Genetic relatedness and phenotypic characteristics of Treponema associated with human periodontal tissues and ruminant foot disease

Treponema have been implicated recently in the pathogenesis of digital dermatitis (DID) and contagious ovine digital dermatitis (CODD) that are infectious diseases of bovine and ovine foot tissues, respectively. Previous analyses of treponemal 16S rDNA sequences, PCR-amplified directly from DID or CODD lesions, have suggested relatedness of animal Treponema to some human oral Treponema species isolated from periodontal tissues. In this study a range of adhesion and virulence-related properties of three animal Treponema isolates have been compared with representative human oral strains of Treponema denticola and Treponema vincentii. In adhesion assays using biotinylated treponemal cells, T denticola cells bound in consistently higher numbers to fibronectin, laminin, collagen type 1, gelatin, keratin and lactoferrin than did T. vincentii or animal Treponema isolates. However, animal DID strains adhered to fibrinogen at equivalent or greater levels than T denticola. All Treponema strains bound to the amino-terminal heparin l/fibrin I domain of fibronectin. 16S rDNA sequence analyses placed ovine strain UB1090 and bovine strain UB1467 within a cluster that was phylogenetically related to T vincentii, while ovine strain UB1466 appeared more closely related to T denticola. These observations correlated with phenotypic properties. Thus, T denticola ATCC 35405, GM-1, and Treponema UB1466 had similar outer-membrane protein profiles, produced chymotrypsin-like protease (CTLP), trypsin-like protease and high levels of proline iminopeptidase, and co-aggregated with human oral bacteria Porphyromonas gingivalis and Streptococcus crista. Conversely, T vincentii ATCC 35580, D2A-2, and animal strains UB1090 and UB1467 did not express CTLP or trypsin-like protease and did not co-aggregate with P. gingivalis or S. crista. Taken collectively, these results suggest that human oral-related Treponema have broad host specificity and that similar control or preventive strategies might be developed for human and animal Treponema-associated infections.

Binding properties and adhesion-mediating regions of the major sheath protein of Treponema denticola ATCC 35405

There is growing evidence that a number of oral Treponema species, in particular Treponema denticola, are associated with the progression of human periodontal disease. The major sheath (or surface) protein (Msp) of T. denticola is implicated in adhesion of bacteria to host cells and tissue proteins and is likely to be an important virulence factor. However, the binding regions of the Msp are not known. We have purified from Escherichia coli recombinant Msp (rMsp) polypeptides corresponding to the following: full-length Msp (rMsp) minus 13 N-terminal amino acid (aa) residues, an amino-terminal fragment (rN-Msp, 189 aa residues), a 57-aa residue segment from the central region (rV-Msp), and a C-terminal fragment (rC-Msp, 272 aa residues). rMsp (530 aa residues) bound to immobilized fibronectin, keratin, laminin, collagen type 1, fibrinogen, hyaluronic acid, and heparin. The N- and V-region polypeptides, but not rC-Msp, also bound to these substrates. Binding of rMsp to fibronectin was targeted to the N-terminal heparin I/fibrin I domain. Antibodies to the N-region or V-region polypeptides, but not antibodies to the rC-Msp fragment, blocked adhesion of T. denticola ATCC 35405 cells to a range of host protein molecules. These results suggest that the N-terminal half of Msp carries epitopes that are surface exposed and that are involved in mediating adhesion. Binding of rMsp onto the cell surface of low-level fibronectin-binding Treponema isolates conferred a 10-fold increase in fibronectin binding. This confirms that Msp functions autonomously as an adhesin and raises the possibility that phenotypic complementation of virulence functions might occur within mixed populations of Treponema species.

Soroepidemiologia de Chlamydia trachomatis, Chlamydia pneumoniae e Treponema pallidum nas aldeias indígenas Bakajá, Apyterewa, Xingu e Mrotidjãm, Altamira, Pará, Brasil

As bactérias do gênero Chlamydia estão associadas à diversas doenças, como cegueira, infecções genitais e pneumonia. Existem poucos dados sobre como a Chlamydia e o Treponema pallidum afetam indígenas na Amazônia brasileira. Este estudo objetivou determinar a soroprevalência das infecções pela Chlamydia trachomatis, Chlamydia pneumoniae e Treponema pallidum nas aldeias indígenas Bakajá, Apyterewa, Xingu e Mrotdidjãm, no município de Altamira, Pará, Brasil. O estudo incluiu 270 amostras de sangue coletadas no ano de 2007. A detecção de anticorpos das classes IgM e IgG anti-Chlamydia foi realizada empregando-se o ensaio imunoenzimático (ELISA), e selecionada de forma aleatória amostragem de 36, entre os positivos, para determinar a sorotipagem pela microimunofluorescência. Para detecção de anticorpos anti-T. pallidum foi utilizado um teste treponêmico (ELISA) e as amostras positivas foram submetidas a um teste não treponêmico (RPR). A prevalência geral de anticorpos anti-Chlamydia foi de 26,7%, com prevalência de 100% para C. trachomatis entre as amostras testadas pela MIF. Para a C. pneumoniae a prevalência foi de 61,1% e a prevalência de anticorpos contra Treponema pallidum foi baixa. As bactérias do estudo circulam nas comunidades indígenas da Amazônia brasileira estudada, o que requer uma resposta urgente das autoridades de saúde pública, pois estas bactérias podem causar doenças graves, mas são sensíveis a tratamento específico, quando diagnosticadas adequadamente.

Soroepidemiologia de Chlamydia trachomatis, Chlamydia pneumoniae e Treponema pallidum em portadores do Vírus da imunodeficiência humana (HIV), no Estado do Pará

A Chlamydia trachomatis e o Treponema pallidum compartilham com o HIV uma importante forma de transmissão: a via sexual. Por conta do comprometimento imunológico dos portadores de HIV, a C. pneumoniae pode apresentar um papel potencial em infecções respiratórias. Este trabalho objetivou a descrição da soroprevalência destes três agentes em portadores de HIV do Estado do Pará, Brasil. Entre setembro de 2007 a junho de 2008, foram coletadas 430 amostras de portadores de HIV em Belém, Pará. Estas foram submetidas a um ELISA para detecção de anticorpo IgG e IgM anti-Chlamydia e, dentre os positivos, uma amostragem aleatória foi escolhida e submetida à microimunofluorescência para sorotipagem. Para a detecção de anticorpos anti-Treponema pallidum foi feito um teste não treponêmico (RPR) e um teste treponêmico (ELISA). Os resultados obtidos foram analisados pelo teste do χ². A prevalência geral de anticorpos anti-Chlamydia foi 64,2% (51,6% para IgG e 4% para IgM). A sorotipagem mostrou uma alta prevalência de C. trachomatis (100% tanto para IgG como IgM), e C. pneumoniae (73,5% IgG e 70,5% IgM), sendo que houve uma larga disseminação dos sorotipos que causam infecções genitais da Chlamydia trachomatis. A prevalência geral de anticorpos contra o Treponema pallidum foi de 34,9%, sendo que 7,3% apresentaram resultado laboratorial indicativo de sífilis. As variáveis que apresentaram associação com a infecção por Chlamydia e Treponema pallidum foram: o gênero masculino, maior idade, baixa escolaridade, número de parceiros por semana, a prática de sexo anal, homossexualismo/bissexualismo, uso de droga não-endovenosa, histórico de IST. Faz-se necessário tanto a conscientização como o monitoramento da população, para impedir a transmissão destes agentes e para a melhoria da qualidade de vida dos indivíduos portadores de HIV.

Molecular detection of Treponema pallidum sp. pallidum in blood samples of VDRL-seroreactive women with lethal pregnancy outcomes: a retrospective observational study in northern Brazi

INTRODUÇÃO: Apesar das medidas de controle da sífilis materna e congênita estarem disponíveis no Brasil, existem dificuldades da rede em prover o diagnóstico laboratorial da infecção durante o pré-natal. O objetivo deste estudo foi confirmar a presença do Treponema pallidum pela PCR em mulheres com sorologia positiva ao VDRL e com resultado letal da gravidez, isto é, aborto, natimorto e neomorto. MÉTODOS: Estudo retrospectivo realizado em mulheres VDRL-sororeativas com resultado negativo da gravidez, admitidas na Fundação Santa Casa de Misericórdia do Pará FSCM-PA entre janeiro e julho de 2004. As amostras de soro e DNA de sangue total foram obtidas no mesmo período da triagem pelo VDRL. Estas amostras foram analisadas pelo ELISA IgG, FTA-Abs IgM e PCR simples (polA). RESULTADOS: Durante o período de estudo, 0,7% (36/4.912) das mulheres com resultado letal da gravidez apresentaram VDRL positivo. O genepolA foi amplificado em 72,7% (24/33) destas mulheres,com 55,6% (20/36) e 94,4% (34/36) apresentando anticorpos tipo IgG e IgM contra o T. pallidum, respectivamente. A comparação destes resultados mostrou uma diferença estatística significativa, sendo que os resultados da PCR versus FTA-Abs Ig Mmostraram-se concordantes, sugerindo que a sífilis materna era uma infecção ativa. A causa básica de morte dos conceptos não foi relatada em 97,2% (35/36) dos casos. Entre as mulheres que foram submetidas ao VDRL no pré-natal, somente quatro das nove soropositivas receberam tratamento. CONCLUSÕES: A elevada frequência de sífilis no grupo de estudo indica a fragilidade do serviço no diagnóstico, tratamento e monitoramento da infecção, comprometendo o controle epidemiológico.

Prevalence of Treponema Species Detected in Endodontic Infections: Systematic Review and Meta-regression Analysis

Introduction: This systematic review and meta-regression analysis aimed to calculate a combined prevalence estimate and evaluate the prevalence of different Treponema species in primary and secondary endodontic infections, including symptomatic and asymptomatic eases. Methods: The MEDLINE/PubMed, Embase, Scielo, Web of Knowledge, and Scopus data-bases were searched without starting date restriction up to and including March 2014. Only reports in English were included. The selected literature was reviewed by 2 authors and classified as suitable or not to be included in this review. Lists were compared, and, in case of disagreements, decisions were made after a discussion based on inclusion and exclusion criteria. A pooled prevalence of Treponema species in endodontic infections was estimated. Additionally, a meta-regression analysis was performed. Results: Among the 265 articles identified in the initial search, only 51 were included in the final analysis. The studies were classified into 2 different groups according to the type of endodontic infection and whether it was an exclusively primary/secondary study (n = 36) or a primary/secondary comparison (n = 15). The pooled prevalence of Treponema species was 41.5% (95% confidence interval, 35.9-47.0). In the multivariate model of meta-regression analysis, primary endodontic infections (P < .001), acute apical abscess, symptomatic apical periodontitis (P < .001), and concomitant presence of 2 or more species (P = .028) explained the heterogeneity regarding the prevalence rates of Treponema species. Conclusions: Our findings suggest that Treponema species are important pathogens involved in endodontic infections, particularly in cases of primary and acute infections.

Complete Genome Sequence of Treponema paraluiscuniculi, Strain Cuniculi A: The Loss of Infectivity to Humans Is Associated with Genome Decay.

Treponema paraluiscuniculi is the causative agent of rabbit venereal spirochetosis. It is not infectious to humans, although its genome structure is very closely related to other pathogenic Treponema species including Treponema pallidum subspecies pallidum, the etiological agent of syphilis. In this study, the genome sequence of Treponema paraluiscuniculi, strain Cuniculi A, was determined by a combination of several high-throughput sequencing strategies. Whereas the overall size (1,133,390 bp), arrangement, and gene content of the Cuniculi A genome closely resembled those of the T. pallidum genome, the T. paraluiscuniculi genome contained a markedly higher number of pseudogenes and gene fragments (51). In addition to pseudogenes, 33 divergent genes were also found in the T. paraluiscuniculi genome. A set of 32 (out of 84) affected genes encoded proteins of known or predicted function in the Nichols genome. These proteins included virulence factors, gene regulators and components of DNA repair and recombination. The majority (52 or 61.9%) of the Cuniculi A pseudogenes and divergent genes were of unknown function. Our results indicate that T. paraluiscuniculi has evolved from a T. pallidum-like ancestor and adapted to a specialized host-associated niche (rabbits) during loss of infectivity to humans. The genes that are inactivated or altered in T. paraluiscuniculi are candidates for virulence factors important in the infectivity and pathogenesis of T. pallidum subspecies.

Characterization of humoral immune responses against Treponema pallidum antigens

The spirochete Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with an estimated 12 million new cases per year worldwide. There is no vaccine currently available for the prevention of syphilis. In the present study, the T. pallidum hypothetical protein TP0693 was examined to determine its cellular location, and its potential for use as a vaccine candidate and immunodiagnostic for syphilis. TP0693 was demonstrated to be strongly reactive with sera from rabbits infected experimentally with T. pallidum for >25 days. Results from proteinase K digestion, immunofluorescence and immunoelectron microscopy were consistent with outer surface localization of TP0693. Serum reactivity against TP0693 was detected in only 68% of syphilis patients, which does not support its use as an immunodiagnostic for syphilis. Immunization of rabbits with TP0693 or three other outer membrane candidates did not alter the course of lesion development atter T. pallidum inoculation. We also examined the T. pallidum proteome by two-dimensional gel electrophoresis coupled with mass spectrometry analysis and immunoblotting. This approach resulted in the identification of 95 unique polypeptides, several of which were reactive with sera from infected rabbits and syphilis patients. The analyses described here enabled us to identify antigens potentially useful as vaccine candidates or diagnostic markers, and may provide insight into host-pathogen interactions during T. pallidum infection. ^

Detection and discrimination of herpes simplex viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmatobacterium (Klebsiella) granulomatis from genital ulcers

Background. Genital ulcer disease (GUD) is commonly caused by pathogens for which suitable therapies exist, but clinical and laboratory diagnoses may be problematic. This collaborative project was undertaken to address the need for a rapid, economical, and sensitive approach to the detection and diagnosis of GUD using noninvasive techniques to sample genital ulcers. Methods. The genital ulcer disease multiplex polymerase chain reaction (GUMP) was developed as an inhouse nucleic acid amplification technique targeting serious causes of GUD, namely, herpes simplex viruses (HSVs), Haemophilus ducreyi, Treponema pallidum, and Klebsiella species. In addition, the GUMP assay included an endogenous internal control. Amplification products from GUMP were detected by enzyme linked amplicon hybridization assay (ELAHA). Results. GUMP-ELAHA was sensitive and specific in detecting a target microbe in 34.3% of specimens, including 1 detection of HSV-1, three detections of HSV-2, and 18 detections of T. pallidum. No H. ducreyi has been detected in Australia since 1998, and none was detected here. No Calymmatobacterium ( Klebsiella) granulomatis was detected in the study, but there were 3 detections during ongoing diagnostic use of GUMP-ELAHA in 2004 and 2005. The presence of C. granulomatis was confirmed by restriction enzyme digestion and nucleotide sequencing of the 16S rRNA gene for phylogenetic analysis. Conclusions. GUMP-ELAHA permitted comprehensive detection of common and rare causes of GUD and incorporated noninvasive sampling techniques. Data obtained by using GUMP-ELAHA will aid specific treatment of GUD and better define the prevalence of each microbe among at-risk populations with a view to the eradication of chancroid and donovanosis in Australia.

Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variation

Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection.

Oral health, smoking and adolescence

The present cross-sectional study examined the effect of smoking on oral health in a birth cohort of 15 to 16-year-old Finnish adolescents. The hypothesis was that oral health parameters were poorer among smoking than non-smoking subjects and that a tobacco intervention program could be effective among the adolescents. The study was conducted in the Kotka Health Center, Kotka, Finland. Altogether 501 out of 545 subjects (15- to 16-year-old boys [n = 258] and girls [n = 243]) were clinically examined in 2004 and 2005. The sample frame was a birth cohort of all subjects in 1989 and 1990, living in Kotka. A structured questionnaire was also filled in by the participants to record their general health and health habits, such as smoking, tooth brushing, and medication used. The participants were classified into nonsmokers, current smokers, and former smokers. Subgingival pooled plaque samples were taken and stimulated salivary samples were also collected. The subjects were asked from which of seven professional groups (doctors, school nurses, dental nurses, general nurses, dentists, teachers and media professionals) they would prefer to receive information about tobacco. The two most popular groups they picked up were dentists and school nurses. Current smokers (n=127) were then randomly assigned into three groups: the dentist group (n =44), the school-nurse group (n =42), and the control group (n =39). The intervention was based on a national recommendation of evidence based guidelines by The Finnish Medical Society Duodecim ( 5A counseling system). Two months after the intervention, a second questionnaire was sent to the smokers in the intervention groups. Smoking cessation, smoking quantity per week, and self-rated addiction for smoking (SRA) were recorded. The results were analyzed using the R-statistical program. The results showed that 15% of the subjects had periodontitis. Smokers (25%) had more periodontitis than non-smokers (66%) (p < 0.001). Smoking boys (24%) also had more caries lesions than non-smokers (69%) (p < 0.001), and they brushed their teeth less frequently than non-smokers. Smoking significantly impaired periodontal health of the subjects, even when the confounding effects of plaque and tooth brushing were adjusted. Smoking pack-years, intensified the effects of smoking. Periodontal bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia and Treponema denticola were more frequently detected among the smokers than non-smokers, especially among smoking girls. Smoking significantly decreased the values of both the salivary periodontal biomarkers MMP-8 (p=0.04) and PMN elastase (p=0.02) in boys. The effect was strengthened by pack years of smoking (MMP-8 p=0.04; elastase p0.01). Of those who participated in the intervention, 19 % quit smoking. The key factors associated with smoking cessation were best friend`s influence, nicotine dependence and diurnal type. When the best friend was not a smoker, the risk ratio (RR) of quit smoking after the intervention was 7.0 (Cl 95% 4.6 10.7). Of the diurnal types, the morning people seemed to be more likely to quit (RR 2.2 [Cl 95% 1.4 3.6]). Nicotine dependence also elicited an opposite effect: those who scored between 3 and 5 dependence scores were less likely to quit. In conclusion, smoking appears to be a major etiological risk factor for oral health. However, the early signs of periodontal disease were mild in the subjects studied. Based on the opinions of the adolescent s, dental professionals may have a key position in their smoking cessation. The harmful effects of smoking on oral health could be used in counselling. Best friend`s influence, nicotine dependence and diurnal type, all factors associated with smoking cessation, should be taken more carefully into account in the prevention programs for adolescents.

Catechol 2,3-dioxygenase-assisted cleavage of aromatics by “anaerobic” termite gut spirochetes and genomic evidence of a complete meta-pathway

The termite hindgut microbial ecosystem functions like a miniature lignocellulose-metabolizing natural bioreactor, has significant implications to nutrient cycling in the terrestrial environment, and represents an array of microbial metabolic diversity. Deciphering the intricacies of this microbial community to obtain as complete a picture as possible of how it functions as a whole, requires a combination of various traditional and cutting-edge bioinformatic, molecular, physiological, and culturing approaches. Isolates from this ecosystem, including Treponema primitia str. ZAS-1 and ZAS-2 as well as T. azotonutricium str. ZAS-9, have been significant resources for better understanding the termite system. While not all functions predicted by the genomes of these three isolates are demonstrated in vitro, these isolates do have the capacity for several metabolisms unique to spirochetes and critical to the termite system’s reliance upon lignocellulose. In this thesis, work culturing, enriching for, and isolating diverse microorganisms from the termite hindgut is discussed. Additionally, strategies of members of the termite hindgut microbial community to defend against O₂-stress and to generate acetate, the “biofuel” of the termite system, are proposed. In particular, catechol 2,3-dioxygenase and other meta-cleavage catabolic pathway genes are described in the “anaerobic” termite hindgut spirochetes T. primitia str. ZAS-1 and ZAS-2, and the first evidence for aromatic ring cleavage in the phylum (division) Spirochetes is also presented. These results suggest that the potential for O₂-dependent, yet nonrespiratory, metabolisms of plant-derived aromatics should be re-evaluated in termite hindgut communities. Potential future work is also illustrated.