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J Adhes Dent ; 23(5): 461-467, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34549929


PURPOSE: To evaluate the microtensile bond strength (µTBS) of a one-step self-etch adhesive (1-SEA) to dentin and its interfacial nanomechanical properties after 8 years of water storage. MATERIALS AND METHODS: Flat coronal dentin surfaces of extracted human third molars were bonded with a 1-SEA (Clearfil S3 Bond Plus, CS3+) and built up with a hybrid resin composite (Clearfil AP-X). After storage in water for 24 h or 8 years, non-trimmed stick-shaped specimens were fabricated from the central part of each bonded tooth and subjected to the µTBS test at a crosshead speed of 1.0 mm/min. Failure modes and the morphology of debonded interfaces were analyzed using a scanning electron microscope (SEM). In addition, the elastic modulus (E) and hardness (H) of the adhesive layer and the resin composite were determined by an instrumented nanoindentation test. The acquired µTBS, E, and H data were statistically analyzed using t-tests to examine the effect of storage time (α = 0.05). RESULTS: The 8-year µTBS was slightly lower than that after 24 h, but the difference was not significant (p = 0.123). The SEM observation of debonded surfaces after 8 years revealed extrusions and lacunas. E and H of the adhesive layer and the resin composite significantly decreased over the 8-year water storage (p < 0.001). CONCLUSIONS: Although 8 years of water storage did not decrease the µTBS of CS3+ significantly, the observed failure mode patterns and significantly decreased nanomechanical properties indicated resin degradation of the adhesive and the resin composite.

Colagem Dentária , Dentina , Resinas Compostas , Cimentos Dentários , Análise do Estresse Dentário , Adesivos Dentinários , Humanos , Teste de Materiais , Cimentos de Resina , Estresse Mecânico , Propriedades de Superfície , Resistência à Tração
mSystems ; 6(4): e0073821, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34342542


Chlamydia trachomatis is an obligate intracellular bacterium whose unique developmental cycle consists of an infectious elementary body and a replicative reticulate body. Progression of this developmental cycle requires temporal control of the transcriptome. In addition to the three chlamydial sigma factors (σ66, σ28, and σ54) that recognize promoter sequences of genes, chlamydial transcription factors are expected to play crucial roles in transcriptional regulation. Here, we investigate the function of GrgA, a Chlamydia-specific transcription factor, in C. trachomatis transcriptomic expression. We show that 10 to 30 min of GrgA overexpression induces 13 genes, which likely comprise the direct regulon of GrgA. Significantly, σ66-dependent genes that code for two important transcription repressors are components of the direct regulon. One of these repressors is Euo, which prevents the expression of late genes during early phases. The other is HrcA, which regulates molecular chaperone expression and controls stress response. The direct regulon also includes a σ28-dependent gene that codes for the putative virulence factor PmpI. Furthermore, overexpression of GrgA leads to decreased expression of almost all tRNAs. Transcriptomic studies suggest that GrgA, Euo, and HrcA have distinct but overlapping indirect regulons. These findings, together with temporal expression patterns of grgA, euo, and hrcA, indicate that a transcriptional regulatory network of these three transcription factors plays critical roles in C. trachomatis growth and development. IMPORTANCE Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen worldwide and is a leading cause of preventable blindness in underdeveloped areas as well as some developed countries. Chlamydia carries genes that encode a limited number of known transcription factors. While Euo is thought to be critical for early chlamydial development, the functions of GrgA and HrcA in the developmental cycle are unclear. Activation of euo and hrcA immediately following GrgA overexpression indicates that GrgA functions as a master transcriptional regulator. In addition, by broadly inhibiting tRNA expression, GrgA serves as a key regulator of chlamydial protein synthesis. Furthermore, by upregulating pmpI, GrgA may act as an upstream virulence determinant. Finally, genes coregulated by GrgA, Euo, and HrcA likely play critical roles in chlamydial growth and developmental control.

Microb Pathog ; 156: 104917, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33940135


The obligate intracellular bacterium Chlamydia trachomatis is an important human pathogen with a biphasic developmental cycle comprised of an infectious elementary body (EB) and a replicative reticulate body (RB). Whereas σ66, the primary sigma factor, is necessary for transcription of most chlamydial genes throughout the developmental cycle, σ28 is required for expression of some late genes. We previously showed that the Chlamydia-specific transcription factor GrgA physically interacts with both of these sigma factors and activates transcription from σ66- and σ28-dependent promoters in vitro. Here, we investigated the organismal functions of GrgA. We show that overexpression of GrgA slows EB-to-RB conversion, decreases RB proliferation, and reduces progeny EB production. In contrast, overexpression of a GrgA variant without the σ28-binding domain shows significantly less severe inhibitory effects, while overexpression of a variant without the σ66-binding domain demonstrates no adverse effects. These findings indicate that GrgA plays important roles in the expression regulation of both σ66-dependent genes and σ28-dependent genes during the chlamydial developmental cycle.

Chlamydia trachomatis , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Humanos , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
Pathog Dis ; 77(9)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32011704


Chlamydiae are common, important pathogens for humans and animals alike. Despite recent advancement in genetics, scientists are still searching for efficient tools to knock out or knock down the expression of chromosomal genes. We attempted to adopt a dCas9-based CRISPR interference (CRISPRi) technology to conditionally knock down gene expression in Chlamydia trachomatis using an anhydrotetracycline (ATC)-inducible expression system. Surprisingly, expression of the commonly used Streptococcus pyogenes dCas9 in C. trachomatis causes strong inhibition in the absence of any guide RNA (gRNA). Staphylococcus aureus dCas9 also shows strong toxicity in the presence of only an empty gRNA scaffold. Toxicity of the S. pyogenes dCas9 is readily observed with as little as 0.2 nM ATC. Growth inhibition by S. aureus dCas9 is evident starting at 1.0 nM ATC. In contrast, C. trachomatis growth was not affected by methionine-tRNA ligase overexpression induced with 10 nM ATC. We conclude that S. pyogenes and S. aureus dCas9 proteins in their current forms have limited utility for chlamydial research and suggest strategies to overcome this problem.

Sistemas CRISPR-Cas , Chlamydia trachomatis/genética , Staphylococcus aureus/genética , Streptococcus pyogenes/genética , Chlamydia trachomatis/metabolismo , RNA Guia/antagonistas & inibidores , RNA Guia/metabolismo
J Bacteriol ; 200(20)2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30061357


The obligate intracellular bacterial pathogen Chlamydia trachomatis has a unique developmental cycle consisting of two contrasting cellular forms. Whereas the primary Chlamydia sigma factor, σ66, is involved in the expression of the majority of chlamydial genes throughout the developmental cycle, expression of several late genes requires the alternative sigma factor, σ28 In prior work, we identified GrgA as a Chlamydia-specific transcription factor that activates σ66-dependent transcription by binding DNA and interacting with a nonconserved region (NCR) of σ66 Here, we extend these findings by showing GrgA can also activate σ28-dependent transcription through direct interaction with σ28 We measure the binding affinity of GrgA for both σ66 and σ28, and we identify regions of GrgA important for σ28-dependent transcription. Similar to results obtained with σ66, we find that GrgA's interaction with σ28 involves an NCR located upstream of conserved region 2 of σ28 Our findings suggest that GrgA is an important regulator of both σ66- and σ28-dependent transcription in C. trachomatis and further highlight NCRs of bacterial RNA polymerase as targets for regulatory factors unique to particular organisms.IMPORTANCE Chlamydia trachomatis is the number one sexually transmitted bacterial pathogen worldwide. A substantial proportion of C. trachomatis-infected women develop infertility, pelvic inflammatory syndrome, and other serious complications. C. trachomatis is also a leading infectious cause of blindness in underdeveloped countries. The pathogen has a unique developmental cycle that is transcriptionally regulated. The discovery of an expanded role for the Chlamydia-specific transcription factor GrgA helps us understand the progression of the chlamydial developmental cycle.

Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Regulação Bacteriana da Expressão Gênica , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Chlamydia trachomatis/metabolismo , Citoplasma/metabolismo , RNA Polimerases Dirigidas por DNA , Escherichia coli/genética , Genes Bacterianos , Humanos , Fator sigma/genética , Fatores de Transcrição/genética