RESUMO
BACKGROUND: RNA sequencing (RNA-Seq) offers profound insights into the complex transcriptomes of diverse biological systems. However, standard differential expression analysis pipelines based on DESeq2 and edgeR encounter challenges when applied to the immediate early transcriptomes of Chlamydia spp., obligate intracellular bacteria. These challenges arise from their reliance on assumptions that do not hold in scenarios characterized by extensive transcriptomic activation and limited repression. RESULTS: Standard analyses using unique chlamydial RNA-Seq reads alone identify nearly 300 upregulated and about 300 downregulated genes, significantly deviating from actual RNA-Seq read trends. By incorporating both chlamydial and host reads or adjusting for total sequencing depth, the revised normalization methods each detected over 700 upregulated genes and 30 or fewer downregulated genes, closely aligned with observed RNA-Seq data. Further validation through qRT-PCR analysis confirmed the effectiveness of these adjusted approaches in capturing the true extent of transcriptomic activation during the immediate early phase of chlamydial infection. CONCLUSIONS: This study highlights the limitations of standard RNA-Seq analysis tools in scenarios with extensive transcriptomic activation, such as in Chlamydia spp. during early infection. Our revised normalization methods, incorporating host reads or total sequencing depth, provide a more accurate representation of gene expression dynamics. These approaches may inform similar adjustments in other systems with unbalanced gene expression dynamics, enhancing the accuracy of transcriptomic analysis.
Assuntos
Chlamydia , Transcriptoma , Chlamydia/genética , Humanos , RNA-Seq/métodos , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/genéticaRESUMO
Motivation: RNA sequencing (RNA-Seq) offers profound insights into the complex transcriptomes of diverse biological systems. However, standard differential expression analysis pipelines based on DESeq2 and edgeR encounter challenges when applied to the immediate early transcriptomes of Chlamydia spp., obligate intracellular bacteria. These challenges arise from their reliance on assumptions that do not hold in scenarios characterized by extensive transcriptomic activation and limited repression. Standard analyses using unique chlamydial RNA-Seq reads alone identify nearly 300 upregulated and about 300 downregulated genes, significantly deviating from actual RNA-Seq read trends. Results: By incorporating both chlamydial and host reads or adjusting for total sequencing depth, the revised normalization methods each detected over 700 upregulated genes and 30 or fewer downregulated genes, closely aligned with observed RNA-Seq data. Further validation through qRT-PCR analysis confirmed the effectiveness of these adjusted approaches in capturing the true extent of transcriptomic activation during the immediate early phase of chlamydial infection. While the strategies employed are developed in the context of Chlamydia, the principles of flexible and context-aware normalization may inform adjustments in other systems with unbalanced gene expression dynamics, such as bacterial spore germination. Availability and implementation: The code for reproducing the presented bioinformatic analysis is available at https://zenodo.org/records/11201379.
Assuntos
Regulação Bacteriana da Expressão Gênica , Cinética , Humanos , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Infecções por Chlamydia/microbiologia , Chlamydia/genética , Chlamydia trachomatis/genética , Chlamydia trachomatis/crescimento & desenvolvimento , Estágios do Ciclo de VidaRESUMO
The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.
Assuntos
Infecções por Chlamydia , Chlamydia trachomatis , Humanos , Células Cultivadas , Sequência de Bases , Transcriptoma , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Chlamydia/genéticaRESUMO
IMPORTANCE: Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.
Assuntos
Infecções por Chlamydia , Chlamydia trachomatis , Humanos , Chlamydia trachomatis/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Fator sigma/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Chlamydia, an obligate intracellular bacterial pathogen, has a unique developmental cycle involving the differentiation of invading elementary bodies (EBs) to noninfectious reticulate bodies (RBs), replication of RBs, and redifferentiation of RBs into progeny EBs. Progression of this cycle is regulated by three sigma factors, which direct the RNA polymerase to their respective target gene promoters. We hypothesized that the Chlamydia-specific transcriptional regulator GrgA, previously shown to activate σ66 and σ28, plays an essential role in chlamydial development and growth. To test this hypothesis, we applied a novel genetic tool known as dependence on plasmid-mediated expression (DOPE) to create Chlamydia trachomatis with conditional GrgA-deficiency. We show that GrgA-deficient C. trachomatis RBs have a growth rate that is approximately half of the normal rate and fail to transition into progeny EBs. In addition, GrgA-deficient C. trachomatis fail to maintain its virulence plasmid. Results of RNA-seq analysis indicate that GrgA promotes RB growth by optimizing tRNA synthesis and expression of nutrient-acquisition genes, while it enables RB-to-EB conversion by facilitating the expression of a histone and outer membrane proteins required for EB morphogenesis. GrgA also regulates numerous other late genes required for host cell exit and subsequent EB invasion into host cells. Importantly, GrgA stimulates the expression of σ54, the third and last sigma factor, and its activator AtoC, and thereby indirectly upregulating the expression of σ54-dependent genes. In conclusion, our work demonstrates that GrgA is a master transcriptional regulator in Chlamydia and plays multiple essential roles in chlamydial pathogenicity.
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Chlamydiae are obligate intracellular Gram-negative bacteria and widespread pathogens in humans and animals. Broad-spectrum antibiotics are currently used to treat chlamydial infections. However, broad-spectrum drugs also kill beneficial bacteria. Recently, two generations of benzal acylhydrazones have been shown to selectively inhibit chlamydiae without toxicity to human cells and lactobacilli, which are dominating, beneficial bacteria in the vagina of reproductive-age women. Here, we report the identification of two acylpyrazoline-based third-generation selective antichlamydials (SACs). With minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of 10-25 µM against Chlamydia trachomatis and Chlamydia muridarum, these new antichlamydials are 2- to 5-fold more potent over the benzal acylhydrazone-based second-generation selective antichlamydial lead SF3. Both acylpyrazoline-based SACs are well tolerated by Lactobacillus, Escherichia coli, Klebsiella, and Salmonella as well as host cells. These third-generation selective antichlamydials merit further evaluation for therapeutic application.
RESUMO
Gene transcription in bacteria is carried out by the multisubunit RNA polymerase (RNAP), which is composed of a catalytic core enzyme and a promoter-recognizing σ factor. The core enzyme comprises two α subunits, one ß subunit, one ß' subunit, and one ω subunit. The ω subunit plays critical roles in the assembly of the core enzyme and other cellular functions, including the regulation of bacterial growth, the stress response, and biofilm formation. However, the identity of an ω subunit for the obligate intracellular bacterium Chlamydia has not previously been determined. Here, we report the identification of the hypothetical protein CTL0286 as the probable chlamydial ω subunit based on sequence, synteny, and AlphaFold and AlphaFold-Multimer three-dimensional-structure predictions. Our findings indicate that CTL0286 functions as the missing ω subunit of chlamydial RNAP. Our extended analysis also indicates that all obligate intracellular bacteria have ω orthologs. IMPORTANCE Chlamydiae are obligate intracellular bacteria that replicate only inside eukaryotic cells. Previously, it has not been possible to identify a candidate gene encoding the chlamydial RNA polymerase ω subunit, and it has been hypothesized that the chlamydial RNA polymerase ω subunit was lost in the evolutionary process through which Chlamydiae reduced their genome size and proteome sizes to adapt to an obligate intracellular lifestyle. Here, we report the identification of the chlamydial RNA polymerase ω subunit, based on conserved sequence, conserved synteny, AlphaFold-predicted conserved three-dimensional structure, and AlfaFold-Multimer-predicted conserved interactions. Our identification of the previously elusive chlamydial RNA polymerase ω subunit sets the stage for investigation of its roles in regulation of gene expression during chlamydial growth, development, and stress responses, and sets the stage for preparation and study of the intact chlamydial RNA polymerase and its interactions with inhibitors.
Assuntos
Chlamydia , RNA Polimerases Dirigidas por DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Bactérias/genética , Sequência Conservada , Chlamydia/genética , Chlamydia/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
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/metabolismoRESUMO
Cells reprogram their transcriptome in response to stress, such as heat shock. In free-living bacteria, the transcriptomic reprogramming is mediated by increased DNA-binding activity of heat shock sigma factors and activation of genes normally repressed by heat-induced transcription factors. In this study, we performed transcriptomic analyses to investigate heat shock response in the obligate intracellular bacterium Chlamydia trachomatis, whose genome encodes only three sigma factors and a single heat-induced transcription factor. Nearly one-third of C. trachomatis genes showed statistically significant (≥1.5-fold) expression changes 30 min after shifting from 37 to 45°C. Notably, chromosomal genes encoding chaperones, energy metabolism enzymes, type III secretion proteins, as well as most plasmid-encoded genes, were differentially upregulated. In contrast, genes with functions in protein synthesis were disproportionately downregulated. These findings suggest that facilitating protein folding, increasing energy production, manipulating host activities, upregulating plasmid-encoded gene expression, and decreasing general protein synthesis helps facilitate C. trachomatis survival under stress. In addition to relieving negative regulation by the heat-inducible transcriptional repressor HrcA, heat shock upregulated the chlamydial primary sigma factor σ66 and an alternative sigma factor σ28. Interestingly, we show for the first time that heat shock downregulates the other alternative sigma factor σ54 in a bacterium. Downregulation of σ54 was accompanied by increased expression of the σ54 RNA polymerase activator AtoC, thus suggesting a unique regulatory mechanism for reestablishing normal expression of select σ54 target genes. Taken together, our findings reveal that C. trachomatis utilizes multiple novel survival strategies to cope with environmental stress and even to replicate. Future strategies that can specifically target and disrupt Chlamydia's heat shock response will likely be of therapeutic value.
RESUMO
OBJECTIVE: This report is a side product of experiments aimed at identifying serum for culturing obligate intracellular bacteria Chlamydia trachomatis and C. muridarum in mouse fibroblast L929 cells. RESULTS: Of five commercial serum samples tested, two showed optimal efficiencies at supporting growth of the human pathogen Chlamydia trachomatis as control fetal bovine serum, whereas two showed modest ~ 40% inhibitions in progeny production, and the remaining one showed a 20% inhibition. Three of the six sera poorly supported growth of the murine pathogen Chlamydia muridarum, resulting in 73-90% reduction in progeny formation. Most significantly, the one with the strongest (90%) C. muridarum inhibition activity showed optimal C. trachomatis-supporting efficiency. These findings indicate that in laboratories that study multiple Chlamydia species, serum samples should be prescreened on a species basis. Considering Chlamydial biology and epidemiology, it may even be necessary to perform serum tests on a serovar- or strain-basis for studying some animal chlamydiae.
Assuntos
Chlamydia muridarum/crescimento & desenvolvimento , Chlamydia trachomatis/crescimento & desenvolvimento , Animais , Linhagem Celular , Chlamydia muridarum/fisiologia , Chlamydia trachomatis/fisiologia , Meios de Cultura , Camundongos , Soro/químicaRESUMO
A transcription factor (TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two σ factors, namely σ66 and σ28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.
Assuntos
Chlamydia/genética , Interferometria/métodos , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Bactérias/metabolismo , Chlamydia/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica , Cinética , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Chlamydia is a common pathogen that can causes serious complications in the reproductive system and eyes. Lack of vaccine and other effective prophylactic measures coupled with the largely asymptomatic nature and unrare clinical treatment failure calls for development of new antichlamydials, particularly selective antichlamydials without adverse effects on humans and the beneficial microbiota. We previously reported that benzal-N-acylhydrazones (BAH) can inhibit chlamydiae without detectable adverse effects on host cells and beneficial lactobacilli that dominate the human vaginal microbiota among reproductive-age women. However, the antichlamydial mechanism of BAH is not known. Whereas 4 single nucleotide polymorphisms (i.e., SNP1-4) were identified in a rare Chlamydia variant with a low level of BAH resistance, termed MCR, previous studies failed to establish a causal effect of any particular SNP(s). In the present work, we performed recombination to segregate the four SNPs. Susceptibility tests indicate that the R51G GrgA allele is both necessary and sufficient for the low level of BAH resistance. Thus, the Chlamydia-specific transcription factor GrgA either is a direct target of BAH or regulates BAH susceptibility. We further confirm an extremely low rate of BAH resistance in Chlamydia. Our findings warrant exploration of GrgA as a therapeutic and prophylactic target for chlamydial infections.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Chlamydia muridarum/genética , Farmacorresistência Bacteriana/genética , Fatores de Transcrição/genética , Animais , Antibacterianos/uso terapêutico , Proteínas de Bactérias/antagonistas & inibidores , Compostos de Benzilideno/farmacologia , Compostos de Benzilideno/uso terapêutico , Linhagem Celular , Infecções por Chlamydia/tratamento farmacológico , Infecções por Chlamydia/microbiologia , Chlamydia muridarum/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Hidrazonas/farmacologia , Hidrazonas/uso terapêutico , Camundongos , Testes de Sensibilidade Microbiana , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/antagonistas & inibidoresRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Chlamydia trachomatis/genética , Staphylococcus aureus/genética , Streptococcus pyogenes/genética , Chlamydia trachomatis/metabolismo , RNA Guia de Cinetoplastídeos/antagonistas & inibidores , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
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.IMPORTANCEChlamydia 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.
Assuntos
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/genéticaRESUMO
The obligate intracellular bacterium Chlamydia is a widespread human pathogen that causes serious problems, including (but not limited to) infertility and blindness. Our search for novel antichlamydial metabolites from marine-derived microorganisms led to the isolation of pyocyanin, a small compound from Pseudomonas aeruginosa Pyocyanin is an effective antichlamydial for all three Chlamydia spp. tested. It has a 50% inhibitory concentration (IC50) of 0.019 to 0.028 µM, which is comparable to the IC50 of tetracycline. At concentrations as low as 0.0039 µM, pyocyanin disables infectivity of the chlamydial elementary body (EB). At 0.5 µM or higher concentrations, the continuous presence of pyocyanin also inhibits chlamydial growth in the inclusion during later stages of the developmental cycle. Oxidative stress, a major known antimicrobial mechanism of pyocyanin, appears to be responsible only for the inhibition of bacterial growth and not for the disinfection of EBs. Pyocyanin is well-tolerated by probiotic vaginal Lactobacillus spp. Our findings suggest that pyocyanin is of therapeutic value for chlamydial infections and can serve as a valuable chemical probe for studying chlamydial biology.
Assuntos
Antibacterianos/farmacologia , Infecções por Chlamydia/microbiologia , Piocianina/farmacologia , Lactobacillus/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estresse Oxidativo/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Tetraciclina/farmacologiaRESUMO
The First Chinese Chlamydia Research Meeting was held in Lanzhou, China in May 2017, 60 years after the disclosure of reproducible isolation of Chlamydia trachomatis by (Fei-fan Tang). We report current state of the Chlamydia research community in China, and briefly review recent progress in Chlamydia vaccinology. The meeting represents a new milestone for Chlamydia research in the country. The Chinese Chlamydia Research Society (CCRS) was formed during the meeting. Future meetings will be held biennially and should facilitate collaboration of Chinese researchers with their domestic and international colleagues.
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Infecções por Chlamydia/virologia , Chlamydia trachomatis/fisiologia , Pesquisa/tendências , Animais , China , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/prevenção & controle , Chlamydia trachomatis/imunologia , Humanos , Vacinas ViraisRESUMO
Sexually transmitted Chlamydia trachomatis is an extremely common infection and often leads to serious complications including infertility and pelvic inflammatory syndrome. Several broad-spectrum antibiotics are currently used to treat C. trachomatis. Although effective, they also kill beneficial vaginal lactobacilli. Two N-acylhydrazones, CF0001 and CF0002, have been shown previously to inhibit chlamydial growth without toxicity to human cells and Lactobacillus spp. Of particular significance, the rate of random mutation leading to resistance of these inhibitors appears to be extremely low. Here, we report three analogs of CF0001 and CF0002 with significantly stronger inhibitory effects on chlamydiae. Even though the new compounds (termed SF1, SF2 and SF3) displayed slightly decreased inhibition efficiencies for a rare Chlamydia variant selected for CF0001 resistance (Chlamydia muridarum MCR), they completely overcame the resistance when used at concentrations of 75-100 µM. Importantly, SF1, SF2 and SF3 did not shown any toxic effect on lactobacilli, whereas SF3 was also well tolerated by human host cells. An effort to isolate SF3-resistant variants was unsuccessful. By comparison, variants resistant to rifampin or spectinomycin were obtained from smaller numbers of chlamydiae. Our findings suggest that SF3 utilizes an antichlamydial mechanism similar to that of CF0001 and CF0002, and will be more difficult for chlamydiae to develop resistance to, potentially making it a more effective antichlamydial agent.
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Antibacterianos/farmacologia , Infecções por Chlamydia/tratamento farmacológico , Chlamydia muridarum/efeitos dos fármacos , Chlamydia trachomatis/efeitos dos fármacos , Lactobacillus/efeitos dos fármacos , Antibacterianos/uso terapêutico , Infecções por Chlamydia/microbiologia , Feminino , Humanos , Vagina/efeitos dos fármacosRESUMO
Chlamydiae are widely distributed pathogens of human populations, which can lead to serious reproductive and other health problems. In our search for novel antichlamydial metabolites from marine derived-microorganisms, one new (1) and two known (2, 3) dimeric indole derivatives were isolated from the sponge-derived actinomycete Rubrobacter radiotolerans. The chemical structures of these metabolites were elucidated by NMR spectroscopic data as well as CD calculations. All three metabolites suppressed chlamydial growth in a concentration-dependent manner. Among them, compound 1 exhibited the most effective antichlamydial activity with IC50 values of 46.6 ~ 96.4 µM in the production of infectious progeny. Compounds appeared to target the mid-stage of the chlamydial developmental cycle by interfering with reticular body replication, but not directly inactivating the infectious elementary body.