Construction and characterization of a<i>Porphyromonas gingivalis htpG</i>disruption mutant

Sweier, Domenica G.; Combs, Allison; Shelburne, Charles E.; Fenno, J. Christopher; Lopatin, Dennis E.

doi:10.1016/s0378-1097(03)00506-8

Cited by 13 publications

(12 citation statements)

References 19 publications

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“…Construction of the other mutant strains was carried out in a similar manner: the BglII, NaeI, and ApaI sites in the wecA, wbaP, and wzt regions, respectively, were utilized for insertion of the ermF-ermAM cassette. Construction of the htpG mutant as a control strain was also performed, since HtpG, which is a homolog of the human heat shock protein Hsp90, was reported to have no effect on the growth of P. gingivalis or on its adherence to other bacterial and human cells (21,36). The PmaCI site in the htpG region was utilized for insertion of the ermF-ermAM cassette.…”

Section: Methodsmentioning

confidence: 99%

Porphyromonas gingivalis galEIs Involved in Lipopolysaccharide O-Antigen Synthesis and Biofilm Formation

2006

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Porphyromonas gingivalis is a crucial component of complex plaque biofilms that form in the oral cavity, resulting in the progression of periodontal disease. To elucidate the mechanism of periodontal biofilm formation, we analyzed the involvement of several genes related to the synthesis of polysaccharides in P. gingivalis. Gene knockout P. gingivalis mutants were constructed by insertion of an ermF-ermAM cassette; among these mutants, the galE mutant showed some characteristic phenotypes involved in the loss of GalE activity. As expected, the galE mutant accumulated intracellular carbohydrates in the presence of 0.1% galactose and did not grow in the presence of galactose at a concentration greater than 1%, in contrast to the parental strain. Lipopolysaccharide (LPS) analysis indicated that the length of the O-antigen chain of the galE mutant was shorter than that of the wild type. It was also demonstrated that biofilms generated by the galE mutant had an intensity 4.5-fold greater than those of the wild type. Further, the galE mutant was found to be significantly susceptible to some antibiotics in comparison with the wild type. In addition, complementation of the galE mutation led to a partial recovery of the parental phenotypes. We concluded that the galE gene plays a pivotal role in the modification of LPS O antigen and biofilm formation in P. gingivalis and considered that our findings of a relationship between the function of the P. gingivalis galE gene and virulence phenotypes such as biofilm formation may provide clues for understanding the mechanism of pathogenicity in periodontal disease.

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Section: Methodsmentioning

confidence: 99%

Porphyromonas gingivalis galEIs Involved in Lipopolysaccharide O-Antigen Synthesis and Biofilm Formation

2006

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“…This provided clues about the gene products that require further study. The gene encoding the P. gingivalis Hsp90 homologue, which was one of the most upregulated genes in this study, had been knocked out in a previous study and, unexpectedly, failed to show any phenotype with respect to bacterial adherence or invasion of cultured human epithelial cells (165).…”

Section: Infection Regulates Stress Protein Synthesis In Parasite Andmentioning

confidence: 64%

Stress Wars: the Direct Role of Host and Bacterial Molecular Chaperones in Bacterial Infection

2006

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“…An E. tarda htpG mutant was attenuated for virulence in Japanese flounder, as demonstrated by diminished recovery of bacteria from blood and reduced fish mortality; the strain also had defects in growth at elevated temperatures (27). In contrast, analysis of a Porphyromonas gingivalis htpG mutant in cell adhesion and invasion assays found that its virulence was not impaired (38). Accordingly, we performed a number of phenotypic assays to investigate possible mechanisms responsible for the attenuation of the leptospiral htpG mutant.…”

Section: Resultsmentioning

confidence: 99%

“…The whole-cell lysates and LPS samples were analyzed by 12.5% SDS-PAGE and immunoblotting using standard methods (37). The immunoblots were blocked and were probed with a rabbit antiserum against either Loa22 (11) (dilution, 1:2,000), whole cells of serovar Manilae (dilution, 1:1,000) (9), or recombinant HtpG from Porphyromonas gingivalis (dilution, 1:1,000) (38) in Tris-buffered saline-Tween (TBS-T). The membranes were washed three times in TBS-T, probed with horseradish peroxidase-conjugated anti-rabbit IgG (diluted 1:5,000) (Millipore), developed using Amersham ECL Western blotting detection reagents (GE Healthcare), and exposed to a LAS3000 chemiluminescence imager (Fujifilm).…”

Section: Sds-page and Immunoblottingmentioning

confidence: 99%

High-Temperature Protein G Is an Essential Virulence Factor of Leptospira interrogans

et al. 2014

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e Leptospira interrogans is a global zoonotic pathogen and is the causative agent of leptospirosis, an endemic disease of humans and animals worldwide. There is limited understanding of leptospiral pathogenesis; therefore, further elucidation of the mechanisms involved would aid in vaccine development and the prevention of infection. HtpG (high-temperature protein G) is the bacterial homolog to the highly conserved molecular chaperone Hsp90 and is important in the stress responses of many bacteria. The specific role of HtpG, especially in bacterial pathogenesis, remains largely unknown. Through the use of an L. interrogans htpG transposon insertion mutant, this study demonstrates that L. interrogans HtpG is essential for virulence in the hamster model of acute leptospirosis. Complementation of the htpG mutant completely restored virulence. Surprisingly, the htpG mutant did not appear to show sensitivity to heat or oxidative stress, phenotypes common in htpG mutants in other bacterial species. Furthermore, the mutant did not show increased sensitivity to serum complement, reduced survival within macrophages, or altered protein or lipopolysaccharide expression. The underlying cause for attenuation thus remains unknown, but HtpG is a novel leptospiral virulence factor and one of only a very small number identified to date.

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Construction and characterization of aPorphyromonas gingivalis htpGdisruption mutant

Cited by 13 publications

References 19 publications

Porphyromonas gingivalis galEIs Involved in Lipopolysaccharide O-Antigen Synthesis and Biofilm Formation

Porphyromonas gingivalis galEIs Involved in Lipopolysaccharide O-Antigen Synthesis and Biofilm Formation

Stress Wars: the Direct Role of Host and Bacterial Molecular Chaperones in Bacterial Infection

High-Temperature Protein G Is an Essential Virulence Factor of Leptospira interrogans

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