BACKGROUND
francisella tularensis  is a gram-negative intracellular pathogen that is the etiological agent of a multi-syndromic disease with a high morbidity/mortality that is referred to as tularemia.
the pneumonic form of tularemia is of particular concern because of the high mortality rate  following inhalation of as few as ten organisms  <cit> .
francisella species are found throughout the northern hemisphere and infect a variety of vertebrate and invertebrate hosts  <cit> .
infections with ft can be contracted from blood sucking insects, such as the deer fly  <cit> , mosquitoes  <cit> , and ticks  <cit> , and by open-wound contact with infected animal tissue  <cit> .
upon entry into a susceptible vertebrate host, ft is readily phagocytized by resident macrophages and dendritic cells and quickly escapes into the cytoplasm  <cit>  where it multiplies.
late in its replicative cycle, ft induces apoptotic death of the host phagocyte, resulting in release of progeny bacteria that can infect new host cells.
recent studies have shown that significant numbers of ft are found in the acellular plasma fraction of mice infected intradermally or intranasally with either ft live vaccine strain   or ft schu s <dig>   <cit> , and intranasally with ft novicida  <cit> .
these findings suggest that, in addition to utilizing the intracellular cytoplasmic niche for replication and protection from humoral immunity, ft may also have a significant extracellular phase.
several studies have shown that deposition of host complement component c <dig> on the surface of ft is required for opsonophagocytosis by activating cr <dig> and cr4-mediated phagocytosis by macrophages and dendritic cells  <cit> .
it is also known that ft is relatively resistant to complement-mediated lysis  <cit> .
a recent report suggested that resistance of ft to membrane attack complex-mediated lysis may be due  to its ability to bind to factor h from host plasma  <cit> .
it is possible that the ability of ft to bind to factor h and potentially to other host plasma components plays a significant role in its pathogenesis.
it has been long established that a broad spectrum of both gram-positive and gram-negative bacterial pathogens gain a survival advantage by interacting with components of the host coagulation/fibrinolytic system in humans  <cit> .
for instance, the ability to acquire surface-associated plasmin has been documented as an important virulence mechanism in group a streptococci  <cit> , borrelia burgdorferi  <cit> , and yersinia pestis  <cit>  by aiding in the organism's ability to penetrate the extracellular matrix and to disseminate to distal sites in the host.
plasminogen  is a 92-kda glycoprotein zymogen that is involved in fibrinolysis.
this precursor protein is converted to an active serine protease  by cleavage of the peptide bond between residues r560and v <dig> in vivo via urokinase-type  and/or tissue-type  plg activators.
plasmin has an important role in blood clot resolution because of its role in the degradation of fibrin polymers.
because plasmin has other substrates that include pro-collagenases, pro-metalloproteinases, and extracellular matrix proteins, such as fibronectin, laminin, and vitronectin, the ability of a bacterium to acquire surface-associated plasmin can result in an enhanced ability of the pathogen to penetrate the extracellular matrix and to disseminate to distal sites in the host  <cit> .
in this report we show that plg binds to the surface of ft in vitro and that surface-bound plg can be converted to the active plasmin form.
in addition, using a combination of far-western blotting analyses coupled with proteomic methodologies, we have identified several ft proteins that can bind to human plg in vitro.
RESULTS
binding of plg from fresh human plasma to the surface of ftlvs
we used an elisa assay to determine that plg in fresh frozen plasma  binds to ftlvs grown to mid-log phase in bhi .
binding was inhibited when ε-aminocaproic acid , known to inhibit binding of plg to lysine groups in proteins, was included in the incubation mixture.
to help eliminate the possibility of non-specific binding of plg due to its high concentrations in human plasma and also to rule out the contributions of other plasma proteins, we used purified human glu-plg  and noted similar results to those observed when ffp was used .
we also found that huplg binds to the highly virulent schu s <dig> strain of ft at moderately higher levels than observed with ftlvs .
we confirmed that binding of huplg to ft is a lysine-dependent interaction by showing that increasing concentrations of εaca can inhibit binding of huplg to ftlvs in a dose-dependent fashion .
when similar concentrations of glycine were used as an inhibitor control, no inhibition of huplg binding was observed .
confocal microscopic analyses suggested that huplg binds to the surface of ft ; however, it is possible that some of the staining observed was the result of huplg penetration into the outer envelope of ft. although is has been reported that culture media composition can have a significant impact of the surface properties and virulence characteristics of ftlvs  <cit> , we observed no differences in the ability of plg to bind to the surface of ftlvs grown in modified mueller-hinton medium vs. brain-heart infusion broth .
plasmin activation on the surface of ft lvs in vitro by a plg activator
in other bacterial systems, surface-bound plg can be converted to its proteolytically active plasmin form that contributes to the organism's virulence  <cit> .
to test whether huplg bound to ftlvs can be converted to plasmin, we used a chromogenic plasmin substrate  to detect proteolytic activity following the addition of tissue plg activator  .
we also found that plasmin on the surface of ft can break down fibronectin , suggesting that ft-bound plasmin can potentially participate in the degradation of extracellular matrices.
identification of putative francisella plg-binding membrane proteins
we used far-western blot methodology and mass spectrometry to identify potential plg receptors in the sarkosyl soluble and insoluble ftlvs membrane fractions .
sarkosyl is a weak anionic detergent in which many outer membrane proteins of gram-negative bacteria are insoluble  <cit> .
we transferred the sarkosyl-treated proteins to a pvdf membrane and incubated the membrane with plg and identified bound plg by reaction with anti-plg mabs .
we used the relative migration rates of the reactive bands to identify the reactive proteins on a duplicate coomassie-stained polyacrylamide gel , which were then excised for proteomic analysis by mass spectrometry.
several prominent plg-binding proteins were noted in the total membrane fraction of ftlvs, all but one of which was found in the sarkosyl insoluble fraction .
the identity of the prominent proteins from this assay  are the products of the following genes: ftl_ <dig> , ftl_ <dig> , ftl_ <dig> , ftl_ <dig> , and ftl_ <dig> .
discussion
until recently ft has been considered an intracellular pathogen whose dissemination to tissues distal to the site of initial infection was highly dependent on its ability survive within host macrophages.
the observation that ft can be found in relatively high numbers in the acellular plasma fraction of its mammalian host  <cit>  suggested that ft may have a significant extracellular component to its life cycle and that interactions between ft and one or more plasma proteins could contribute to its ability to disseminate within the host.
there are a number of examples of bacterial pathogens that utilize interactions with host plasma components to enhance their ability to colonize and to penetrate the extracellular matrices of host cells/tissues.
a wide range of bacterial pathogens  subvert the destructive mechanisms of the complement cascade by acquiring surface-bound complement control proteins  <cit> .
moreover, a number of gram-positive bacterial pathogens including streptococcal spp.
<cit> , staphylococcal spp.
<cit> , and bacillus anthracis  <cit> , as well as gram-negative bacteria such as pseudomonas aeruginosa  <cit>  have been shown to augment their invasive capacity by interacting with fibrinogen, fibronectin, and/or plg.
yersinia pestis is probably the best-characterized example of a pathogen that exploits the host fibrinolytic system to penetrate host tissues.
yersinia expresses a surface serine protease  whose substrates include several complement components, plg, and alpha2-antiplasmin .
pla also has adhesin activity and binds to laminin .
because pla upregulates plasmin activity, and because laminin is a substrate of plasmin, yersinia can very efficiently penetrate basement membranes of host tissues .
clearly, interaction with plasma components is a strategy that is used by many bacterial pathogens to gain a survival advantage within their hosts.
the goal of the studies described here was to determine whether ft has the potential to use the host fibrinolytic system  to enhance its ability to penetrate/disseminate following infection of a mammalian host.
our results indicate that both ftlvs and ftschus <dig> are able to acquire surface bound plg in vitro and that this zymogen can be converted by a host-derived plg activator into its active serine protease form  while bound to ftlvs.
the ability of plg to bind its ligands typically involves its lysine-binding kringle domains.
this specific interaction between plg and exposed lysine residues can be inhibited with the lysine-analogue εaca and, to a lesser extent, with free lysine.
our findings revealed that binding of plg to the surface of ftlvs could be inhibited by εaca in a dose-dependent fashion.
moreover, we showed that plasmin bound to the surface of ft could degrade fibronectin.
this finding supports our hypothesis that the ability of ft to bind to serum plasmin may enhance its ability to penetrate extracellular matrices, enhancing its ability to disseminate in vivo.
using a ligand-blotting technique coupled with proteomic methodologies we identified five ftlvs proteins that were able to bind to plg, each of which are highly conserved among the various ft type a and b strains.
three of these proteins are lipoproteins .
two of the lipoproteins are unique to ft, while the third, peptidoglycan-associated lipoprotein , is highly conserved among gram-negative bacteria.
the specific use of surface-exposed lipoproteins as receptors for host plg is not unusual and has been well documented in other human bacterial pathogens, such as some members of the genus borrelia and treponema.
several members of the genus borrelia use complement regulator-acquiring surface proteins  to bind both plg and complement factor h to aid in the ability of the organism to both disseminate and to resist innate immunity  <cit> .
an additional example of a plg-binding lipoprotein is oppa of treponema denticola, which has been suggested to play a role in periodontal disease in humans  <cit> .
with this in mind, there lies the possibility that lipoproteins of francisella species may have the capacity to bind multiple host-derived proteins in addition to plg.
here we have shown that ft can bind to plg and that surface-bound plg can be activated by tpa to its proteolytic form .
the binding of plg on the surface of ft could play a role in several phases of tularemia, including the initial entry into the host through insect bites and/or broken skin where active fibrinolytic processes would provide an early opportunity for ft to acquire proteolytic activity that might augment the establishment or dissemination of infection.
during later phases of tularemia the acquisition of plasmin on the cell surface may contribute to its pathogenicity by degrading host innate effector molecules and extracellular matrix components.
based on the new report that ft-bound plasmin can degrade immunoglobulins  <cit> , as well as the established ability of ft to acquire surface-bound factor h  <cit> , it also appears likely that ft uses plasma components to interfere with host humoral immune mechanisms throughout the course of ft infection.
future studies to identify additional plasma components that can be surface acquired by ft may uncover additional virulence mechanisms used by this pathogen during its extracellular life cycle.
CONCLUSIONS
ft interacts with at least two serum components , and it seems likely that ft also uses interactions with additional host serum components to gain a survival advantage.
our lab is examining ft interactions with additional targets, including fibrinogen and fibronectin, both of which are substrates for plasmin and are host components that are known to be exploited by numerous pathogens for adhesion to and penetration of extracellular matrix layers.
the interaction of ft with host serum components may play a significant role in the survival and dissemination of this highly pathogenic bacterium.
gaining a better understanding of these interactions could be a critical step in the development of therapeutic and prophylactic interventions for tularemic disease.

 francisella tularensis is a gram-negative intracellular pathogen that is the etiological agent of a multi-syndromic disease with a high morbidity/mortality that is referred to as tularemia. the pneumonic form of tularemia is of particular concern because of the high mortality rate following inhalation of as few as ten organisms. francisella species are found throughout the northern hemisphere and infect a variety of vertebrate and invertebrate hosts. infections with ft can be contracted from blood sucking insects, such as the deer fly, mosquitoes, and ticks, and by open-wound contact with infected animal tissue. upon entry into a susceptible vertebrate host, ft is readily phagocytized by resident macrophages and dendritic cells and quickly escapes into the cytoplasm where it multiplies. late in its replicative cycle, ft induces apoptotic death of the host phagocyte, resulting in release of progeny bacteria that can infect new host cells. recent studies have shown that significant numbers of ft are found in the acellular plasma fraction of mice infected intradermally or intranasally with either ft live vaccine strain or ft schu s, and intranasally with ft novicida. these findings suggest that, in addition to utilizing the intracellular cytoplasmic niche for replication and protection from humoral immunity, ft may also have a significant extracellular phase. several studies have shown that deposition of host complement component c on the surface of ft is required for opsonophagocytosis by activating cr and cr4-mediated phagocytosis by macrophages and dendritic cells. it is also known that ft is relatively resistant to complement-mediated lysis. a recent report suggested that resistance of ft to membrane attack complex-mediated lysis may be due to its ability to bind to factor h from host plasma. it is possible that the ability of ft to bind to factor h and potentially to other host plasma components plays a significant role in its pathogenesis. it has been long established that a broad spectrum of both gram-positive and gram-negative bacterial pathogens gain a survival advantage by interacting with components of the host coagulation/fibrinolytic system in humans. for instance, the ability to acquire surface-associated plasmin has been documented as an important virulence mechanism in group a streptococci, borrelia burgdorferi, and yersinia pestis by aiding in the organism's ability to penetrate the extracellular matrix and to disseminate to distal sites in the host. plasminogen is a 92-kda glycoprotein zymogen that is involved in fibrinolysis. this precursor protein is converted to an active serine protease by cleavage of the peptide bond between residues r560and v in vivo via urokinase-type and/or tissue-type plg activators. plasmin has an important role in blood clot resolution because of its role in the degradation of fibrin polymers. because plasmin has other substrates that include pro-collagenases, pro-metalloproteinases, and extracellular matrix proteins, such as fibronectin, laminin, and vitronectin, the ability of a bacterium to acquire surface-associated plasmin can result in an enhanced ability of the pathogen to penetrate the extracellular matrix and to disseminate to distal sites in the host. in this report we show that plg binds to the surface of ft in vitro and that surface-bound plg can be converted to the active plasmin form. in addition, using a combination of far-western blotting analyses coupled with proteomic methodologies, we have identified several ft proteins that can bind to human plg in vitro.  binding of plg from fresh human plasma to the surface of ftlvs we used an elisa assay to determine that plg in fresh frozen plasma binds to ftlvs grown to mid-log phase in bhi. binding was inhibited when ε-aminocaproic acid, known to inhibit binding of plg to lysine groups in proteins, was included in the incubation mixture. to help eliminate the possibility of non-specific binding of plg due to its high concentrations in human plasma and also to rule out the contributions of other plasma proteins, we used purified human glu-plg and noted similar results to those observed when ffp was used. we also found that huplg binds to the highly virulent schu s strain of ft at moderately higher levels than observed with ftlvs. we confirmed that binding of huplg to ft is a lysine-dependent interaction by showing that increasing concentrations of εaca can inhibit binding of huplg to ftlvs in a dose-dependent fashion. when similar concentrations of glycine were used as an inhibitor control, no inhibition of huplg binding was observed. confocal microscopic analyses suggested that huplg binds to the surface of ft; however, it is possible that some of the staining observed was the result of huplg penetration into the outer envelope of ft. although is has been reported that culture media composition can have a significant impact of the surface properties and virulence characteristics of ftlvs, we observed no differences in the ability of plg to bind to the surface of ftlvs grown in modified mueller-hinton medium vs. brain-heart infusion broth. plasmin activation on the surface of ft lvs in vitro by a plg activator in other bacterial systems, surface-bound plg can be converted to its proteolytically active plasmin form that contributes to the organism's virulence. to test whether huplg bound to ftlvs can be converted to plasmin, we used a chromogenic plasmin substrate to detect proteolytic activity following the addition of tissue plg activator. we also found that plasmin on the surface of ft can break down fibronectin, suggesting that ft-bound plasmin can potentially participate in the degradation of extracellular matrices. identification of putative francisella plg-binding membrane proteins we used far-western blot methodology and mass spectrometry to identify potential plg receptors in the sarkosyl soluble and insoluble ftlvs membrane fractions. sarkosyl is a weak anionic detergent in which many outer membrane proteins of gram-negative bacteria are insoluble. we transferred the sarkosyl-treated proteins to a pvdf membrane and incubated the membrane with plg and identified bound plg by reaction with anti-plg mabs. we used the relative migration rates of the reactive bands to identify the reactive proteins on a duplicate coomassie-stained polyacrylamide gel, which were then excised for proteomic analysis by mass spectrometry. several prominent plg-binding proteins were noted in the total membrane fraction of ftlvs, all but one of which was found in the sarkosyl insoluble fraction. the identity of the prominent proteins from this assay are the products of the following genes: ftl_, ftl_, ftl_, ftl_, and ftl_. discussion until recently ft has been considered an intracellular pathogen whose dissemination to tissues distal to the site of initial infection was highly dependent on its ability survive within host macrophages. the observation that ft can be found in relatively high numbers in the acellular plasma fraction of its mammalian host suggested that ft may have a significant extracellular component to its life cycle and that interactions between ft and one or more plasma proteins could contribute to its ability to disseminate within the host. there are a number of examples of bacterial pathogens that utilize interactions with host plasma components to enhance their ability to colonize and to penetrate the extracellular matrices of host cells/tissues. a wide range of bacterial pathogens subvert the destructive mechanisms of the complement cascade by acquiring surface-bound complement control proteins. moreover, a number of gram-positive bacterial pathogens including streptococcal spp., staphylococcal spp., and bacillus anthracis, as well as gram-negative bacteria such as pseudomonas aeruginosa have been shown to augment their invasive capacity by interacting with fibrinogen, fibronectin, and/or plg. yersinia pestis is probably the best-characterized example of a pathogen that exploits the host fibrinolytic system to penetrate host tissues. yersinia expresses a surface serine protease whose substrates include several complement components, plg, and alpha2-antiplasmin. pla also has adhesin activity and binds to laminin. because pla upregulates plasmin activity, and because laminin is a substrate of plasmin, yersinia can very efficiently penetrate basement membranes of host tissues. clearly, interaction with plasma components is a strategy that is used by many bacterial pathogens to gain a survival advantage within their hosts. the goal of the studies described here was to determine whether ft has the potential to use the host fibrinolytic system to enhance its ability to penetrate/disseminate following infection of a mammalian host. our results indicate that both ftlvs and ftschus are able to acquire surface bound plg in vitro and that this zymogen can be converted by a host-derived plg activator into its active serine protease form while bound to ftlvs. the ability of plg to bind its ligands typically involves its lysine-binding kringle domains. this specific interaction between plg and exposed lysine residues can be inhibited with the lysine-analogue εaca and, to a lesser extent, with free lysine. our findings revealed that binding of plg to the surface of ftlvs could be inhibited by εaca in a dose-dependent fashion. moreover, we showed that plasmin bound to the surface of ft could degrade fibronectin. this finding supports our hypothesis that the ability of ft to bind to serum plasmin may enhance its ability to penetrate extracellular matrices, enhancing its ability to disseminate in vivo. using a ligand-blotting technique coupled with proteomic methodologies we identified five ftlvs proteins that were able to bind to plg, each of which are highly conserved among the various ft type a and b strains. three of these proteins are lipoproteins. two of the lipoproteins are unique to ft, while the third, peptidoglycan-associated lipoprotein, is highly conserved among gram-negative bacteria. the specific use of surface-exposed lipoproteins as receptors for host plg is not unusual and has been well documented in other human bacterial pathogens, such as some members of the genus borrelia and treponema. several members of the genus borrelia use complement regulator-acquiring surface proteins to bind both plg and complement factor h to aid in the ability of the organism to both disseminate and to resist innate immunity. an additional example of a plg-binding lipoprotein is oppa of treponema denticola, which has been suggested to play a role in periodontal disease in humans. with this in mind, there lies the possibility that lipoproteins of francisella species may have the capacity to bind multiple host-derived proteins in addition to plg. here we have shown that ft can bind to plg and that surface-bound plg can be activated by tpa to its proteolytic form. the binding of plg on the surface of ft could play a role in several phases of tularemia, including the initial entry into the host through insect bites and/or broken skin where active fibrinolytic processes would provide an early opportunity for ft to acquire proteolytic activity that might augment the establishment or dissemination of infection. during later phases of tularemia the acquisition of plasmin on the cell surface may contribute to its pathogenicity by degrading host innate effector molecules and extracellular matrix components. based on the new report that ft-bound plasmin can degrade immunoglobulins, as well as the established ability of ft to acquire surface-bound factor h, it also appears likely that ft uses plasma components to interfere with host humoral immune mechanisms throughout the course of ft infection. future studies to identify additional plasma components that can be surface acquired by ft may uncover additional virulence mechanisms used by this pathogen during its extracellular life cycle.  ft interacts with at least two serum components, and it seems likely that ft also uses interactions with additional host serum components to gain a survival advantage. our lab is examining ft interactions with additional targets, including fibrinogen and fibronectin, both of which are substrates for plasmin and are host components that are known to be exploited by numerous pathogens for adhesion to and penetration of extracellular matrix layers. the interaction of ft with host serum components may play a significant role in the survival and dissemination of this highly pathogenic bacterium. gaining a better understanding of these interactions could be a critical step in the development of therapeutic and prophylactic interventions for tularemic disease. 
this observation suggests that the interaction between ft and host plasma components may play an important role in survival and dissemination of the bacterium during the course of infection. we show that plg binds to the surface of ft and that surface-bound plg can be activated to plasmin in the presence of tissue plg activator in vitro. francisella tularensis is a gram-negative facultative intracellular coccobacillus and is the causal agent of a life-threatening zoonotic disease known as tularemia. the ability of ft to acquire surface bound plg that can be activated on its surface may be an important virulence mechanism that results in an increase in initial infectivity, survival, and/or dissemination of this bacterium in vivo. a number of both gram-positive and gram-negative bacterial pathogens have the ability to bind to plg, giving them a survival advantage by increasing their ability to penetrate extracellular matrices and cross tissue barriers.
 francisella tularensis is a gram-negative facultative intracellular coccobacillus and is the causal agent of a life-threatening zoonotic disease known as tularemia. although ft preferentially infects phagocytic cells of the host, recent evidence suggests that a significant number of bacteria can be found extracellularly in the plasma fraction of the blood during active infection. this observation suggests that the interaction between ft and host plasma components may play an important role in survival and dissemination of the bacterium during the course of infection. plasminogen is a protein zymogen that is found in abundance in the blood of mammalian hosts. a number of both gram-positive and gram-negative bacterial pathogens have the ability to bind to plg, giving them a survival advantage by increasing their ability to penetrate extracellular matrices and cross tissue barriers.  we show that plg binds to the surface of ft and that surface-bound plg can be activated to plasmin in the presence of tissue plg activator in vitro. in addition, using far-western blotting assays coupled with proteomic analyses of ft outer membrane preparations, we have identified several putative plg-binding proteins of ft.  the ability of ft to acquire surface bound plg that can be activated on its surface may be an important virulence mechanism that results in an increase in initial infectivity, survival, and/or dissemination of this bacterium in vivo. 