RESUMO
At the end of 2022 into early 2023, the UK Health Security Agency reported unusually high levels of scarlet fever and invasive disease caused by Streptococcus pyogenes (StrepA or group A Streptococcus). During this time, we collected and genome-sequenced 341 non-invasive throat and skin S. pyogenes isolates identified during routine clinical diagnostic testing in Sheffield, a large UK city. We compared the data with that obtained from a similar collection of 165 isolates from 2016 to 2017. Numbers of throat-associated isolates collected peaked in early December 2022, reflecting the national scarlet fever upsurge, while skin infections peaked later in December. The most common emm-types in 2022-2023 were emm1 (28.7â%), emm12 (24.9â%) and emm22 (7.7â%) in throat and emm1 (22â%), emm12 (10â%), emm76 (18â%) and emm49 (7â%) in skin. While all emm1 isolates were the M1UK lineage, the comparison with 2016-2017 revealed diverse lineages in other emm-types, including emm12, and emergent lineages within other types including a new acapsular emm75 lineage, demonstrating that the upsurge was not completely driven by a single genotype. The analysis of the capsule locus predicted that only 51â% of throat isolates would produce capsule compared with 78% of skin isolates. Ninety per cent of throat isolates were also predicted to have high NADase and streptolysin O (SLO) expression, based on the promoter sequence, compared with only 56% of skin isolates. Our study has highlighted the value in analysis of non-invasive isolates to characterize tissue tropisms, as well as changing strain diversity and emerging genomic features which may have implications for spillover into invasive disease and future S. pyogenes upsurges.
Assuntos
Infecções Estreptocócicas , Streptococcus pyogenes , Streptococcus pyogenes/genética , Streptococcus pyogenes/classificação , Streptococcus pyogenes/isolamento & purificação , Humanos , Reino Unido , Infecções Estreptocócicas/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Antígenos de Bactérias/genética , Faringe/microbiologia , Escarlatina/microbiologia , Escarlatina/epidemiologia , Proteínas de Transporte/genética , Estreptolisinas/genética , Sequenciamento Completo do Genoma/métodos , Proteínas de Bactérias/genética , Filogenia , Criança , Adulto , NAD+ Nucleosidase/genética , NAD+ Nucleosidase/metabolismo , Pele/microbiologia , Pré-Escolar , MasculinoRESUMO
Epithelial colonization is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilize several host factors to associate with cells, including α5ß1 integrin and heparan sulfate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process, while integrins are only critical for post-adhesion entry into human epithelial cells. Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion, whereas blockade of ß1 integrins did not affect adhesion but did reduce the subsequent internalization of adhered bacteria. CD9 disruption or deletion increased ß1 integrin-mediated internalization, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalization. CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organization to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogs could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. IMPORTANCE Staphylococcus aureus infection is a significant cause of disease and morbidity. Staphylococci utilize multiple adhesion pathways to associate with epithelial cells, including interactions with proteoglycans or ß1 integrins through a fibronectin bridge. Interference with another host protein, tetraspanin CD9, halves staphylococcal adherence to epithelial cells, although CD9 does not interact directly with bacteria. Here, we define the role of CD9 in staphylococcal adherence and uptake, observing that CD9 coordinates syndecan-1, fibronectin, and ß1 integrins to allow efficient staphylococcal infection. Two treatments that disrupt this action are effective and may provide an alternative to antibiotics. We provide insights into the mechanisms that underlie staphylococcal infection of host cells, linking two known adhesion pathways together through CD9 for the first time.
Assuntos
Infecções Estafilocócicas , Sindecana-1 , Humanos , Sindecana-1/genética , Fibronectinas/metabolismo , Adesão Celular , Integrinas , Proteínas de Membrana , Integrina beta1/metabolismo , Heparina , Tetraspaninas , Tetraspanina 29RESUMO
Streptococcus pyogenes is a leading cause of human morbidity and mortality, especially in resource-limited settings. The development of a vaccine against S. pyogenes is a global health priority to reduce the burden of postinfection rheumatic heart disease. To support this, molecular characterization of circulating S. pyogenes isolates is needed. We performed whole-genome analyses of S. pyogenes isolates from skin and soft tissue infections in Sukuta, The Gambia, a low-income country (LIC) in West Africa where there is a high burden of such infections. To act as a comparator to these LIC isolates, skin infection isolates from Sheffield, United Kingdom (a high-income country [HIC]), were also sequenced. The LIC isolates from The Gambia were genetically more diverse (46 emm types in 107 isolates) than the HIC isolates from Sheffield (23 emm types in 142 isolates), with only 7 overlapping emm types. Other molecular markers were shared, including a high prevalence of the skin infection-associated emm pattern D and the variable fibronectin-collagen-T antigen (FCT) types FCT-3 and FCT-4. Fewer of the Gambian LIC isolates carried prophage-associated superantigens (64%) and DNases (26%) than did the Sheffield HIC isolates (99% and 95%, respectively). We also identified streptococcin genes unique to 36% of the Gambian LIC isolates and a higher prevalence (48%) of glucuronic acid utilization pathway genes in the Gambian LIC isolates than in the Sheffield HIC isolates (26%). Comparison to a wider collection of HIC and LIC isolate genomes supported our findings of differing emm diversity and prevalence of bacterial factors. Our study provides insight into the genetics of LIC isolates and how they compare to HIC isolates. IMPORTANCE The global burden of rheumatic heart disease (RHD) has triggered a World Health Organization response to drive forward development of a vaccine against the causative human pathogen Streptococcus pyogenes. This burden stems primarily from low- and middle-income settings where there are high levels of S. pyogenes skin and soft tissue infections, which can lead to RHD. Our study provides much needed whole-genome-based molecular characterization of isolates causing skin infections in Sukuta, The Gambia, a low-income country (LIC) in West Africa where infection and RHD rates are high. Although we identified a greater level of diversity in these LIC isolates than in isolates from Sheffield, United Kingdom (a high-income country), there were some shared features. There were also some features that differed by geographical region, warranting further investigation into their contribution to infection. Our study has also contributed data essential for the development of a vaccine that would target geographically relevant strains.
Assuntos
Cardiopatia Reumática , Infecções dos Tecidos Moles , Infecções Estreptocócicas , Humanos , Streptococcus pyogenes/genética , Infecções dos Tecidos Moles/epidemiologia , Infecções Estreptocócicas/microbiologia , Antígenos de Bactérias , GenômicaRESUMO
Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The abundance of genetically distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made the development of virus-specific treatments challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical TLR7 agonist suppressed both the local and subsequent systemic course of infection with a variety of arboviruses from the Alphavirus, Flavivirus, and Orthobunyavirus genera. Clinical outcome was improved in mice after infection with a model alphavirus. In the absence of treatment, antiviral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, stimulating the more populous skin-resident macrophages with a TLR7 agonist elicited protective responses in key cellular targets of virus that otherwise proficiently replicated virus. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have identified a putative new strategy for limiting disease after infection with a variety of genetically distinct arboviruses.
Assuntos
Infecções por Arbovirus/imunologia , Infecções por Arbovirus/metabolismo , Arbovírus/imunologia , Arbovírus/patogenicidade , Macrófagos/metabolismo , Pele/citologia , Alphavirus/imunologia , Alphavirus/patogenicidade , Animais , Flavivirus/imunologia , Flavivirus/patogenicidade , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Orthobunyavirus/imunologia , Orthobunyavirus/patogenicidade , Receptor 7 Toll-Like/metabolismoRESUMO
Chikungunya virus (CHIKV) is a re-emerging, pathogenic alphavirus that is transmitted to humans by Aedes spp. mosquitoes-causing fever and debilitating joint pain, with frequent long-term health implications and high morbidity. The CHIKV lifecycle is poorly understood and specific antiviral therapeutics or vaccines are lacking. In this study, we investigated the role of host-cell chloride (Cl-) channels on CHIKV replication.We demonstrate that specific pharmacological Cl- channel inhibitors significantly inhibit CHIKV replication in a dose-dependent manner, suggesting that Cl-channels are pro-viral factors in human cells. Further analysis of the effect of the inhibitors on CHIKV attachment, entry, viral protein expression and replicon replication demonstrated that Cl- channels are specifically required for efficient CHIKV genome replication. This was conserved in mosquito cells, where CHIKV replication and genome copy number was significantly reduced following Cl- channel inhibition. siRNA silencing identified chloride intracellular channels 1 and 4 (CLIC1 and CLIC4, respectively) as required for efficient CHIKV replication and protein affinity chromatography showed low levels of CLIC1 in complex with CHIKV nsP3, an essential component of the viral replication machinery. In summary, for the first time we demonstrate that efficient replication of the CHIKV genome depends on cellular Cl- channels, in both human and mosquito cells and identifies CLIC1 and CLIC4 as agonists of CHIKV replication in human cells. We observe a modest interaction, either direct or indirect, between CLIC1 and nsP3 and hypothesize that CLIC1 may play a role in the formation/maintenance of CHIKV replication complexes. These findings advance our molecular understanding of CHIKV replication and identify potential druggable targets for the treatment and prevention of CHIKV mediated disease.
Assuntos
Febre de Chikungunya/metabolismo , Vírus Chikungunya/fisiologia , Canais de Cloreto/metabolismo , Genoma Viral , Replicação Viral , Aedes/genética , Aedes/metabolismo , Aedes/virologia , Animais , Febre de Chikungunya/genética , Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Canais de Cloreto/genética , Interações Hospedeiro-Parasita , Humanos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Chikungunya virus (CHIKV) is a re-emerging, pathogenic Alphavirus transmitted to humans by Aedes spp. mosquitoes. We have mapped the RNA structure of the 5' region of the CHIKV genome using selective 2'-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, we identified six RNA replication elements essential to efficient CHIKV genome replication - including novel elements, either not previously analysed in other alphaviruses or specific to CHIKV. Importantly, through a reverse genetic approach we demonstrate that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome. Comparative analysis in human and mosquito-derived cell lines reveal that a novel element within the 5'UTR is essential for efficient replication in both host systems, while those in the adjacent nsP1 encoding region are specific to either vertebrate or invertebrate host cells. In addition to furthering our knowledge of fundamental aspects of the molecular virology of this important human pathogen, we foresee that results from this study will be important for rational design of a genetically stable attenuated vaccine.
Assuntos
Febre de Chikungunya/genética , Vírus Chikungunya/genética , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Aedes/virologia , Animais , Febre de Chikungunya/virologia , Vírus Chikungunya/patogenicidade , Genoma Viral/genética , Humanos , RNA Viral/genética , Replicon/genéticaRESUMO
BACKGROUND: Anaemia in pregnancy is linked with an increased risk of preterm delivery, low birth weight, perinatal and maternal mortality and it is related with variable histo-morphological changes in placenta which show a reflection for the poor foetal outcome. The objective of this study is to assess the micro-morphometery of intervillous space and villous membrane of placenta in anaemic mothers. METHODS: This case control study was carried out at Army Medical College, National University of Sciences and Technology Islamabad in collaboration with Department of Obstetrics and Gynaecology, Military Hospital, Rawalpindi, Pakistan, from December 2011 to November 2012. A total of 75 placentas were included, that were divided into study and control group. In control group (n = 15) placentas were taken from mothers having normal haemoglobin levels and study group (n = 60) included placentas from anaemic mothers having haemoglobin less than 11g/dl. Study group was subdivided into three groups, i.e., mildly (10.0-10.9 g/dl), moderately (7.0-9.9 g/dl) and severely (< 7 g/dl) anaemic group. Three representative sections were taken from placenta, i.e., one close to umbilical cord (A), one from periphery (C) and one midway between A and C (B).Ocular micro-meter was used to measure intervillous space and villous membrane thickness. RESULTS: Intervillous space was prominent in study groups (41.26 ± 16.33 ìm) as compared to control group (15.98 ± 3.81 ìm) (p < 0.05). Thickness of villous membrane was significantly less in study group as compared to control group (2.97 ± 0.70) (p < 0.05). CONCLUSION: The present study showed wide intervillous space and thin villous membrane in study group as compared to control group reflecting adaptive changes in response to hypoxiain maternal anaemia.