Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia ...from March to August 2020. We show that two consecutive mutations (R203K/G204R) in the nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found increased interaction of GSK3A kinase simultaneously with hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein. Furthermore, the host cell transcriptome analysis suggests that the mutant N protein produces dysregulated interferon response genes. Here, we provide crucial information in linking the R203K/G204R mutations in the N protein to modulations of host-virus interactions and underline the potential of the nucleocapsid protein as a drug target during infection.
Objective
The Saudi government requires that all pilgrims receive a quadrivalent meningococcal vaccine at least 10 days before the Hajj. We conducted a study to determine the uptake of meningococcal ...vaccine and antibiotic use. We also investigated risk factors of meningococcal carriage and carriage of Neisseria meningitidis pathogenic serogroups A, C, W and Y.
Methods
A cross‐sectional oropharyngeal carriage survey was conducted in 2973 Hajj pilgrims in September 2017. A real‐time polymerase chain reaction (rt‐PCR) assay was used to identify N. meningitidis from the oropharyngeal swabs. A questionnaire investigated potential risk factors for carriage of N. meningitidis.
Results
Two thousand two hundred forty nine oropharyngeal swabs were obtained. The overall prevalence of carriage of N. meningitidis was 4.6% (95% CI: 3.4%–6%). Carriage of pathogenic serogroups was not associated significantly with any of the meningococcal risk factors evaluated. 77% of pilgrims were vaccinated but 22.58 % said they were carrying unofficial vaccination cards.
Conclusion
Carriage of serogroups A, C, W and Y was not significantly associated with any of the risk factors investigated. Almost a quarter of pilgrims were unlikely to have been vaccinated, highlighting a need to strengthen compliance with the current policy of vaccination to prevent meningococcal disease outbreaks during and after the Hajj.
Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 h suggesting a circadian basis, but the mechanism controlling this periodicity is unknown. Combining ...in vivo and in vitro approaches utilizing rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit daily rhythms in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 6% of Plasmodium falciparum genes show 24 h rhythms in expression under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a 24 h transcriptional rhythm and disrupting it in rodent malaria parasites shortens the IDC by 2-3 h; (v) Multiple processes including DNA replication, and the ubiquitin and proteasome pathways, are affected by loss of coordination with host rhythms and by disruption of SR10. Our results reveal malaria parasites are at least partly responsible for scheduling the IDC and coordinating their development with host daily rhythms.
The final step during cell division is the separation of daughter cells, a process that requires the coordinated delivery and assembly of new membrane to the cleavage furrow. While most eukaryotic ...cells replicate by binary fission, replication of apicomplexan parasites involves the assembly of daughters (merozoites/tachyzoites) within the mother cell, using the so-called Inner Membrane Complex (IMC) as a scaffold. After de novo synthesis of the IMC and biogenesis or segregation of new organelles, daughters bud out of the mother cell to invade new host cells. Here, we demonstrate that the final step in parasite cell division involves delivery of new plasma membrane to the daughter cells, in a process requiring functional Rab11A. Importantly, Rab11A can be found in association with Myosin-Tail-Interacting-Protein (MTIP), also known as Myosin Light Chain 1 (MLC1), a member of a 4-protein motor complex called the glideosome that is known to be crucial for parasite invasion of host cells. Ablation of Rab11A function results in daughter parasites having an incompletely formed IMC that leads to a block at a late stage of cell division. A similar defect is observed upon inducible expression of a myosin A tail-only mutant. We propose a model where Rab11A-mediated vesicular traffic driven by an MTIP-Myosin motor is necessary for IMC maturation and to deliver new plasma membrane to daughter cells in order to complete cell division.
The emergence of programmatically incurable tuberculosis threatens to destabilise control efforts. The aim of this study was to collect prospective patient-level data to inform treatment and ...containment strategies.
In a prospective cohort study, 273 South African patients with extensively drug-resistant tuberculosis, or resistance beyond extensively drug-resistant tuberculosis, were followed up over a period of 6 years. Transmission dynamics, infectiousness, and drug susceptibility were analysed in a subset of patients from the Western Cape using whole-genome sequencing (WGS; n=149), a cough aerosol sampling system (CASS; n=26), and phenotypic testing for 18 drugs (n=179).
Between Oct 1, 2008, and Oct 31, 2012, we enrolled and followed up 273 patients for a median of 20·3 months (IQR 9·6-27·8). 203 (74%) had programmatically incurable tuberculosis and unfavourable outcomes (treatment failure, relapse, default, or death despite treatment with a regimen based on capreomycin, aminosalicylic acid, or both). 172 (63%) patients were discharged home, of whom 104 (60%) had an unfavourable outcome. 54 (31%) home-discharged patients had failed treatment, with a median time to death after discharge of 9·9 months (IQR 4·2-17·4). 35 (20%) home-discharged cases were smear-positive at discharge. Using CASS, six (23%) of 26 home-discharged cases with data available expectorated infectious culture-positive cough aerosols in the respirable range (<5 μm), and most reported inter-person contact with suboptimal protective mask usage. WGS identified 17 (19%) of the 90 patients (with available sequence data) that were discharged home before the diagnosis of 20 downstream cases of extensively drug-resistant tuberculosis with almost identical sequencing profiles suggestive of community-based transmission (five or fewer single nucleotide polymorphisms different and with identical resistance-encoding mutations for 14 drugs). 11 (55%) of these downstream cases had HIV co-infection and ten (50%) had died by the end of the study. 22 (56%) of 39 isolates in patients discharged home after treatment failure were resistant to eight or more drugs. However, five (16%) of 31 isolates were susceptible to rifabutin and more than 90% were likely to be sensitive to linezolid, bedaquiline, and delamanid.
More than half of the patients with programmatically incurable tuberculosis were discharged into the community where they remained for an average of 16 months, were at risk of expectorating infectious cough aerosols, and posed a threat of transmission of extensively drug-resistant tuberculosis. Urgent action, including appropriate containment strategies, is needed to address this situation. Access to delamanid, bedaquiline, linezolid, and rifabutin, when appropriate, must be accelerated along with comprehensive drug susceptibility testing.
UK Medical Research Council, South African Medical Research Council, South African National Research Foundation, European & Developing Countries Clinical Trials Partnership, Oppenheimer Foundation, Newton Fund, Biotechnology and Biological Sciences Research Council, King Abdullah University of Science & Technology.
Theileria annulata is a tick‐transmitted apicomplexan parasite that infects and transforms bovine leukocytes into disseminating tumours that cause a disease called tropical theileriosis. Using ...comparative transcriptomics we identified genes transcriptionally perturbed during Theileria‐induced leukocyte transformation. Dataset comparisons highlighted a small set of genes associated with Theileria‐transformed leukocyte dissemination. The roles of Granzyme A (GZMA) and RAS guanyl‐releasing protein 1 (RASGRP1) were verified by CRISPR/Cas9‐mediated knockdown. Knocking down expression of GZMA and RASGRP1 in attenuated macrophages led to a regain in their dissemination in Rag2/γC mice confirming their role as dissemination suppressors in vivo. We further evaluated the roles of GZMA and RASGRP1 in human B lymphomas by comparing the transcriptome of 934 human cancer cell lines to that of Theileria‐transformed bovine host cells. We confirmed dampened dissemination potential of human B lymphomas that overexpress GZMA and RASGRP1. Our results provide evidence that GZMA and RASGRP1 have a novel tumour suppressor function in both T. annulata‐infected bovine host leukocytes and in human B lymphomas.
Summarising the main findings of this study that identified new players in dissemination and oxidative stress regulation of Theileria‐transformed leukocytes and provided evidence for similar roles for GZMA and RASGRP1 in transcriptionally matched human B lymphoma cell lines.
Theileria annulata is an apicomplexan parasite that infects and transforms bovine macrophages that disseminate throughout the animal causing a leukaemia-like disease called tropical theileriosis. ...Using deep RNAseq of T. annulata-infected B cells and macrophages we identify a set of microRNAs induced by infection, whose expression diminishes upon loss of the hyper-disseminating phenotype of virulent transformed macrophages. We describe how infection-induced upregulation of miR-126-5p ablates JIP-2 expression to release cytosolic JNK to translocate to the nucleus and trans-activate AP-1-driven transcription of mmp9 to promote tumour dissemination. In non-disseminating attenuated macrophages miR-126-5p levels drop, JIP-2 levels increase, JNK1 is retained in the cytosol leading to decreased c-Jun phosphorylation and dampened AP-1-driven mmp9 transcription. We show that variation in miR-126-5p levels depends on the tyrosine phosphorylation status of AGO2 that is regulated by Grb2-recruitment of PTP1B. In attenuated macrophages Grb2 levels drop resulting in less PTP1B recruitment, greater AGO2 phosphorylation, less miR-126-5p associated with AGO2 and a consequent rise in JIP-2 levels. Changes in miR-126-5p levels therefore, underpin both the virulent hyper-dissemination and the attenuated dissemination of T. annulata-infected macrophages.
Klebsiella pneumoniae is a major bacterial and opportunistic human pathogen, increasingly recognized as a healthcare burden globally. The convergence of resistance and virulence in K. pneumoniae ...strains has led to the formation of hypervirulent and multidrug-resistant strains with dual risk, limiting treatment options. K. pneumoniae clones are known to emerge locally and spread globally. Therefore, an understanding of the dynamics and evolution of the emerging strains in hospitals is warranted to prevent future outbreaks.
In this study, we conducted an in-depth genomic analysis on a large-scale collection of 328 multidrug-resistant (MDR) K. pneumoniae strains recovered from 239 patients from a single major hospital in the western coastal city of Jeddah in Saudi Arabia from 2014 through 2022. We employed a broad range of phylogenetic and phylodynamic methods to understand the evolution of the predominant clones on epidemiological time scales, virulence and resistance determinants, and their dynamics. We also integrated the genomic data with detailed electronic health record (EHR) data for the patients to understand the clinical implications of the resistance and virulence of different strains.
We discovered a diverse population underlying the infections, with most strains belonging to Clonal Complex 14 (CC14) exhibiting dominance. Specifically, we observed the emergence and continuous expansion of strains belonging to the dominant ST2096 in the CC14 clade across hospital wards in recent years. These strains acquired resistance mutations against colistin and extended spectrum β-lactamase (ESBL) and carbapenemase genes, namely bla
and bla
, located on three distinct plasmids, on epidemiological time scales. Strains of ST2096 exhibited a high virulence level with the presence of the siderophore aerobactin (iuc) locus situated on the same mosaic plasmid as the ESBL gene. Integration of ST2096 with EHR data confirmed the significant link between colonization by ST2096 and the diagnosis of sepsis and elevated in-hospital mortality (p-value < 0.05).
Overall, these results demonstrate the clinical significance of ST2096 clones and illustrate the rapid evolution of an emerging hypervirulent and MDR K. pneumoniae in a clinical setting.
Background information
The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, ...therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized.
Results
Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt‐interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab‐interactome using putative parasite Rab‐effectors identified by homology to Ypt‐effectors. We demonstrate its potential by wet‐bench testing three predictions; that casein kinase‐1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP‐dependent protein kinase A (PfPKA‐C) is a PfRab5A and PfRab7 effector.
Conclusions
The establishment of a shared set of physical Ypt/PfRab‐effector proteins sheds light on a core set Plasmodium Rab‐interactants shared with yeast. The PfRab‐interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA‐C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab‐recruited kinases potentially play a role in early and late endosome function in malaria parasites.
Rabs are important regulators of vesicular traffic via their recruitment of specific effectors. To identify Rab effectors for the human malaria parasite Plasmodium falciparum we made a yeast Ypt‐interactome and exploited this to build a parasite Rab‐interactome. The kinase PfCK1 was shown to be a specific PfRab5B effector, whereas the catalytic subunit of PfPKA‐C an effector of both PfRab5A and PfRab7. The recruitment of PfPKA to PfRab5A+ and PfRab7+ vesicles suggests that the cAMP‐dependent kinase potentially plays a role in early and late endosome function in malaria parasites.
MicroRNAs (miRNAs) are small noncoding RNAs that can play critical roles in regulating various cellular processes, including during many parasitic infections. Here, we report a regulatory role for ...miR-34c-3p in cAMP-independent regulation of host cell protein kinase A (PKA) activity in
-infected bovine leukocytes. We identified
(cAMP-dependent protein kinase A type II-beta regulatory subunit) as a novel miR-34c-3p target gene and demonstrate how infection-induced upregulation of miR-34c-3p repressed PRKAR2B expression to increase PKA activity. As a result, the disseminating tumorlike phenotype of
-transformed macrophages is enhanced. Finally, we extend our observations to Plasmodium falciparum-parasitized red blood cells, where infection-induced augmentation in miR-34c-3p levels led to a drop in the amount of
mRNA and increased PKA activity. Collectively, our findings represent a novel cAMP-independent way of regulating host cell PKA activity in infections by
and
parasites.
Small microRNA levels are altered in many diseases, including those caused by parasites. Here, we describe how infection by two important animal and human parasites, Theileria annulata and Plasmodium falciparum, induce changes in infected host cell miR-34c-3p levels to regulate host cell PKA kinase activity by targeting mammalian
. Infection-induced changes in miR-34c-3p levels provide a novel epigenetic mechanism for regulating host cell PKA activity independent of fluxes in cAMP to both aggravate tumor dissemination and improve parasite fitness.
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