BACKGROUND
yersinia pestis is a gram-negative bacterium with a zoonotic life cycle that occasionally results in human infections leading to plague  <cit> .
plague manifests in two major forms, the most common being bubonic plague.
bubonic plague is transmitted to humans through the bite of an infected flea, resulting in intradermal inoculation of the bacterium, which progresses to form the characteristic lymphadenitis  of plague  <cit> .
the less common but contagious form of the disease is pneumonic plague, which is a severe pneumonia resulting either from inhalation of infectious respiratory droplets, or secondary to bubonic or septicemic plague.
pneumonic plague is highly aggressive and if untreated, is able to kill the host within 2- <dig> days post-exposure, with mortality rates that approach 100%  <cit> .
we and others have begun to analyze the immune response to primary pneumonic plague using wild-type strains of y. pestis in an effort to understand the interactions of y. pestis with its host  <cit> early during the development of disease.
the rapid progression of the disease and associated mortality suggests that subversion of innate immunity plays a key role in disease development  <cit> .
our previous studies indicate a delay in the host inflammatory response to infection, resulting in an opportunity for the bacterium to replicate to high numbers and potentially overwhelm the host immune system, rapidly resulting in death  <cit> .
modulation of host-immune responses is a common pathogenic mechanism among the virulent species of yersinia and much work has been done to understand the molecular mechanisms underlying the virulence factors involved in this process  <cit> .
the  <dig> kb virulence plasmid , which is essential for virulence, contains all of the machinery and effector proteins for a type-three secretion system   <cit> .
the secreted effector proteins are also known as the yersinia outer proteins  and are transported from the bacterial cytosol through the ttss into the cytoplasm of a host cell to facilitate infection.
all six of the effector yops have been studied, and at least one function has been assigned to each based on in vitro studies.
several are involved in manipulating the host cytoskeleton ; these yops interfere with the rho family of gtpases and other host proteins involved in the regulation of the cytoskeleton, and are therefore important in the modulation of phagocytosis  <cit> .
others are involved in the tempering of the host immune response, in particular, yoph and yopj, and to a lesser extent, yope and yopm have been shown to impact inflammatory cytokine expression in vitro and in vivo respectively  <cit> .
yoph is a protein tyrosine phosphatase, which is known to interact with p130cas and fak to impair invasion of epithelial cells by yersinia pseudotuberculosis  <cit> .
yoph blocks cytoskeletal rearrangement upon injection into the host cell, contributing to the decreased ability of macrophages and other immune cells to phagocytose the bacteria  <cit> .
distinct from its ability to interact with the cytoskeleton, yoph is also known to impact host cell signaling by inactivation of the pi3k pathway through unknown mechanisms  <cit> .
the pi3k pathway plays an important role in the macrophage response to infection including generation of the oxidative burst and nitric oxide production, phagosome formation, and has a role in a negative regulation of il- <dig> production, to help keep the inflammatory response in check  <cit> .
numerous studies have determined that mutations in yoph severely impact the virulence of the yersiniae  <cit> .
recently we reported that co92Δyoph is severely attenuated in both an intranasal and subcutaneous models of y. pestis infection, which makes co92Δyoph a very good live-attenuated vaccine strain  <cit> .
the ability of co92Δyoph to provide significant protection against virulent challenge suggests that a robust protective immune response is generated during primary infection with this strain.
however, the role of yoph in the pathogenesis of plague pneumonia or its impact on the immune response in the lung has not been examined.
in this study we test the impact of the co92Δyoph mutant on both the virulence and inflammatory response using a mouse model of primary pneumonic plague.
RESULTS
y. pestis co92Δyoph is severely attenuated in an intranasal model of primary pneumonic plague
the survival of cd <dig> mice after in infection with co <dig> or co92Δyoph was determined and the survival curves analyzed by log-rank analysis.
mice infected in with ~ <dig> cfu co92Δyoph appeared normal at all time points post infection and showed no outward signs of disease whereas animals infected with ~ <dig> cfu co <dig> were severely debilitated two days post-infection and succumbed to disease by day  <dig> post-infection .
altogether, these data revealed that mice were able to survive both intranasal and intradermal infection with co92Δyoph at doses of ~ <dig> cfu .
our results indicate a significant difference in survival  when co <dig> is compared to co92Δyoph and that at the highest dose tested  co92Δyoph is avirulent in cd <dig> mice.
attenuation of co92Δyoph is due to the loss of yoph and yoph enzymatic activity
we tested the ability of the co92Δyoph mutant complemented with a wild type copy of the yoph gene to secrete yoph in vitro.
as shown in figure 2a when the ttss is induced in vitro, the co92Δyoph mutant complemented with yoph secretes similar levels of yoph into the culture supernatant as co <dig>  due to the yoph cleavage products in secreted protein preparations, we also evaluated the levels of yoph in whole cell extracts after induction.
similar levels of yoph were detected in the whole cell extracts and were consistent with the secreted protein profile .
to test if the attenuation phenotype of co92Δyoph is linked to the yoph mutation, mice were infected with the co92Δyoph mutant carrying the pamc- <dig> complementing plasmid.
complementation of yoph expressed from the pamc- <dig> plasmid restores virulence to the levels observed with co <dig> carrying the empty pcr <dig>  plasmid .
however, virulence is lost when the co92Δyoph mutant is complemented with the yoph-c403a gene expressed from the pamc- <dig> plasmid .
the c403a mutation abolishes the yoph tyrosine phophatase activity, strongly suggesting that the attenuation of the co92Δyoph mutant is due to loss of the yoph phosphatase activity and these data are consistent with previous studies of other yersinia species and attenuated y. pestis strains which determined that yoph mutants are highly attenuated  <cit> .
y. pestis co92Δyoph is able to persist in the lungs but does not spread systemically
the severe attenuation of y. pestis co92Δyoph could be due to an inability to colonize the lung, disseminate, or replicate in the mouse.
to further investigate the pathogenesis of co92Δyoph, we determined the changes in bacterial burdens over time.
mice were infected in with 2× <dig> or 1× <dig> cfu y. pestis co <dig> or co92Δyoph respectively and sacrificed at the indicated times.
lungs, livers, and spleens were processed as described in the methods and dilutions plated to determine the cfu-bacteria/g tissue at each time point post-infection .
all of the mice infected with wild-type co <dig> were dead by  <dig> hours post-infection making collection of data at  <dig> h and beyond impossible for this group.
our results suggest that co92Δyoph is able to persist in the lungs through  <dig> h post-infection .
bacterial burdens in the lungs of mice infected with co <dig> were significantly higher when compared to co92Δyoph at both  <dig> h and  <dig> h post-infection  .
these data indicate that the co92Δyoph mutant was unable to replicate at the site of infection.
in addition, we were unable to detect dissemination of the co92Δyoph bacterium to the spleen or liver at any time post-infection, while wild-type co <dig> was present in the livers and spleens in large numbers at  <dig> h post-infection consistent with what we have reported previously  <cit> .
intranasal infection with co92Δyoph induces tnf-α and il-1β early during infection
in mouse models of primary pneumonic plague, mice infected with co <dig> do not mount an inflammatory cytokine response until  <dig> h to  <dig> h post-infection  <cit> .
we hypothesized that the attenuation of co92Δyoph could be partially due to a strong inflammatory response to the presence of the bacterium.
further, due to the delayed arrival of neutrophils during plague pneumonia, we suspected that tnf-α and il-1β might be involved  <cit> .
to test this hypothesis, we measured the concentrations of tnf-α and il-1β in the bronchiolar alveolar lavage fluid  after in infection with co <dig> or co92Δyoph.
mice were infected with 2× <dig> cfu of either y. pestis co <dig> or co92Δyoph and sacrificed  <dig> or  <dig> hours post-infection.
bal was performed on these mice, and then elisa analyzed the balf for the concentration of tnf-α and il-1β.
mice infected with co92Δyoph elicited a robust pro-inflammatory cytokine response at  <dig> h post-infection, characterized by  <dig> ±  <dig> pg/ml of tnf-α and  <dig> ±  <dig> pg/ml of il-1β in the balf of these mice  <dig> h post-infection .
by  <dig> h post-infection the levels of both cytokines had decreased but remained significant with concentrations of tnf-α =  <dig> ±  <dig> pg/ml, and il-1β =  <dig> ±  <dig> pg/ml .
bal from mice infected for  <dig> hours with wild-type co <dig> had tnf-α and il-1β levels approximately equal to the levels in the balf from uninfected mice.
however, at  <dig> h post-infection, both cytokines were detected in the balf of these mice with concentrations of tnf-α =  <dig> ±  <dig> pg/ml, and il-1β =  <dig> ±  <dig> pg/ml .
the timing of cytokine expression and the levels of cytokine induced during a co <dig> are consistent with what we have observed previously .
altogether, these data suggest that yoph is involved in delaying or inhibiting the expression of tnf-α and il-1β during plague pneumonia and may be involved in the overall delayed inflammatory response to this infection.
mice lacking proinflammatory cytokines are more sensitive to co92Δyoph infection
if tnf-α and il-1β were critical host-components leading to the attenuation observed in mice infected with co92Δyoph, then it would not be unreasonable to predict that mice deficient in these molecules would be more sensitive to infection with co92Δyoph.
to test this hypothesis, we used antibody mediated cytokine depletion followed by in challenge with co92Δyoph or co <dig>  briefly, mice were treated with monoclonal antibodies to tnf-α, il-1β, tnf-α+il-1β, or the appropriate control igg one day prior to infection and then every third day for the course of the infection as we have described previously  <cit> .
mice treated with il-1β depleting antibodies were not significantly more sensitive to co92Δyoph infection than the control mice  and only 10% of the mice treated with the tnf-α depleting antibodies succumbed to infection .
however, 30% of the mice treated with both the il-1β and the tnf-α depleting antibodies succumbed to co92Δyoph infection .
these data did not reach significance  but suggest that proinflammatory cytokines expressed during co92Δyoph infection help control this infection.
because the site of the infection was the mucosal surface of the nasal cavity and lung, it was possible that systemically administered igg poorly depletes cytokines at this site.
to test our hypothesis in another way, we infected tnf-α deficient mice  and control mice  with co92Δyoph or co <dig>  as predicted, the control mice and the tnf-α-/- mice rapidly succumbed to infection with co <dig>  consistent with what was observed with out-bred cd <dig> mice, the b6129sf2/j control mice were completely resistant to infection with  <dig> cfu of co92Δyoph .
however, when the isogenic tnf-α-/- mice were infected with  <dig> ×  <dig> cfu co92Δyoph, significantly , 40% of the mice succumbed to infection within  <dig> days .
altogether, these data suggest that the co92Δyoph mutant of yersinia pestis is attenuated in multiple strains of wild type mice including cd- <dig>  b6129sf2/j, and c57bl6/j but is partially virulent in mice lacking tnf-α .
these data further suggest that yoph mediated suppression of pulmonary tnf-α is an important virulence promoting mechanism .
histopathology of co92Δyophinfection
recently, we and others described the development of primary pneumonic plague in the mouse following infection with co <dig>  <cit> .
the most striking feature of primary plague pneumonia is a 36-hour delay in the inflammatory response to infection, which is characterized by rapid increases in bacterial burden, a lack of inflammatory cytokine and chemokine expression, and scant evidence of inflammation in histopathological examination of lung tissues.
co92Δyoph infection presents a significantly different picture: 1) there is little bacterial replication in the lungs , 2) co92Δyoph is severely attenuated in both bubonic and pneumonic plague models , and 3) tnf-α and il-1β are readily detectable in the balf of mice infected with co92Δyoph at  <dig> hours post-infection  suggesting that co92Δyoph induces a detectable inflammatory response in the lung.
altogether, these data would suggest that the histopathology of co92Δyoph might be different than that observed during a co <dig> infection.
to evaluate any differences in histopathology, cd <dig> mice were infected in with  <dig> ×  <dig> cfu of co <dig> or co92Δyoph.
twenty-four and  <dig> h post-infection lungs were harvested, fixed, embedded in paraffin, and stained with hematoxylin and eosin.
tissues were examined for inflammatory changes as we have described previously  <cit> .
consistent with our previous findings, infection with co <dig> for  <dig> h leads to very subtle changes including congestion  and hyperplasia of the bronchial epithelium  with little apparent tissue damage.
however, infection with co92Δyoph for  <dig> h leads to wide spread vacuolization of the bronchial epithelium , and infiltration of the parenchyma with inflammatory cells.
all of the mice infected with co92Δyoph had extensive vacuolization of the bronchial epithelium as shown in figure 7c.
although there is extensive damage to the bronchial epithelium, the majority of conducting airways remain relatively clear of debris with the exception of the occasional macrophage in the lumen of the airways .
consistent with what is seen with the wild-type infection at  <dig> h, the lower airways and alveolar spaces of the co92Δyoph infected animals were characterized by congestion .
cd <dig> mice were infected in with  <dig> ×  <dig> cfu of co <dig> or co92Δyoph for  <dig> or  <dig> hours.
representative 20× images of lungs are presented.
a) mouse mock infected for  <dig> hours with saline.
b) mouse infected with co <dig> for  <dig> hours.
c) mouse infected with co92Δyoph for  <dig> hours with black arrow pointing out vacuolated bronchial epithelium.
d) mouse infected with co <dig> for  <dig> hours.
black arrows denote robust neutrophilic inflammation, bacteria, and dead cells.
e) 100× image of free bacteria in the airways of mice infected with co <dig> for  <dig> hours .
f-h) mouse infected with co92Δyoph for  <dig> hrs.
f) vacuolization in conjunction with exudates denoted by the double black arrows and an area of sub-mucosal necrosis denoted by the single black arrow.
g) large infiltration of pmns adjacent to bronchiole.
note that there are no bacteria evident at this time point.
h) 100× image of infiltration of the bronchial epithelium with pmns.
data is representative images of 8- <dig> mice per time point.
at  <dig> h post infection the histopathology of the co92Δyoph infection remains very different than that observed during a co <dig> infection.
the wild-type infection is characterized by large areas of pulmonary consolidation , a robust inflammatory response composed of mostly of pmns , fibrin, exudates in conducting airways , and abundant free bacteria .
the inflammatory response to the co <dig> infection at  <dig> h post-infection also includes multiple areas of overt extracellular bacterial growth interspersed with a large number of dead host-cells and cellular debris.
these findings were observed in 100% of the mice infected with co <dig> for  <dig> h. co92Δyoph exhibits several prominent findings at the  <dig> h time point .
large pronounced inflammatory lesions composed mostly of pmns were present in 50% of the mice investigated at this time point .
these lesions are different than those observed in the wild-type infection in that bacterial growth is not apparent , there is less cell death and cellular debris , and there is a pronounced infiltration of the bronchial mucosa with inflammatory cells .
the other type of lesion common in the co92Δyoph infected mice is exemplified in figure 7f and consists of severe vacuolization of the bronchial epithelium.
in contrast to what is observed at  <dig> h , necrosis of the bronchial sub-mucosa is apparent in areas adjacent to vacuolated epithelium in 50% of the animals examined .
unlike the clear airways observed  <dig> h after co92Δyoph infection, at  <dig> h post-infection, there are exudates composed of pmns, macrophages, and sloughed epithelial cells .
however, bacteria were not evident in the airways of any of the animals infected with co92Δyoph.
altogether these data suggest that co92Δyoph induces a robust inflammatory response prior to what is observed with co <dig> infection.
further, co92Δyoph causes significant vacuolization of the bronchial epithelium that is only occasionally observed during a co <dig> infection.
discussion
we investigated the effect of yoph deletion on the virulence of y. pestis using a murine model of primary pneumonic plague.
yoph, a type-three secretion system effector protein, is a protein tyrosine phosphatase that is involved in inhibiting phagocytosis  <cit> , as well as blocking the pi3-kinase pathway  <cit> .
investigators have studied the effect of yoph deletion using multiple species of yersinia, including y. pestis yoph pgm double mutants utilizing several routes of infection  <cit> .
to date most yoph mutants tested have exhibited decreased virulence and interestingly, lung infections of balb/c mice with a yoph mutant of the closely related yersinia pseudotuberculosis resulted in a strong attenuation phenotype  <cit> .
however, this has never been tested in a murine model of primary pneumonic plague using wt y. pestis strains.
the attenuation of co92Δyoph is due to the loss of the protein because virulence can be restored when the co92Δyoph mutant is complemented with a wild type copy of the yoph gene.
further, it is the actual phosphatase activity of yoph that is required for virulence since a co92Δyoph mutant complemented with a yoph gene containing an enzymatic inactivating mutation  is completely attenuated.
this is an intriguing finding suggesting that small molecule inhibitors of the yoph phosphatase activity might be useful therapeutics for the treatment of plague.
in addition to evaluating the virulence of co92Δyoph, we examined the host response to pulmonary infection by testing for differences in pro-inflammatory cytokines expression and lung histopathology.
we hypothesized that there could be a host component that contributed to the attenuation of the co92Δyophmutant.
it has been observed by us and others that infection with y. pestis co <dig> leads to a delayed inflammatory response, which results in very high bacterial loads in the lungs, liver and spleen, ultimately leading to the death of the host  <cit> .
we speculated that alleviation of the anti-inflammatory mechanisms of y. pestis would provide protection for the host.
in the absence of yoph, the host responds to the presence of y. pestis with an early pro-inflammatory response, namely, an increase in tnf-α and il-1β in the balf at  <dig> h post-infection, which diminishes in magnitude by  <dig> h post-infection.
this is different than what is observed with co <dig> infection, where an inflammatory response is not observed until  <dig> h post-infection  <cit> .
in addition, the kinetics of bacterial growth following an intranasal infection with co92Δyoph is different than the kinetics of co <dig> intranasal infection.
first, co92Δyoph is able to persist in the lungs of infected mice, although the bacterial burdens observed are significantly lower than those of co <dig> at both at  <dig> h and  <dig> h post-infection.
it appears that the co92Δyoph is unable to propagate in the lungs of the host, as demonstrated by the fact that the amount of bacteria detected in the lungs remains approximately the same at  <dig> h and  <dig> h post-infection.
these data are similar to what is observed with a pulmonary infection with the y. pseudotuberculosis yoph mutant  <cit> .
additionally, co92Δyoph is unable to disseminate from the site of infection to either the liver or spleen at  <dig> h,  <dig> h,  <dig> h, or  <dig> h post-infection.
in contrast, infection with wild-type co <dig> results in dissemination and high bacterial burdens in the liver and spleen at  <dig> h post-infection, and the majority of the animals are dead by  <dig> h post-infection  <cit> .
viable co92Δyoph is cleared from the host by  <dig> h post-infection being undetectable in the lung, liver, or spleen of mice  <dig> h and  <dig> h post-infection .
in light of these data, we hypothesize that the early pro-inflammatory response observed during infection with co92Δyoph is key in allowing the host to adequately control the bacteria and prevent a fulminate infection.
additional evidence for this conclusion lies in the differences observed in the histopathology of lungs from mice infected with either wild type co <dig> or co92Δyoph at both  <dig> h and  <dig> h post-infection.
the lung histopathology of these two infections is strikingly different and provides some insight into the dissimilar courses that these infections follow.
at  <dig> hours post-infection with wild-type y. pestis, there are only subtle changes in the lung tissue with very little tissue damage.
infection with co92Δyoph at this same time post-infection leads to widespread vacuolization of bronchial epithelium and infiltration of inflammatory cells.
it is unclear what leads to the vacuolization, but it is possible that the cyto-toxic effects of yope and yopo are amplified in the yoph mutant  <cit> .
after  <dig> hours of infection with co <dig>  evidence of severe lung pathology is evident including pulmonary consolidation and a heavy infiltration of inflammatory cells comprised predominantly of pmns.
in addition, free bacteria are readily observed throughout the lung tissue.
in co92Δyoph infection, at  <dig> h post-infection, about 50% of mice exhibit large lesions with pmns, but strikingly, no bacteria are observed.
this provides further evidence that an early inflammatory response to infection with co92Δyoph might be protective, and contribute to the clearance of y. pestis prior to outgrowth and dissemination of the bacteria.
other investigators recently observed that when innate immunity and specifically inflammatory immune responses are intact, infection with y. pestis does not cause severe disease.
for example, y. pestis modifies its los when growing at 37°c, using a tetra-acylated lipid a, which only weakly induces a pro-inflammatory response in the host.
at 21-27°c, y. pseudotuberculosis produces a hexa-acylated lipid a, the form of lipid a which is able to elicit potent inflammatory responses from the host  <cit> .
the tetra-acylated lipid a has poor toll-like receptor  <dig>  stimulating activity and therefore does not elicit the same robust pro-inflammatory response induced by hexa-acylated lipid a  <cit> .
when montminy et al genetically modified y. pestis to produce the more potent hexa-acylated lipid a at 37°c, they found that mice did not develop bubonic plague when infected subcutaneously even at doses approaching  <dig> mean lethal doses  <cit> .
these data indicate that the tlr- <dig> response is critical in overcoming infection with y. pestis and that by avoiding this response through lps modification, y. pestis is able to cause disease in the host  <cit> .
a second study demonstrating the importance of an active inflammatory response early in y. pestis infection showed that when mice were latently infected with gamma-herpes virus there is a significant increase in activated circulating macrophages and the severity of subsequent infection with fully virulent y. pestis was decreased compared to mice which were mock infected with virus  <cit> .
consistent with the data presented in this study, tlr- <dig> signaling and activated macrophages are capable of producing and responding to il-1β and tnf-α to fight against bacterial infection.
yoph is a complex virulence factor that impacts many aspects of the pathogenesis of yersinia sp.
given that yoph is a potent protein tyrosine phosphatase, it is well suited to disrupting host signal transduction pathways.
it is interesting to note that the majority of evidence obtained studying the enteropathogenic yersinia suggests that yoph acts to diminish integrin signaling following invasin binding  <cit> .
the result of this is that y. enterocolitica and y. pseudotuberculosis are predominantly extracellular pathogens.
in contrast, y. pestis co <dig> does not express invasin  <cit> , and is a facultative intracellular pathogen but yoph is a required virulence factor suggesting it has additional roles in y. pestis pathogenesis.
we hypothesize that the anti-host activities of yoph are beneficial to the survival of y. pestis within the mammalian host and that yoph contributes to the virulence of the organism by preventing an early pro-inflammatory response at the site of infection; thereby allowing the bacteria to rapidly replicate in the organs of infected mice, overwhelming and killing the host.
yoph directly impacts these key aspects of the pathogenesis of plague pneumonia.
however, given the number of potential host-pathways that could be targeted by yoph, it is likely that this virulence factor impacts multiple aspects of y. pestis pathogenesis.
CONCLUSIONS
pathogenic yersinia have evolved numerous mechanisms to survive in their hosts and many of the proteins that support survival in the host are virulence factors.
many of the most potent virulence factors are encoded on the  <dig> kb virulence plasmid pcd- <dig> and several of them have been shown to be critical for causing disease.
amongst the essential plasmid-encoded virulence factors is yoph a protein tyrosine phosphatase that has been shown to be critical for the inhibition of invasion by the enteropathogenic yersiniae.
interestingly, y. pestis is a facultative intracellular pathogen that does not express the inv protein suggesting that y. pestis yoph plays additional roles in the pathogenesis of this pathogen.
a mutation in yoph leads to severe attenuation of virulence in mouse models of primary plague pneumonia.
in this study, our data suggests that yoph inhibits the production of il-1β and tnf-α during the first  <dig> hours post-infection.
the inhibition of il-1β and tnf-α is a critical step in the pathogenesis of infection because mice deficient in these molecules are partially sensitive to infection with the yoph mutant.
further, the yoph phosphatase activity is essential for virulence because mice infected with the co92Δyoph strain complemented with a wild type copy of the yoph gene succumb to infection with kinetics similar to co <dig> infection but complementation with the yoph-c403a gene restores complete attenuation.
altogether our data suggests that yoph plays an important role in the subversion of innate immunity during plague pneumonia.

 yersinia pestis is a gram-negative bacterium with a zoonotic life cycle that occasionally results in human infections leading to plague. plague manifests in two major forms, the most common being bubonic plague. bubonic plague is transmitted to humans through the bite of an infected flea, resulting in intradermal inoculation of the bacterium, which progresses to form the characteristic lymphadenitis of plague. the less common but contagious form of the disease is pneumonic plague, which is a severe pneumonia resulting either from inhalation of infectious respiratory droplets, or secondary to bubonic or septicemic plague. pneumonic plague is highly aggressive and if untreated, is able to kill the host within 2- days post-exposure, with mortality rates that approach 100%. we and others have begun to analyze the immune response to primary pneumonic plague using wild-type strains of y. pestis in an effort to understand the interactions of y. pestis with its host early during the development of disease. the rapid progression of the disease and associated mortality suggests that subversion of innate immunity plays a key role in disease development. our previous studies indicate a delay in the host inflammatory response to infection, resulting in an opportunity for the bacterium to replicate to high numbers and potentially overwhelm the host immune system, rapidly resulting in death. modulation of host-immune responses is a common pathogenic mechanism among the virulent species of yersinia and much work has been done to understand the molecular mechanisms underlying the virulence factors involved in this process. the kb virulence plasmid, which is essential for virulence, contains all of the machinery and effector proteins for a type-three secretion system. the secreted effector proteins are also known as the yersinia outer proteins and are transported from the bacterial cytosol through the ttss into the cytoplasm of a host cell to facilitate infection. all six of the effector yops have been studied, and at least one function has been assigned to each based on in vitro studies. several are involved in manipulating the host cytoskeleton; these yops interfere with the rho family of gtpases and other host proteins involved in the regulation of the cytoskeleton, and are therefore important in the modulation of phagocytosis. others are involved in the tempering of the host immune response, in particular, yoph and yopj, and to a lesser extent, yope and yopm have been shown to impact inflammatory cytokine expression in vitro and in vivo respectively. yoph is a protein tyrosine phosphatase, which is known to interact with p130cas and fak to impair invasion of epithelial cells by yersinia pseudotuberculosis. yoph blocks cytoskeletal rearrangement upon injection into the host cell, contributing to the decreased ability of macrophages and other immune cells to phagocytose the bacteria. distinct from its ability to interact with the cytoskeleton, yoph is also known to impact host cell signaling by inactivation of the pi3k pathway through unknown mechanisms. the pi3k pathway plays an important role in the macrophage response to infection including generation of the oxidative burst and nitric oxide production, phagosome formation, and has a role in a negative regulation of il- production, to help keep the inflammatory response in check. numerous studies have determined that mutations in yoph severely impact the virulence of the yersiniae. recently we reported that co92Δyoph is severely attenuated in both an intranasal and subcutaneous models of y. pestis infection, which makes co92Δyoph a very good live-attenuated vaccine strain. the ability of co92Δyoph to provide significant protection against virulent challenge suggests that a robust protective immune response is generated during primary infection with this strain. however, the role of yoph in the pathogenesis of plague pneumonia or its impact on the immune response in the lung has not been examined. in this study we test the impact of the co92Δyoph mutant on both the virulence and inflammatory response using a mouse model of primary pneumonic plague.  y. pestis co92Δyoph is severely attenuated in an intranasal model of primary pneumonic plague the survival of cd mice after in infection with co or co92Δyoph was determined and the survival curves analyzed by log-rank analysis. mice infected in with ~ cfu co92Δyoph appeared normal at all time points post infection and showed no outward signs of disease whereas animals infected with ~ cfu co were severely debilitated two days post-infection and succumbed to disease by day post-infection. altogether, these data revealed that mice were able to survive both intranasal and intradermal infection with co92Δyoph at doses of ~ cfu. our results indicate a significant difference in survival when co is compared to co92Δyoph and that at the highest dose tested co92Δyoph is avirulent in cd mice. attenuation of co92Δyoph is due to the loss of yoph and yoph enzymatic activity we tested the ability of the co92Δyoph mutant complemented with a wild type copy of the yoph gene to secrete yoph in vitro. as shown in figure 2a when the ttss is induced in vitro, the co92Δyoph mutant complemented with yoph secretes similar levels of yoph into the culture supernatant as co due to the yoph cleavage products in secreted protein preparations, we also evaluated the levels of yoph in whole cell extracts after induction. similar levels of yoph were detected in the whole cell extracts and were consistent with the secreted protein profile. to test if the attenuation phenotype of co92Δyoph is linked to the yoph mutation, mice were infected with the co92Δyoph mutant carrying the pamc- complementing plasmid. complementation of yoph expressed from the pamc- plasmid restores virulence to the levels observed with co carrying the empty pcr plasmid. however, virulence is lost when the co92Δyoph mutant is complemented with the yoph-c403a gene expressed from the pamc- plasmid. the c403a mutation abolishes the yoph tyrosine phophatase activity, strongly suggesting that the attenuation of the co92Δyoph mutant is due to loss of the yoph phosphatase activity and these data are consistent with previous studies of other yersinia species and attenuated y. pestis strains which determined that yoph mutants are highly attenuated. y. pestis co92Δyoph is able to persist in the lungs but does not spread systemically the severe attenuation of y. pestis co92Δyoph could be due to an inability to colonize the lung, disseminate, or replicate in the mouse. to further investigate the pathogenesis of co92Δyoph, we determined the changes in bacterial burdens over time. mice were infected in with 2× or 1× cfu y. pestis co or co92Δyoph respectively and sacrificed at the indicated times. lungs, livers, and spleens were processed as described in the methods and dilutions plated to determine the cfu-bacteria/g tissue at each time point post-infection. all of the mice infected with wild-type co were dead by hours post-infection making collection of data at h and beyond impossible for this group. our results suggest that co92Δyoph is able to persist in the lungs through h post-infection. bacterial burdens in the lungs of mice infected with co were significantly higher when compared to co92Δyoph at both h and h post-infection. these data indicate that the co92Δyoph mutant was unable to replicate at the site of infection. in addition, we were unable to detect dissemination of the co92Δyoph bacterium to the spleen or liver at any time post-infection, while wild-type co was present in the livers and spleens in large numbers at h post-infection consistent with what we have reported previously. intranasal infection with co92Δyoph induces tnf-α and il-1β early during infection in mouse models of primary pneumonic plague, mice infected with co do not mount an inflammatory cytokine response until h to h post-infection. we hypothesized that the attenuation of co92Δyoph could be partially due to a strong inflammatory response to the presence of the bacterium. further, due to the delayed arrival of neutrophils during plague pneumonia, we suspected that tnf-α and il-1β might be involved. to test this hypothesis, we measured the concentrations of tnf-α and il-1β in the bronchiolar alveolar lavage fluid after in infection with co or co92Δyoph. mice were infected with 2× cfu of either y. pestis co or co92Δyoph and sacrificed or hours post-infection. bal was performed on these mice, and then elisa analyzed the balf for the concentration of tnf-α and il-1β. mice infected with co92Δyoph elicited a robust pro-inflammatory cytokine response at h post-infection, characterized by ± pg/ml of tnf-α and ± pg/ml of il-1β in the balf of these mice h post-infection. by h post-infection the levels of both cytokines had decreased but remained significant with concentrations of tnf-α = ± pg/ml, and il-1β = ± pg/ml. bal from mice infected for hours with wild-type co had tnf-α and il-1β levels approximately equal to the levels in the balf from uninfected mice. however, at h post-infection, both cytokines were detected in the balf of these mice with concentrations of tnf-α = ± pg/ml, and il-1β = ± pg/ml. the timing of cytokine expression and the levels of cytokine induced during a co are consistent with what we have observed previously. altogether, these data suggest that yoph is involved in delaying or inhibiting the expression of tnf-α and il-1β during plague pneumonia and may be involved in the overall delayed inflammatory response to this infection. mice lacking proinflammatory cytokines are more sensitive to co92Δyoph infection if tnf-α and il-1β were critical host-components leading to the attenuation observed in mice infected with co92Δyoph, then it would not be unreasonable to predict that mice deficient in these molecules would be more sensitive to infection with co92Δyoph. to test this hypothesis, we used antibody mediated cytokine depletion followed by in challenge with co92Δyoph or co briefly, mice were treated with monoclonal antibodies to tnf-α, il-1β, tnf-α+il-1β, or the appropriate control igg one day prior to infection and then every third day for the course of the infection as we have described previously. mice treated with il-1β depleting antibodies were not significantly more sensitive to co92Δyoph infection than the control mice and only 10% of the mice treated with the tnf-α depleting antibodies succumbed to infection. however, 30% of the mice treated with both the il-1β and the tnf-α depleting antibodies succumbed to co92Δyoph infection. these data did not reach significance but suggest that proinflammatory cytokines expressed during co92Δyoph infection help control this infection. because the site of the infection was the mucosal surface of the nasal cavity and lung, it was possible that systemically administered igg poorly depletes cytokines at this site. to test our hypothesis in another way, we infected tnf-α deficient mice and control mice with co92Δyoph or co as predicted, the control mice and the tnf-α-/- mice rapidly succumbed to infection with co consistent with what was observed with out-bred cd mice, the b6129sf2/j control mice were completely resistant to infection with cfu of co92Δyoph. however, when the isogenic tnf-α-/- mice were infected with × cfu co92Δyoph, significantly, 40% of the mice succumbed to infection within days. altogether, these data suggest that the co92Δyoph mutant of yersinia pestis is attenuated in multiple strains of wild type mice including cd- b6129sf2/j, and c57bl6/j but is partially virulent in mice lacking tnf-α. these data further suggest that yoph mediated suppression of pulmonary tnf-α is an important virulence promoting mechanism. histopathology of co92Δyophinfection recently, we and others described the development of primary pneumonic plague in the mouse following infection with co. the most striking feature of primary plague pneumonia is a 36-hour delay in the inflammatory response to infection, which is characterized by rapid increases in bacterial burden, a lack of inflammatory cytokine and chemokine expression, and scant evidence of inflammation in histopathological examination of lung tissues. co92Δyoph infection presents a significantly different picture: 1) there is little bacterial replication in the lungs, 2) co92Δyoph is severely attenuated in both bubonic and pneumonic plague models, and 3) tnf-α and il-1β are readily detectable in the balf of mice infected with co92Δyoph at hours post-infection suggesting that co92Δyoph induces a detectable inflammatory response in the lung. altogether, these data would suggest that the histopathology of co92Δyoph might be different than that observed during a co infection. to evaluate any differences in histopathology, cd mice were infected in with × cfu of co or co92Δyoph. twenty-four and h post-infection lungs were harvested, fixed, embedded in paraffin, and stained with hematoxylin and eosin. tissues were examined for inflammatory changes as we have described previously. consistent with our previous findings, infection with co for h leads to very subtle changes including congestion and hyperplasia of the bronchial epithelium with little apparent tissue damage. however, infection with co92Δyoph for h leads to wide spread vacuolization of the bronchial epithelium, and infiltration of the parenchyma with inflammatory cells. all of the mice infected with co92Δyoph had extensive vacuolization of the bronchial epithelium as shown in figure 7c. although there is extensive damage to the bronchial epithelium, the majority of conducting airways remain relatively clear of debris with the exception of the occasional macrophage in the lumen of the airways. consistent with what is seen with the wild-type infection at h, the lower airways and alveolar spaces of the co92Δyoph infected animals were characterized by congestion. cd mice were infected in with × cfu of co or co92Δyoph for or hours. representative 20× images of lungs are presented. a) mouse mock infected for hours with saline. b) mouse infected with co for hours. c) mouse infected with co92Δyoph for hours with black arrow pointing out vacuolated bronchial epithelium. d) mouse infected with co for hours. black arrows denote robust neutrophilic inflammation, bacteria, and dead cells. e) 100× image of free bacteria in the airways of mice infected with co for hours. f-h) mouse infected with co92Δyoph for hrs. f) vacuolization in conjunction with exudates denoted by the double black arrows and an area of sub-mucosal necrosis denoted by the single black arrow. g) large infiltration of pmns adjacent to bronchiole. note that there are no bacteria evident at this time point. h) 100× image of infiltration of the bronchial epithelium with pmns. data is representative images of 8- mice per time point. at h post infection the histopathology of the co92Δyoph infection remains very different than that observed during a co infection. the wild-type infection is characterized by large areas of pulmonary consolidation, a robust inflammatory response composed of mostly of pmns, fibrin, exudates in conducting airways, and abundant free bacteria. the inflammatory response to the co infection at h post-infection also includes multiple areas of overt extracellular bacterial growth interspersed with a large number of dead host-cells and cellular debris. these findings were observed in 100% of the mice infected with co for h. co92Δyoph exhibits several prominent findings at the h time point. large pronounced inflammatory lesions composed mostly of pmns were present in 50% of the mice investigated at this time point. these lesions are different than those observed in the wild-type infection in that bacterial growth is not apparent, there is less cell death and cellular debris, and there is a pronounced infiltration of the bronchial mucosa with inflammatory cells. the other type of lesion common in the co92Δyoph infected mice is exemplified in figure 7f and consists of severe vacuolization of the bronchial epithelium. in contrast to what is observed at h, necrosis of the bronchial sub-mucosa is apparent in areas adjacent to vacuolated epithelium in 50% of the animals examined. unlike the clear airways observed h after co92Δyoph infection, at h post-infection, there are exudates composed of pmns, macrophages, and sloughed epithelial cells. however, bacteria were not evident in the airways of any of the animals infected with co92Δyoph. altogether these data suggest that co92Δyoph induces a robust inflammatory response prior to what is observed with co infection. further, co92Δyoph causes significant vacuolization of the bronchial epithelium that is only occasionally observed during a co infection. discussion we investigated the effect of yoph deletion on the virulence of y. pestis using a murine model of primary pneumonic plague. yoph, a type-three secretion system effector protein, is a protein tyrosine phosphatase that is involved in inhibiting phagocytosis, as well as blocking the pi3-kinase pathway. investigators have studied the effect of yoph deletion using multiple species of yersinia, including y. pestis yoph pgm double mutants utilizing several routes of infection. to date most yoph mutants tested have exhibited decreased virulence and interestingly, lung infections of balb/c mice with a yoph mutant of the closely related yersinia pseudotuberculosis resulted in a strong attenuation phenotype. however, this has never been tested in a murine model of primary pneumonic plague using wt y. pestis strains. the attenuation of co92Δyoph is due to the loss of the protein because virulence can be restored when the co92Δyoph mutant is complemented with a wild type copy of the yoph gene. further, it is the actual phosphatase activity of yoph that is required for virulence since a co92Δyoph mutant complemented with a yoph gene containing an enzymatic inactivating mutation is completely attenuated. this is an intriguing finding suggesting that small molecule inhibitors of the yoph phosphatase activity might be useful therapeutics for the treatment of plague. in addition to evaluating the virulence of co92Δyoph, we examined the host response to pulmonary infection by testing for differences in pro-inflammatory cytokines expression and lung histopathology. we hypothesized that there could be a host component that contributed to the attenuation of the co92Δyophmutant. it has been observed by us and others that infection with y. pestis co leads to a delayed inflammatory response, which results in very high bacterial loads in the lungs, liver and spleen, ultimately leading to the death of the host. we speculated that alleviation of the anti-inflammatory mechanisms of y. pestis would provide protection for the host. in the absence of yoph, the host responds to the presence of y. pestis with an early pro-inflammatory response, namely, an increase in tnf-α and il-1β in the balf at h post-infection, which diminishes in magnitude by h post-infection. this is different than what is observed with co infection, where an inflammatory response is not observed until h post-infection. in addition, the kinetics of bacterial growth following an intranasal infection with co92Δyoph is different than the kinetics of co intranasal infection. first, co92Δyoph is able to persist in the lungs of infected mice, although the bacterial burdens observed are significantly lower than those of co at both at h and h post-infection. it appears that the co92Δyoph is unable to propagate in the lungs of the host, as demonstrated by the fact that the amount of bacteria detected in the lungs remains approximately the same at h and h post-infection. these data are similar to what is observed with a pulmonary infection with the y. pseudotuberculosis yoph mutant. additionally, co92Δyoph is unable to disseminate from the site of infection to either the liver or spleen at h, h, h, or h post-infection. in contrast, infection with wild-type co results in dissemination and high bacterial burdens in the liver and spleen at h post-infection, and the majority of the animals are dead by h post-infection. viable co92Δyoph is cleared from the host by h post-infection being undetectable in the lung, liver, or spleen of mice h and h post-infection. in light of these data, we hypothesize that the early pro-inflammatory response observed during infection with co92Δyoph is key in allowing the host to adequately control the bacteria and prevent a fulminate infection. additional evidence for this conclusion lies in the differences observed in the histopathology of lungs from mice infected with either wild type co or co92Δyoph at both h and h post-infection. the lung histopathology of these two infections is strikingly different and provides some insight into the dissimilar courses that these infections follow. at hours post-infection with wild-type y. pestis, there are only subtle changes in the lung tissue with very little tissue damage. infection with co92Δyoph at this same time post-infection leads to widespread vacuolization of bronchial epithelium and infiltration of inflammatory cells. it is unclear what leads to the vacuolization, but it is possible that the cyto-toxic effects of yope and yopo are amplified in the yoph mutant. after hours of infection with co evidence of severe lung pathology is evident including pulmonary consolidation and a heavy infiltration of inflammatory cells comprised predominantly of pmns. in addition, free bacteria are readily observed throughout the lung tissue. in co92Δyoph infection, at h post-infection, about 50% of mice exhibit large lesions with pmns, but strikingly, no bacteria are observed. this provides further evidence that an early inflammatory response to infection with co92Δyoph might be protective, and contribute to the clearance of y. pestis prior to outgrowth and dissemination of the bacteria. other investigators recently observed that when innate immunity and specifically inflammatory immune responses are intact, infection with y. pestis does not cause severe disease. for example, y. pestis modifies its los when growing at 37°c, using a tetra-acylated lipid a, which only weakly induces a pro-inflammatory response in the host. at 21-27°c, y. pseudotuberculosis produces a hexa-acylated lipid a, the form of lipid a which is able to elicit potent inflammatory responses from the host. the tetra-acylated lipid a has poor toll-like receptor stimulating activity and therefore does not elicit the same robust pro-inflammatory response induced by hexa-acylated lipid a. when montminy et al genetically modified y. pestis to produce the more potent hexa-acylated lipid a at 37°c, they found that mice did not develop bubonic plague when infected subcutaneously even at doses approaching mean lethal doses. these data indicate that the tlr- response is critical in overcoming infection with y. pestis and that by avoiding this response through lps modification, y. pestis is able to cause disease in the host. a second study demonstrating the importance of an active inflammatory response early in y. pestis infection showed that when mice were latently infected with gamma-herpes virus there is a significant increase in activated circulating macrophages and the severity of subsequent infection with fully virulent y. pestis was decreased compared to mice which were mock infected with virus. consistent with the data presented in this study, tlr- signaling and activated macrophages are capable of producing and responding to il-1β and tnf-α to fight against bacterial infection. yoph is a complex virulence factor that impacts many aspects of the pathogenesis of yersinia sp. given that yoph is a potent protein tyrosine phosphatase, it is well suited to disrupting host signal transduction pathways. it is interesting to note that the majority of evidence obtained studying the enteropathogenic yersinia suggests that yoph acts to diminish integrin signaling following invasin binding. the result of this is that y. enterocolitica and y. pseudotuberculosis are predominantly extracellular pathogens. in contrast, y. pestis co does not express invasin, and is a facultative intracellular pathogen but yoph is a required virulence factor suggesting it has additional roles in y. pestis pathogenesis. we hypothesize that the anti-host activities of yoph are beneficial to the survival of y. pestis within the mammalian host and that yoph contributes to the virulence of the organism by preventing an early pro-inflammatory response at the site of infection; thereby allowing the bacteria to rapidly replicate in the organs of infected mice, overwhelming and killing the host. yoph directly impacts these key aspects of the pathogenesis of plague pneumonia. however, given the number of potential host-pathways that could be targeted by yoph, it is likely that this virulence factor impacts multiple aspects of y. pestis pathogenesis.  pathogenic yersinia have evolved numerous mechanisms to survive in their hosts and many of the proteins that support survival in the host are virulence factors. many of the most potent virulence factors are encoded on the kb virulence plasmid pcd- and several of them have been shown to be critical for causing disease. amongst the essential plasmid-encoded virulence factors is yoph a protein tyrosine phosphatase that has been shown to be critical for the inhibition of invasion by the enteropathogenic yersiniae. interestingly, y. pestis is a facultative intracellular pathogen that does not express the inv protein suggesting that y. pestis yoph plays additional roles in the pathogenesis of this pathogen. a mutation in yoph leads to severe attenuation of virulence in mouse models of primary plague pneumonia. in this study, our data suggests that yoph inhibits the production of il-1β and tnf-α during the first hours post-infection. the inhibition of il-1β and tnf-α is a critical step in the pathogenesis of infection because mice deficient in these molecules are partially sensitive to infection with the yoph mutant. further, the yoph phosphatase activity is essential for virulence because mice infected with the co92Δyoph strain complemented with a wild type copy of the yoph gene succumb to infection with kinetics similar to co infection but complementation with the yoph-c403a gene restores complete attenuation. altogether our data suggests that yoph plays an important role in the subversion of innate immunity during plague pneumonia. 
here, we report that, intranasal infection of mice with co delta yoph mutant results in an early pro-inflammatory response in the lungs characterized by an increase in the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin one-beta hours post-infection. altogether, our results suggest that yoph can inhibit an early pro-inflammatory response in the lungs of mice and that this is an important step in the pathogenesis of infection. yersinia pestis is the causative agent of pneumonic plague; recently, we and others reported that during the first 24- hours after pulmonary infection with y. pestis pro-inflammatory cytokine expression is undetectable in lung tissues. mice deficient in tnf-alpha are more susceptible to co delta yoph infection with 40% of the mice succumbing to infection. co rapidly disseminates to the liver and spleen resulting in high bacterial burdens in these tissues ultimately cumulating in death 72- h post-infection.
 yersinia pestis is the causative agent of pneumonic plague; recently, we and others reported that during the first 24- hours after pulmonary infection with y. pestis pro-inflammatory cytokine expression is undetectable in lung tissues.  here, we report that, intranasal infection of mice with co delta yoph mutant results in an early pro-inflammatory response in the lungs characterized by an increase in the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin one-beta hours post-infection. co delta yoph colonizes the lung but does not disseminate to the liver or spleen and is cleared from the host within hours post-infection. this is different from what is observed in a wild-type co infection, where pro-inflammatory cytokine expression and immune cell infiltration into the lungs is not detectable until 36- h post-infection. co rapidly disseminates to the liver and spleen resulting in high bacterial burdens in these tissues ultimately cumulating in death 72- h post-infection. mice deficient in tnf-alpha are more susceptible to co delta yoph infection with 40% of the mice succumbing to infection.  altogether, our results suggest that yoph can inhibit an early pro-inflammatory response in the lungs of mice and that this is an important step in the pathogenesis of infection. 