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In JoVE (1)
Other Publications (7)
Articles by Joanna B. Goldberg in JoVE
JoVE Immunology and Infection
Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Department of Microbiology, Immunology, & Cancer Biology, University of Virginia Health System
We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.
Other articles by Joanna B. Goldberg on PubMed
Construction and Characterization of a Live, Attenuated AroA Deletion Mutant of Pseudomonas Aeruginosa As a Candidate Intranasal Vaccine
Antibodies to the lipopolysaccharide O antigen of Pseudomonas aeruginosa mediate high-level immunity, but protective epitopes have proven to be poorly immunogenic, while nonprotective or minimally protective O-antigen epitopes often elicit the best immune responses. With the goal of developing a broadly protective P. aeruginosa vaccine, we used a gene replacement system based on the Flp recombinase to construct an unmarked aroA deletion mutant of the P. aeruginosa serogroup O2/O5 strain PAO1. The resultant aroA deletion mutant of PAO1 is designated PAO1 Delta aroA. The aroA deletion was confirmed by both PCR and failure of the mutant to grow on minimal media lacking aromatic amino acids. When evaluated for safety and immunogenicity in mice, PAO1 Delta aroA could be applied either intranasally or intraperitoneally at doses up to 5 x 10(9) CFU per mouse without adverse effects. No dissemination of PAO1 Delta aroA to blood, liver, or spleen was detected after intranasal application, and histological evidence of pneumonia was minimal. Intranasal immunization of mice and rabbits elicited high titers of immunoglobulin G to whole bacterial cells and to heat-stable bacterial antigens of all seven prototypic P. aeruginosa serogroup O2/O5 strains. The mouse antisera mediated potent phagocytic killing of most of the prototypic serogroup O2/O5 strains, while the rabbit antisera mediated phagocytic killing of several serogroup-heterologous strains in addition to killing all O2/O5 strains. This live, attenuated P. aeruginosa strain PAO1 Delta aroA appears to be safe for potential use as an intranasal vaccine and elicits high titers of opsonic antibodies against multiple strains of the P. aeruginosa O2/O5 serogroup.
Protection Against Fatal Pseudomonas Aeruginosa Pneumonia in Mice After Nasal Immunization with a Live, Attenuated AroA Deletion Mutant
Studies of immunity to Pseudomonas aeruginosa have indicated that a variety of potential immunogens can elicit protection in animal models, utilizing both antibody- and cell-mediated immune effectors for protection. To attempt to optimize delivery of multiple protective antigens and elicit a broad range of immune effectors, we produced an aroA deletion mutant of the P. aeruginosa serogroup O2/O5 strain PAO1, designated PAO1deltaaroA. Previously, we reported that this strain elicits high levels of opsonic antibody directed against many serogroup O2/O5 strains after nasal immunization of mice and rabbits. Here, we assessed the protective efficacy of immunization with PAO1deltaaroA against acute fatal pneumonia in mice. After active immunization, high levels of protection were achieved against an ExoU-expressing cytotoxic variant of the parental strain PAO1 at doses up to 1,000-fold greater than the 50% lethal dose. Significant protection against PAO1 and two of four other serogroup O2/O5 strains was also found, but there was no protection against serogroup-heterologous strains. The serogroup O2/O5 strains not protected against were killed in opsonophagocytic assays as efficiently as the strains with which protection was seen, indicating a lack of correlation of protection and opsonic killing within the serogroup. In passive immunization experiments using challenge with wild-type PAO1 or other noncytotoxic members of the O2/O5 serogroup, there was no protection despite the presence of high levels of opsonic antibody in the mouse sera. However, passive immunization did prevent mortality from pneumonia due to the cytotoxic PAO1 variant at low-challenge doses. These data suggest that a combination of humoral and cellular immunity is required for protection against P. aeruginosa lung infections, that such immunity can be elicited by using aroA deletion mutants, and that a multivalent P. aeruginosa vaccine composed of aroA deletion mutants of multiple serogroups holds significant promise.
The GalU Gene of Pseudomonas Aeruginosa is Required for Corneal Infection and Efficient Systemic Spread Following Pneumonia but Not for Infection Confined to the Lung
Acute pneumonias and corneal infections due to Pseudomonas aeruginosa are typically caused by lipopolysaccharide (LPS)-smooth strains. In cystic fibrosis patients, however, LPS-rough strains of P. aeruginosa, which lack O antigen, can survive in the lung and cause chronic infection. It is not clear whether an LPS-rough phenotype affects cytotoxicity related to the type III secretion system (TTSS). We previously reported that interruption of the galU gene in P. aeruginosa results in production of a rough LPS and truncated LPS core. Here we evaluated the role of the galU gene in the pathogenesis of murine lung and eye infections and in cytotoxicity due to the TTSS effector ExoU. We studied galU mutants of strain PAO1, of its cytotoxic variant expressing ExoU from a plasmid, and of the inherently cytotoxic strain PA103. The galU mutants were more serum sensitive than the parental strains but remained cytotoxic in vitro. In a corneal infection model, the galU mutants were significantly attenuated. In an acute pneumonia model, the 50% lethal doses of the galU mutants were higher than those of the corresponding wild-type strains, yet these mutants could cause mortality and severe pneumonia, as judged by histology, even with minimal systemic spread. These findings suggest that the galU gene is required for corneal infection and for efficient systemic spread following lung infection but is not required for infection confined to the lung. Host defenses in the lung appear to be insufficient to control infection with LPS-rough P. aeruginosa when local bacterial levels are high.
Intranasal Immunization with Heterologously Expressed Polysaccharide Protects Against Multiple Pseudomonas Aeruginosa Infections
Surface-expressed bacterial polysaccharides are often immunodominant, protective antigens. However, these antigens are chemically and serologically highly heterogeneous, and conjugation to protein carriers is often necessary to enhance their immunogenicity. Here we show the efficacy of intranasal immunization of mice with attenuated Salmonella enterica serovar Typhimurium expressing the O antigen portion of Pseudomonas aeruginosa lipopolysaccharide. P. aeruginosa is an ideal model system because it can cause a myriad of localized and systemic infections. In particular, this bacterium is a leading cause of hospital-acquired pneumonia and is responsible for infections after burns and after eye injury. In addition, there are mouse models of infection that mimic the clinical manifestations of P. aeruginosa infections. Immunized mice were highly protected against infection, with long-lasting immunity to acute P. aeruginosa pneumonia, whereas mice immunized with Salmonella containing only the cloning vector or PBS were not. Prophylactic and therapeutic administration of sera from vaccinated animals protected naive mice. Intranasal vaccination also provided complete protection from infections after burns and reduced pathology after corneal abrasions. These results indicate that intranasal delivery of heterologously expressed polysaccharide antigens provides protection at distinct sites of infection. This approach for the expression and delivery of polysaccharide antigens as recombinant immunogens could be easily adapted to develop vaccines for many infectious agents, without the need for complicated purification and conjugation procedures.
Pilus-mediated Epithelial Cell Death in Response to Infection with Burkholderia Cenocepacia
Burkholderia cenocepacia is an opportunistic pathogen that can cause serious infections in cystic fibrosis (CF) patients. The ET12 lineage appears particularly virulent in CF; however, its pathogenesis is poorly understood and may be associated with host response. To help characterize this response, the ability of B. cenocepacia to induce cytotoxicity and apoptosis in an epithelial cell model was examined. Upon infection with B. cenocepacia strain K56-2, A549 human lung epithelial cells underwent significant cell death; propidium iodine staining and DNA fragmentation assays suggested apoptosis. Initiation of cell death was independent of the type III secretion system, biofilm formation, and secreted bacterial cytotoxins. However, the frequency of cell death was lower in cells infected with a non-piliated mutant, K56-2 cblA::Tp. Furthermore, purified cbl pili were found to directly induce cytotoxicity in A549 cells and activate caspase-9, -8, -7, and -3, the major cysteine proteinases involved in apoptosis. It appears that B. cenocepacia cbl pili, which are a distinctive feature of the ET12 lineage, act as an initiator of cytotoxicity and apoptosis. Understanding the role of cbl pili in the pathogenesis of B. cenocepacia infections offers the potential for decreasing the virulence of these potentially life-threatening organisms in CF patients.
IL-17 is a Critical Component of Vaccine-induced Protection Against Lung Infection by Lipopolysaccharide-heterologous Strains of Pseudomonas Aeruginosa
In a murine model of acute fatal pneumonia, we previously showed that nasal immunization with a live-attenuated aroA deletant of Pseudomonas aeruginosa strain PAO1 elicited LPS serogroup-specific protection, indicating that opsonic Ab to the LPS O Ag was the most important immune effector. Because P. aeruginosa strain PA14 possesses additional virulence factors, we hypothesized that a live-attenuated vaccine based on PA14 might elicit a broader array of immune effectors. Thus, an aroA deletant of PA14, denoted PA14DeltaaroA, was constructed. PA14DeltaaroA-immunized mice were protected against lethal pneumonia caused not only by the parental strain but also by cytotoxic variants of the O Ag-heterologous P. aeruginosa strains PAO1 and PAO6a,d. Remarkably, serum from PA14DeltaaroA-immunized mice had very low levels of opsonic activity against strain PAO1 and could not passively transfer protection, suggesting that an antibody-independent mechanism was needed for the observed cross-serogroup protection. Compared with control mice, PA14DeltaaroA-immunized mice had more rapid recruitment of neutrophils to the airways early after challenge. T cells isolated from P. aeruginosa DeltaaroA-immunized mice proliferated and produced IL-17 in high quantities after coculture with gentamicin-killed P. aeruginosa. Six hours following challenge, PA14DeltaaroA-immunized mice had significantly higher levels of IL-17 in bronchoalveolar lavage fluid compared with unimmunized, Escherichia coli-immunized, or PAO1DeltaaroA-immunized mice. Antibody-mediated depletion of IL-17 before challenge or absence of the IL-17 receptor abrogated the PA14DeltaaroA vaccine's protection against lethal pneumonia. These data show that IL-17 plays a critical role in antibody-independent vaccine-induced protection against LPS-heterologous strains of P. aeruginosa in the lung.
Mucosal Vaccination with a Multivalent, Live-attenuated Vaccine Induces Multifactorial Immunity Against Pseudomonas Aeruginosa Acute Lung Infection
Many animal studies investigating adaptive immune effectors important for protection against Pseudomonas aeruginosa have implicated opsonic antibody to the antigenically variable lipopolysaccharide (LPS) O antigens as a primary effector. However, active and passive vaccination of humans against these antigens has not shown clinical efficacy. We hypothesized that optimal immunity would require inducing multiple immune effectors targeting multiple bacterial antigens. Therefore, we evaluated a multivalent live-attenuated mucosal vaccination strategy in a murine model of acute P. aeruginosa pneumonia to assess the contributions to protective efficacy of various bacterial antigens and host immune effectors. Vaccines combining 3 or 4 attenuated strains having different LPS serogroups were associated with the highest protective efficacy compared to vaccines with fewer components. Levels of opsonophagocytic antibodies, which were directed not only to the LPS O antigens but also to the LPS core and surface proteins, correlated with protective immunity. The multivalent live-attenuated vaccines overcame prior problems involving immunologic interference in the development of O-antigen-specific antibody responses when closely related O antigens were combined in multivalent vaccines. Antibodies to the LPS core were associated with in vitro killing and in vivo protection against strains with O antigens not expressed by the vaccine strains, whereas antibodies to the LPS core and surface proteins augmented the contribution of O-antigen-specific antibodies elicited by vaccine strains containing a homologous O antigen. Local CD4 T cells in the lung also contributed to vaccine-based protection when opsonophagocytic antibodies to the challenge strain were absent. Thus, multivalent live-attenuated vaccines elicit multifactorial protective immunity to P. aeruginosa lung infections.
