Fimbrial Protein Research Articles

Sublethal concentrations of antibiotics and bacterial adhesion.

Various antibiotics in sublethal concentrations markedly impair adhesion of Streptococcus pyogenes and Escherichia coli to human cells. In streptococcal cells penicillin G caused an enhances loss of lipoteichoic acid, the ligand (adhesion) that binds the organism to host cells, with consequent loss of their adhesive properties. In E coli sublethal concentrations of penicillin prevented the surface expression of the mannose-specific adhesion by distorting cell wall biosynthesis. In contrast to streptococci, E coli cells could not be made to lose their adhesions once their adhesions once they had been formed. Streptomycin in subinhibitory concentration similarly suppressed the acquisition of mannose-binding and adhesive activities in several strains of antibiotic-sensitive E. coli but not in isogenic derivatives with ribosomal mutation to high-level streptomycin resistance, rpsL, or in bacteria in the stationary phase of growth, suggesting that streptomycin exerted its sublethal suppressive effects by classic mechanisms of action on the bacterial ribosome. Strain VL2, derived from one streptomycin-resistant mutant, retained a high level (1000 microgram/ml) of resistance to streptomycin but reacquired sensitivity to the sublethal effect; growth in 30 microgram streptomycin/ml suppressed mannose-sensitive haemagglutination (less than 1% of control) as well as mannose-sensitive adhesion to epithelial cells (42%) or leucocytes (7%). Although these streptomycin-treated bacteria demonstrated an unaltered degree of fimbriation their fimbriae were significantly longer than those on the untreated bacteria. Furthermore, in contrast to the untreated bacteria, the fimbriae isolated from the drug-treated bacteria were found to lack mannose-binding activity as measured by haemagglutination. It therefore, appears that streptomycin can cause even resistant bacteria to produce an aberrant fimbrial protein, presumably by causing misreading in "competent" ribosomes. These studies indicate that the use of sublethal doses of certain antibiotics whose mode of action is well known may shed light on the genetic and chemical modulation of bacterial factors involved in mucosal colonization.

HrpG regulates type II secretory proteins in Xanthomonas axonopodis pv. citri

HrpG, a two-component response regulator-like protein, is a key regulator of the type III secretion system (T3SS) in Xanthomonas spp. In X. campestris pv. vesicatoria, HrpG with a single amino acid substitution (HrpG*) gains the ability to induce the expression of T3SS-related genes even under nutrient-rich conditions. In this study, we investigated the role of HrpG in the synthesis of the secretory protein using HrpG* in strain NA-1 of X. axonopodis pv. citri (Xac NA-1), a causal agent of citrus canker. Eleven proteins secreted via a type II secretion system (T2SS) were induced by HrpG*. In proteomic analyses, six of the 11 proteins were identified as extracellular enzymes, and the others as a fimbrial biogenesis-related protein, a type IV-related protein, two hypothetical proteins, and a conserved hypothetical protein. Further analysis of these proteins revealed that the genes coding all 11 proteins were upregulated by HrpG*, even though they had different expression patterns for HrpXct-dependency. The data indicated that HrpG, a key regulator of T3SS, also acts as a positive regulator of certain proteins secreted via a T2SS in Xac NA-1.

Rapid Detection of Salmonella enteritidis by PCR Amplification of The SefA Gene and It`s Cloning

The emergence of Salmonella enteritidis as an important food-borne pathogenesis in humans, demands the development of novel detection and intervention strategies. It is generally accepted that fimbriae are an important factor in bacterial survival and persistence in the host. This study is directed towards the method of amplifying and cloning the SefA gene, which encode Salmonella enteritidis fimbrial protein. Strains used for these studies were S. enteritidis (E3), which were collected from Kermanshah region. Chromosomal DNA was extracted by boiling method and PCR reaction was performed and single band of 511 bp amplified by SefA-F and SefA-R primers. The resulting PCR product was inserted into the cloning vector (pTZ57R/T). In order to amplify the recombinant plasmid, E. coli DH5 alpha bacteria were transformed with SefA-pTZ57R/T. Recombinant clones were identified by blue/white selection and purified recombinant plasmids were indicated by an alkaline lysis procedure. Identity of the SefA-pTZ57R/T product was confirmed by RFLP and sequencing. Nucleotide and protein alignment with BLAST software showed that the sequence of the SefA gene derived from S. enteritidis (E3), which was cloned in the pTZ57R/T vector, was 99% identical to that of the Genbank (L11008). The sequence of the SefA gene from S. enteritidis (E3) differed only in two nucleotides and one amino acid. The cloned SefA gene from S. enteritidis (E3) was submitted to the NCBI Genbank (EF553334).

Characterisation of cisplatin coordination sites in cellular Escherichia coli DNA-binding proteins by combined biphasic liquid chromatography and ESI tandem mass spectrometry

Combined multidimensional liquid chromatography and electrospray ionisation tandem mass spectrometry was employed to analyse platinated tryptic peptides from Escherichia coli cells treated with the anticancer drug cis-[PtCl2(NH3)2] at pH 7.0. Prerequisites for the LC/LC/MS/MS analysis of protein targets that are fulfilled by cisplatin are (a) that the original protein binding sites have a high kinetic stability over the range 2.3 < pH < 8.5, and (b) that the metal fragment remains coordinated to a significant number of b+ and y+ peptide ions under MS/MS fragmentation conditions. Matching the MS/MS spectra of the platinated tryptic peptides to sequences of proteins in the E. coli database enabled the identification of 31 protein targets for cisplatin. Whereas six of these are high-abundance enzymes and ribosomal proteins in E. coli cells, five low-abundance DNA-binding proteins were also identified as specific targets. These include the DNA mismatch repair protein mutS, the DNA helicase II (uvrD) and topoisomerase I (top1). Two efflux proteins (acrD, mdtA), the redox regulator thioredoxin 1 (thiO) and the external filament-like type-1 fimbrial protein A chain (fimA1) were also characterised as specific cisplatin-binding proteins. Kinetically favoured carboxylate (D, E) and hydroxy (S, T, Y) O atoms were identified as the Pt coordination sites in 18 proteins and methionyl S atoms in 9 proteins.

Role of the Clp System in Stress Tolerance, Biofilm Formation, and Intracellular Invasion inPorphyromonas gingivalis

Clp proteases and chaperones are ubiquitous among prokaryotes and eukaryotes, and in many pathogenic bacteria the Clp stress response system is also involved in regulation of virulence properties. In this study, the roles of ClpB, ClpC, and ClpXP in stress resistance, homotypic and heterotypic biofilm formation, and intracellular invasion in the oral opportunistic pathogen Porphyromonas gingivalis were investigated. Absence of ClpC and ClpXP, but not ClpB, resulted in diminished tolerance to high temperatures. Response to oxidative stress was not affected by the loss of any of the Clp proteins. The clpC and clpXP mutants demonstrated elevated monospecies biofilm formation, and the absence of ClpXP also enhanced heterotypic P. gingivalis-Streptococcus gordonii biofilm formation. All clp mutants adhered to gingival epithelial cells to the same level as the wild type; however, ClpC and ClpXP were found to be necessary for entry into host epithelial cells. ClpB did not play a role in entry but was required for intracellular replication and survival. ClpXP negatively regulated the surface exposure of the minor fimbrial (Mfa) protein subunit of P. gingivalis, which stimulates biofilm formation but interferes with epithelial cell entry. Collectively, these results show that the Clp protease complex and chaperones control several processes that are important for the colonization and survival of P. gingivalis in the oral cavity.

Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis

Porphyromonas gulae is black-pigmented anaerobic bacteria associated with canine periodontitis. There is little information available about the specific identify and relative occurrence of pigmented anaerobes in companion animals. Our aim was to clarify the factor involved in the adherence and colonization of the organism in the oral cavity. Fimbrial protein was purified from P. gulae ATCC 51700. The molecular mass of this protein was approximately 41 kDa as estimated by SDS-PAGE. An antibody against 41-kDa fimbrial protein from P. gingivalis ATCC 33277 reacted with fimbrillin of P. gulae ATCC 51700. Immunogold electron microscopy revealed that the anti-41 kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. Thus, fimbrial protein of P. gulae ATCC 51700 had the same size and antigenicity as 41-kDa fimbriae of P. gingivalis ATCC 33277. The nucleotide sequence of the fimA gene from P. gulae ATCC 51700 showed 94% homology with that of P. gingivalis ATCC 33277. Moreover, the deduced amino acid sequences have 96.8% identity. P. gulae has adherent ability to gingival epithelial cells. The properties of P. gulae fimbriae are similar to those of P. gingivalis fimbriae. We suggest that the surface structure of P. gulae may play a role in the colonization of this organism in periodontal pockets in companion animals.

Fimbrial Proteins of Porphyromonas gingivalis Mediate In Vivo Virulence and Exploit TLR2 and Complement Receptor 3 to Persist in Macrophages

Porphyromonas gingivalis is an oral/systemic pathogen implicated in chronic conditions, although the mechanism(s) whereby it resists immune defenses and persists in the host is poorly understood. The virulence of this pathogen partially depends upon expression of fimbriae comprising polymerized fimbrillin (FimA) associated with quantitatively minor proteins (FimCDE). In this study, we show that isogenic mutants lacking FimCDE are dramatically less persistent and virulent in a mouse periodontitis model and express shorter fimbriae than the wild type. Strikingly, native fimbriae allowed P. gingivalis to exploit the TLR2/complement receptor 3 pathway for intracellular entry, inhibition of IL-12p70, and persistence in macrophages. This virulence mechanism also required FimCDE; indeed, mutant strains exhibited significantly reduced ability to inhibit IL-12p70, invade, and persist intracellularly, attributable to failure to interact with complement receptor 3, although not with TLR2. These results highlight a hitherto unknown mechanism of immune evasion by P. gingivalis that is surprisingly dependent upon minor constituents of its fimbriae, and support the concept that pathogens evolved to manipulate innate immunity for promoting adaptive fitness and thus their capacity to cause disease.

Glycosylation of type-IV fimbriae of Dichelobacter nodosus

Dichelobacter nodosus is the causative agent of ovine footrot and the type-IV fimbriae on this bacterium are essential for maintaining its virulence. In this study, we reveal that these fimbriae are glycosylated. This was demonstrated in several ways: by the detection of carbohydrate on fimbrial protein using periodic acid Schiff reagent (PAS) staining of SDS-PAGE gels and by demonstrating enzymatic deglycosylation and by analysis of the amino acid sequences derived from the fimA gene, whereby the gene from isolates of D. nodosus that appeared to be glycosylated had potential glycosylation sites both inside and outside of the variable region of fimA. The results would also explain the observation that the calculated molecular weight of fimA from some D. nodosus serotypes does not correlate with the apparent size determined from electrophoretic mobility.

Expression and subcellular targeting of a model subunit vaccine in transgenic soybean (41.13)

Abstract Enterotoxigenic Escherichia coli (ETEC) strains are the leading cause of enteric diseases affecting humans and livestock. Edible transgenic plants producing E. coli fimbrial subunit proteins have the potential to vaccinate against these diseases by delivering naturally encapsulated antigens to effecter sites lining the gastrointestinal tract and eliciting mucosal as well as systemic immunity. Plants, however, have not reached their full potential as a renewable source of oral vaccines, due in part to insufficient levels of recombinant protein accumulation. In an effort to increase the relative amount of subunit vaccine candidate in a practical host system, we targeted the agricultural antigen, FanC, to soybean chloroplasts. We monitored FanC mRNA expression and protein production, demonstrated proper chloroplast accumulation of the protein, and compared the amounts of this chloroplast targeted-protein to accumulation of cytosol-targeted FanC. Surprisingly, cytosol-targeted FanC accumulation was 5-fold greater than chloroplast-targeted FanC accumulation, despite similar FanC mRNA transcription levels in cytosol and chloroplast targeted events. Intraperitoneal vaccination of mice with the chloroplast-derived antigen induced an anti-FanC IgG immune response similar to that generated by vaccination with cytosol-derived and recombinant FanC.

Intranasal immunisation with recombinant Lactococcus lactis displaying either anchored or secreted forms of Proteus mirabilis MrpA fimbrial protein confers specific immune response and induces a significant reduction of kidney bacterial colonisation in mice

Proteus mirabilis, a common cause of urinary tract infections in humans, can express different fimbriae. MR/P fimbriae may contribute to bacterial colonisation, and its structural protein MrpA represents a promising candidate antigen for mucosal vaccination. Commercial complex vaccines have limited, short-lived protection and are incapable of eliciting mucosal responses against putative antigens related to virulence. The development of mucosal live vaccines using food-grade lactic acid bacterium Lactococcus lactis as antigen vehicle is an attractive alternative and a safe vaccination strategy against P. mirabilis infection. Here, we report the construction of L. lactis strains modified to produce MrpA via two cellular locations, cell wall-anchored and secreted. Protection assays against P. mirabilis infection and evaluation of the immune response generated after immunisation were conducted in a mouse model. MrpA protein was efficiently expressed by L. lactis strain and caused a significant induction of specific serum IgG and IgA in the animals immunised with L. lactis pSEC: mrpA and L. lactis pCWA: mrpA respectively. A significant reduction of renal bacterial colonisation was observed in both groups of mice ( P < 0.05) after P. mirabilis challenge. This is the first example of a P. mirabilis fimbrial antigen expressed in a food-grade live strain with promising applications in vaccine design.

Chloroplast targeting of FanC, the major antigenic subunit of Escherichia coli K99 fimbriae, in transgenic soybean

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of enteric diseases affecting livestock and humans. Edible transgenic plants producing E. coli fimbrial subunit proteins have the potential to vaccinate against these diseases, but have not reached their full potential as a renewable source of oral vaccines due in part to insufficient levels of recombinant protein accumulation. Previously, we reported that cytosol targeting of the E. coli K99 fimbrial subunit antigen resulted in FanC accumulation to approximately 0.4% of total soluble protein in soybean leaves (Piller et al. in Planta 222:6-18, 2005). In this study, we report on the subcellular targeting of FanC to chloroplasts. Twenty-two transgenic T1 progeny derived from seven individual T0 transformation events were characterized, and 17 accumulated transgenic FanC. All of the characterized events displayed relatively low T-DNA complexity, and all exhibited proper targeting of FanC to the chloroplast. Accumulation of chloroplast-targeted FanC was approximately 0.08% of total soluble leaf protein, or approximately 5-fold less than cytosol-targeted FanC. Protein analysis of leaves at various stages of maturity suggested stability of chloroplast-targeted FanC throughout leaf maturation. Furthermore, mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing chloroplast-targeted FanC developed significant antibody titers against FanC. This is the first report of subcellular targeting of a vaccine subunit antigen in soybean.

Inhibition of enterotoxigenic Escherichia coli (ETEC) adhesion to Caco-2 cells by human milk and its immunoglobulin and non-immunoglobulin fractions

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea in children in developing countries and among travelers to ETEC endemic areas. ETEC diarrhea is caused by colonization of the small intestine mediated by colonization factor (CF) antigens, and subsequent elaboration of enterotoxins. Breast feeding has been related to protection against enteric infections. The protective effect of human milk can be ascribed to its immunoglobulin content, specially secretory immunoglobulin A (sIgA), and to non-immunoglobulin components such as free oligosaccharides, glycoproteins and glycolipids. In this study we investigated the effect of whole human milk and its fractions immunoglobulin and non-immunoglobulin on the adherence of ETEC strains possessing different CFs to Caco-2 cells, as well as the ability of sIgA and free secretory component (fSC) to bind to bacterial superficial proteins. Pooled human milk from three donors were fractionated by gel filtration and analyzed by SDS-PAGE. Our results revealed that whole human milk and its proteins fractions, containing sIgA and fSC, inhibited adhesion ETEC strains harboring different colonization factors antigens. We also verified that sIgA and fSC, using immunoblotting and immunogold labeling assays, bound to some fimbrial proteins and other material present in bacterial surface. Our findings suggest that whole human milk and its fractions may contribute to protection against ETEC infections by blocking bacterial adhesion mediated by different colonization antigens.

Contamination of a Recombinant Hepatitis B Virus Nucleocapsid Preparation with a Human B-Cell Activator

The hepatitis B virus nucleocapsid is a very immunogenic structure (7) that activates mouse B cells in a T-cell-independent manner (3). We reported a similar observation with human B cells: nucleocapsids induced capsid-binding immunoglobulin M molecules when purified B cells of unprimed humans were transferred into the spleens of NOD/Scid mice (2). However, recent results which we report in this letter suggest that an immunostimulatory contaminant in the HBcAg preparation rather than the nucleocapsids themselves were likely responsible for the observed T-cell-independent B-cell activation. This emerged when four different nucleocapsid preparations were used to study the stimulatory potential of nucleocapsids for purified human B cells. HBcAg-c1 (Diasorin), HBcAg-c2 (Biodesign), and HBcAg-c3 (Biodesign) were produced in Escherichia coli. HBcAg-y was produced in Saccharomyces cerevisiae (GlaxoSmithKline Biologicals, Belgium). HBcAg-c1 nucleocapsids lack the first 2 amino acids and contain 11 foreign amino acids, 8 of which are derived from β-galactosidase (4). HBcAg-c1, HBcAg-y, and HBcAg-c2 contain encapsidated RNA, while HBcAg-c3 nucleocapsids do not, as these lack amino acids 145 to 183. The lipopolysaccharide (LPS) contents of HBcAg-c1, -y, -c2, and -c3 were 136, 0.422, 9.48, and 0.17 endotoxin units LPS/μg nucleocapsid, respectively. We first investigated whether nucleocapsids induced or enhanced the proliferation of purified B cells. Neither HBcAg-c1, HBcAg-y, nor HBcAg-c2 induced thymidine (TdR) incorporation (Fig. ​(Fig.1A).1A). The addition of HBcAg-c1 to B cells stimulated with interleukin-4 (IL-4), anti-CD40 plus IL-2, and anti-CD40 plus IL-4 had a clear synergistic effect on incorporation of TdR. This was not observed with HBcAg-y and HBcAg-c2. Next, we observed that HBcAg-c1 caused a clear up-regulation of CD86 and CD80 (Fig. ​(Fig.1B).1B). This was never observed with HBcAg-y, HBcAg-c2, and HBcAg-c3 (data not shown). A goat anti-human Fab-specific F(ab)2 fragment induced only the expression of CD86, indicating that the intracellular pathways triggered by HBcAg-c1 and the anti-Fab were different. Because only HBcAg-c1 stimulated B cells, we concluded that a contaminant was present. Total purified human circulating B cells do not respond to different toll-like receptor 2 (TLR2), TLR4, TLR2/TLR6, and TLR7/8 ligands (1), suggesting that HBcAg-c1-encapsidated RNA and contaminating LPS and TLR2 ligands (5, 6) were not responsible for the activation of B cells. The capacity of HBcAg-c1 to induce CD80 and CD86 was not destroyed by boiling for 30 min (data not shown), demonstrating indeed that HBcAg-c1 nucleocapsids themselves were not responsible for the stimulation of B cells. Unluckily, the use of HBcAg-c1 in our experiments suggested that nucleocapsids behaved as T-cell-independent B-cell antigens (2). We suggested previously that the stimulatory capacities of nucleocapsids for monocytes and dendritic cells might be attributed to contaminating TLR4 and TLR2 ligands (5, 6). Our data again highlight that caution is needed when studying the stimulatory capacities of nucleocapsids, especially when produced in a bacterium. Proteins produced in E. coli inherently contain products like LPS, bacterial DNA, porins, lipid A-associated proteins, fimbrial proteins, protein A, and lipoproteins. All these can activate cells (8). FIG. 1. (A) HBcAg-c1 enhances B-cell proliferation induced by IL-4 and anti-CD40 plus IL-2 or IL-4. Purified B cells were incubated for 3 days with 1 μg/ml of HBcAg-c1 (black), HBcAg-y (dark gray), or HBcAg-c2 (light gray) or without nucleocapsids (white), ...

Characterization of Adhesion Threads of Deinococcus geothermalis as Type IV Pili

Deinococcus geothermalis E50051 forms tenuous biofilms on paper machine surfaces. Field emission electron microscopy analysis revealed peritrichous appendages which mediated cell-to-surface and cell-to-cell interactions but were absent in planktonically grown cells. The major protein component of the extracellular extract of D. geothermalis had an N-terminal sequence similar to the fimbrial protein pilin annotated in the D. geothermalis DSM 11300 draft sequence. It also showed similarity to the type IV pilin sequence of D. radiodurans and several gram-negative pathogenic bacteria. Other proteins in the extract had N-terminal sequences identical to D. geothermalis proteins with conservative motifs for serine proteases, metallophosphoesterases, and proteins whose function is unknown. Periodic acid-Schiff staining for carbohydrates indicated that these extracellular proteins may be glycosylated. A further confirmation for the presence of glycoconjugates on the cell surface was obtained by confocal laser scanning imaging of living D. geothermalis cells stained with Amaranthus caudatus lectin, which specifically binds to galactose residues. The results indicate that the thread-like appendages of D. geothermalis E50051 are glycosylated type IV pili, bacterial attachment organelles which have thus far not been described for the genus Deinococcus.

The Cytotoxic Fimbrial Structural Subunit of Xenorhabdus nematophila Is a Pore-Forming Toxin

We have purified a fimbrial shaft protein (MrxA) of Xenorhabdus nematophila. The soluble monomeric protein lysed larval hemocytes of Helicoverpa armigera. Osmotic protection of the cells with polyethylene glycol suggested that the 17-kDa MrxA subunit makes pores in the target cell membrane. The internal diameter of the pores was estimated to be >2.9 nm. Electron microscopy confirmed the formation of pores by the fimbrial subunit. MrxA protein oligomerized in the presence of liposomes. Electrophysiological studies demonstrated that MrxA formed large, voltage-gated passive-diffusion channels in lipid bilayers.

Preliminary Evaluation of the Use of thesefA Fimbrial Gene to Elicit Immune Response Against Salmonella enterica Serotype Enteritidis in Chickens

In the last 2 decades, the prevalence of Salmonella enterica serotype Enteritidis (Salmonella Enteritidis) has dramatically increased worldwide, becoming the leading cause of food-borne illnesses and an important public health issue. Many studies have suggested the role of the SEF14 fimbrial protein in the adhesion of Salmonella Enteritidis to the host. In the present study, the sefA gene, which encodes the main subunit of the SEF14 fimbrial protein, was cloned into a temperature-sensitive expression vector and transformed into a nonpathogenic, avirulent strain of Escherichia coli. The recombinant strain was used as a vaccine to elicit specific immune response against the SefA protein of Salmonella Enteritidis in 1-day-old chickens. The recombinant strain was reisolated from the intestines of treated birds for up to 21 days posttreatment, demonstrating its ability to colonize the intestinal tracts of 1-day-old chickens. In addition, immunoglobulin A (IgA) against the SefA protein was detected in intestinal secretions from treated birds at 7 days posttreatment and in bile samples from 14 to 21 days posttreatment by enzyme-linked immunosorbent assay. Nontreated birds did not show any evidence of intestinal colonization by the recombinant strain or anti-SefA IgA response in their bile or intestinal secretions. Preliminary evaluation of the recombinant strain showed a potential use of this strain to elicit protection against Salmonella Enteritidis infection in chickens. Further experiments are needed to study the ability of the recombinant strain to protect birds against Salmonella Enteritidis colonization.

Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities

BackgroundActinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galβ binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking.ResultsHere we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galβ-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8–66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (>97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA.ConclusionThe genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated.

Effects of Various Growth Conditions in a Chemostat on Expression of Virulence Factors inPorphyromonas gingivalis

Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide-limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.

Identification of Proteins Differentially Expressed in Human Monocytes Exposed toPorphyromonas gingivalisand Its Purified Components by High-Throughput Immunoblotting

To characterize the roles of Porphyromonas gingivalis and its components in disease processes, we investigated the cytokine profiles induced by live P. gingivalis, its lipopolysaccharide (LPS), and its major fimbrial protein, fimbrillin (FimA). A cytokine antibody array revealed that human monocyte-derived macrophages were induced to produce chemokines (e.g., monocyte chemoattractant protein 1, macrophage inflammatory protein 1beta [MIP-1beta], and MIP-3alpha) as early as 1 h after exposure to P. gingivalis, with production declining after 24 h of exposure. As expected, an extensive repertoire of inflammatory mediators increased subsequent to infection, most predominantly tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor. The induction of cytokines by P. gingivalis was not triggered simply by bacterial cell surface components, since purified P. gingivalis LPS and FimA induced similar patterns of cytokines, while the pattern of cytokines induced by live P. gingivalis was significantly different, indicating that the host defense system senses live bacteria differently than it does the cell surface components LPS and FimA. To further understand the mechanisms by which live P. gingivalis and its components exert their effects, we used a high-throughput immunoblot screening approach (Becton-Dickinson PowerBlot) to analyze intracellular proteins involved in P. gingivalis infection in human macrophages. Exposure of human macrophages to either live P. gingivalis, its LPS, or its FimA protein led to the up-regulation of 12, 8, and 10 proteins and the down-regulation of 15, 8, and 17 proteins, respectively. The expression of proteins involved in gene transcription (e.g., monocyte enhancer factor 2D [MEF2D], signal transducer and activator of transcription 1 [STAT1], STAT3, STAT6, and IL enhancer binding factors [ILF3]), of protein kinases (e.g., mitogen-activated protein kinase 3 [MAPK3], MAP3K8, double-stranded RNA-activated protein kinase [PRKR], and MAP2K4), and of proteins involved in immune responses (e.g., TNF super family member 6 [TNFSF6] and interferon-induced protein with tetratricopeptide repeat 4 [IFIT4]), apoptosis (e.g., genes associated with retinoid interferon-induced mortality 19 [GRIM19]), and other fundamental cellular processes (e.g., clathrin heavy-chain polypeptide, culreticulin, and Ras-associated protein RAB27A) was found to be modulated differentially by P. gingivalis, LPS, and FimA. These differential changes are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P. gingivalis infection.

Gene Expression Profiling of Intrinsic Thermotolerance in Escherichia coli

DNA microarrays were employed to compare gene expression in a thermotolerant, nalidixic acid-resistant mutant of Escherichia coli with that of the parental strain. When grown at 37 degrees C, up-regulated genes in the mutant included those coding for multiple antibiotic resistance proteins and enzymes for the degradation of small molecules, whereas among the down-regulated genes were those coding for fimbrial, flagellar, and outer membrane proteins as well as sigma 38. When the mutant grown at 42 degrees C was compared to the mutant grown at 37 degrees C, enhanced expression of several genes coding for flagellar proteins was detected. Reverse transcriptase-polymerase chain reaction analysis of selected genes confirmed results obtained with microarrays.