RESUMO
Advances in the molecular epidemiological studies of the Human Immunodeficiency Virus (HIV) at the Robert Koch Institute (RKI) by laboratory and bioinformatic automation should allow the processing of larger numbers of samples and more comprehensive and faster data analysis in order to provide a higher resolution of the current HIV infection situation in near real-time and a better understanding of the dynamic of the German HIV epidemic. The early detection of the emergence and transmission of new HIV variants is important for the adaption of diagnostics and treatment guidelines. Likewise, the molecular epidemiological detection and characterization of spatially limited HIV outbreaks or rapidly growing sub-epidemics is of great importance in order to interrupt the transmission pathways by regionally adapting prevention strategies. These aims are becoming even more important in the context of the SARS-CoV2 pandemic and the Ukrainian refugee movement, which both have effects on the German HIV epidemic that should be monitored to identify starting points for targeted public health measures in a timely manner. To this end, a next level integrated genomic surveillance of HIV is to be established.
Assuntos
Infecções por HIV , Humanos , Infecções por HIV/epidemiologia , Infecções por HIV/prevenção & controle , HIV , RNA Viral , Epidemiologia Molecular , Alemanha/epidemiologia , GenômicaRESUMO
Numbers of infections with Neisseria gonorrhoeae are among the top three sexually transmitted infections (STI) worldwide. In addition, the emergence and spread of antimicrobial resistance (AMR) in Neisseria gonorrhoeae pose an important public-health issue. The integration of genomic, phenotypic and epidemiological data to monitor Neisseria gonorrhoeae fosters our understanding of the emergence and spread of AMR in Neisseria gonorrhoeae and helps to inform therapy guidelines and intervention strategies. Thus, the Gonococcal resistance surveillance (Go-Surv-AMR) was implemented at the Robert Koch Institute in Germany in 2021 to obtain molecular, phenotypic and epidemiological data on Neisseria gonorrhoeae isolated in Germany. Here, we describe the structure and aims of Go-Surv-AMR. Furthermore, we point out future directions of Go-Surv-AMR to improve the integrated genomic surveillance of Neisseria gonorrhoeae. In this context we discuss current and prospective sequencing approaches and the information derived from their application. Moreover, we highlight the importance of combining phenotypic and WGS data to monitor the evolution of AMR in Neisseria gonorrhoeae in Germany. The implementation and constant development of techniques and tools to improve the genomic surveillance of Neisseria gonorrhoeae will be important in coming years.
Assuntos
Gonorreia , Neisseria gonorrhoeae , Humanos , Neisseria gonorrhoeae/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Estudos Prospectivos , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Alemanha/epidemiologiaRESUMO
Within the German Gonococcal Resistance Network's (GORENET) Neisseria gonorrhoeae (NG) sample collection, azithromycin-resistant NG isolates increased from 4.3% in 2016 to 9.2% in 2018. We aim to understand this observed increase using whole genome sequencing of NG isolates combined with epidemiological and clinical data. Reduced susceptibility to azithromycin in 2018 was predominately clonal (NG multiantigen sequence typing G12302) and could mainly be attributed to the recently described mosaic-like mtr locus. Our data suggest that, together with horizontal gene transfer of resistance determinants and well-established point mutations, international spread of resistant lineages plays a major role regarding azithromycin resistance in Germany.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Antibacterianos/farmacologia , Azitromicina/farmacologia , Farmacorresistência Bacteriana/genética , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/efeitos dos fármacos , Alemanha/epidemiologia , Gonorreia/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/isolamento & purificação , Filogenia , Sequenciamento Completo do GenomaRESUMO
We monitored antimicrobial susceptibility developments of Neisseria gonorrhoeae in Germany from January 2014 to May 2021. The proportion of isolates with azithromycin minimum inhibitory concentrations above the epidemiological cut-off increased substantially, from 1.3% in 2014 to 12.2% in 2020. Preliminary data from 2021 showed a further rise (January to May: 20.7%). Therefore, azithromycin as part of the recommended dual therapy in Germany for non-adherent patients is challenged. Antimicrobial susceptibility testing in clinical practice is crucial and continuous susceptibility surveillance indispensable.
Assuntos
Gonorreia , Neisseria gonorrhoeae , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Azitromicina/farmacologia , Ceftriaxona/farmacologia , Farmacorresistência Bacteriana , Alemanha/epidemiologia , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/genéticaRESUMO
Ceramide transfer protein (CERT) mediates non-vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate-limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non-homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT-mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96-well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Ceramidas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Transferência Ressonante de Energia de Fluorescência , Preparações Farmacêuticas/análise , Animais , Transporte Biológico/efeitos dos fármacos , HumanosRESUMO
BACKGROUND: Worldwide, an increase in antimicrobial resistance (AMR) of Neisseria gonorrhoeae has been observed. Until now, no protocol for an external quality assessment (EQA) has been available for Germany. The German gonococcal resistance network (GORENET) performed an EQA of primary laboratories in Germany in order to assess quality of antibiotic susceptibility testing, to gain information about laboratory procedures and to assess the impact of these procedures on test results. METHODS: Laboratories assessed drug susceptibility to cefixime, ceftriaxone, azithromycin, penicillin and ciprofloxacin for five N. gonorrhoeae strains, using their standard laboratory protocols. Minimal inhibitory concentrations (MICs) were compared to World Health Organisation (WHO) consensus results (or, if not available, reference laboratory results), while deviation by +/- one doubling dilution was accepted. Data on laboratory procedures were collected via a standardised questionnaire. Generalized linear models and conditional inference trees (CTREE) were used to assess relationships between laboratory procedures and testing outcomes. RESULTS: Twenty-one primary laboratories participated in the EQA in June 2018. 96% of ciprofloxacin MICs were reported within accepted deviations, as well as 88% for cefixime, 85% for ceftriaxone, 79% for penicillin and 70% for azithromycin. The use of interpretation standards and general laboratory procedures like agar base, incubation settings or the use of control strains strongly differed between laboratories. In statistical analysis, incubation time of cultures < 24 h was associated with correct measurements. Additionally, a 5% CO2 concentration was associated with correct results regarding azithromycin compared to 3%. CTREE analysis showed that incubation time, humidity and CO2 concentration had the greatest influence on the average deviation from consensus results. CONCLUSIONS: In conclusion, we report the development of a protocol for N. gonorrhoeae antimicrobial susceptibility testing in Germany. While testing results were in accordance with the expected consensus results in 70-96%, depending on the antibiotic agent, laboratory methodology was heterogeneous and may significantly affect the testing quality. We therefore recommend the development of a standard operating procedure (SOP) for N. gonorrhoeae susceptibility testing in Germany.
Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/efeitos dos fármacos , Gonorreia/tratamento farmacológico , Laboratórios/normas , Ensaio de Proficiência Laboratorial , Neisseria gonorrhoeae/efeitos dos fármacos , Antibacterianos/farmacologia , Azitromicina/farmacologia , Azitromicina/uso terapêutico , Cefixima/farmacologia , Cefixima/uso terapêutico , Ceftriaxona/farmacologia , Ceftriaxona/uso terapêutico , Ciprofloxacina/farmacologia , Ciprofloxacina/uso terapêutico , Alemanha , Gonorreia/microbiologia , Humanos , Ensaio de Proficiência Laboratorial/métodos , Testes de Sensibilidade Microbiana , Penicilinas/farmacologia , Penicilinas/uso terapêutico , Controle de Qualidade , Padrões de Referência , Inquéritos e QuestionáriosRESUMO
BackgroundEmerging antimicrobial resistance (AMR) challenges gonorrhoea treatment and requires surveillance.AimThis observational study describes the genetic diversity of Neisseria gonorrhoeae isolates in Germany from 2014 to 2017 and identifies N. gonorrhoeae multi-antigen sequence typing (NG-MAST) genogroups associated with AMR or some patient demographics.Methods1,220 gonococcal isolates underwent AMR testing and NG-MAST. Associations between genogroups and AMR or sex/age of patients were statistically assessed.ResultsPatients' median age was 32 years (interquartile range: 25-44); 1,078 isolates (88.4%) originated from men. In total, 432 NG-MAST sequence types including 156 novel ones were identified, resulting in 17 major genogroups covering 59.1% (721/1,220) of all isolates. Genogroups G1407 and G10557 (G7072) were significantly associated with decreased susceptibility to cefixime (Kruskal-Wallis chi-squared: 549.3442, df: 16, p < 0.001). Their prevalences appeared to decline during the study period from 14.2% (15/106) to 6.2% (30/481) and from 6.6% (7/106) to 3.1% (15/481) respectively. Meanwhile, several cefixime susceptible genogroups' prevalence seemed to increase. Proportions of isolates from men differed among genogroups (Fisher's exact test, p < 0.001), being e.g. lower for G25 (G51) and G387, and higher for G5441 and G2992. Some genogroups differed relative to each other in affected patients' median age (Kruskal-Wallis chi-squared: 47.5358, df: 16, p < 0.001), with e.g. G25 (G51) and G387 more frequent among ≤ 30 year olds and G359 and G17420 among ≥ 40 year olds.ConclusionAMR monitoring with molecular typing is important. Dual therapy (ceftriaxone plus azithromycin) recommended in 2014 in Germany, or only the ceftriaxone dose of this therapy, might have contributed to cefixime-resistant genogroups decreasing.
Assuntos
Cefixima/farmacologia , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Adulto , Cefixima/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Alemanha/epidemiologia , Gonorreia/epidemiologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Neisseria gonorrhoeae/efeitos dos fármacos , Filogenia , PrevalênciaRESUMO
Chlamydia trachomatis is an important human pathogen. This obligate intracellular bacterium grows inside the eukaryotic cell in a membrane-bound compartment, the inclusion. Recent global approaches describe the interactions of C. trachomatis with its host cell and indicate the inclusion is an intracellular trafficking hub embedded into the cellular vesicular trafficking pathways recruiting subunits of the retromer protein complex of the host cell. Here we review these recent developments in deciphering Chlamydia-host cell interactions with emphasis on the role of the retromer complex.
Assuntos
Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/metabolismo , Interações Hospedeiro-Patógeno , Animais , Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/crescimento & desenvolvimento , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/microbiologia , Transporte Proteico , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Chlamydiaceae are bacterial pathogens that cause diverse diseases in humans and animals. Despite their broad host and tissue tropism, all Chlamydia species share an obligate intracellular cycle of development and have evolved sophisticated mechanisms to interact with their eukaryotic host cells. Here, we have analysed interactions of the zoonotic pathogen Chlamydia psittaci with a human epithelial cell line. We found that C. psittaci recruits the ceramide transport protein (CERT) to its inclusion. Chemical inhibition and CRISPR/Cas9-mediated knockout of CERT showed that CERT is a crucial factor for C. psittaci infections thereby affecting different stages of the infection including inclusion growth and infectious progeny formation. Interestingly, the uptake of fluorescently labelled sphingolipids in bacteria inside the inclusion was accelerated in CERT-knockout cells indicating that C. psittaci can exploit CERT-independent sphingolipid uptake pathways. Moreover, the CERT-specific inhibitor HPA-12 strongly diminished sphingolipid transport to inclusions of infected CERT-knockout cells, suggesting that other HPA-12-sensitive factors are involved in sphingolipid trafficking to C. psittaci. Further analysis is required to decipher these interactions and to understand their contributions to bacterial development, host range, tissue tropism, and disease outcome.
Assuntos
Chlamydophila psittaci/metabolismo , Chlamydophila psittaci/patogenicidade , Proteínas Serina-Treonina Quinases/metabolismo , Esfingolipídeos/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Ceramidas/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , HumanosRESUMO
Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.
Assuntos
Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/patogenicidade , Vacúolos/metabolismo , Separação Celular , Chlamydia trachomatis/metabolismo , Citometria de Fluxo , Células HeLa , Humanos , Corpos de Inclusão/metabolismo , Transporte Proteico , Proteoma , Proteômica , RNA Interferente Pequeno , Espectrometria de Massas em Tandem , TransfecçãoRESUMO
For membrane-bound intracellular pathogens, the surrounding vacuole is the portal of communication with the host cell. The parasitophorous vacuole (PV) harboring intrahepatocytic Plasmodium parasites satisfies the parasites' needs of nutrition and protection from host defenses to allow the rapid parasite growth that occurs during the liver stage of infection. In this study, we visualized the PV membrane (PVM) and the associated tubovesicular network (TVN) through fluorescent tagging of two PVM-resident Plasmodium berghei proteins, UIS4 and IBIS1. This strategy revealed previously unrecognized dynamics with which these membranes extend throughout the host cell. We observed dynamic vesicles, elongated clusters of membranes and long tubules that rapidly extend and contract from the PVM in a microtubule-dependent manner. Live microscopy, correlative light-electron microscopy and fluorescent recovery after photobleaching enabled a detailed characterization of these membranous features, including velocities, the distribution of UIS4 and IBIS1, and the connectivity of PVM and TVN. Labeling of host cell compartments revealed association of late endosomes and lysosomes with the elongated membrane clusters. Moreover, the signature host autophagosome protein LC3 was recruited to the PVM and TVN and colocalized with UIS4. Together, our data demonstrate that the membranes surrounding intrahepatic Plasmodium are involved in active remodeling of host cells.
Assuntos
Fígado/parasitologia , Plasmodium/metabolismo , Animais , Membrana Celular/metabolismo , Interações Hospedeiro-Parasita , Plasmodium/patogenicidadeRESUMO
Influenza A virus, being responsible for seasonal epidemics and reoccurring pandemics, represents a worldwide threat to public health. High mutation rates facilitate the generation of viral escape mutants, rendering vaccines and drugs directed against virus-encoded targets potentially ineffective. In contrast, targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape. Here we report the discovery of 287 human host cell genes influencing influenza A virus replication in a genome-wide RNA interference (RNAi) screen. Using an independent assay we confirmed 168 hits (59%) inhibiting either the endemic H1N1 (119 hits) or the current pandemic swine-origin (121 hits) influenza A virus strains, with an overlap of 60%. Notably, a subset of these common hits was also essential for replication of a highly pathogenic avian H5N1 strain. In-depth analyses of several factors provided insights into their infection stage relevance. Notably, SON DNA binding protein (SON) was found to be important for normal trafficking of influenza virions to late endosomes early in infection. We also show that a small molecule inhibitor of CDC-like kinase 1 (CLK1) reduces influenza virus replication by more than two orders of magnitude, an effect connected with impaired splicing of the viral M2 messenger RNA. Furthermore, influenza-virus-infected p27(-/-) (cyclin-dependent kinase inhibitor 1B; Cdkn1b) mice accumulated significantly lower viral titres in the lung, providing in vivo evidence for the importance of this gene. Thus, our results highlight the potency of genome-wide RNAi screening for the dissection of virus-host interactions and the identification of drug targets for a broad range of influenza viruses.
Assuntos
Fatores Biológicos , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Influenza Humana/genética , Influenza Humana/virologia , Interferência de RNA , Replicação Viral/fisiologia , Animais , Fatores Biológicos/genética , Fatores Biológicos/metabolismo , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Inibidor de Quinase Dependente de Ciclina p27/deficiência , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Epiteliais/virologia , Genoma Humano/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genéticaRESUMO
The obligate intracellular bacterium Chlamydia trachomatis survives and replicates within a membrane-bound vacuole, termed the inclusion, which intercepts host exocytic pathways to obtain nutrients. Like many other intracellular pathogens, C. trachomatis has a marked requirement for host cell lipids, such as sphingolipids and cholesterol, produced in the endoplasmic reticulum and the Golgi apparatus. However, the mechanisms by which intracellular pathogens acquire host cell lipids are not well understood. In particular, no host cell protein responsible for transporting Golgi-derived lipids to the chlamydial inclusions has yet been identified. Here we show that Chlamydia infection in human epithelial cells induces Golgi fragmentation to generate Golgi ministacks surrounding the bacterial inclusion. Ministack formation is triggered by the proteolytic cleavage of the Golgi matrix protein golgin-84. Inhibition of golgin-84 truncation prevents Golgi fragmentation, causing a block in lipid acquisition and maturation of C. trachomatis. Golgi fragmentation by means of RNA-interference-mediated knockdown of distinct Golgi matrix proteins before infection enhances bacterial maturation. Our data functionally connect bacteria-induced golgin-84 cleavage, Golgi ministack formation, lipid acquisition and intracellular pathogen growth. We show that C. trachomatis subverts the structure and function of an entire host cell organelle for its own advantage.
Assuntos
Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydia trachomatis/patogenicidade , Complexo de Golgi/microbiologia , Complexo de Golgi/patologia , Chlamydia muridarum/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Técnicas de Silenciamento de Genes , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Interferência de RNA , Proteínas de Transporte VesicularRESUMO
Chlamydia trachomatis is a medically important human pathogen causing different diseases, including trachoma, the leading cause of preventable blindness in developing countries, and sexually transmitted infections that can lead to infertility and ectopic pregnancies. There is no vaccine against C. trachomatis at present. Broad-spectrum antibiotics are used as standard therapy to treat the infection but have unwanted side effects, such as inducing persistent or recurring infections and affecting the host microbiome, necessitating the development of novel anti-Chlamydia therapies. Here, we describe the establishment of a robust, fast, and simple plaque assay using liquid overlay medium (LOM) for the identification of anti-Chlamydia compounds. Using the LOM plaque assay, we identified nitrobenzoxadiazole (NBD)-labeled 1-O-methyl-ceramide-C16 as a compound that efficiently inhibits C. trachomatis replication without affecting the viability of the host cell. Further detailed analyses indicate that 1-O-methyl-NBD-ceramide-C16 acts outside the inclusion. Thereby, 1-O-methyl-NBD-ceramide-C16 represents a lead compound for the development of novel anti-Chlamydia drugs and furthermore constitutes an agent to illuminate sphingolipid trafficking pathways in Chlamydia infections.
Assuntos
Antibacterianos/farmacologia , Ceramidas/farmacologia , Chlamydia trachomatis/efeitos dos fármacos , Técnicas de Laboratório Clínico/métodos , Animais , Antibacterianos/uso terapêutico , Transporte Biológico , Linhagem Celular Tumoral , Ceramidas/uso terapêutico , Infecções por Chlamydia/tratamento farmacológico , Descoberta de Drogas/métodos , Células HeLa , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Esfingolipídeos/metabolismoRESUMO
The distinctive and unique features of the avian and mammalian zoonotic pathogen Chlamydia (C.) psittaci include the fulminant course of clinical disease, the remarkably wide host range and the high proportion of latent infections that are not leading to overt disease. Current knowledge on associated diseases is rather poor, even in comparison to other chlamydial agents. In the present paper, we explain and summarize the major findings of a national research network that focused on the elucidation of host-pathogen interactions in vitro and in animal models of C. psittaci infection, with the objective of improving our understanding of genomics, pathology, pathophysiology, molecular pathogenesis and immunology, and conceiving new approaches to therapy. We discuss new findings on comparative genome analysis, the complexity of pathophysiological interactions and systemic consequences, local immune response, the role of the complement system and antigen presentation pathways in the general context of state-of-the-art knowledge on chlamydial infections in humans and animals and single out relevant research topics to fill remaining knowledge gaps on this important yet somewhat neglected pathogen.
Assuntos
Chlamydophila psittaci/genética , Chlamydophila psittaci/imunologia , Interações Hospedeiro-Patógeno , Patologia Clínica , Psitacose/imunologia , Psitacose/patologia , Animais , Chlamydophila psittaci/patogenicidade , Modelos Animais de Doenças , Genômica , Humanos , Psitacose/microbiologiaRESUMO
Chlamydiae are obligate intracellular bacterial pathogens that cause trachoma, sexually transmitted diseases and respiratory infections in humans. Fragmentation of the host cell Golgi apparatus (GA) is essential for chlamydial development, whereas the consequences for host cell functions, including cell migration are not well understood. We could show that Chlamydia trachomatis-infected cells display decelerated migration and fail to repopulate monolayer scratch wounds. Furthermore, infected cells lost the ability to reorient the fragmented GA or the microtubule organization centre (MTOC) after a migratory stimulus. Silencing of golgin-84 phenocopied this defect in the absence of the infection. Interestingly, GA stabilization via knockdown of Rab6A and Rab11A improved its reorientation in infected cells and it was fully rescued after inhibition of Golgi fragmentation with WEHD-fmk. These results show that C. trachomatis infection perturbs host cell migration on multiple levels, including the alignment of GA and MTOC.
Assuntos
Movimento Celular , Polaridade Celular , Chlamydia trachomatis/patogenicidade , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Interações Hospedeiro-Patógeno , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Microtúbulos/metabolismoRESUMO
The egress of intracellular bacteria from host cells and cellular tissues is a critical process during the infection cycle. This process is essential for bacteria to spread inside the host and can influence the outcome of an infection. For the obligate intracellular Gram-negative zoonotic bacterium Chlamydia psittaci, little is known about the mechanisms resulting in bacterial egress from the infected epithelium. Here, we describe and characterize Chlamydia-containing spheres (CCSs), a novel and predominant type of non-lytic egress utilized by Chlamydia spp. CCSs are spherical, low-phase contrast structures surrounded by a phosphatidylserine-exposing membrane with specific barrier functions. They contain infectious progeny and morphologically impaired cellular organelles. CCS formation is a sequential process starting with the proteolytic cleavage of a DEVD tetrapeptide-containing substrate that can be detected inside the chlamydial inclusions, followed by an increase in the intracellular calcium concentration of the infected cell. Subsequently, blebbing of the plasma membrane begins, the inclusion membrane destabilizes, and the proteolytic cleavage of a DEVD-containing substrate increases rapidly within the whole infected cell. Finally, infected, blebbing cells detach and leave the monolayer, thereby forming CCS. This sequence of events is unique for chlamydial CCS formation and fundamentally different from previously described Chlamydia egress pathways. Thus, CCS formation represents a major, previously uncharacterized egress pathway for intracellular pathogens that could be linked to Chlamydia biology in general and might influence the infection outcome in vivo.IMPORTANCEHost cell egress is essential for intracellular pathogens to spread within an organism and for host-to-host transmission. Here, we characterize Chlamydia-containing sphere (CCS) formation as a novel and predominant non-lytic egress pathway of the intracellular pathogens Chlamydia psittaci and Chlamydia trachomatis. CCS formation is fundamentally different from extrusion formation, the previously described non-lytic egress pathway of C. trachomatis. CCS formation is a unique sequential process, including proteolytic activity, followed by an increase in intracellular calcium concentration, inclusion membrane destabilization, plasma membrane blebbing, and the final detachment of a whole phosphatidylserine-exposing former host cell. Thus, CCS formation represents an important and previously uncharacterized egress pathway for intracellular pathogens that could possibly be linked to Chlamydia biology, including host tropism, protection from host cell defense mechanisms, or bacterial pathogenicity.
Assuntos
Chlamydophila psittaci , Chlamydophila psittaci/genética , Chlamydophila psittaci/fisiologia , Humanos , Células HeLa , Corpos de Inclusão/microbiologia , Corpos de Inclusão/metabolismo , Membrana Celular/metabolismo , Interações Hospedeiro-Patógeno , Células Epiteliais/microbiologia , Infecções por Chlamydia/microbiologiaRESUMO
BACKGROUND: Regular quality-assured whole-genome sequencing linked to antimicrobial resistance (AMR) and patient metadata is imperative to elucidate the shifting gonorrhoea epidemiology, both nationally and internationally. We aimed to examine the gonococcal population in the European Economic Area (EEA) in 2020, elucidate emerging and disappearing gonococcal lineages associated with AMR and patient metadata, compare with 2013 and 2018 whole-genome sequencing data, and explain changes in gonococcal AMR and gonorrhoea epidemiology. METHODS: In this retrospective genomic surveillance study, we analysed consecutive gonococcal isolates that were collected in EEA countries through the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) in 2020, and made comparisons with Euro-GASP data from 2013 and 2018. All isolates had linked AMR data (based on minimum inhibitory concentration determination) and patient metadata. We performed whole-genome sequencing and molecular typing and AMR determinants were derived from quality-checked whole-genome sequencing data. Links between genomic lineages, AMR, and patient metadata were examined. FINDINGS: 1932 gonococcal isolates collected in 2020 in 21 EEA countries were included. The majority (81·2%, 147 of 181 isolates) of azithromycin resistance (present in 9·4%, 181 of 1932) was explained by the continued expansion of the Neisseria gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) clonal complexes (CCs) 63, 168, and 213 (with mtrD/mtrR promoter mosaic 2) and the novel NG-STAR CC1031 (semi-mosaic mtrD variant 13), associated with men who have sex with men and anorectal or oropharyngeal infections. The declining cefixime resistance (0·5%, nine of 1932) and negligible ceftriaxone resistance (0·1%, one of 1932) was largely because of the progressive disappearance of NG-STAR CC90 (with mosaic penA allele), which was predominant in 2013. No known resistance determinants for novel antimicrobials (zoliflodacin, gepotidacin, and lefamulin) were found. INTERPRETATION: Azithromycin-resistant clones, mainly with mtrD mosaic or semi-mosaic variants, appear to be stabilising at a relatively high level in the EEA. This mostly low-level azithromycin resistance might threaten the recommended ceftriaxone-azithromycin therapy, but the negligible ceftriaxone resistance is encouraging. The decreased genomic population diversity and increased clonality could be explained in part by the COVID-19 pandemic resulting in lower importation of novel strains into Europe. FUNDING: European Centre for Disease Prevention and Control and Örebro University Hospital.
Assuntos
Antibacterianos , Farmacorresistência Bacteriana , Gonorreia , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae , Sequenciamento Completo do Genoma , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Humanos , Estudos Retrospectivos , Europa (Continente)/epidemiologia , Gonorreia/epidemiologia , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Masculino , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Feminino , Adulto , Genoma Bacteriano/genética , Pessoa de Meia-Idade , Adulto Jovem , Genômica , Azitromicina/farmacologia , Azitromicina/uso terapêutico , AdolescenteRESUMO
Fretful novelty: We developed two novel doubly labelled fluorescent ceramide analogues that exhibit significant FRET and undergo hydrolysis by ceramidases. We present a fluorescent sphingolipid FRET probe that allows homogeneous ratiometric determination of enzyme activity in real-time.