BACKGROUND
burkholderia cenocepacia is a member of the b. cepacia complex , which comprises at least  <dig> closely related bacterial species  <cit> .
ubiquitous in nature, bcc bacteria thrive in the rhizosphere, fresh and marine water, and in association with amoebae, fungi, insects, plants and animals  <cit> .
members of the complex have biopesticidal and plant growth-promoting properties  <cit> , and due to their metabolic diversity, also have the ability to degrade environmental pollutants  <cit> .
the beneficial properties of bcc bacteria, however, are offset by their opportunistic pathogenicity in immunocompromised individuals, especially in cystic fibrosis  patients  <cit> .
bcc-infected cf patients suffer a rapid decline in lung function  <cit> , and some develop "cepacia syndrome", a fatal condition characterized by rapid lung deterioration, acute, necrotizing pneumonia, and septicemia  <cit> .
resistance to almost all clinically relevant antibiotics  <cit>  and patient-to-patient transmission further complicate treatment and prevention  <cit> .
nearly all species of the bcc have been identified in the sputum of cf patients, but b. cenocepacia and b. multivorans are the most prevalent  <cit> .
of these, b. cenocepacia is associated with epidemic spread and more severe infection  <cit> , and is thus the focus of this study.
environmental strains of b. cenocepacia have been isolated from cf patients, indicating a single strain can occupy multiple niches  <cit> .
this suggests an adaptive capacity of these bacteria to different and potentially stressful environments, which likely requires gene transcription.
b. cenocepacia has a very large genome  arranged in three chromosomes and one plasmid  <cit> .
the genome encodes many putative virulence factors, but none has been shown to be completely necessary for survival .
therefore, little is known about the mechanisms by which b. cenocepacia causes disease.
bacterial survival within host cells, including epithelial cells  <cit>  and macrophages  <cit> , may potentially provide a reservoir for bacterial persistence  <cit> .
intracellular b. cenocepacia delay phagosomal maturation  <cit>  and assembly of the nadph oxidase  <cit> , and alter the actin cytoskeleton  <cit> .
furthermore, intracellular b. cenocepacia can replicate within macrophages  <cit> .
the bacteria-containing phagosome fuses with the lysosome and becomes acidified by  <dig> h post-infection  <cit> , suggesting that b. cenocepacia may be able to replicate in an acidic environment.
to gain a full picture of bacterial adaption to the macrophage cell host we investigated the gene expression of intracellular bacteria upon internalization into macrophages.
the study of gene expression by intracellular bacteria is challenging since bacterial mrna, which has a short half-life, represents only a small fraction of total bacterial rna, which itself is only a small fraction of the total rna in infected host cells.
selective capture of transcribed sequences  is a technique developed to address these challenges and identify bacterial genes expressed during infection.
previous studies in other bacteria have shown that scots enriches microbial transcripts without introducing a significant bias in gene expression data  <cit> .
this technique has been used to identify genes expressed by several intracellular pathogens, including mycobacterium tuberculosis  <cit> , salmonella enterica serovar typhi  <cit> , escherichia coli o157:h <dig>  <cit> , legionella pneumophila  <cit> , and ehrlichia ruminantium  <cit> .
in this study, we characterized gene expression by intracellular b. cenocepacia during an early post-infection stage in an attempt to identify gene products involved in bacterial adaptive responses to the phagosomal environment.
we demonstrate that b. cenocepacia alters gene expression post-internalization, and that various upregulated genes are involved in bacterial entry, intracellular replication, and cytotoxicity towards the macrophage host.
RESULTS
low abundance transcripts identified by competitive enrichment
scots was used to examine gene expression by intracellular b. cenocepacia in murine macrophages at  <dig> h post-infection.
this interval was selected because at  <dig> h post-infection most intracellular bacteria reside in phagosomes that do not fuse with the lysosome  <cit> ; infected macrophages also display observable alterations of the actin cytoskeleton caused by the bacterial type vi secretion system   <cit> , which is poorly expressed in lb medium  <cit> .
therefore,  <dig> h post-infection provides a good window to document early changes in bacterial gene expression in response to the intracellular environment.
as a control, we used bacteria grown for  <dig> h in macrophage growth medium .
to isolate and enrich bacterial transcripts, three rounds of scots were performed on both intracellular and control bacterial cdna samples.
the resultant cdna pools were subjected to competitive enrichment for transcripts specific to intra-macrophage bacteria.
this was accomplished by pre-blocking the chromosome with both rdna and control cdna from non macrophage-exposed bacteria prior to capturing the cdna from intracellular bacteria.
the procedure facilitates capture of bacterial transcripts that are preferentially expressed within the phagosome.
after three rounds of competitive enrichment, intracellular cdna sequences were cloned into puc <dig> and introduced into e. coli dh5α by transformation.
dna inserts from colonies were individually amplified by pcr and screened by southern blot for differential hybridization to digoxigenin-labeled cdna libraries.
many of the recombinant plasmids obtained hybridized to labeled intracellular cdna but not to non macrophage-exposed cdna .
one hundred eighteen unique sequences, encoding  <dig> proteins, were mapped to the b. cenocepacia genome ; of these sequences, four were secondary sequences within an already identified gene, two were within a duplicated region on chromosome one, and one was a transposase gene with nine identical copies distributed throughout the genome.
identified sequences were proportionally represented in each of the three chromosomes and the plasmid:  <dig> sequences mapped to chromosome one,  <dig> to chromosome two,  <dig> to chromosome three, and  <dig> to the plasmid.
identified genes were classified into functional categories based on clusters of orthologous genes  designations .
almost half  of identified genes belonged to the category of poorly characterized genes, of which two-thirds had no cog classification.
of the remaining genes, half were involved in metabolism, one-third in cellular processes and signaling, and the remainder in information storage and processing.
several major cogs were not represented in the set, including genes encoding functions of nucleotide metabolism and transport, trafficking, secretion and transport, posttranslational modification and chaperones, and defense mechanisms.
in contrast, genes classified as encoding cell motility functions were overrepresented relative to genome content.
competitive enrichment and downstream screening do not require specialized equipment, significant financial outlay, or additional knowledge of the genome, and are thus easily and rapidly applicable to any bacterium of interest.
applying competitive enrichment to scots-derived cdna identified trends in the intracellular behavior of b. cenocepacia, as well as specific gene targets for further study.
however, analysis of global changes in gene expression requires a high-throughput screening system.
global intracellular gene expression identified by microarray analysis
to determine if the trends of intra-macrophage expressed genes are representative of the entire cdna library, scots-derived cdna pools were applied to b. cenocepacia-specific microarrays.
each cdna pool was hybridized against a reference sample of genomic dna, allowing comparison between samples.
of the  <dig> sequences initially identified by competitive enrichment,  <dig> percent showed higher expression by intracellular bacteria than non-macrophage-exposed bacteria, validating the use of competitive enrichment as a preliminary investigation technique.
twenty percent of sequences identified by competitive enrichment showed significant intracellular upregulation  of the gene itself or putative transcriptionally associated gene.
in a global comparison of gene expression between intracellular and non-macrophage-exposed bacteria,  <dig> genes or intergenic sequences demonstrated significant changes in expression  , from which  <dig> and  <dig> showed increased and decreased expression, respectively.
the gene distribution among the three chromosomes and the large plasmid of b. cenocepacia was relatively proportionate to the size of the genetic element , with the exception of the plasmid, where 20% of all plasmid-encoded genes showed decreased expression by intracellular bacteria.
to validate our results we first determined how many of the genes obtained by the scots method were previously identified by signature-tagged mutagenesis  as essential for in vivo survival.
in the previous study we mapped  <dig> unique transposon insertions in genes that were associated with attenuation of bacterial survival in a rat agar bead model of chronic lung infection  <cit> .
four of the stm genes were identified directly by scots using competitive enrichment, and another three scots-identified genes were found in genes located within a putative transcriptional unit with an stm insertion .
the microarray analysis identified an additional  <dig> of the  <dig> stm genes as being more highly expressed by intracellular bacteria than non-macrophage-exposed bacteria.
these results were considered significant since bacterial survival in a rat agar bead model involves more that just intra-macrophage survival.
a more direct validation of the scots gene set was performed by quantitative rt-pcr using a subset of genes.
because bacterial rna accounts for a very small proportion of the total rna isolated from infected cells, an internal control was necessary to allow comparison across samples.
sigma factor bcam <dig>  or rpod  was chosen due to stable expression across growth phases and conditions  <cit> .
despite reverse transcribing equal amounts of rna for each sample, intracellular samples averaged 60-fold less rpod transcripts than in non macrophage-exposed samples, demonstrating the scarcity of bacterial rna relative to eukaryotic content in the intracellular sample.
to account for this problem, expression of the gene of interest was first related to the internal control, and this ratio was compared between samples to give fold increase in expression in intracellular bacteria.
the test genes included bcam <dig> , bcam <dig> , bcam <dig> , and bcas <dig> , all of which show high expression in intracellular bacteria.
bcam <dig>  was chosen as an example of a gene with decreased expression in intracellular bacteria.
qrt-pcr confirmed higher expression of bcam <dig>  bcam <dig>  bcam <dig>  and bcas <dig> ; expression of negative control bcam <dig>  though detectable in both samples, was far lower in intracellular bacteria than in non-macrophage-exposed bacteria, with a fold change ratio of  <dig>  × 10- <dig> ±  <dig>  × 10- <dig>  based on the comparison of the scots results with stm data and the results of qrt-pcr, we concluded that the set of genes identified by microarray provides a good representation of the b. cenocepacia transcriptome in the phagosome at  <dig> h post infection.
differentially regulated genes identified by microarray were classified into functional categories based on cog designations .
globally, our results indicate that internalization of b. cenocepacia by macrophages causes large changes in the expression of genes associated with signal transduction mechanisms and transcription, showing that b. cenocepacia senses the phagosomal environment and activates mechanisms necessary for survival in a new niche.
adaptive gene expression is a continual process, as transcription-associated genes remain among the most-affected at  <dig> h post-infection .
metabolic adaptation accounts for one-third of differentially regulated genes in intracellular bacteria.
in particular, genes involved in carbohydrate transport and metabolism were prominently upregulated in intracellular bacteria, accounting for 6% of all differentially upregulated genes .
in leishmania, a protozoan parasite that delays phagosomal maturation and replicates in an acidified phagolysosome, the phagolysosome contains various carbon sources and essential nutrients, but lacks carbohydrates  <cit> , which could explain the upregulation of carbohydrate metabolism pathways.
we speculate that in similar fashion, the upregulation by intracellular b. cenocepacia of genes involved in carbohydrate metabolism is a consequence of the low carbohydrate content in the phagosomal lumen.
this also agrees with the observation that some of the most highly expressed genes in intracellular b. cenocepacia are those involved in amino acid transport .
similar findings were reported for other pathogens, which upregulate amino acid transport systems during intracellular growth  <cit> , suggesting amino acid acquisition from the host may be a general strategy of intracellular bacteria to obtain carbon and nitrogen.
however, with nearly 40% of the genes most affected by internalization falling into the poorly characterized category , it is likely that b. cenocepacia also employs novel survival strategies.
intracellular bacteria showed decreased expression of genes involved in siderophore-mediated iron acquisition, including those encoding the ornibactin receptor, the transcriptional activator of the pyochelin receptor, and a putative pyochelin biosynthesis protein .
ornibactin contributes to colonization, bacterial persistence, and lung pathology in murine models of acute and chronic lung infection  <cit> .
ornibactin, however, may not be required for iron uptake by intracellular bacteria, as the phagosome appears to be rich in iron content  <cit> .
the large number of genes in the burkholderia genus, and specifically in b. cenocepacia  <cit> , endows these bacteria with redundant nutrient uptake systems to function under different conditions and perhaps even exploit multiple forms of similar metabolites.
thus, the changes observed in the expression of metabolic genes may reflect the ability of b. cenocepacia to utilize resources present in the phagolysosome.
since intracellular bacteria only delay phagolysosomal fusion and reside in a phagosome that remains in contact with the endocytic pathway  <cit> , it would be reasonable to propose that the phagosomal maturation delay allows b. cenocepacia to become adapted to survive in low ph environment.
the results of our scots analysis support the notion that intracellular b. cenocepacia may adapt to survive in a low ph environment.
indeed, bcas <dig>  one of the two genes encoding the squalene-hopene cyclase for the synthesis of hopanoids, was strongly upregulated by intracellular bacteria.
production of cholesterol-like hopanoids in certain bacteria, including b. cenocepacia  <cit>  contributes to membrane stability under acidic environments  <cit> .
furthermore, the upregulation of a putative sodium:solute symporter and a na+/h+ antiporter suggests that proton-driven high-affinity uptake by metabolite:proton symporters is important for intracellular b. cenocepacia to bring nutrients into the cell under acidic conditions  <cit> .
scots-identified genes are involved in bacterial entry, intracellular replication, and macrophage cytotoxicity
because survival within macrophages may contribute to the pathogenesis of b. cenocepacia, the role of several scots-identified genes in intracellular survival was examined.
ten genes, chosen based on their high intracellular expression by microarray data or identification by competitive enrichment, were selected for deletion in the gentamicin-sensitive b. cenocepacia mh1k.
this strain is isogenic with k56- <dig> but allows intracellular bacterial survival to be assessed via the gentamicin protection assay  <cit> .
the ten deleted genes were: bcal <dig> , bcas <dig> , bcam0434- <dig>  , bcam <dig> , bcal <dig> , bcam <dig> , bcam <dig> , bcal <dig> , bcam <dig>  , and bcam <dig> .
the ability of mutant strains to enter and replicate within macrophages was assessed relative to the parental strain.
only bcal <dig>  and bcam <dig>  showed statistically significant defects in macrophage entry, while deletion of bcal <dig> resulted in a significant increase in macrophage entry .
none of the deletions negatively affected intracellular survival, but deletions in bcal <dig> and bcam <dig> increased the ability of the bacteria to replicate intracellularly .
with the exception of Δbcal <dig>  statistically significant differences in entry and intracellular survival could be complemented by introducing the deleted gene encoded in a plasmid under the control of a constitutive promoter .
the lack of complementation of Δbcal <dig> suggests the possibility of a secondary mutation.
alternatively, because bcal <dig> is predicted to be the first in a four-gene operon, the deletion may affect downstream genes, thereby preventing possible complementation by bcal <dig> alone.
the results of the gentamicin protection assay may be misleading if the survival of the host cell is not considered, as bacterial cytotoxicity causing membrane damage to the host cell would allow entry of the antibiotic and result in the killing of intracellular bacteria.
the viability of burkholderia-infected macrophages was assayed by flow cytometry using the impermeant fluorescent dye 7-aad, which binds dna only in macrophages with permeabilized membranes.
macrophages were infected with gfp-expressing bacteria and assessed on three different parameters: entry, cytotoxicity, and spread.
parental b. cenocepacia was found in 3% of all macrophages, and was cytotoxic to macrophages at a 1: <dig> ratio, killing approximately 3% of all cells .
the only mutant displaying statistically significant differences from the parental isolate was Δbcam2837; despite infecting nine-fold better than the parental isolate, Δbcam <dig> was also cytotoxic at a 1: <dig> ratio, killing 27% of all macrophages.
some mutants, including Δbcam <dig> and Δbcam <dig>  appeared to prevent permeabilization of the macrophage membrane, decreasing cytotoxicity below the level seen in uninfected macrophages .
of the ten mutants, only four were cytotoxic, defined as permeabilization of the host cellular membrane greater than seen in uninfected cells.
the normalization of intracellular replication quantified by the gentamicin protection assay against the rate of cytotoxicity quantified by flow cytometry provided a better overall assessment of the potential role of the genes examined in our study.
normalization revealed significant intracellular replication by Δbcal <dig> .
normalization also showed that Δbcal <dig> replicates at a rate similar to the parental isolate, but is protected from gentamicin because it is less cytotoxic.
in contrast, Δbcal <dig> is more cytotoxic than the parental strain , and, despite initially infecting at a higher level, has likely been killed by gentamicin entering dying host cells.
mutants that displayed a cytotoxic ratio  less than zero were not normalized, as in the absence of membrane permeability defects, intracellular bacteria would not be inhibited by gentamicin.
flow cytometry also permitted investigation of possible intercellular spread of b. cenocepacia in an unchecked infection.
after killing extracellular bacteria, infected macrophages were maintained in antibiotic-free media.
under these conditions any bacteria escaping the macrophage would be able to infect neighboring macrophages.
the results of this experiment showed that the number of macrophages infected with the parental strain increased six-fold , suggesting that b. cenocepacia was able to escape the macrophage and be engulfed by neighboring macrophages.
with the exception of Δbcam <dig>  the mutant strains spread at least as well as the parental isolate; both Δbcal <dig> and Δbcam <dig> showed significantly increased spread relative to the parental strain, more than doubling the number of infected macrophages.
the ratio of dead:infected cells generally remained approximately 1: <dig> in an unchecked infection, suggesting that viable b. cenocepacia escaped from dead macrophages rather than by an active process of exocytosis.
the two exceptions to this were Δbcal <dig>  which killed  <dig>  cells for every cell infected at the time of analysis, and Δbcam <dig>  which killed only 30% of infected cells.
the phenotypes seen in individual gene deletions underscore several of the trends seen in the global characterization of intracellular gene expression.
the importance of cellular motility suggested by the upregulation of such genes by intracellular bacteria is supported by the phenotypes seen in Δbcal <dig> and Δbcam <dig>  bcal <dig> encodes flhd, a subunit of the flagellar regulon master regulator necessary for expression of the flagellar structural components  <cit> .
bcam <dig>  a pseudogene in sequenced type strain j <dig>  is annotated as the response regulator component of a two-component regulatory system.
bioinformatic analysis shows that the protein consists of two distinct domains, one homologous to the chemotaxis protein chec, and the second homologous to the response regulator chey.
che proteins are typically involved in chemotaxis  <cit> , where chec is a phosphatase that dephosphorylates chey  <cit> .
the genetic organization of bcam <dig> suggests that this gene is cotranscribed with genes encoding putative diguanylate cyclase and histidine kinase proteins.
b. cenocepacia carries three other chey homologues  <cit> , which are expressed in intracellular bacteria while there is one chec-like gene, bcal <dig>  which is not well expressed intracellularly.
therefore, we speculate that the product of bcam <dig> is part of a complex signal transduction cascade required for bacteria to move away from the phagocytic cell, which may also affect bacterial escape from dying macrophages.
the mutant strain lacking bcal <dig> is non-motile, while Δbcam <dig> is capable of swimming, but is defective in swarming .
the two mutants are significantly different in their uptake by macrophages, suggesting that the presence of flagella may be important to stimulate phagocytosis.
loss of the bacterial flagella through deletion of flicd also results in a severe defect in bacterial internalization by macrophages .
the requirement of flagellin for efficient macrophage engulfment was previously noted in b. pseudomallei  <cit>  and p. aeruginosa  <cit> .
in contrast, bacteria with a motility defect may lose the ability to actively evade phagocytosis, resulting in increased uptake as seen in Δbcam <dig>  the two mutants also vary in their rates of intracellular replication, host cytotoxicity, and cell spread.
b. cenocepacia lacking flagellin is unable to activate nfκb or elicit secretion of il- <dig> through tlr signaling  <cit> .
therefore, it is possible that Δbcal <dig> bacteria avoid activation of classical proinflammatory signaling cascades, allowing undetected intracellular replication until overwhelming the macrophage and escaping to re-infect neighboring cells.
in contrast, the decreased ability to spread and lower cytotoxicity suggest that Δbcam <dig> may kill and escape macrophages more slowly, potentially resulting from a defect in swarming motility.
the importance of the flagella to the interaction between b. cenocepacia and macrophages is not unique, as upregulation of flagellar genes is also seen in intracellular l. pneumophila  <cit> .
genes involved in membrane biogenesis and modification, as well as trafficking across the membrane were highly regulated by intracellular bacteria .
general systems involved in the biogenesis of membrane proteins such as the first gene of the tat system, several genes in the sec system, and the signal recognition particle rna showed decreased expression by intracellular bacteria.
in contrast, increased expression of t6ss genes was seen in intracellular bacteria.
deletion of bcal <dig>  encoding a putative lipoprotein central to the t6ss cluster, promotes replication of intracellular bacteria  but does not inactivate the t6ss, as evidenced by the appearance of actin-rich protrusions in infected macrophages, a typical phenotype of a functionally active t6ss.
in a previous study, three transposon mutants within the t6ss cluster showed attenuated survival in an in vivo rat model of chronic lung infection  <cit> , corroborating the importance of a functional t6ss to infection.
genes involved in modification of the membrane were also highly expressed by intracellular bacteria.
bcam <dig> encodes a putative lysylphosphatidylglycerol  synthetase which adds lysine to pg, thereby lowering the net negative charge of the cellular envelope, already lowered in b. cenocepacia by the constitutive presence of 4-amino-4-deoxy-l-arabinose linked to lipid a  <cit> .
lys-pg may also decrease permeability to cations and protons by raising the surface potential of the membrane, increase membrane fluidity, inhibit genomic dna replication, and decrease activity of the host defense factor phospholipase a <dig>  .
deletion of bcam <dig> caused extensive spread in an unchecked infection .
since secreted pla <dig> can hydrolyze the membrane of cells undergoing early stages of apoptosis  <cit> , it is possible that macrophages infected with Δbcam <dig> undergo apoptosis and intracellular bacteria could escape from apoptotic cells to re-infect neighboring cells.
previous studies have demonstrated that b. cenocepacia is cytotoxic to epithelial cells  <cit>  and causes necrosis of dendritic cells  <cit> .
our results indicate that the same is true for macrophages.
the mechanism of macrophage cell death induction by b. cenocepacia is under investigation in our laboratory  <cit> .
pilin-mediated apoptosis induced by b. cenocepacia in infected epithelial cells has been documented and shown to depend on the bacterial load  <cit> .
however, production of il-1β by infected macrophages indicates inflammasome-mediated caspase- <dig> activation, which can lead to pyroptosis  <cit> .
pyroptosis also induces lysosome exocytosis, whereby any bacteria residing within a lysosome are freely delivered to the extracellular milieu as the lysosome fuses with the macrophage plasma membrane, which would explain the mechanism of bacterial release and reinfection of neighboring macrophages.
CONCLUSIONS
our results indicate that the intracellular survival of b. cenocepacia in macrophages is associated with profound changes in metabolic activity and motility rather than the expression of more traditional "virulence-specific" genes.
these observations are consistent with the ability of b. cenocepacia to adapt and survive in a broad range of niches.
our data also show that the delay of phagosomal maturation provides sufficient time for intracellular bacteria to undergo changes in gene expression that likely provide a growth advantage in the phagolysosomal compartment.
therefore, b. cenocepacia is an opportunistic pathogen with the capacity to adapt to survive intracellularly until bacteria can escape back to the extracellular milieu.

 burkholderia cenocepacia is a member of the b. cepacia complex, which comprises at least closely related bacterial species. ubiquitous in nature, bcc bacteria thrive in the rhizosphere, fresh and marine water, and in association with amoebae, fungi, insects, plants and animals. members of the complex have biopesticidal and plant growth-promoting properties, and due to their metabolic diversity, also have the ability to degrade environmental pollutants. the beneficial properties of bcc bacteria, however, are offset by their opportunistic pathogenicity in immunocompromised individuals, especially in cystic fibrosis patients. bcc-infected cf patients suffer a rapid decline in lung function, and some develop "cepacia syndrome", a fatal condition characterized by rapid lung deterioration, acute, necrotizing pneumonia, and septicemia. resistance to almost all clinically relevant antibiotics and patient-to-patient transmission further complicate treatment and prevention. nearly all species of the bcc have been identified in the sputum of cf patients, but b. cenocepacia and b. multivorans are the most prevalent. of these, b. cenocepacia is associated with epidemic spread and more severe infection, and is thus the focus of this study. environmental strains of b. cenocepacia have been isolated from cf patients, indicating a single strain can occupy multiple niches. this suggests an adaptive capacity of these bacteria to different and potentially stressful environments, which likely requires gene transcription. b. cenocepacia has a very large genome arranged in three chromosomes and one plasmid. the genome encodes many putative virulence factors, but none has been shown to be completely necessary for survival. therefore, little is known about the mechanisms by which b. cenocepacia causes disease. bacterial survival within host cells, including epithelial cells and macrophages, may potentially provide a reservoir for bacterial persistence. intracellular b. cenocepacia delay phagosomal maturation and assembly of the nadph oxidase, and alter the actin cytoskeleton. furthermore, intracellular b. cenocepacia can replicate within macrophages. the bacteria-containing phagosome fuses with the lysosome and becomes acidified by h post-infection, suggesting that b. cenocepacia may be able to replicate in an acidic environment. to gain a full picture of bacterial adaption to the macrophage cell host we investigated the gene expression of intracellular bacteria upon internalization into macrophages. the study of gene expression by intracellular bacteria is challenging since bacterial mrna, which has a short half-life, represents only a small fraction of total bacterial rna, which itself is only a small fraction of the total rna in infected host cells. selective capture of transcribed sequences is a technique developed to address these challenges and identify bacterial genes expressed during infection. previous studies in other bacteria have shown that scots enriches microbial transcripts without introducing a significant bias in gene expression data. this technique has been used to identify genes expressed by several intracellular pathogens, including mycobacterium tuberculosis, salmonella enterica serovar typhi, escherichia coli o157:h, legionella pneumophila, and ehrlichia ruminantium. in this study, we characterized gene expression by intracellular b. cenocepacia during an early post-infection stage in an attempt to identify gene products involved in bacterial adaptive responses to the phagosomal environment. we demonstrate that b. cenocepacia alters gene expression post-internalization, and that various upregulated genes are involved in bacterial entry, intracellular replication, and cytotoxicity towards the macrophage host.  low abundance transcripts identified by competitive enrichment scots was used to examine gene expression by intracellular b. cenocepacia in murine macrophages at h post-infection. this interval was selected because at h post-infection most intracellular bacteria reside in phagosomes that do not fuse with the lysosome; infected macrophages also display observable alterations of the actin cytoskeleton caused by the bacterial type vi secretion system, which is poorly expressed in lb medium. therefore, h post-infection provides a good window to document early changes in bacterial gene expression in response to the intracellular environment. as a control, we used bacteria grown for h in macrophage growth medium. to isolate and enrich bacterial transcripts, three rounds of scots were performed on both intracellular and control bacterial cdna samples. the resultant cdna pools were subjected to competitive enrichment for transcripts specific to intra-macrophage bacteria. this was accomplished by pre-blocking the chromosome with both rdna and control cdna from non macrophage-exposed bacteria prior to capturing the cdna from intracellular bacteria. the procedure facilitates capture of bacterial transcripts that are preferentially expressed within the phagosome. after three rounds of competitive enrichment, intracellular cdna sequences were cloned into puc and introduced into e. coli dh5α by transformation. dna inserts from colonies were individually amplified by pcr and screened by southern blot for differential hybridization to digoxigenin-labeled cdna libraries. many of the recombinant plasmids obtained hybridized to labeled intracellular cdna but not to non macrophage-exposed cdna. one hundred eighteen unique sequences, encoding proteins, were mapped to the b. cenocepacia genome; of these sequences, four were secondary sequences within an already identified gene, two were within a duplicated region on chromosome one, and one was a transposase gene with nine identical copies distributed throughout the genome. identified sequences were proportionally represented in each of the three chromosomes and the plasmid: sequences mapped to chromosome one, to chromosome two, to chromosome three, and to the plasmid. identified genes were classified into functional categories based on clusters of orthologous genes designations. almost half of identified genes belonged to the category of poorly characterized genes, of which two-thirds had no cog classification. of the remaining genes, half were involved in metabolism, one-third in cellular processes and signaling, and the remainder in information storage and processing. several major cogs were not represented in the set, including genes encoding functions of nucleotide metabolism and transport, trafficking, secretion and transport, posttranslational modification and chaperones, and defense mechanisms. in contrast, genes classified as encoding cell motility functions were overrepresented relative to genome content. competitive enrichment and downstream screening do not require specialized equipment, significant financial outlay, or additional knowledge of the genome, and are thus easily and rapidly applicable to any bacterium of interest. applying competitive enrichment to scots-derived cdna identified trends in the intracellular behavior of b. cenocepacia, as well as specific gene targets for further study. however, analysis of global changes in gene expression requires a high-throughput screening system. global intracellular gene expression identified by microarray analysis to determine if the trends of intra-macrophage expressed genes are representative of the entire cdna library, scots-derived cdna pools were applied to b. cenocepacia-specific microarrays. each cdna pool was hybridized against a reference sample of genomic dna, allowing comparison between samples. of the sequences initially identified by competitive enrichment, percent showed higher expression by intracellular bacteria than non-macrophage-exposed bacteria, validating the use of competitive enrichment as a preliminary investigation technique. twenty percent of sequences identified by competitive enrichment showed significant intracellular upregulation of the gene itself or putative transcriptionally associated gene. in a global comparison of gene expression between intracellular and non-macrophage-exposed bacteria, genes or intergenic sequences demonstrated significant changes in expression, from which and showed increased and decreased expression, respectively. the gene distribution among the three chromosomes and the large plasmid of b. cenocepacia was relatively proportionate to the size of the genetic element, with the exception of the plasmid, where 20% of all plasmid-encoded genes showed decreased expression by intracellular bacteria. to validate our results we first determined how many of the genes obtained by the scots method were previously identified by signature-tagged mutagenesis as essential for in vivo survival. in the previous study we mapped unique transposon insertions in genes that were associated with attenuation of bacterial survival in a rat agar bead model of chronic lung infection. four of the stm genes were identified directly by scots using competitive enrichment, and another three scots-identified genes were found in genes located within a putative transcriptional unit with an stm insertion. the microarray analysis identified an additional of the stm genes as being more highly expressed by intracellular bacteria than non-macrophage-exposed bacteria. these results were considered significant since bacterial survival in a rat agar bead model involves more that just intra-macrophage survival. a more direct validation of the scots gene set was performed by quantitative rt-pcr using a subset of genes. because bacterial rna accounts for a very small proportion of the total rna isolated from infected cells, an internal control was necessary to allow comparison across samples. sigma factor bcam or rpod was chosen due to stable expression across growth phases and conditions. despite reverse transcribing equal amounts of rna for each sample, intracellular samples averaged 60-fold less rpod transcripts than in non macrophage-exposed samples, demonstrating the scarcity of bacterial rna relative to eukaryotic content in the intracellular sample. to account for this problem, expression of the gene of interest was first related to the internal control, and this ratio was compared between samples to give fold increase in expression in intracellular bacteria. the test genes included bcam, bcam, bcam, and bcas, all of which show high expression in intracellular bacteria. bcam was chosen as an example of a gene with decreased expression in intracellular bacteria. qrt-pcr confirmed higher expression of bcam bcam bcam and bcas; expression of negative control bcam though detectable in both samples, was far lower in intracellular bacteria than in non-macrophage-exposed bacteria, with a fold change ratio of × 10- ± × 10- based on the comparison of the scots results with stm data and the results of qrt-pcr, we concluded that the set of genes identified by microarray provides a good representation of the b. cenocepacia transcriptome in the phagosome at h post infection. differentially regulated genes identified by microarray were classified into functional categories based on cog designations. globally, our results indicate that internalization of b. cenocepacia by macrophages causes large changes in the expression of genes associated with signal transduction mechanisms and transcription, showing that b. cenocepacia senses the phagosomal environment and activates mechanisms necessary for survival in a new niche. adaptive gene expression is a continual process, as transcription-associated genes remain among the most-affected at h post-infection. metabolic adaptation accounts for one-third of differentially regulated genes in intracellular bacteria. in particular, genes involved in carbohydrate transport and metabolism were prominently upregulated in intracellular bacteria, accounting for 6% of all differentially upregulated genes. in leishmania, a protozoan parasite that delays phagosomal maturation and replicates in an acidified phagolysosome, the phagolysosome contains various carbon sources and essential nutrients, but lacks carbohydrates, which could explain the upregulation of carbohydrate metabolism pathways. we speculate that in similar fashion, the upregulation by intracellular b. cenocepacia of genes involved in carbohydrate metabolism is a consequence of the low carbohydrate content in the phagosomal lumen. this also agrees with the observation that some of the most highly expressed genes in intracellular b. cenocepacia are those involved in amino acid transport. similar findings were reported for other pathogens, which upregulate amino acid transport systems during intracellular growth, suggesting amino acid acquisition from the host may be a general strategy of intracellular bacteria to obtain carbon and nitrogen. however, with nearly 40% of the genes most affected by internalization falling into the poorly characterized category, it is likely that b. cenocepacia also employs novel survival strategies. intracellular bacteria showed decreased expression of genes involved in siderophore-mediated iron acquisition, including those encoding the ornibactin receptor, the transcriptional activator of the pyochelin receptor, and a putative pyochelin biosynthesis protein. ornibactin contributes to colonization, bacterial persistence, and lung pathology in murine models of acute and chronic lung infection. ornibactin, however, may not be required for iron uptake by intracellular bacteria, as the phagosome appears to be rich in iron content. the large number of genes in the burkholderia genus, and specifically in b. cenocepacia, endows these bacteria with redundant nutrient uptake systems to function under different conditions and perhaps even exploit multiple forms of similar metabolites. thus, the changes observed in the expression of metabolic genes may reflect the ability of b. cenocepacia to utilize resources present in the phagolysosome. since intracellular bacteria only delay phagolysosomal fusion and reside in a phagosome that remains in contact with the endocytic pathway, it would be reasonable to propose that the phagosomal maturation delay allows b. cenocepacia to become adapted to survive in low ph environment. the results of our scots analysis support the notion that intracellular b. cenocepacia may adapt to survive in a low ph environment. indeed, bcas one of the two genes encoding the squalene-hopene cyclase for the synthesis of hopanoids, was strongly upregulated by intracellular bacteria. production of cholesterol-like hopanoids in certain bacteria, including b. cenocepacia contributes to membrane stability under acidic environments. furthermore, the upregulation of a putative sodium:solute symporter and a na+/h+ antiporter suggests that proton-driven high-affinity uptake by metabolite:proton symporters is important for intracellular b. cenocepacia to bring nutrients into the cell under acidic conditions. scots-identified genes are involved in bacterial entry, intracellular replication, and macrophage cytotoxicity because survival within macrophages may contribute to the pathogenesis of b. cenocepacia, the role of several scots-identified genes in intracellular survival was examined. ten genes, chosen based on their high intracellular expression by microarray data or identification by competitive enrichment, were selected for deletion in the gentamicin-sensitive b. cenocepacia mh1k. this strain is isogenic with k56- but allows intracellular bacterial survival to be assessed via the gentamicin protection assay. the ten deleted genes were: bcal, bcas, bcam0434-, bcam, bcal, bcam, bcam, bcal, bcam, and bcam. the ability of mutant strains to enter and replicate within macrophages was assessed relative to the parental strain. only bcal and bcam showed statistically significant defects in macrophage entry, while deletion of bcal resulted in a significant increase in macrophage entry. none of the deletions negatively affected intracellular survival, but deletions in bcal and bcam increased the ability of the bacteria to replicate intracellularly. with the exception of Δbcal statistically significant differences in entry and intracellular survival could be complemented by introducing the deleted gene encoded in a plasmid under the control of a constitutive promoter. the lack of complementation of Δbcal suggests the possibility of a secondary mutation. alternatively, because bcal is predicted to be the first in a four-gene operon, the deletion may affect downstream genes, thereby preventing possible complementation by bcal alone. the results of the gentamicin protection assay may be misleading if the survival of the host cell is not considered, as bacterial cytotoxicity causing membrane damage to the host cell would allow entry of the antibiotic and result in the killing of intracellular bacteria. the viability of burkholderia-infected macrophages was assayed by flow cytometry using the impermeant fluorescent dye 7-aad, which binds dna only in macrophages with permeabilized membranes. macrophages were infected with gfp-expressing bacteria and assessed on three different parameters: entry, cytotoxicity, and spread. parental b. cenocepacia was found in 3% of all macrophages, and was cytotoxic to macrophages at a 1: ratio, killing approximately 3% of all cells. the only mutant displaying statistically significant differences from the parental isolate was Δbcam2837; despite infecting nine-fold better than the parental isolate, Δbcam was also cytotoxic at a 1: ratio, killing 27% of all macrophages. some mutants, including Δbcam and Δbcam appeared to prevent permeabilization of the macrophage membrane, decreasing cytotoxicity below the level seen in uninfected macrophages. of the ten mutants, only four were cytotoxic, defined as permeabilization of the host cellular membrane greater than seen in uninfected cells. the normalization of intracellular replication quantified by the gentamicin protection assay against the rate of cytotoxicity quantified by flow cytometry provided a better overall assessment of the potential role of the genes examined in our study. normalization revealed significant intracellular replication by Δbcal. normalization also showed that Δbcal replicates at a rate similar to the parental isolate, but is protected from gentamicin because it is less cytotoxic. in contrast, Δbcal is more cytotoxic than the parental strain, and, despite initially infecting at a higher level, has likely been killed by gentamicin entering dying host cells. mutants that displayed a cytotoxic ratio less than zero were not normalized, as in the absence of membrane permeability defects, intracellular bacteria would not be inhibited by gentamicin. flow cytometry also permitted investigation of possible intercellular spread of b. cenocepacia in an unchecked infection. after killing extracellular bacteria, infected macrophages were maintained in antibiotic-free media. under these conditions any bacteria escaping the macrophage would be able to infect neighboring macrophages. the results of this experiment showed that the number of macrophages infected with the parental strain increased six-fold, suggesting that b. cenocepacia was able to escape the macrophage and be engulfed by neighboring macrophages. with the exception of Δbcam the mutant strains spread at least as well as the parental isolate; both Δbcal and Δbcam showed significantly increased spread relative to the parental strain, more than doubling the number of infected macrophages. the ratio of dead:infected cells generally remained approximately 1: in an unchecked infection, suggesting that viable b. cenocepacia escaped from dead macrophages rather than by an active process of exocytosis. the two exceptions to this were Δbcal which killed cells for every cell infected at the time of analysis, and Δbcam which killed only 30% of infected cells. the phenotypes seen in individual gene deletions underscore several of the trends seen in the global characterization of intracellular gene expression. the importance of cellular motility suggested by the upregulation of such genes by intracellular bacteria is supported by the phenotypes seen in Δbcal and Δbcam bcal encodes flhd, a subunit of the flagellar regulon master regulator necessary for expression of the flagellar structural components. bcam a pseudogene in sequenced type strain j is annotated as the response regulator component of a two-component regulatory system. bioinformatic analysis shows that the protein consists of two distinct domains, one homologous to the chemotaxis protein chec, and the second homologous to the response regulator chey. che proteins are typically involved in chemotaxis, where chec is a phosphatase that dephosphorylates chey. the genetic organization of bcam suggests that this gene is cotranscribed with genes encoding putative diguanylate cyclase and histidine kinase proteins. b. cenocepacia carries three other chey homologues, which are expressed in intracellular bacteria while there is one chec-like gene, bcal which is not well expressed intracellularly. therefore, we speculate that the product of bcam is part of a complex signal transduction cascade required for bacteria to move away from the phagocytic cell, which may also affect bacterial escape from dying macrophages. the mutant strain lacking bcal is non-motile, while Δbcam is capable of swimming, but is defective in swarming. the two mutants are significantly different in their uptake by macrophages, suggesting that the presence of flagella may be important to stimulate phagocytosis. loss of the bacterial flagella through deletion of flicd also results in a severe defect in bacterial internalization by macrophages. the requirement of flagellin for efficient macrophage engulfment was previously noted in b. pseudomallei and p. aeruginosa. in contrast, bacteria with a motility defect may lose the ability to actively evade phagocytosis, resulting in increased uptake as seen in Δbcam the two mutants also vary in their rates of intracellular replication, host cytotoxicity, and cell spread. b. cenocepacia lacking flagellin is unable to activate nfκb or elicit secretion of il- through tlr signaling. therefore, it is possible that Δbcal bacteria avoid activation of classical proinflammatory signaling cascades, allowing undetected intracellular replication until overwhelming the macrophage and escaping to re-infect neighboring cells. in contrast, the decreased ability to spread and lower cytotoxicity suggest that Δbcam may kill and escape macrophages more slowly, potentially resulting from a defect in swarming motility. the importance of the flagella to the interaction between b. cenocepacia and macrophages is not unique, as upregulation of flagellar genes is also seen in intracellular l. pneumophila. genes involved in membrane biogenesis and modification, as well as trafficking across the membrane were highly regulated by intracellular bacteria. general systems involved in the biogenesis of membrane proteins such as the first gene of the tat system, several genes in the sec system, and the signal recognition particle rna showed decreased expression by intracellular bacteria. in contrast, increased expression of t6ss genes was seen in intracellular bacteria. deletion of bcal encoding a putative lipoprotein central to the t6ss cluster, promotes replication of intracellular bacteria but does not inactivate the t6ss, as evidenced by the appearance of actin-rich protrusions in infected macrophages, a typical phenotype of a functionally active t6ss. in a previous study, three transposon mutants within the t6ss cluster showed attenuated survival in an in vivo rat model of chronic lung infection, corroborating the importance of a functional t6ss to infection. genes involved in modification of the membrane were also highly expressed by intracellular bacteria. bcam encodes a putative lysylphosphatidylglycerol synthetase which adds lysine to pg, thereby lowering the net negative charge of the cellular envelope, already lowered in b. cenocepacia by the constitutive presence of 4-amino-4-deoxy-l-arabinose linked to lipid a. lys-pg may also decrease permeability to cations and protons by raising the surface potential of the membrane, increase membrane fluidity, inhibit genomic dna replication, and decrease activity of the host defense factor phospholipase a. deletion of bcam caused extensive spread in an unchecked infection. since secreted pla can hydrolyze the membrane of cells undergoing early stages of apoptosis, it is possible that macrophages infected with Δbcam undergo apoptosis and intracellular bacteria could escape from apoptotic cells to re-infect neighboring cells. previous studies have demonstrated that b. cenocepacia is cytotoxic to epithelial cells and causes necrosis of dendritic cells. our results indicate that the same is true for macrophages. the mechanism of macrophage cell death induction by b. cenocepacia is under investigation in our laboratory. pilin-mediated apoptosis induced by b. cenocepacia in infected epithelial cells has been documented and shown to depend on the bacterial load. however, production of il-1β by infected macrophages indicates inflammasome-mediated caspase- activation, which can lead to pyroptosis. pyroptosis also induces lysosome exocytosis, whereby any bacteria residing within a lysosome are freely delivered to the extracellular milieu as the lysosome fuses with the macrophage plasma membrane, which would explain the mechanism of bacterial release and reinfection of neighboring macrophages.  our results indicate that the intracellular survival of b. cenocepacia in macrophages is associated with profound changes in metabolic activity and motility rather than the expression of more traditional "virulence-specific" genes. these observations are consistent with the ability of b. cenocepacia to adapt and survive in a broad range of niches. our data also show that the delay of phagosomal maturation provides sufficient time for intracellular bacteria to undergo changes in gene expression that likely provide a growth advantage in the phagolysosomal compartment. therefore, b. cenocepacia is an opportunistic pathogen with the capacity to adapt to survive intracellularly until bacteria can escape back to the extracellular milieu. 
we examined gene expression by intracellular b. cenocepacia using selective capture of transcribed sequences combined with microarray analysis. deletion of various scots-identified genes affected bacterial entry into macrophages and intracellular replication. affected genes represented all aspects of cellular life including information storage and processing, cellular processes and signaling, and metabolism. we identified genes with significantly different levels of expression by intracellular bacteria, of which showed increased expression and showed decreased expression. burkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in patients with cystic fibrosis. in particular, genes involved in bacterial motility, cobalamin biosynthesis, the type vi secretion system, and membrane modification contributed greatly to macrophage entry and subsequent intracellular behavior of b. cenocepacia. in general, intracellular gene expression demonstrated a pattern of environmental sensing, bacterial response, and metabolic adaptation to the phagosomal environment. we hypothesize that b. cenocepacia undergoes changes in gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation. b. cenocepacia enters macrophages, adapts to the phagosomal environment, replicates within a modified phagosome, and exhibits cytotoxicity towards the host cells. the analysis of the transcriptomic response of intracellular b. cenocepacia reveals that metabolic adaptation to a new niche plays a major role in the survival of b. cenocepacia in macrophages. we also show that intracellular b. cenocepacia is cytotoxic towards the macrophage host, and capable of spread to neighboring cells, a role dependent on scots-identified genes. the bacterium survives within macrophages by interfering with endocytic trafficking and delaying the maturation of the b. cenocepacia-containing phagosome. further investigation into the remaining scots-identified genes will provide a more complete picture of the adaptive response of b. cenocepacia to the host cell environment.
 burkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in patients with cystic fibrosis. the bacterium survives within macrophages by interfering with endocytic trafficking and delaying the maturation of the b. cenocepacia-containing phagosome. we hypothesize that b. cenocepacia undergoes changes in gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation.  we examined gene expression by intracellular b. cenocepacia using selective capture of transcribed sequences combined with microarray analysis. we identified genes with significantly different levels of expression by intracellular bacteria, of which showed increased expression and showed decreased expression. affected genes represented all aspects of cellular life including information storage and processing, cellular processes and signaling, and metabolism. in general, intracellular gene expression demonstrated a pattern of environmental sensing, bacterial response, and metabolic adaptation to the phagosomal environment. deletion of various scots-identified genes affected bacterial entry into macrophages and intracellular replication. we also show that intracellular b. cenocepacia is cytotoxic towards the macrophage host, and capable of spread to neighboring cells, a role dependent on scots-identified genes. in particular, genes involved in bacterial motility, cobalamin biosynthesis, the type vi secretion system, and membrane modification contributed greatly to macrophage entry and subsequent intracellular behavior of b. cenocepacia.  b. cenocepacia enters macrophages, adapts to the phagosomal environment, replicates within a modified phagosome, and exhibits cytotoxicity towards the host cells. the analysis of the transcriptomic response of intracellular b. cenocepacia reveals that metabolic adaptation to a new niche plays a major role in the survival of b. cenocepacia in macrophages. this adaptive response does not require the expression of any specific virulence-associated factor, which is consistent with the opportunistic nature of this microorganism. further investigation into the remaining scots-identified genes will provide a more complete picture of the adaptive response of b. cenocepacia to the host cell environment. 