Periodontal disease (PD) is a polymicrobial infection characterized by inflammation of the gingiva, alveolar bone resorption, and tooth loss (edentulism). Treponema denticola along with Porphyromonas gingivalis and Tannerella forsythia are among the “Red Complex” and are main etiological agents in PD. Treponemes are a member of the Spirochaeta phylum and are obligate anaerobes, that express pyruvate ferredoxin oxidoreductase (PFOR). The enzyme catalyzes the oxidation of pyruvate to acetyl-CoA and reduced ferredoxin. Amixicile is a novel bacteriostatic derivative of nitazoxanide and an inhibitor of PFOR. In light of the fact that Treponemes express PFOR, this study was conducted to investigate the susceptibility of oral Treponemes to AMX. All oral Treponemes tested were susceptible to AMX and the MIC values were determined ranging of 1.5-4.5 μg mL-1 for an initial starting cell concentration of 1.9x106 cells mL-1. Other potentially therapeutic effects for AMX for T. denticola were investigated: motility, hydrogen sulfide production, and serum sensitivity. AMX reduced overall spirochete motility by 50% at sub-MIC concentrations. There was a dose dependent decrease in H2S production in T. denticola at sub-MIC and MIC values. Furthermore, prior exposure of AMX led to increases in serum sensitivity. Taking into account the fact that other periodontal red complex bacteria express PFOR, AMX could serve as a new selective adjunctive treatment for periodontal disease.

A criptococose é uma infecção fúngica causada pela levedura encapsulada Cryptococcus neoformans que acomete freqüentemente o meningoencéfalo de pacientes imunocomprometidos. No Brasil, cerca de 6% dos pacientes com AIDS desenvolvem a criptococose. A construção da resposta imune na criptococose envolve fagócitos e linfócitos T e B presentes no granuloma. Atualmente, são descritos três subtipos de linfócitos B: linfócitos B-1 a, B-1 b e B-2, o último conhecido como linfócito B \"convencional\". Os linfócitos B-1 (B-1a e B-1b) expressam concomitantemente imunofenótipo de macrófagos, células B e células T. Estes linfócitos receberam o nome de fagócitos mononucleares derivados de linfócitos B-1 e possuem a capacidade de migrar do peritônio até um foco inflamatório não específico, fagocitarem, expressarem antígeno e secretarem anticorpos e citocinas. Portanto, avaliamos a participação destes linfócitos B-1 na criptococose através de ensaios in vitro e in vivo com duas cepas diferentes de Cryptococcus neoformans variedade neoformans sorotipo A. Os linfócitos B-1 fagocitaram e destruiram as leveduras de Cryptococcus neoformans, principalmente na presença de complemento. Altas produções de NO encontradas no sobrenadante de cultura do ensaio de fagocitose associada à alta atividade peroxidásica e capacidade destes linfócitos em gerar EROs, poderiam justificar a alta atividade de \"killing\" encontrada nestes linfócitos B-1. Ainda, quando estimuladas com o fungo, os linfócitos B-1 secretaram principalmente IL-12 e IFN-γ, enquanto a IL-10 diminuiu significativamente. Na infecção experimental os camundongos BALB/xid, desprovidos de linfócitos B-1, apresentaram maior susceptibilidade à criptococose, com ambas as cepas, quando comparados aos camundongos BALB controle (BALB/c). Estes animais BALB/xid apresentaram maior carga fúngica nos órgãos baço, fígado e pulmão e morreram precocemente. A concentração de IL-10 foi maior nos órgãos dos camundongos BALB/xid, enquanto a IL-4 aumentou nos camundongos BALB/c. A concentração das citocinas de padrão Th1 (IL-12, IFN-γ e IL-2). consideradas protetoras na criptococose foi maior nos camundongos BALB/c. Além· da produção de citocinas protetoras, os órgãos dos camundongos BALB/c infectados com a cepa de menor cápsula apresentaram um maior população de linfócitos B-1. Estes linfócitos provavelmente estavam participando do processo inflamatório e contribuindo para a cura da infecção. Embora outras investigações sejam necessárias para melhor compreendermos a participação de linfócitos B-1 na criptococose, podemos considerar que estas células interagem com leveduras de Cryptococcus neoformans e podem ser decisivas para uma resposta protetora na criptococose experimental. / Abstract not available.

A criptococose é uma infecção fúngica causada pela levedura encapsulada Cryptococcus neoformans que acomete freqüentemente o meningoencéfalo de pacientes imunocomprometidos. No Brasil, cerca de 6% dos pacientes com AIDS desenvolvem a criptococose. A construção da resposta imune na criptococose envolve fagócitos e linfócitos T e B presentes no granuloma. Atualmente, são descritos três subtipos de linfócitos B: linfócitos B-1 a, B-1 b e B-2, o último conhecido como linfócito B \"convencional\". Os linfócitos B-1 (B-1a e B-1b) expressam concomitantemente imunofenótipo de macrófagos, células B e células T. Estes linfócitos receberam o nome de fagócitos mononucleares derivados de linfócitos B-1 e possuem a capacidade de migrar do peritônio até um foco inflamatório não específico, fagocitarem, expressarem antígeno e secretarem anticorpos e citocinas. Portanto, avaliamos a participação destes linfócitos B-1 na criptococose através de ensaios in vitro e in vivo com duas cepas diferentes de Cryptococcus neoformans variedade neoformans sorotipo A. Os linfócitos B-1 fagocitaram e destruiram as leveduras de Cryptococcus neoformans, principalmente na presença de complemento. Altas produções de NO encontradas no sobrenadante de cultura do ensaio de fagocitose associada à alta atividade peroxidásica e capacidade destes linfócitos em gerar EROs, poderiam justificar a alta atividade de \"killing\" encontrada nestes linfócitos B-1. Ainda, quando estimuladas com o fungo, os linfócitos B-1 secretaram principalmente IL-12 e IFN-γ, enquanto a IL-10 diminuiu significativamente. Na infecção experimental os camundongos BALB/xid, desprovidos de linfócitos B-1, apresentaram maior susceptibilidade à criptococose, com ambas as cepas, quando comparados aos camundongos BALB controle (BALB/c). Estes animais BALB/xid apresentaram maior carga fúngica nos órgãos baço, fígado e pulmão e morreram precocemente. A concentração de IL-10 foi maior nos órgãos dos camundongos BALB/xid, enquanto a IL-4 aumentou nos camundongos BALB/c. A concentração das citocinas de padrão Th1 (IL-12, IFN-γ e IL-2). consideradas protetoras na criptococose foi maior nos camundongos BALB/c. Além· da produção de citocinas protetoras, os órgãos dos camundongos BALB/c infectados com a cepa de menor cápsula apresentaram um maior população de linfócitos B-1. Estes linfócitos provavelmente estavam participando do processo inflamatório e contribuindo para a cura da infecção. Embora outras investigações sejam necessárias para melhor compreendermos a participação de linfócitos B-1 na criptococose, podemos considerar que estas células interagem com leveduras de Cryptococcus neoformans e podem ser decisivas para uma resposta protetora na criptococose experimental. / Abstract not available.

As a foodborne pathogen, Listeria monocytogenes (Lm) encounters many barriers to invasion and dissemination in the host that may change the nature of host response. Lm has been most commonly studied using intravenous (i.v.) inoculation, however, a method that delivers a bolus of bacteria directly to the bloodstream. Thus, little is known about what systemic and local mediators are triggered during the natural course of infection and how these may impact susceptibility. Our laboratory used foodborne transmission of Lm in mice to assess whether the method of transmission and the specific organ microenvironment could affect infection-induced secretion of type I interferon or prostaglandin E2. Type I interferon is a pro-inflammatory effector secreted in response to viruses that has been proposed to paradoxically down-regulate innate immunity to intracellular bacteria. In contrast to i.v. infection, type I interferon was not detrimental to the immune response when Lm were acquired orally. In fact, most of the anti-inflammatory effects of type I interferon in the spleen were attributable to i.v. but not foodborne infection. Importantly however, downregulation of the receptor for interferon gamma (IFNGR1), previously ascribed to the type I interferon response, was found to be a consequence of infection and unrelated to type I interferon. In the liver, robust recruitment and activation of neutrophils (PMN) is thought to be required for initiation of Lm immunity. Prostaglandin E2 (PGE2) is a lipid mediator most commonly associated with pain and fever that has also been demonstrated to have anti-inflammatory or tolerogenic effects. It is unknown, however, whether foodborne infection induces PGE2 in the liver and if PGE2 then down-regulates PMN activities. Recruitment of PMN to the liver following foodborne infection was robust in both susceptible and resistant animals. Bone marrow PMN from each killed Lm ex vivo with similar efficiency, thus suggesting that if PMN were dysfunctional during the course of natural infection, they were responding to cues in the microenvironment. Accordingly, significantly more PGE2 was made ex vivo by cells from the livers of susceptible animals than from resistant animals. When PGE2 was applied to naïve PMN prior to exposure to Lm, it consistently dampened the killing efficiency of these cells, suggesting that this lipid better known for its pro-inflammatory roles might have anti-inflammatory effects during Lm infection. Overall, these studies indicate that mediators produced as a result of infection may have very different roles dependent on route of inoculation, timing, and the specific organ examined.

Porphyromonas gingivalis, a Gram negative anaerobe implicated in the progression of periodontal disease, is capable of surviving and causing infection despite high levels of reactive nitrogen species found in the oral cavity due to its efficient nitrosative stress response. HcpR is an important sensor-regulator that plays a vital step in the initiation of the nitrosative stress response in many Gram negative anaerobic bacteria. We employ a combination of X-ray crystallography, SAXS, resonance Raman spectroscopy, UV-Vis spectroscopy, and molecular biology techniques to better understand this key regulator. Knockout of the hcpR gene in W83 P. gingivalis results in the inability of the bacteria to grow in physiological concentrations of nitrite and complementation of hcpR using the novel plasmid Pg108 rescues this phenotype. HcpR causes a drastic, dose dependent upregulation of PG0893, a gene coding for a putative NO reductase, when exposed to nitrite or nitric oxide. Full transcriptome sequencing reveals that hcp is the only significantly upregulated gene when P. gingivalis is exposed to nitrite and knockout of hcp resulted in a phenotype that is similar to that of the hcpR deficient strain. HcpR directly regulates the expression of hcp via direct binding to an inverted repeat sequence in the promoter region of the hcp gene. We present a 2.6 Å crystal structure of the N-terminal sensing domain of HcpR and show that it is FNR-CRP regulator. A putative hydrophobic heme binding pocket was identified in the junction between the N-terminal domain and the dimerization helix. Mutation of two methionine residues (Met68 and Met145) in this pocket abrogates activation of HcpR thus verifying the binding site. Heme bound to HcpR exhibits heme iron as a hexa-coordinate system in the absence of nitric oxide (NO) and upon nitrosylation transitions to a penta-coordinated system. Finally, Small Angle X-ray Scattering experiments of the full length HcpR reveal that the C-terminal DNA binding domain of HcpR has a high degree of interdomain flexibility.

Streptococcus sanguinis, an abundant and benign inhabitant of the oral cavity, is an important etiologic agent of infective endocarditis, particularly in people with pre-disposing cardiac valvular damage. Although commonly isolated from patients with IE, little is known about the factors that make any particular S. sanguinis isolate more virulent than another or, indeed, whether significant differences in virulence exist among isolates. To investigate the virulence of multiple isolates, a variation of the Bar-seq (barcode sequencing) method was employed. A conserved chromosomal site was identified for subsequent insertion of a barcode identifier, unique for each strain. Barcode insertion did not affect growth in vitro or in a rabbit model of endocarditis. Pooling of these strains and inoculation into rabbits demonstrated that all strains were capable of causing disease; however, virulence varied widely among strains. Genomic comparisons of the more virulent strains versus less virulent strains failed to conclusively identify any single gene responsible for virulence. Given this result, we continued our examination of the manganese transport system SsaACB, which is present in every strain of S. sanguinis examined. Although its contribution to virulence has not been confirmed in any strain other than SK36, it has been shown to be required for virulence in multiple species of streptococci, making it a candidate for emerging targeted therapies. In S. sanguinis strain SK36, previous studies have confirmed that loss of the manganese transport protein SsaB is tantamount to loss of virulence. Moreover, ssaB-deficient mutants are deficient for serum growth—a phenotype we have previously found to be associated with virulence. Our in vitro studies of manganese transporter-deficient strain SK36 supported this, but also revealed the emergence of suppressor mutants. In each suppressor mutant that was isolated, mutations were identified that mapped to a common gene, SSA_0696. Deletion of SSA_0696 resulted in restored in vitro growth in the ssaACB-deficient background, unearthing a novel mechanism for bacterial growth under manganese limitation. Fortunately, the suppressor mutant phenotype was not maintained in vivo; however, the combined results of these experiments suggest the efficacy of future therapeutics may require consideration of virulence at the species level and the incorporation of multiple targets.

The opportunistic fungus, Aspergillus fumigatus, is a leading cause of morbidity and mortality among the immunocompromised population. Experimental and clinical findings have established that phagocytic defenses are critical in the recognition and clearance of A. fumigatus. Previous studies found that Toll-like receptors (TLRs), specifically TLR2 and TLR4, were essential in the detection of the mold. Furthermore, one study found that mice deficient in TLR9 lived longer than their wild-type counterparts following challenge with A. fumigatus. We sought to determine the role of TLR9 during A. fumigatus infection. Our results show that A. fumigatus contains unmethylated CpG DNA, the natural ligand of TLR9. Furthermore, A. fumigatus DNA stimulates a potent pro-inflammatory response in mouse bone marrow derived dendritic cells (BMDCs) and human plasmacytoid dendritic cells (pDCs). A genome wide analysis showed that A. fumigatus DNA contains 87 human and 23 mouse putative immunostimulatory motifs. The response to A. fumigatus DNA is TLR9-dependent, as BMDCs from TLR9-/- mice were unresponsive to the fungal DNA. In addition, HEK293 cells cotransfected with human TLR9 and NFκB driven Luciferase conferred responsiveness to A. fumigatus CpG-rich sequences found in the fungal DNA. Our results show that TLR9 detects A. fumigatus DNA, resulting in the secretion of proinflammatory cytokines. While pDCs secrete IFNα in response to A. fumigatus DNA, these cells have been mainly described to play critical roles in the antiviral responses. The role of pDCs during fungal infections remains to be elucidated. Our data show that CD304+ peripheral blood pDCs challenged with A. fumigatus hyphae secrete large concentrations of IFNα and TNFα in response to infection. Furthermore, the response appears to be TLR9- independent. However, pDCs spread over the hyphae and inhibit fungal growth. Furthermore, pDCs undergo cell lysis upon incubation with A. fumigatus. The antifungal activity of the pDCs was retained in the cell lysates, suggesting that this response was mediated by an intracellular factor. Addition of exogenous Zn2+, but not Fe3+, partially restores hyphal growth. In addition, western blot of pDC lysates show that these cells have the Zn2+-binding protein calprotectin. Over 60% cell death is observed in the pDC population following a 2 hour incubation with A. fumigatus. The observed pDC cell death can be partially attributed to gliotoxin, as pDCs challenged with A. fumigatus stains deficient in production of the mycotoxin result in decreased pDC cytotoxicity. Furthermore, pDC cell death occurs independent of contact with the mold, confirming that pDC cell death is mediated by a secreted fungal factor. In addition, our results show that pDCs are required for the host response against A. fumigatus. Mice depleted of their pDCs are more susceptible to A. fumigatus infection than the control counterparts, suggesting that pDCs play a role in the antifungal response. Also, we observe a 5-fold increase in the pDC population in the lungs of infected mice. Therefore, the possibility of these cells playing a role in recruiting and communicating with other immune cells cannot be eliminated. Upon maturation, pDCs acquire characteristics of conventional DCs (cDCs) such as upregulation of major histocompatability complex (MHC) and becoming more phagocytic. Whether mature pDCs are involved in the detection of and responses against fungal pathogens remains to be determined. Here we show that mature pDC secrete IFNα and TNFα in response to A. fumigatus conidia as early as 6 hours post-challenge. While cytokine secretion of mature pDCs against A. fumigatus does not require opsonization, it requires for A. fumigatus being alive and growing. Furthermore, supernatants from conidial growth induced cytokine secretion by the mature pDCs. The work presented in this thesis establishes that the nucleic acids in A. fumigatus serve as a pathogen associated molecular pattern (PAMP) that can induce a TLR9- dependent response. Furthermore, I show that pDCs secrete cytokines and induce an antifungal response against A. fumigatus conidia and hyphae. While the pDC population in the blood appears to be small, our work shows that these cells could be intimately involved in the antifungal responses against A. fumigatus.

Orientador: Fernanda Lourenção Brighenti / Resumo: Este trabalho está dividido em 2 publicações cujos objetivos foram: a) realizar uma revisão sistemática da literatura, seguida de uma metanálise sobre a eficácia da terapia fotodinâmica (TFD) nos micro-organismos responsáveis pela cárie dentária (Publicação 1); b) avaliar o potencial in vitro dos óleos essenciais de Coffea arabica, Matricaria recutita e Eugenia uniflora e dos extratos vegetais de Senna splendida, Senna reticulata e Senna Macranthera para serem utilizados como monoterapia na terapia fotodinâmica sobre suspensões de micro-organismos de interesse médico-odontológico. Publicação 1: A questão de pesquisa e as palavras-chave foram construídas de acordo com a estratégia do PICO. A pesquisa do artigo foi realizada nas bases de dados Embase, Lilacs, Scielo, Medline, Scopus, Cochrane Library, Web of Science, Science Direct e Pubmed. Ensaios clínicos randomizados e estudos in vitro foram selecionados na revisão. O estudo foi conduzido de acordo com as diretrizes do PRISMA para revisão sistemática. Publicação 2: Foram utilizadas as seguintes cepas de referência em suspensão: Cutibacterium acnes ATCC 6919, Streptococcus mutans ATCC 35688, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 e Candida albicans ATCC 90028. Os materiais vegetais foram testados nas concentrações de 50 μg/mL (extratos) ou 2% (óleos essenciais). Foram estudados cinco grupos: controle negativo, material vegetal sem exposição a luz (FS-luz), material vegetal com exposição a luz (FS+luz),... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. Precursors of these cytokines are expressed downstream of TLR signaling and are then enzymatically processed into mature bioactive forms, typically by caspase-1 which is activated through a process dependent on multi-molecular structures called inflammasomes. Y. pestis evades immune detection in part by using a Type three secretion system (T3SS) to inject effector proteins (Yops) into host cells and suppress IL-1β and IL-18 production. We investigated the cooperation between two effectors, YopM and YopJ, in regulating inflammasome activation, and found that Y. pestis lacking both YopM and YopJ triggers robust caspase-1 activation and IL-1Β/IL-18 production in vitro. Furthermore, this strain is attenuated in a manner dependent upon caspase-1, IL-1β and IL-18 in vivo, yet neither effector appears essential for full virulence. We then demonstrate that YopM fails to inhibit NLRP3/NLRC4 mediated caspase-1 activation and is not a general caspase-1 inhibitor. Instead, YopM specifically prevents the activation of a Pyrin-dependent inflammasome by the Rho-GTPase inhibiting effector YopE. Mutations rendering Pyrin hyperactive are implicated in the autoinflammatory disease Familial Mediterranean Fever (FMF) in humans, and we discuss the potential significance of this disease in relation to plague. Altogether, the Y. pestis T3SS activates and inhibits several inflammasome pathways, and the fact that so many T3SS components are involved in manipulating IL-1β/IL-18 underscores the importance of these mechanisms in plague.

Host cell attachment by pathogenic bacteria can play very different roles in the course of infection. The pathogenic spirochetes Borrelia hermsii and Borrelia burgdorferi sensu lato which cause relapsing fever and Lyme disease, respectively, are transmitted by the bite of infected ticks. After transmission, these spirochetes can cause systemic infection. Relapsing fever spirochetes remain largely in the bloodstream causing febrile episodes, while Lyme disease will often colonize a variety of tissues, such as the heart, joint and nervous system, resulting in a chronic multisystemic disorder. Borrelia species have the ability to bind to various cell types, a process which plays a crucial role in pathogenesis and may influence spirochetal clearance from the bloodstream. Colonization of multiple tissues and cell types is likely promoted by the ability to bind to components found in target tissues, and many B. burgdorferi adhesins have been shown to promote attachment to a wide variety of cells and extracellular matrix components. Different Lyme disease strains have been shown to preferentially colonize certain tissues, although the basis of this tissue tropism is not well understood. In this study we found that among different Lyme disease strains, allelic variation of the adhesin DbpA contributes to variation in its in vitro binding activities raising the possibility that this variation contributes to tissue tropism in vivo. In studying B. hermsii infection, we found evidence by both histological and fluorescence in situ hybridization (FISH) analysis of tissues that indicated that red blood cells were removed by tissue resident macrophages in infected mice. Spirochetes in the spleen and liver were often visualized associated with RBCs, lending support to the hypothesis that direct interaction of B. hermsii spirochetes with RBCs leads to clearance of bacteria from the bloodstream. Our findings indicate that host cell attachment play a key role in the establishment of Lyme disease infection, and in contrast contributes to the clearance of relapsing fever infection.