Ali Khammanivong

ANTI-INFECTIVE PROTECTIVE PROPERTIES OF S100 CALGRANULINS.

The calgranulins are a subgroup of proteins in the S100 family (calgranulin A, S100A8; calgranulin B, S100A9 and calgranulin C, S100A12) that provide protective anti-infective and anti-inflammatory functions for the mammalian host. In this review, we discuss the structure-function relationships whereby S100A8 and S100A9, and for comparison, S100A12, provide intra- and extracellular protection during the complex interplay between infection and inflammation and how the calgranulins are regulated to optimally protect the host. Ideally located to support epithelial barrier function, calprotectin, a complex of S100A8/S100A9, is expressed in squamous mucosal keratinocytes and innate immune cells present at mucosal surfaces. The calgranulins are also abundantly produced in neutrophils and monocytes, whereas expression is induced in epidermal keratinocytes, gastrointestinal epithelial cells and fibroblasts during inflammation. The calgranulins show species-specific expression and function. For example, S100A8 is chemotactic in rodents but not in humans. In humans, S100A12 appears to serve as a functional chemotactic homolog to murine S100A8. Transition metal-binding and oxidation sites within calgranulins are able to create structural changes that may orchestrate new protective functions or binding targets. The calgranulins thus appear to adopt a variety of roles to protect the host. In addition to serving as a leukocyte chemoattractant, protective functions include oxidant scavenging, antimicrobial activity, and chemokine-like activities. Each function may reflect the concentration of the calgranulin, post-transcriptional modifications, oligomeric forms, and the proximal intracellular or extracellular environments. Calprotectin and the calgranulins are remarkable as multifunctional proteins dedicated to protecting the intra- and extracellular environments during infection and inflammation.

Involvement of Streptococcus gordonii Beta-Glucoside Metabolism Systems in Adhesion, Biofilm Formation, and In Vivo Gene Expression

Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides. Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci. Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.

Oral Streptococci and Cardiovascular Disease: Searching for the Platelet Aggregation-Associated Protein Gene and Mechanisms of Streptococcus sanguis-Induced Thrombosis

BACKGROUND Pathogenic mechanisms in infective endocarditis, disseminated intravascular coagulation, and cardiovascular events involve the aggregation of platelets into thrombi. Attendant infection by oral bacteria contributes to these diseases. We have been studying how certain oral streptococci induce platelet aggregation in vitro and in vivo. Streptococcus sanguis expresses a platelet aggregation-associated protein (PAAP), which contributes little to adhesion to platelets. When specific antibodies or peptides block PAAP, S. sanguis fails to induce platelet aggregation in vitro or in vivo. METHODS We used subtractive hybridization to identify the gene encoding for PAAP. RESULTS After subtraction of strain L50 (platelet aggregation-negative), four strain 133-79 specific sequences were characterized. Sequence agg4 encoded a putative collagen-binding protein (CbpA), which was predicted to contain two PAAP collagen-like octapeptide sequences. S. sanguis CbpA- mutants were constructed and tested for induction of platelet aggregation in vitro. Platelet aggregation was substantially inhibited when compared to the wild-type using platelet-rich plasma from the principal donor, but adhesion was unaffected. Other donor platelets responded normally to the CbpA- strain, suggesting additional mechanisms of response to S. sanguis. In contrast, CshA- and methionine sulfoxide reductase-negative (MsrA-) strains neither adhered nor induced platelet aggregation. CONCLUSIONS CbpA was suggested to contribute to site 2 interactions in our two-site model of platelet aggregation in response to S. sanguis. Platelet polymorphisms were suggested to contribute to the thrombogenic potential of S. sanguis.

Streptococcus gordonii Hsa Environmentally Constrains Competitive Binding by Streptococcus sanguinis to Saliva-Coated Hydroxyapatite

Competition between pioneer colonizing bacteria may determine polymicrobial succession during dental plaque development, but the ecological constraints are poorly understood. For example, more Streptococcus sanguinis than Streptococcus gordonii organisms are consistently isolated from the same intraoral sites, yet S. gordonii fails to be excluded and survives as a species over time. To explain this observation, we hypothesized that S. gordonii could compete with S. sanguinis to adhere to saliva-coated hydroxyapatite (sHA), an in vitro model of the tooth surface. Both species bound similarly to sHA, yet 10- to 50-fold excess S. gordonii DL1 reduced binding of S. sanguinis SK36 by 85 to >95%. S. sanguinis, by contrast, did not significantly compete with S. gordonii to adhere. S. gordonii competed with S. sanguinis more effectively than other species of oral streptococci and depended upon the salivary film on HA. Next, putative S. gordonii adhesins were analyzed for contributions to interspecies competitive binding. Like wild-type S. gordonii, isogenic mutants with mutations in antigen I/II polypeptides (sspAB), amylase-binding proteins (abpAB), and Csh adhesins (cshAB) competed effectively against S. sanguinis. By contrast, an hsa-deficient mutant of S. gordonii showed significantly reduced binding and competitive capabilities, while these properties were restored in an hsa-complemented strain. Thus, Hsa confers a selective advantage to S. gordonii over S. sanguinis in competitive binding to sHA. Hsa expression may, therefore, serve as an environmental constraint against S. sanguinis, enabling S. gordonii to persist within the oral cavity, despite the greater natural prevalence of S. sanguinis in plaque and saliva.

S100A8/A9 (Calprotectin) Negatively Regulates G2/M Cell Cycle Progression and Growth of Squamous Cell Carcinoma

Malignant transformation results in abnormal cell cycle regulation and uncontrolled growth in head and neck squamous cell carcinoma (HNSCC) and other cancers. S100A8/A9 (calprotectin) is a calcium-binding heterodimeric protein complex implicated in cell cycle regulation, but the specific mechanism and role in cell cycle control and carcinoma growth are not well understood. In HNSCC, S100A8/A9 is downregulated at both mRNA and protein levels. We now report that downregulation of S100A8/A9 correlates strongly with a loss of cell cycle control and increased growth of carcinoma cells. To show its role in carcinogenesis in an in vitro model, S100A8/A9 was stably expressed in an S100A8/A9-negative human carcinoma cell line (KB cells, HeLa-like). S100A8/A9 expression increases PP2A phosphatase activity and p-Chk1 (Ser345) phosphorylation, which appears to signal inhibitory phosphorylation of mitotic p-Cdc25C (Ser216) and p-Cdc2 (Thr14/Tyr15) to inactivate the G2/M Cdc2/cyclin B1 complex. Cyclin B1 expression then downregulates and the cell cycle arrests at the G2/M checkpoint, reducing cell division. As expected, S100A8/A9-expressing cells show both decreased anchorage-dependent and -independent growth and mitotic progression. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increases growth and survival and reduces Cdc2 inhibitory phosphorylation at Thr14/Tyr15. The level of S100A8/A9 endogenous expression correlates strongly with the reduced p-Cdc2 (Thr14/Tyr14) level in HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B. S100A8/A9-mediated control of the G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may suggest new therapeutic approaches.

Inactivation of Streptococcus gordonii SspAB alters expression of multiple adhesin genes.

ABSTRACT SspA and SspB (antigen I/II family proteins) can bind Streptococcusgordonii to other oral bacteria and also to salivary agglutinin glycoprotein, a constituent of the salivary film or pellicle that coats the tooth. To learn if SspA and SspB are essential for adhesion and initial biofilm formation on teeth, S. gordonii DL1 was incubated with saliva-coated hydroxyapatite (sHA) for 2 h in Todd-Hewitt broth with 20% saliva to develop initial biofilms. Sessile cells attached to sHA, surrounding planktonic cells, and free-growing cells were recovered separately. Free-growing cells expressed more sspA-specific mRNA and sspB-specific mRNA than sessile cells. Free-growing cells expressed the same levels of sspA and sspB as planktonic cells. Surprisingly, an SspA− SspB− mutant strain showed 2.2-fold greater biofilm formation on sHA than wild-type S. gordonii DL1. To explain this observation, we tested the hypothesis that inactivation of sspA and sspB genes altered the expression of other adhesin genes during initial biofilm formation in vitro. When compared to wild-type cells, expression of scaA and abpB was significantly up-regulated in the SspA− SspB− strain in sessile, planktonic, and free-growing cells. Consistent with this finding, ScaA antigen was also overexpressed in planktonic and free-growing SspA− SspB− cells compared to the wild type. SspA/B adhesins, therefore, were strongly suggested to be involved in the regulation of multiple adhesin genes.

Identification of a Novel Two-Component System in Streptococcus gordonii V288 Involved in Biofilm Formation

ABSTRACT Streptococcus gordonii is a pioneer colonizer of the teeth, contributing to the initiation of the oral biofilm called dental plaque. To identify genes that may be important in biofilm formation, a plasmid integration library of S. gordonii V288 was used. After screening for in vitro biofilm formation on polystyrene, a putative biofilm-defective mutant was isolated. In this mutant, pAK36 was inserted into a locus encoding a novel two-component system (bfr [biofilm formation related]) with two cotranscribed genes that form an operon. bfrA encodes a putative response regulator, while bfrB encodes a receptor histidine kinase. The bfr mutant and wild-type strain V288 showed similar growth rates in Todd-Hewitt broth (THB). A bfr-cat fusion strain was constructed. During growth in THB, the reporter activity (chloramphenicol acetyltransferase) was first detected in mid-log phase and reached a maximum in stationary phase, suggesting that transcription of bfr was growth stage dependent. After being harvested from THB, the bfr mutant adhered less effectively than did wild-type strain V288 to saliva-coated hydroxyapatite (sHA). To simulate pioneer colonization of teeth, S. gordonii V288 was incubated with sHA for 4 h in THB with 10% saliva to develop biofilms. RNA was isolated, and expression of bfrAB was estimated. In comparison to that of cells grown in suspension (free-growing cells), bfr mRNA expression by sessile cells on sHA was 1.8-fold greater and that by surrounding planktonic cells was 3.5-fold greater. Therefore, bfrAB is a novel two-component system regulated in association with S. gordonii biofilm formation in vitro.

The two-component system BfrAB regulates expression of ABC transporters in Streptococcus gordonii and Streptococcus sanguinis

The putative two-component system BfrAB is involved in Streptococcus gordonii biofilm development. Here, we provide evidence that BfrAB regulates the expression of bfrCD and bfrEFG, which encode two ATP-binding cassette (ABC) transporters, and bfrH, which encodes a CAAX amino-terminal protease family protein. BfrC and BfrE are ATP-binding proteins, and BfrD, BfrF and BfrG are homologous membrane-spanning polypeptides. Similarly, BfrABss, the BfrAB homologous system in Streptococcus sanguinis, controls the expression of two bfrCD-homologous operons (bfrCDss and bfrXYss), a bfrH-homologous gene (bfrH1ss) and another CAAX amino-terminal protease family protein gene (bfrH2ss). Furthermore, we demonstrate that the purified BfrA DNA-binding domain from S. gordonii binds to the promoter regions of bfrCD, bfrEFG, bfrH, bfrCDss, bfrXYss and bfrH1ss in vitro. Finally, we show that the BfrA DNA-binding domain recognizes a conserved DNA motif with a consensus sequence of TTTCTTTAGAAATATTTTAGAATT. These data suggest, therefore, that S. gordonii BfrAB controls biofilm formation by regulating multiple ABC-transporter systems.

Light pulses suppress responsiveness within the mouse photic entrainment pathway.

We have characterized a decrease in photic responsiveness of the mammalian circadian entrainment pathway caused by light stimulation. Phase delays of the running-wheel activity rhythm were used to quantify the photic responsiveness of the circadian system in mice (C57BL/6J). In an initial experiment, the authors measured the responsiveness to single “saturating” light pulses (“white” fluorescent light; [.approxequal]1876 [.mu]W; 15 min). In two additional experiments, the authors measured responses to this stimulus at several time points following a saturating pulse at CT 14 or CT 16. Data from these experiments were analyzed in two manners. Experiment 2 was analyzed assuming that the phase of the circadian pacemaker was unchanged by an initial pulse, and Experiment 3 was analyzed assuming that the initial pulse induced an instantaneous phase delay. Results reveal a significant reduction in responsivity to light that persists for at least 2 h and possibly up to 4 h after the initial stimulus. Immediately after the stimulus, the responsiveness of the photic entrainment pathway was reduced to levels [.lessequal] 12% of normal. After 2 h, the responsiveness was [.lessequal] 42% of normal, and by 4 h, responsiveness had recovered to levels that were [.lessequal] 60% of normal (levels not statistically different from controls). By the following circadian cycle, responsiveness was more completely recovered, although the magnitude of some phase delays remained [.lessequal] 85% of normal. These major reductions in the magnitude of phase delays (and phase response curve amplitude) caused by saturating light pulses confound interpretations of two-pulse experiments designed to measure the rate of circadian phase delays. In addition, the time course for this reduced responsiveness may reflect the time course of cellular and molecular events that underlie light-induced resetting of the mammalian circadian pacemaker.

IL-1 receptor regulates S100A8/A9-dependent keratinocyte resistance to bacterial invasion.

Previously, we reported that epithelial cells respond to exogenous interleukin (IL)-1α by increasing expression of several genes involved in the host response to microbes, including the antimicrobial protein complex calprotectin (S100A8/A9). Given that S100A8/A9 protects epithelial cells against invading bacteria, we studied whether IL-1α augments S100A8/A9-dependent resistance to bacterial invasion of oral keratinocytes. When inoculated with Listeria monocytogenes, human buccal epithelial (TR146) cells expressed and released IL-1α. Subsequently, IL-1α-containing media from Listeria-infected cells increased S100A8/A9 gene expression in naïve TR146 cells an IL-1 receptor (IL-1R)-dependent manner. Incubation with exogenous IL-1α decreased Listeria invasion into TR146 cells, whereas invasion increased with IL-1R antagonist. Conversely, when S100A8/A9 genes were knocked down using short hairpin RNA (shRNA), TR146 cells responded to exogenous IL-1α with increased intracellular bacteria. These data strongly suggest that infected epithelial cells release IL-1α to signal neighboring keratinocytes in a paracrine manner, promoting S100A8/A9-dependent resistance to invasive L. monocytogenes.