Antimicrobial antisense RNA delivery to F-pili producing multidrug-resistant bacteria via a genetically engineered bacteriophage.

Multidrug-resistant bacteria are a growing issue worldwide. This study developed a convenient and effective method to downregulate the expression of a specific gene to produce a novel antimicrobial tool using a small (140 nucleotide) RNA with a 24-nucleotide antisense (as) region from an arabinose-inducible expression phagemid vector in Escherichia coli. Knockdown effects of rpoS encoding RNA polymerase sigma factor were observed using this inducible artificial asRNA approach. asRNAs targeting several essential E. coli genes produced significant growth defects, especially when targeted to acpP and ribosomal protein coding genes rplN, rplL, and rpsM. Growth inhibited phenotypes were facilitated in hfq- conditions. Phage lysates were prepared from cells harboring phagemids as a lethal-agent delivery tool. Targeting the rpsM gene by phagemid-derived M13 phage infection of E. coli containing a carbapenem-producing F-plasmid and multidrug-resistant Klebsiella pneumoniae containing an F-plasmid resulted in the death of over 99.99% of infected bacteria. This study provides a possible strategy for treating bacterial infection and can be applied to any F-pilus producing bacterial species.

Initiation of phage infection by partial unfolding and prolyl isomerization.

Infection of Escherichia coli by the filamentous phage fd starts with the binding of the N2 domain of the phage gene-3-protein to an F pilus. This interaction triggers partial unfolding of the gene-3-protein, cis → trans isomerization at Pro-213, and domain disassembly, thereby exposing its binding site for the ultimate receptor TolA. The trans-proline sets a molecular timer to maintain the binding-active state long enough for the phage to interact with TolA. We elucidated the changes in structure and local stability that lead to partial unfolding and thus to the activation of the gene-3-protein for phage infection. Protein folding and TolA binding experiments were combined with real-time NMR spectroscopy, amide hydrogen exchange measurements, and phage infectivity assays. In combination, the results provide a molecular picture of how a local unfolding reaction couples with prolyl isomerization not only to generate the activated state of a protein but also to maintain it for an extended time.

Sequence determination and analysis of three plasmids of Pseudomonas sp. GLE121, a psychrophile isolated from surface ice of Ecology Glacier (Antarctica).

Pseudomonas sp. GLE121 (a psychrophilic Antarctic strain) carries three plasmids: pGLE121P1 (6899 bp), pGLE121P2 (8330 bp) and pGLE121P3 (39,583 bp). Plasmids pGLE121P1 and pGLE121P2 show significant sequence similarity to members of the IncP-9 and IncP-7 incompatibility groups, respectively, while the largest replicon, pGLE121P3, is highly related to plasmid pNCPPB880-40 of Pseudomonas syringae pathovar tomato NCPPB880. All three plasmids have a narrow host range, limited to members of the genus Pseudomonas. Plasmid pGLE121P3 encodes a conjugal transfer system, while pGLE121P1 carries only a putative MOB module, conserved in many mobilizable plasmids. Plasmid pGLE121P3 contains an additional load of genetic information, including a pair of genes with homology to the rulAB operon, responsible for ultraviolet radiation (UVR) tolerance. Given the increasing UV exposure in Antarctic regions, the expression of these genes is likely to be an important adaptive response.

Characterization of periodontal biofilm in Down syndrome patients: a comparative study.

UNLABELLED: The aim of this study was to characterize the main periodontal bacterial species in Down syndrome (DS) patients with and without periodontitis. METHOD: This cross-sectional study involved 75 DS patients, 45 with and 30 without periodontitis. Informed consent, health and dental questionnaires and periodontitis diagnosis were performed PCR and LAMP assays were performed on subgingival dental plaque sample. RESULTS: Tannerella forsythia was the most frequent bacteria detected in the group with and without periodontitis (95.5 and 63.3%) followed by Treponema denticola (88.8 and 50%) and Porphyromonas gingivalis (53.3 and 25% respectively). There were statistical differences between groups (p < 0.05). Pg fimA type I was the most frequent Porphyromonas gingivalis genotype. Two different sets of primers (Aa-F/Aa-R and ltx3/ltx4) were used to detect Aggregatibacter actinomycetemcomitans and different frequencies were obtained, (68% and 14.6% respectively), they had a weak correlation (Cohen Kappa = 0.16). After sequencing of PCR products, ltx3/ltx4 showed more specificity. JP2 clone of A. actinomycetemcomitans was not detected in any sample. CONCLUSIONS: The composition of oral biofilm is fundamental for the development of periodontal disease independently of immunological alterations associated with DS. The frequency of detection of A. actinomycetemcomitans reported in the literature has a wide range, because the primers and probes applied

Characterization of aortic aneurysms in cardiovascular disease patients harboring Porphyromonas gingivalis.

OBJECTIVE: Porphyromonas gingivalis was recently shown to cause intimal hyperplasia in a mouse model by a novel cholesterol-independent mechanism, suggesting to be a pathogen-specific feature of cardiovascular diseases. The aim of this study was to characterize the clinical and histopathological features of aortic aneurysms in cardiovascular disease patients harboring oral P. gingivalis. SUBJECT AND METHODS: Aortic aneurysm specimens were collected from 76 Japanese patients who underwent surgery, of whom dental plaque specimens were also collected from 31 patients. Bacterial DNA was extracted from each specimen to detect P. gingivalis by polymerase chain reaction. Histopathological analyses of the aortic aneurysm specimens, including immunohistochemical staining for embryonic myosin heavy chain isoform (SMemb) and S100 calcium-binding protein A9 (S100A9), were also performed. RESULTS: The number of aneurysms occurring in the distal aorta was significantly higher in subjects positive for P. gingivalis in dental plaque compared with those who were negative. The expressions of S100A9 and SMemb were also significantly greater in the subjects positive for P. gingivalis in dental plaque. On the other hand, there were no significant differences in adipocellular accumulation between the groups. CONCLUSIONS: These results suggest that aortic aneurysms in patients harboring oral P. gingivalis have greater expression of S100A9 and proliferative smooth muscle cells, which was different from the present patients without oral P. gingivalis.

fimA genotypes and PFGE profile patterns in Porphyromonas gingivalis isolates from subjects with periodontitis.

BACKGROUND AND OBJECTIVES: Porphyromonas gingivalis is frequently identified to type by evaluation of fimA polymorphisms and less often by pulsed-field gel electrophoresis (PFGE) because of the technical intricacies of PFGE. To compare these techniques, we genotyped P. gingivalis clinical isolates as to (i) their fimA type and (ii) their whole genome restriction profile (PFGE analysis). MATERIAL AND METHODS: Thirty-two P. gingivalis strains were isolated from 16 unrelated periodontitis patients. Two strains were isolated from each patient. Strains were subjected to a fimA-typing polymerase chain reaction (PCR) assay. Strains that could not be typed by PCR were submitted to sequencing of the entire fimA gene. The PFGE profiles of clinical strains were compared using bioinformatic analysis. RESULTS: Seven of the 32 isolates were not typeable by PCR and so their entire fimA gene was sequenced. The sequencing identified each strain as belonging to a single fimA type. In one case, sequencing of the fimA gene did not agree with the result obtained using fimA PCR typing. With the exception of one patient, each patient presented isolates bearing the same fimA type. However, in three patients, isolates with the same fimA type presented different PFGE pulsotypes. CONCLUSION: The P. gingivalis typing using fimA PCR has limitations in typeability and discriminatory power. A typing technique for P. gingivalis that is easy to perform but that presents adequate typeability and discriminatory power is needed if we want to better understand the epidemiology of periodontal disease.

Homotypic biofilm structure of Porphyromonas gingivalis is affected by FimA type variations.

INTRODUCTION: Porphyromonas gingivalis is a periodontal pathogen whose long fimbriae (FimA) are classified into six genotypes (types I-V and Ib) based on the diversity of the fimA genes. FimA variations were previously shown to be related to the onset and development of adult periodontitis in a general population, while FimA were recently found to be critical mediators of initial biofilm formation. However, it is unclear if FimA variations have effects on biofilm features. Here, we compare the characteristic structures of homotypic biofilms developed by P. gingivalis strains with different FimA types. METHODS: Biofilms were formed on saliva-coated glass bottom wells in phosphate-buffered saline and their structures were analysed using confocal laser scanning microscopy. Furthermore, the biovolumes of the biofilms were quantified with a three-dimensional fluorophotometric method. RESULTS: Biofilm structures formed by the six representative FimA-type strains apparently differed. Type I and Ib P. gingivalis formed biofilms with a dense basal monolayer and dispersed microcolonies, whereas those formed by types II, III and IV strains had markedly luxuriant biofilms filled with widely clumped and tall colonies, and their biovolumes were significantly greater than those of types I and Ib. These characteristic features were confirmed to be closely related to FimA type in assays that utilized fimA-substituted mutants from type I to II and those from type II to I. CONCLUSION: Our results suggest that FimA variations have effects on the structures of biofilms formed by P. gingivalis, which may be an important factor in the pathogenesis of periodontitis.

Porphyromonas gingivalis fimbriae-dependent interleukin-6 autocrine regulation by increase of gp130 in endothelial cells.

BACKGROUND AND OBJECTIVE: Local persistent infection by Porphyromonas gingivalis leads to inflammatory systemic diseases, such as atherosclerosis. We have reported previously that avirulent P. gingivalis fimbriae-dependent invasion into endothelial cells might be involved in progression of atherosclerosis. Although interleukin-6 (IL-6) regulates progression of atherosclerosis, little is known about the relationship of P. gingivalis fimbriae-dependent invasion to IL-6 regulation in endothelial cells. MATERIAL AND METHODS: We examined the secretion of IL-6 and the expression of the IL-6 signal transducer gp130 in human umbilical vein endothelial cells (HUVEC) infected with the wild-type FDC381 strain of P. gingivalisand a fimbriae-deficient mutant (fimA) by enzyme-linked immunosorbent assay, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry (fluorescence-activated cell sorting, FACS) analysis. RESULTS: Coculture of HUVEC with P. gingivalis resulted in increase of IL-6 secretion at 24 h postinfection. Interestingly, the increase was inhibited significantly in HUVEC infected with the P. gingivalis fimA mutant. In addition, the increase of IL-6 secretion induced by P. gingivalis infection was significantly impaired by the meiosis specific kinase 1 inhibitor, PD98059, or the nuclear factor kappaB inhibitor, Bay11-7082. Furthermore, we demonstrated that gp130 expression increased with P. gingivalis infection. Importantly, gp130 expression was significantly impaired by P gingivalis fimA mutant infection compared with wild-type P. gingivalis infection, as assessed by both quantitative RT-PCR and FACS analysis. CONCLUSION: Our findings indicate that P. gingivalis fimbriae are important factors in the autocrine regulation of IL-6, by increasing gp130 in endothelial cells.

Quantification of Porphyromonas gingivalis and fimA genotypes in smoker chronic periodontitis.

AIM: Porphyromonas gingivalis fimA genotypes were associated with virulence factors in vitro, but little evidence of an association with disease severity were shown in humans. We aimed to correlate levels of P. gingivalis fimA genotypes II and IV and probing depth in smoker-chronic periodontitis subjects. MATERIAL AND METHODS: One hundred and sixty eight subgingival samples of 20 smokers non-treated chronic periodontitis subjects obtained from sites with different probing depths [shallow (< or =3 mm), intermediate (4-6 mm), deep (> or =7 mm)] were analysed by real-time PCR for P. gingivalis and genotypes fimA II and IV. RESULTS: P. gingivalis and fimA IV were detected in all subjects, whereas fimA II was detected in 18 subjects (90%). One hundred and fifty two sites (90.5%) harboured P. gingivalis. Genotypes II and IV were detected in 28% and 69.6% of sites, respectively. The proportions of genotypes II and IV in relation to P. gingivalis levels were similar in shallow, intermediate and deep probing sites (2.4%, 4.6%, 1.4% for genotype II and 15.5%, 17.7%, 11.7% for genotype IV, respectively), indicating that other non-tested genotypes were more abundant. Increased levels of genotype IV were associated with increasing probing depth, but not of genotype II. CONCLUSIONS: The data suggested an association between P. gingivalis genotype fimA IV and disease severity in smoker-chronic periodontitis subjects.

Sex pilus specific bacteriophage to drive bacterial population towards antibiotic sensitivity.

Antimicrobial resistance (AMR) is now a major global problem largely resulting from the overuse of antibiotics in humans and livestock. In some AMR bacteria, resistance is encoded by conjugative plasmids expressing sex-pili that can readily spread resistance through bacterial populations. The aim of this study was to use sex pilus-specific (SPS) phage to reduce the carriage of AMR plasmids. Here, we demonstrate that SPS phage can kill AMR Escherichia coli and select for AMR plasmid loss in vitro. For the first time, we also demonstrate that SPS phage can both prevent the spread of AMR Salmonella Enteritidis infection in chickens and shift the bacterial population towards antibiotic sensitivity.

Tying rings for sex.

The primary component of the sex pilus encoded by IncP (RP4) and Ti plasmids has been identified as a circular pilin protein with a peptide bond between the amino and carboxyl terminus. Here, we review the key experiments that led to this discovery, and the present mechanistic model for pilin-precursor processing and the cyclization reaction. In addition, we discuss the implications for horizontal gene transfer in bacterial conjugation.

Selective phage infection mediated by epitope expression on F pilus.

Proteins and peptides can be displayed on bacterial and bacteriophage surfaces as fusions to bacterial integral membrane proteins or phage coat proteins. We now report on the expression of peptide antigens on the surface of F pili, elaborated by F+ strains of Escherichia coli. The peptides were expressed as fusions to F pilin, the building block of the F pilus that is encoded by the traA gene on the F plasmid. Filamentous bacteriophage infection of E. coli is normally mediated by phage binding to pilin at the F pili tip. Expression of 13 to 15 amino acid long peptides on the F pilus completely blocked infection by derivatives of wild-type infectious M13 phage. However, when a phage displaying a specific recombinant antibody fragment was allowed to interact with F pili displaying an antigenic peptide a bacterial infection could be demonstrated. This infection, mediated by the antibody-antigen interaction, resulted in bacterial cells containing plasmids encoding both the protein and the ligand. In a model library, where a scFv antibody against the human cytomegalovirus AD-2 epitope was selected we achieved an enrichment of 2500 of phage carrying the specific antibody, indicating an efficient selective infection.

Neisserial pilin genes display extensive interspecies diversity.

All Neisseria live in association with host cells, however, little is known about the genetic potential of nonpathogenic Neisseria species to express attachment factors such as pili. In this study, we demonstrate that type IV pilin-encoding genes are present in a wide range of Neisseria species. N. sicca, N. subflava, and N. elongata each contain two putative pilE genes arranged in tandem, while single genes were identified in N. polysaccharea, N. mucosa, and N. denitrificans. Neisserial pilE genes are highly diverse and display features consistent with a history of horizontal gene transfer.

Genetics of Natural Competence in Vibrio cholerae and other Vibrios.

Many Gram-positive and Gram-negative bacteria can become naturally competent to take up extracellular DNA from the environment via a dedicated uptake apparatus. The genetic material that is acquired can (i) be used for nutrients, (ii) aid in genome repair, and (iii) promote horizontal gene transfer when incorporated onto the genome by homologous recombination, the process of "transformation." Recent studies have identified multiple environmental cues sufficient to induce natural transformation in Vibrio cholerae and several other Vibrio species. In V. cholerae, nutrient limitation activates the cAMP receptor protein regulator, quorum-sensing signals promote synthesis of HapR-controlled QstR, chitin stimulates production of TfoX, and low extracellular nucleosides allow CytR to serve as an additional positive regulator. The network of signaling systems that trigger expression of each of these required regulators is well described, but the mechanisms by which each in turn controls competence apparatus genes is poorly understood. Recent work has defined a minimal set of genes that encode apparatus components and begun to characterize the architecture of the machinery by fluorescence microscopy. While studies with a small set of V. cholerae reference isolates have identified regulatory and competence genes required for DNA uptake, future studies may identify additional genes and regulatory connections, as well as revealing how common natural competence is among diverse V. cholerae isolates and other Vibrio species.

Type IV pilus gene homologs pilABCD are required for natural transformation in Actinobacillus actinomycetemcomitans.

Some clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans are naturally competent for DNA uptake. In this study, we examined the sequence and the function of a type IV pilus-like pilABCD gene cluster and its downstream region in a naturally transformable A. actinomycetemcomitans strain D7S. Specific knockout mutants of pilABCD of strain D7S were constructed by replacing individual genes with an antibiotic resistance cassette. The transformation frequency of chromosome markers in the wildtype strain D7S was approximately 10(-3) per CFU. In contrast, the delta pilA, delta pilB, delta pilC, delta pilBC or delta pilD mutants were non-transformable (transformation frequency <10(-8)). Disruption of an ORF downstream of pilD had no apparent effect on the transformability of this bacterium. The pilA or pilBC deletion did not seem to affect fimbria expression or cell surface structure in either rough or smooth strains as determined by scanning and transmission electron microscopy examinations. RT-PCR analysis showed that pilA was expressed in strain D7S under a competence-inducing growth condition. The expression of pilA was barely detectable in strain D7S cultured under a non-competence-inducing condition or in the non-transformable strain JP-2. The results indicate that pilABCD are required for competence but are apparently not involved in fimbria expression of A. actinomycetemcomitans.

The pathogenic neisseriae contain an inactive rpoN gene and do not utilize the pilE sigma54 promoter.

The sigma54 promoter (P3) upstream of the pilE gene in Neisseria gonorrhoeae was shown to be non-functional by transcriptional analysis of a PpilE::lacZ fusion containing only P3. A region on the chromosome of N. gonorrhoeae strain MS11-A was identified that potentially encodes a protein with a significant similarity to the Escherichia coli RpoN protein. However, this region (designated RLS for rpoN-like sequence) does not contain a single open reading frame (ORF) capable of encoding a functional RpoN protein. It appears that RLS may have arisen from an ancestral rpoN homologue that underwent a deletion removing the sequence encoding the essential helix-turn-helix (HTH) motif, and changing the subsequent reading frame. An RLS has been identified in several strains of N. gonorrhoeae and N. meningitidis. A 90-kDa gonococcal protein has previously been shown to react with a monoclonal antibody raised against the RpoN from Salmonella typhimurium. However, mutagenesis and Western blot analysis confirmed that the gene encoding this protein is not contained within RLS.

Regions of the CFA/I promoter involved in the activation by the transcriptional activator CfaD and repression by the histone-like protein H-NS.

Expression of CFA/I fimbriae of Escherichia coli requires the transcriptional activator CfaD. The mechanism by which CfaD activates the CFA/I promoter is to overcome the repression by H-NS, one of the histone-like proteins in E coli. This study addresses the question of which sequences in the promoter region of CFA/I interact with CfaD and H-NS. In order to determine this, deletion mutants of the CFA/I promoter were constructed and cloned upstream of the promoterless lacZ gene. The effect of CfaD and H-NS on the expression of these constructs was determined.

The genome of Neisseria gonorrhoeae retains the remnants of a two-component regulatory system that once controlled piliation.

An intact activator-binding site upstream of the sigma(54) promoter of the pilin-encoding pilE gene of Neisseria gonorrhoeae suggests gonococci produce a protein capable of binding this sequence. We cloned a chimeric gene, rsp, that has sequence similarity to both the pilS and pilR genes of Pseudomonas aeruginosa encoding a two-component regulatory system that controls piliation. This gene is transcribed in N. gonorrhoeae and indirect evidence suggests that Rsp binds to the activator-binding site of the pilE gene. Despite this, mutation of rsp has no effect on piliation in N. gonorrhoeae, suggesting that the remnants of this regulatory system have persisted in the genome, despite the loss of its original function.

Optimisation of ISO 10705-1 on enumeration of F-specific bacteriophages.

During the European project 'Bacteriophages in bathing waters' (January 1996-June 1999), research was carried out to optimise the method for detection and enumeration of F-specific (RNA) phages in water. It was evaluated whether further optimisation would be possible/needed for the procedure as described in the standard method of the International Organisation for Standardisation (ISO) 10705-1. The research focused mainly on optimisation of the different steps for culturing the host strain WG49 Salmonella Typhimurium. It was concluded that all steps described in ISO 10705-1 are necessary and, if followed carefully, using a culture of host strain WG49 Salmonella Typhimurium of good quality, reliable results could be obtained for the enumeration of F-specific RNA phages.

Prevalence of specific genotypes of Porphyromonas gingivalis fimA and periodontal health status.

Porphyromonas gingivalis fimA gene encoding fimbrillin, a subunit of fimbriae, has been classified into 5 genotypes (types I to V) based on their nucleotide sequences. Here, we investigated the relationship between the prevalence of these fimA genotypes and periodontal health status in adults. Dental plaque specimens obtained from 380 periodontally healthy adults and 139 periodontitis patients were analyzed by the PCR method. P. gingivalis was detected in 36.8% of the healthy subjects and in 87.1% of the periodontitis patients. Among the P. gingivalis-positive healthy adults, the most prevalent fimA type was type I (76.1%), followed by type V. In contrast, a majority of the periodontitis patients carried type II fimA organisms (66.1%), followed by type IV. The univariate analysis illustrated that periodontitis was associated with the occurrences of type I fimA (OR 0.16), type II (OR 44.44), type III (1.96), type IV (13.87), and type V (1.40). These findings clearly indicate that there are both disease-associated and non-disease-associated strains of P. gingivalis, and that their infectious traits influencing periodontal health status could be differentiated based on the clonal variation of fimA genes.