Resistance of a Tn4351-generated polysaccharide mutant of Porphyromonas gingivalis to polymorphonuclear leukocyte killing

C. A. Genco, R. E. Schifferle, T. Njoroge, R. Y. Forng, Christopher W Cutler

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Abstract

In this study, we describe the development of an efficient transpositional mutagenesis system for Porphyromonas gingivalis using the Bacteroides fragilis transposon Tn4351. Using this system, we have isolated and characterized a Tn4351-generated mutant of P. gingivalis A7436, designated MSM-1, which exhibits enhanced resistance to polymorphonuclear leukocyte (PMN) phagocytosis and killing. P. gingivalis MSM-1 was initially selected based on its colony morphology; MSM-1 appeared as a mucoid, beige-pigmented colony. Analysis of P. gingivalis MSM-1 by electron microscopy and staining with ruthenium red revealed the presence of a thick ruthenium red-staining layer that was twice the thickness of this layer observed in the parent strain. P. gingivalis MSM-1 was found to be more hydrophilic than strain A7436 by hydrocarbon partitioning. Analysis of phenol-water extracts prepared from P. gingivalis A7436 and MSM-1 by Western (immunoblot) analysis and immunodiffusion with hyperimmune sera raised against A7436 and MSM-1 revealed the loss of a high-molecular-weight anionic polysaccharide component in extracts prepared from MSM-1. P. gingivalis MSM-1 was also found to be more resistant to PMN phagocytosis and intracellular killing than the parent strain, as assessed in a fluorochrome phagocytosis microassay. These differences were statistically significant (P < 0.05) when comparing PMN phagocytosis in nonimmune serum and intracellular killing in nonimmune and immune sera. P. gingivalis MSM-1 was also more resistant to killing by crude granule extracts from PMNs than was P. gingivalis A7436. These results indicate that the increased evasion of PMN phagocytosis and killing exhibited by P. gingivalis MSM-1 may result from alterations in polysaccharide- containing antigens.

Original languageEnglish (US)
Pages (from-to)393-401
Number of pages9
JournalInfection and Immunity
Volume63
Issue number2
StatePublished - Jan 1 1995
Externally publishedYes

Fingerprint

Porphyromonas gingivalis
Polysaccharides
Neutrophils
Phagocytosis
Ruthenium Red
Staining and Labeling
Bacteroides fragilis
Immunodiffusion
Hydrocarbons
Phenol
Complex Mixtures
Serum
Fluorescent Dyes
Mutagenesis
Immune Sera
Electron Microscopy
Molecular Weight
Western Blotting
Antigens

ASJC Scopus subject areas

  • Immunology

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Resistance of a Tn4351-generated polysaccharide mutant of Porphyromonas gingivalis to polymorphonuclear leukocyte killing. / Genco, C. A.; Schifferle, R. E.; Njoroge, T.; Forng, R. Y.; Cutler, Christopher W.

In: Infection and Immunity, Vol. 63, No. 2, 01.01.1995, p. 393-401.

Research output: Contribution to journalArticle

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abstract = "In this study, we describe the development of an efficient transpositional mutagenesis system for Porphyromonas gingivalis using the Bacteroides fragilis transposon Tn4351. Using this system, we have isolated and characterized a Tn4351-generated mutant of P. gingivalis A7436, designated MSM-1, which exhibits enhanced resistance to polymorphonuclear leukocyte (PMN) phagocytosis and killing. P. gingivalis MSM-1 was initially selected based on its colony morphology; MSM-1 appeared as a mucoid, beige-pigmented colony. Analysis of P. gingivalis MSM-1 by electron microscopy and staining with ruthenium red revealed the presence of a thick ruthenium red-staining layer that was twice the thickness of this layer observed in the parent strain. P. gingivalis MSM-1 was found to be more hydrophilic than strain A7436 by hydrocarbon partitioning. Analysis of phenol-water extracts prepared from P. gingivalis A7436 and MSM-1 by Western (immunoblot) analysis and immunodiffusion with hyperimmune sera raised against A7436 and MSM-1 revealed the loss of a high-molecular-weight anionic polysaccharide component in extracts prepared from MSM-1. P. gingivalis MSM-1 was also found to be more resistant to PMN phagocytosis and intracellular killing than the parent strain, as assessed in a fluorochrome phagocytosis microassay. These differences were statistically significant (P < 0.05) when comparing PMN phagocytosis in nonimmune serum and intracellular killing in nonimmune and immune sera. P. gingivalis MSM-1 was also more resistant to killing by crude granule extracts from PMNs than was P. gingivalis A7436. These results indicate that the increased evasion of PMN phagocytosis and killing exhibited by P. gingivalis MSM-1 may result from alterations in polysaccharide- containing antigens.",
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N2 - In this study, we describe the development of an efficient transpositional mutagenesis system for Porphyromonas gingivalis using the Bacteroides fragilis transposon Tn4351. Using this system, we have isolated and characterized a Tn4351-generated mutant of P. gingivalis A7436, designated MSM-1, which exhibits enhanced resistance to polymorphonuclear leukocyte (PMN) phagocytosis and killing. P. gingivalis MSM-1 was initially selected based on its colony morphology; MSM-1 appeared as a mucoid, beige-pigmented colony. Analysis of P. gingivalis MSM-1 by electron microscopy and staining with ruthenium red revealed the presence of a thick ruthenium red-staining layer that was twice the thickness of this layer observed in the parent strain. P. gingivalis MSM-1 was found to be more hydrophilic than strain A7436 by hydrocarbon partitioning. Analysis of phenol-water extracts prepared from P. gingivalis A7436 and MSM-1 by Western (immunoblot) analysis and immunodiffusion with hyperimmune sera raised against A7436 and MSM-1 revealed the loss of a high-molecular-weight anionic polysaccharide component in extracts prepared from MSM-1. P. gingivalis MSM-1 was also found to be more resistant to PMN phagocytosis and intracellular killing than the parent strain, as assessed in a fluorochrome phagocytosis microassay. These differences were statistically significant (P < 0.05) when comparing PMN phagocytosis in nonimmune serum and intracellular killing in nonimmune and immune sera. P. gingivalis MSM-1 was also more resistant to killing by crude granule extracts from PMNs than was P. gingivalis A7436. These results indicate that the increased evasion of PMN phagocytosis and killing exhibited by P. gingivalis MSM-1 may result from alterations in polysaccharide- containing antigens.

AB - In this study, we describe the development of an efficient transpositional mutagenesis system for Porphyromonas gingivalis using the Bacteroides fragilis transposon Tn4351. Using this system, we have isolated and characterized a Tn4351-generated mutant of P. gingivalis A7436, designated MSM-1, which exhibits enhanced resistance to polymorphonuclear leukocyte (PMN) phagocytosis and killing. P. gingivalis MSM-1 was initially selected based on its colony morphology; MSM-1 appeared as a mucoid, beige-pigmented colony. Analysis of P. gingivalis MSM-1 by electron microscopy and staining with ruthenium red revealed the presence of a thick ruthenium red-staining layer that was twice the thickness of this layer observed in the parent strain. P. gingivalis MSM-1 was found to be more hydrophilic than strain A7436 by hydrocarbon partitioning. Analysis of phenol-water extracts prepared from P. gingivalis A7436 and MSM-1 by Western (immunoblot) analysis and immunodiffusion with hyperimmune sera raised against A7436 and MSM-1 revealed the loss of a high-molecular-weight anionic polysaccharide component in extracts prepared from MSM-1. P. gingivalis MSM-1 was also found to be more resistant to PMN phagocytosis and intracellular killing than the parent strain, as assessed in a fluorochrome phagocytosis microassay. These differences were statistically significant (P < 0.05) when comparing PMN phagocytosis in nonimmune serum and intracellular killing in nonimmune and immune sera. P. gingivalis MSM-1 was also more resistant to killing by crude granule extracts from PMNs than was P. gingivalis A7436. These results indicate that the increased evasion of PMN phagocytosis and killing exhibited by P. gingivalis MSM-1 may result from alterations in polysaccharide- containing antigens.

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