Peptidoglycan from Bacillus anthracis Inhibits Human Macrophage Efferocytosis in Part by Reducing Cell Surface Expression of MERTK and TIM-3.

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. In this study, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163+CD206+ MΦ to PGN for 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVß5, CD36, and TIM-3, whereas TIM-1, αVß3, CD300b, CD300f, STABILIN-1, and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant, suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.

Anthrax lethal toxin and tumor necrosis factor-α synergize on intestinal epithelia to induce mouse death.

Bacillus anthracis lethal toxin (LT) is a determinant of lethal anthrax. Its function in myeloid cells is required for bacterial dissemination, and LT itself can directly trigger dysfunction of the cardiovascular system. The interplay between LT and the host responses is important in the pathogenesis, but our knowledge on this interplay remains limited. Tumor necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine induced by bacterial infections. Since LT accumulates and cytokines, predominantly TNF, amass during B. anthracis infection, co-treatment of TNF + LT in mice was used to mimic in vivo conditions for LT to function in inflamed hosts. Bone marrow transplantation and genetically engineered mice showed unexpectedly that the death of intestinal epithelial cells (IECs) rather than that of hematopoietic cells led to LT + TNF-induced lethality. Inhibition of p38α mitogen-activated protein kinase (MAPK) signaling by LT in IECs promoted TNF-induced apoptosis and necroptosis of IECs, leading to intestinal damage and mouse death. Consistently, p38α inhibition by LT enhanced TNF-mediated cell death in human colon epithelial HT-29 cells. As intestinal damage is one of the leading causes of lethality in anthrax patients, the IEC damage caused by LT + TNF would most likely be a mechanism underneath this clinical manifestation and could be a target for interventions.

Modeling gastrointestinal anthrax disease.

Bacillus anthracis is a spore-forming microbe that persists in soil and causes anthrax disease. The most natural route of infection is ingestion by grazing animals. Gastrointestinal (GI) anthrax also occurs in their monogastric predators, including humans. Exposure of carcasses to oxygen triggers sporulation and contamination of the surrounding soil completing the unusual life cycle of this microbe. The pathogenesis of GI anthrax is poorly characterized. Here, we use B. anthracis carrying the virulence plasmids pXO1 and pXO2, to model gastrointestinal disease in Guinea pigs and mice. We find that spores germinate in the GI tract and precipitate disease in a dose-dependent manner. Inoculation of vegetative bacilli also results in GI anthrax. Virulence is impacted severely by the loss of capsule (pXO2-encoded) but only moderately in absence of toxins (pXO1-encoded). Nonetheless, the lack of toxins leads to reduced bacterial replication in infected hosts. B. cereus Elc4, a strain isolated from a fatal case of inhalational anthrax-like disease, was also found to cause GI anthrax. Because transmission to new hosts depends on the release of large numbers of spores in the environment, we propose that the acquisition of pXO1- and pXO2-like plasmids may promote the successful expansion of members of the Bacillus cereus sensu lato group able to cause anthrax-like disease.

A Stochastic Intracellular Model of Anthrax Infection With Spore Germination Heterogeneity.

We present a stochastic mathematical model of the intracellular infection dynamics of Bacillus anthracis in macrophages. Following inhalation of B. anthracis spores, these are ingested by alveolar phagocytes. Ingested spores then begin to germinate and divide intracellularly. This can lead to the eventual death of the host cell and the extracellular release of bacterial progeny. Some macrophages successfully eliminate the intracellular bacteria and will recover. Here, a stochastic birth-and-death process with catastrophe is proposed, which includes the mechanism of spore germination and maturation of B. anthracis. The resulting model is used to explore the potential for heterogeneity in the spore germination rate, with the consideration of two extreme cases for the rate distribution: continuous Gaussian and discrete Bernoulli. We make use of approximate Bayesian computation to calibrate our model using experimental measurements from in vitro infection of murine peritoneal macrophages with spores of the Sterne 34F2 strain of B. anthracis. The calibrated stochastic model allows us to compute the probability of rupture, mean time to rupture, and rupture size distribution, of a macrophage that has been infected with one spore. We also obtain the mean spore and bacterial loads over time for a population of cells, each assumed to be initially infected with a single spore. Our results support the existence of significant heterogeneity in the germination rate, with a subset of spores expected to germinate much later than the majority. Furthermore, in agreement with experimental evidence, our results suggest that most of the spores taken up by macrophages are likely to be eliminated by the host cell, but a few germinated spores may survive phagocytosis and lead to the death of the infected cell. Finally, we discuss how this stochastic modelling approach, together with dose-response data, allows us to quantify and predict individual infection risk following exposure.

Complement C5 inhibition protects against hemolytic anemia and acute kidney injury in anthrax peptidoglycan-induced sepsis in baboons.

Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of Bacillus anthracis, leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections.

Structural Integrity of the Alveolar-Capillary Barrier in Cynomolgus Monkeys Challenged with Fully Virulent and Toxin-Deficient Strains of Bacillus anthracis.

Inhalational anthrax, a disease caused by inhaling Bacillus anthracis spores, leads to respiratory distress, vascular leakage, high-level bacteremia, and often death within days. Anthrax lethal toxin and edema toxin, which are composed of protective antigen (PA) plus either lethal factor (LF) or edema factor (EF), respectively, play an important yet incompletely defined role in the pulmonary pathophysiology. To better understand their contribution, we examined the structural integrity of the alveolar-capillary barrier in archival formalin-fixed lungs of cynomolgus monkeys challenged with the fully virulent B. anthracis Ames wild-type strain or the isogenic toxin-deficient mutants ΔEF, ΔLF, and ΔPA. Pulmonary spore challenge with the wild-type strain caused high mortality, intra-alveolar hemorrhages, extensive alveolar septal sequestration of bacteria and neutrophils, diffuse destabilization of epithelial and endothelial junctions, increased markers of coagulation and complement activation (including tissue factor and C5a), and multifocal intra-alveolar fibrin deposition. ΔEF challenge was lethal and showed similar alveolar-capillary alterations; however, intra-alveolar hemorrhages, bacterial deposition, and markers of coagulation or complement were absent or markedly lower. In contrast, ΔLF or ΔPA challenges were nonlethal and showed no signs of alveolar bacterial deposition or alveolar-capillary changes. These findings provide evidence that lethal toxin plays a determinative role in bacterial dissemination and alveolar-capillary barrier dysfunction, and edema toxin may significantly exacerbate pulmonary pathologies in a systemic infection.

Human anthrax in Kyrgyz Republic: Epidemiology and clinical features.

BACKGROUND: Bacillus anthracis infects both humans and animals which leads to anthrax disease. The disease is still a global issue as it occurs naturally and has a potential use for bioterrorism/bio-weapons agents. This study aimed to investigate the clinical and epidemiologic features of human anthrax that has been seen in Kyrgyzstan. METHODS: This study was carried out at the Osh Regional Hospital that is located in the southern regions of Kyrgyz Republic. Medical records of patient's diagnosed with cutaneous anthrax between 2005 and 2015 were reviewed. Epidemiology and clinical features of the infection and the effectiveness of antibiotic therapy were investigated. RESULTS: Two hundred thirty four anthrax cases were reviewed. Among the patients, men in the 31-50 age group prevailed. Infection episodes that sought medical attention occurred mostly during the summer and autumn. Analysis of epidemiological data shows that anthrax contamination occurred as a result of direct contact with mainly farm animals, slaughtering, butchering and the selling of the meat. Cutaneous lesions were localized mostly in the upper extremities. Among the observed patients, a majority of the cases presented a mild form of the disease. The isolated strains of B. anthracis were mostly susceptible to amoxiclav, ofloxacin, 3rd generation of cephalosporins, doxycycline, ciprofloxacin, rifampicin, penicillin and streptomycin. A majority of the isolates were found to be resistant to ampicillin, gentamicin, chloramphenicol. CONCLUSION: Anthrax is still an endemic disease that has lead to outbreaks in the Kyrgyz Republic. The transmission of human infection is seen by direct contact with ill animals or dead animal products. The common form seen in the clinical setting is cutaneous anthrax. Future studies should focus on the surveillance and infection control measurements associated with the anthrax.

Sporadic human cutaneous anthrax outbreak in Shaanxi Province, China: report of two cases from 2018

ABSTRACT China's compulsory annual livestock anthrax vaccination policy has remarkably reduced but not completely eradicated human anthrax infections. Herein we describe a sporadic human cutaneous anthrax outbreak involving two cases in 2018 in Shaanxi Province, both involving herdsman who dealt with unvaccinated and potentially sick cattle. Both patients showed Bacillus anthracis-positive blister smear and blood culture. Treatment with penicillin was followed by uneventful recovery for both. The prompt performance of the prophylactic measures successfully interrupted the further transmission of this sporadic human cutaneous anthrax outbreak.

Sporadic human cutaneous anthrax outbreak in Shaanxi Province, China: report of two cases from 2018.

China's compulsory annual livestock anthrax vaccination policy has remarkably reduced but not completely eradicated human anthrax infections. Herein we describe a sporadic human cutaneous anthrax outbreak involving two cases in 2018 in Shaanxi Province, both involving herdsman who dealt with unvaccinated and potentially sick cattle. Both patients showed Bacillus anthracis-positive blister smear and blood culture. Treatment with penicillin was followed by uneventful recovery for both. The prompt performance of the prophylactic measures successfully interrupted the further transmission of this sporadic human cutaneous anthrax outbreak.

Knowledge, attitudes, and practices related to anthrax and animal care: A case-control study in Georgia.

INTRODUCTION: Anthrax is endemic in Georgia and recent outbreaks prompted a livestock-handler case-control study with a component to evaluate anthrax knowledge, attitudes, and practices (KAP) among livestock handlers or owners. METHODS: Cases were handlers of livestock with confirmed animal anthrax from June 2013-May 2015. Handlers of four matched unaffected animals were selected as controls, two from the same village as the case animal ("village control") and two from 3-10 km away ("area control"). Descriptive statistics were reported and conditional logistic regression was performed to estimate the magnitude of the association of cases with specific study KAP factors. RESULTS: Cases were more likely male, had lower level college education, less animal care experience, and provided more animal care to their cattle. Cases had lower odds of burying a suddenly dead animal compared to all controls (Odds Ratio [OR]: 0.32, 95% Confidence interval [CI]:0.12, 0.88) and area controls (OR: 0.32, 95% CI: 0.11, 0.91). On an 8-point knowledge scale, cases having an animal with anthrax had a 1.31 times greater knowledge score compared to all controls (95% CI: 1.03, 1.67). Cases had higher odds of ever having human anthrax or knowing another person who had anthrax compared to all controls (OR: 4.56, 95% CI: 1.45, 14.30) and area controls (OR: 7.16, 95% CI: 1.52, 33.80). DISCUSSION: Cases were more knowledgeable of anthrax and had better anthrax prevention practices, but these are likely a result of the case investigation and ring vaccination following the death of their animal. CONCLUSIONS: The findings reveal a low level of knowledge and practices related to anthrax control and prevention, and will guide educational material development to fill these gaps.

Development of a novel chimeric PA-LF antigen of Bacillus anthracis, its immunological characterization and evaluation as a future vaccine candidate in mouse model.

Tremendous efforts are being made to develop an anthrax vaccine with long term protection. The main component of traditional anthrax vaccine is protective antigen (PA) with the trace amount of other proteins and bacterial components. In this study, we developed a recombinant PA-LF chimera antigen of Bacillus anthracis by fusing the PA domain 2-4 with lethal factor (LF) domain 1 and evaluated its protective potential against B. anthracis in mouse model. The anti-PA-LF chimera serum reacted with both PA and LF antigen, individually. The chimera elicited a strong antibody titer in mice with predominance of IgG1 isotype followed by IgG2b, IgG2a and IgG3. Cytokines were assessed in splenocytes of immunized mice and a significant up-regulation in the expression of IL-4, IL-10, IFN-γ and TNF-α was observed. The PA-LF chimera immunized mice exhibited 80% survival after challenge with virulent spores of B. anthracis. Pathological studies showed normal architecture in vital organs (spleen, lung, liver and kidney) of recovered immunized mice on 20 DPI after spore challenge. These findings suggested that PA-LF chimera of B. anthracis elicited good humoral as well as cell mediated immune response in mice, and thus, can be a potent vaccine candidate against anthrax.

An unusual case of bovine anthrax in the canton of Jura, Switzerland in 2017.

BACKGROUND: Anthrax caused by Bacillus anthracis is a zoonotic disease mainly affecting herbivores. The last Swiss outbreak was over 20 years ago. We describe a recent anthrax outbreak involving two cows from the same herd. One cow was designated as a peracute clinical case with sudden death and typical lung lesions, while the other cow presented with protracted fever and abortion. CASE PRESENTATION: On April 29th 2017, a 3.5-year-old Montbéliard dairy cow was found dead while out at pasture with haemorrhage from the nose. The veterinarian suspected pneumonia and performed a necropsy on site. Subsequently, a lung and liver sample were sent to the laboratory. Unexpectedly, Bacillus anthracis was isolated, a pathogen not found in Switzerland for decades. Several days later, a second cow from the same farm showed signs of abortion after protracted fever. Since these symptoms are not typical for anthrax, and the bacteria could not be demonstrated in blood samples from this animal, a necropsy was performed under appropriate biosafety measures. Subsequently, Bacillus anthracis could be isolated from the placenta and the sublumbal lymph nodes but not from the blood, liver, spleen and kidney. The outbreak strain (17OD930) was shown to belong to the lineage B.Br.CNEVA, the same as Swiss strains from previous outbreaks in the region. We speculate that the disease came from a temporarily opened cave system that is connected to an old carcass burial site and was flushed by heavy rainfall preceding the outbreak. CONCLUSION: Even in countries like Switzerland, where anthrax is very rare, new cases can occur after unusual weather conditions or ground disturbance. It is important for public officials to be aware of this risk to avoid possible spread.

Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax.

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.

Infection with a Nonencapsulated Bacillus anthracis Strain in Rabbits-The Role of Bacterial Adhesion and the Potential for a Safe Live Attenuated Vaccine.

Nonencapsulated (∆pXO2) Bacillus anthracis strains are commonly used as vaccines and for anthrax research, mainly in the mouse model. Previously, we demonstrated that the infection of rabbits, intranasally or subcutaneously, with the spores of a fully virulent strain results in the systemic dissemination of the bacteria, meningitis, and death, whereas ∆pXO2 strains are fully attenuated in this animal model. We used the intravenous inoculation of rabbits to study the pathogenicity of the ∆pXO2 strain infection. Bacteremia, brain bacterial burden, and pathology were used as criteria to compare the Vollum∆pXO2 disease to the wild type Vollum infection. To test the role of adhesion in the virulence of Vollum∆pXO2, we deleted the major adhesion protein BslA and tested the virulence and immunogenicity of this mutant. We found that 50% of the rabbits succumb to Vollum∆pXO2 strain following i.v. infection, a death that was accompanied with significant neurological symptoms. Pathology revealed severe brain infection coupled with an atypical massive bacterial growth into the parenchyma. Contrary to the Vollum strain, deletion of the bslA gene fully attenuated the ∆pXO2 strain. Though the Vollum∆pXO2 cannot serve as a model for B. anthracis pathogenicity in rabbits, deletion of the bslA gene prevents central nervous system (CNS) infections, possibly leading to the generation of a safer vaccine.

A case of Anthrax in two captive pumas (Puma concolor).

In this study, we aimed to report anthrax cases in two pumas, brought to the Pathology Department, Faculty of Veterinary Medicine, Erciyes University for suspected poisoning upon their sudden death at the Kayseri Zoo, in Turkey. In the necropsy, enlargement and malacia were observed in the spleens. The cut surfaces of the spleens were in extreme red-blackish color. Bacillus anthracis was isolated as a pure culture from both samples which belong to dead pumas. B. anthracis isolates had pXO1 and pXO2 plasmids. Both isolates were found to be sensitive to eight antibacterials tested. This study demonstrates that feeding of the wild carnivorous kept in any zoo with the appropriate meats which belongs to healthy animals is extremely important.

Peptidoglycan induces disseminated intravascular coagulation in baboons through activation of both coagulation pathways.

Anthrax infections exhibit progressive coagulopathies that may contribute to the sepsis pathophysiology observed in fulminant disease. The hemostatic imbalance is recapitulated in primate models of late-stage disease but is uncommon in toxemic models, suggesting contribution of other bacterial pathogen-associated molecular patterns (PAMPs). Peptidoglycan (PGN) is a bacterial PAMP that engages cellular components at the cross talk between innate immunity and hemostasis. We hypothesized that PGN is critical for anthrax-induced coagulopathies and investigated the activation of blood coagulation in response to a sterile PGN infusion in primates. The PGN challenge, like the vegetative bacteria, induced a sepsis-like pathophysiology characterized by systemic inflammation, disseminated intravascular coagulation (DIC), organ dysfunction, and impaired survival. Importantly, the hemostatic impairment occurred early and in parallel with the inflammatory response, suggesting direct engagement of coagulation pathways. PGN infusion in baboons promoted early activation of contact factors evidenced by elevated protease-serpin complexes. Despite binding to contact factors, PGN did not directly activate either factor XII (FXII) or prekallikrein. PGN supported contact coagulation by enhancing enzymatic function of active FXII (FXIIa) and depressing its inhibition by antithrombin. In parallel, PGN induced de novo monocyte tissue factor expression in vitro and in vivo, promoting extrinsic clotting reactions at later stages. Activation of platelets further amplified the procoagulant state during PGN challenge, leading to DIC and subsequent ischemic damage of peripheral tissues. These data indicate that PGN may be a major cause for the pathophysiologic progression of Bacillus anthracis sepsis and is the primary PAMP behind the pathogen-induced coagulopathy in late-stage anthrax.

Treating Anthrax-Induced Meningitis in Rabbits.

Treatment of anthrax is challenging, especially during the advanced stages of the disease. Recently, the Centers for Disease Control and Prevention (CDC) updated its recommendations for postexposure prophylaxis and treatment of exposed populations (before and after symptom onset). These recommendations distinguished, for the first time, between systemic disease with and without meningitis, a common and serious complication of anthrax. The CDC considers all systemic cases meningeal unless positively proven otherwise. The treatment of patients suffering from systemic anthrax with suspected or confirmed meningitis includes the combination of three antibiotics, i.e., a fluoroquinolone (levofloxacin or ciprofloxacin), a ß-lactam (meropenem or imipenem), and a protein synthesis inhibitor (linezolid or clindamycin). In addition, treatment with an antitoxin (anti-protective antigen antibodies) and dexamethasone should be applied. Since the efficacy of most of these treatments has not been demonstrated, especially in animal meningitis models, we developed an anthrax meningitis model in rabbits and tested several of these recommendations. We demonstrated that, in this model, ciprofloxacin, linezolid, and meropenem were ineffective as single treatments, while clindamycin was highly effective. Furthermore, combined treatments of ciprofloxacin and linezolid or ciprofloxacin and dexamethasone failed in treating rabbits with meningitis. We demonstrated that dexamethasone actually hindered blood-brain barrier penetration by antibiotics, reducing the effectiveness of antibiotic treatment of anthrax meningitis in this rabbit model.

Gastric pH and Toxin Factors Modulate Infectivity and Disease Progression After Gastrointestinal Exposure to Bacillus anthracis.

Gastrointestinal (GI) anthrax is the most prevalent form of naturally acquired Bacillus anthracis infection, which is associated with exposure to vegetative bacteria in infected meat (carnivores) or to fermented rumen contents (herbivores). We assessed whether key host and pathogen factors modulate infectivity and progression of infection using a mouse model of GI infection. Gastric acid neutralization increases infectivity, but 30%-40% of mice succumb to infection without neutralization. Mice either fed or fasted before exposure showed similar infectivity rates. Finally, the pathogen's anthrax lethal factor is required to establish lethal infection, whereas its edema factor modulates progression and dissemination of infection.

Epidemiology of Human Anthrax in China, 1955-2014.

Using national surveillance data for 120,111 human anthrax cases recorded during 1955-2014, we analyzed the temporal, seasonal, geographic, and demographic distribution of this disease in China. After 1978, incidence decreased until 2013, when it reached a low of 0.014 cases/100,000 population. The case-fatality rate, cumulatively 3.6% during the study period, has also decreased since 1990. Cases occurred throughout the year, peaking in August. Geographic distribution decreased overall from west to east, but the cumulative number of affected counties increased during 2005-2014. The disease has shifted from industrial to agricultural workers; 86.7% of cases occurred in farmers and herdsmen. Most (97.7%) reported cases were the cutaneous form. Although progress has been made in reducing incidence, this study highlights areas that need improvement. Adequate laboratory diagnosis is lacking; only 7.6% of cases received laboratory confirmation. Geographic expansion of the disease indicates that livestock control programs will be essential in eradicating anthrax.

S-nitrosylation of peroxiredoxin 1 contributes to viability of lung epithelial cells during Bacillus anthracis infection.

BACKGROUND: Using Bacillus anthracis as a model gram-positive bacterium, we investigated the effects of host protein S-nitrosylation during bacterial infection. B. anthracis possesses a bacterial nitric oxide synthase (bNOS) that is important for its virulence and survival. However, the role of S-nitrosylation of host cell proteins during B. anthracis infection has not been determined. METHODS: Nitrosoproteomic analysis of human small airway epithelial cells (HSAECs) infected with toxigenic B. anthracis Sterne was performed, identifying peroxiredoxin 1 (Prx1) as one predominant target. Peroxidase activity of Prx during infection was measured using 2-Cys-Peroxiredoxin activity assay. Chaperone activity of S-nitrosylated Prx1 was measured by insulin aggregation assay, and analysis of formation of multimeric species using Native PAGE. Griess assay and DAF-2DA fluorescence assay were used to measure NO production. Cell viability was measured using the Alamar Blue assay and the ATPlite assay (Perkin Elmer). RESULTS: S-nitrosylation of Prx1 in Sterne-infected HSAECs leads to a decrease in its peroxidase activity while enhancing its chaperone function. Treatment with bNOS inhibitor, or infection with bNOS deletion strain, reduces S-nitrosylation of Prx1 and decreases host cell survival. Consistent with this, siRNA knockdown of Prx1 lowers bNOS-dependent protection of HSAEC viability. CONCLUSIONS: Anthrax infection results in S-nitrosylation of multiple host proteins, including Prx1. The nitrosylation-dependent decrease in peroxidase activity of Prx1 and increase in its chaperone activity is one factor contributing to enhancing infected cell viability. GENERAL SIGNIFICANCE: These results provide a new venue of mechanistic investigation for inhalational anthrax that could lead to novel and potentially effective countermeasures.