Construction and Characterization of a Live, Attenuated<i>aroA</i>Deletion Mutant of<i>Pseudomonas aeruginosa</i>as a Candidate Intranasal Vaccine

Priebe, Gregory P.; Brinig, Mary M.; Hatano, K; Grout, Martha; Coleman, Fadie T.; Pier, Gerald B.; Goldberg, Joanna B.

doi:10.1128/iai.70.3.1507-1517.2002

Cited by 64 publications

(68 citation statements)

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“…6). We previously reported similar pathology following infection with the wild-type strain PAO1 (38). As another marker of lung injury, we also assessed the weights of the lungs of mice infected with the galU mutants compared with those of mice infected with the wild-type strains.…”

Section: Vol 72 2004 P Aeruginosa Galu In Keratitis and Pneumonia mentioning

confidence: 87%

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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

et al. 2004

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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.

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Section: Vol 72 2004 P Aeruginosa Galu In Keratitis and Pneumonia mentioning

confidence: 87%

“…Histopathology was performed as previously described with lungs fixed in 1% paraformaldehyde in PBS instilled via the trachea after euthanasia (38). For survival analyses, mice were monitored daily for 10 days to assess mortality.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2004

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“…Mutations in the basic branch of the aromatic amino acid biosynthesis pathway proved to be efficient in attenuating virulence, e.g., in Listeria monocytogenes (57), Shigella dysenteriae (62), Pseudomonas aeruginosa (44), Neisseria gonorrhoeae (9), and Bacillus anthracis (27), but they failed to do so in Mycobacterium tuberculosis (41). The main objective of this study was to ascertain whether an aro mutant of S. aureus could be attenuated and immunogenic.…”

Section: Discussionmentioning

confidence: 99%

“…Several virulent strains have been attenuated by inactivation of genes in the aromatic amino acid biosynthesis pathway. Aromatic-dependent mutants of Salmonella enterica serovar Typhimurium (38), Yersinia pestis (40), Bordetella pertussis (50), Corynebacterium pseudotuberculosis (53), Pseudomonas aeruginosa (44), and Listeria monocytogenes (1) have been shown to be avirulent and to stimulate protective immunity in different hosts. Requirement of p-aminobenzoic acid (PABA), a precursor of folic acid that is not synthesized by mammals, has been singled out as the likely reason for reduced virulence of these bacterial strains (25).…”

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confidence: 99%

Attenuation and Persistence of and Ability To Induce Protective Immunity to a Staphylococcus aureus aroA Mutant in Mice

et al. 2006

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Staphylococcus aureus is the most important etiological agent of bovine mastitis, a disease that causes significant economic losses to the dairy industry. Several vaccines to prevent the disease have been tested, with limited success. The aim of this study was to obtain a suitable attenuated aro mutant of S. aureus by transposon mutagenesis and to demonstrate its efficacy as a live vaccine to induce protective immunity in a murine model of intramammary infection. To do this, we transformed S. aureus RN6390 with plasmid pTV1ts carrying Tn917. After screening of 3,493 erythromycin-resistant colonies, one mutant incapable of growing on plates lacking phenylalanine, tryptophan, and tyrosine was isolated and characterized. Molecular characterization of the mutant showed that the affected gene was aroA and that the insertion occurred 756 bp downstream of the aroA start codon. Complementation of the aroA mutant with a plasmid carrying aroA recovered the wild-type phenotype. The mutant exhibited a 50% lethal dose (1 ؋ 10 6 CFU/mouse) higher than that of the parental strain (4.3 ؋ 10 4 CFU/mouse). The aroA mutant showed decreased ability to persist in the lungs, spleens, and mammary glands of mice. Intramammary immunization with the aroA mutant stimulated both Th1 and Th2 responses in the mammary gland, as ascertained by reverse transcription-PCR, and induced significant protection from challenge with either the parental wild-type or a heterologous strain isolated from a cow with mastitis.

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“…We also determined the IgG titers in the sera of the immunized mice against whole cells of P. aeruginosa strain PAO1 by ELISA using plates coated with live PAO1 grown in type III secretion-inducing conditions. As shown in Figure 4B, no reactivity was observed in the antisera from mice immunized with the protein antigens compared with antisera to PAO1DaroA (34). This suggests that the epitopes recognized by the antigen-specific antibodies are not accessible on the bacterial surface, which likely contributes to the lack of opsonic killing activity.…”

Section: Opsonic Killing Activities and Recognition Of P Aeruginosa mentioning

confidence: 94%

Th17-stimulating Protein Vaccines Confer Protection against Pseudomonas aeruginosa Pneumonia

Jin

Duan

et al. 2012

Am J Respir Crit Care Med

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Rationale: New vaccine approaches are needed for Pseudomonas aeruginosa, which continues to be a major cause of serious pulmonary infections. Although Th17 cells can protect against gram-negative pathogens at mucosal surfaces, including the lung, the bacterial proteins recognized by Th17 cells are largely unknown and could be potential new vaccine candidates. Objectives: We describe a strategy to identify Th17-stimulating protein antigens of Pseudomonas aeruginosa to assess their efficacy as vaccines against pneumonia.Methods: Using a library of in vitro transcribed and translated P. aeruginosa proteins, we screened for Th17-stimulating antigens by coculturing the library proteins with splenocytes from mice immunized with a live-attenuated P. aeruginosa vaccine that is protective via Th17-based immunity. We measured antibody and Th17 responses after intranasal immunization of mice with the purified proteins mixed with the Th17 adjuvant curdlan, and we tested the protective efficacy of vaccination in a murine model of acute pneumonia. Measurements and Main Results: The proteins PopB, FpvA, FptA, OprL, and PilQ elicited strong IL-17 secretion in the screen, and purified versions of PopB, FpvA, and OprL stimulated high IL-17 production from immune splenocytes. Immunization with PopB, which is a highly conserved component of the type III secretion system and a known virulence factor, elicited Th17 responses and also enhanced clearance of P. aeruginosa from the lung and spleen after challenge. PopB-immunized mice were protected from lethal pneumonia in an antibody-independent, IL-17-dependent manner. Conclusions: Screening for Th17-stimulating protein antigens identified PopB as a novel and promising vaccine candidate for P. aeruginosa.Keywords: vaccine; pneumonia; Th17; IL-17; Pseudomonas aeruginosa Pseudomonas aeruginosa is a major cause of serious, often antibiotic-resistant, lung infections in humans, particularly in patients receiving mechanical ventilation and those with cystic fibrosis (1, 2). Most P. aeruginosa vaccines developed to date, including those based on the LPS O antigen (3), the outer membrane proteins F and I (4, 5), or the type III secretion system component PcrV (6), have relied on conventional protective mechanisms-namely, antibody-mediated opsonophagocytic killing and/or antibody-mediated toxin inhibition. Although LPS O antigen-based vaccines can mediate high levels of immunity to P. aeruginosa, the protection is limited to strains having the same LPS serogroup (3). The failure of the Federal Hyperimmune Immunoglobulin Trial (7), which found no benefit in critically ill adults passively immunized with P. aeruginosa LPS O antigenspecific IgG, perhaps best illustrates that antibody-mediated protective mechanisms are not sufficient.Th17 cells have recently been shown to mediate antibodyindependent host defense against Klebsiella pneumoniae (8), although the bacterial proteins recognized by the Th17 cells in those studies were not fully characterized. In our own evaluations of live-attenuated P. ...

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Construction and Characterization of a Live, AttenuatedaroADeletion Mutant ofPseudomonas aeruginosaas a Candidate Intranasal Vaccine

Cited by 64 publications

References 60 publications

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

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

Attenuation and Persistence of and Ability To Induce Protective Immunity to a Staphylococcus aureus aroA Mutant in Mice

Th17-stimulating Protein Vaccines Confer Protection against Pseudomonas aeruginosa Pneumonia

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