Longus,a Type IV Pilus of Enterotoxigenic Escherichia coli,Is Involved in Adherence to Intestinal Epithelial Cells

Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of diarrhea in the developing world, as well as the most common cause of traveler''s diarrhea. The main hallmarks of this type of bacteria are the expression of one or more enterotoxins and fimbriae used for attachment to host intestinal cells. Longus is a pilus produced by ETEC. These bacteria grown in pleuropneumonia-like organism (PPLO) broth at 37°C and in 5% CO₂ produced longus, showing that the assembly and expression of the pili depend on growth conditions and composition of the medium. To explore the role of longus in the adherence to epithelial cells, quantitative and qualitative analyses were done, and similar levels of adherence were observed, with values of 111.44 × 10⁴ CFU/ml in HT-29, 101.33 × 10⁴ CFU/ml in Caco-2, and 107.11 × 10⁴ CFU/ml in T84 cells. In addition, the E9034AΔlngA strain showed a significant reduction in longus adherence of 32% in HT-29, 22.28% in Caco-2, and 21.68% in T84 cells compared to the wild-type strain. In experiments performed with nonintestinal cells (HeLa and HEp-2 cells), significant differences were not observed in adherence between E9034A and derivative strains. Interestingly, the E9034A and E9034AΔlngA(pLngA) strains were 30 to 35% more adherent in intestinal cells than in nonintestinal cells. Twitching motility experiments were performed, showing that ETEC strains E9034A and E9034AΔlngA(pLngA) had the capacity to form spreading zones while ETEC E9034AΔlngA does not. In addition, our data suggest that longus from ETEC participates in the colonization of human colonic cells.Enterotoxigenic Escherichia coli (ETEC) is an important cause of infant diarrhea in developing countries, a leading cause of traveler''s diarrhea, and a reemergent diarrheal pathogen in the United States (1, 25, 29, 33, 38, 40, 41, 44, 51, 52, 55). ETEC strains were first recognized as a cause of diarrheal disease in animals, especially in piglets and calves, where the disease continues to cause lethal infection in newborn animals (3, 37). Studies of ETEC in piglets first elucidated the mechanisms of disease, including the presence of two plasmid-encoded enterotoxins. In humans, the clinical appearance of ETEC infection is identical to that of cholera, with severe dehydrating illness not commonly seen in adults (38, 46). DuPont et al. (12) subsequently showed that ETEC strains were able to cause diarrhea in adult volunteers. ETEC strains cause watery diarrhea similar to that caused by Vibrio cholerae through the action of two enterotoxins, the cholera-like heat-labile and heat-stable enterotoxins (LT and ST, respectively) (38). These strains may express an LT only, an ST only, or both LT and ST. To cause diarrhea, ETEC strains must first adhere to small bowel enterocytes, an event mediated by a variety of surface fimbrial appendages called colonization factor antigens (CFAs), coli surface antigens (CSs), and putative colonization factors (PCF) (22, 33, 38). Transmission electron microscopy (TEM) of ETEC strains typically reveals many peritrichously arranged fimbriae around the bacterium; often, multiple fimbrial morphologies can be visualized on the same bacterium (6, 19, 31, 38). ETEC strains also express the K99 fimbriae, which are pathogenic for calves, lambs, and pigs, whereas K88-expressing organisms are able to cause disease only in pigs (8). Human ETEC strains possess their own array of colonization fimbriae, the CFAs usually encoded in plasmids (10). Currently, more than 20 CFAs known in human ETEC infections have been described (17). The CFAs can be subdivided based on their morphological characteristics. Three major morphological varieties exist: rigid rods (CFA I), bundle-forming flexible rods (CFA III), and thin, flexible, wiry structures (CFA II and CFA IV) (7, 8, 26, 30, 49, 53, 54).A high proportion of human ETEC strains contain a plasmid-encoded type IV pilus (T4P) antigen (CS20) also called longus for its length (19, 21). Longus is a T4P composed of a repeating structural subunit called LngA of 22 kDa, and its N-terminal amino acid sequences shares similarities with the class B type IV pili. These pili include the CFA III pilin subunit CofA of ETEC, the toxin-coregulated pilin (TCP) of V. cholerae, and the bundle-forming pilin (BFP) found in enteropathogenic E. coli (EPEC) and in a small percentage in other Gram-negative pathogens (21, 23). The lngA gene, which encodes the longus pilus in ETEC strains, is widely distributed in different geographic regions such Bangladesh, Chile, Brazil, Egypt, and Mexico (23). Interestingly, the lngA gene has been observed in association with ETEC strain producers of LT and ST (23). Sequence analysis of the fimbrial genes provided insight into the evolutionary history of longus. It appears that the highly conserved nonstructural lngA genes evolved in a similar manner to that of housekeeping genes.Recently, another important adherence factor called E. coli common pilus (ECP) has been identified; it is composed of a 21-kDa pilin subunit whose amino acid sequence corresponds to the product of the yagZ (renamed ecpA) gene present in all E. coli genomes sequenced to date (47). ECP production was demonstrated in strains representing intestinal (enterohemorrhagic E. coli [EHEC], EPEC, and ETEC) and extraintestinal pathogenic E. coli as well as normal-flora E. coli.In this study we report that longus plays an important role in the adherence to colonic epithelial cells. In addition to mediating cell adherence, longus is also associated with other pathogenicity attributes exhibited by other Gram-negative pathogenic bacteria producing T4P, which can contribute in part to the virulence of ETEC.     

Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin

The Gram-negative type II secretion (T2S) system is a multiprotein complex mediating the release of virulence factors from a number of pathogens. While an understanding of the function of T2S components is emerging, little is known about what identifies substrates for export. To investigate T2S substrate recognition, we compared mutations affecting the secretion of two highly homologous substrates: heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae. Each toxin consists of one enzymatic A subunit and a ring of five B subunits mediating the toxin''s secretion. Here, we report two mutations in LT''s B subunit (LTB) that reduce its secretion from ETEC without global effects on the toxin. The Q3K mutation reduced levels of secreted LT by half, and as with CT (T. D. Connell, D. J. Metzger, M. Wang, M. G. Jobling, and R. K. Holmes, Infect. Immun. 63:4091-4098, 1995), the E11K mutation impaired LT secretion. Results in vitro and in vivo show that these mutants are not degraded more readily than wild-type LT. The Q3K mutation did not significantly affect CT B subunit (CTB) secretion from V. cholerae, and the E11A mutation altered LT and CTB secretion to various extents, indicating that these toxins are identified as secretion substrates in different ways. The levels of mutant LTB expressed in V. cholerae were low or undetectable, but each CTB mutant expressed and secreted at wild-type levels in ETEC. Therefore, ETEC''s T2S system seems to accommodate mutations in CTB that impair the secretion of LTB. Our results highlight the exquisitely fine-tuned relationship between T2S substrates and their coordinate secretion machineries in different bacterial species.Gram-negative bacteria have evolved a number of methods to secrete proteins into the extracellular milieu, with at least six specific secretion systems currently described (14, 30). Type II secretion (T2S), or the main terminal branch of the general secretory pathway, is a feature of a number of proteobacteria and has been shown to be required for pathogenesis and maintenance of environmental niches in a large number of species (5). The T2S system is a multiprotein complex of 12 to 15 components that spans the inner and outer membranes, allowing for the controlled release of certain folded proteins that have been directed to the periplasm through the Sec or Tat machinery (21). Aside from providing a means of exporting freely released virulence factors from plant, animal, and human pathogens (5), the T2S system has been shown to export surface-associated virulence factors (18), fimbrial components (46), outer membrane cytochromes (36), and a surfactant required for sliding motility in Legionella pneumophila (39), among other substrates.While an increasing number of studies have focused on understanding the structure and function of the components of the T2S system itself, little is known about what identifies a periplasmic protein as a substrate for secretion (21, 32). Because proteins secreted from the same bacterial species need not share any obvious structural homology, it is not even clear how much of a T2S substrate interacts with the secretion machinery (32). Analysis of two similar substrates that can each be secreted by the T2S systems of two distinct species would provide information about species-specific identification of T2S substrates and, by extension, the nature of the “secretion motif” identifying those substrates. Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae represent one such pair of substrates.ETEC and V. cholerae are enteric pathogens causing significant morbidity and mortality worldwide (33). The causative agents of traveler''s diarrhea and cholera, respectively, these two pathogens share a number of similarities, including the nature of their disease symptoms (38). Each pathogen secretes an AB₅ toxin important for colonization and the induction of water and electrolyte efflux from intestinal epithelial cells (1, 29). These toxins, LT and CT, are both encoded by two-gene operons. After sec-dependent transport to the periplasm, holotoxin formation occurs spontaneously (13), with one catalytic A subunit (LTA or CTA) assembling with five B subunits (LTB or CTB), which are responsible for the binding properties of the toxins. Export of fully folded and assembled LT or CT is then accomplished by the T2S system (34, 40). In ETEC, this system is encoded by gspC to -M (40), while in V. cholerae, these genes are found in the eps operon (34).LT and CT are very similar in structure, sharing approximately 80% sequence homology and 83% identity in the mature B subunit (16, 24). ETEC is thought to have acquired the genes for CT through horizontal transfer, with the toxins evolving over time to possess slight differences (45). As such, these toxins share the same primary host receptor, the monosialoganglioside G_M1, and catalyze the same ADP-ribosylation reaction within host cells (38). However, LT is able to bind other host sphingolipids in addition to G_M1 and to interact with sugar residues from the A-type blood antigen, which CT cannot bind (16, 41). Both LT and CT are able to associate with sugar residues in lipopolysaccharide (LPS) on the surface of E. coli cells (17). Binding to each of these substrates can be impaired by point mutation (26, 43).In this study, we report point mutations impairing the release of LT from ETEC and CT from V. cholerae. We analyzed the specificity of the defects in substrate recognition by comparing the effects of substituting charged and neutral residues in key regions of LTB and CTB. To confirm that the identified mutations resulted specifically in a secretion defect, we tested the effect of the mutations on (i) ligand binding by each toxin, (ii) toxin stability, and (iii) formation of secretion-competent B-subunit pentamers. By introducing comparable mutations into both toxins, including one previously reported to impair the secretion of CT (6), and exchanging toxin substrates between the two species, we have revealed species-dependent differences in T2S substrate recognition. Although wild-type LT and CT can be heterologously expressed and secreted from V. cholerae and ETEC, respectively, the substrate residues identified by the secretion machinery in each species are distinct. Together, our results demonstrate that highly homologous T2S substrates are recognized in different ways when secreted by two distinct systems.     

Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies (Enterobacter sakazakii) in Powdered Infant Formula

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.Cronobacter strains were originally described as Enterobacter sakazakii (12), but they are now known to comprise a novel genus consisting of six separate genomospecies (20, 21). These opportunistic pathogens are ubiquitous in the environment and various types of food and are occasionally found in the normal human flora (11, 12, 16, 32, 47). Based on case reports, Cronobacter infections in adults are generally less severe than Cronobacter infections in newborn infants, with which a high fatality rate is associated (24).The ability to detect Cronobacter and trace possible sources of infection is essential as a means of limiting the impact of these organisms on neonatal health and maintaining consumer confidence in powdered infant formula (PIF). Conventional methods, involving isolation of individual colonies followed by biochemical identification, are more time-consuming than molecular methods, and the reliability of some currently proposed culture-based methods has been questioned (28). Recently, several PCR-based techniques have been described (23, 26, 28-31, 38). These techniques are reported to be efficient even when low levels of Cronobacter cells are found in a sample (0.36 to 66 CFU/100 g). However, PCR requires DNA extraction and does not allow direct, in situ visualization of the bacterium in a sample.Fluorescence in situ hybridization (FISH) is a method that is commonly used for bacterial identification and localization in samples. This method is based on specific binding of nucleic acid probes to particular DNA or RNA target regions (1, 2). rRNA has been regarded as the most suitable target for bacterial FISH, allowing differentiation of potentially viable cells. Traditionally, FISH methods are based on the use of conventional DNA oligonucleotide probes, and a commercial system, VIT-E sakazakii (Vermicon A.G., Munich, Germany), has been developed based on this technology (25). However, a recently developed synthetic DNA analogue, peptide nucleic acid (PNA), has been shown to provide improved hybridization performance compared to DNA probes, making FISH procedures easier and more efficient (41). Taking advantage of the PNA properties, FISH using PNA has been successfully used for detection of several clinically relevant microorganisms (5, 15, 17, 27, 34-36).     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Prophylactic Administration of Bacterially Derived Immunomodulators Improves the Outcome of Influenza Virus Infection in a Murine Model

Prophylactic or therapeutic immunomodulation is an antigen-independent strategy that induces nonspecific immune system activation, thereby enhancing host defense to disease. In this study, we investigated the effect of prophylactic immunomodulation on the outcome of influenza virus infection using three bacterially derived immune-enhancing agents known for promoting distinct immunological profiles. BALB/c mice were treated nasally with either cholera toxin (CT), a mutant form of the CT-related Escherichia coli heat-labile enterotoxin designated LT(R192G), or CpG oligodeoxynucleotide. Mice were subsequently challenged with a lethal dose of influenza A/PR/8/34 virus 24 h after the last immunomodulation treatment and either monitored for survival or sacrificed postchallenge for viral and immunological analysis. Treatment with the three immunomodulators prevented or delayed mortality and weight loss, but only CT and LT(R192G) significantly reduced initial lung viral loads as measured by plaque assay. Analysis performed 4 days postinfection indicated that prophylactic treatments with CT, LT(R192G), or CpG resulted in significantly increased numbers of CD4 T cells, B cells, and dendritic cells and altered costimulatory marker expression in the airways of infected mice, coinciding with reduced expression of pulmonary chemokines and the appearance of inducible bronchus-associated lymphoid tissue-like structures in the lungs. Collectively, these results suggest that, despite different immunomodulatory mechanisms, CT, LT(R192G), and CpG induce an initial inflammatory process and enhance the immune response to primary influenza virus challenge while preventing potentially damaging chemokine expression. These studies provide insight into the immunological parameters and immune modulation strategies that have the potential to enhance the nonspecific host response to influenza virus infection.Influenza viruses cause acute, contagious respiratory disease. Despite the availability of vaccines and antiviral therapies, influenza virus infections cause considerable morbidity and mortality each year. It is estimated that during seasonal epidemics 10% of the world population is infected, resulting in 2 to 3 million severe cases and up to 500,000 deaths (1). The failure of conventional methods to prevent illness and death from influenza is attributed to the continuous antigenic variability of the virus due to mutations (antigen drift) and reassortments (antigenic shift). The inadequacy of current anti-influenza virus treatments is particularly concerning in the case of influenza pandemics with new viral strains for which effective vaccines would not be initially available. Thus, an antigen-independent prophylactic treatment that could nonspecifically enhance immune responses to negate or inhibit the progression of influenza virus infection would provide invaluable benefits.Several recent studies have explored the use of immunomodulation strategies as prophylaxis or therapeutic treatments to modify the immune response to influenza virus infection, thereby preventing or decreasing viral burden, disease symptoms, and mortality. These strategies have one of two distinct immunologic goals: either to increase immune system activation and/or Th1 responses specific against influenza virus, or alternatively, decrease inflammation and immunopathology. The first strategy has been demonstrated in animal models by administering host proteins/glycoproteins that function in immune defense, such as the pattern recognition receptor (PRR) mindin (28), milk-derived glycoproteins (61), and virally delivered interferon (IFN) cytokines (27). Immunomodifiers of microbial origin have also been used to enhance host response to infection, including the binding subunit of cholera toxin (CT-B) (49), Th1-promoting Toll-like receptor (TLR) agonists CpG oligodeoxynucleotides (ODN) (15, 82), poly(I:C) (81), 3 M-011 (23), and synthetic lipid A analogs (11). Immunomodulators used in the second strategy, with the aim to prevent detrimental inflammation, have been associated with improved infection outcomes and include enterotoxin mutant LT(S63K) (80) and anti-inflammatory COX-2 inhibitors (84). However, immunomodulation does not always result in beneficial responses to infection. Administration of Δ⁹-THC, an immunosuppressive compound, decreased cellular infiltration and increased viral load when given prior to and during influenza virus infection (7). Similarly, sphingosine 1-phosphate (S1P) analog, an immunotherapeutic agent, was found to suppress induction of T-cell responses to influenza virus (46). Lastly, fish oil-fed mice demonstrated reduced lung inflammation, cellular infiltration, and cytokine secretion but increased mortality during influenza virus infection (60).These studies highlight the need for experiments that clarify the consequences of various immunomodulation strategies on influenza virus infection and the particular requirements for generating a protective response. Furthermore, very little attention has been given to the mechanisms by which different immunomodulators with unique effector functions modulate the host response when evaluated in the same infection model. To address these questions and increase our understanding of the consequences brought about by prophylactic immunomodulation in pulmonary disease, we chose to compare the effects of pulmonary delivery of three well-characterized vaccine adjuvants on the outcome of influenza virus infection in a murine model. The immunomodulators used in this study are CpG, a nontoxic protein designated LT(R192G) that was derived from the cholera-related heat-labile enterotoxin produced by Escherichia coli, and CT. These bacterially derived agents, known to promote distinct effector functions, are excellent immunomodulators, as they induce strong immune activation and have been previously evaluated as components of influenza vaccines (29, 42, 49, 53, 56, 58). CpG ODNs are synthetic unmethylated oligodeoxynucleotides containing CpG motifs that trigger a TLR9-dependent MyD88 signaling pathway. CpG treatment results in potent Th1 cytokine expression (IFNs and interleukin-12 [IL-12]), activation of dendritic cells (DCs), NK cells, and B cells, and induction of Th1 cells and a Th1 antibody profile (30, 35, 83). CpG has been extensively studied in animal models of systemic and pulmonary infectious diseases caused by influenza virus (15, 82) and other bacterial, fungal, and parasitic pathogens (3, 9, 15, 17, 25, 34, 51, 77).Bacterially derived ADP-ribosylating enterotoxins, including CT from Vibrio cholerae and LT from E. coli, are robust systemic and mucosal adjuvants. Both in vitro and in vivo studies have demonstrated that CT induces secretion of Th2 cytokines (IL-4, IL-5, IL-6, and IL-10) by immune system cells, maturation of DCs, generation of Th2 and T-regulatory cells, and active suppression of Th1 responses (2, 32, 38, 39, 47, 49, 53, 56). Studies in vivo have also shown that intranasal delivery of CT-B, the binding subunit of the enterotoxin, combined with minimal levels of CT holotoxin, induces protective effects in influenza virus-infected mice (49). In contrast to CpG and CT, LT and LT(R192G) induce a more balanced cytokine and antibody subclass profile indicative of a mixed Th1/Th2 immune response (16, 45, 73). LT(R192G) has yet to be evaluated as a prophylactic immunomodulator, but another LT mutant, LT(S63K), has demonstrated some protective effects against influenza virus, respiratory syncytial virus (RSV), and Cryptococcus neoformans infections (80). Although safety concerns limit the use of native enterotoxins for intranasal or intrapulmonary use in humans (54, 76), animal model studies are warranted because they enhance our understanding of the initial responses that can ultimately lead to protection of the host against infection. In addition, the use of these enterotoxins in laboratory research has the potential to be translated into clinical application by using mutated low-toxinogenic derivatives that retain their immunomodulatory properties.In this study we used a comprehensive approach to evaluate the effects of intrapulmonary delivery of three strong immunomodulators prior to influenza virus infection in a murine model. We hypothesized that the unique immunologic effects induced by prophylactic treatment with CT, LT(R192G), or CpG would differentially affect survival, viral loads, and immune responses of BALB/c mice to influenza A/PR/8/34 (H1N1) virus infection. The relevance of this study to influenza virus disease pathogenesis and infectious disease immunomodulation strategies is discussed.     

Inhibition of Selenium Metabolism in the Oral Pathogen Treponema denticola

In this report we provide evidence that the antimicrobial action of stannous salts and a gold drug, auranofin, against Treponema denticola is mediated through inhibition of the metabolism of selenium for synthesis of selenoproteins.The biological use of selenium as a catalyst, incorporated into proteins as selenocysteine, is broad. It plays an essential role in energy metabolism, redox balance, and reproduction in a variety of organisms, from bacterial pathogens to eukaryotic parasites to humans. The results of several epidemiological studies indicate that higher levels of selenium in the mammalian diet can have a negative effect on dental health (2, 17-19, 39). Although the impact of selenium is attributed to its influence on the physical properties of the enamel surface (10), the role of selenium in supporting the oral microbial community has not been studied.The oral cavity is a highly complex microbiome, with a large proportion of its residents uncharacterized due to their fastidious nature and resistance to traditional culture methods (11). Analysis of whole saliva indicates that bacterial metabolism influences the amino acid composition and indicates a role for amino acid fermentation (38). Curtis et al. demonstrated the occurrence of Stickland reactions in dental plaque (9). These reactions were first described in clostridia (35-37). They involve the coupled fermentation of amino acids in which one amino acid is oxidized (Stickland donor) and another (Stickland acceptor) is reduced (29). Treponema denticola, an established resident of the oral cavity, performs Stickland reactions via the selenoprotein glycine reductase (32). Glycine reductase is composed of a multiprotein complex that contains two separate selenoproteins, termed selenoprotein A and selenoprotein B (1, 7, 8, 15, 16). This complex of proteins converts glycine to acetyl phosphate by using inorganic phosphate and the reducing potential from thioredoxin. For the organisms that use this complex, this is a vital source of ATP. Thus far, the requirement for selenocysteine at the active site of this enzyme complex is universally conserved, even though all other selenoproteins that have been identified using computational techniques have a putative cysteine homologue (24).Treponema denticola is considered one of the primary pathogens responsible for periodontitis, a chronic inflammatory disease that is the major cause of adult tooth loss (11, 27, 33). It is the best-studied oral spirochete, commonly found with other spirochetes within the periodontal pocket. It expresses a variety of virulence factors and is capable of adhering to and penetrating endothelial cell monolayers (31). Its health impact may reach beyond the oral cavity. A recent study linked periodontitis with peripheral arterial disease and detected T. denticola, along with other periodontal pathogens, in atherosclerotic plaque (3). Sequence analysis indicates the presence of several selenoproteins in addition to glycine reductase within the genome of T. denticola (24). This organism exhibits a strict growth requirement for selenium (32).A significant literature exists that clearly demonstrates the antimicrobial activity of fluoride compounds against microorganisms associated with dental decay and periodontitis. Both sodium fluoride and stannous fluoride, as well as stannous ions alone, inhibit the growth of T. denticola (21). The inhibitory effect of stannous salts on T. denticola''s growth is unexplained. It should be noted that toothpastes containing stannous fluoride are more effective in reducing gingivitis and plaque (28, 30).Tin, as well as several other trace elements, modulates the effects of acute selenium toxicity (20). Conversely, selenium affects the activity of tin in animal models (4-6). In this study, we examine the possibility that stannous ions interfere with selenium metabolism in T. denticola.