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Polymorphonuclear neutrophils (PMNs)-derived ROS are involved in the regulation of multiple functions of PMNs critical in both inflammation and its timely resolution. Selenium is an essential trace element that functions as a gatekeeper of cellular redox homeostasis in the form of selenoproteins. Despite their well-studied involvement in regulating functions of various immune cells, limited studies have focused on the regulation of selenoproteins in PMN and their associated functions. Ex-vivo treatment of murine primary bone marrow derived PMNs with bacterial endotoxin lipopolysaccharide (LPS) indicated temporal regulation of several selenoprotein genes at the mRNA level. However, only glutathione peroxidase 4 (Gpx4) was significantly upregulated, while Selenof, Selenow, and Gpx1 were significantly downregulated in a temporal manner at the protein level. Exposure of PMNs isolated from tRNA

Plasma medicine is a rapidly expanding field that utilizes nonequilibrium plasma discharges at atmospheric conditions or in liquids for clinical applications. There is a significant interest in the production of plasma in the liquid phase for wastewater treatment, agricultural applications, and medical purposes. However, little investigation has been done about the effects of dielectric coatings on submerged electrodes, which is of significant interest to limit electrical current flow in the liquid. This article investigates the effect of different dielectric coatings, including aluminum oxide, parylene C, and bi-layer combinations, on plasma discharge characteristics in phosphate-buffered saline ( $\mathbf {\sigma = 18}$ mS/cm) from nanosecond high-voltage pulses. Observed results for aluminum oxide are consistent with past works, including micron-sized clusters of holes generated in the layer due to dielectric breakdown. A bi-layer combination of parylene C on top of aluminum oxide resulted in a longer lifetime for electrodes, possibly due to the melting/solidification behavior of the polymer, which may have a “healing” effect. The use of a thick parylene C layer resulted in a different, “creeping,” discharge regime, which is hypothesized to be similar to triple-gap discharge observed in space plasma physics and high-voltage insulators, in which the electric field is enhanced at the boundary of a conductor, dielectric, and a vacuum/fluid, resulting in discharge at this junction point. Temporally-resolved and high-spatial-resolution imaging are required for verification.

Coronavirus disease 2019 (Covid-19) has caused over 5.5 million deaths worldwide, and viral mutants continue to ravage communities with limited access to injectable vaccines or high rates of vaccine hesitancy. Inhalable vaccines have the potential to address these distribution and compliance issues as they are less likely to require cold storage, avoid the use of needles, and can elicit localized immune responses with only a single dose. Alveolar macrophages represent attractive targets for inhalable vaccines as they are abundant within the lung mucosa (up to 95% of all immune cells) and are important mediators of mucosal immunity, and evidence suggests that they may be key cellular players in early Covid-19 pathogenesis. Here, we report inhalable coronavirus mimetic particles (CoMiP) designed to rapidly bind to, and be internalized by, alveolar macrophages to deliver nucleic acid-encoded viral antigens. Inspired by the SARS-CoV-2 virion structure, CoMiP carriers package nucleic acid cargo within an endosomolytic peptide envelope that is wrapped in a macrophage-targeting glycosaminoglycan coating. Through this design, CoMiP mimic several important features of the SARS-CoV-2 virion, particularly surface topography and macromolecular chemistry. As a result, CoMiP effect pleiotropic transfection of macrophages and lung epithelial cells in vitro with multiple antigen-encoding plasmids. In vivo immunization yields increased mucosal IgA levels within the respiratory tract of CoMiP vaccinated mice.

Despite nearly a century of clinical use as a blood thinner, heparin's rapid serum clearance and potential to induce severe bleeding events continue to urge the development of more effective controlled delivery strategies. Subcutaneous depots that steadily release the anticoagulant into circulation represent a promising approach to reducing overdose frequency, sustaining therapeutic concentrations of heparin in plasma, and prolonging anticoagulant activity in a safe and effective manner. Subcutaneously deliverable heparin-peptide nanogranules that allow for long-lasting heparin bioavailability in the circulatory system, while enabling on-demand activation of heparin's anticoagulant effects in the thrombus microenvironment, are reported. Biophysical studies demonstrate this responsive behavior is due to the sequestration of heparin within self-assembling peptide nanofibrils and its mechanically actuated decoupling to elicit antithrombotic effects at the clotting site. In vivo studies show these unique properties converge to allow subcutaneous nanogranule depots to extend heparin serum concentrations for an order of magnitude longer than standard dosing regimens while enabling prolonged and controlled anticoagulant activity. This biohybrid delivery system demonstrates a potentially scalable platform for the development of safer, easier to administer, and more effective antithrombotic nanotechnologies.

Enterobactin (Ent), a prototypical bacterial siderophore known for its unparalleled affinity for iron, is widely conserved among members of the Enterobacteriaceae family of Gram-negative bacteria. ...

Francisella tularensis (Ft) is a Gram-negative, facultative intracellular coccobacillus that is the etiological agent of tularemia. Interestingly, the disease tularemia has variable clinical presentations that are dependent upon the route of infection with Ft. Two of the most likely routes of Ft infection include intranasal and intradermal, which result in pneumonic and ulceroglandular tularemia, respectively. While there are several differences between these two forms of tularemia, the most notable disparity is between mortality rates: the mortality rate following pneumonic tularemia is over ten times that of the ulceroglandular disease. Understanding the differences between intradermal and intranasal Ft infections is important not only for clinical diagnoses and treatment but also for the development of a safe and effective vaccine. However, the immune correlates of protection against Ft, especially within the context of infection by disparate routes, are not yet fully understood. Recent advances in different animal models have revealed new insights in the complex interplay of innate and adaptive immune responses, indicating dissimilar patterns in both responses following infection with Ft via different routes. Further investigation of these differences will be crucial to predicting disease outcomes and inducing protective immunity via vaccination or natural infection.

To determine the clinical utility of preoperative urine cultures in asymptomatic men undergoing prostate needle biopsy (PNB). One hundred fifty asymptomatic men had urine cultures obtained 14-days prior to PNB. As per study protocol, positive cultures were not treated. Antibiotic prophylaxis prior to PNB included ciprofloxacin 500 mg the night before and morning of the biopsy. Repeat urine cultures were obtained immediately prior to PNB with colony-forming units (CFUs) annotated. Infectious complications post-biopsy were recorded. Of the 150 men, six patients (4%) had evidence of asymptomatic bacteriuria with > 10,000 CFU/mL on office urine culture. Repeat urine cultures on morning of biopsy in all 150 patients noted a mean bacterial count of 55 CFU/mL (range 0–1000). All six patients with positive office urine cultures had

We report that IgA−/− mice exhibit specific defects in IgG antibody responses to various polysaccharide vaccines (Francisella tularensis LPS and Pneumovax), but not protein vaccines such as Fluzone. This defect further included responses to polysaccharide-protein conjugate vaccines (Prevnar and Haemophilus influenzae type b-tetanus toxoid vaccine). In agreement with these findings, IgA−/− mice were protected from pathogen challenge with protein- but not polysaccharide-based vaccines. Interestingly, after immunization with live bacteria, IgA+/+ and IgA−/− mice were both resistant to lethal challenge and their IgG anti-polysaccharide antibody responses were comparable. Immunization with live bacteria, but not purified polysaccharide, induced production of serum B cell-activating factor (BAFF), a cytokine important for IgG class switching; supplementing IgA−/− cell cultures with BAFF enhanced in vitro polyclonal IgG production. Taken together, these findings show that IgA deficiency impairs IgG class switching following vaccination with polysaccharide antigens and that live bacterial immunization can overcome this defect. Since IgA deficient patients also often show defects in antibody responses following immunization with polysaccharide vaccines, our findings could have relevance to the clinical management of this population.

Emergence and spread of antibiotic resistance calls for development of non-chemical treatment options for bacterial infections. Plasma medicine applies low-temperature plasma (LTP) physics to address biomedical problems such as wound healing and tumor suppression. LTP has also been used for surface disinfection. However, there is still much to be learned regarding the effectiveness of LTP on bacteria in suspension in liquids, and especially on porous surfaces. We investigated the efficacy of LTP treatments against bacteria using an atmospheric-pressure plasma jet and show that LTP treatments have the ability to inhibit both gram-positive (S. aureus) and gram-negative (E. coli) bacteria on solid and porous surfaces. Additionally, both direct LTP treatment and plasma-activated media were effective against the bacteria suspended in liquid culture. Our data indicate that reactive oxygen species are the key mediators of the bactericidal effects of LTP and hydrogen peroxide is necessary but not sufficient for antibacterial effects. In addition, our data suggests that bacteria exposed to LTP do not develop resistance to further treatment with LTP. These findings suggest that this novel atmospheric-pressure plasma jet could be used as a potential alternative to antibiotic treatments in vivo.

The trace element selenium is an essential micronutrient that plays an important role in maintaining homeostasis of several tissues including the immune system of mammals. The vast majority of the biological functions of selenium are mediated via selenoproteins, proteins which incorporate the selenium-containing amino acid selenocysteine. Several bacterial infections of humans and animals are associated with decreased levels of selenium in the blood and an adjunct therapy with selenium often leads to favorable outcomes. Many pathogenic bacteria are also capable of synthesizing selenocysteine suggesting that selenoproteins may have a role in bacterial physiology. Interestingly, the composition of host microbiota is also regulated by dietary selenium levels. Therefore, bacterial pathogens, microbiome, and host immune cells may be competing for a limited supply of selenium. Elucidating how selenium, in particular selenoproteins, may regulate pathogen virulence, microbiome diversity, and host immune response during a bacterial infection is critical for clinical management of infectious diseases.

Bacterial ribosomes frequently translate to the 3’ end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these non-stop ribosomes are rescued usingtrans-translation. In some species, genes required fortrans-translation are essential, but other species can survive withouttrans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB.Francisella tularensiscells lackingtrans-translation are viable, butF. tularensisdoes not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT.arfTcan be deleted in wild-type cells but not in cells that lacktrans-translation activity. Over-expression of ArfT suppresses the slow growth phenotype in cells lackingtrans-translation and counteracts growth arrest caused bytrans-translation inhibitors, indicating that ArfT rescues non-stop ribosomesin vivo. Ribosome rescue assaysin vitroshow that ArfT promotes hydrolysis of peptidyl-tRNA on non-stop ribosomes in conjunction withF. tularensisrelease factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB.ImportanceFrancisella tularensisis a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated.F. tularensishas also been classified as a potential bioterrorism agent with the greatest risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity againstF. tularensisand other important pathogens. Like all bacteria that have been studied,F. tularensisusestrans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria,F. tularensiscan survive without any of the known factors for ribosome rescue. Our work identifies aF. tularensisprotein, ArfT, that rescues stalled ribosomes in the absence oftrans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential inF. tularensisand suggest that ribosome rescue activity might be essential in all bacteria.

Bacteria require at least one pathway to rescue ribosomes stalled at the ends of mRNAs. The primary pathway for ribosome rescue is trans -translation, which is conserved in >99% of sequenced bacterial genomes. Some species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient trans -translation activity. Small-molecule inhibitors of ribosome rescue have broad-spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against the tier 1 select agent Francisella tularensis to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL-10 and KKL-40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 or KKL-40 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis , addition of KKL-10 or KKL-40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis . Neither KKL-10 nor KKL-40 was cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for F. tularensis growth at all stages of its infection cycle and suggest that KKL-10 and KKL-40 are good lead compounds for antibiotic development.

Tularemia is an endemic zoonotic disease in many parts of the world including Asia. A cross-sectional study was conducted to determine genome-based prevalence of Francisella tularensis (Ft) in soil, assess an association between its occurrence in soil and likely predictors i.e., macro and micro-nutrients and several categorical variables, and determine seroconversion in small and large ruminants. The study included a total of 2,280 soil samples representing 456 villages in eight districts of the Punjab Province of Pakistan followed by an analysis of serum antibodies in 707 ruminants. The genome of Ft was detected in 3.25% (n = 74, 95% CI: 2.60–4.06) of soil samples. Soluble salts (OR: 1.276, 95% CI: 1.043–1.562, p = 0.015), Ni (OR: 2.910, 95%CI: 0.795–10.644, p = 0.106), Mn (OR:0.733, 95% CI:0.565–0.951, p = 0.019), Zn (OR: 4.922, 95% CI:0.929–26.064, p = 0.061) and nutrients clustered together as PC-1 (OR: 4.76, 95% CI: 2.37–9.54, p = 0.000) and PC-3 (OR: 0.357, 95% CI: 0.640, p = 0.001) were found to have a positive association for the presence of Ft in soil. The odds of occurrence of Ft DNA in soil were higher at locations close to a water source, including canals, streams or drains, [χ2 = 6.7, OR = 1.19, 95% CI:1.05–3.09, p = 0.004] as well as places where animals were present [χ2 = 4.09, OR = 2.06, 95% CI: 1.05–4.05, p = 0.02]. The seroconversion was detected in 6.22% (n = 44, 95% CI: 4.67–8.25) of domestic animals. An occurrence of Ft over a wide geographical region indicates its expansion to enzootic range, and demonstrates the need for further investigation among potential disease reservoirs and at-risk populations, such as farmers and veterinarians.

To report outcomes 5 years after a resident quality initiative incorporated topical rectal antiseptic into our ultrasound-guided prostate needle biopsy (TRUS PNB) protocol. A chart review was conducted on 1007 men who underwent TRUS PNB between 2010 and 2017. Comparison groups include those who received a topical rectal antiseptic (N = 437) compared to those who did not (N = 570). Povidone-iodine (N = 303) or 4% chlorhexidine solution without alcohol (N = 134) were topical agents. Outcomes of interest included post-biopsy infection (urinary tract infection and/or sepsis), hospital admission, and need for ICU monitoring. Median age and PSA of men included in this study were 64 years and 12 ng/mL. Almost 90% of patients were Caucasian, 13% had diabetes, 3% were on immunosuppression, 32% had at least one prior biopsy, 14% received antibiotics, and 7% were hospitalized in the past 6 months. 22 patients (2.2%) developed a post-biopsy infection with a significant reduction in the group receiving topical rectal antiseptic (0.8 vs. 3.3%, p = 0.01). Post-biopsy UTI rates (p = 0.04) and hospital admission (p = 0.03) were also lower in the topical antiseptic group with trends to reduction in sepsis and need for ICU monitoring. What started as a resident quality safety project 5 years ago has demonstrated a reduction in infections and hospital admissions following TRUS PNB. Our institutional practice now routinely uses povidone-iodine or chlorhexidine as an adjunct to oral quinolones for TRUS PNB perioperative prophylaxis.

Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to generate this knowledge. CD8+ T cells are essential for protective immunity against virulent strains of Francisella tularensis, but to-date, it has not been possible to study these cells in an antigen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) in F. tularensis, which allows for the study of CD8+ T cell responses to the bacterium. We demonstrate that in response to intranasal infection with the F. tularensis Live Vaccine Strain, adoptively transferred OVA-specific CD8+ T cells expand after the first week and produce IFN-γ but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably, OVA-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more antigen-specific CD8+ T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8+ T cell response to F. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.

Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to generate this knowledge. CD8+T cells are essential for protective immunity against virulent strains ofFrancisella tularensis, but to-date, it has not been possible to study these cells in a pathogen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) inF. tularensis, which allows for the study of CD8+T cell responses to the bacterium. We demonstrate that in response to intranasal infection with theF. tularensisLive Vaccine Strain, pathogen-specific CD8+T cells expand after the first week and produce IFN-γ but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably,F. tularensis-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more pathogen-specific CD8+T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8+T cell response toF. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.

Francisella tularensis subsp. holarctica is found in North America and much of Europe and causes the disease tularemia in humans and animals. An aquatic cycle has been described for this subspecies, which has caused waterborne outbreaks of tularemia in at least 10 countries. In this study, we sought to identify the mechanosensitive channel(s) required for the bacterium to survive the transition from mammalian hosts to freshwater, which is likely essential for the transmission of the bacterium between susceptible hosts. A single 165-amino-acid MscS-type mechanosensitive channel ( Ft MscS) was found to protect F. tularensis subsp. holarctica from hypoosmotic shock, despite lacking much of the cytoplasmic vestibule domain found in well-characterized MscS proteins from other organisms. The deletion of this channel did not affect virulence within the mammalian host; however, Ft MscS was required to survive the transition from the host niche to freshwater. The deletion of Ft MscS did not alter the sensitivity of F. tularensis subsp. holarctica to detergents, H 2 O 2 , or antibiotics, suggesting that the role of Ft MscS is specific to protection from hypoosmotic shock. The deletion of Ft MscS also led to a reduced average cell size without altering gross cell morphology. The mechanosensitive channel identified and characterized in this study likely contributes to the transmission of tularemia between hosts by allowing the bacterium to survive the transition from mammalian hosts to freshwater. IMPORTANCE The contamination of freshwater by Francisella tularensis subsp. holarctica has resulted in a number of outbreaks of tularemia. Invariably, the contamination originates from the carcasses or excreta of infected animals and thus involves an abrupt osmotic downshock as the bacteria enter freshwater. How F. tularensis survives this drastic change in osmolarity has not been clear, but here we report that a single mechanosensitive channel protects the bacterium from osmotic downshock. This channel is functional despite lacking much of the cytoplasmic vestibule domain that is present in better-studied organisms such as Escherichia coli ; this report builds on previous studies that have suggested that parts of this domain are dispensable for downshock protection. These findings extend our understanding of the aquatic cycle and ecological persistence of F. tularensis , with further implications for mechanosensitive channel biology.

The purpose of the study was to evaluate whether a peri-procedural povidone-iodine rectal preparation (PIRP) prior to transrectal ultrasound-guided prostate needle biopsy (TRUS PNB) can reduce microorganism colony counts and infectious complications. Our institutional TRUS PNB database was reviewed to identify infectious post-biopsy complications (defined as fever >38.5 °C with positive culture). The last 570 biopsy patients were divided into those administered only preoperative oral and/or parenteral antibiotics (n = 456; chronologically cohorts A–D) versus men receiving peri-procedural PIRP in conjunction with standard preoperative antibiotics (n = 114; cohort E). Rectal cultures were obtained in the PIRP cohort to quantify changes in microorganism colony counts. Mean baseline PSA for patients was 11.6 ng/ml, 63 % were undergoing an initial biopsy, and 17 % had documented use of antibiotic therapy within the previous 6 months. A reduction in infectious complications was observed when comparing the conventional antibiotic (cohorts A–D) versus PIRP (cohort E) group (1.8 vs. 0 %), with the largest magnitude of decline occurring in the concurrent contemporary cohorts (cohort D—5.3 % vs. cohort E—0 %, p = 0.03). Rectal cultures obtained in 92 men before and after PIRP administration noted a 97 % reduction in microorganism colonies (2.1 × 105 vs. 6.3 × 103 CFU/ml, p

OBJECTIVES: Selenium (Se), which exerts its effects via selenoproteins, is an essential micronutrient that has been shown to be required for optimal functioning of the immune system. Although numerous studies have examined the effects of selenoproteins on various aspects of the immune response, few studies have investigated the effects of Se on antigen presentation. Moreover, these studies have provided conflicting results indicating the need for further research regarding the effect of Se on Antigen Presenting Cells (APCs). Our goal is to identify the role of selenoproteins in antigen presentation in professional APCs. METHODS: Mice deficient in selenoproteins in a cell specific manner were generated by knocking out the trspgene, which encodes for an essential tRNA for selenocysteine synthesis, utilizing the cre/lox system. The resulting mice are referred to asTrsp(DC)and Trsp(B)representing selenoprotein deficiency specifically in dendritic cells (DCs) and B cells, respectively. Additional studies were done utilizing a diet model of Se deficiency to isolate Se-deficient DCs and B cells. Rate of endocytosis by specific receptors and expression of various surface molecule characteristics of APCs were determined by flow cytometry. Degradation of antigens by APCs in the presence or absence of selenoproteins was measured by western blot. RESULTS: In B cells, selenoprotein deficiency results in a decrease in the number of B220 + Splenocytes, B Cell Receptor (BCR) endocytosis, calcium flux following antigen stimulation, and BCR degradation following internalization. Similarly, selenoprotein deficiency in DCs results in decreased DEC-205 (a collectin-like receptor for multiple antigens) endocytosis; and a reduction in the expression of MHCII, CD80, and CD86. These results suggest a role for selenoproteins during the processing and presentation of peptides to T cells. CONCLUSIONS: Loss of selenoproteins results in the decreased functionality of both B cells and DCs, which suggests that Se and selenoproteins may be involved in the regulation of antigen presentation. Further studies are needed to elucidate the underlying mechanism by which selenoproteins regulate antigen presentation. Such studies are likely to reveal novel targets for enhancing the immune response to vaccines and other specific antigens. FUNDING SOURCES: NIH/NIAID to GK; NIH/GM to SS; NIH/NIDDK to KSP.

Francisella tularensis , the causative agent of tularemia, is most deadly in the pneumonic form; therefore, mucosal immunity is an important first line of defense against this pathogen. We have now evaluated the lethality of primary F. tularensis live vaccine strain (LVS) pulmonary infection in mice that are defective in IgA (IgA −/− mice), the predominant mucosal Ig isotype. The results showed that IgA −/− mice were more susceptible than IgA +/+ mice to intranasal F. tularensis LVS infection, despite developing higher levels of LVS-specific total, IgG, and IgM antibodies in the bronchoalveolar lavage specimens following infection. In addition, the absence of IgA resulted in a significant increase in bacterial loads and reduced survival. Interestingly, IgA −/− mice had lower pulmonary gamma interferon (IFN-γ) levels and decreased numbers of IFN-γ-secreting CD4 + and CD8 + T cells in the lung on day 9 postinfection compared to IgA +/+ mice. Furthermore, IgA −/− mice displayed reduced interleukin 12 (IL-12) levels at early time points, and supplementing IgA −/− mice with IL-12 prior to LVS challenge induced IFN-γ production by NK cells and rescued them from mortality. Thus, IgA −/− mice are highly susceptible to primary pulmonary LVS infections not only because of IgA deficiency but also because of reduced IFN-γ responses.

INTRODUCTION AND OBJECTIVES: Infectious complications following transrectal prostate needle biopsy (PNB) continue to rise. These infections are thought to occur from translocation of bacteria in the rectal vault into the highly vascular prostate. The contribution (if any) of pre-biopsy bacteriuria on infectious sequelae following the procedure remains unclear. We investigate the utility of pre-operative urine culture in a cohort of patients undergoing PNB. METHODS: Between March 2013 and August 2015, 150 asymptomatic male patients were prospectively enrolled in an IRB approved study investigating infections following PNB. Routine office urine cultures were obtained 14-days prior to PNB with reports of 10,000 flora or greater recorded. Per study protocol, positive cultures were not treated. Antibiotic prophylaxis regimen prior to PNB included Ciprofloxacin 500mg the day before and morning of the biopsy. Urine cultures were obtained immediately prior to PNB with plating onto MuellerHinton agar plates with colony forming units (CFUs) determined after incubation at 37 C for 48 hours. Infectious complications post-biopsy were recorded. RESULTS: 150 men with a mean PSA of 11.4 ng/mL were included. 64% were undergoing an initial biopsy and 19% had a history of antibiotic use within the previous 6 months. Six patients (4%) had evidence of asymptomatic bacteriuria on office urine culture including 2 with > 100,000 CFU/mL E.Coli, 1 with > 100,000 CFU/mL Klebsiella, 1 with 10,000 CFU/mL E.Coli, and 2 with 10,000 CFU/mL mixed flora. Just prior to PNB, repeat urine cultures in all 150 patients noted a mean bacterial count of 55 CFU/mL with the highest urine bacterial count being 1000 CFU/mL. All 6 patients with positive office urine cultures had < 100 CFU/mL just prior to PNB. Following PNB, 4 patients (2.7%) developed an infectious complication including 2 with sepsis and 2 with culture positive UTIs. The causative organism in all cases was quinolone resistant E. Coli. None of the 6 patients with preoperative positive urine cultures developed an infectious complication following PNB. CONCLUSIONS: In this prospective study of 150 patients, < 5% had positive office cultures prior to PNB. Repeat urine culture on the morning of biopsy showed resolution in these patients and none developed post-biopsy infectious complications. Routine office urine culture in the asymptomatic male prior to PNB is likely unnecessary

Inhalation of Yersinia pestis causes pneumonic plague, which rapidly progresses to death. A previously licensed killed whole-cell vaccine is presently unavailable due to its reactogenicity and inconclusive evidence of efficacy. The present study now shows that vaccination intranasally (i.n.) with inactivated Y. pestis CO92 (iYp) adjuvanted with interleukin-12 (IL-12) followed by an i.n. challenge with a lethal dose of Y. pestis CO92 prevented bacterial colonization and protected 100% of mice from pneumonic plague. Survival of the vaccinated mice correlated with levels of systemic and lung antibodies, reduced pulmonary pathology and proinflammatory cytokines, and the presence of lung lymphoid cell aggregates. Protection against pneumonic plague was partially dependent upon Fc receptors and could be transferred to naïve mice with immune mouse serum. On the other hand, protection was not dependent upon complement, and following vaccination, depletion of CD4 and/or CD8 T cells before challenge did not affect survival. In summary, the results demonstrate the safety, immunogenicity, and protective efficacy of i.n. administered iYp plus IL-12 in a mouse model of pneumonic plague.

Francisella tularensis is a highly pathogenic gram negative bacterium that infects multiple sites in a host, including the skin and the respiratory tract, which can lead to the onset of a deadly disease with a 50% mortality rate. The live vaccine strain (LVS) of F. tularensis, while attenuated in humans but still virulent in mice, is not an option for vaccine use in the United States due to safety concerns, and currently no FDA approved vaccine exists. The purpose of the present work was to assess the ability of recombinant Francisella outer membrane protein A (FopA) to induce a protective response in mice. The gene encoding FopA from F. tularensis LVS was cloned and expressed in Escherichia coli. The resulting recombinant protein was affinity-purified from the E. coli outer membrane, incorporated into liposomes and administered to mice via multiple routes. FopA-immunized mice produced FopA-specific antibodies and were protected against both lethal intradermal and intranasal challenges with F. tularensis LVS. The vaccinated mice had reduced bacterial numbers in their lungs, livers and spleens during infection, and complete bacterial clearance was observed by day 28 post infection. Passive transfer of FopA-immune serum protected naïve mice against lethal F. tularensis LVS challenge, showing that humoral immunity played an important role in vaccine efficacy. FopA-immunization was unable to protect against challenge with the fully virulent SchuS4 strain of F. tularensis; however, the findings demonstrate proof of principle that an immune response generated against a component of a subunit vaccine is protective against lethal respiratory and intradermal tularemia.

Vaccination of mice with Francisella tularensis live vaccine strain (LVS) mutants described so far have failed to induce protection in C57BL/6 mice against challenge with the virulent strain F. tularensis SchuS4. We have previously reported that a mutant of F. tularensis LVS deficient in iron superoxide dismutase (sodB(Ft)) is hypersensitive to oxidative stress and attenuated for virulence in mice. Herein, we evaluated the efficacy of this mutant as a vaccine candidate against respiratory tularemia caused by F. tularensis SchuS4. C57BL/6 mice were vaccinated intranasally (i.n.) with the sodB(Ft) mutant and challenged i.n. with lethal doses of F. tularensis SchuS4. The level of protection against SchuS4 challenge was higher in sodB(Ft) vaccinated group as compared to the LVS vaccinated mice. sodB(Ft) vaccinated mice following SchuS4 challenge exhibited significantly reduced bacterial burden in lungs, liver and spleen, regulated production of pro-inflammatory cytokines and less severe histopathological lesions compared to the LVS vaccinated mice. The sodB(Ft) vaccination induced a potent humoral immune response and protection against SchuS4 required both CD4 and CD8 T cells in the vaccinated mice. sodB(Ft) mutants revealed upregulated levels of chaperonine proteins DnaK, GroEL and Bfr that have been shown to be important for generation of a potent immune response against Francisella infection. Collectively, this study describes an improved live vaccine candidate against respiratory tularemia that has an attenuated virulence and enhanced protective efficacy than the LVS.

A recent resurgence in the number of cases of whooping cough, and other respiratory diseases caused by members of the bordetellae, in vaccinated populations has demonstrated the need for a thorough understanding of vaccine-induced immunity to facilitate more intelligent vaccine design. In this work, we use a murine model of respiratory infection using the highly successful animal pathogen, Bordetella bronchiseptica. Since previously infected animals have been shown to resist re-infection by B. bronchiseptica, we sought to examine the differences between vaccine-induced immunity and infection-induced immunity. Both prior infection and vaccination conferred nearly complete protection in the lungs, however, only prior infection resulted in significant protection in the upper respiratory tract. While immunity induced by prior infection offered significant protection even in the absence of complement or FcgammaRs, vaccination-induced protection required both complement and FcgammaRs. Although vaccination induced higher titers of B. bronchiseptica-specific antibodies, this serum was less effective than infection-induced serum in clearing bacteria from the lower respiratory tract. Together these findings highlight substantial differences between the mechanisms involved in vaccine- and infection-induced protective immunity.

Single institution and population-based studies highlight that infectious complications following transrectal ultrasound guided prostate needle biopsy (TRUS PNB) are increasing. Such infections are largely attributable to quinolone resistant microorganisms which colonize the rectal vault and are translocated into the bloodstream during the biopsy procedure. A povidone iodine rectal preparation (PIRP) at time of biopsy is a simple, reproducible method to reduce rectal microorganism colony counts and therefore resultant infections following TRUS PNB. All patients are administered three days of oral antibiotic therapy prior to biopsy. The PIRP technique involves initially positioning the patient in the standard manner for a TRUS PNB. Following digital rectal examination, 15 ml of a 10% solution of commercially available povidone iodine is mixed with 5 ml of 1% lidocaine jelly to create slurry. A 4 cmx4 cm sterile gauze is soaked in this slurry and then inserted into the rectal vault for 2 min after which it is removed. Thereafter, a disposable cotton gynecologic swab is used to paint both the perianal area and the rectal vault to a distance of 3 cm from the anus. The povidone iodine solution is then allowed to dry for 2-3 min prior to proceeding with standard transrectal ultrasonography and subsequent biopsy. This PIRP technique has been in practice at our institution since March of 2012 with an associated reduction of post-biopsy infections from 4.3% to 0.6% (p=0.02). The principal advantage of this prophylaxis regimen is its simplicity and reproducibility with use of an easily available, inexpensive agent to reduce infections. Furthermore, the technique avoids exposing patients to additional systemic antibiotics with potential further propagation of multi-drug resistant organisms. Usage of PIRP at TRUS PNB, however, is not applicable for patients with iodine or shellfish allergies.

Although the antibacterial effects of Abs are well studied in in vitro systems, the in vivo effects of Abs cannot always be accurately predicted. Complicated cross-talk between different effector functions of Abs and various arms of the immune system can affect their activities in vivo. Using the mouse respiratory pathogen Bordetella bronchiseptica, we examined the mechanisms of Ab-mediated clearance of bacteria from the respiratory tract. Interestingly, although TLR4 was not necessary for protective immunity following infection, it was required for rapid bacterial clearance in mice that were vaccinated or adoptively transferred Abs. TLR4 was important for the rapid recruitment of neutrophils that are necessary for Ab-mediated bacterial clearance via a mechanism that requires both FcγR and CR3. These data are consistent with a model in which TLR4-mediated inflammatory responses aid in the recruitment of neutrophils, which phagocytose Ab- and complement-opsonized bacteria via FcγRs and CR3. Although pattern recognition receptors are known to be involved in innate immunity and the generation of adaptive immunity, their contributions to specific adaptive immune functions should be considered in ongoing efforts to improve vaccine-induced protective immunity.

Bordetella parapertussisandBordetella pertussisare closely related species that cause whooping cough, an acute, immunizing disease. Their coexistence in the same host populations at the same time and vaccine studies showing thatB. pertussisvaccines have little effect onB. parapertussisinfection or disease suggest that the protective immunity induced by each does not efficiently cross protect against the other. Although the mechanisms of protective immunity toB. pertussishave been well studied, those ofB. parapertussishave not. The present study explores the mechanism by whichB. parapertussisis cleared from the lower respiratory tract by anamnestic immunity. Serum antibodies are necessary and sufficient for elimination of this bacterium, and CD4+T cells, complement, and neutrophils are required for serum antibody-mediated clearance. Mice lacking immunoglobulin A had no defect in their ability to control or clear infection. Interestingly, serum antibody-mediated clearance ofB. parapertussisdid not require Fc receptors that are required for antibody-mediated clearance ofB. pertussis. Together these data support a model for the mechanism of protective immunity toB. parapertussisthat is similar but distinct from that ofB. pertussis.

To assess the contribution of complement to respiratory immunity in the context of a natural bacterial infection, we used mice genetically deficient in complement components and the murine pathogen Bordetella bronchiseptica. Complement component C3 was not required for the control of bacterial infection or for the generation of infection-induced protective immunity. However, C3-deficient (C3(-/-)) mice were severely defective, compared to wild type, in vaccine-induced protective immunity. Adoptively transferred immune serum from convalescent wild-type or C3(-/-) animals rapidly cleared B. bronchiseptica from the lungs of wild-type mice but did not affect its growth in C3(-/-) mice, indicating that the defect is not in the generation of protective immunity, but in its function. Immune serum was effective in C5-deficient mice but had little effect in the lungs of mice lacking either Fcgamma receptors (FcgammaR) or CR3, suggesting bacterial clearance is not via direct complement-mediated lysis. Together, these data indicate that complement is required for antibody-mediated clearance of Bordetella and suggest the mechanism involves C3 opsonization of bacteria for phagocytosis that is both CR3- and FcgammaR-dependent.

The persistence of Bordetella pertussis and B . parapertussis within vaccinated populations and the reemergence of associated disease highlight the need to better understand protective immunity. The present study examined host immunity to bordetellae and addressed potential concerns about the mouse model by using a comparative approach including the closely related mouse pathogen B . bronchiseptica . As previously observed with B . pertussis , all three organisms persisted throughout the respiratory tracts of B-cell-deficient mice, indicating that B cells are required for bacterial clearance. However, adoptively transferred antibodies rapidly cleared B . bronchiseptica but not human pathogens. These results obtained with the mouse model are consistent with human clinical observations, including the lack of correlation between antibody titers and protection, as well as the limited efficacy of intravenous immunoglobulin treatments against human disease. Together, this evidence suggests that the mouse model accurately reflects substantial differences between immunities to these organisms. Although both B . pertussis and B . parapertussis are more closely related to B . bronchiseptica than they are to each other, they share the ability to resist rapid clearance from the lower respiratory tract by adoptively transferred antibodies, an adaptation that correlates with their emergence as human pathogens that circulate within vaccinated populations.

There has been a growing appreciation for the influence of dietary nutrients on the microbiome as well as host response to infectious disease. The micronutrient selenium (Se) is known to regulate immune functions via selenoproteins, a class of proteins that contain the 21st amino acid selenocysteine. However, the mechanisms by which selenoproteins regulate immune functions during an acute infection are not clear. Therefore, we investigated the role of macrophage (Mac) selenoproteins during an acute bacterial infection. Francisella tularensis (Ft.), the causative agent of tularemia, is a gram-negative intracellular bacterium. Since Ft. infects and replicates primarily in Macs, we measured the bacterial replication in Macs derived from TrspM mice that are unable to synthesize selenoproteins. TrspM Macs had uncontrolled replication of Ft. compared to wild-type (WT) Macs. Moreover, TrspM mice were more susceptible to Ft. infection and harbored significantly higher levels of bacteria in their livers and spleens as compared to WT mice, which suggests that Mac selenoproteins are essential for restricting bacterial replication and promoting host survival. Since Ft. replication is closely associated with autophagy, we investigated if the Mac selenoproteins had an effect on autophagy. In fact, our data indicates that selenoproteins inhibit autophagy, which may limit the availability of nutrients to the bacteria. Future studies will seek to establish the influence of selenoproteins on the microbiome and how these changes impact the infectious disease process. Our studies demonstrate that nutrients affect the outcome of an infection by influencing host immune response, and reveal potential novel targets for antibacterial therapies.

We investigated the role of interleukin-10 (IL-10) in cutaneous and pulmonary infection with Francisella tularensis . We found that after intradermal challenge of mice with the live vaccine strain (LVS) of F. tularensis , splenic IL-10 levels increased rapidly and reached a peak 5 days after infection. However, IL-10 expression after infection was detrimental, since IL-10 −/− mice showed increased bacterial clearance and were resistant to an infectious dose (>10 6 CFU/mouse) that was uniformly lethal for IL-10 +/+ mice. Furthermore, IL-10 +/+ mice treated with neutralizing anti-IL-10R monoclonal antibody were able to survive lethal cutaneous LVS challenge. The presence of IL-10 appeared to restrain the expression of IL-17, since high levels of splenic IL-17 were observed after intradermal LVS infection only in IL-10 −/− mice. Furthermore, treatment with neutralizing anti-IL-17R antibody ablated the enhanced survival observed in IL-10 −/− mice. However, neutralization of IL-10 activity in IL-17R −/− mice failed to provide protection. Thus, IL-10 suppresses a protective IL-17 response that is necessary for resistance to cutaneous LVS infection. Surprisingly, however, IL-10 −/− mice were significantly more susceptible to pulmonary infection with LVS. Finally, although IL-10 is a critical and novel regulator of immunity to F. tularensis LVS infection, its effects were masked during infection with the highly virulent SchuS4 strain. Taken together, these findings suggest that differentially regulating expression of the IL-10 pathway in various tissues could ultimately have prophylactic and therapeutic benefits for protection against tularemia.

The citrulline ureidase (CTU) activity has been shown to be associated with highly virulentFrancisella tularensisstrains, including Schu S4, while it is absent in avirulent or less virulent strains. A definitive role of thectugene in virulence and pathogenesis ofF. tularensisSchu S4 has not been assessed; thus, an understanding of the significance of this phenotype is long overdue. CTU is a carbon-nitrogen hydrolase encoded by the citrulline ureidase (ctu) gene (FTT0435) on theF. tularensisSchu S4 genome. In the present study, we evaluated the contribution of thectugene in the virulence of category A agentF. tularensisSchu S4 by generating a nonpolar deletion mutant, the Δctumutant. The deletion of thectugene resulted in loss of CTU activity, which was restored by transcomplementing thectugene. The Δctumutant did not exhibit any growth defect under acellular growth conditions; however, it was impaired for intramacrophage growth in resting as well as gamma interferon-stimulated macrophages. The Δctumutant was further tested for its virulence attributes in a mouse model of respiratory tularemia. Mice infected intranasally with the Δctumutant showed significantly reduced bacterial burden in the lungs, liver, and spleen compared to wild-type (WT) Schu S4-infected mice. The reduced bacterial burden in mice infected with the Δctumutant was also associated with significantly lower histopathological scores in the lungs. Mice infected with the Δctumutant succumbed to infection, but they survived longer and showed significantly extended median time to death compared to that shown by WT Schu S4-infected mice. To conclude, this study demonstrates thatctucontributes to intracellular survival, in vivo growth, and pathogenesis. However,ctuis not an absolute requirement for the virulence ofF. tularensisSchu S4 in mice.

Francisella tularensis can cause fatal respiratory tularemia in humans and animals and is increasingly being isolated in the United States and several European countries. The correlates of protective immunity against this intracellular bacterium are not known, and currently there are no licensed vaccines available for human use. Cell-mediated immunity has long been believed to be critical for protection, and the importance of humoral immunity is also now recognized. Furthermore, synergy between antibodies, T cell-derived cytokines, and phagocytes appears to be critical to achieve sterilizing immunity against F. tularensis. Thus, novel vaccine approaches should be designed to induce robust antibody and cell-mediated immune responses to this pathogen.

The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.

The genus Bordetella includes a group of closely related mammalian pathogens that cause a variety of respiratory diseases in a long list of animals ( B. bronchiseptica ) and whooping cough in humans ( B. pertussis and B. parapertussis ). While past research has examined how these pathogens are eliminated from the lower respiratory tract, the host factors that control and/or clear the bordetellae from the upper respiratory tract remain unclear. We hypothesized that immunoglobulin A (IgA), the predominant mucosal antibody isotype, would have a protective role against these mucosal pathogens. IgA −/− mice were indistinguishable from wild-type mice in their control and clearance of B. pertussis or B. parapertussis , suggesting that IgA is not crucial to immunity to these organisms. However, naïve and convalescent IgA −/− mice were defective in reducing the numbers of B. bronchiseptica in the upper respiratory tract compared to wild-type controls. Passively transferred serum from convalescent IgA −/− mice was not as effective as serum from convalescent wild-type mice in clearing this pathogen from the tracheae of naive recipient mice. IgA induced by B. bronchiseptica infection predominantly recognized lipopolysaccharide-containing O-antigen, and antibodies against O-antigen were important to bacterial clearance from the trachea. Since an IgA response contributes to the control of B. bronchiseptica infection of the upper respiratory tract, immunization strategies aimed at inducing B. bronchiseptica -specific IgA may be beneficial to preventing the spread of this bacterium among domestic animal populations.

The role of Abs in protection against respiratory infection with the intracellular bacterium Francisella tularensis is not clear. To investigate the ability of Abs to clear bacteria from the lungs and prevent systemic spread, immune serum was passively administered i.p. to naive mice before intranasal F. tularensis live vaccine strain infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcγR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro experiments demonstrated that macrophages were more readily infected by Ab-opsonized bacteria but became highly efficient in killing upon activation by IFN-γ. Consistent with this finding, in vivo Ab-mediated protection was found to be dependent upon IFN-γ. SCID mice were not protected by passive Ab transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-γ. These data suggest that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis. Of considerable interest was the finding that serum Abs were capable of conferring protection against lethal respiratory tularemia when given 24–48 h postexposure. Thus, this study provides the first evidence for the therapeutic use of Abs in Francisella-infected individuals.

Respiratory infection with Francisella tularensis is the deadliest form of disease and represents the most likely route to be used by bioterrorists. Although mucosal surfaces represent the first line of defense against respiratory tularemia, and in fact, against the great majority of human pathogens, little is known about protective immunity at these sites. The objective of this chapter is to review recent data examining the importance of various pulmonary immune mechanisms in defense against F. tularensis infection and to evaluate potential strategies for induction of protective lung immunity. Aerosol and intranasal mouse infection models have yielded essentially equivalent results and have implicated an important role for Th1-type immune responses in protection, including IFN-gamma, TNF-alpha, and IL-12. The cells responsible for protection in the lung are not well-characterized but NK cells are an early target for activation after infection although it appears that CD8 T cells might be most critical for host resistance. In addition, it is becoming increasingly clear that antibodies can provide prophylactic and therapeutic protection against pulmonary infection but only in the presence of active cell-mediated immunity. In fact, in vitro exposure of resting macrophages to antibody-coated bacteria in the absence of IFN-gamma can actually enhance infection. Although various immune mechanisms can be shown to be important for protection against attenuated strains such as LVS, the real challenge for the future is to design efficacious approaches to prevent disease by highly virulent strains such as SchuS4.

Whooping cough is considered a childhood disease, although there is growing evidence that children are infected by adult carriers. Additionally, increasing numbers of vaccinated adults are being diagnosed with Bordetella pertussis disease. Thus it is critical to understand how B. pertussis remains endemic even in highly vaccinated or immune populations. Here we used the mouse model to examine the nature of sterilizing immunity to B. pertussis. Antibodies were necessary to control infection but did not rapidly clear B. pertussis from the lungs. However, antibodies affected B. pertussis after a delay of at least a week by a mechanism that involved neutrophils and Fc receptors, suggesting that neutrophils phagocytose and clear antibody-opsonized bacteria via Fc receptors. B. pertussis blocked migration of neutrophils and inhibited their recruitment to the lungs during the first week of infection by a pertussis toxin-dependent (PTx-dependent) mechanism; a PTx mutant of B. pertussis induced rapid neutrophil recruitment and was rapidly cleared from the lungs by adoptively transferred antibodies. Depletion of neutrophils abrogated the defects of the PTx mutant. Together these results indicate that PTx inhibits neutrophil recruitment, which consequently allows B. pertussis to avoid rapid antibody-mediated clearance and therefore successfully infect immune hosts.

Many pathogens are able to manipulate the signaling pathways responsible for the generation of host immune responses. Here we examine and model a respiratory infection system in which disruption of host immune functions or of bacterial factors changes the dynamics of the infection. We synthesize the network of interactions between host immune components and two closely related bacteria in the genus Bordetellae. We incorporate existing experimental information on the timing of immune regulatory events into a discrete dynamic model, and verify the model by comparing the effects of simulated disruptions to the experimental outcome of knockout mutations. Our model indicates that the infection time course of both Bordetellae can be separated into three distinct phases based on the most active immune processes. We compare and discuss the effect of the species-specific virulence factors on disrupting the immune response during their infection of naive, antibody-treated, diseased, or convalescent hosts. Our model offers predictions regarding cytokine regulation, key immune components, and clearance of secondary infections; we experimentally validate two of these predictions. This type of modeling provides new insights into the virulence, pathogenesis, and host adaptation of disease-causing microorganisms and allows systems-level analysis that is not always possible using traditional methods., Author Summary The immune response is a complex network of processes activated in a host upon infection. Pathogens seek to disrupt or evade these processes to ensure their own survival and proliferation. This article provides a systems-level analysis of the immune response against two related bacterial species in the Bordetella genus, B. bronchiseptica and B. pertussis. B. pertussis, the causative agent of whooping cough, has lost many of the virulence factors of its B. bronchiseptica–like progenitor, and is using different strategies for the modulation of the immune system. We have synthesized two separate network models for the interaction of these pathogens with their hosts. Each network is then translated into a predictive dynamic model and is validated by comparison with available experimental data. The model offers predictions regarding cytokine regulation and the effects of perturbations of the immune system, as well as the time course of infections in hosts that had previously encountered the pathogens. We experimentally validate the prediction that convalescent hosts can rapidly clear both pathogens, while antibody transfer cannot substantially reduce the duration of a B. pertussis infection. This type of modeling provides new insights into the virulence, pathogenesis, and host adaptation of disease-causing microorganisms and can be readily extended to other pathogens.