Drusilla L. Burns

Importance of srtA and srtB for growth of Bacillus anthracis in macrophages.

ABSTRACT We examined the effect of mutation of two sortase genes of Bacillus anthracis, srtA and srtB, on the ability of the bacterium to grow in J774A.1 cells, a mouse macrophage-like cell line. While disruption of either srtA or srtB had no effect on the ability of the bacteria to grow in rich culture media, mutations in each of these genes dramatically attenuated growth of the bacterium in J774A.1 cells. Complementation of the mutation restored the ability of bacteria to grow in the cells. Since the initial events in inhalation anthrax are believed to be uptake of B. anthracis spores by alveolar macrophages followed by germination of the spores and growth of the bacteria within the macrophages, these results suggest that two sortases of B. anthracis may be critical in the early stages of inhalation anthrax.

Pertussis Toxin-Catalyzed ADP-Ribosylation: Effects on the Coupling of Inhibitory Receptors to the Adenylate Cyclase System

The adenylate cyclase system consists of stimulatory and inhibitory hormone and drug receptors coupled through different GTP-binding proteins to a catalytic unit, responsible for the synthesis of cAMP from ATP. Pertussis toxin blocks the effect of inhibitory agonists on the catalytic unit by enzymatically inactivating the inhibitory GTP-binding protein (Gi). Study of the inhibitory arm of the cyclase system has been facilitated by the dissection of the overall process of hormonal inhibition of cAMP formation into a series of reactions characteristic of the individual protein components of this complex system; pertussis toxin has proven to be a useful tool with which to study these individual reactions. Exposure of cells or membranes to pertussis toxin in the presence of NAD results in ADP-ribosylation of a 41,000 Da subunit of Gi. ADP-ribosylation of Gi has a number of effects on the overall and partial reactions of the cyclase system, including a loss of a) hormonal inhibition of cAMP formation, b) hormonal stimulation of GTPase and c) agonist-induced release of membrane-bound guanyl nucleotides. In addition, in toxin-treated membranes, the affinity of inhibitory receptors for agonist but not antagonist is decreased with no significant change in receptor number.

Membrane Localization of the S1 Subunit of Pertussis Toxin in Bordetella pertussis and Implications for Pertussis Toxin Secretion

ABSTRACT Pertussis toxin is secreted from Bordetella pertussis with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type B. pertussis, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of B. pertussis that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of B. pertussis. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of Escherichia coli (J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of B. pertussis. Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system.

Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays

ABSTRACT Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fcγ) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcγ receptor-dependent neutralization was observed in the J774A.1 cell-based assay than in the RAW 264.7 cell-based assay, a finding that could be due to the larger numbers of Fcγ receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcγ receptor blocking monoclonal antibodies, we found that at least three murine Fcγ receptor classes, IIB, III, and IV, can contribute to Fcγ receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcγ receptor-dependent neutralization observed in the J774A.1 cell-based assay was dependent on the concentration of protective antigen utilized in the assay. Our results suggest that the characteristics of the antibodies analyzed in the assay (e.g., species of origin, isotype, and subclass), as well as the assay design (e.g., cell type and protective antigen concentration), could significantly influence the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by anthrax toxin neutralization assays. These findings should be considered when interpreting anthrax toxin neutralization assay output.

Requirements for Assembly of PtlH with the Pertussis Toxin Transporter Apparatus of Bordetella pertussis

ABSTRACT PtlH is an essential component of the Ptl system, the type IV transporter responsible for secretion of pertussis toxin (PT) across the outer membrane of Bordetella pertussis. The nine Ptl proteins are believed to interact to form a membrane-spanning apparatus through which the toxin is secreted. In this study, we monitored the subcellular localization of PtlH in strains of B. pertussis lacking PT, lacking other Ptl proteins, or from which ATP has been depleted in order to gain insight into the requirements for assembly of PtlH with the remainder of the Ptl transporter complex that is thought to be tightly embedded in the membrane. We found that PtlH is exclusively localized to the inner membrane fraction of the cell in a wild-type strain of B. pertussis. In contrast, PtlH localized to both the cytoplasmic and inner membrane fractions of a mutant strain of B. pertussis that does not produce PT. In comparison to how it localized in wild-type strains of B. pertussis, PtlH exhibited aberrant localization in strains lacking PtlD, PtlE, PtlF, and PtlG. We also found that localization of PtlH was perturbed in B. pertussis strains that were treated with carbonyl cyanide m-chlorophenylhydrazone and sodium arsenate, which are capable of depleting cellular ATP levels, and in strains of B. pertussis that produce an altered form of PtlH that lacks ATPase activity. When taken together, these results indicate that tight association of PtlH with the membrane, likely through interactions with components of the transporter-PT complex, requires the toxin substrate, a specific subset of the Ptl proteins, and ATP. Based on these data, a model for the assembly of the Ptl transporter-PT complex is presented.

Comparison of Three Anthrax Toxin Neutralization Assays

ABSTRACT Different types of anthrax toxin neutralization assays have been utilized to measure the antibody levels elicited by anthrax vaccines in both nonclinical and clinical studies. In the present study, we sought to determine whether three commonly used toxin neutralization assays—J774A.1 cell-, RAW 264.7 cell-, and CHO cell-based assays—yield comparable estimates of neutralization activities for sera obtained after vaccination with anthrax vaccines composed of recombinant protective antigen (rPA). In order to compare the assays, sera were assayed alongside a common reference serum sample and the neutralization titers were expressed relative to the titer for the reference sample in each assay. Analysis of sera from rabbits immunized with multiple doses of the rPA vaccine showed that for later bleeds, the quantitative agreement between the assays was good; however, for early bleeds, some heterogeneity in relative neutralization estimates was observed. Analysis of serum samples from rabbits, nonhuman primates, and humans immunized with the rPA vaccine showed that the relative neutralization estimates obtained in the different assays agreed to various extents, depending on the species of origin of the sera examined. We identified differences in the magnitudes of the Fc receptor-mediated neutralization associated with the J774A.1 cell- and RAW 264.7 cell-based assays, which may account for some of the species dependence of the assays. The differences in the relative neutralization estimates among the assays were relatively small and were always less than 2.5-fold. However, because toxin neutralization assays will likely be used to establish the efficacies of new anthrax vaccines, our findings should be considered when assay outputs are interpreted.

Recent advances in the development of pertussis vaccines.

Publisher Summary This chapter describes Bordetella pertussis and discusses recent advances in the development of pertussis vaccines. Bordetella pertussis is the pathogen that causes the human respiratory disease known as whooping cough, or pertussis. It is a fastidious Gram-negative bacillus that has a particular tropism for the ciliated epithelium of the respiratory tract. Pertussis begins as a mild upper respiratory infection that generally lasts 7–10 days, with nonspecific symptoms, usually in the absence of fever. During this initial and most infectious stage of disease, known as the catarrhal stage, the organism can be cultured from the nasopharynx. The disease usually progresses to the paroxysmal stage, typified by episodes of paroxysmal coughing, which, in young children, may be accompanied by an inspiratory whoop although this is often absent in infants younger than six months of age. Paroxysmal episodes may be followed by vomiting. The chapter also discusses new pertussis vaccines by examining the properties of the current whole cell vaccine. There is little doubt that whole cell pertussis vaccine (WCPV) has controlled pertussis in the communities where it has been adequately prepared and used under recommended protocols.

Analysis of antibody responses to protective antigen-based anthrax vaccines through use of competitive assays.

ABSTRACT The licensed anthrax vaccine and many of the new anthrax vaccines being developed are based on protective antigen (PA), a nontoxic component of anthrax toxin. For this reason, an understanding of the immune response to PA vaccination is important. In this study, we examined the antibody response elicited by PA-based vaccines and identified the domains of PA that contribute to that response in humans as well as nonhuman primates (NHPs) and rabbits, animal species that will be used to generate efficacy data to support approval of new anthrax vaccines. To this end, we developed a competitive enzyme-linked immunosorbent assay (ELISA), using purified recombinant forms of intact PA and its individual domains. We found that PA-based vaccines elicited IgG antibodies to each of the four PA domains in all three species. We also developed a competitive toxin neutralization assay, which showed that rabbits, NHPs, and humans all have functional antibody populations that bind to domains 1, 3, and 4. While the domain specificities of the antibody responses elicited by PA-based vaccines were similar in humans, NHPs, and rabbits, competitive assays suggested that humans may have a more significant secondary population of IgG antibodies that bind to partially unfolded or incorrectly folded PA. These findings provide information that will be useful when linking animal protection data to humans via an antibody bridge to establish efficacy of new anthrax vaccines.

Stabilization of the Pertussis Toxin Secretion Apparatus by the C Terminus of PtlD

Pertussis toxin (PT) is secreted from Bordetella pertussis by a type IV secretion system, known as the Ptl transporter, that comprises nine different proteins, PtlA to PtlI. In this study, we found that PtlD is required for the stability of three Ptl proteins, PtlE, PtlF, and PtlH. A region limited to the C-terminal 72 amino acids of PtlD (amino acids 392 to 463) was sufficient for maintaining the stability of PtlE, PtlF, and PtlH, although this region was not sufficient to support secretion of the toxin. Further analysis demonstrated that a stretch of 10 amino acids at the C-terminal end of PtlD (amino acids 425 to 434) contributes to transporter stability.

Analysis of subassemblies of pertussis toxin subunits in vivo and their interaction with the ptl transport apparatus.

ABSTRACT Pertussis toxin (PT) has an AB5 structure that is typical of many bacterial protein toxins; however, this toxin is more complex than many toxins since it is composed of five different subunit types, subunits S1 to S5. Little is known about how PT assembles in vivo and how and when it interacts with its secretion apparatus, known as the Ptl transporter. In order to better understand these events, we expressed subsets of the genes encoding the S1, S2, and/or S4 subunits of PT in strains of Bordetella pertussis that either did or did not produce the Ptl proteins. We found evidence to suggest that certain subassemblies of the toxin, including subassemblies consisting of the S1 subunit and incomplete forms of the B oligomer, can form in vivo, at least transiently. These results suggest that the B oligomer of the toxin does not need to completely form before interactions between the S1 subunit and B-oligomer subunits can occur in vivo. All subassemblies localized primarily to the membrane fraction of the cell. Moreover, we found that Ptl-mediated secretion occurs in a strain that produces S1 and an incomplete complement of B-oligomer subunits. These results indicate that subassemblies of the toxin consisting of the S1 subunit and a partial B oligomer can interact with the Ptl system.