In the present study, we have established the ability of the IFN-inducible ELR− CXC chemokines CXCL9, CXCL10, and CXCL11 to exert direct antimicrobial effects against B. anthracis spores and bacilli in a concentration-dependent manner. The abilities of these host chemokines to inhibit spore germination and reduce spore viability are, to our knowledge, the first description of direct chemokine-mediated antimicrobial effects on bacterial spores and may represent an important mechanism for promoting host defense during the initial stages of inhalational anthrax. In support of this conclusion, we have demonstrated that the induction of CXCL9, CXCL10, and CXCL11 within the lungs of spore-challenged mice was associated with a substantial reduction of spore germination and subsequent disease progression. Taken together, these results open a new avenue of research for examining host chemokines as potential sporicidal/bactericidal agents.
The ability of the IFN-inducible ELR−
CXC chemokines to prevent spore germination and directly mediate sporicidal effects represents novel antimicrobial roles for host chemokines. Defining spore-specific targets and the molecular mechanism(s) of chemokine-mediated antimicrobial activity, however, is complicated by a lack of understanding concerning the mechanistic details of the signaling pathways and molecular interactions involved in spore germination (48
). Whereas little data on the inhibition of B. anthracis
spore germination exist, numerous compounds capable of inhibiting spore germination in Bacillus
species have been described and include germinant molecule analogs (1
), alkyl alcohols (62
), ion channel blockers (45
), protease inhibitors (8
), sulfydryl reagents (22
), and a variety of other compounds (15
). While incompletely defined, the mechanisms by which these compounds inhibit spore germination appear to rely on their ability to disrupt germinant receptor engagement and/or the subsequent signaling activities of one or more nutrient receptors (15
). Consequently, DPA release (which is essential in triggering cortex hydrolysis during nutrient-mediated germination) is prevented, and spore germination is blocked prior to cortex degradation (15
). Notably, the abilities of the above-described compounds to inhibit germination are largely reversible and do not affect spore viability (colony-forming ability), suggesting that subsequent mediators of germination remain functional (15
Although the effects of CXCL9, CXCL10, and CXCL11 on initial germination events and the possible role of these events in spore susceptibility remain to be determined, TEM visualization of CXCL10-treated B. anthracis spores established that the inhibition of spore germination and reduction in spore viability occur prior to spore coat and cortex degradation, demonstrating that CXCL10 directly targets spores and not newly emerging vegetative bacilli. Since ungerminated spores are metabolically inactive, a chemokine-mediated inhibition of macromolecular synthesis as a mechanism for antimicrobial activity is unlikely. These data suggest that CXCL10 exerts its antimicrobial effects by targeting a process inherent, and likely essential, to spore germination and the maintenance of spore viability.
Immunoelectron microscopy demonstrated that CXCL10 associates with the outermost spore layers including the exosporium, spore coat, and spore coat-cortex interface by 1 h, and this localization does not rely on the presence of germinants in the treatment medium. The nonessential nature of the exosporium in spore germination and viability makes it an unlikely target of chemokine-mediated antimicrobial activity. We cannot exclude, however, a possible role for the exosporium, especially since it is the outermost spore layer and is rich in negatively charged carboxylate and phosphate groups (30
) that may facilitate initial interactions with the positively charged C-terminal regions of the ELR−
CXC chemokines. Although the roles of the spore coat and outer membrane (located at the spore coat-cortex interface) in the process of germination are incompletely defined, studies investigating spore germination in Bacillus subtilis
and Bacillus cereus
have identified several components associated with spore germination that localize to these structures and have orthologs in B. anthracis
. For example, cortex-lytic enzymes such as CwlJ, located in the spore coat (3
), and SleB, located at the spore coat-cortex interface (49
) and inner membrane (10
), are responsible for mediating cortex degradation during spore germination. Although the inhibition of these enzymes is consistent with the mechanism of action for several inhibitors of spore germination (2
), a recent study suggested that the inhibition of cortex-lytic enzymes is likely an indirect effect and not the primary site of action for these inhibitors (15
). Studies of B. subtilis
and B. cereus
have also identified a gerP
operon that encodes proteins thought to be structural components of the spore coat that are important in influencing spore coat permeability and thereby facilitating germinant access to the inner membrane (6
). Interestingly, mutational inactivation of the gerP
locus results in germination defects similar to those seen in CXCL10-treated B. anthracis
spores, including a block in spore germination prior to cortex hydrolysis, a lower rate of germination, and a reduction in colony-forming ability (6
It is not currently known whether CXCL10 is able to interact with spore structures in addition to the exosporium, spore coat, and spore coat-cortex interface. Potential CXCL10 localization at the germ cell wall and/or inner membrane may not be identified using immunoelectron microscopy since these sites are likely inaccessible to the relatively large labeling reagents required for visualization. Given the proposed ability of antimicrobial chemokines to interact with bacterial membranes and cell wall components (described below), it is possible that CXCL9, CXCL10, and CXCL11 exert their antimicrobial effects against B. anthracis
spores by altering the structure of the germ cell wall and/or spore membranes. As a result, the proper functioning of membrane-associated spore components may be prevented, leading to the irreversible inhibition of spore germination. The possibility that changes in membrane structure and/or membrane protein function can have deleterious effects on spore germination is suggested by tetracaine- and procaine-mediated inhibition of spore germination (45
). The mechanisms by which these germination inhibitors act are thought to reflect a disruption in ion efflux and/or DPA release due to increased disordering of the lipid bilayer hydrocarbon interior (15
). This type of mechanism would help to explain how chemokine treatment results in the reduction of spore viability; however, it remains to be determined whether the decrease in spore viability upon chemokine treatment occurs via the same mechanism as germination inhibition or whether these effects act through separate, independent mechanisms.
Similar to the abilities of the IFN-inducible ELR−
CXC chemokines to mediate direct antimicrobial effects against B. anthracis
spores, all three CXC chemokines were found to exhibit direct antimicrobial activity against B. anthracis
bacilli. These data support a growing body of literature demonstrating the ability of host chemokines to target vegetative bacteria through a similar, as-yet-undefined, mechanism. In this regard, chemokines share several biophysical properties, including cationicity and amphipathicity, with antimicrobial peptides that function in innate host defense against infection (72
). Furthermore, the C-terminal helical region of antimicrobial chemokines, which is thought to mediate the direct antimicrobial activity of host chemokines, has a structure and amino acid composition similar to those of classical α-helical antimicrobial peptides (72
). Due to such similarities, several groups have proposed that chemokines may mediate antimicrobial activity through a similar mechanism in which electrostatic interactions between the cationic host molecule and the anionic microbial surface facilitate interactions resulting in membrane permeabilization and cell lysis (12
In support of this proposed mechanism of action, CXCL9- and CXCL10-treated vegetative bacilli were found to undergo bacterial chain segmentation with subsequent disruption, consistent with the generation of defects in cell wall and/or cell membrane integrity. These effects on the structural integrity of bacillus chains mimic those seen when vegetative bacilli were treated with penicillin G, which disrupts bacterial cell wall synthesis and results in the loss of bacterial cell integrity (data not shown). In addition, CXCL10-treated bacilli demonstrated a complete loss of cellular integrity as determined through TEM visualization. Although we cannot exclude the possibility that the loss of cellular integrity is a consequence of bactericidal activity rather than the cause, our data do not contradict the current hypothesis that chemokine-mediated antimicrobial activity against vegetative bacteria results from a direct disruption of bacterial membranes and/or cell wall integrity.
The studies described here were performed using the toxigenic, unencapsulated Sterne strain of B. anthracis (pXO1+ pXO2−). Although B. anthracis strain differences are thought to be minimal or absent with regard to spores, the presence of the poly-d-glutamic acid capsule may interfere with chemokine-mediated antimicrobial activity against vegetative bacilli. In order to confirm the susceptibility of encapsulated bacilli to CXCL9, CXCL10, and CXCL11, the findings described here will need to be further examined using an encapsulated strain such as the nontoxigenic, encapsulated Pasteur strain (pXO1− pXO2+) or the toxigenic, encapsulated Ames strain (pXO1+ pXO2+).
In order to begin investigating possible protective roles of chemokine-mediated antimicrobial activity during B. anthracis infection in vivo, we used a murine model of inhalational anthrax. The lungs of spore-challenged A/J mice, which are susceptible to infection by B. anthracis Sterne strain and succumb to inhalational anthrax, did not demonstrate elevated levels of CXCL9, CXCL10, or CXCL11 compared to sham-challenged controls, and extensive spore germination was observed within the lungs at all time points examined, as determined by bioluminescent measurement. In contrast, the lungs of spore-challenged C57BL/6 mice, which are resistant to infection and survive intranasal spore challenge, were found to have significantly higher levels of CXCL9, CXCL10, and CXCL11 than sham-challenged controls; increased chemokine levels were associated with a substantial reduction (1 h) or absence (6 h, 24 h, and 48 h) of detectable spore germination within the lungs of C57BL/6 mice. Although these data support a potential role for chemokine-mediated antimicrobial activity in promoting host defense during infection, they do not differentiate between direct antimicrobial effects and indirect effects resulting from immune cell recruitment to sites of infection. Differentiation of these activities and characterization of potential direct chemokine-mediated effects in vivo will require an infection model in which cellular infiltration in response to these chemokines is prevented. While host cell recruitment to CXCL9, CXCL10, and CXCL11 can be disrupted by using mice deficient in the chemokine receptor CXCR3, this lack of cellular infiltration will likely disrupt the positive-feedback loop whereby recruited cells produce factors (e.g., IFN-γ) that promote the induction of these chemokines. Therefore, future experiments will focus on the development of an in vivo model in which indirect chemokine-mediated effects are reduced and the overall chemokine production remains comparable to that seen during infection. When discussing the in vivo data presented in this paper, it is also important to recognize known differences among the mouse strains used as well as differences between in vitro and in vivo chemokine concentrations.
Previous studies have attributed differences in susceptibility to B. anthracis
infection among inbred murine strains to a deficiency in complement. Specifically, host resistance to Sterne strain infection has been shown to be associated with the host's ability to produce complement component 5 (C5), and complement depletion in normally resistant mice renders them highly susceptible to inhalational Sterne strain infection (29
). Although a direct role for C5 in controlling Sterne strain infection has not been determined, several explanations have been proposed, including increased phagocytic killing and the promotion of immune cell infiltration to sites of infection (66
). In the present context, the latter explanation is particularly interesting since a defect in C5a, a chemotactic cleavage product of C5, would likely disrupt host cell recruitment during inhalational B. anthracis
infection and may prevent the induction of the interferon-inducible ELR−
CXC chemokines. Along these lines, C5a neutralization has been shown to significantly reduce CXC and CC chemokine production by alveolar macrophages in vivo (17
). Similarly, C5 depletion has been found to inhibit production of IFN-γ, a potent inducer of the ELR−
CXC chemokines, and prevent the induction of proinflammatory cytokines and chemokines during experimental sepsis (21
All antimicrobial chemokines characterized to date, including those presented here, mediate direct antimicrobial effects within a concentration range that is higher than that required for inducing directed cell migration (12
). In the present study, the concentrations of CXCL9, CXCL10, and CXCL11 measured from the lungs of spore-challenged animals were lower than the concentrations used in the in vitro studies. The notion that direct chemokine-mediated antimicrobial activity is likely to be relevant during infection is supported by several published studies, including that (i) the stimulation of peripheral blood mononuclear cells with IFN-γ induces the production of CXCL9 and CXCL10 to levels calculated to be capable of exerting direct antimicrobial effects against E. coli
); (ii) supernatants from IFN-γ/tumor necrosis factor alpha-stimulated normal human bronchial epithelial cells demonstrate IFN-inducible ELR−
CXC chemokine concentrations of several hundred nanograms per milliliter, with CXCL10 levels approaching 1 μg/ml (56
); and (iii) markedly elevated concentrations of CXCL9 (>170 ng/ml) and CXCL10 (>400 ng/ml) have been shown to be present in the plasma of patients with melioidosis and correlate with the severity and outcome of infection (38
). While the above-described studies support that the ELR−
CXC chemokines are part of the innate immune response to bacterial infections, these concentrations are lower than the effective in vitro concentrations used in this study. Local chemokine concentrations at sites of infection, however, are likely higher than those measured in whole-tissue filtrates or cell culture supernatants and may reach levels sufficient for exerting antimicrobial effects through the individual or additive effects of these CXC chemokines.
Further support for a potential role of direct chemokine-mediated antimicrobial activity during infection comes from studies examining the effector functions of antimicrobial peptides, including defensins. The α-, β-, and θ-defensins exhibit antimicrobial activity in vitro at concentrations in the microgram-per-milliliter range (57
), much like the direct chemokine-mediated antimicrobial activity described in the present study. Despite relatively high effective concentrations in vitro, defensins have been shown to play a critical role in innate host defense against bacterial challenge. Several transgenic mouse studies have provided evidence for defensin-mediated antibacterial effector functions in vivo and include (i) delayed clearance of Haemophilus influenzae
from the lungs of mice deficient in mouse β-defensin-1 (50
), (ii) reductions in bacterial burden and increased survival rates following challenge with Salmonella enterica
serovar Typhimurium in mice expressing human defensin 5 (53
), and (iii) increased virulence of E. coli
in mice deficient in Paneth cell α-defensins (68
). Taken together, these in vivo studies provide evidence for a physiological function of defensins in promoting host defense and suggest that the chemokine concentrations presented in the current study are not so high as to preclude them from exerting antimicrobial activity in vivo.
Given ongoing concerns about the threat posed by weaponized B. anthracis spores and the inability of current treatment options to prevent the establishment of anthrax, novel therapeutic strategies capable of effectively targeting the early stages of B. anthracis infection are required. The ability of CXCL9, CXCL10, and CXCL11 to affect both B. anthracis spores and bacilli establishes a novel antimicrobial effect of these chemokines. Also, their induction by the administration of exogenous IFN-γ may offer an effective way of augmenting the production of protective CXCL9, CXCL10, and CXCL11 levels in the host lungs. By understanding the mechanism(s) by which these chemokines target B. anthracis spores and vegetative bacilli and their ability to promote host defense during infection, it is likely that innovative therapeutic strategies can be devised for effectively treating and/or preventing inhalational B. anthracis infection. In addition, these findings will likely have a therapeutic impact on infections caused by a range of pathogenic and potentially multidrug-resistant bacteria including other spore-forming organisms such as Clostridium difficile.