BACKGROUND
francisella tularensis is a gram-negative bacterium that is pathogenic to humans  <cit> .
this organism causes mortality in up to 60% of infected individuals if untreated  <cit> .
based on the potential to weaponize this organism, the center for disease control and prevention has classified f. tularensis as a category a biodefense agent  <cit> .
it is therefore vital to understand how this organism responds to environmental and host signals, and how these cues alter expression of virulence determinants.
during the course of a natural francisella infection, this bacterium may transition from an amoeba  <cit>  or an arthropod host  <cit>  to colonize human cells.
accompanying this transition, it is likely that chemical and physical signals alert francisella that it has entered a mammalian host.
the manner by which f. tularensis integrates environmental stimuli to regulate gene expression is fundamental to the success of this organism as an intracellular pathogen.
the most well-studied virulence factors of f. tularensis are encoded in the francisella pathogenicity island   <cit> .
the amount of one of the virulence proteins encoded in this cluster, iglc, increases in response to growth in macrophages and hydrogen peroxide  <cit> .
in addition, iron limitation has been shown to induce transcription and protein levels of iglacd and pdpb  <cit>  as well as stimulate siderophore production by f. tularensis  <cit> .
previously, we have shown that differing culture conditions greatly influence host-pathogen interactions and the ability of f. tularensis live vaccine strain  to activate macrophages  <cit> .
prior to the current study, there has only been a single published report characterizing the global transcriptional francisella response to an environmental cue, specifically iron limitation  <cit> .
important insights into the regulation of virulence factors like iglc were defined in this analysis.
however, numerous genes associated with virulence by other studies are not affected by different iron concentrations  <cit> .
because f. tularensis may transition between hosts, mammalian body temperature is another signal that is likely to be critical for this pathogen.
pathogenic bacteria that encounter a shift in temperature during their life cycle sometimes respond with enhanced virulence factor expression  <cit> .
however, there are discrepancies among the specific groups of genes that are affected by temperature and the mechanism of regulation between organisms.
for example, shigella increases production of its type iii secretion system in response to mammalian temperature  <cit> .
the homologous secretion apparatus in pathogenic salmonella, however, is not regulated by temperature  <cit> .
regarding the mechanism of regulation, genes involved in the heat-shock response are often induced at mammalian temperatures relative to those of the environment.
this regulation is usually due to the presence of a conserved inverted repeat regulatory structure in the promoter region  <cit> , or control by a σ32-type heat shock sigma factor  <cit> .
in contrast, the bacterium responsible for whooping cough, bordetella pertussis, uses a two-component system comprised of bvgs and bvga to alter transcription of genes in response to temperature.
following induction of bvgas at 37°c, phosphorylation by bvgs allows bvga-binding to promoter regions of virulence genes, such as the adhesin, fimx  <cit> .
given the uncertainty of target temperature-regulated genes and differences in mechanism among bacteria, it is necessary to define the temperature regulon in individual species.
studying gene expression changes induced by a shift to mammalian temperature could provide valuable insight into francisella virulence.
a few previous studies have investigated the francisella response to temperature at the molecular level.
high temperature  synonymous with heat stress has been shown to have dramatic effects on f. tularensis gene and protein expression  <cit>  including increased production of the heat shock proteins groel, groes, dnak, and clpb  <cit> .
heat stress has also been shown to enhance the virulence of a mutant form of f. tularensis lvs  <cit> .
interestingly, f. novicida has been shown to alter its outer membrane at 25°c versus 37°c by differentially modifying the lipid a component of the lipopolysaccharide  <cit> .
in addition, it has been observed that virulent f. tularensis clinical isolates increase the mannose modification of their lipopolysaccharide when cultivated at temperatures less than 25°c  <cit> .
the results from these previous studies suggest that a global transcript analysis following a shift to mammalian body temperature should reveal gene regulation that is critical to francisella pathogenesis.
here we show that a shift from environmental to mammalian body temperature significantly alters the transcriptome of f. tularensis lvs.
many genes that we identified as significantly induced at 37°c have been previously implicated in francisella virulence, supporting the notion that this temperature shift is important for the regulation of pathogenesis.
we provide evidence that the product of a gene encoding the hypothetical protein most profoundly induced by mammalian temperature contributes to virulence and intracellular growth.
moreover, data presented here indicate that a locus up-regulated at 37°c was required for optimal uptake by eukaryotic cells.
this is the first f. tularensis gene identified to be involved in the entry of this organism into both phagocytic and non-phagocytic host cells.
the results from this study support a model where detection of mammalian body temperature by f. tularensis is important for regulation of physiology necessary for successful infection.
RESULTS
f. tularensis lvs global temperature regulation
to study francisella gene expression changes associated with exposure to mammalian temperature, we conducted a microarray analysis of lvs as a model f. tularensis strain .
gene expression of lvs cultured at 26°c  was compared to bacteria shifted to 37°c .
labeled cdna target was produced from rna isolated from f. tularensis lvs and was subsequently hybridized to a custom agilent francisella microarray.
global gene expression data were analyzed with a j <dig> statistical test, which was selected to limit the number of false positives  <cit> .
this analysis identified  <dig> genes with significantly increased expression and  <dig> genes with decreased expression in response to a shift to mammalian body temperature .
collectively, this represents approximately ~11% of the genes in the entire lvs genome.
genes with a significant change in expression were examined with the gene pattern program by hierarchical clustering .
the clustered data exhibited a distinct pattern of transcript induction and repression in response to mammalian body temperature .
together, these data indicate that this temperature shift has a broad impact on f. tularensis transcription.
strain, plasmid,
the microarray data were confirmed using quantitative real time pcr , in which eight differentially-expressed genes were analyzed, representing transcripts that were both significantly up- and down-regulated .
induced genes chosen for validation were the carbamoyl-phosphate synthase large chain, carb , a hypothetical lipoprotein , a dimethyladenosine transferase annotated to function in kasugamycin resistance, ksga , and phosphopentomutase, deob .
the down-regulated genes tested by q-pcr included two hypothetical proteins , a cold shock protein, cspc , and a metal ion transporter, tlyc .
a similar pattern of expression was observed in both q-pcr and in the microarray experiments .
a correlation plot  showed a strong, positive association between both data sets .
this indicated the microarray platform and the subsequent statistical analysis were robust compared to the sensitive, though low-throughput q-pcr approach.
to analyze the differentially-regulated genes more thoroughly, we categorized their presumed protein products based on their clusters of orthologous groups  category.
as expected, many of the induced and repressed genes were involved in central biological functions such as metabolism, transcription, translation, dna replication, and rna genes .
heat shock proteins belong to the category labeled "posttranslational modification/protein turnover/chaperones", and as anticipated, these genes were induced at the higher temperature, providing additional support to our microarray analysis .
the heat shock protein result was confirmed by analyzing hsp <dig> protein quantity by western blotting, which showed significantly more hsp <dig> in bacteria shifted to 37°c versus f. tularensis lvs cultured at 26°c .
cog categories found only among the up-regulated transcripts included genes functioning in secretion and cell division, suggesting that host temperature may trigger these cellular processes and reflect, in part, enhanced growth rate .
in addition, the cog for bacterial defense mechanisms, which includes type i site-specific restriction-modification systems, was uniquely down-regulated.
this response may have evolved to allow this bacterium to better contend with environmental stresses, such as invading bacteriophages.
we observed that a large percentage of both up- and down-regulated genes  did not belong to a known cog category .
proteins with an unknown cog category may have a novel biological role in f. tularensis associated with their temperature regulation.
f. tularensis lvs temperature regulation of genes necessary for infection
we hypothesized that the shift from environmental to mammalian temperature may be used to regulate genes important for infection.
surprisingly, none of the genes encoded in the fpi were significantly up-regulated at 37°c in the microarray analysis.
we also confirmed, by immunoblotting, that iglc protein levels were equivalent in bacteria grown at the two different temperatures .
upon further scrutiny, we did notice that one gene in the fpi, pdpc , was near the statistical threshold for induction at 37°c.
we therefore analyzed pdpc transcript levels by q-pcr as before, which confirmed that this gene was induced at mammalian body temperature .
we next compared our list of genes induced at 37°c with those previously shown to be necessary for francisella infection, or postulated to be involved in the pathogenesis of this organism .
forty percent of the protein-coding genes significantly up-regulated at 37°c have been reported or predicted to be important for intracellular growth and/or virulence of f. tularensis .
the list depicted in table  <dig> included an assortment of metabolic genes, chaperones, genes encoding hypothetical proteins, and others.
the data from table  <dig> and the pdpc expression results in fig.
2a further supported our hypothesis that genes important for francisella infection are regulated by mammalian body temperature.
ftl_ <dig> is associated with f. tularensis lvs virulence
ftl_ <dig>  annotated as a hypothetical lipoprotein, was induced by mammalian temperature more profoundly than any other hypothetical protein gene in the f. tularensis lvs genome .
we selected this gene for further study to test our hypothesis that temperature regulates genes necessary for infection.
a blast search  <cit>  against the national center for biotechnology information database of non-redundant sequences did not identify proteins from organisms other than francisella that had >30% identity to ftl_ <dig> .
this suggested that ftl_ <dig> may have a function unique to f. tularensis.
the primary structure of ftl_ <dig> was further examined by a prosite analysis  <cit>  which indicated this protein likely contained a lipoprotein signal sequence  consistent with its annotation.
this analysis also revealed that the signal sequence overlapped with a motif similar to that of the enterobacterial trat complement resistance protein .
although the initial blast search did not retrieve homologous proteins with high degrees of identity, it did show that ftl_ <dig> contained a domain with 20–24% identity and 40–44% similarity with the vacuolating cytotoxin   <cit>  and paralogs  of helicobacter pylori j <dig> .
because ftl_ <dig> contained domains similar to other proteins that had a role in pathogenesis, we hypothesized that this protein may contribute to the virulence of f. tularensis.
we disrupted ftl_ <dig> in lvs, producing strain 1581d, to determine if this gene was associated with f. tularensis virulence.
to assess the virulence of 1581d, we utilized a competition assay based on the chicken embryo infection model  <cit> .
the chicken embryo produces a robust innate immune response comprised of complement, phagocytic cells, and cytokine production  <cit> .
here, chicken embryos were infected with a ~1: <dig> mixture of lvs and 1581d.
at days  <dig>   <dig>  and  <dig> post-infection, wild-type lvs exhibited superior survival compared to 1581d  .
this result was not due to a growth defect since 1581d grew identically to wild-type lvs when cultivated in bacterial growth medium .
in a separate experiment in which chicken embryos were infected only with 1581d, isolates from homogenates were all resistant to hygromycin, indicating that this mutant did not revert to wild type during infection .
since 1581d was attenuated compared to lvs, the results suggest the function of the ftl_ <dig> gene product contributes to f. tularensis virulence.
we wanted to determine if the virulence attenuation of 1581d in the chicken embryo infection model was due to a reduced ability to inactivate complement, as ftl_ <dig> contained a putative complement resistance domain .
therefore, we subjected lvs and 1581d to serum sensitivity assays.
there was equivalent survival of wild-type lvs and 1581d when cultured in media containing 20% serum or 20% heat-inactivated serum for up to  <dig> h .
to ensure that the serum complement was functional, e. coli dh5α was used as a control.
here, the cfu from the serum-treated e. coli exhibited a reduction of  <dig> logs relative to the input or heat-inactivated serum groups after  <dig> min .
this suggested that the disruption in 1581d does not affect complement resistance.
we determined if ftl_ <dig> contributed to growth in a macrophage environment.
primary human monocyte-derived macrophages were infected in vitro with either lvs or 1581d.
at various time-points, macrophages were lysed, and the lysates were diluted and plated to enumerate viable cfu.
we observed attenuated growth of 1581d in macrophages at  <dig> h post-infection  .
when ftl_ <dig> was complemented in trans in 1581d, wild-type level of growth was restored .
this complementation confirmed that the reduced intracellular fitness of 1581d was due to the inactivation of ftl_ <dig>  and not due to polar effects or alternate mutations.
this experiment  also suggested that the attenuation of 1581d in the chicken embryo model  was likely due to a defect in intracellular survival.
together, the data presented here implicate ftl_ <dig>  a francisella orf induced by mammalian body temperature that has no obvious homologs, with virulence and intracellular survival.
therefore, we propose that ftl_ <dig> be named temperature-induced, virulence-associated locus a, or tiva.
involvement of ftl_ <dig>  in uptake of f. tularensis lvs
many of the genes critical for francisella infection involved metabolism , a major cog category induced at 37°c .
this suggested that physiology vital for the success of f. tularensis as a pathogen was regulated by the temperature shift.
therefore, we were interested in determining the contribution of temperature-regulated metabolic genes toward francisella pathogenesis.
previously, a microarray-based negative selection screen of f. novicida transposon mutants identified the phosphopentomutase, deob, as a gene contributing to growth and/or survival in mice  <cit> .
in other bacteria, proteins encoded by deob homologs normally catalyze the reversible reaction between ribose-1-phosphate and ribose-5-phosphate or between deoxyribose-1-phosphate and deoxyribose-5-phosphate  <cit> .
an f. tularensis lvs chromosomal disruption mutant of deob, ftl_ <dig>  was constructed .
this mutant had a cellular and colony morphology similar to its wild-type parent strain .
also, 1664d grew similarly to wild-type lvs in bacterial culture medium .
we employed the chicken embryo infection model  <cit>  to confirm that deob contributes to lvs pathogenesis, as it did in f. novicida  <cit> .
here, chicken embryos that had been infected with f. tularensis lvs 1664d exhibited significantly enhanced survival compared to those infected with wild-type lvs over a  <dig> day period   corroborating the deob data from f. novicida  <cit> .
accessing the host cytoplasm to replicate intracellularly is a hallmark of francisella pathogenesis.
therefore, we next tested if the virulence attenuation of 1664d in the chicken embryo infection model was due to a defect in entering host cells.
at two hours post-infection in vitro, human, monocyte-derived macrophages were treated with gentamicin to kill extracellular bacteria, followed by extensive washing.
we consistently observed that substantially fewer 1664d cells were phagocytosed relative to wild-type lvs  .
importantly, trans complementation of 1664d  rescued the uptake defect .
this suggested deob is involved in a bacterial mechanism that enhances uptake of f. tularensis.
this finding was extended by conducting similar uptake assays in primary human dendritic cells .
in both phagocytic cell types, the uptake defect of 1664d was reproduced and complemented .
we then qualitatively assessed uptake into phagocytes by microscopy to confirm the quantitative results obtained with cfu measurements.
lvs, 1664d, and 1664d/pftl_ <dig> were treated with the green fluorescent stain, syto- <dig>  and then incubated with raw  <dig>  cells, a murine macrophage-like cell line.
after a two hour incubation and washes, cells were observed by fluorescence microscopy.
here, raw  <dig>  cells infected with lvs or 1664d/pftl_ <dig> exhibited bright green fluorescence .
in contrast, the raw  <dig>  cells infected with 1664d produced considerably less fluorescence despite comparable inocula to the cultures .
the results obtained by microscopy suggested the discrepancy in cfu between wildtype and 1664d was not due to killing in the early phagosome.
rather, the levels of fluorescence in fig.
5b were consistent with the cfu uptake data presented in fig.
5a and confirmed that the temperature-regulated deob is important for optimal uptake by mammalian phagocytes.
to determine if deob contributed to entry into non-phagocytic cells, we exposed the human embryonic kidney cell line hek- <dig> to either lvs, 1664d, or 1664d/pftl_ <dig>  at two hours post-infection, these cells were treated with gentamicin to kill extracellular bacteria followed by extensive washing.
subsequently, hek- <dig> cells were lysed and the lysates were diluted and plated to enumerate cfu.
here we observed that 1664d showed reduced entry into the non-phagocytic hek- <dig> cells .
the uptake defect was again rescued by trans complementation .
these data suggest that deob contributes significantly to entry into both phagocytic  and non-phagocytic cells .
discussion
in this paper, we provide evidence that f. tularensis lvs undergoes significant gene expression changes in response to mammalian body temperature .
we hypothesized that genes important for infection of mammals may be induced during transition to this higher temperature.
although some genes, such as ribosomal rna and trna genes, may reflect an increased growth rate at 37°c, our overall hypothesis was supported by the finding that 40% of the protein coding genes induced at 37°c have been shown or implicated to be important for successful francisella infection .
in addition, we showed that two specific genes induced at 37°c contribute to the fitness of f. tularensis lvs during infection.
while infection of a mammalian host exerts a complex set of signals on f. tularensis in addition to temperature, it is likely that many of the unexplored genes induced at mammalian temperature have a role in pathogenesis of f. tularensis.
differential responses to temperature among virulent and less virulent f. tularensis strains will also provide a focused list of relevant candidate virulence determinants to investigate further.
the products of temperature-regulated genes with central roles in physiology and virulence could be targets for novel therapeutics or mutation to generate defined live attenuated vaccines.
one gene induced at 37°c, tiva , encodes a protein with little overall identity to other proteins.
it does, however, contain regions of meager similarity to the domains of proteins involved in the pathogenesis of other bacteria .
we showed that this gene was necessary for full f. tularensis lvs virulence in the chicken embryo model, most likely because it contributed to optimal replication in primary human macrophages .
in a previous high-throughput microarray-based, negative-selection screen for f. novicida genes important for a murine infection, the tiva allele of f. novicida  was not identified  <cit> .
there are two possible explanations for this discrepancy.
there may be intrinsic differences between f. tularensis lvs and f. novicida pathogenesis  <cit>  and their reliance on tiva.
a more probable explanation is that f. tularensis lvs and the more pathogenic f. tularensis schu s <dig> contain a single copy of this gene.
in contrast, f. novicida has two homologs of tiva in its genome, suggesting the possibility of functional redundancy.
these alternate forms  may have compensated for a mutation of tiva  during the negative-selection screen  <cit> , thereby masking any effects on pathogenesis.
this possibility underscores the value of analyzing several different strains when probing f. tularensis pathogenesis.
strain 1664d, a disruption mutant of deob, exhibited significant reduction in uptake into mammalian cells  indicating that this gene's product is involved in a mechanism that enhances entry.
it would be advantageous for francisella to couple expression of a gene important for uptake  with mammalian temperature, a cue encountered early in infection.
augmenting entry would improve access to the intracellular environment in which f. tularensis proliferates  <cit> .
a previous study showed that pyrb  from f. tularensis schu s <dig> was important for invasion in the human hepg <dig> hepatocellular carcinoma line  <cit> .
however, a schu s <dig> mutant of pyrb was not defective in uptake by j <dig>  macrophage-like cells  <cit> , suggesting that the francisella mechanisms of optimal uptake by phagocytes and invasion into hepatocytes are separate phenomena.
in another prior study, mutants of mgla, a key regulator of the fpi  <cit> , and mutants of six genes controlled by this protein were not defective for cell entry  <cit> .
this suggests that francisella has evolved separate regulatory mechanisms for enhancing uptake and for intracellular survival.
however, based on our data, genes critical for both phenomena are affected by mammalian temperature indicating that this cue is an important signal for multiple regulatory networks in francisella.
the uptake defect we have shown with the deob mutant in both phagocytic and non-phagocytic cells has not been described previously.
in our system, deob may have a direct or indirect role in the uptake of francisella.
deob may influence the lps structure of francisella, secondarily improving interactions with host cell receptors.
importantly, complement receptors and mannose receptors are crucial for optimal francisella phagocytosis  <cit> .
this model is consistent with the fact that deob is induced at 37°c and the finding that francisella lps structure is different at lower temperature versus mammalian temperature  <cit> .
alternatively, deob may directly promote francisella entry into host cells as this protein may have an additional function aside from being a phosphopentomutase.
another possibility is that mutation of metabolic genes in pathogenic bacteria can yield pleiotropic effects, resulting in defects in virulence mechanisms, including invasion  <cit> .
further investigation is necessary to determine the precise mechanism of francisella deob in host cell entry.
the data presented here suggest that some genes important for francisella during infection are induced by mammalian temperature.
although we showed by q-pcr that pdpc transcripts were induced at 37°c, none of the other loci in the fpi  <cit>  were up-regulated at mammalian body temperature.
this is consistent with other findings suggesting that iglc protein levels were not induced by a shift from 37°c to 42°c  <cit> .
because many of the loci in the fpi are essential for intra-amoeba growth  <cit> , which would occur at lower, ambient temperatures, it seems logical that these genes are not regulated by temperature.
therefore, the virulence associated-genes induced by mammalian temperature that we have identified are on a separate regulon than most of the genes of the fpi.
this suggests francisella possesses an intricate regulatory circuit to maximize its success in diverse environments.
CONCLUSIONS
f. tularensis lvs undergoes significant gene expression changes in response to mammalian temperature.
this temperature shift is important for the regulation of pathogenesis.
our study characterizes a previously underappreciated environmental cue that regulates the expression of f. tularensis genes associated with virulence in other studies.
importantly, the collection of temperature-regulated genes also defines a rich set of novel candidate virulence determinants, including tiva .
detailed investigation of tiva and deob  revealed unknown or unrecognized roles of these genes in intracellular survival and entry into mammalian cells, respectively.

 francisella tularensis is a gram-negative bacterium that is pathogenic to humans. this organism causes mortality in up to 60% of infected individuals if untreated. based on the potential to weaponize this organism, the center for disease control and prevention has classified f. tularensis as a category a biodefense agent. it is therefore vital to understand how this organism responds to environmental and host signals, and how these cues alter expression of virulence determinants. during the course of a natural francisella infection, this bacterium may transition from an amoeba or an arthropod host to colonize human cells. accompanying this transition, it is likely that chemical and physical signals alert francisella that it has entered a mammalian host. the manner by which f. tularensis integrates environmental stimuli to regulate gene expression is fundamental to the success of this organism as an intracellular pathogen. the most well-studied virulence factors of f. tularensis are encoded in the francisella pathogenicity island. the amount of one of the virulence proteins encoded in this cluster, iglc, increases in response to growth in macrophages and hydrogen peroxide. in addition, iron limitation has been shown to induce transcription and protein levels of iglacd and pdpb as well as stimulate siderophore production by f. tularensis. previously, we have shown that differing culture conditions greatly influence host-pathogen interactions and the ability of f. tularensis live vaccine strain to activate macrophages. prior to the current study, there has only been a single published report characterizing the global transcriptional francisella response to an environmental cue, specifically iron limitation. important insights into the regulation of virulence factors like iglc were defined in this analysis. however, numerous genes associated with virulence by other studies are not affected by different iron concentrations. because f. tularensis may transition between hosts, mammalian body temperature is another signal that is likely to be critical for this pathogen. pathogenic bacteria that encounter a shift in temperature during their life cycle sometimes respond with enhanced virulence factor expression. however, there are discrepancies among the specific groups of genes that are affected by temperature and the mechanism of regulation between organisms. for example, shigella increases production of its type iii secretion system in response to mammalian temperature. the homologous secretion apparatus in pathogenic salmonella, however, is not regulated by temperature. regarding the mechanism of regulation, genes involved in the heat-shock response are often induced at mammalian temperatures relative to those of the environment. this regulation is usually due to the presence of a conserved inverted repeat regulatory structure in the promoter region, or control by a σ32-type heat shock sigma factor. in contrast, the bacterium responsible for whooping cough, bordetella pertussis, uses a two-component system comprised of bvgs and bvga to alter transcription of genes in response to temperature. following induction of bvgas at 37°c, phosphorylation by bvgs allows bvga-binding to promoter regions of virulence genes, such as the adhesin, fimx. given the uncertainty of target temperature-regulated genes and differences in mechanism among bacteria, it is necessary to define the temperature regulon in individual species. studying gene expression changes induced by a shift to mammalian temperature could provide valuable insight into francisella virulence. a few previous studies have investigated the francisella response to temperature at the molecular level. high temperature synonymous with heat stress has been shown to have dramatic effects on f. tularensis gene and protein expression including increased production of the heat shock proteins groel, groes, dnak, and clpb. heat stress has also been shown to enhance the virulence of a mutant form of f. tularensis lvs. interestingly, f. novicida has been shown to alter its outer membrane at 25°c versus 37°c by differentially modifying the lipid a component of the lipopolysaccharide. in addition, it has been observed that virulent f. tularensis clinical isolates increase the mannose modification of their lipopolysaccharide when cultivated at temperatures less than 25°c. the results from these previous studies suggest that a global transcript analysis following a shift to mammalian body temperature should reveal gene regulation that is critical to francisella pathogenesis. here we show that a shift from environmental to mammalian body temperature significantly alters the transcriptome of f. tularensis lvs. many genes that we identified as significantly induced at 37°c have been previously implicated in francisella virulence, supporting the notion that this temperature shift is important for the regulation of pathogenesis. we provide evidence that the product of a gene encoding the hypothetical protein most profoundly induced by mammalian temperature contributes to virulence and intracellular growth. moreover, data presented here indicate that a locus up-regulated at 37°c was required for optimal uptake by eukaryotic cells. this is the first f. tularensis gene identified to be involved in the entry of this organism into both phagocytic and non-phagocytic host cells. the results from this study support a model where detection of mammalian body temperature by f. tularensis is important for regulation of physiology necessary for successful infection.  f. tularensis lvs global temperature regulation to study francisella gene expression changes associated with exposure to mammalian temperature, we conducted a microarray analysis of lvs as a model f. tularensis strain. gene expression of lvs cultured at 26°c was compared to bacteria shifted to 37°c. labeled cdna target was produced from rna isolated from f. tularensis lvs and was subsequently hybridized to a custom agilent francisella microarray. global gene expression data were analyzed with a j statistical test, which was selected to limit the number of false positives. this analysis identified genes with significantly increased expression and genes with decreased expression in response to a shift to mammalian body temperature. collectively, this represents approximately ~11% of the genes in the entire lvs genome. genes with a significant change in expression were examined with the gene pattern program by hierarchical clustering. the clustered data exhibited a distinct pattern of transcript induction and repression in response to mammalian body temperature. together, these data indicate that this temperature shift has a broad impact on f. tularensis transcription. strain, plasmid, the microarray data were confirmed using quantitative real time pcr, in which eight differentially-expressed genes were analyzed, representing transcripts that were both significantly up- and down-regulated. induced genes chosen for validation were the carbamoyl-phosphate synthase large chain, carb, a hypothetical lipoprotein, a dimethyladenosine transferase annotated to function in kasugamycin resistance, ksga, and phosphopentomutase, deob. the down-regulated genes tested by q-pcr included two hypothetical proteins, a cold shock protein, cspc, and a metal ion transporter, tlyc. a similar pattern of expression was observed in both q-pcr and in the microarray experiments. a correlation plot showed a strong, positive association between both data sets. this indicated the microarray platform and the subsequent statistical analysis were robust compared to the sensitive, though low-throughput q-pcr approach. to analyze the differentially-regulated genes more thoroughly, we categorized their presumed protein products based on their clusters of orthologous groups category. as expected, many of the induced and repressed genes were involved in central biological functions such as metabolism, transcription, translation, dna replication, and rna genes. heat shock proteins belong to the category labeled "posttranslational modification/protein turnover/chaperones", and as anticipated, these genes were induced at the higher temperature, providing additional support to our microarray analysis. the heat shock protein result was confirmed by analyzing hsp protein quantity by western blotting, which showed significantly more hsp in bacteria shifted to 37°c versus f. tularensis lvs cultured at 26°c. cog categories found only among the up-regulated transcripts included genes functioning in secretion and cell division, suggesting that host temperature may trigger these cellular processes and reflect, in part, enhanced growth rate. in addition, the cog for bacterial defense mechanisms, which includes type i site-specific restriction-modification systems, was uniquely down-regulated. this response may have evolved to allow this bacterium to better contend with environmental stresses, such as invading bacteriophages. we observed that a large percentage of both up- and down-regulated genes did not belong to a known cog category. proteins with an unknown cog category may have a novel biological role in f. tularensis associated with their temperature regulation. f. tularensis lvs temperature regulation of genes necessary for infection we hypothesized that the shift from environmental to mammalian temperature may be used to regulate genes important for infection. surprisingly, none of the genes encoded in the fpi were significantly up-regulated at 37°c in the microarray analysis. we also confirmed, by immunoblotting, that iglc protein levels were equivalent in bacteria grown at the two different temperatures. upon further scrutiny, we did notice that one gene in the fpi, pdpc, was near the statistical threshold for induction at 37°c. we therefore analyzed pdpc transcript levels by q-pcr as before, which confirmed that this gene was induced at mammalian body temperature. we next compared our list of genes induced at 37°c with those previously shown to be necessary for francisella infection, or postulated to be involved in the pathogenesis of this organism. forty percent of the protein-coding genes significantly up-regulated at 37°c have been reported or predicted to be important for intracellular growth and/or virulence of f. tularensis. the list depicted in table included an assortment of metabolic genes, chaperones, genes encoding hypothetical proteins, and others. the data from table and the pdpc expression results in fig. 2a further supported our hypothesis that genes important for francisella infection are regulated by mammalian body temperature. ftl_ is associated with f. tularensis lvs virulence ftl_ annotated as a hypothetical lipoprotein, was induced by mammalian temperature more profoundly than any other hypothetical protein gene in the f. tularensis lvs genome. we selected this gene for further study to test our hypothesis that temperature regulates genes necessary for infection. a blast search against the national center for biotechnology information database of non-redundant sequences did not identify proteins from organisms other than francisella that had >30% identity to ftl_. this suggested that ftl_ may have a function unique to f. tularensis. the primary structure of ftl_ was further examined by a prosite analysis which indicated this protein likely contained a lipoprotein signal sequence consistent with its annotation. this analysis also revealed that the signal sequence overlapped with a motif similar to that of the enterobacterial trat complement resistance protein. although the initial blast search did not retrieve homologous proteins with high degrees of identity, it did show that ftl_ contained a domain with 20–24% identity and 40–44% similarity with the vacuolating cytotoxin and paralogs of helicobacter pylori j. because ftl_ contained domains similar to other proteins that had a role in pathogenesis, we hypothesized that this protein may contribute to the virulence of f. tularensis. we disrupted ftl_ in lvs, producing strain 1581d, to determine if this gene was associated with f. tularensis virulence. to assess the virulence of 1581d, we utilized a competition assay based on the chicken embryo infection model. the chicken embryo produces a robust innate immune response comprised of complement, phagocytic cells, and cytokine production. here, chicken embryos were infected with a ~1: mixture of lvs and 1581d. at days and post-infection, wild-type lvs exhibited superior survival compared to 1581d. this result was not due to a growth defect since 1581d grew identically to wild-type lvs when cultivated in bacterial growth medium. in a separate experiment in which chicken embryos were infected only with 1581d, isolates from homogenates were all resistant to hygromycin, indicating that this mutant did not revert to wild type during infection. since 1581d was attenuated compared to lvs, the results suggest the function of the ftl_ gene product contributes to f. tularensis virulence. we wanted to determine if the virulence attenuation of 1581d in the chicken embryo infection model was due to a reduced ability to inactivate complement, as ftl_ contained a putative complement resistance domain. therefore, we subjected lvs and 1581d to serum sensitivity assays. there was equivalent survival of wild-type lvs and 1581d when cultured in media containing 20% serum or 20% heat-inactivated serum for up to h. to ensure that the serum complement was functional, e. coli dh5α was used as a control. here, the cfu from the serum-treated e. coli exhibited a reduction of logs relative to the input or heat-inactivated serum groups after min. this suggested that the disruption in 1581d does not affect complement resistance. we determined if ftl_ contributed to growth in a macrophage environment. primary human monocyte-derived macrophages were infected in vitro with either lvs or 1581d. at various time-points, macrophages were lysed, and the lysates were diluted and plated to enumerate viable cfu. we observed attenuated growth of 1581d in macrophages at h post-infection. when ftl_ was complemented in trans in 1581d, wild-type level of growth was restored. this complementation confirmed that the reduced intracellular fitness of 1581d was due to the inactivation of ftl_ and not due to polar effects or alternate mutations. this experiment also suggested that the attenuation of 1581d in the chicken embryo model was likely due to a defect in intracellular survival. together, the data presented here implicate ftl_ a francisella orf induced by mammalian body temperature that has no obvious homologs, with virulence and intracellular survival. therefore, we propose that ftl_ be named temperature-induced, virulence-associated locus a, or tiva. involvement of ftl_ in uptake of f. tularensis lvs many of the genes critical for francisella infection involved metabolism, a major cog category induced at 37°c. this suggested that physiology vital for the success of f. tularensis as a pathogen was regulated by the temperature shift. therefore, we were interested in determining the contribution of temperature-regulated metabolic genes toward francisella pathogenesis. previously, a microarray-based negative selection screen of f. novicida transposon mutants identified the phosphopentomutase, deob, as a gene contributing to growth and/or survival in mice. in other bacteria, proteins encoded by deob homologs normally catalyze the reversible reaction between ribose-1-phosphate and ribose-5-phosphate or between deoxyribose-1-phosphate and deoxyribose-5-phosphate. an f. tularensis lvs chromosomal disruption mutant of deob, ftl_ was constructed. this mutant had a cellular and colony morphology similar to its wild-type parent strain. also, 1664d grew similarly to wild-type lvs in bacterial culture medium. we employed the chicken embryo infection model to confirm that deob contributes to lvs pathogenesis, as it did in f. novicida. here, chicken embryos that had been infected with f. tularensis lvs 1664d exhibited significantly enhanced survival compared to those infected with wild-type lvs over a day period corroborating the deob data from f. novicida. accessing the host cytoplasm to replicate intracellularly is a hallmark of francisella pathogenesis. therefore, we next tested if the virulence attenuation of 1664d in the chicken embryo infection model was due to a defect in entering host cells. at two hours post-infection in vitro, human, monocyte-derived macrophages were treated with gentamicin to kill extracellular bacteria, followed by extensive washing. we consistently observed that substantially fewer 1664d cells were phagocytosed relative to wild-type lvs. importantly, trans complementation of 1664d rescued the uptake defect. this suggested deob is involved in a bacterial mechanism that enhances uptake of f. tularensis. this finding was extended by conducting similar uptake assays in primary human dendritic cells. in both phagocytic cell types, the uptake defect of 1664d was reproduced and complemented. we then qualitatively assessed uptake into phagocytes by microscopy to confirm the quantitative results obtained with cfu measurements. lvs, 1664d, and 1664d/pftl_ were treated with the green fluorescent stain, syto- and then incubated with raw cells, a murine macrophage-like cell line. after a two hour incubation and washes, cells were observed by fluorescence microscopy. here, raw cells infected with lvs or 1664d/pftl_ exhibited bright green fluorescence. in contrast, the raw cells infected with 1664d produced considerably less fluorescence despite comparable inocula to the cultures. the results obtained by microscopy suggested the discrepancy in cfu between wildtype and 1664d was not due to killing in the early phagosome. rather, the levels of fluorescence in fig. 5b were consistent with the cfu uptake data presented in fig. 5a and confirmed that the temperature-regulated deob is important for optimal uptake by mammalian phagocytes. to determine if deob contributed to entry into non-phagocytic cells, we exposed the human embryonic kidney cell line hek- to either lvs, 1664d, or 1664d/pftl_ at two hours post-infection, these cells were treated with gentamicin to kill extracellular bacteria followed by extensive washing. subsequently, hek- cells were lysed and the lysates were diluted and plated to enumerate cfu. here we observed that 1664d showed reduced entry into the non-phagocytic hek- cells. the uptake defect was again rescued by trans complementation. these data suggest that deob contributes significantly to entry into both phagocytic and non-phagocytic cells. discussion in this paper, we provide evidence that f. tularensis lvs undergoes significant gene expression changes in response to mammalian body temperature. we hypothesized that genes important for infection of mammals may be induced during transition to this higher temperature. although some genes, such as ribosomal rna and trna genes, may reflect an increased growth rate at 37°c, our overall hypothesis was supported by the finding that 40% of the protein coding genes induced at 37°c have been shown or implicated to be important for successful francisella infection. in addition, we showed that two specific genes induced at 37°c contribute to the fitness of f. tularensis lvs during infection. while infection of a mammalian host exerts a complex set of signals on f. tularensis in addition to temperature, it is likely that many of the unexplored genes induced at mammalian temperature have a role in pathogenesis of f. tularensis. differential responses to temperature among virulent and less virulent f. tularensis strains will also provide a focused list of relevant candidate virulence determinants to investigate further. the products of temperature-regulated genes with central roles in physiology and virulence could be targets for novel therapeutics or mutation to generate defined live attenuated vaccines. one gene induced at 37°c, tiva, encodes a protein with little overall identity to other proteins. it does, however, contain regions of meager similarity to the domains of proteins involved in the pathogenesis of other bacteria. we showed that this gene was necessary for full f. tularensis lvs virulence in the chicken embryo model, most likely because it contributed to optimal replication in primary human macrophages. in a previous high-throughput microarray-based, negative-selection screen for f. novicida genes important for a murine infection, the tiva allele of f. novicida was not identified. there are two possible explanations for this discrepancy. there may be intrinsic differences between f. tularensis lvs and f. novicida pathogenesis and their reliance on tiva. a more probable explanation is that f. tularensis lvs and the more pathogenic f. tularensis schu s contain a single copy of this gene. in contrast, f. novicida has two homologs of tiva in its genome, suggesting the possibility of functional redundancy. these alternate forms may have compensated for a mutation of tiva during the negative-selection screen, thereby masking any effects on pathogenesis. this possibility underscores the value of analyzing several different strains when probing f. tularensis pathogenesis. strain 1664d, a disruption mutant of deob, exhibited significant reduction in uptake into mammalian cells indicating that this gene's product is involved in a mechanism that enhances entry. it would be advantageous for francisella to couple expression of a gene important for uptake with mammalian temperature, a cue encountered early in infection. augmenting entry would improve access to the intracellular environment in which f. tularensis proliferates. a previous study showed that pyrb from f. tularensis schu s was important for invasion in the human hepg hepatocellular carcinoma line. however, a schu s mutant of pyrb was not defective in uptake by j macrophage-like cells, suggesting that the francisella mechanisms of optimal uptake by phagocytes and invasion into hepatocytes are separate phenomena. in another prior study, mutants of mgla, a key regulator of the fpi, and mutants of six genes controlled by this protein were not defective for cell entry. this suggests that francisella has evolved separate regulatory mechanisms for enhancing uptake and for intracellular survival. however, based on our data, genes critical for both phenomena are affected by mammalian temperature indicating that this cue is an important signal for multiple regulatory networks in francisella. the uptake defect we have shown with the deob mutant in both phagocytic and non-phagocytic cells has not been described previously. in our system, deob may have a direct or indirect role in the uptake of francisella. deob may influence the lps structure of francisella, secondarily improving interactions with host cell receptors. importantly, complement receptors and mannose receptors are crucial for optimal francisella phagocytosis. this model is consistent with the fact that deob is induced at 37°c and the finding that francisella lps structure is different at lower temperature versus mammalian temperature. alternatively, deob may directly promote francisella entry into host cells as this protein may have an additional function aside from being a phosphopentomutase. another possibility is that mutation of metabolic genes in pathogenic bacteria can yield pleiotropic effects, resulting in defects in virulence mechanisms, including invasion. further investigation is necessary to determine the precise mechanism of francisella deob in host cell entry. the data presented here suggest that some genes important for francisella during infection are induced by mammalian temperature. although we showed by q-pcr that pdpc transcripts were induced at 37°c, none of the other loci in the fpi were up-regulated at mammalian body temperature. this is consistent with other findings suggesting that iglc protein levels were not induced by a shift from 37°c to 42°c. because many of the loci in the fpi are essential for intra-amoeba growth, which would occur at lower, ambient temperatures, it seems logical that these genes are not regulated by temperature. therefore, the virulence associated-genes induced by mammalian temperature that we have identified are on a separate regulon than most of the genes of the fpi. this suggests francisella possesses an intricate regulatory circuit to maximize its success in diverse environments.  f. tularensis lvs undergoes significant gene expression changes in response to mammalian temperature. this temperature shift is important for the regulation of pathogenesis. our study characterizes a previously underappreciated environmental cue that regulates the expression of f. tularensis genes associated with virulence in other studies. importantly, the collection of temperature-regulated genes also defines a rich set of novel candidate virulence determinants, including tiva. detailed investigation of tiva and deob revealed unknown or unrecognized roles of these genes in intracellular survival and entry into mammalian cells, respectively. 
f. tularensis lvs undergoes considerable gene expression changes in response to mammalian body temperature. this is the first study identifying a francisella gene that contributes to uptake into both phagocytic and non-phagocytic host cells. after infecting a mammalian host, the facultative intracellular bacterium, francisella tularensis, encounters an elevated environmental temperature. the ftl_ mutant was attenuated in a chicken embryo infection model, which was likely attributable to a defect in survival within macrophages. our results provide new insight into mechanisms of francisella virulence regulation and pathogenesis. interestingly, the deob mutant showed diminished entry into mammalian cells compared to wild-type lvs, including primary human macrophages and dendritic cells, the macrophage-like raw line, and non-phagocytic hek- cells. microarray analysis of f. tularensis lvs shifted from 26°c to 37°c showed ~11% of this bacterium's genes were differentially-regulated. an analysis of tiva and deob revealed that these genes contribute to intracellular survival and entry into mammalian cells, respectively. importantly, the compilation of temperature-regulated genes also defines a rich collection of novel candidate virulence determinants, including tiva. importantly, 40% of the protein-coding genes that were induced at 37°c have been previously implicated in virulence or intracellular growth of francisella in other studies, associating the bacterial response to this temperature shift with pathogenesis. forty-four percent of the genes induced at 37°c encode proteins of unknown function, suggesting novel francisella virulence traits are regulated by mammalian temperature.
 after infecting a mammalian host, the facultative intracellular bacterium, francisella tularensis, encounters an elevated environmental temperature. we hypothesized that this temperature change may regulate genes essential for infection.  microarray analysis of f. tularensis lvs shifted from 26°c to 37°c showed ~11% of this bacterium's genes were differentially-regulated. importantly, 40% of the protein-coding genes that were induced at 37°c have been previously implicated in virulence or intracellular growth of francisella in other studies, associating the bacterial response to this temperature shift with pathogenesis. forty-four percent of the genes induced at 37°c encode proteins of unknown function, suggesting novel francisella virulence traits are regulated by mammalian temperature. to explore this possibility, we generated two mutants of loci induced at 37°c. the ftl_ mutant was attenuated in a chicken embryo infection model, which was likely attributable to a defect in survival within macrophages. ftl_ encodes a novel hypothetical protein that we suggest naming temperature-induced, virulence-associated locus a, tiva. interestingly, the deob mutant showed diminished entry into mammalian cells compared to wild-type lvs, including primary human macrophages and dendritic cells, the macrophage-like raw line, and non-phagocytic hek- cells. this is the first study identifying a francisella gene that contributes to uptake into both phagocytic and non-phagocytic host cells.  our results provide new insight into mechanisms of francisella virulence regulation and pathogenesis. f. tularensis lvs undergoes considerable gene expression changes in response to mammalian body temperature. this temperature shift is important for the regulation of genes that are critical for the pathogenesis of francisella. importantly, the compilation of temperature-regulated genes also defines a rich collection of novel candidate virulence determinants, including tiva. an analysis of tiva and deob revealed that these genes contribute to intracellular survival and entry into mammalian cells, respectively. 