O diagnóstico de neurosífilis é freqüentemente dependente dos resultados dos testes serológicos e alterações no líquido cefalorraquidiano, mas a confiabilidade desses resultados em pacientes com infecção pelo HIV-1 tem sido questionada especialmente em pacientes assintomáticos com sífilis latente. O estudo se propõe avaliar a presença de DNA do T. pallidum no LCR de pacientes assintomáticos infectados pelo HIV, com o diagnóstico de sífilis. Amostras de LCR foram coletadas de 12 pacientes infectados pelo HIV atendidos em um terciário localizado no sul do Brasil , durante o período de 2012 a 2013. A presença de DNA do T. pallidum foram analisadas nas amostras de LCR pelo método de PCR “seminested”. Dados demográficos dos pacientes, parâmetros bioquímicos, celularidade e VDRL do LCR e linfócitos T-CD4 também foram analisados. Nas amostras de LCR de cinco dos 12 pacientes (40%) foram detectados o DNA do T. pallidum . Inesperadamente, nestes doentes, os níveis de contagem de células, proteína e glicose no LCR foram normais. Além disso , nenhuma destas cinco amostras de CSF apresentou uma reacção positiva VDRL. Os títulos de VDRL no soro foram semelhantes entre pacientes positivos e negativos para a presença T. pallidum DNA no LCR. A maioria dos pacientes com DNA de T. pallidum detectável apresentaram baixos títulos de VDRL no soro. O VDRL sérico elevado com título de 1:64 foi observada em apenas um paciente. Nossos resultados demostraram que os pacientes assintomáticos infectados pelo HIV com evidência de sífilis latente e LCR normais podem apresentar DNA de T. pallidum detectável no LCR. A detecção do DNA do T. pallidum pelo nosso seminested PCR pode fornecer informações adicionais além da análise convencional do LCR para o diagnóstico de neurossífilis. presença do DNA de T. pallidum no LCR em pacientes infectados pelo HIV com sífilis latente e resultados de LCR normais pode determinar uma mudança terapêutica do uso de penicilana benzatina intramuscular para o de penicilina cristalina intravenosa aquosa para o tratamento da sífilis. / Neurosyphilis diagnosis is frequently dependent upon the results of serological tests and cerebrospinal fluid abnormalities, but the reliability of findings in patients with HIV-1 infection has been questioned, especially asymptomatic patients with latent syphilis, We present the data on the presence of T. pallidum DNA in CSF from asymptomatic HIV-infected patients with the diagnosis of syphilis. CSF and serum samples were collected from 12 HIV-infected patients attending a tertiary care located in southern Brazil, during the period 2012 to 2013. In CSF samples from five of 12 patients (40%), we detected T. pallidum DNA. Unexpectedly, in these patients, CSF cell count, protein and glucose levels were normal. In addition, none of these 5 CSF samples presented a positive VDRL reaction. Serum VDRL titers were similar between patients with positive and negative CSF T. pallidum DNA. Most patients with detectable T. pallidum DNA presented low serum VDRL titers. Serum VDRL titer of 1:64 was observed in one patient. Our results have shown that asymptomatic HIV-infected patients with evidence of latent syphilis and normal CSF might present detectable T. pallidum DNA in the CSF. The detection of T. pallidum DNA by our seminested PCR provide additional information beyond conventional CSF analysis for diagnosis of neurosyphilis. The detection of T. pallidum DNA in the CSF despite normal CSF findings in HIV-infected patients could also provide a different therapeutic approach including the use of intravenous aqueous crystalline penicillin.

This study had two objectives: to achieve suspension cultivation of Sf1Ep cells and to develop procedures for achieving the replication of T. pallidum in those cell cultures. Sf1Ep cells have been the sole cell line used for the in vitro cultivation of T. pallidum. A study was undertaken to determine if other cell lines can support growth of T. pallidum. Rabbit skin fibroblasts (RAB-9), nude mouse ear (NME) cells, and normal rebbit testis fibroblasts (RT) were compared to Sf1Ep cells for their ability to support in vitro multiplication of T. pallidum. RAB-9 cells supported multiplication of treponemes equal to that of Sf1Ep cells. NME and RT cells also supported growth but to a lesser extent than Sf1Ep cells. Utilization of alternative cell lines may lead to improved in vitro growth of T. pallidum including possible serial passage.

Periodontitis is a polymicrobially-induced, chronic inflammatory disease of the tissues that support and surround the tooth. While greater than 500 organisms are found in dental plaque, Treponema denticola is one of only a few species associated with disease. It has been hypothesized that oral bacteria disrupt host homeostasis through manipulation of the innate immune system. In this study, we examine the impact of binding and subsequent cleavage of factor H, a complement regulator, by T. denticola in the evasion and subversion of complement. The molecular interaction between the sole FH-binding protein, FhbB, and FH was detailed by x-ray crystallography, site-directed mutagenesis, and inhibition analyses. Negatively-charged amino acids of FhbB formed salt-bridges with positively-charged residues of FH within the complement control protein domains (CCP) 6 and 7. In support of its critical role in disease, FhbB was universal among T. denticola isolates, although, different strains produce highly divergent FhbB proteins. Despite extensive sequence variation, predominantly within the FH-binding determinant, all FhbB proteins have similar structure and interact with FH using nearly identical molecular mechanisms. In addition to differences in FhbB, many isolates of T. denticola displayed significant variation in the activity of the chymotrypsin-like protease, dentilisin. Neither FhbB type produced nor dentilisin activity influenced serum resistance, as all strains tested were highly serum resistant. While sequence diversity of FhbB did not influence the interaction with FH or serum resistance, FhbB proteins elicited type-specific antibodies that blocked FH binding. Collectively, these analyses indicate that FH binding is an essential complement evasion mechanism of T. denticola and characterizes the uniquely complex interaction between T. denticola and complement. The data presented here provide novel insight into the pathogenesis of disease and begins to explore a hypothetical molecular mechanism by which this key periopathogen disrupts the host innate immune system, leading to periodontitis.

Periodontitis is a chronic inflammatory disease that affects over 116 million adults in the United States. A shift in the normal microflora occurs as periodontal disease develops resulting in a larger number of Gram-negative anaerobes and spirochetes. An increase in the oral spirochete, Treponema denticola, is highly correlated with periodontal disease progression and severity. The ability of this periopathogen to thrive in the subgingival crevice is dependent on complement evasion mechanisms. Earlier analyses demonstrated that the primary mechanism of T. denticola serum resistance is binding of the human complement regulatory protein, Factor H (FH), to the factor H-binding protein (FhbB). FH serves as cofactor in the Factor-I mediated cleavage of C3b and accelerates the decay of the C3 convertase complex, leading to downregulation of C3b production. Several pathogens bind FH, and a number of these bacterial binding proteins have been shown to bind plasminogen. Plasminogen is a plasma glycoprotein that circulates as a zymogen. Its active form, plasmin, degrades components of the extracellular matrix and cleaves complement proteins C3b and C5 inhibiting complement pathway progression. Through molecular and biochemical analyses, this study demonstrates that FhbB simultaneously binds plasminogen and FH to residues located on its positively and negatively charged surfaces, respectively, and that the two ligands do not compete for binding. This study also shows that the surface-bound plasminogen is available for proteolytic cleavage into the active serine protease plasmin. The activated plasmin could break down components of the periodontal tissue leading to increased nutrient availability and creation of a larger anaerobic environment where the bacteria can flourish, thereby promoting periodontal disease.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 77-93).

EVALUATION AND DETERMINATION OF THE SENSITIVITY AND SPECIFICITY OF A Treponema pallidum DRIED BLOOD SPOT (DBS) METHOD FOR SEROLOGIC DIAGNOSIS OF SYPHILIS Background: Syphilis is a sexually transmitted infection (STI) caused by Treponema pallidum subspecies pallidum. Syphilis is known as the "great imitator" due to the similarity of clinical signs and symptoms to other infectious diseases. The primary diagnosis of syphilis relies on clinical findings, including the examination of treponemal lesions, and/or serologic tests. Serologic tests are divided into nontreponemal and treponemal tests. Nontreponemal tests are useful for screening, while treponemal tests are used as confirmatory tests. Methods: A total of 200 serum and DBS specimens collected from patients at the Los Angeles Municipal Sexually Transmitted Disease Clinics were tested by the DBS and enzyme immunoassay (EIA) methods. These samples were sent to the Syphilis Diagnostics Laboratory, Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia for testing. Samples were blindly evaluated by the TREP-SPOTTM DBS and the TREP- SURETM EIA methods for the detection of anti-treponemal IgG- and IgM-class antibodies. Results: The sensitivity of the DBS method was 83% (95% CI, 73.89 - 89.50) and specificity was 100% (95% CI, 95.39 - 100)). The positive predictive value and negative predictive values were 100% (95% CI, 94.48 - 100) and 85% (95% CI, 77.43 - 91.0), respectively. The efficiency of the DBS method was 91.5%. The kappa value for the agreement between the DBS method and EIA assay was 0.83 (95% CI, 0.754 - 0.906). The correlation coefficient (r2) between the anti-treponemal antibody assay results obtained from DBS and serum samples was 0.94. Conclusion: DBS is an optimal choice to be used as a screening tool for the detection of anti-treponemal antibodies for the diagnosis of syphilis. The detection of anti-treponemal antibodies (TREP-SPOTTM DBS EIA) compared favorably to the results of serum-base assay (TREP-SURETM EIA), with an overall concordance of 91.5%. Dried blood spots are technically easier to obtain and are suitable blood samples for primary health care centers.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 112-126).

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [141]-172).

Selenoproteins are unique proteins in which selenocysteine is inserted into the polypeptide chain by highly specialized translational machinery. They exist within all three kingdoms of life. The functions of these proteins in biology are still being defined. In particular, the importance of selenoproteins in pathogenic microorganisms has received little attention. We first established that a nosocomial pathogen, Clostridium difficile, utilizes a selenoenzyme dependent pathway for energy metabolism. Following this initial characterization, we demonstrate that this pathway is linked to production of toxins by this organism. Finally, we show that interruption of selenium metabolism is a viable pathway for development of antimicrobials against this, and other selenoprotein dependent pathogens. We investigated whether Stickland reactions (paired amino acid fermentation) might be at the heart of C. difficile's bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases; glycine and D-proline reductase. These selenoenzymes were expressed upon addition of the corresponding Stickland acceptor (glycine, proline or hydroxyproline). Purification of the selenoenzyme D-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys containing) proteins. D-proline reductase utilized only D-proline but not L-hydroxyproline, even in the presence of an expressed and purified proline racemase. The enzyme was found to be independent of divalent cations, and zinc was a potent inhibitor. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of D-proline reductase may differ significantly from similar enzymes from non-pathogenic species. C. difficile pathogenesis is due to the production of toxins, A and B, members of the large clostridial cytotoxin family. Previous studies have shown that toxin production by this organism is influenced by the composition of the growth medium. We examined the impact of Stickland acceptor amino acids (Stickland acceptors; glycine, proline and hydroxyproline) on growth kinetics and yield, protein synthesis, toxin production and gene expression. Although addition of Stickland acceptors moderately increases growth yield and total protein synthesis, there does not appear to be a clear impact on entry into stationary phase. Glycine dramatically increases the amount of toxin released into the growth medium. Conversely, the addition of hydroxyproline suppresses toxin production. We examine possible mechanisms of regulation and demonstrate that CodY, a regulator of toxin gene transcription does not appear to mediate this effect. Given the importance of selenium dependent Stickland reactions to C. difficile growth and toxin production we aimed to examine the efficacy of blocking such pathways as a means of antimicrobial development. Selenide is the only known substrate for selenophosphate synthetase, the first enzyme involved in the specific incorporation of selenium into selenoproteins. We have identified a stable complex formed upon reaction of auranofin (a gold containing drug) with selenide in vitro. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (75Se) in selenoproteins in both E. coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth media significantly reduces the inhibitory action of auranofin on the growth of C. difficile. Based on these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. The antimicrobial potential of blocking selenium metabolism is further demonstrated in the dental pathogen Treponema denticola. We show that auranofin blocks the growth this organism which also participates in Stickland fermentation. In addition, we provide evidence that the antimicrobial action of stannous salts against T. denticola is also mediated through inhibition of the metabolism of selenium. These studies clearly show that, at least in a subset of microbes that use selenium for the synthesis of selenoproteins, the need for this metalloid can be a useful target for future antimicrobial development.