Alterations in Sarcopenia Index are Associated with Inflammation, Gut and Oral Microbiota among Heart Failure, Left Ventricular Assist Device and Heart Transplant Patients.

BACKGROUND: Sarcopenia, characterized by loss of muscle mass and function, is prevalent in heart failure (HF) and predicts poor outcomes. We investigated alterations in sarcopenia index (SI), a surrogate for skeletal muscle mass, in HF, left ventricular assist device (LVAD) and heart transplant (HT), and assessed its relationship with inflammation and digestive tract (gut and oral) microbiota. METHODS: We enrolled 460 HF, LVAD and HT patients. Repeated measures pre/post procedures were obtained prospectively in a subset of LVAD and HT patients. SI (serum Creatinine/Cystatin C) and inflammatory biomarkers (C-reactive protein, interleukin-6, tumor necrosis factor-alpha) were measured in 271 and 622 blood samples, respectively. Gut and saliva microbiota were assessed via 16S rRNA sequencing among 335 stool and 341 saliva samples. Multivariable regression assessed the relationship between SI and i) New York Heart Association class; ii) pre- vs. post-LVAD or HT; iii) biomarkers of inflammation and microbial diversity. RESULTS: Median (interquartile range) natural logarithm (ln)-SI was -0.13 (-0.32,0.05). Ln-SI decreased across worsening HF class, further declined at 1-month after LVAD and HT and rebounded over time. Ln-SI was correlated with inflammation (r=-0.28, p<0.01), and gut (r=0.28, p<0.01) and oral microbial diversity (r=0.24, p<0.01), these associations remained significant after multivariable adjustment in the combined cohort but not for all individual cohorts. Presence of the gut taxa Roseburia inulinivorans was associated with increased SI. CONCLUSIONS: SI levels decreased in symptomatic HF and remained decreased long-term after LVAD and HT. In the combined cohort, SI levels covaried with inflammation in a similar fashion and significantly related to overall microbial (gut and oral) diversity, including specific taxa compositional changes.

Processing-bias correction with DEBIAS-M improves cross-study generalization of microbiome-based prediction models.

Every step in common microbiome profiling protocols has variable efficiency for each microbe. For example, different DNA extraction kits may have different efficiency for Gram-positive and -negative bacteria. These variable efficiencies, combined with technical variation, create strong processing biases, which impede the identification of signals that are reproducible across studies and the development of generalizable and biologically interpretable prediction models. "Batch-correction" methods have been used to alleviate these issues computationally with some success. However, many make strong parametric assumptions which do not necessarily apply to microbiome data or processing biases, or require the use of an outcome variable, which risks overfitting. Lastly and importantly, existing transformations used to correct microbiome data are largely non-interpretable, and could, for example, introduce values to features that were initially mostly zeros. Altogether, processing bias currently compromises our ability to glean robust and generalizable biological insights from microbiome data. Here, we present DEBIAS-M (Domain adaptation with phenotype Estimation and Batch Integration Across Studies of the Microbiome), an interpretable framework for inference and correction of processing bias, which facilitates domain adaptation in microbiome studies. DEBIAS-M learns bias-correction factors for each microbe in each batch that simultaneously minimize batch effects and maximize cross-study associations with phenotypes. Using benchmarks of HIV and colorectal cancer classification from gut microbiome data, and cervical neoplasia prediction from cervical microbiome data, we demonstrate that DEBIAS-M outperforms batch-correction methods commonly used in the field. Notably, we show that the inferred bias-correction factors are stable, interpretable, and strongly associated with specific experimental protocols. Overall, we show that DEBIAS-M allows for better modeling of microbiome data and identification of interpretable signals that are reproducible across studies.

Periodontitis and brain magnetic resonance imaging markers of Alzheimer's disease and cognitive aging.

INTRODUCTION: We examined the association of clinical, microbiological, and host response features of periodontitis with MRI markers of atrophy/cerebrovascular disease in the Washington Heights Inwood Columbia Aging Project (WHICAP) Ancillary Study of Oral Health. METHODS: We analyzed 468 participants with clinical periodontal data, microbial plaque and serum samples, and brain MRIs. We tested the association of periodontitis features with MRI features, after adjusting for multiple risk factors for Alzheimer's disease/Alzheimer's disease-related dementia (AD/ADRD). RESULTS: In fully adjusted models, having more teeth was associated with lower odds for infarcts, lower white matter hyperintensity (WMH) volume, higher entorhinal cortex volume, and higher cortical thickness. Higher extent of periodontitis was associated with lower entorhinal cortex volume and lower cortical thickness. Differential associations emerged between colonization by specific bacteria/serum antibacterial IgG responses and MRI outcomes. DISCUSSION: In an elderly cohort, clinical, microbiological, and serological features of periodontitis were associated with MRI findings related to ADRD risk. Further investigation of causal associations is warranted.

Mapping the genomic landscape of multidrug resistance in Plasmodium falciparum and its impact on parasite fitness.

Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance in vitro and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping using 34 recombinant haplotypes, and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.

Role of Microbiome in the Outcomes Following Surgical Repair of Perianal Fistula: Prospective Cohort Study Design and Preliminary Results.

BACKGROUND: Anal fistulae are common, predominantly cryptoglandular, and almost invariably require surgical treatment. Recurrences are common for procedures other than fistulotomy regardless of technique and adequacy of repair. Growing evidence supports the pivotal role of specific intestinal bacteria in anastomotic failures after bowel resection. Anal crypts harbor colonic microbiota suggesting that similar mechanisms to anastomotic healing might prevail after anal fistula repair and hence influence healing. This study aims at assessing the potential role of the intestinal microbiome in the clinical outcomes after surgical repair of cryptoglandular anal fistula. METHODS: This is a pilot prospective cohort study enrolling patients with anal fistula undergoing endoanal advancement flap. For microbiome analysis, stool samples are taken via rectal swab before the procedure; additionally, a portion of the fistula is collected intraoperatively after fistulectomy. Samples from groups with treatment failure are compared to samples from patients who healed after surgical repair. Alpha and beta diversities and differential abundance of microbial taxa are determined and compared between groups with DADA2 analytical pipeline. RESULTS: Five patients have been enrolled to date (one female, four male). At median follow-up of 6 months (2-11), one patient experienced disease recurrence at 3 months. DNA from the 5 rectal swab and tissue samples was extracted, showing increased relative abundance of Enterococcus faecalis in samples from the patient who developed a recurrent fistula but not in those without recurrence. CONCLUSION: These very preliminary data suggest that intestinal microbiome may represent a crucial determinant of the surgical outcomes after anal fistula surgery.

Mapping the genomic landscape of multidrug resistance in Plasmodium falciparum and its impact on parasite fitness.

Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.

Generation of a mutator parasite to drive resistome discovery in Plasmodium falciparum.

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5-8 fold elevation in the mutation rate, with an increase of 13-28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an "irresistible" compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this "mutator" parasite can be leveraged to drive P. falciparum resistome discovery.

The abundance of the potential pathogen Staphylococcus hominis in the air microbiome in a dental clinic and its susceptibility to far-UVC light.

The dental clinic air microbiome incorporates microbes from the oral cavity and upper respiratory tract (URT). This study aimed to establish a reliable methodology for air sampling in a dental clinic setting and quantify the abundance of culturable mesophilic aerobic bacteria present in these samples using regression modeling. Staphylococcus hominis, a potentially pathogenic bacterium typically found in the human oropharynx and URT, was consistently isolated. S. hominis was the most abundant species of aerobic bacteria (22%-24%) and comprised 60%-80% of all Staphylococcus spp. The study also assessed the susceptibility of S. hominis to 222 nm-far-UVC light in laboratory experiments, which showed an exponential surface inactivation constant of k = 0.475 cm2 /mJ. This constant is a critical parameter for future on-site use of far-UVC light as a technique for reducing pathogenic bacterial load in dental clinics.

Tradeoff between lag time and growth rate drives the plasmid acquisition cost.

Conjugative plasmids drive genetic diversity and evolution in microbial populations. Despite their prevalence, plasmids can impose long-term fitness costs on their hosts, altering population structure, growth dynamics, and evolutionary outcomes. In addition to long-term fitness costs, acquiring a new plasmid introduces an immediate, short-term perturbation to the cell. However, due to the transient nature of this plasmid acquisition cost, a quantitative understanding of its physiological manifestations, overall magnitudes, and population-level implications, remains unclear. To address this, here we track growth of single colonies immediately following plasmid acquisition. We find that plasmid acquisition costs are primarily driven by changes in lag time, rather than growth rate, for nearly 60 conditions covering diverse plasmids, selection environments, and clinical strains/species. Surprisingly, for a costly plasmid, clones exhibiting longer lag times also achieve faster recovery growth rates, suggesting an evolutionary tradeoff. Modeling and experiments demonstrate that this tradeoff leads to counterintuitive ecological dynamics, whereby intermediate-cost plasmids outcompete both their low and high-cost counterparts. These results suggest that, unlike fitness costs, plasmid acquisition dynamics are not uniformly driven by minimizing growth disadvantages. Moreover, a lag/growth tradeoff has clear implications in predicting the ecological outcomes and intervention strategies of bacteria undergoing conjugation.

A Distinct Nasal Microbiota Signature in Peritoneal Dialysis Patients.

Rationale & Objective: The nasal passages harbor both commensal and pathogenic bacteria. In this study, we sought to characterize the anterior nasal microbiota in PD patients using 16S rRNA gene sequencing. Study Design: Cross-sectional. Setting & Participants: We recruited 32 PD patients, 37 kidney transplant (KTx) recipients, 22 living donor/healthy control (HC) participants and collected anterior nasal swabs at a single point in time. Predictors: We performed 16S rRNA gene sequencing of the V4-V5 hypervariable region to determine the nasal microbiota. Outcomes: Nasal microbiota profiles were determined at the genus level as well as the amplicon sequencing variant level. Analytical Approach: We compared nasal abundance of common genera among the 3 groups using Wilcoxon rank sum testing with Benjamini-Hochberg adjustment. DESeq2 was also utilized to compare the groups at the ASV levels. Results: In the entire cohort, the most abundant genera in the nasal microbiota included: Staphylococcus, Corynebacterium, Streptococcus , and Anaerococcus . Correlational analyses revealed a significant inverse relationship between the nasal abundance of Staphylococcus and that of Corynebacterium . PD patients have a higher nasal abundance of Streptococcus than KTx recipients and HC participants. PD patients have a more diverse representation of Staphylococcus and Streptococcus than KTx recipients and HC participants. PD patients who concurrently have or who developed future Staphylococcus peritonitis had a numerically higher nasal abundance of Staphylococcus than PD patients who did not develop Staphylococcus peritonitis. Limitations: 16S RNA gene sequencing provides taxonomic information to the genus level. Conclusions: We find a distinct nasal microbiota signature in PD patients compared to KTx recipients and HC participants. Given the potential relationship between the nasal pathogenic bacteria and infectious complications, further studies are needed to define the nasal microbiota associated with these infectious complications and to conduct studies on the manipulation of the nasal microbiota to prevent such complications.

Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome.

The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic ß2 and ß5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the ß5 P1 binding pocket, compromising compound binding. Dual targeting of P. falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria.

Estimation of COVID-19 mRNA Vaccine Effectiveness and COVID-19 Illness and Severity by Vaccination Status During Omicron BA.4 and BA.5 Sublineage Periods.

Importance: Recent SARS-CoV-2 Omicron variant sublineages, including BA.4 and BA.5, may be associated with greater immune evasion and less protection against COVID-19 after vaccination. Objectives: To evaluate the estimated vaccine effectiveness (VE) of 2, 3, or 4 doses of COVID-19 mRNA vaccination among immunocompetent adults during a period of BA.4 or BA.5 predominant circulation; and to evaluate the relative severity of COVID-19 in hospitalized patients across Omicron BA.1, BA.2 or BA.2.12.1, and BA.4 or BA.5 sublineage periods. Design, Setting, and Participants: This test-negative case-control study was conducted in 10 states with data from emergency department (ED) and urgent care (UC) encounters and hospitalizations from December 16, 2021, to August 20, 2022. Participants included adults with COVID-19-like illness and molecular testing for SARS-CoV-2. Data were analyzed from August 2 to September 21, 2022. Exposures: mRNA COVID-19 vaccination. Main Outcomes and Measures: The outcomes of interest were COVID-19 ED or UC encounters, hospitalizations, and admission to the intensive care unit (ICU) or in-hospital death. VE associated with protection against medically attended COVID-19 was estimated, stratified by care setting and vaccine doses (2, 3, or 4 doses vs 0 doses as the reference group). Among hospitalized patients with COVID-19, demographic and clinical characteristics and in-hospital outcomes were compared across sublineage periods. Results: During the BA.4 and BA.5 predominant period, there were 82 229 eligible ED and UC encounters among patients with COVID-19-like illness (median [IQR] age, 51 [33-70] years; 49 682 [60.4%] female patients), and 19 114 patients (23.2%) had test results positive for SARS-CoV-2; among 21 007 hospitalized patients (median [IQR] age, 71 [58-81] years; 11 209 [53.4%] female patients), 3583 (17.1 %) had test results positive for SARS-CoV-2. Estimated VE against hospitalization was 25% (95% CI, 17%-32%) for receipt of 2 vaccine doses at 150 days or more after receipt, 68% (95% CI, 50%-80%) for a third dose 7 to 119 days after receipt, and 36% (95% CI, 29%-42%) for a third dose 120 days or more (median [IQR], 235 [204-262] days) after receipt. Among patients aged 65 years or older who had received a fourth vaccine dose, VE was 66% (95% CI, 53%-75%) at 7 to 59 days after vaccination and 57% (95% CI, 44%-66%) at 60 days or more (median [IQR], 88 [75-105] days) after vaccination. Among hospitalized patients with COVID-19, ICU admission or in-hospital death occurred in 21.4% of patients during the BA.1 period vs 14.7% during the BA.4 and BA.5 period (standardized mean difference: 0.17). Conclusions and Relevance: In this case-control study of COVID-19 vaccines and illness, VE associated with protection against medically attended COVID-19 illness was lower with increasing time since last dose; estimated VE was higher after receipt of 1 or 2 booster doses compared with a primary series alone.

Contamination source modeling with SCRuB improves cancer phenotype prediction from microbiome data.

Sequencing-based approaches for the analysis of microbial communities are susceptible to contamination, which could mask biological signals or generate artifactual ones. Methods for in silico decontamination using controls are routinely used, but do not make optimal use of information shared across samples and cannot handle taxa that only partially originate in contamination or leakage of biological material into controls. Here we present Source tracking for Contamination Removal in microBiomes (SCRuB), a probabilistic in silico decontamination method that incorporates shared information across multiple samples and controls to precisely identify and remove contamination. We validate the accuracy of SCRuB in multiple data-driven simulations and experiments, including induced contamination, and demonstrate that it outperforms state-of-the-art methods by an average of 15-20 times. We showcase the robustness of SCRuB across multiple ecosystems, data types and sequencing depths. Demonstrating its applicability to microbiome research, SCRuB facilitates improved predictions of host phenotypes, most notably the prediction of treatment response in melanoma patients using decontaminated tumor microbiome data.

Capsular Polysaccharide Is Essential for the Virulence of the Antimicrobial-Resistant Pathogen Enterobacter hormaechei.

Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.

Dietary tryptophan deficiency promotes gut RORγt+ Treg cells at the expense of Gata3+ Treg cells and alters commensal microbiota metabolism.

Micronutrient deficiency is a major cause of disease throughout the world. Yet, how perturbations influence the immune-microbiome interface remains poorly understood. Here, we report that loss of dietary tryptophan (Trp) reshapes intestinal microbial communities, including the depletion of probiotic L. reuteri, drives transcriptional changes to immune response genes in the intestinal ileum, and reshapes the regulatory T cell (Treg) compartment. Dietary Trp deficiency promotes expansion of RORγt+ Treg cells and the loss of Gata3+ Tregs in a microbiota-dependent manner. In the absence of dietary Trp, provision of the AhR ligand indole-3-carbinol is sufficient to restore the Treg compartment. Together, these data show that dietary Trp deficiency perturbs the interaction between the host and its bacterial symbionts to regulate Treg homeostasis via the deprivation of bacterially derived Trp metabolites. Our findings highlight an essential role for immune-microbiome crosstalk as a key homeostatic regulator during nutrient deficiency.

Optimization of 2,3-Dihydroquinazolinone-3-carboxamides as Antimalarials Targeting PfATP4.

There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na+ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.

The gut microbiome, short chain fatty acids, and related metabolites in cystic fibrosis patients with and without colonic adenomas.

BACKGROUND: Adults with cystic fibrosis (CF) are at increased risk for colon cancer. CF patients have reductions in intestinal bacteria that produce short chain fatty acids (SCFAs), although it is unclear whether this corresponds with intestinal SCFA levels and the presence of colonic neoplasia. The aim of this study was to compare gut microbiome and SCFA composition in patients with and without CF, and to assess associations with colonic adenomas. METHODS: Colonic aspirates were obtained from adults with and without CF undergoing colon cancer screening or surveillance colonoscopy. Microbiome characterization was performed by 16S rRNA V3-V4 sequencing. Targeted profiling of SCFAs and related metabolites was performed by LC-MS. RESULTS: 42 patients (21 CF, 21 control) were enrolled. CF patients had significantly reduced alpha diversity and decreased relative abundance of many SCFA-producing taxa. There were no significant differences in SCFA levels in CF patients, although there were reduced levels of branched chain fatty acids (BCFAs) and related metabolites. CF patients with adenomas, but not controls with adenomas, had significantly increased relative abundance of Bacteroides fragilis. CF microbiome composition was significantly associated with isovalerate concentration and the presence of adenomas. CONCLUSIONS: CF patients have marked disturbances in the gut microbiome, and CF patients with adenomas had notably increased relative abundance of B. fragilis, a pathogen known to promote colon cancer. Reductions in BCFAs but not SCFAs were found in CF. Further studies are warranted to evaluate the role of B. fragilis as well the biological significance of reductions in BCFAs in CF.

Staphylococcal phosphatidylglycerol antigens activate human T cells via CD1a.

Expressed on epidermal Langerhans cells, CD1a presents a range of self-lipid antigens found within the skin; however, the extent to which CD1a presents microbial ligands from bacteria colonizing the skin is unclear. Here we identified CD1a-dependent T cell responses to phosphatidylglycerol (PG), a ubiquitous bacterial membrane phospholipid, as well as to lysylPG, a modified PG, present in several Gram-positive bacteria and highly abundant in Staphylococcus aureus. The crystal structure of the CD1a-PG complex showed that the acyl chains were buried within the A'- and F'-pockets of CD1a, while the phosphoglycerol headgroup remained solvent exposed in the F'-portal and was available for T cell receptor contact. Using lysylPG and PG-loaded CD1a tetramers, we identified T cells in peripheral blood and in skin that respond to these lipids in a dose-dependent manner. Tetramer+CD4+ T cell lines secreted type 2 helper T cell cytokines in response to phosphatidylglycerols as well as to co-cultures of CD1a+ dendritic cells and Staphylococcus bacteria. The expansion in patients with atopic dermatitis of CD4+ CD1a-(lysyl)PG tetramer+ T cells suggests a response to lipids made by bacteria associated with atopic dermatitis and provides a link supporting involvement of PG-based lipid-activated T cells in atopic dermatitis pathogenesis.

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful against COVID-19 (refs. 1,2). However, because SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here we examined this possibility by in vitro passaging of SARS-CoV-2 in nirmatrelvir using two independent approaches, including one on a large scale. Indeed, highly resistant viruses emerged from both and their sequences showed a multitude of 3CL protease mutations. In the experiment peformed with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Nevertheless, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones showed that these mutations mediated only low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (around 100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next-generation protease inhibitors.