Tobacco use increases susceptibility to bacterial infection

Active smokers and those exposed to secondhand smoke are at increased risk of bacterial infection. Tobacco smoke exposure increases susceptibility to respiratory tract infections, including tuberculosis, pneumonia and Legionnaires disease; bacterial vaginosis and sexually transmitted diseases, such as chlamydia and gonorrhoea; Helicobacter pylori infection; periodontitis; meningitis; otitis media; and post-surgical and nosocomial infections. Tobacco smoke compromises the anti-bacterial function of leukocytes, including neutrophils, monocytes, T cells and B cells, providing a mechanistic explanation for increased infection risk. Further epidemiological, clinical and mechanistic research into this important area is warranted

Tobacco Smoke Augments Porphyromonas gingivalis - Streptococcus gordonii Biofilm Formation

Smoking is responsible for the majority of periodontitis cases in the US and smokers are more susceptible than non-smokers to infection by the periodontal pathogen Porphyromonas gingivalis. P. gingivalis colonization of the oral cavity is dependent upon its interaction with other plaque bacteria, including Streptococcus gordonii. Microarray analysis suggested that exposure of P. gingivalis to cigarette smoke extract (CSE) increased the expression of the major fimbrial antigen (FimA), but not the minor fimbrial antigen (Mfa1). Therefore, we hypothesized that CSE promotes P. gingivalis-S. gordonii biofilm formation in a FimA-dependent manner. FimA total protein and cell surface expression were increased upon exposure to CSE whereas Mfa1 was unaffected. CSE exposure did not induce P. gingivalis auto-aggregation but did promote dual species biofilm formation, monitored by microcolony numbers and depth (both, p<0.05). Interestingly, P. gingivalis biofilms grown in the presence of CSE exhibited a lower pro-inflammatory capacity (TNF-α, IL-6) than control biofilms (both, p<0.01). CSE-exposed P. gingivalis bound more strongly to immobilized rGAPDH, the cognate FimA ligand on S. gordonii, than control biofilms (p<0.001) and did so in a dose-dependent manner. Nevertheless, a peptide representing the Mfa1 binding site on S. gordonii, SspB, completely inhibited dual species biofilm formation. Thus, CSE likely augments P. gingivalis biofilm formation by increasing FimA avidity which, in turn, supports initial interspecies interactions and promotes subsequent high affinity Mfa1-SspB interactions driving biofilm growth. CSE induction of P. gingivalis biofilms of limited pro-inflammatory potential may explain the increased persistence of this pathogen in smokers. These findings may also be relevant to other biofilm-induced infectious diseases and conditions

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

Structural and Functional Variation within the Alanine-Rich Repetitive Domain of Streptococcal Antigen I/II

Members of the antigen I/II family of cell surface proteins are highly conserved, multifunctional adhesins that mediate interactions of oral streptococci with other oral bacteria, with cell matrix proteins (e.g., type I collagen), and with salivary glycoproteins, e.g., gp340. The interaction of gp340 (formerly designated salivary agglutinin) with Streptococcus mutans requires an alanine-rich repetitive domain (A region) of antigen I/II that is highly conserved in all members of this family of proteins. In this report, we show that the A regions from the two Streptococcus gordonii M5 antigen I/II proteins (SspA and SspB) interact differently with the salivary gp340 glycoprotein and appear to be structurally distinct. Recombinant polypeptides encompassing the A region of SspA or from a highly related S. mutans antigen I/II protein (SpaP) competitively inhibited the interaction of gp340 with intact S. gordonii and S. mutans cells, respectively. In contrast, an A region polypeptide from SspB was inactive, and furthermore, it did not bind to purified gp340 in vitro. Circular dichroism spectra suggested that all three polypeptides were highly α-helical and may form coiled-coil structures. However, the A region of SspB underwent a conformational change and exhibited reduced α-helical structure at pH 8.5, whereas the A region polypeptides from SspA and SpaP were relatively stable under these conditions. Melt curves also indicated that at physiological pH, the A region of SspB lost α-helical structure more rapidly than that of SspA or SpaP when the temperature was increased from 10 to 40°C. Furthermore, the SspB A region polypeptide denatured completely at a temperature that was 7 to 9°C lower than that required for the A region polypeptide of SspA or SpaP. The full-length SspB protein and the three A region peptides migrated in native gel electrophoresis and column chromatography with apparent molecular masses that were approximately 2- to 2.5-fold greater than their predicted molecular masses. However, sedimentation equilibrium ultracentrifugation data showed that the A region peptides sedimented as monomers, suggesting that the peptides may form nonglobular intramolecular coiled-coil structures under the experimental conditions used. Taken together, our results suggest that the A region of SspB is less stable than the corresponding A regions of SspA and SpaP and that this structural difference may explain, at least in part, the functional variation observed in their interactions with salivary gp340

Positive and Negative cis-Acting Regulatory Sequences Control Expression of Leukotoxin in Actinobacillus actinomycetemcomitans 652

Integration of IS1301 into an AT-rich inverted repeat located upstream of the ltx operon was previously shown to confer a hyperleukotoxic phenotype in Actinobacillus actinomycetemcomitans IS1 (T. He, T. Nishihara, D. R. Demuth, and I. Ishikawa, J. Periodontol. 70:1261-1268, 1999), but the mechanism leading to increased leukotoxin production was not determined. We show that an IS1 ltx promoter::lacZ reporter construct expresses 12-fold higher levels of β-galactosidase activity than a reporter containing the ltx promoter from A. actinomycetemcomitans 652, suggesting that IS1301 increases transcription of the ltx operon. Examination of the IS1301 sequence identified a potential outwardly directed promoter. However, site-specific mutagenesis of the −35 element of the putative promoter had no effect on the transcriptional activity of the IS1 reporter construct. Furthermore, reverse transcriptase PCR and real-time PCR experiments did not detect a transcript that was initiated within IS1301. These results suggest that increased expression of leukotoxin in strain IS1 does not arise from an outwardly directed IS1301 promoter. To determine how IS1301 alters transcriptional regulation of the ltx operon, cis-acting sequences that regulate leukotoxin expression were identified. The AT-rich sequence that resides downstream from the site of IS1301 insertion was shown to function as a positive cis-acting regulator of leukotoxin expression. This sequence resembles an UP element in its location, AT-rich content, and activity and is homologous to the consensus UP element sequence. In addition, a negative cis-acting sequence was identified upstream from the site of IS1301 insertion, and deletion of this region increased promoter activity by fourfold. Mobility shift experiments showed that this region bound to a protein(s) in extracts from A. actinomycetemcomitans 652. The specific sequences required for this interaction were localized to a 26-nucleotide segment of the ltx promoter that resides 17 bp upstream from the site of IS1301 insertion. Together, these results suggest that positive and negative cis-acting sequences regulate leukotoxin expression and that IS1301 may increase transcription of the ltx operon in A. actinomycetemcomitans IS1 by displacing a negative cis-acting regulator approximately 900 bp upstream from the basal elements of the ltx promoter

Exposure of Lymphocytes to High Doses of Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Induces Rapid Onset of Apoptosis-Mediated DNA Fragmentation

We have shown previously that treatment of human lymphocytes with the Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) results in dose-dependent G(2) arrest, followed 24 h later by apoptotic cell death. Here we demonstrated that for Jurkat cells exposed to high concentrations of Cdt (>0.2 ng/ml) there was a dose-dependent increase in the level of S-phase cells and a concomitant decrease in the level of G(2) cells. Fluorescence-activated cell sorter analysis demonstrated that the S-phase cells did not incorporate BrdU and likely represented cells that arrested in G(2) and underwent significant DNA fragmentation. Analysis of the kinetics of the appearance of both S-phase cells and apoptotic cells supported this interpretation. Cells exposed to low doses of toxin exhibited G(2) arrest at 24 h, but at 48 and 72 h there were also decreases in the level of G(2) cells and concomitant increases in the levels of S, G(0)/G(1), and sub-G(0) cells; these changes were paralleled by increased numbers of apoptotic cells. Cells exposed to high doses of toxin exhibited these changes 24 to 48 h earlier. We also examined the relationship between G(2) arrest, DNA fragmentation, and activation of the apoptotic cascade. We employed two inhibitors of apoptosis, overexpression of Bcl-2 and the caspase-3 inhibitor zvad. Both inhibitors blocked Cdt-induced apoptosis, Cdt-induced DNA fragmentation, and phosphorylation of the histone H2AX. However, the cells retained the ability to undergo G(2) arrest in the presence of the toxin. Thus, it appears that high doses of Cdt induce rapid onset of DNA degradation resulting from activation of the apoptotic cascade

luxS and arcB Control Aerobic Growth of Actinobacillus actinomycetemcomitans under Iron Limitation

LuxS is responsible for the production of autoinducer 2 (AI-2), which functions in Vibrio harveyi as a quorum-sensing signal that controls the cell density-dependent expression of the lux operon. In nonluminescent organisms, the physiologic role of AI-2 is not clear. We report that inactivation of luxS in Actinobacillus actinomycetemcomitans JP2 results in reduced growth of the mutant, but not the wild-type organism, under aerobic, iron-limited conditions. Stunted cultures of the luxS mutant A. actinomycetemcomitans JP2-12 grew to high cell density when subcultured under iron-replete conditions. In addition, the mutant strain grew to high cell density under iron limitation after transformation with a plasmid containing a functional copy of luxS. Results of real-time PCR showed that A. actinomycetemcomitans JP2-12 exhibited significantly reduced expression of afuA (eightfold), fecBCDE (10-fold), and ftnAB (>50-fold), which encode a periplasmic ferric transport protein, a putative ferric citrate transporter, and ferritin, respectively. The expressions of putative receptors for transferrin, hemoglobin, and hemophore binding protein were also reduced at more modest levels (two- to threefold). In contrast, expressions of sidD and frpB (encoding putative siderophore receptors) were increased 10- and 3-fold, respectively, in the luxS mutant. To better understand the mechanism of the AI-2 response, the A. actinomycetemcomitans genome was searched for homologs of the V. harveyi signal transduction proteins, LuxP, LuxQ, LuxU, and LuxO. Interestingly, ArcB was found to be most similar to LuxQ sensor/kinase. To determine whether arcB plays a role in the response of A. actinomycetemcomitans to AI-2, an arcB-deficient mutant was constructed. The isogenic arcB mutant grew poorly under anaerobic conditions but grew normally under aerobic iron-replete conditions. However, the arcB mutant failed to grow aerobically under iron limitation, and reverse transcriptase PCR showed that inactivation of arcB resulted in decreased expression of afuA and ftnAB. Thus, isogenic luxS and arcB mutants of A. actinomycetemcomitans exhibit similar phenotypes when cultured aerobically under iron limitation, and both mutants exhibit reduced expression of a common set of genes involved in the transport and storage of iron. These results suggest that LuxS and ArcB may act in concert to control the adaptation of A. actinomycetemcomitans to iron-limiting conditions and its growth under such conditions