Antibacterial role for natural killer cells in host defense to Bacillus anthracis.

Natural killer (NK) cells have innate antibacterial activity that could be targeted for clinical interventions for infectious disease caused by naturally occurring or weaponized bacterial pathogens. To determine a potential role for NK cells in immunity to Bacillus anthracis, we utilized primary human and murine NK cells, in vitro assays, and in vivo NK cell depletion in a murine model of inhalational anthrax. Our results demonstrate potent antibacterial activity by human NK cells against B. anthracis bacilli within infected autologous monocytes. Surprisingly, NK cells also mediate moderate antibacterial effects on extracellular vegetative bacilli but do not have activity against extracellular or intracellular spores. The immunosuppressive anthrax lethal toxin impairs NK gamma interferon (IFN-γ) expression, but neither lethal nor edema toxin significantly alters the viability or cytotoxic effector function of NK cells. Compared to human NK cells, murine NK cells have a similar, though less potent, activity against intracellular and extracellular B. anthracis. The in vivo depletion of murine NK cells does not alter animal survival following intranasal infection with B. anthracis spores in our studies but significantly increases the bacterial load in the blood of infected animals. Our studies demonstrate that NK cells participate in the innate immune response against B. anthracis and suggest that immune modulation to augment NK cell function in early stages of anthrax should be further explored in animal models as a clinical intervention strategy.

Testing the efficacy and toxicity of adenylyl cyclase inhibitors against enteric pathogens using in vitro and in vivo models of infection.

Enterotoxigenic Escherichia coli (ETEC) produces the ADP-ribosyltransferase toxin known as heat-labile enterotoxin (LT). In addition to the toxic effect of LT resulting in increases of cyclic AMP (cAMP) and disturbance of cellular metabolic processes, this toxin promotes bacterial adherence to intestinal epithelial cells (A. M. Johnson, R. S. Kaushik, D. H. Francis, J. M. Fleckenstein, and P. R. Hardwidge, J. Bacteriol. 191:178-186, 2009). Therefore, we hypothesized that the identification of a compound that inhibits the activity of the toxin would have a suppressive effect on the ETEC colonization capabilities. Using in vivo and in vitro approaches, we present evidence demonstrating that a fluorenone-based compound, DC5, which inhibits the accumulation of cAMP in intoxicated cultured cells, significantly decreases the colonization abilities of adenylyl cyclase toxin-producing bacteria, such as ETEC. These findings established that DC5 is a potent inhibitor both of toxin-induced cAMP accumulation and of ETEC adherence to epithelial cells. Thus, DC5 may be a promising compound for treatment of diarrhea caused by ETEC and other adenylyl cyclase toxin-producing bacteria.

Comparative Global Gene Expression Profiles of Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant at Flea and Human Body Temperatures.

Braun/murein lipoprotein (Lpp) is involved in inflammatory responses and septic shock. We previously characterized a Deltalpp mutant of Yersinia pestis CO92 and found that this mutant was defective in surviving in macrophages and was attenuated in a mouse inhalation model of plague when compared to the highly virulent wild-type (WT) bacterium. We performed global transcriptional profiling of WT Y. pestis and its Deltalpp mutant using microarrays. The organisms were cultured at 26 and 37 degrees Celsius to simulate the flea vector and mammalian host environments, respectively. Our data revealed vastly different effects of lpp mutation on the transcriptomes of Y. pestis grown at 37 versus 26 degrees C. While the absence of Lpp resulted mainly in the downregulation of metabolic genes at 26 degrees C, the Y. pestis Deltalpp mutant cultured at 37 degrees C exhibited profound alterations in stress response and virulence genes, compared to WT bacteria. We investigated one of the stress-related genes (htrA) downregulated in the Deltalpp mutant relative to WT Y. pestis. Indeed, complementation of the Deltalpp mutant with the htrA gene restored intracellular survival of the Y. pestis Deltalpp mutant. Our results support a role for Lpp in Y. pestis adaptation to the host environment, possibly via transcriptional activation of htrA.

Comparative Analyses of Transcriptional Profiles in Mouse Organs Using a Pneumonic Plague Model after Infection with Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant.

We employed Murine GeneChips to delineate the global transcriptional profiles of the livers, lungs, and spleens in a mouse pneumonic plague infection model with wild-type (WT) Y. pestis CO92 and its Braun lipoprotein (Deltalpp) mutant with reduced virulence. These organs showed differential transcriptional responses to infection with WT Y. pestis, but the overall host functional processes affected were similar across all three tissues. Gene expression alterations were found in inflammation, cytokine signaling, and apoptotic cell death-associated genes. Comparison of WT and Deltalpp mutant-infected mice indicated significant overlap in lipopolysaccharide- (LPS-) associated gene expression, but the absence of Lpp perturbed host cell signaling at critical regulatory junctions resulting in altered immune response and possibly host cell apoptosis. We generated a putative signaling pathway including major inflammatory components that could account for the synergistic action of LPS and Lpp and provided the mechanistic basis of attenuation caused by deletion of the lpp gene from Y. pestis in a mouse model of pneumonic plague.

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections.

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 µL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. This platform has great potential in both medical diagnostics and research applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.

Centrifugal sedimentation immunoassays for multiplexed detection of enteric bacteria in ground water.

Waterborne pathogens pose significant threat to the global population and early detection plays an important role both in making drinking water safe, as well as in diagnostics and treatment of water-borne diseases. We present an innovative centrifugal sedimentation immunoassay platform for detection of bacterial pathogens in water. Our approach is based on binding of pathogens to antibody-functionalized capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk. Beads at the distal end of the disk are imaged to quantify the fluorescence and determine the bacterial concentration. Our platform is fast (20 min), can detect as few as ∼10 bacteria with minimal sample preparation, and can detect multiple pathogens simultaneously. The platform was used to detect a panel of enteric bacteria (Escherichia coli, Salmonella typhimurium, Shigella, Listeria, and Campylobacter) spiked in tap and ground water samples.

Telemetric left ventricular monitoring using wireless telemetry in the rabbit model.

BACKGROUND: Heart failure is a critical condition that affects many people and often results from left ventricular dysfunction. Numerous studies investigating this condition have been performed using various model systems. To do so, investigators must be able to accurately measure myocardial performance in order to determine the degree of left ventricular function. In this model development study, we employ a wireless telemetry system purchased from Data Sciences International to continuously assess left ventricular function in the rabbit model. FINDINGS: We surgically implanted pressure-sensitive catheters fitted to wireless radio-transmitters into the left ventricle of Dutch-belted rabbits. Following recovery of the animals, we continuously recorded indices of cardiac contractility and ventricular relaxation at baseline for a given time period. The telemetry system allowed us to continuously record baseline left ventricular parameters for the entire recording period. During this time, the animals were unrestrained and fully conscious. The values we recorded are similar to those obtained using other reported methods. CONCLUSIONS: The wireless telemetry system can continuously measure left ventricular pressure, cardiac contractility, and cardiac relaxation in the rabbit model. These results, which were obtained just as baseline levels, substantiate the need for further validation in this model system of left ventricular assessment.

Hemodynamic effects of anthrax toxins in the rabbit model and the cardiac pathology induced by lethal toxin.

Anthrax lethal toxin (LeTx) and edema toxin (EdTx) have been shown to alter hemodynamics in the rodent model, while LeTx primarily is reported to induce extensive tissue pathology. However, the rodent model has limitations when used for comparison to higher organisms such as humans. The rabbit model, on the other hand, has gained recognition as a useful model for studying anthrax infection and its pathophysiological effects. In this study, we assessed the hemodynamic effects of lethal toxin (LeTx) and edema toxin (EdTx) in the rabbit model using physiologically relevant amounts of the toxins. Moreover, we further examine the pathological effects of LeTx on cardiac tissue. We intravenously injected Dutch-belted rabbits with either low-dose and high-dose recombinant LeTx or a single dose of EdTx. The animals' heart rate and mean arterial pressure were continuously monitored via telemetry until either 48 or 72 h post-challenge. Additional animals challenged with LeTx were used for cardiac troponin I (cTnI) quantitation, cardiac histopathology, and echocardiography. LeTx depressed heart rate at the lower dose and mean arterial pressure (MAP) at the higher dose. EdTx, on the other hand, temporarily intensified heart rate while lowering MAP. Both doses of LeTx caused cardiac pathology with the higher dose having a more profound effect. Lastly, left-ventricular dilation due to LeTx was not apparent at the given time-points. Our study demonstrates the hemodynamic effects of anthrax toxins, as well as the pathological effects of LeTx on the heart in the rabbit model, and it provides further evidence for the toxins' direct impact on the heart.

Emergence of anthrax edema toxin as a master manipulator of macrophage and B cell functions.

Anthrax edema toxin (ET), a powerful adenylyl cyclase, is an important virulence factor of Bacillus anthracis. Until recently, only a modest amount of research was performed to understand the role this toxin plays in the organism's immune evasion strategy. A new wave of studies have begun to elucidate the effects this toxin has on a variety of host cells. While efforts have been made to illuminate the effect ET has on cells of the adaptive immune system, such as T cells, the greatest focus has been on cells of the innate immune system, particularly the macrophage. Here we discuss the immunoevasive activities that ET exerts on macrophages, as well as new research on the effects of this toxin on B cells.

Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis.

Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 µg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities.

Recombinant Sindbis virus vectors designed to express protective antigen of Bacillus anthracis protect animals from anthrax and display synergy with ciprofloxacin.

Recombinant Sindbis viruses were engineered to express alternative forms of the protective antigen (PA) of Bacillus anthracis. The recombinant viruses induced PA-specific immunoglobulin G and neutralizing antibodies in Swiss Webster mice. Vaccination with the recombinant viruses induced immunity that offered some protection from a lethal Ames strain spore challenge and synergized the protective effects of ciprofloxacin.

Levofloxacin rescues mice from lethal intra-nasal infections with virulent Francisella tularensis and induces immunity and production of protective antibody.

The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with F. tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72h post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to re-challenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4-specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis.

Transcriptional profiling of murine organ genes in response to infection with Bacillus anthracis Ames spores.

Bacillus anthracis is the Gram-positive, spore-forming etiological agent of anthrax, an affliction studied because of its importance as a potential bioweapon. Although in vitro transcriptional responses of macrophages to either spore or anthrax toxins have been previously reported, little is known regarding the impact of infection on gene expression in host tissues. We infected Swiss-Webster mice intranasally with 5 LD(50) of B. anthracis-virulent Ames spores and observed the global transcriptional profiles of various tissues over a 48 h time period. RNA was extracted from spleen, lung, and heart tissues of infected and control mice and examined by Affymetrix GeneChip analysis. Approximately 580 host genes were significantly over or under expressed among the lung, spleen, and heart tissues at 8 and 48 h time points. Expression of genes encoding for surfactant and major histocompatibility complex (MHC) presentation was diminished during the early phase of infection in lungs. By 48 h, a significant number of genes were modulated in the heart, including up-regulation of calcium-binding-related gene expression, and down-regulation of multiple genes related to cell adhesion, formation of the extracellular matrix, and the cell cytoskeleton. Interestingly, the spleen 8h post-infection showed striking increases in the expression of genes that encode hydrolytic enzymes, and these levels remained elevated throughout infection. Further, genes involving antigen presentation and interferon responses were down-regulated in the spleen at 8 h. In late stages of infection, splenic genes related to the inflammatory response were up-regulated. This study is the first to describe the in vivo global transcriptional response of multiple organs during inhalational anthrax. Although numerous genes related to the host immunological response and certain protection mechanisms were up-regulated in these organs, a vast list of genes important for fully developing and maintaining this response were decreased. Additionally, the lung, spleen, and heart showed differential responses to the infection, further validating the demand for a better understanding of anthrax pathogenesis in order to design therapies against novel targets.