A quest for novel antimicrobial targets: Inhibition of Asp-tRNAAsn/Glu-tRNAGln amidotransferase (GatCAB) by synthetic analogs of aminoacyl-adenosine in vitro and live bacteria.

The Asp-tRNAAsn/Glu-tRNAGln amidotransferase (GatCAB) has been proposed as a novel antibacterial drug target due to its indispensability in prominent human pathogens. While several inhibitors with in vitro activity have been identified, none have been demonstrated to have potent activity against live bacteria. In this work, seven non-hydrolyzable transition state mimics of GatCAB were synthesized and tested as the transamidase inhibitors against GatCAB from the human pathogen Helicobacter pylori. Notably, the methyl sulfone analog of glutamyl-adenosine significantly reduced GatCAB's transamination rate. Additionally, four lipid-conjugates of these mimics displayed antibacterial activity against Bacillus subtilis, likely due to enhanced cell permeability. Inhibitory activity against GatCAB in live bacteria was confirmed using a sensitive gain-of-function dual luciferase reporter in Mycobacterium bovis-BCG. Only the lipid-conjugated methyl sulfone analog exhibited a significant increase in mistranslation rate, highlighting its cell permeability and inhibitory potential. This study provides insights for developing urgently needed novel antibacterial agents amidst emerging antimicrobial drug resistance.

A teenage girl with altered mental status and paraparesis.

A teenage girl presented with fever and altered mental status. MRI showed diffuse leptomeningeal enhancement of the brain and spine. She was diagnosed by a positive cerebrospinal fluid (CSF) culture with tuberculous (TB) meningitis and was started on anti-TB medications and corticosteroids. Her mental status improved, but she was noted to have proximal weakness of the lower extremities. In the course of tapering corticosteroids at week 11 of anti-TB therapy, she became acutely confused and febrile. MRI demonstrated interval development of tuberculomas in the brain and a mass lesion in the thoracic spine causing cord compression. Given the clinical picture was suggestive of a paradoxical reaction, the dose of corticosteroids was increased. Infliximab was added when repeat MRI revealed enlargement of the mass lesion in the spine with worsening cord compression. She was successfully tapered off of corticosteroids. Over several months, the patient's motor function recovered fully, and she returned to ambulating without assistance.

Mechanical morphotype switching as an adaptive response in mycobacteria.

Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report the discovery of a virulence mechanism by which changes to the mechanical stiffness of the mycobacterial cell surface confer refraction to killing during infection. Long-term time-lapse atomic force microscopy was used to reveal a process of "mechanical morphotype switching" in mycobacteria exposed to host intracellular stress. A "soft" mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Both pharmacologic treatment, with bedaquiline, and a genetic mutant lacking uvrA modified the basal mechanical state of mycobacteria into a soft mechanical morphotype, enhancing survival in macrophages. Our study proposes microbial cell mechanical adaptation as a critical axis for surviving host-mediated stressors.

Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates: an in-vitro longitudinal study.

Antibiotic tolerance in Mycobacterium tuberculosis leads to less effective bacterial killing, poor treatment responses and resistant emergence. There is limited understanding of antibiotic tolerance in clinical isolates of M. tuberculosis. Therefore, we investigated the rifampicin tolerance of M. tuberculosis isolates, with or without pre-existing isoniazid-resistance. In-vitro rifampicin survival fractions determined by minimum duration of killing assay in isoniazid susceptible (n=119) and resistant (n=84) M. tuberculosis isolates. Rifampicin tolerance was correlated with bacterial growth, rifampicin minimum inhibitory concentrations (MICs) and isoniazid-resistant mutations. The longitudinal isoniazid-resistant isolates were analyzed for rifampicin tolerance based on collection time from patients and associated emergence of genetic variants. The median duration of rifampicin exposure reducing the M. tuberculosis surviving fraction by 90% (minimum duration of killing-MDK90) increased from 1.23 (95%CI 1.11; 1.37) and 1.31 (95%CI 1.14; 1.48) to 2.55 (95%CI 2.04; 2.97) and 1.98 (95%CI 1.69; 2.56) days, for IS and IR respectively, during 15 to 60 days of incubation respectively. Increase in MDK90 time indicated the presence of fast and slow growing tolerant sub-populations. A range of 6 log10-fold survival fraction enabled classification of tolerance as low, medium or high and revealed isoniazid-resistance association with increased tolerance with faster growth (OR=2.68 for low vs. medium, OR=4.42 for low vs. high, P-trend=0.0003). The high tolerance in longitudinal isoniazid-resistant isolates was specific to those collected during rifampicin treatment in patients and associated with bacterial genetic microvariants. Our study identifies a range of rifampicin tolerance and reveals that isoniazid resistance is associated with higher tolerance with growth fitness. Furthermore, rifampicin treatment may select isoniazid-resistant isolate microvariants with higher rifampicin tolerance, with survival potential similar to multi-drug resistant isolates. These findings suggest that isoniazid-resistant tuberculosis needs to be evaluated for rifampicin tolerance or needs further improvement in treatment regimen. It is made available under a CC-BY 4.0 International license.

An anti-LpqH human monoclonal antibody from an asymptomatic individual mediates protection against Mycobacterium tuberculosis.

Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (Mtb). Whilst a functional role for humoral immunity in Mtb protection remains poorly defined, previous studies have suggested that antibodies can contribute towards host defense. Thus, identifying the critical components in the antibody repertoires from immune, chronically exposed, healthy individuals represents an approach for identifying new determinants for natural protection. In this study, we performed a thorough analysis of the IgG/IgA memory B cell repertoire from occupationally exposed, immune volunteers. We detail the identification and selection of a human monoclonal antibody that exhibits protective activity in vivo and show that it targets a virulence factor LpqH. Intriguingly, protection in both human ex vivo and murine challenge experiments was isotype dependent, with most robust protection being mediated via IgG2 and IgA. These data have important implications for our understanding of natural mucosal immunity for Mtb and highlight a new target for future vaccine development.

Navigating a Path to Rifampicin Resistance in Tuberculosis.

For model bacteria, genetic drug resistance usually arises from antibiotic-tolerant subpopulations, but whether this is true for the globally important pathogen Mycobacterium tuberculosis-the cause of tuberculosis-is not known. Here, we discuss a recent article by Sebastian et al. (J. Sebastian, A. Thomas, C. Levine, R. Shrestha, et al., mBio 14:e0279522, 2023, 10.1128/mbio.02795-22) which leverages a robotic transwell microtiter experimental system coupled with deep sequencing of a barcoded library of M. tuberculosis to answer this question for rifampicin resistance. The authors investigate two distinct forms of antibiotic-tolerant subpopulations-classical tolerance, characterized by prolonged minimum duration of killing, and "differentially detectable" (DD) bacilli that are viable but can be recovered only in liquid medium as opposed to plating. They demonstrate that, indeed, resistance arises preferentially from both rifampicin-tolerant subpopulations, though earlier in the DD population. Use of barcoded libraries and parallel culture systems shows promise in investigating phenotypes mediated by minority subpopulations of bacteria such as development of antibiotic resistance.

Therapeutic Vaccines for Tuberculosis: An Overview.

Tuberculosis (TB), caused by Mycobacterium tuberculosis is the world's deadliest bacterial infection, resulting in more than 1.4 million deaths annually. The emergence of drug-resistance to first-line antibiotic therapy poses a threat to successful treatment, and novel therapeutic options are required, particularly for drug-resistant tuberculosis. One modality emerging for TB treatment is therapeutic vaccination. As opposed to preventative vaccination - the aim of which is to prevent getting infected by M. tuberculosis or developing active tuberculosis, the purpose of therapeutic vaccination is as adjunctive treatment of TB or to prevent relapse following cure. Several candidate therapeutic vaccines, using killed whole-cell or live attenuated mycobacteria, mycobacterial fragments and viral vectored vaccines are in current clinical trials. Other modes of passive immunization, including monoclonal antibodies directed against M. tuberculosis antigens are in various pre-clinical stages of development. Here, we will discuss these various therapeutics and their proposed mechanisms of action. Although the full clinical utility of therapeutic vaccination for the treatment of tuberculosis is yet to be established, they hold potential as useful adjunct therapies.

Harnessing Twitter data to survey public attention and attitudes towards COVID-19 vaccines in the UK.

Attitudes to COVID-19 vaccination vary considerably within and between countries. Although the contribution of socio-demographic factors to these attitudes has been studied, the role of social media and how it interacts with news about vaccine development and efficacy is uncertain. We examined around 2 million tweets from 522,893 persons in the UK from November 2020 to January 2021 to evaluate links between Twitter content about vaccines and major scientific news announcements about vaccines. The proportion of tweets with negative vaccine content varied, with reductions of 20-24% on the same day as major news announcement. However, the proportion of negative tweets reverted back to an average of around 40% within a few days. Engagement rates were higher for negative tweets. Public health messaging could consider the dynamics of Twitter-related traffic and the potential contribution of more targeted social media campaigns to address vaccine hesitancy.

Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity.

Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampin tolerance. However, the mechanisms by which these clinically derived mutations in gatA impact GatCAB function were unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity. IMPORTANCE Most bacteria use a two-step indirect pathway to aminoacylate tRNAGln and tRNAAsn, despite the fact that the indirect pathway consumes more energy and is error prone. We have previously shown that the higher protein synthesis errors from this indirect pathway in mycobacteria allow adaptation to hostile environments such as antibiotic treatment through generation of novel alternate proteins not coded by the genome. However, the precise mechanisms of how translational fidelity is tuned were not known. Here, we biochemically and biophysically characterize the critical enzyme of the Mycobacterium tuberculosis indirect pathway, GatCAB, as well as two mutant enzymes previously identified from clinical isolates that were associated with increased mistranslation. We show that the mutants dysregulate the pathway via destabilizing the enzyme complex. Importantly, increasing stability improves translational fidelity in both wild-type and mutant bacteria, demonstrating a mechanism by which mycobacteria may tune mistranslation rates.

Direct and indirect evidence of efficacy and safety of rapid exercise tests for exertional desaturation in Covid-19: a rapid systematic review.

BACKGROUND: Even when resting pulse oximetry is normal in the patient with acute Covid-19, hypoxia can manifest on exertion. We summarise the literature on the performance of different rapid tests for exertional desaturation and draw on this evidence base to provide guidance in the context of acute Covid-19. MAIN RESEARCH QUESTIONS: 1. What exercise tests have been used to assess exertional hypoxia at home or in an ambulatory setting in the context of Covid-19 and to what extent have they been validated? 2. What exercise tests have been used to assess exertional hypoxia in other lung conditions, to what extent have they been validated and what is the applicability of these studies to acute Covid-19? METHOD: AMED, CINAHL, EMBASE MEDLINE, Cochrane and PubMed using LitCovid, Scholar and Google databases were searched to September 2020. Studies where participants had Covid-19 or another lung disease and underwent any form of exercise test which was compared to a reference standard were eligible. Risk of bias was assessed using QUADAS 2. A protocol for the review was published on the Medrxiv database. RESULTS: Of 47 relevant papers, 15 were empirical studies, of which 11 described an attempt to validate one or more exercise desaturation tests in lung diseases other than Covid-19. In all but one of these, methodological quality was poor or impossible to fully assess. None had been designed as a formal validation study (most used simple tests of correlation). Only one validation study (comparing a 1-min sit-to-stand test [1MSTST] with reference to the 6-min walk test [6MWT] in 107 patients with interstitial lung disease) contained sufficient raw data for us to calculate the sensitivity (88%), specificity (81%) and positive and negative predictive value (79% and 89% respectively) of the 1MSTST. The other 4 empirical studies included two predictive studies on patients with Covid-19, and two on HIV-positive patients with suspected pneumocystis pneumonia. We found no studies on the 40-step walk test (a less demanding test that is widely used in clinical practice to assess Covid-19 patients). Heterogeneity of study design precluded meta-analysis. DISCUSSION: Exertional desaturation tests have not yet been validated in patients with (or suspected of having) Covid-19. A stronger evidence base exists for the diagnostic accuracy of the 1MSTST in chronic long-term pulmonary disease; the relative intensity of this test may raise safety concerns in remote consultations or unstable patients. The less strenuous 40-step walk test should be urgently evaluated.

Human antibodies targeting a Mycobacterium transporter protein mediate protection against tuberculosis.

Mycobacterium tuberculosis (Mtb) exposure drives antibody responses, but whether patients with active tuberculosis elicit protective antibodies, and against which antigens, is still unclear. Here we generate monoclonal antibodies from memory B cells of one patient to investigate the B cell responses during active infection. The antibodies, members of four distinct B cell clones, are directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 reduce Mycobacterium bovis-BCG and Mtb levels in an ex vivo human whole blood growth inhibition assay in an FcR-dependent manner; meanwhile, germline versions of p4-36 and p4-163 do not bind Mtb. Crystal structures of p4-36 and p4-170, complexed to PstS1, are determined at 2.1 Å and 2.4 Å resolution, respectively, to reveal two distinctive PstS1 epitopes. Lastly, a prophylactic p4-36 and p4-163 treatment in Mtb-infected Balb/c mice reduces bacterial lung burden by 50%. Our study shows that inhibitory anti-PstS1 B cell responses arise during active tuberculosis.

Most-Probable-Number-Based Minimum Duration of Killing Assay for Determining the Spectrum of Rifampicin Susceptibility in Clinical Mycobacterium tuberculosis Isolates.

Accurate antibiotic susceptibility testing is essential for successful tuberculosis treatment. Recent studies have highlighted the limitations of MIC-based phenotypic susceptibility methods in detecting other aspects of antibiotic susceptibilities in bacteria. Duration and peak of antibiotic exposure, at or above the MIC required for killing the bacterial population, has emerged as another important factor for determining antibiotic susceptibility. This is broadly defined as antibiotic tolerance. Antibiotic tolerance can further facilitate the emergence of antibiotic resistance. Currently, there are limited methods to quantify antibiotic tolerance among clinical M. tuberculosis isolates. In this study, we develop a most-probable-number (MPN)-based minimum duration of killing (MDK) assay to quantify the spectrum of M. tuberculosis rifampicin susceptibility within subpopulations based on the duration of rifampicin exposure required for killing the bacterial population. MDK90-99 and MDK99.99 were defined as the minimum duration of antibiotic exposure at or above the MIC required for killing 90 to 99% and 99.99% of the initial (pretreatment) bacterial population, respectively. Results from the rifampicin MDK assay applied to 28 laboratory and clinical M. tuberculosis isolates showed that there is variation in rifampicin susceptibility among isolates. The rifampicin MDK99/99.99 time for isolates varied from less than 2 to 10 days. MDK was correlated with larger subpopulations of M. tuberculosis from clinical isolates that were rifampicin tolerant. Our study demonstrates the utility of MDK assays to measure the variation in antibiotic tolerance among clinical M. tuberculosis isolates and further expands clinically important aspects of antibiotic susceptibility testing.

Managing outbreaks of highly contagious diseases in prisons: a systematic review.

BACKGROUND: There are reports of outbreaks of COVID-19 in prisons in many countries. Responses to date have been highly variable and it is not clear whether public health guidance has been informed by the best available evidence. We conducted a systematic review to synthesise the evidence on outbreaks of highly contagious diseases in prison. METHODS: We searched seven electronic databases for peer-reviewed articles and official reports published between 1 January 2000 and 28 July 2020. We included quantitative primary research that reported an outbreak of a given contagious disease in a correctional facility and examined the effects of interventions. We excluded studies that did not provide detail on interventions. We synthesised common themes using the Synthesis Without Meta-analysis (SWiM) guideline, identified gaps in the literature and critically appraised the effectiveness of various containment approaches. RESULTS: We identified 28 relevant studies. Investigations were all based in high-income countries and documented outbreaks of tuberculosis, influenza (types A and B), varicella, measles, mumps, adenovirus and COVID-19. Several themes were common to these reports, including the public health implications of infectious disease outbreaks in prison, and the role of interagency collaboration, health communication, screening for contagious diseases, restriction, isolation and quarantine, contact tracing, immunisation programmes, epidemiological surveillance and prison-specific guidelines in addressing any outbreaks. DISCUSSION: Prisons are high-risk settings for the transmission of contagious diseases and there are considerable challenges in managing outbreaks in them. A public health approach to managing COVID-19 in prisons is required. PROSPERO REGISTRATION NUMBER: CRD42020178827.

Forward Genetics Reveals a gatC-gatA Fusion Polypeptide Causes Mistranslation and Rifampicin Tolerance in Mycobacterium smegmatis.

Most bacteria, including mycobacteria, utilize a two-step indirect tRNA aminoacylation pathway to generate correctly aminoacylated glutaminyl and asparaginyl tRNAs. This involves an initial step in which a non-discriminatory aminoacyl tRNA synthetase misacylates the tRNA, followed by a second step in which the essential amidotransferase, GatCAB, amidates the misacylated tRNA to its correct, cognate form. It had been previously demonstrated that mutations in gatA can mediate increased error rates specifically of glutamine to glutamate or asparagine to aspartate in protein synthesis. However, the role of mutations in gatB or gatC in mediating mistranslation are unknown. Here, we applied a forward genetic screen to enrich for mistranslating mutants of Mycobacterium smegmatis. The majority (57/67) of mutants had mutations in one of the gatCAB genes. Intriguingly, the most common mutation identified was an insertion in the 3' of gatC, abolishing its stop codon, and resulting in a fused GatC-GatA polypeptide. Modeling the effect of the fusion on GatCAB structure suggested a disruption of the interaction of GatB with the CCA-tail of the misacylated tRNA, suggesting a potential mechanism by which this mutation may mediate increased translational errors. Furthermore, we confirm that the majority of mutations in gatCAB that result in increased mistranslation also cause increased tolerance to rifampicin, although there was not a perfect correlation between mistranslation rates and degree of tolerance. Overall, our study identifies that mutations in all three gatCAB genes can mediate adaptive mistranslation and that mycobacteria are extremely tolerant to perturbation in the indirect tRNA aminoacylation pathway.

Selective translation by alternative bacterial ribosomes.

Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of M. smegmatis in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.

ADA2 deficiency complicated by EBV-driven lymphoproliferative disease.

A 29-year old male with recurrent respiratory and skin infections, anaemia and neutropaenia during childhood required immunoglobulin replacement for antibody deficiency from age 16. He remained relatively well until age 28 when he presented with a two-week history of fatigue, sore throat, fever and productive cough. He was found to have EBV viraemia and splenomegaly and a diagnosis of EBV-driven lymphoproliferative disease was made following bone marrow trephine. Family history was notable with three siblings: a healthy sister and two brothers with anaemia and neutropaenia; one who succumbed to septicaemia secondary to neutropaenic enterocolitis age 5 and another who developed intestinal vasculitis and antibody deficiency and had a successful haemopoetic stem cell transplant. The proband's DNA underwent targeted sequencing of 279 genes associated with immunodeficiency (GRID panel). The best candidates were two ADA2 variants, p.Arg169Gln (R169Q) and p.Asn370Lys (N370K). Sanger sequencing and co-segregation of variants in the parents, unaffected sister and all three affected brothers was fully consistent with compound heterozygous inheritance. Subsequent whole genome sequencing of the proband identified no other potential causal variants. ADA2 activity was consistent with a diagnosis of ADA2 deficiency in affected family members. This is the first description of EBV-driven lymphoproliferative disease in ADA2 deficiency. ADA2 deficiency may cause susceptibility to severe EBV-induced disease and we would recommend that EBV status and viral load is monitored in patients with this diagnosis and allogeneic SCT is considered at an early stage for patients whose ADA2 deficiency is associated with significant complications.

Premature termination codon readthrough in Drosophila varies in a developmental and tissue-specific manner.

Despite their essential function in terminating translation, readthrough of stop codons occurs more frequently than previously supposed. However, little is known about the regulation of stop codon readthrough by anatomical site and over the life cycle of animals. Here, we developed a set of reporters to measure readthrough in Drosophila melanogaster. A focused RNAi screen in whole animals identified upf1 as a mediator of readthrough, suggesting that the stop codons in the reporters were recognized as premature termination codons (PTCs). We found readthrough rates of PTCs varied significantly throughout the life cycle of flies, being highest in older adult flies. Furthermore, readthrough rates varied dramatically by tissue and, intriguingly, were highest in fly brains, specifically neurons and not glia. This was not due to differences in reporter abundance or nonsense-mediated mRNA decay (NMD) surveillance between these tissues. Readthrough rates also varied within neurons, with cholinergic neurons having highest readthrough compared with lowest readthrough rates in dopaminergic neurons. Overall, our data reveal temporal and spatial variation of PTC-mediated readthrough in animals, and suggest that readthrough may be a potential rescue mechanism for PTC-harboring transcripts when the NMD surveillance pathway is inhibited.