipolysis, cytotoxicity, adherence, biofilm formation, survival within eukaryotic cells and intracellular motility. these proteins share an n-terminal extracellular passenger domain that specifies the biological activity of the autotransporter and a c-terminus containing several β-strands, which tether the molecule to the om. based on the structure of this membrane-anchoring domain, autotransporters can be classified as conventional or trimeric. one of the best characterized trimeric autotransporters is the y. enterocolitica adhesin yada. this protein, along with structurally-related adherence proteins such as m. catarrhalis hag and h. influenzae hia, are often referred to as oligomeric coiled-coil adhesins. tiyawisutsri and colleagues previously reported that the published genomic sequences of b. pseudomallei k and b. mallei atcc contain several orfs encoding putative trimeric autotransporters. of these, only bima has been functionally characterized and shown to be required for actin-based motility of the organisms inside eukaryotic cells. in the present study, we identified the boaa orf based on similarities to the oca proteins y. enterocolitica yada and m. catarrhalis hag. specifically, we searched the genome of b. mallei atcc for gene products specifying n-terminal aig β-roll motifs, a transporter module containing β-strands, and a yada-like c-terminal domain. we demonstrated that when expressed by e. coli, boaa increases adherence to the human epithelial cell lines hep and a grown as monolayers in submerged cultures. though these cell types are relevant to the aerosol route of infection by b. mallei and b. pseudomallei, they lack important features of the airway mucosa such as cilia and mucociliary activity. the ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving and contribute to preventing colonization by pathogens. for these reasons, we also measured the adherence of e. coli expressing boaa to cultures of normal human bronchial epithelium grown in an air-liquid interface system. these cultures mimic the structure and function of the airway mucosa more accurately as they are fully differentiated, form a pseudostratified epithelium with tight junctions, contain ciliated and mucus-producing goblet cells, and exhibit mucociliary activity. quantitative attachment assays utilizing this culture system revealed that boaa expression increases adherence to nhbe cultures. in addition to showing that boaa specifies adhesive properties when expressed in the heterologous genetic background of e. coli, we determined that disruption of the boaa gene in the genome of b. mallei atcc reduces adherence of the organism to monolayers of hep and a cells and to nhbe cultures, therefore substantiating the function of boaa as an adhesin. database searches using the ncbi genomic blast service identified boaa in several b. pseudomallei and b. mallei isolates and we demonstrated that inactivation of boaa in the b. pseudomallei strain dd also decreases attachment to hep laryngeal cells, a pneumocytes, and nhbe cultures. together, our data indicate that boaa is an adhesin common to b. mallei and b. pseudomallei and mediates adherence to host cells relevant to pathogenesis by the organisms. these findings are consistent with the recent inclusion of boaa in the virulome of b. mallei and b. pseudomallei, which consists of a set of putative virulence genes that are shared by b. pseudomallei and b. mallei but are not present in five closely-related non-pathogenic burkholderia species. comparative genomic analyses revealed that several b. pseudomallei isolates possess a second oca-like gene product highly similar to boaa, which we termed boab. the c-terminus of boab is strikingly similar to that of boaa and the predicted passenger domains of the molecules contain numerous matching serine-rich slst motifs. the proteins are also functionally related as they mediate adherence to the same types of host cells. therefore, it is tempting to speculate that boaa and boab are the result of gene duplication. this hypothesis would be consistent with the genomic organization of the genes. in b. pseudomallei strains k 1710b, and mshr the boab gene is located on chromosome while boaa is on chromosome moreover, the boab gene in all these isolates is preceded by two orfs specifying an invertase and a transposase. these genes may be the remnants of mobile genetic elements possibly involved in gene duplication. database searches also revealed that b. mallei isolates do not possess a boab gene, which was likely lost during evolution of the organism into a host-adapted pathogen. interestingly, the closely-related bacterium burkholderia thailandensis has been reported by others to bind poorly to epithelial cells. this organism exhibits high genomic similarities to b. pseudomallei and b. mallei and, like b. pseudomallei, is a natural inhabitant of the tropical soil environment. however, b. thailandensis is not considered pathogenic to humans or higher animals. this difference in virulence can be attributed to the fact that b. thailandensis does not produce a capsule and lacks the genes comprising the aforementioned virulome of b. mallei and b. pseudomallei. analysis of the published genome of the b. thailandensis strain e indicated that it contains neither the boaa nor the boab gene. b. pseudomallei dd and b. mallei atcc do not produce detectable amounts of the boaa and boab proteins under the conditions tested. these results are consistent with qrt-pcr experiments demonstrating that the organisms express very low levels of the boa genes relative to the burkholderia reca control. similar observations were made by druar and colleagues while studying expression of the burkholderia type secretion system- proteins bipb and bipd. these proteins were not detected in lysates of b. pseudomallei or b. mallei grown under different conditions, even though the antibodies used in their western blot experiments recognized recombinant forms of bipb and bipd. the authors concluded that these two t3ss- molecules must be expressed in detectable amounts only under very specific in vitro conditions. using a gfp reporter strain, burtnick et al recently showed that the b. mallei type secretion system- gene tsse is not expressed at detectable levels when bacteria are grown in lslb or tissue culture medium, but is expressed upon phagocytosis of the organisms by murine macrophages. the protein preparations tested in our studies were obtained from bacteria cultured on lslb agar plates at 37°c, conditions which may not be optimal for expression of the boaa and boab proteins. additionally, chantratita and colleagues reported that growth of b. pseudomallei under various conditions triggers a complex adaptive process altering the expression of surface molecules. this process, termed phenotypic plasticity, was correlated with changes in the morphology of b. pseudomallei colonies grown on agar plates and appears to modulate the environmental fitness, as well as virulence, of the organism. given their surface location and likely role in virulence, it is possible that boaa and boab are subject to phenotypic plasticity and are expressed in detectable amounts only under very specific in vitro conditions. in concordance, the reduced adherence phenotype of the boaa and boab mutant strains suggests increased level of expression of the genes when burkholderia is incubated with epithelial cells. however, efforts to detect protein expression under these conditions have been unsuccessful. of further note, studies have shown that sera from horses infected with b. mallei and sera from melioidosis patients contain antibodies reacting with boaa and with boab, respectively, which indicates expression of the autotransporters in vivo. determining the conditions and mechanisms that modulate expression of the boa adhesins, and their influence on the binding of b. pseudomallei and b. mallei to host surfaces, represent key areas for future study. disruption of boaa and boab in the b. pseudomallei double mutant strain dd boaa.boab was found to have a significant effect on the growth of the organism within murine macrophages. at present, it is not clear whether boaa and boab play a direct role in intracellular replication. it is possible that the absence of both boa proteins in the om of dd boaa.boab affects the proper surface display of another molecule involved in this phenotypic trait. one candidate is lps, as this molecule was previously shown to play an important role in the ability of b. pseudomallei to grow inside host cells. b. pseudomallei produces multiple t3ss and t6ss that are involved in the intracellular lifestyle of the organism. these specialized secretion apparatuses are used to inject bacterial effector proteins inside host cells where they exert cytopathic effects or manipulate signaling pathways. one important step in this process is the proper docking of bacteria to the host cell to deliver the effectors. given their roles in adherence, it is possible that the lack of expression of the boaa and boab gene products interferes with the delivery of t3ss and/or t6ss cell-altering effectors, which in turn reduces the intracellular fitness of the double mutant strain dd boaa.boab. the yersinia pestis om adhesin ail was recently shown to affect delivery of yop effector proteins to hep cells and macrophages in such a manner. alternatively, the reduced intracellular growth of the double boaa boab mutant may be due to a greater sensitivity to immune effectors produced by the macrophages. the molecular basis for this phenotype is currently being investigated.  the present study reports the identification of b. pseudomallei and b. mallei gene products mediating adherence to epithelial cells. because of their classification as select agents, there is currently a shortage of tools for genetic studies in b. pseudomallei and b. mallei, which precluded us from complementing mutants. our ability to express boaa and boab in e. coli, however, conclusively demonstrates that the proteins directly mediate binding to epithelial cells. these results, along with our analyses of the mutant strains, clearly establish that these molecules participate in adherence by b. pseudomallei and b. mallei. adherence is an essential step in pathogenesis by most infectious agents because it is necessary for colonization and precedes invasion of host cells by intracellular pathogens. thus, continued investigation of boaa and boab will yield important information regarding the biology and virulence of these organisms. 
the genomes of the b. pseudomallei strains k and dd were also found to contain boaa and inactivation of the gene in dd considerably decreased binding to monolayers of hep and a cells and to nhbe cultures. a second yada-like gene product highly similar to boaa was identified in the published genomic sequence of b. pseudomallei strain k. additionally, a b. pseudomallei strain lacking expression of both boaa and boab was impaired in its ability to thrive inside j774a. the boaa and boab genes specify adhesins that mediate adherence to epithelial cells of the human respiratory tract. a well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. comparative sequence analyses identified a gene product in the published genome of b. mallei strain atcc that resembles the well-characterized yersinia enterocolitica autotransporter adhesin yada. the boaa gene product is shared by b. pseudomallei and b. mallei whereas boab appears to be a b. pseudomallei-specific adherence factor. in contrast, the means by which b. pseudomallei and b. mallei adhere to cells are poorly defined. the gene specifying this protein, termed boab, appears to be b. pseudomallei-specific. moreover, a boab mutant of b. pseudomallei dd showed decreased adherence to these respiratory cells. consistent with these findings, disruption of the boaa gene in b. mallei atcc reduced adherence to all three cell types by ~50%. murine macrophages, suggesting a possible role for these proteins in survival within professional phagocytic cells. burkholderia pseudomallei and burkholderia mallei cause the diseases melioidosis and glanders, respectively. the gene encoding this b. mallei protein, designated boaa, was expressed in escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically hep and a, as well as to cultures of normal human bronchial epithelium. quantitative attachment assays demonstrated that recombinant e. coli expressing boab displayed greater binding to a pneumocytes, hep cells and nhbe cultures.
 burkholderia pseudomallei and burkholderia mallei cause the diseases melioidosis and glanders, respectively. a well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. in contrast, the means by which b. pseudomallei and b. mallei adhere to cells are poorly defined. the purpose of this study was to identify adherence factors expressed by these organisms.  comparative sequence analyses identified a gene product in the published genome of b. mallei strain atcc that resembles the well-characterized yersinia enterocolitica autotransporter adhesin yada. the gene encoding this b. mallei protein, designated boaa, was expressed in escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically hep and a, as well as to cultures of normal human bronchial epithelium. consistent with these findings, disruption of the boaa gene in b. mallei atcc reduced adherence to all three cell types by ~50%. the genomes of the b. pseudomallei strains k and dd were also found to contain boaa and inactivation of the gene in dd considerably decreased binding to monolayers of hep and a cells and to nhbe cultures. a second yada-like gene product highly similar to boaa was identified in the published genomic sequence of b. pseudomallei strain k. the gene specifying this protein, termed boab, appears to be b. pseudomallei-specific. quantitative attachment assays demonstrated that recombinant e. coli expressing boab displayed greater binding to a pneumocytes, hep cells and nhbe cultures. moreover, a boab mutant of b. pseudomallei dd showed decreased adherence to these respiratory cells. additionally, a b. pseudomallei strain lacking expression of both boaa and boab was impaired in its ability to thrive inside j774a. murine macrophages, suggesting a possible role for these proteins in survival within professional phagocytic cells.  the boaa and boab genes specify adhesins that mediate adherence to epithelial cells of the human respiratory tract. the boaa gene product is shared by b. pseudomallei and b. mallei whereas boab appears to be a b. pseudomallei-specific adherence factor. 