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1.  Toxin Inhibition of Antimicrobial Factors Induced by Bacillus anthracis Peptidoglycan in Human Blood 
Infection and Immunity  2013;81(10):3693-3702.
Here, we describe the capacity of Bacillus anthracis peptidoglycan (BaPGN) to trigger an antimicrobial response in human white blood cells (WBCs). Analysis of freshly isolated human blood cells found that monocytes and neutrophils, but not B and T cells, were highly responsive to BaPGN and produced a variety of cytokines and chemokines. This BaPGN-induced response was suppressed by anthrax lethal toxin (LT) and edema toxin (ET), with the most pronounced effect on human monocytes, and this corresponded with the higher levels of anthrax toxin receptor 1 (ANTXR1) in these cells than in neutrophils. The supernatant from BaPGN-treated cells altered the growth of B. anthracis Sterne, and this effect was blocked by LT, but not by ET. An FtsX mutant of B. anthracis known to be resistant to the antimicrobial effects of interferon-inducible Glu-Leu-Arg (ELR)-negative CXC chemokines was not affected by the BaPGN-induced antimicrobial effects. Collectively, these findings describe a system in which BaPGN triggers expression of antimicrobial factors in human WBCs and reveal a distinctive role, not shared with ET, in LT's capacity to suppress this response.
doi:10.1128/IAI.00709-13
PMCID: PMC3811742  PMID: 23876807
2.  TcdB from Hypervirulent Clostridium difficile Exhibits Increased Efficiency of Autoprocessing 
Molecular Microbiology  2012;84(1):66-76.
Summary
TcdB, an intracellular bacterial toxin that inactivates small GTPases, is a major Clostridium difficile virulence factor. Recent studies have found that TcdB produced by emerging/hypervirulent strains of C. difficile is more potent than TcdB from historical strains, and in the current work, studies were performed to investigate the underlying mechanisms for this change in TcdB toxicity. Using a series of biochemical analyses we found that TcdB from a hypervirulent strain (TcdBHV) was more efficient at autoprocessing than TcdB from a historical strain (TcdBHIST). TcdBHV and TcdBHIST were activated by similar concentrations of IP6; however, the overall efficiency of processing was 20% higher for TcdBHV. Using an activity based fluorescent probe (AWP19) an intermediate, activated but uncleaved, form of TcdBHIST was identified, while only a processed form of TcdBHV could be detected under the same conditions. Using a much higher concentration (200 µM) of the probe revealed an activated uncleaved form of TcdBHV, indicating a preferential and more efficient engagement of intramolecular substrate than TcdBHIST. Futhermore, a peptide-based inhibitor (Ac-GSL-AOMK), was found to block the cytotoxicity of TcdBHIST at a lower concentration than required to inhibit TcdBHV. These findings suggest that TcdBHV may cause increased cytotoxicity due to more efficient autoprocessing.
doi:10.1111/j.1365-2958.2012.08009.x
PMCID: PMC3313004  PMID: 22372854
Clostridium difficile; Toxin B; NAP1; cysteine protease
3.  Clostridium difficile Infection 
Clostridium difficile is the leading cause of hospital-acquired diarrhea in Europe and North America and is a serious re-emerging pathogen. Recent outbreaks have led to increasing morbidity and mortality and have been associated with a new strain (BI/NAP1/027) of C. difficile that produces more toxin than historical strains. With the increasing incidence of C. difficile infection, clinicians have also seen a change in the epidemiology with increased infections in previously low-risk populations. This chapter highlights the current knowledge on C. difficile virulence, human disease, epidemic outbreaks, and optimal treatment strategies.
doi:10.1097/MAJ.0b013e3181e939d8
PMCID: PMC2935936  PMID: 20697257
Clostridium difficile; toxin
4.  Clostridium difficile 027/BI/NAP1 Encodes a Hypertoxic and Antigenically Variable Form of TcdB 
PLoS Pathogens  2013;9(8):e1003523.
The Clostridium difficile exotoxin, TcdB, which is a major virulence factor, varies between strains of this pathogen. Herein, we show that TcdB from the epidemic BI/NAP1/027 strain of C. difficile is more lethal, causes more extensive brain hemorrhage, and is antigenically variable from TcdB produced by previously studied strains of this pathogen (TcdB003). In mouse intoxication assays, TcdB from a ribotype 027 strain (TcdB027) was at least four fold more lethal than TcdB003. TcdB027 caused a previously undescribed brain hemorrhage in mice and this correlated with a heightened sensitivity of brain microvascular endothelial cells to the toxin. TcdB003 and TcdB027 also differed in their antigenic profiles and did not share cross-neutralizing epitopes in a major immunogenic region of the protein. Solid phase humoral mapping of epitopes in the carboxy-terminal domains (CTD) of TcdB027 and TcdB003 identified 11 reactive epitopes that varied between the two forms of TcdB, and 13 epitopes that were shared or overlapping. Despite the epitope differences and absence of neutralizing epitopes in the CTD of TcdB027, a toxoid form of this toxin primed a strong protective response. These findings indicate TcdB027 is a more potent toxin than TcdB003 as measured by lethality assays and pathology, moreover the sequence differences between the two forms of TcdB alter antigenic epitopes and reduce cross-neutralization by antibodies targeting the CTD.
Author Summary
During the past decade, the C. difficile BI/NAP1/027 strain has emerged and in some settings predominated as the cause of C. difficile infection. Moreover, in some reports C. difficile BI/NAP1/027 has been associated with more severe disease. The reasons for association of this strain with more severe disease and relapse are poorly understood. We compared the toxicity and antigenic profiles of the major C. difficile virulence factor, TcdB, from a previously studied reference strain and a BI/NAP1/027 strain. The results indicate TcdB027, the toxin from the BI/NAP1/027 strain, is more lethal and causes more extensive brain hemorrhaging than TcdB003, the toxin produced by a reference strain of C. difficile. Furthermore, the results show that the antigenic carboxy-terminal domain (CTD) encodes at least 11 epitopes that differ between the two forms of TcdB. In line with this, experiments demonstrate that antiserum against the CTD does not cross-neutralize TcdB003 and TcdB027 toxicity against CHO cells, and TcdB027 appears to be devoid of neutralizing epitopes in this domain. These findings indicate differences in TcdB003 and TcdB027 contribute to increased virulence of C. difficile BI/NAP1/027 and reduce the likelihood of acquired immunity providing cross-protection against infection by these strains.
doi:10.1371/journal.ppat.1003523
PMCID: PMC3731247  PMID: 23935501
5.  Serum Amyloid A Protects Murine Macrophages from Lethal Toxin-Mediated Death1 
Cellular immunology  2011;272(2):175-181.
Lethal toxin, a key virulence factor produced by Bacillus anthracis, induces cell death, in part by disrupting numerous signaling pathways, in mouse macrophages. However, exposure to sublethal doses of lethal toxin allows some cells to survive. Because these pro-survival signaling events occur within a few hours after exposure to sublethal doses, we hypothesized that acute phase proteins might influence macrophage survival. Our data show that serum amyloid A (SAA) is produced in response to lethal toxin treatment. Moreover, pre-treatment of macrophages with exogenous SAA protected macrophages from lethal toxin-mediated death. Exogenous SAA activated the p38 mitogen activated protein kinase (MAP) kinase pathway, while lethal toxin mutants incapable of p38 activation were incapable of causing cell death. Chemical inhibition of the p38 activation pathway abrogated the protective effects of SAA. These data show that SAA affords protection against lethal toxin in mouse macrophages and link this response to the p38 pathway.
doi:10.1016/j.cellimm.2011.10.014
PMCID: PMC3244522  PMID: 22082566
anthrax lethal toxin; acute phase proteins; serum amyloid A
6.  Anthrax Vaccination Induced Anti-Lethal Factor IgG: Fine Specificity and Neutralizing Capacity 
Vaccine  2011;29(20):3670-3678.
The efficacy biomarker of the currently licensed anthrax vaccine (AVA) is based on quantity and neutralizing capacity of anti-Protective Antigen (anti-PA) antibodies. However, animal studies have demonstrated that antibodies to Lethal Factor (LF) can provide protection against in vivo bacterial spore challenges. Improved understanding of the fine specificities of humoral immune responses that provide optimum neutralization capacity may enhance the efficacy of future passive immune globulin preparations to treat and prevent inhalation anthrax morbidity and mortality. This study (n = 1000) was designed to identify AVA vaccinated individuals who generate neutralizing antibodies and to determine what specificities correlate with protection. The number of vaccine doses, years post vaccination, and PA titer were associated with in vitro neutralization, reinforcing previous reports. In addition, African American individuals had lower serologic neutralizing activity than European Americans, suggesting a genetic role in the generation of these neutralizing antibodies. Of the vaccinated individuals, only 69 (6.9%) had moderate levels of anti-LF IgG compared to 244 (24.4%) with low and 687 (68.7%) with extremely low levels of IgG antibodies to LF. Using overlapping decapeptide analysis, we identified six common LF antigenic regions targeted by those individuals with moderate levels of antibodies to LF and high in vitro toxin neutralizing activity. Affinity purified antibodies directed against antigenic epitopes within the PA binding and ADP-ribotransferase-like domains of LF were able to protect mice against lethal toxin challenge. Findings from these studies have important implications for vaccine design and immunotherapeutic development.
doi:10.1016/j.vaccine.2011.03.011
PMCID: PMC3233230  PMID: 21420416
Bacillus anthracis; Anthrax; Anthrax Vaccine Adsorbed; Lethal Factor; Protective Antigen; correlate of protection
7.  Determination of Serum Antibodies to Clostridium difficile Toxin B in Patients with Inflammatory Bowel Disease 
Gastroenterology & Hepatology  2012;8(5):313-317.
Clostridium difficile infection has increased in prevalence among patients with inflammatory bowel disease (IBD). Serum antibodies against C. difficile toxins have been detected in susceptible populations and may be protective; however, such antibodies have not been previously characterized in IBD patients. This study measured immunoglobulin G antibody levels to C. difficile toxin B in serum from IBD patients in remission and IBD patients in relapse. IBD patients demonstrated significantly higher antibody levels than non-IBD patients. In addition, a higher proportion of IBD patients in remission had positive antibody levels compared to IBD patients in relapse. Further characterization of antibody responses may elucidate understanding of susceptibility to C. difficile infection among IBD patients.
PMCID: PMC3424425  PMID: 22933861
Serum; Clostridium difficile; inflammatory bowel disease; antibodies
8.  Glycogen Synthase Kinase 3 Activation Is Important for Anthrax Edema Toxin-Induced Dendritic Cell Maturation and Anthrax Toxin Receptor 2 Expression in Macrophages ▿  
Infection and Immunity  2011;79(8):3302-3308.
Anthrax edema toxin (ET) is one of two binary toxins produced by Bacillus anthracis that contributes to the virulence of this pathogen. ET is an adenylate cyclase that generates high levels of cyclic AMP (cAMP), causing alterations in multiple host cell signaling pathways. We previously demonstrated that ET increases cell surface expression of the anthrax toxin receptors (ANTXR) in monocyte-derived cells and promotes dendritic cell (DC) migration toward the lymph node-homing chemokine MIP-3β. In this work, we sought to determine if glycogen synthase kinase 3 (GSK-3) is important for ET-induced modulation of macrophage and DC function. We demonstrate that inhibition of GSK-3 dampens ET-induced maturation and migration processes of monocyte-derived dendritic cells (MDDCs). Additional studies reveal that the ET-induced expression of ANTXR in macrophages was decreased when GSK-3 activity was disrupted with chemical inhibitors or with small interfering RNA (siRNA) targeting GSK-3. Further examination of the ET induction of ANTXR revealed that a dominant negative form of CREB could block the ET induction of ANTXR, suggesting that CREB or a related family member was involved in the upregulation of ANTXR. Because CREB and GSK-3 activity appeared to be important for ET-induced ANTXR expression, the impact of GSK-3 on ET-induced CREB activity was examined in RAW 264.7 cells possessing a CRE-luciferase reporter. As with ANTXR expression, the ET induction of the CRE reporter was decreased by reducing GSK-3 activity. These studies not only provide insight into host pathways targeted by ET but also shed light on interactions between GSK-3 and CREB pathways in host immune cells.
doi:10.1128/IAI.05070-11
PMCID: PMC3147554  PMID: 21576335
9.  Autoregulatory Characteristics of a Bacillus anthracis Serine/Threonine Kinase▿ 
Journal of Bacteriology  2011;193(8):1833-1842.
BA-Stk1 is a serine/threonine kinase (STK) expressed by Bacillus anthracis. In previous studies, we found that BA-Stk1 activity is modulated through dephosphorylation by a partner phosphatase, BA-Stp1. In this study, we identified critical phosphorylation regions of BA-Stk1 and determined the contributions of these phosphodomains to autophosphorylation and substrate phosphorylation. The data indicate that BA-Stk1 undergoes trans-autophosphorylation within a regulatory domain, referred to as the activation loop, which carries eight putative regulatory serine and threonine residues. We identified activation loop mutants that impacted kinase activity in three different manners: regulation of autophosphorylation (T162), regulation of substrate phosphorylation (T159 and S169), and regulation of overall kinase activity (T163). Tandem mass spectrometry (MS/MS) analysis of the phosphorylation profile of each mutant revealed a second site of phosphorylation on the kinase that was influenced by the phosphorylation status of the activation loop. This second region of the kinase contained a single phosphorylation residue, S214. Previous work has shown S214 to be necessary for downstream substrate phosphorylation, and we have shown that this residue is subject to dephosphorylation by BA-Stp1. These findings indicate a connection between the phosphorylation status of the activation loop and phosphorylation of S214, and this suggests a previously undescribed model for how a bacterial STK shifts from a state of autophosphorylation to targeting downstream substrates.
doi:10.1128/JB.01401-10
PMCID: PMC3133050  PMID: 21296958
10.  Regulation of Anthrax Toxin-Specific Antibody Titers by Natural Killer T Cell-Derived IL-4 and IFNγ 
PLoS ONE  2011;6(8):e23817.
Activation of Natural Killer-like T cells (NKT) with the CD1d ligand α-GC leads to enhanced production of anthrax toxin protective Ag (PA)-neutralizing Abs, yet the underlying mechanism for this adjuvant effect is not known. In the current study we examined the role of Th1 and Th2 type responses in NKT-mediated enhancement of antibody responses to PA. First, the contribution of IL-4 and IFNγ to the production of PA-specific toxin-neutralizing Abs was examined. By immunizing C57Bl/6 controls IL-4−/− mice and IFNγ−/− mice and performing passive serum transfer experiments, it was observed that sera containing PA-specific IgG1, IgG2b and IgG2c neutralized toxin in vitro and conferred protection in vivo. Sera containing IgG2b and IgG2c neutralized toxin in vitro but were not sufficient for protection in vivo. Sera containing IgG1 and IgG2b neutralized toxin in vitro and conferred protection in vivo. IgG1 therefore emerged as a good correlate of protection. Next, C57Bl/6 mice were immunized with PA alone or PA plus a Th2-skewing α-GC derivative known as OCH. Neutralizing PA-specific IgG1 responses were modestly enhanced by OCH in C57Bl/6 mice. Conversely, IgG2b and IgG2c were considerably enhanced in PA/OCH-immunized IL-4−/− mice but did not confer protection. Finally, bone marrow chimeras were generated such that NKT cells were unable to express IL-4 or IFNγ. NKT-derived IL-4 was required for OCH-enhanced primary IgG1 responses but not recall responses. NKT-derived IL-4 and IFNγ also influenced primary and recall IgG2b and IgG2c titers. These data suggest targeted skewing of the Th2 response by α-GC derivatives can be exploited to optimize anthrax vaccination.
doi:10.1371/journal.pone.0023817
PMCID: PMC3157475  PMID: 21858226
11.  Select human anthrax protective antigen (PA) epitope-specific antibodies provide protection from lethal toxin challenge 
The Journal of infectious diseases  2010;202(2):251-260.
Bacillus anthracis remains a serious bioterrorism concern, and the currently licensed vaccine remains an incomplete solution for population protection from inhalation anthrax and has been associated with concerns regarding efficacy and safety. Thus, understanding how to generate long lasting protective immunity with reduced immunizations or providing protection through post exposure immunotherapeutics are long sought goals. Through evaluation of a large military cohort, we characterized the levels of antibodies against protective antigen and found that over half of anthrax vaccinees had low levels of in vitro toxin neutralization capacity in their sera. Using solid phase epitope mapping and confirmatory assays, we identified several neutralization-associated humoral epitopes and demonstrated that select anti-peptide responses mediated protection in vitro. Finally, passively transferred antibodies specific for select epitopes provided protection in an in vivo lethal toxin mouse model. Identification of these antigenic regions has important implications for vaccine design and the development of directed immunotherapeutics.
doi:10.1086/653495
PMCID: PMC2891133  PMID: 20533877
anthrax; vaccination; antibodies; protective antigen
12.  Inflammatory Cytokine Response to Bacillus anthracis Peptidoglycan Requires Phagocytosis and Lysosomal Trafficking▿  
Infection and Immunity  2010;78(6):2418-2428.
During advanced stages of inhalation anthrax, Bacillus anthracis accumulates at high levels in the bloodstream of the infected host. This bacteremia leads to sepsis during late-stage anthrax; however, the mechanisms through which B. anthracis-derived factors contribute to the pathology of infected hosts are poorly defined. Peptidoglycan, a major component of the cell wall of Gram-positive bacteria, can provoke symptoms of sepsis in animal models. We have previously shown that peptidoglycan of B. anthracis can induce the production of proinflammatory cytokines by cells in human blood. Here, we show that biologically active peptidoglycan is shed from an active culture of encapsulated B. anthracis strain Ames in blood. Peptidoglycan is able to bind to surfaces of responding cells, and internalization of peptidoglycan is required for the production of inflammatory cytokines. We also show that the peptidoglycan traffics to lysosomes, and lysosomal function is required for cytokine production. We conclude that peptidoglycan of B. anthracis is initially bound by an unknown extracellular receptor, is phagocytosed, and traffics to lysosomes, where it is degraded to a product recognized by an intracellular receptor. Binding of the peptidoglycan product to the intracellular receptor causes a proinflammatory response. These findings provide new insight into the mechanism by which B. anthracis triggers sepsis during a critical stage of anthrax disease.
doi:10.1128/IAI.00170-10
PMCID: PMC2876538  PMID: 20308305
13.  Resistance of Human Alveolar Macrophages to Bacillus anthracis Lethal Toxin1 
The etiologic agent of inhalational anthrax, B. anthracis, produces virulence toxins that are important in the disease pathogenesis. Current studies suggest that mouse and human macrophages are susceptible to immunosuppressive effects of one of the virulence toxins, lethal toxin (LT). Thus a paradigm has emerged that holds that the alveolar macrophage (AM) does not play a significant role in the innate immune response to B. anthracis or defend against the pathogen as it is disabled by LT. This is inconsistent with animal models and autopsy studies that show minimal disease at the alveolar surface. We examined whether AM are immunosuppressed by LT. We found that human AM (HAM) were relatively resistant to LT-mediated innate immune cytokine suppression, MEK cleavage, and induction of apoptosis as compared to mouse RAW 264.7 macrophages. Mouse AM (MAM) and murine bone marrow derived macrophages (BMDM) were also relatively resistant to LT-mediated apoptosis despite intermediate sensitivity to MEK cleavage. The binding component of LT, protective antigen (PA), does not attach to HAM, although it did bind to MAM, murine BMDM and RAW 264.7 macrophages. HAM do not produce significant amounts of the PA receptors anthrax toxin receptor 1 (TEM8/ANTXR1) and anthrax toxin receptor 2 (CMG2/ANTXR2). Thus, mature and differentiated AM are relatively resistant to the effects of LT as compared to mouse RAW 264.7 macrophages. AM resistance to LT may enhance clearance of the pathogen from the alveolar surface and explain why this surface is relatively free of B. anthracis in animal models and autopsy studies.
doi:10.4049/jimmunol.0803406
PMCID: PMC2770336  PMID: 19812208
human; macrophage; lung; apoptosis; anthrax
14.  CD1d-Dependent B-Cell Help by NK-Like T Cells Leads to Enhanced and Sustained Production of Bacillus anthracis Lethal Toxin-Neutralizing Antibodies▿  
Infection and Immunity  2010;78(4):1610-1617.
The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (α-galactosylceramide [α-GC]) at the time of immunization improves PA-specific Ab responses. We observed that α-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d−/− mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jα281−/− mice expressing CD1d but lacking type I α-GC-reactive NKT cells, α-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.
doi:10.1128/IAI.00002-10
PMCID: PMC2849421  PMID: 20123711
15.  Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile 
PLoS Pathogens  2010;6(8):e1001061.
Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdBHV) was more cytotoxic in vitro than TcdB from historical strains (TcdBHIST). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdBHV and TcdBHIST. Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdBHV were found to be substantially broader and more pronounced than those caused by TcdBHIST. The increased toxicity of TcdBHV was related to the toxin's ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdBHIST. The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdBHV undergoes acid-induced conformational changes at a pH much higher than that of TcdBHIST. Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile.
Author Summary
Clostridium difficile is a spore-forming bacterium that contaminates hospitals and infects patients undergoing antibiotic therapy. C. difficile is now the leading cause of hospital-acquired diarrhea in developed countries. Most concerning has been the recent increase in mortality of C. difficile patients due to the emergence of a hypervirulent strain of this pathogen. Results from the current study suggest this change in disease severity may be due to new strains producing a variant form of C. difficile's major virulence factor, TcdB. The findings indicate TcdB from hypervirulent strains targets a much broader range of cells in vivo and is able to translocate into target cells more quickly than TcdB from historical strains of C. difficile. The more rapid cell entry by TcdB from hypervirulent C. difficile appears to be due to the toxin's capacity to undergo conformational changes necessary for membrane translocation at a higher pH than TcdB from historical strains. To date, very little has been learned about the underlying reasons for the increased virulence of emerging C. difficile strains. These findings provide insight into this problem and suggest variations in TcdB activity could be an important contributing factor to the hypervirulence of emerging strains of C. difficile.
doi:10.1371/journal.ppat.1001061
PMCID: PMC2924371  PMID: 20808849
16.  Regulatory Interactions of a Virulence-Associated Serine/Threonine Phosphatase-Kinase Pair in Bacillus anthracis▿  
Journal of Bacteriology  2009;192(2):400-409.
In the current study, we examined the regulatory interactions of a serine/threonine phosphatase (BA-Stp1), serine/threonine kinase (BA-Stk1) pair in Bacillus anthracis. B. anthracis STPK101, a null mutant lacking BA-Stp1 and BA-Stk1, was impaired in its ability to survive within macrophages, and this correlated with an observed reduction in virulence in a mouse model of pulmonary anthrax. Biochemical analyses confirmed that BA-Stp1 is a PP2C phosphatase and dephosphorylates phosphoserine and phosphothreonine residues. Treatment of BA-Stk1 with BA-Stp1 altered BA-Stk1 kinase activity, indicating that the enzymatic function of BA-Stk1 can be influenced by BA-Stp1 dephosphorylation. Using a combination of mass spectrometry and mutagenesis approaches, three phosphorylated residues, T165, S173, and S214, in BA-Stk1 were identified as putative regulatory targets of BA-Stp1. Further analysis found that T165 and S173 were necessary for optimal substrate phosphorylation, while S214 was necessary for complete ATP hydrolysis, autophosphorylation, and substrate phosphorylation. These findings provide insight into a previously undescribed Stp/Stk pair in B. anthracis.
doi:10.1128/JB.01221-09
PMCID: PMC2805330  PMID: 19915022
17.  The Major Neutralizing Antibody Responses to Recombinant Anthrax Lethal and Edema Factors Are Directed to Non-Cross-Reactive Epitopes▿ †  
Infection and Immunity  2009;77(11):4714-4723.
Anthrax lethal and edema toxins (LeTx and EdTx, respectively) form by binding of lethal factor (LF) or edema factor (EF) to the pore-forming moiety protective antigen (PA). Immunity to LF and EF protects animals from anthrax spore challenge and neutralizes anthrax toxins. The goal of the present study is to identify linear B-cell epitopes of EF and to determine the relative contributions of cross-reactive antibodies of EF and LF to LeTx and EdTx neutralization. A/J mice were immunized with recombinant LF (rLF) or rEF. Pools of LF or EF immune sera were tested for reactivity to rLF or rEF by enzyme-linked immunosorbent assays, in vitro neutralization of LeTx and EdTx, and binding to solid-phase LF and EF decapeptides. Cross-reactive antibodies were isolated by column absorption of EF-binding antibodies from LF immune sera and by column absorption of LF-binding antibodies from EF immune sera. The resulting fractions were subjected to the same assays. Major cross-reactive epitopes were identified as EF amino acids (aa) 257 to 268 and LF aa 265 to 274. Whole LF and EF immune sera neutralized LeTx and EdTx, respectively. However, LF sera did not neutralize EdTx, nor did EF sera neutralize LeTx. Purified cross-reactive immunoglobulin G also failed to cross-neutralize. Cross-reactive B-cell epitopes in the PA-binding domains of whole rLF and rEF occur and have been identified; however, the major anthrax toxin-neutralizing humoral responses to these antigens are constituted by non-cross-reactive epitopes. This work increases understanding of the immunogenicity of EF and LF and offers perspective for the development of new strategies for vaccination against anthrax.
doi:10.1128/IAI.00749-09
PMCID: PMC2772542  PMID: 19720758
18.  Correction: Bacillus anthracis Lethal Toxin Disrupts TCR Signaling in CD1d-Restricted NKT Cells Leading to Functional Anergy 
PLoS Pathogens  2009;5(10):10.1371/annotation/cfc9c388-05fa-4b1c-8c5f-99835278a458.
doi:10.1371/annotation/cfc9c388-05fa-4b1c-8c5f-99835278a458
PMCID: PMC2771656
19.  Bacillus anthracis Lethal Toxin Disrupts TCR Signaling in CD1d-Restricted NKT Cells Leading to Functional Anergy 
PLoS Pathogens  2009;5(9):e1000588.
Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation of type-1 natural killer–like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis–derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.
Author Summary
The bacterium Bacillus anthracis is the causative agent of anthrax infection. Anthrax is a life-threatening disease caused by inhalation or ingestion of spores, or transmission through wounds and abrasions. The bacterium secretes toxins, proteins that enter numerous cell types in an infected individual altering their function and contributing to the disease process. Consequently, we and other researchers are dedicated to identifying cells affected by anthrax toxins and to understanding the implications for the function of those cells. We have observed that anthrax toxins adversely affect a type of cell in the immune system known as a natural killer–like T (NKT) cell. NKT cells are found in the bone marrow, blood, spleen, lymph nodes and liver and are required for optimal immune responses. We report that anthrax toxins effectively shut down NKT cells, preventing them from functioning normally. We propose that this has serious consequences because anyone infected with Bacillus anthracis will be less able to mount an immune response against it.
doi:10.1371/journal.ppat.1000588
PMCID: PMC2742733  PMID: 19779559
20.  The Mechanism of Bacillus anthracis Intracellular Germination Requires Multiple and Highly Diverse Genetic Loci▿  
Infection and Immunity  2008;77(1):23-31.
In an effort to better understand the mechanisms by which Bacillus anthracis establishes disease, experiments were undertaken to identify the genes essential for intracellular germination. Eighteen diverse genetic loci were identified via an enrichment protocol using a transposon-mutated library of B. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated with B. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions in gerHA, gerHB, gerHC, and pagA, respectively, each of which has been previously associated with germination or survival of B. anthracis within macrophages. Strain MIGD101 (named for macrophage intracellular germination defective 101) was of particular interest, since this mutant contained a transposon insertion in an intergenic region between BAs2807 and BAs2808, and was the most highly represented mutant in the enrichment. Analysis of B. anthracis MIGD101 by confocal microscopy and differential heat sensitivity following macrophage infection revealed ungerminated spores within the cell. Moreover, B. anthracis MIGD101 was attenuated in cell killing relative to the parent strain. Further experimental analysis found that B. anthracis MIGD101 was defective in five known B. anthracis germination pathways, supporting a mechanism wherein the intergenic region between BAs2807 and BAs2808 has a global affect on germination of this pathogen. Collectively, these findings provide insight into the mechanisms supporting B. anthracis germination within host cells.
doi:10.1128/IAI.00801-08
PMCID: PMC2612280  PMID: 18936179
21.  Sequential B-Cell Epitopes of Bacillus anthracis Lethal Factor Bind Lethal Toxin-Neutralizing Antibodies▿  
Infection and Immunity  2008;77(1):162-169.
The bipartite anthrax lethal toxin (LeTx) consisting of protective antigen (PA) and lethal factor (LF) is a major virulence factor contributing to death from systemic Bacillus anthracis infection. The current vaccine elicits antibodies directed primarily to PA; however, in experimental settings serologic responses to LF can neutralize LeTx and contribute to protection against infection. The goals of the present study were to identify sequential B-cell epitopes of LF and to determine the capacity of these determinants to bind neutralizing antibodies. Sera of recombinant LF-immunized A/J mice exhibited high titers of immunoglobulin G anti-LF reactivity that neutralized LeTx in vitro 78 days after the final booster immunization and protected the mice from in vivo challenge with 3 50% lethal doses of LeTx. These sera bound multiple discontinuous epitopes, and there were major clusters of reactivity on native LF. Strikingly, all three neutralizing, LF-specific monoclonal antibodies tested bound specific peptide sequences that coincided with sequential epitopes identified in polyclonal antisera from recombinant LF-immunized mice. This study confirms that LF induces high-titer protective antibodies in vitro and in vivo. Moreover, the binding of short LF peptides by LF-specific neutralizing monoclonal antibodies suggests that generation of protective antibodies by peptide vaccination may be feasible for this antigen. This study paves the way for a more effective anthrax vaccine by identifying discontinuous peptide epitopes of LF.
doi:10.1128/IAI.00788-08
PMCID: PMC2612257  PMID: 18981257
22.  Inhibition of Bacillus anthracis Spore Outgrowth by Nisin▿  
Antimicrobial Agents and Chemotherapy  2008;52(12):4281-4288.
The lantibiotic nisin has previously been reported to inhibit the outgrowth of spores from several Bacillus species. However, the mode of action of nisin responsible for outgrowth inhibition is poorly understood. By using B. anthracis Sterne 7702 as a model, nisin acted against spores with a 50% inhibitory concentration (IC50) and an IC90 of 0.57 μM and 0.90 μM, respectively. Viable B. anthracis organisms were not recoverable from cultures containing concentrations of nisin greater than the IC90. These studies demonstrated that spores lose heat resistance and become hydrated in the presence of nisin, thereby ruling out a possible mechanism of inhibition in which nisin acts to block germination initiation. Rather, germination initiation is requisite for the action of nisin. This study also revealed that nisin rapidly and irreversibly inhibits growth by preventing the establishment of oxidative metabolism and the membrane potential in germinating spores. On the other hand, nisin had no detectable effects on the typical changes associated with the dissolution of the outer spore structures (e.g., the spore coats, cortex, and exosporium). Thus, the action of nisin results in the uncoupling of two critical sequences of events necessary for the outgrowth of spores: the establishment of metabolism and the shedding of the external spore structures.
doi:10.1128/AAC.00625-08
PMCID: PMC2592879  PMID: 18809941
23.  Bacillus anthracis Edema Toxin Activates Nuclear Glycogen Synthase Kinase 3β▿  
Infection and Immunity  2008;76(11):4895-4904.
Bacillus anthracis edema toxin (ET) generates high levels of cyclic AMP and impacts a complex network of signaling pathways in targeted cells. In the current study, we sought to identify kinase signaling pathways modulated by ET to better understand how this toxin alters cell physiology. Using a panel of small-molecule inhibitors of mammalian kinases, we found that inhibitors of glycogen synthase kinase 3 beta (GSK-3β) protected cells from ET-induced changes in the cell cycle. GSK-3β inhibitors prevented declines in cellular levels of cyclin D1 and c-Jun following treatment of macrophages with ET. Strikingly, cell fractionation experiments and confocal immunofluorescence microscopy revealed that ET activates a compartmentalized pool of GSK-3β residing in the nuclei, but not in the cytoplasm, of macrophages. To investigate the outcome of this event, we examined the cellular location and activation state of β-catenin, a critical substrate of GSK-3β, and found that the protein was inactivated within the nucleus following intoxication with ET. To determine if ET could overcome the effects of stimuli that inactivate GSK-3β, we examined the impact of the toxin on the Wnt signaling pathway. The results of these experiments revealed that by targeting GSK-3β residing in the nucleus, ET circumvents the upstream cytoplasmic inactivation of GSK-3β, which occurs following exposure to Wnt-3A. These findings suggest ET arrests the cell cycle by a mechanism involving activation of GSK-3β residing in the nucleus, and by using this novel mechanism of intoxication, ET avoids cellular systems that would otherwise reverse the effects of the toxin.
doi:10.1128/IAI.00889-08
PMCID: PMC2573379  PMID: 18765729
24.  High-Throughput, Single-Cell Analysis of Macrophage Interactions with Fluorescently Labeled Bacillus anthracis Spores▿  
Applied and Environmental Microbiology  2008;74(16):5201-5210.
The engulfment of Bacillus anthracis spores by macrophages is an important step in the pathogenesis of inhalational anthrax. However, from a quantitative standpoint, the magnitude to which macrophages interact with and engulf spores remains poorly understood, in part due to inherent limitations associated with commonly used assays. To analyze phagocytosis of spores by RAW264.7 macrophage-like cells in a high-throughput, nonsubjective manner, we labeled B. anthracis Sterne 7702 spores prior to infection with an Alexa Fluor 488 amine-reactive dye in a manner that did not alter their germination, growth kinetics, and heat resistance. Using flow cytometry, large numbers of cells exposed to labeled spores were screened to concurrently discriminate infected from uninfected cells and surface-associated from internalized spores. These experiments revealed that spore uptake was not uniform, but instead, highly heterogeneous and characterized by subpopulations of infected and uninfected cells, as well as considerable variation in the number of spores associated with individual cells. Flow cytometry analysis of infections demonstrated that spore uptake was independent of the presence or absence of fetal bovine serum, a germinant that, while routinely used in vitro, complicates the interpretation of the outcome of infections. Two commonly used macrophage cell lines, RAW264.7 and J774A.1 cells, were compared, revealing significant disparity between these two models in the rates of phagocytosis of labeled spores. These studies provide the experimental framework for investigating mechanisms of spore phagocytosis, as well as quantitatively evaluating strategies for interfering with macrophage binding and uptake of spores.
doi:10.1128/AEM.02890-07
PMCID: PMC2519261  PMID: 18552183
25.  Effects of Endogenous d-Alanine Synthesis and Autoinhibition of Bacillus anthracis Germination on In Vitro and In Vivo Infections▿  
Infection and Immunity  2007;75(12):5726-5734.
Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous d-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous d-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with d-alanine production, was prevented by an alanine racemase inhibitor, and required l-alanine. Interestingly, endogenous production of inhibitory levels of d-alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of d-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of d-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.
doi:10.1128/IAI.00727-07
PMCID: PMC2168361  PMID: 17923523

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