Effect of Resveratrol on biofilm formation and virulence factor gene expression of Porphyromonas gingivalis in periodontal disease.

Periodontal disease is an oral inflammatory disease that destroys the tooth supporting periodontal tissues resulting in tooth loss. Porphyromonas gingivalis is a keystone pathogen that plays a significant role in periodontitis. In previous studies, resveratrol has shown significant results by targeting inflammatory and adhesive markers. Virulence factors of P. gingivalis play an important role in the bacterial adhesion and colonization. In this study, we aimed to demonstrate the anti-biofilm and anti-bacterial activity of resveratrol and also study the effect of resveratrol on the expression of virulence factor genes of P. gingivalis using reverse transcriptase polymerase chain reaction (RT-PCR). The anti-microbial and anti-biofilm activity of resveratrol on P. gingivalis was carried out by broth microdilution assay and biofilm adhesion reduction-crystal violet assay, respectively. We carried out the gene expression analysis by RT-PCR with the P. gingivalis treated compound to analyze the change in the expression of virulence factors: fimbriae and gingipain. Minimal inhibitory concentrations (MIC) of resveratrol against P. gingivalis and other clinical strains are in the range of 78.12-156.25 µg/mL. Resveratrol dose-dependently prevented the biofilm formation and also attenuated the virulence of P. gingivalis by reducing the expression of virulence factor genes such as fimbriae (type II and IV) and proteinases (kgp and rgpA). Resveratrol demonstrated superior anti-bacterial and anti-biofilm activity against P. gingivalis. There was significant reduction in the expression of fimbriae and gingipain with the resveratrol-treated compound. The results suggest that resveratrol, due to its multiple actions, may become a simple and inexpensive therapeutic strategy for treating periodontal disease.

An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants.

Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280 nm and their emission spectra collected from 290 to 400 nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4 µg/mL and limit of linearity between 100 and 200 µg/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing.

A Potential New Risk Factor for Stroke: Streptococcus Mutans With Collagen-Binding Protein.

BACKGROUND: Among human oral bacteria, particular kinds of Streptococcus mutans (SM) known as dental caries pathogens contain a collagen-binding protein, Cnm, and show platelet aggregation inhibition and matrix metalloproteinase-9 activation. We have previously reported that these strains may be a risk factor for intracerebral hemorrhage. As a major sample-providing hospital, we report the clinical details, including intracranial aneurysms and ischemic stroke. METHODS: After the study received approval from the Ethical Committee, 429 samples of whole saliva were obtained from patients who were admitted to or visited our hospital between February 16, 2010, and February 28, 2011. The study cohort comprised 48 patients with cardioembolic stroke (CES), 151 with non-CES infarct, 54 with intracerebral hemorrhage (ICH), 43 with ruptured intracranial aneurysm (RIA), and 97 with unruptured intracranial aneurysm (UIA). Cultured SM was identified as Cnm-positive when the corresponding gene was positive. The results were compared with those from 79 healthy volunteers. Relationships between Cnm-positive SM and known risk factors, including hypertension, diabetes, hyperlipidemia, smoking, and alcohol consumption, were analyzed. RESULTS: A statistically significant high Cnm-positive rate was observed in patients with CES, non-CES infarct, ICH, and RIA (P = 0.002, 0.039, 0.013, and 0.009, respectively). There were no relationships between Cnm-positive SM and known risk factors. CONCLUSIONS: Specific types of oral SM can be a risk factor for cardioembolic infarct, intracerebral hemorrhage, and intracranial aneurysm rupture. Further study is needed.

Placental and colostral transfer of antibodies reactive with enteropathogenic Escherichia coli intimins α, ß, or γ / Transferência placentária e colostral de anticorpos reativos a Escherichia coli enteropatogênica com expressão das intiminas α, ß, or γ

Abstract Objective: Intimins are protein adhesins of enteropathogenic Escherichia coli and enterohemorrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodies are important for the protection from enteropathogenic E. coli and enterohemorrhagic E. coli infections because these antibodies inhibit bacterial adhesion and impair the initial step of the pathogenesis. We studied the transfer of maternal anti-intimin antibodies from healthy Brazilian mothers to their newborns through the placenta and colostrum. Methods: Serum immunoglobulin G and secretory immunoglobulin A antibodies against conserved and variable regions of intimins α, β, and γ were analyzed using an enzyme linked-immunosorbent assay in the blood and colostrum from 45 healthy women as well as cord blood serum samples from their newborns. Results: The concentrations of antibodies reactive with α intimin were significantly lower than those of anti-γ and anti-conserved intimin antibodies in the colostrum samples. IgG serum antibodies reactive with all the subtypes of intimins were transferred to the newborns, but the concentrations of anti-conserved intimin serum antibodies were significantly higher in mothers and newborns than concentrations of antibodies against variable regions. The patterns of IgG transfer from mothers to newborns were similar for all anti-intimin antibodies. These values are similar to the percentage transference of total IgG. Conclusions: Anti-intimin antibodies are transferred from mothers to newborns through the placenta, and reinforce the protection provided by breastfeeding against diarrheagenic E. coli infections.

Resumo Objetivo: As intiminas são adesinas proteicas de Escherichia coli enteropatogênicas (EPEC) e enterro-hemorrágicas (EHEC) capazes de induzir as lesões attaching and effacing nos enterócitos. Anticorpos anti-intiminas são importantes para a proteção contra infecções por EPEC e EHEC porque esses anticorpos inibem a adesão bacteriana e impedem o passo inicial do mecanismo patogênico dessas bactérias. Nós estudamos a transferência de anticorpos maternos anti-intiminas de mães brasileiras saudáveis para os seus recém-nascidos através da placenta e do colostro. Métodos: Anticorpos séricos da classe IgG e secretórios da classe IgA (SIgA) reativos com as porções conservada (cons) e variáveis das intiminas α (vα), β (vβ) e γ (vγ) foram analisados pelo teste de ELISA no sangue e no colostro de 45 parturientes saudáveis e no sangue de cordão umbilical dos seus respectivos recém-nascidos. Resultados: As concentrações de anticorpos reativos com intimina vα foram significativamente mais baixas que as dos anticorpos anti-vγ e anti-cons nas amostras de colostro. Anticorpos IgG séricos reativos com todas as intiminas foram transferidos para os recém-nascidos, mas as concentrações de anti-cons foram significativamente mais altas tanto nas mães como nos recém-nascidos do que os anticorpos reativos com as regiões variáveis das intiminas. O padrão de transferência de IgG das mães para os recém-nascidos foi muito semelhante para todos os anticorpos anti-intiminas. Os valores de porcentagem de transferência foram semelhantes à transferência de IgG total. Conclusões: Anticorpos anti-intimina são transferidos das mães para os recém-nascidos pela placenta e corroboram a proteção contra infecções por Escherichia coli diarreiogênicas (DEC) conferida pelo aleitamento materno.

PAR-2 expression in the gingival crevicular fluid reflects chronic periodontitis severity.

Recent studies investigating protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. It is known that gingipain, a bacterial protease secreted by the important periodontopathogen Porphyromonas gingivalis can activate PAR-2. Previous studies by our group found that PAR-2 is overexpressed in the gingival crevicular fluid (GCF) of patients with moderate chronic periodontitis (MP). The present study aimed at evaluating whether PAR-2 expression is associated with chronic periodontitis severity. GCF samples and clinical parameters, including plaque and bleeding on probing indices, probing pocket depth and clinical attachment level, were collected from the control group (n = 19) at baseline, and from MP patients (n = 19) and severe chronic periodontitis (SP) (n = 19) patients before and 6 weeks after periodontal non-surgical treatment. PAR-2 and gingipain messenger RNA (mRNA) in the GCF of 4 periodontal sites per patient were evaluated by Reverse Transcription Polymerase Chain Reaction (RT-qPCR). PAR-2 and gingipain expressions were greater in periodontitis patients than in control group patients. In addition, the SP group presented increased PAR-2 and gingipain mRNA levels, compared with the MP group. Furthermore, periodontal treatment significantly reduced (p <0.05) PAR-2 expression in patients with periodontitis. In conclusion, PAR-2 is associated with chronic periodontitis severity and with gingipain levels in the periodontal pocket, thus suggesting that PAR-2 expression in the GCF reflects the severity of destruction during periodontal infection.

PAR-2 expression in the gingival crevicular fluid reflects chronic periodontitis severity

Abstract Recent studies investigating protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. It is known that gingipain, a bacterial protease secreted by the important periodontopathogen Porphyromonas gingivalis can activate PAR-2. Previous studies by our group found that PAR-2 is overexpressed in the gingival crevicular fluid (GCF) of patients with moderate chronic periodontitis (MP). The present study aimed at evaluating whether PAR-2 expression is associated with chronic periodontitis severity. GCF samples and clinical parameters, including plaque and bleeding on probing indices, probing pocket depth and clinical attachment level, were collected from the control group (n = 19) at baseline, and from MP patients (n = 19) and severe chronic periodontitis (SP) (n = 19) patients before and 6 weeks after periodontal non-surgical treatment. PAR-2 and gingipain messenger RNA (mRNA) in the GCF of 4 periodontal sites per patient were evaluated by Reverse Transcription Polymerase Chain Reaction (RT-qPCR). PAR-2 and gingipain expressions were greater in periodontitis patients than in control group patients. In addition, the SP group presented increased PAR-2 and gingipain mRNA levels, compared with the MP group. Furthermore, periodontal treatment significantly reduced (p <0.05) PAR-2 expression in patients with periodontitis. In conclusion, PAR-2 is associated with chronic periodontitis severity and with gingipain levels in the periodontal pocket, thus suggesting that PAR-2 expression in the GCF reflects the severity of destruction during periodontal infection.

A novel high-throughput assay to quantify the vaccine-induced inhibition of Bordetella pertussis adhesion to airway epithelia.

BACKGROUND: Pertussis or whooping cough is an acute respiratory illness caused by the Gram-negative pathogen Bordetella pertussis. Despite high vaccination coverage whooping cough is currently re-emerging in many developed countries. Although the causes of pertussis resurgence are matter of debate, emerging evidences suggest that acellular vaccines efficiently protect against the hallmark symptoms of pertussis disease but fail to prevent colonization. This presumably impacts on increased risk of bacterial transmission and consequent spread throughout the population. These evidences suggest that improved vaccines may be required for efficient bacterial clearance in the upper respiratory tract. Consequently, there is a need for novel bioassays to evaluate at pre-clinical or clinical level the impact of different vaccines on B. pertussis colonization. RESULTS: We developed a high-throughput bacterial adhesion inhibition (BAI) assay based on human respiratory cell lines and on live bacteria chemically conjugated to a fluorescent dye. Employing A549 cells as model, we evaluated the impact of antibodies elicited by acellular (aP) and whole cell (wP) vaccines on B. pertussis adhesion in vitro. Moreover, we settled the method also on polarized Calu-3 cells grown at air-liquid interface (ALI), showing that this assay can be extended to more complex cell models mimicking the airway epithelium. CONCLUSIONS: We proved that this method is a sensitive, rapid and reproducible system to evaluate the anti-adhesive properties of vaccine-induced antibodies and can be employed to assess improved pertussis vaccines.

Quantifying implant-associated biofilms: Comparison of microscopic, microbiologic and biochemical methods.

Biofilm-associated infections pose severe problems in modern implant medicine. Screening for new implant materials with antibacterial properties requires reliable quantification of colonizing bacteria. There are many different methods to quantify biofilms on solid surfaces in vitro, employing different (bio-)chemical/microbiological reference parameters. It is therefore difficult to compare studies with different quantification techniques. Here, we have evaluated commonly used microscopic, microbiologic and biochemical methods to quantify bacterial biofilms, in order to clarify their comparability and applicability. Two bacterial species frequently involved in biofilm-associated infections, Staphylococcus aureus and Aggregatibacter actinomycetemcomitans, were used as model organisms; their initial adhesion and biofilm formation on titanium and on antibacterial copper were analyzed using the following methods: LIVE/DEAD fluorescence staining and confocal laser-scanning microscopy, ultrasonic or a newly developed enzymatic detachment followed by standard plate counting (CFU method), a resazurin-based assay, the BacTiter-Glo™ assay and crystal violet staining. The methods differed greatly in complexity, reliability and the applicability to initial adhesion and biofilm formation. To screen biofilm formation on a multitude of surfaces, the resazurin-based and the BacTiterGlo™ assay are well suited. LIVE/DEAD staining and confocal laser-scanning microscopy can be applied for a more detailed analysis of both, initial adhesion and biofilm formation. When using the CFU method for screening purposes, the introduced enzymatic detachment procedure is to be favored over ultrasonic detachment. There is not one single method, which is suitable for all purposes. The appropriate biofilm quantification method has to be chosen on the basis of the specific scientific question.

Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms.

In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host-pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus-pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.

The expression of heterologous MAM-7 in Lactobacillus rhamnosus reduces its intrinsic capacity to inhibit colonization of pathogen Vibrio parahaemolyticus in vitro.

BACKGROUND: Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative, halophilic bacterium recognized as one of the most important foodborne pathogen. When ingested, V. parahaemolyticus causes a self-limiting illness (Vibriosis), characterized mainly by watery diarrhoea. Treatment is usually oral rehydration and/or antibiotics in complicated cases. Since 1996, the pathogenic and pandemic V. parahaemolyticus O3:K6 serotype has spread worldwide, increasing the reported number of vibriosis cases. Thus, the design of new strategies for pathogen control and illness prevention is necessary. Lactobacillus sp. grouped Gram positive innocuous bacteria, part of normal intestinal microbiota and usually used as oral vaccines for several diarrheic diseases. Recombinants strains of Lactobacillus (RL) expressing pathogen antigens can be used as part of an anti-adhesion strategy where RL block the pathogen union sites in host cells. Thus, we aimed to express MAM-7 V. parahaemolyticus adhesion protein in Lactobacillus sp. to generate an RL that prevents pathogen colonization. RESULTS: We cloned the MAM-7 gene from V. parahaemolyticus RIMD 2210633 in Lactobacillus expression vectors. Recombinant strains (Lactobacillus rhamnosus pSEC-MAM7 and L. rhamnosus pCWA-MAM7) adhered to CaCo-2 cells and competed with the pathogen. However, the L. rhamnosus wild type strain showed the best capacity to inhibit pathogen colonization in vitro. In addition, LDH-assay showed that recombinant strains were cytotoxic compared with the wild type isogenic strain. CONCLUSIONS: MAM-7 expression in lactobacilli reduces the intrinsic inhibitory capacity of L. rhamnosus against V. parahaemolyticus.

The expression of heterologous MAM-7 in Lactobacillus rhamnosus reduces its intrinsic capacity to inhibit colonization of pathogen Vibrio parahaemolyticus in vitro

BACKGROUND: Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative, halophilic bacterium recognized as one of the most important foodborne pathogen. When ingested, V. parahaemolyticus causes a self-limiting illness (Vibriosis), characterized mainly by watery diarrhoea. Treatment is usually oral rehydration and/or antibiotics in complicated cases. Since 1996, the pathogenic and pandemic V. parahaemolyticus O3:K6 serotype has spread worldwide, increasing the reported number of vibriosis cases. Thus, the design of new strategies for pathogen control and illness prevention is necessary. Lactobacillus sp. grouped Gram positive innocuous bacteria, part of normal intestinal microbiota and usually used as oral vaccines for several diarrheic diseases. Recombinants strains of Lactobacillus (RL) expressing pathogen antigens can be used as part of an anti-adhesion strategy where RL block the pathogen union sites in host cells. Thus, we aimed to express MAM-7 V. parahaemolyticus adhesion protein in Lactobacillus sp. to generate an RL that prevents pathogen colonization RESULTS: We cloned the MAM-7 gene from V. parahaemolyticus RIMD 2210633 in Lactobacillus expression vectors. Recombinant strains (Lactobacillus rhamnosus pSEC-MAM7 and L. rhamnosus pCWA-MAM7) adhered to CaCo-2 cells and competed with the pathogen. However, the L. rhamnosus wild type strain showed the best capacity to inhibit pathogen colonization in vitro. In addition, LDH-assay showed that recombinant strains were cytotoxic compared with the wild type isogenic strain CONCLUSIONS: MAM-7 expression in lactobacilli reduces the intrinsic inhibitory capacity of L. rhamnosus against V. parahaemolyticus.

Relationship between phylogenetic groups, antibiotic resistance and patient characteristics in terms of adhesin genes in cystitis and pyelonephritis isolates of Escherichia coli.

Extraintestinal pathogenic Escherichia coli (E. coli) is considered as the main causative agent of urinary tract infections worldwide. The relationship between antimicrobial resistance, phylogenetic groups, patient characteristics and adhesin virulence genes are complex and not fully understood. In this study, among 146 urinary isolates of E. coli, phylogenetic groups and various adhesin virulence genes were examined with multiplex Polymerase Chain Reaction methods. Patient characteristics divided into sex, cystitis and pyelonephritis; community-acquired and hospital-acquired; complicated and uncomplicated infection. Antimicrobial resistance was also determined. The papAH gene was seen more often in pyelonephritis than cystitis and female than male patients. iha gene was more frequent in hospital-acquired infections than in community-acquired infections. sfa/focDE was more frequent in ampicillin, amikacin, gentamicin, nalidixic acid, norfloxacin, cefuroxime, ceftriaxone, cefazolin, cefotaxime, ciprofloxacin and trimethoprim/sulfamethoxazole susceptible and extended-spectrum ß-lactamase (ESBL) and multi-drug resistance (MDR) negative isolates. focG was seen more often in nalidixic acid, norfloxacin, cefuroxime, ceftriaxone, ciprofloxacin susceptible and MDR negative isolates. fimH and papAH were more commonly observed in amoxicillin/clavulanic acid and cefotaxime susceptible isolates, respectively. iha and afa/draBC genes were more frequent in resistant isolates than the susceptible ones; for iha, in ampicillin, amoxicillin/clavulanic acid, nalidixic acid, cefuroxime, ceftriaxone resistant and ESBL and MDR positive isolates; for afa/draBC, in cefotaxime, cefuroxime, ciprofloxacin, trimethoprim/sulfamethoxazole resistant and ESBL and MDR positive isolates, this trend was observed. ST 131 E. coli virulence gene pattern has a direct effect on resistance profile. Isolates belong to that clonal group has MDR and commonly harbour afa/draBC and iha genes. Our findings may provide new insights into the relationships between pathogenesis, patient characteristics and resistance of E. coli UTI.

Prevalence of Panton-Valentine leucocidin and phenotypic and genotypic characterization of biofilm formation among Staphylococcus aureus strains isolated from children with adenoid hypertrophy.

Adenoids as a first line of host defense against respiratory microbes play an important role in majority of upper airway infectious and noninfectious illnesses. Bacterial pathogen can colonize on the adenoid tissue and probably act as a reservoir for them. To determine phenotypic and genotypic characterization of biofilm forming capacity of Staphylococcus aureus isolates from children with adenoid hypertrophy and prevalence of Panton-Valentine leukocidin (PVL) gene we collected 17 consecutive, clinically significant S. aureus isolates from children with adenoid hypertrophy undergoing adenoidectomy with one or more of the upper airway obstruction symptoms, nasal obstruction, mouth breathing, snoring, or sleep apnea. Biofilm formation was evaluated by colorimetric microtiter plate's assay. Gene encoding PVL and adhesion- or biofilm formation-encoding genes were targeted by polymerase chain reaction (PCR) assay. According to the results, all strains produced biofilm. Seven (41.2%) isolates produced strong biofilm whereas 7 (41.2%) isolates produced week and 3 (17.6%) isolates produced medium biofilm. Regarding the adhesion- or biofilm formation-encoding genes, 16 (94.1%) isolates were positive for the gene eno, 13(76.4%) for icaA, 13 (76.4%) for icaD, 10 (58.8%) for fib, 10 (58.8%) for fnbB, 4(23.5%) for can, and 1(5.8%) for fnbA. The high prevalence of genes encoding biofilms and adhesins and phenotypic ability to form a biofilm by S. aureus strains emphasizes the pathogenic character of strains isolated from children with adenoid hypertrophy.

Pneumococcal Adhesins PavB and PspC Are Important for the Interplay with Human Thrombospondin-1.

The human matricellular glycoprotein thrombospondin-1 (hTSP-1) is released by activated platelets and mediates adhesion of Gram-positive bacteria to various host cells. In staphylococci, the adhesins extracellular adherence protein (Eap) and autolysin (Atl), both surface-exposed proteins containing repeating structures, were shown to be involved in the acquisition of hTSP-1 to the bacterial surface. The interaction partner(s) on the pneumococcal surface was hitherto unknown. Here, we demonstrate for the first time that pneumococcal adherence and virulence factor B (PavB) and pneumococcal surface protein C (PspC) are key players for the interaction of Streptococcus pneumoniae with matricellular hTSP-1. PavB and PspC are pneumococcal surface-exposed adhesins and virulence factors exhibiting repetitive sequences in their core structure. Heterologously expressed fragments of PavB and PspC containing repetitive structures exhibit hTSP-1 binding activity as shown by ELISA and surface plasmon resonance studies. Binding of hTSP-1 is charge-dependent and inhibited by heparin. Importantly, the deficiency in PavB and PspC reduces the recruitment of soluble hTSP-1 by pneumococci and decreases hTSP-1-mediated pneumococcal adherence to human epithelial cells. Platelet activation assays suggested that PavB and PspC are not involved in the activation of purified human platelets by pneumococci. In conclusion, this study indicates a pivotal role of PavB and PspC for pneumococcal recruitment of soluble hTSP-1 to the bacterial surface and binding of pneumococci to host cell-bound hTSP-1 during adhesion.

LL-37 in periodontal health and disease and its susceptibility to degradation by proteinases present in gingival crevicular fluid.

AIM: To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease. MATERIALS AND METHODS: Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. RESULTS: The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites. CONCLUSIONS: LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis.

Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

Cleavage of IgG1 in gingival crevicular fluid is associated with the presence of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVES: Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of Porphyromonas gingivalis and other periodontopathogens. MATERIAL AND METHODS: GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis and five periodontally healthy individuals. The bacterial loads of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia were analysed by real-time polymerase chain reaction, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA. RESULTS: Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. Porphyromonas gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (P < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of Tannerella forsythia and Prevotella intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of Porphyromonas gingivalis (r = 0.425, P < 0.01). The presence of Kgp (range 0.07-10.98 ng/mL) was associated with proteolytic fragments of IgG1 (P < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp. CONCLUSION: In patients with periodontitis, cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by Porphyromonas gingivalis.

Neutrophils alter epithelial response to Porphyromonas gingivalis in a gingival crevice model.

A gingival crevice model (epithelial cell-Porphyromonas gingivalis-neutrophil) was established and used to profile gingipain, matrix metalloproteinase (MMP), MMP mediators [neutrophil gelatinase-associated lipocalin (NGAL) and tissue inhibitor of metalloproteinases 1 (TIMP-1)] and cytokine networks. Smoking is the primary environmental risk factor for periodontitis. Therefore, the influence of cigarette smoke extract (CSE) was also monitored in the same model. Porphyromonas gingivalis alone induced low levels of interleukin-1ß and interleukin-8 from epithelial cells, but high levels of both cytokines were produced on the addition of neutrophils. Exposure to CSE (100 and 1000 ng ml(-1) nicotine equivalency) significantly compromised P. gingivalis-induced cytokine secretion (both P < 0.05). P. gingivalis induced impressive secretion of NGAL (P < 0.05) that was not influenced by CSE. The influence of CSE on gingipain production was strain-specific. Purified gingipains effectively and rapidly degraded both TIMP-1 and MMP-9. Induction of large amounts of NGAL, degradation of TIMP-1, and increased gingipain activity would each be expected to prolong collagen degradation and promote disease progression. However, gingipains also degrade MMP-9. Hence, P. gingivalis exerts a complex influence on the proteolytic balance of a gingival crevice model. Exposure to CSE reduces the proinflammatory cytokine burden, which may be expected to promote P. gingivalis survival. In addition to novel findings that provide mechanistic insight into periodontal disease progression, these results are in keeping with the recognized clinical dogma of decreased inflammation/increased disease in smokers. This straightforward gingival crevice model is established as a suitable vehicle for the elucidation of mechanisms that contribute to susceptibility to periodontitis.

Treponema denticola improves adhesive capacities of Porphyromonas gingivalis.

Porphyromonas gingivalis, an important etiological agent of periodontal disease, is frequently found associated with Treponema denticola, an anaerobic spirochete, in pathogenic biofilms. However, interactions between these two bacteria are not well understood at the molecular level. In this study, we seek to link the influence of T. denticola on the expression of P. gingivalis proteases with its capacities to adhere and to form biofilms. The P. gingivalis genes encoding Arg-gingipain A (RgpA), Lys-gingipain (Kgp), and hemagglutinin A (HagA) were more strongly expressed after incubation with T. denticola compared with P. gingivalis alone. The amounts of the three resulting proteins, all of which contain hemagglutinin adhesion domains, were increased in culture supernatants. Moreover, incubation of P. gingivalis with T. denticola promoted static and dynamic biofilm formation, primarily via a time-dependent enhancement of P. gingivalis adhesion capacities on bacterial partners such as Streptococcus gordonii. Adhesion of P. gingivalis to human cells was also increased. These results showed that interactions of P. gingivalis with other bacterial species, such as T. denticola, induce increased adhesive capacities on various substrata by hemagglutinin adhesion domain-containing proteins.

Identificação de adesinas de Leptospira interrogans por shotgun phage display / Identification of Leptospira interrogans adhesins by shotgun phage display

Em Leptospira interrogans algumas proteínas com capacidade de ligação aos componentes de matriz extracelular foram identificadas e, em sua maioria, são fatores de virulência. Phage display é considerada uma técnica poderosa na identificação de novos ligantes, inclusive de moléculas adesinas, importantes no primeiro estágio de infecção do hospedeiro. A técnica de shotgun phage display foi utilizada visando à obtenção de ligantes à células de mamíferos. Quatro bibliotecas, por inserção de fragmentos aleatórios obtidos por sonicação do DNA de L. interrogans nos fagomídeos pG8SAET (BBT1 e BBT2) e pG3DSS (BBT5 e BBT6), foram construídas. As bibliotecas BBT1 e BBT5 contém insertos maiores e as BBT2 e BBT6 contém insertos menores, com tamanhos médios de 1500 pb e 350 pb, respectivamente. Após ensaio de panning da BBT5 contra células de mamíferos e soro fetal bovino, as sequências de clones selecionados foram analisadas quanto a orientação correta e se a fusão estava em fase com a proteína pIII. As proteínas codificadas pelos genes LIC11719, LIC10769, LIC13143 e LIC12976 foram selecionadas com estas características. Os genes que codificam a LIC12976, LIC10768, LIC10769 e LIC13418, tiveram sua conservação avaliada em diferentes sorovares da espécie patogênica L. interrogans e no sorovar Patoc da espécie de vida livre L. biflexa. As proteínas LIC12976 (selecionada pela técnica de phage display) e LIC13418 (selecionada por ferramentas de bioinformática) tiveram suas sequências amplificadas por PCR, clonadas em pGEM T easy, subclonadas em vetor de expressão pAE e expressas na fração celular correspondente ao corpúsculo de inclusão em E. coli BL21 (DE3) Star pLysS e E. coli BL21 SI, respectivamente. Após renaturação e purificação destas proteínas por cromatografia de afinidade a metal bivalente, um grupo de cinco animais BALB/c fêmeas foi imunizado. Ambas as proteínas se mostraram imunogênicas com títulos dos soros policlonais 1:256000 e 1:512000, respectivamente. Em ensaio de Western Blot os soros foram específicos no reconhecimento das proteínas recombinantes e as proteínas nativas foram verificadas em extratos de sorovares patogênicos de L. interrogans. Em ensaios de adesão, as proteínas recombinantes aderiram às células A31, LLC-PK1 e Vero e especificamente à laminina. Em ensaios de interferência em células usando laminina houve um aumento da adesão das proteínas recombinantes, o que pode ser explicado pela ligação da laminina às células e uma maior ligação das LICs estudadas. Em ensaio de localização celular usando imunofluorescência e microscopia eletrônica, foi observado que ambas as proteínas se encontram na superfície da L. interrogans. No experimento de desafio animal, a LIC12976 e a LIC13418 não se mostraram protetoras. Este trabalho contribuiu para a identificação das novas adesinas LIC13418 e LIC12976 que podem participar da virulência de leptospiras patogênicas envolvendo a primeira etapa da infecção na interação patógeno-hospedeiro

In Leptospira interrogans, proteins capable to bind to extracellular matrix components have been identified and most of them are important virulence factors. Phage display is a powerful technique to identify new ligands, including adhesin molecules that are important in the first stage of host infection. A shotgun phage display technique was used in order to obtain cell ligands. Four libraries were constructed by inserting random fragments obtained by sonication of L. interrogans DNA into phagemids pG8SAET (BBT1 and BBT2) and pG3DSS (BBT5 and BBT6). The libraries BBT1 and BBT5 contain larger inserts and BBT2 and BBT6 contain smaller inserts, with 1500 bp and 350 bp average sizes, respectively. After panning of BBT5 against mammalian cells and bovine fetal serum, the sequences of selected clones were analyzed for correct orientation and fusion with pIII protein. The proteins encoded by genes LIC11719, LIC10769, LIC13143 and LIC12976 were selected. The genes LIC12976, LIC10768, LIC10769 and LIC13418 were evaluated for their conservation in different pathogenic serovars of L. interrogans and free-living L. biflexa serovar Patoc. Proteins LIC12976 (selected by phage display technique) and also LIC13418 that was selected by bioinformatic tools, were amplified by PCR, cloned into pGEM T easy, subcloned into expression vector pAE and expressed in cellular fraction corresponding to the inclusion body in E. coli BL21 (DE3) Star pLysS and E. coli BL21 SI, respectively. After protein renaturation protocol and purification by affinity chromatography, a group of five BALB/c mice was immunized with the purified proteins. Both proteins were shown to be immunogenic with 1:256000 and 1:512000 polyclonal sera titers, respectively. In Western blot the sera were specific to recognize recombinant proteins and native proteins were detected in pathogenic L. interrogans serovars extracts. In binding assays, recombinant proteins bind to A31, LLC-PK1 and Vero cells and specifically to laminin. In interference cell assay using laminin there was an increase of recombinant protein bindings, which can be explained by the laminin binding to cells and further binding of the recombinant LICs. In cellular localization assay using immunofluorescence and electron microscopy, it was observed that both are surface proteins of L. interrogans. In the animal challenge, the LIC12976 and LIC13418 were not protective. As a whole, this work contributed to the identification of LIC12976 and LIC13418 as new adhesins and they can participate in the virulence of pathogenic Leptospira in the first stage of host pathogen interaction.