Daniel N. Wolfe

Comparative Toll-Like Receptor 4-Mediated Innate Host Defense to Bordetella Infection

ABSTRACT Bordetella pertussis, B. parapertussis, and B. bronchiseptica are closely related species associated with respiratory disease in humans and other mammals. While B. bronchiseptica has a wide host range, B. pertussis and B. parapertussis evolved separately from a B. bronchiseptica-like progenitor to naturally infect only humans. Despite very different doubling times in vitro, all three establish similar levels of infection in the mouse lung within 72 h. Recent work has revealed separate roles for Toll-like receptor 4 (TLR4) in immunity to B. pertussis and B. bronchiseptica, while no role for TLR4 during B. parapertussis infection has been described. Here we compared the requirement for TLR4 in innate host defense to these organisms using the same mouse infection model. While B. bronchiseptica causes lethal disease in TLR4-deficient mice, B. pertussis and B. parapertussis do not. Correspondingly, TLR4 is critical in limiting B. bronchiseptica but not B. pertussis or B. parapertussis bacterial numbers during the first 72 h. Interestingly, B. bronchiseptica induces a TLR4-dependent cytokine response that is considerably larger than that induced by B. pertussis or B. parapertussis. Analysis of their endotoxins using RAW cells suggests that B. bronchiseptica lipopolysaccharide (LPS) is 10- and 100-fold more stimulatory than B. pertussis or B. parapertussis LPS, respectively. The difference in LPS stimulus is more pronounced when using HEK293 cells expressing human TLR4. Thus, it appears that in adapting to infect humans, B. pertussis and B. parapertussis independently modified their LPS to reduce TLR4-mediated responses, which may compensate for slower growth rates and facilitate host colonization.

Visualizing genomic information across chromosomes with PhenoGram

BackgroundWith the abundance of information and analysis results being collected for genetic loci, user-friendly and flexible data visualization approaches can inform and improve the analysis and dissemination of these data. A chromosomal ideogram is an idealized graphic representation of chromosomes. Ideograms can be combined with overlaid points, lines, and/or shapes, to provide summary information from studies of various kinds, such as genome-wide association studies or phenome-wide association studies, coupled with genomic location information. To facilitate visualizing varied data in multiple ways using ideograms, we have developed a flexible software tool called PhenoGram which exists as a web-based tool and also a command-line program.ResultsWith PhenoGram researchers can create chomosomal ideograms annotated with lines in color at specific base-pair locations, or colored base-pair to base-pair regions, with or without other annotation. PhenoGram allows for annotation of chromosomal locations and/or regions with shapes in different colors, gene identifiers, or other text. PhenoGram also allows for creation of plots showing expanded chromosomal locations, providing a way to show results for specific chromosomal regions in greater detail. We have now used PhenoGram to produce a variety of different plots, and provide these as examples herein. These plots include visualization of the genomic coverage of SNPs from a genotyping array, highlighting the chromosomal coverage of imputed SNPs, copy-number variation region coverage, as well as plots similar to the NHGRI GWA Catalog of genome-wide association results.ConclusionsPhenoGram is a versatile, user-friendly software tool fostering the exploration and sharing of genomic information. Through visualization of data, researchers can both explore and share complex results, facilitating a greater understanding of these data.

The O Antigen Enables Bordetella parapertussis To Avoid Bordetella pertussis-Induced Immunity

ABSTRACT Bordetella pertussis and Bordetella parapertussis are closely related endemic human pathogens which cause whooping cough, a disease that is reemerging in human populations. Despite how closely related these pathogens are, their coexistence and the limited efficacy of B. pertussis vaccines against B. parapertussis suggest a lack of cross-protective immunity between the two. We sought to address the ability of infection-induced immunity against one of these pathogens to protect against subsequent infection by the other using a mouse model of infection. Immunity induced by B. parapertussis infection protected against subsequent infections by either species. However, immunity induced by B. pertussis infection prevented subsequent B. pertussis infections but did not protect against B. parapertussis infections. The O antigen of B. parapertussis inhibited binding of antibodies to the bacterial surface and was required for B. parapertussis to colonize mice convalescent from B. pertussis infection. Thus, the O antigen of B. parapertussis confers asymmetrical cross-immunity between the causative agents of whooping cough. We propose that these findings warrant investigation of the relative role of B. parapertussis in the resurgence of whooping cough.

O Antigen Protects Bordetella parapertussis from Complement

ABSTRACT Bordetella pertussis, a causative agent of whooping cough, expresses BrkA, which confers serum resistance, but the closely related human pathogen that also causes whooping cough, Bordetella parapertussis, does not. Interestingly, B. parapertussis, but not B. pertussis, produces an O antigen, a factor shown in other models to confer serum resistance. Using a murine model of infection, we determined that O antigen contributes to the ability of B. parapertussis to colonize the respiratory tract during the first week of infection, but not thereafter. Interestingly, an O antigen-deficient strain of B. parapertussis was not defective in colonizing mice lacking the complement cascade. O antigen prevented both complement component C3 deposition on the surface and complement-mediated killing of B. parapertussis. In addition, O antigen was required for B. parapertussis to systemically spread in complement-sufficient mice, but not complement-deficient mice. These data indicate that O antigen enables B. parapertussis to efficiently colonize the lower respiratory tract by protecting against complement-mediated control and clearance.

Genomic analyses with biofilter 2.0: knowledge driven filtering, annotation, and model development

AbstractBackgroundThe ever-growing wealth of biological information available through multiple comprehensive database repositories can be leveraged for advanced analysis of data. We have now extensively revised and updated the multi-purpose software tool Biofilter that allows researchers to annotate and/or filter data as well as generate gene-gene interaction models based on existing biological knowledge. Biofilter now has the Library of Knowledge Integration (LOKI), for accessing and integrating existing comprehensive database information, including more flexibility for how ambiguity of gene identifiers are handled. We have also updated the way importance scores for interaction models are generated. In addition, Biofilter 2.0 now works with a range of types and formats of data, including single nucleotide polymorphism (SNP) identifiers, rare variant identifiers, base pair positions, gene symbols, genetic regions, and copy number variant (CNV) location information.ResultsBiofilter provides a convenient single interface for accessing multiple publicly available human genetic data sources that have been compiled in the supporting database of LOKI. Information within LOKI includes genomic locations of SNPs and genes, as well as known relationships among genes and proteins such as interaction pairs, pathways and ontological categories. Via Biofilter 2.0 researchers can: • Annotate genomic location or region based data, such as results from association studies, or CNV analyses, with relevant biological knowledge for deeper interpretation• Filter genomic location or region based data on biological criteria, such as filtering a series SNPs to retain only SNPs present in specific genes within specific pathways of interest• Generate Predictive Models for gene-gene, SNP-SNP, or CNV-CNV interactions based on biological information, with priority for models to be tested based on biological relevance, thus narrowing the search space and reducing multiple hypothesis-testing.ConclusionsBiofilter is a software tool that provides a flexible way to use the ever-expanding expert biological knowledge that exists to direct filtering, annotation, and complex predictive model development for elucidating the etiology of complex phenotypic outcomes.

Clearance of Bordetella parapertussis from the Lower Respiratory Tract Requires Humoral and Cellular Immunity

ABSTRACT Bordetella parapertussis and Bordetella pertussis are 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 that B. pertussis vaccines have little effect on B. parapertussis infection or disease suggest that the protective immunity induced by each does not efficiently cross protect against the other. Although the mechanisms of protective immunity to B. pertussis have been well studied, those of B. parapertussis have not. The present study explores the mechanism by which B. parapertussis is 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 of B. parapertussis did not require Fc receptors that are required for antibody-mediated clearance of B. pertussis. Together these data support a model for the mechanism of protective immunity to B. parapertussis that is similar but distinct from that of B. pertussis.

Inefficient Toll-Like Receptor-4 Stimulation Enables Bordetella parapertussis to Avoid Host Immunity

The recognition of bacterial lipopolysaccharide (LPS) by host Toll-like receptor (TLR)4 is a crucial step in developing protective immunity against several gram negative bacterial pathogens. Bordetella bronchiseptica and B. pertussis stimulate robust TLR4 responses that are required to control the infection, but a close relative, B. parapertussis, poorly stimulates this receptor, and TLR4 deficiency does not affect its course of infection. This led us to hypothesize that inefficient TLR4 stimulation enables B. parapertussis to evade host immunity. In a mouse model of infection, B. parapertussis grew rapidly in the lungs, but no measurable increase in TLR4-mediated cytokine, chemokine, or leukocyte responses were observed over the first few days of infection. Delivery of a TLR4 stimulant in the inoculum resulted in a robust inflammatory response and a 10- to 100-fold reduction of B. parapertussis numbers. As we have previously shown, B. parapertussis grows efficiently during the first week of infection even in animals passively immunized with antibodies. We show that this evasion of antibody-mediated clearance is dependent on the lack of TLR4 stimulation by B. parapertussis as co-inoculation with a TLR4 agonist resulted in 10,000-fold lower B. parapertussis numbers on day 3 in antibody-treated wild type, but not TLR4-deficient, mice. Together, these results indicate that inefficient TLR4 stimulation by B. parapertussis enables it to avoid host immunity and grow to high numbers in the respiratory tract of naïve and immunized hosts.

Comparative Role of Immunoglobulin A in Protective Immunity against the Bordetellae

ABSTRACT 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.

Delayed Role of Tumor Necrosis Factor—α in Overcoming the Effects of Pertussis Toxin

Bordetella pertussis causes whooping cough, an endemic respiratory disease that is increasing in prevalence despite vaccination efforts. Although host immunity is modulated by virulence factors of this pathogen, it is unclear what host factors are required to overcome their effects. Here, we investigate an apparent relationship between the effects of pertussis toxin and tumor necrosis factor (TNF)–a. B. pertussis grows efficiently and causes moderate pathology in wild-type mice, whereas TNF-a / mice have higher numbers of bacteria and leukocytes in lungs, experience more airway resistance, and die of the infection. Interestingly, an isogenic B. pertussis strain lacking pertussis toxin did not induce these effects in TNF-a / mice and behaved similarly in wild-type and TNF-a–deficient hosts. Together, these results indicate that TNF-a is essential for the host to overcome the effects of pertussis toxin, allowing both control of B. pertussis numbers and regulation of the inflammation induced by infection.

Of mice and men: asymmetric interactions between Bordetella pathogen species

In a recent experiment, we found that mice previously infected with Bordetella pertussis were not protected against a later infection with Bordetella parapertussis, while primary infection with B. parapertussis conferred cross-protection. This challenges the common assumption made in most mathematical models for pathogenic strain dynamics that cross-immunity between strains is symmetric. Here we investigate the potential consequences of this pattern on the circulation of the two pathogens in human populations. To match the empirical dominance of B. pertussis, we made the additional assumption that B. parapertussis pays a cost in terms of reduced fitness. We begin by exploring the range of parameter values that allow the coexistence of the two pathogens, with or without vaccination. We then track the dynamics of the system following the introduction of anti-pertussis vaccination. Our results suggest that (1) in order for B. pertussis to be more prevalent than B. parapertussis, the former must have a strong competitive advantage, possibly in the form of higher infectivity, and (2) because of asymmetric cross-immunity, the introduction of anti-pertussis vaccination should have little effect on the absolute prevalence of B. parapertussis. We discuss the evidence supporting these predictions, and the potential relevance of this model for other pathogens.