Isolation and characterization of transposon-induced mutants of deficient in fimbriation

Watanabe-Kato, Tomomi; Hayashi, Jun-ichiro; Terazawa, Yoshihiro; Hoover, Charles I.; Nakayama, Koji; Hibi, E; Kawakami, Naoko; Ikeda, Takeshi; Nakamura, Hiroshi; Noguchi, Toshihide; Yoshimura, Fuminobu

doi:10.1006/mpat.1997.0170

Cited by 33 publications

(22 citation statements)

References 24 publications

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“…In addition, the molecular mechanism involved in the regulation of fimA expression is beginning to be understood. A two-component system (FimS-FimR) that regulates expression has been identified (Hayashi et al, 2000;Watanabe-Kato et al, 1998;Watanabe et al, 1996), and recently it was shown that the response regulator of this two-component system (FimR) controls the expression of these genes at the transcription level (Nishikawa et al, 2004). In regard to mfa1, expression of this gene has been shown to be affected by cell-to-cell contact with a variety of oral streptococci; either upregulation or downregulation was observed, depending on the species (Park et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

A streptococcal effector protein that inhibits Porphyromonas gingivalis biofilm development

Christopher¹,

Arndt

Cugini³

et al. 2010

Microbiology

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Dental plaque formation is a developmental process involving cooperation and competition within a diverse microbial community, approximately 70 % of which is composed of an array of streptococci during the early stages of supragingival plaque formation. In this study, 79 cell-free culture supernatants from a variety of oral streptococci were screened to identify extracellular compounds that inhibit biofilm formation by the oral anaerobe Porphyromonas gingivalis strain 381. The majority of the streptococcal supernatants (61 isolates) resulted in lysis of P. gingivalis cells, and some (17 isolates) had no effect on cell viability, growth or biofilm formation. One strain, however, produced a supernatant that abolished biofilm formation without affecting growth rate. Analysis of this activity led to the discovery that a 48 kDa protein was responsible for the inhibition. Protein sequence identification and enzyme activity assays identified the effector protein as an arginine deiminase. To identify the mechanism(s) by which this protein inhibits biofilm formation, we began by examining the expression levels of genes encoding fimbrial subunits; surface structures known to be involved in biofilm development. Quantitative RT-PCR analysis revealed that exposure of P. gingivalis cells to this protein for 1 h resulted in the downregulation of genes encoding proteins that are the major subunits of two distinct types of thin, single-stranded fimbriae (fimA and mfa1). Furthermore, this downregulation occurred in the absence of arginine deiminase enzymic activity. Hence, our data indicate that P. gingivalis can sense this extracellular protein, produced by an oral streptococcus (Streptococcus intermedius), and respond by downregulating expression of cell-surface appendages required for attachment and biofilm development. INTRODUCTIONHigh cell density and microbial diversity are fundamental to the development and function of the oral biofilm (Kolenbrander et al., 2002). The close proximity of cells within the biofilm facilitates cell-cell interactions, while the diversity offers a wide variety of metabolic functions, allowing the community to acquire nutrients and persist. During plaque formation, organisms affect the activities of one another via physical associations and through the release of extracellular molecules; the effects of these interactions range from mutualistic to antagonistic (Davey & Costerton, 2006;Socransky & Haffajee, 2000). Molecules that are excreted by bacteria into the environment or directly into host cells (through either non-classical or conventional secretion pathways), affecting gene expression of another organism, are often referred to as effectors. The primary objective of this study was to identify effectors produced by oral streptococci that modulate biofilm formation by the oral anaerobe Porphyromonas gingivalis. P. gingivalis is a Gram-negative anaerobe that persists within the oral biofilm community. Outgrowth of this normally commensal organism is associated with severe periodontal disease, r...

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Section: Discussionmentioning

confidence: 99%

A streptococcal effector protein that inhibits Porphyromonas gingivalis biofilm development

Christopher¹,

Arndt

Cugini³

et al. 2010

Microbiology

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“…Transposon-mediated mutagenesis in P. gingivalis has been successfully used to identify novel genes involved in the regulation of gingipain anchoring and expression as well as fimbriation, suggesting that it is a simple and powerful strategy to identify novel virulence factors in this bacterium (Watanabe-Kato et al, 1998;Simpson et al, 1999;Shoji et al, 2002). The utility of the Tn4400' transposon system for generating random genomic mutations in P. gingivalis was previously demonstrated (Chen et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Porphyromonas gingivalis Resistance to Polymyxin B Is Determined by the Lipid A 4′‐Phosphatase, PGN_0524

Coats

Jain

et al. 2009

Int J Oral Sci

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). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 µg⋅mL -1 ). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P.gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate. Conclusion The combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR4 sensing.

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“…The in vivo signal that triggers fimbrial expression is not known; however, it has been shown that expression of fimA, which encodes the fimbrillin protein subunit, is affected by environmental and nutritional parameters, since expression was repressed under elevated temperatures or hemin limitation (4,58,88). A two-component system (FimS-FimR) that regulates the expression of fimA was recently discovered (33,81,83), and it has been shown that the response regulator FimR controls expression at the level of transcription (65). Further studies of the function of PG0106 and the regulation of the expression of capsule are required to determine if the expressions of fimbriae and capsule are coordinated.…”

Section: Discussionmentioning

confidence: 99%

Enhanced Biofilm Formation and Loss of Capsule Synthesis: Deletion of a Putative Glycosyltransferase inPorphyromonas gingivalis

Davey¹,

Duncan²

2006

J Bacteriol

112

104

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Periodontitis is a biofilm-mediated disease. Porphyromonas gingivalis is an obligate anaerobe consistently associated with severe manifestations of this disease. As an opportunistic pathogen, the ability to proliferate within and disseminate from subgingival biofilm (plaque) is central to its virulence. Here, we report the isolation of a P. gingivalis transposon insertion mutant altered in biofilm development and the reconstruction and characterization of this mutation in three different wild-type strains. The mutation responsible for the altered biofilm phenotype was in a gene with high sequence similarity (ϳ61%) to a glycosyltransferase gene. The gene is located in a region of the chromosome that includes up to 16 genes predicted to be involved in the synthesis and transport of capsular polysaccharide. The phenotype of the reconstructed mutation in all three wild-type backgrounds is that of enhanced biofilm formation. In addition, in strain W83, a strain that is encapsulated, the glycosyltransferase mutation resulted in a loss of capsule. Further experiments showed that the W83 mutant strain was more hydrophobic and exhibited increased autoaggregation. Our results indicate that we have identified a gene involved in capsularpolysaccharide synthesis in P. gingivalis and that the production of capsule prevented attachment and the initiation of in vitro biofilm formation on polystyrene microtiter plates.The clinical importance of dental plaque and its accessibility for in vivo research makes it one of most studied and bestunderstood biofilm communities. It is now well documented that gram-negative anaerobic bacteria play a significant role in the development of periodontitis, with Porphyromonas gingivalis being implicated as one of the major players in the progression of this chronic disease (10,19,30,61). A number of factors are associated with the virulence of the organism, including a variety of proteases, endotoxins, and collegenases and the production of surface structures, such as fimbriae and capsular polysaccharide (38). This opportunistic pathogen also attaches to and invades human epithelial cells (18,53,56,57), connective tissue, and endothelial cells (15,17,55), and invasion has been shown to be mediated by the expression of fimbriae and a variety of surface adhesins. It is evident that the growth of P. gingivalis within the subgingival plaque is central to the disease process; however, although there have been numerous studies of the pathogenicity of the organism and its interactions with other organisms within the biofilm community, little is known about the molecular-genetic basis of biofilm formation in P. gingivalis.Research on oral biofilms has focused on determining the spatial organization and complex development of plaque by examining the succession of organisms in the growing biofilm. By monitoring the development of the microbial community on the tooth surface after professional cleaning, studies have demonstrated that the early colonizers are primarily grampositive organisms and the late, o...

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Isolation and characterization of transposon-induced mutants of deficient in fimbriation

Cited by 33 publications

References 24 publications

A streptococcal effector protein that inhibits Porphyromonas gingivalis biofilm development

A streptococcal effector protein that inhibits Porphyromonas gingivalis biofilm development

Porphyromonas gingivalis Resistance to Polymyxin B Is Determined by the Lipid A 4′‐Phosphatase, PGN_0524

Enhanced Biofilm Formation and Loss of Capsule Synthesis: Deletion of a Putative Glycosyltransferase inPorphyromonas gingivalis

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