Tobacco Upregulates P. gingivalis Fimbrial Proteins Which Induce TLR2 Hyposensitivity

Tobacco smokers are more susceptible to periodontitis than non-smokers but exhibit reduced signs of clinical inflammation. The underlying mechanisms are unknown. We have previously shown that cigarette smoke extract (CSE) represents an environmental stress to which P. gingivalis adapts by altering the expression of several virulence factors - including major and minor fimbrial antigens (FimA and Mfa1, respectively) and capsule - concomitant with a reduced pro-inflammatory potential of intact P. gingivalis.We hypothesized that CSE-regulation of capsule and fimbrial genes is reflected at the ultrastructural and functional levels, alters the nature of host-pathogen interactions, and contributes to the reduced pro- inflammatory potential of smoke exposed P. gingivalis. CSE induced ultrastructural alterations were determined by electron microscopy, confirmed by Western blot and physiological consequences studied in open-flow biofilms. Inflammatory profiling of specific CSE-dysregulated proteins, rFimA and rMfa1, was determined by quantifying cytokine induction in primary human innate and OBA-9 cells. CSE up-regulates P. gingivalis FimA at the protein level, suppresses the production of capsular polysaccharides at the ultrastructural level, and creates conditions that promote biofilm formation. We further show that while FimA is recognized by TLR2/6, it has only minimal inflammatory activity in several cell types. Furthermore, FimA stimulation chronically abrogates the pro-inflammatory response to subsequent TLR2 stimulation by other TLR-2-specific agonists (Pam3CSK4, FSL, Mfa1) in an IkappaBalpha- and IRAK-1-dependent manner.These studies provide some of the first information to explain, mechanistically, how tobacco smoke changes the P. gingivalis phenotype in a manner likely to promote P. gingivalis colonization and infection while simultaneously reducing the host response to this major mucosal pathogen

Mutations in DivL and CckA Rescue a divJ Null Mutant of Caulobacter crescentus by Reducing the Activity of CtrA

Polar development and cell division in Caulobacter crescentus are controlled and coordinated by multiple signal transduction proteins. divJ encodes a histidine kinase. A null mutation in divJ results in a reduced growth rate, cell filamentation, and mislocalized stalks. Suppressor analysis of divJ identified mutations in genes encoding the tyrosine kinase (divL) and the histidine kinase (cckA). The divL and cckA suppressor alleles all have single amino acid substitutions, some of which confer a temperature-sensitive phenotype, particularly in a wild-type background. Analysis of transcription levels from several positively regulated CtrA-dependent promoters reveals high expression in the divJ mutant, suggesting that DivJ normally serves to reduce CtrA activity. The divL and cckA suppressors reduce the amount of transcription from promoters positively regulated by CtrA, indicating that the mutations in divL and cckA are suppressing the defects of the divJ mutant by reducing the abnormally high level of CtrA activity. Immunoblotting showed no major perturbations in the CtrA protein level in any of these strains, suggesting that the high amount of CtrA activity seen in the divJ mutant and the reduced amount of activity in the suppressors are regulated at the level of activation and not transcription, translation, or degradation. In vivo phosphorylation assays confirmed that divJ mutants have elevated levels of CtrA phosphorylation and that this level is reduced in the suppressors with mutations in divL

Host Adhesive Activities and Virulence of Novel Fimbrial Proteins of Porphyromonas gingivalis▿

The fimbriae of Porphyromonas gingivalis mediate critical roles in host colonization and evasion of innate defenses and comprise polymerized fimbrilin (FimA) associated with quantitatively minor accessory proteins (FimCDE) of unknown function. We now show that P. gingivalis fimbriae lacking FimCDE fail to interact with the CXC-chemokine receptor 4 (CXCR4), and bacteria expressing FimCDE-deficient fimbriae cannot exploit CXCR4 in vivo for promoting their persistence, as the wild-type organism does. Consistent with these loss-of-function experiments, purified FimC and FimD (but not FimE) were shown to interact with CXCR4. However, significantly stronger binding was observed when a combination of all three proteins was allowed to interact with CXCR4. In addition, FimC and FimD bound to fibronectin and type 1 collagen, whereas FimE failed to interact with these matrix proteins. These data and the fact that FimE is required for the association of FimCDE with P. gingivalis fimbriae suggest that FimE may recruit FimC and FimD into a functional complex, rather than directly binding host proteins. Consistent with this notion, FimE was shown to bind both FimC and FimD. In summary, the FimCDE components cooperate and impart critical adhesive and virulence properties to P. gingivalis fimbriae

FimA induced tolerance reduces Iκ-Bα degradation.

<p>(A) Human PBMCs were pre-treated with 1 µg/ml rFimA for 24 hrs before stimulation with 1 µg/ml of rMfa1 for various timepoints. Immunoblots (25 µg protein per well) were probed for IκBα and re-probed β-actin to ensure equal loading. (B) Mean (s.e.) normalized band intensities.</p

Quantitative characteristics of <i>P. gingivalis</i> biofilms.

<p>Composite image data were analyzed using Matlab softwares to obtain biomass, average thickness; and substratum coverage of 48 h homotypic <i>P. gingivalis</i> biofilms formed with GAM and GAM-CSE.</p><p>**<i>p</i><0.01; ***<i>p</i><0.001.</p

rFimA and rMfa1 signal preferentially through TLR2/6.

<p>(A) HEK 293 cells stably expressing TLR2, 4, 2/1, 2/6 or TLR4-CD14-MD2 were stimulated with 1 µg/ml of rFimA, rMfa1, the classic TLR2-specific agonist, Pam3CSK4 or the classic TLR4-specific agonist <i>E. coli</i> LPS. IL-8 release was quantified in 20 hr cell-free supernatants by ELISA. n.d.  =  not detected (below assay threshold); *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. (B) THP-1 Blue cells are stably transfected with a reporter plasmid expressing secreted embryonic alkaline phosphatase (SEAP) gene under the control of a NF-κB-inducible promoter. THP-1 Blue cells were stimulated with 1 µg/ml of rFimA, rMfa1, Pam3CSK4 or <i>E. coli</i> LPS. Relative expression levels of SEAP (reflecting NF-κB) in cell-free supernatants were determined by spectrophotometric analysis of SEAP activity at 655 nm. Unstimulated cells represent the 100% control. Here we show that all TLR-agonists employed are equally capable of inducing NF-κB. ***<i>p</i><0.001 compared to unstimulated cells.</p