Successful colonization and replication of pathogens in the host requires evasion and/or manipulation of the host immune response. The most infectious pathogens have developed a number of mechanisms to avoid detection or destruction by both innate and adaptive host effector mechanisms. One such effector mechanism is antibody mediated clearance of pathogens. Evasion of antibody mediated immunity is an important hurdle for both extra- and intracellular pathogens to overcome. For example, opsonization of Mycobacteria
by antibody enhances uptake of the bacterium by phagocytic cells, activates the cells, and ultimately results in destruction of the microbe (22
Antibody has also recently been shown to be an important component for optimal protective responses against F. tularensis
. Transfer of immune serum or monoclonal antibodies uniformly protects animals against challenge with the attenuated strains of F. tularensis
such as LVS (1
). In contrast, although transfer of immune serum or purified antibodies extends the mean time to death, these treatments fail to increase overall survival following challenge with virulent F. tularensis
). There are a number of explanations for the failure of antibody to protect against infections with virulent F. tularensis
. These include poor affinity or avidity of antibody directed against the bacterium, low abundance of the antigen targeted by the antibody associated with virulent organisms, direct suppression of pro-inflammatory responses typically elicited following antibody mediated uptake of bacteria by phagocytes, or direct interference with antibody opsonization.
Data presented herein suggests the inability of antibody to efficiently protect against virulent F. tularensis infection is not due to the failure of antibody to bind bacteria under normal tissue culture conditions (). Nor is the ineffectiveness of antibody mediated protection due to the lack of production of pro-inflammatory cytokines following uptake of opsonized F. tularensis (). Rather, data presented herein suggest that the inability of antibody to adequately protect against virulent F. tularensis infections is directly related to the ability of virulent, but not attenuated, F. tularensis to more efficiently harness the proteolytic capability of host plasmin for degradation of Francisella specific antibody (–).
The interaction of SchuS4 and LVS with plasmin and antibody revealed a previously unappreciated difference in the ability of these bacteria to modulate the host immune response. For example, antibody opsonization of SchuS4 resulted in both greater numbers of infected macrophages and greater numbers of cells infected with 3–10 and >10 bacteria per cell compared to LVS (). This increase in phagocytosis may be a result of SchuS4 binding the anti-FT antibody with greater affinity than LVS. Alternatively, SchuS4 may have slightly greater amounts of the antigen recognized by the antibody used in these studies and, thus, bound more antibody on their surfaces. Experiments depicted in suggest that SchuS4 was capable of binding modestly larger amounts of anti-FT than LVS. Regardless of the reason for increased phagocytosis of antibody opsonized SchuS4 versus LVS, this data inferred that antibody mediated clearance would be more efficient for SchuS4 infections than LVS. However, previous reports demonstrate that passive transfer of immune serum or monoclonal antibodies fails to protect infection with SchuS4 (1
). Thus, it is likely that functional differences in antibody mediated uptake (outside of phagocytosis) may contribute to control of LVS, but not SchuS4, infections.
In this report we demonstrate that one such functional difference is the induction of pro-inflammatory cytokines following uptake of antibody opsonized Francisella. Although more macrophages were infected with antibody opsonized SchuS4 than opsonized LVS, production of TNF-α and IL-6 from cells infected with opsonized LVS was significantly greater than that observed in cultures infected with opsonized SchuS4 (). This disparity in cytokine production between infections with either opsonized SchuS4 and LVS was even more dramatic in cultures infected with plasmin coated, antibody opsonized, bacteria. uPA+Plg treated, antibody opsonized LVS induced less TNF-α and IL-6 than PBS treated, opsonized LVS. However, the concentration of cytokines detected in plasmin treated, opsonized LVS cultures was significantly higher than that detected in any SchuS4 culture. In contrast, the presence of plasmin on the surface of SchuS4 resulted in nearly complete reversal of the antibody mediated cytokine production observed in PBS treated, opsonized SchuS4 infections. Thus, although the number of opsonized SchuS4 engulfed by macrophages was either greater or equivalent to that observed with opsonized LVS, virulent SchuS4 was less efficient at inducing cytokine responses. Furthermore, the presence of plasmin had a more profound effect on the induction of cytokines by antibody opsonized SchuS4 compared to opsonized LVS.
We have previously demonstrated that SchuS4, in the absence of antibody, is capable of actively suppressing cytokine responses in various host target cells and tissues (6
). It has also been reported that LVS interferes with cytokine production in host cells (24
). This suggested that SchuS4 and LVS are similar in their disruption of innate immune responses However, data presented herein clearly demonstrates that (unlike LVS) in addition to disrupting innate immunity, SchuS4 is capable of profoundly modulating cytokine responses that are associated with adaptive immune responses as well, i.e. production of cytokines following uptake of antibody opsonized SchuS4. These data reveal an important advance in our understanding of the interplay between host immune responses and virulent Francisella
. Furthermore, they describe an important, fundamental difference in LVS and SchuS4 mediated infections of host cells. Together this data confirms that antibody mediated uptake is likely an important component of protective immunity against virulent Francisella
infections, and that the bacterium has specific mechanisms in place to resist this host response. Moreover, our data also suggests that antibody mediated protection against virulent F. tularensis
may be improved following inhibition of specific components of the host plasminogen activating system (PAS).
Typically, bacteria interacting with the PAS via binding of Plg also efficiently bind plasmin (as reviewed, (5
)). Thus, our observation that both LVS and SchuS4 bound Plg, but only SchuS4 bound active plasmin was unexpected. To our knowledge, this is the first demonstration of a direct comparison of an attenuated and virulent strain of bacteria, with nearly equivalent ability to interact with Plg, to interact with plasmin in such a disparate way. The most likely explanation of the dissimilar nature in which LVS and SchuS4 interact with plasmin is that the molecule responsible for binding plasmin to the surface of the bacterium is either absent or present in low abundance on LVS compared to SchuS4. In a recent study, the only protein present in outer membrane fractions of SchuS4 that was absent in LVS was a homologue to the yersinia autotransporter protein (Yap) designated as YapH-N and YapH-C (26
). In Yersinia
, these proteins function as adhesins similarly to the plasminogen activator protein (Pla) that is also found on the surface of Yersinia
). Pla is well known for its ability to interact with Plg and plasmin (31
). However, the potential interaction of Yap with Plg and plasmin has not been examined. Thus, it is tempting to speculate that YapH-N and YapH-C proteins present on the surface of SchuS4 may play an important role in securing host plasmin to the bacterial surface.
In addition to differences in the species of outer membrane proteins present on LVS and SchuS4, it is also possible that small changes in the overall structure of LPS on these two related bacteria contributes to conversion of bound Plg to plasmin on SchuS4, but not LVS. Previous studies conducted with Yersinia
have shown that the structure of LPS associated with these organisms, specifically the ability of LPS to interact with arginine residues located in Yersinia
proteins interacting with Plg (Pla and PgtE, respectively), was critical for the optimal conversion of Plg to plasmin on the surface of these bacteria (32
). Although, LPS associated with LVS and SchuS4 are both poorly inflammatory, a direct comparison of their molecular structure and their ability to bind arginine residues located in outer membrane proteins has not been performed (6
). Therefore, in addition to specific differences in plasmin binding proteins, small variations in the structure of SchuS4 LPS may contribute to the ability of outer membrane proteins present in this virulent bacterium to bind plasmin.
In conclusion, our data demonstrate a novel role for plasmin associated with virulent, but not attenuated, bacterial pathogens. With regard to Francisella
specific immunity, we demonstrate that antibody mediated opsonization and phagocytosis of virulent F. tularensis
results in the elicitation of pro-inflammatory cytokines by infected macrophages. This supports the hypothesis that Francisella
specific antibody plays an important role in protective immunity against this bacterium. Further, our data provide a potential mechanism by which antibody may aid in the control of Francisella
infections and how virulent strains utilize host systems to modulate this response. Our data also have important implications for many other bacterial diseases. Specifically, the majority of studies examining the role of Plg and plasmin in bacterial infections have focused on the contribution these compounds make toward dissemination of the pathogens via degradation of extracellular matrix proteins. A direct demonstration of plasmin mediated degradation of pathogen specific antibody and the consequence of this activity for control of infection has not been addressed. Furthermore, a direct comparison of the ability of attenuated and virulent strains of a bacterial pathogen to harness the proteolytic potential of plasmin has not been reported. Considering the number of pathogens known to interact with plasminogen and plasmin, e.g. Neisseria, Haemophilus
, it is tempting to speculate that this strategy of plasmin mediated degradation of immunoglobulin for interference of antibody mediated opsonization and uptake by macrophages may occur for a number of pathogenic microbes (9
). Recognition and identification of the specific function of plasmin during bacterial infections will undoubtedly be a critical step in the development of effective therapeutics and vaccines directed against many infectious diseases, including those mediated by F. tularensis
, where antibody is important.