Background Antimicrobial resistance (AMR) poses a major threat to human health around the world. Previous publications have estimated the effect of AMR on incidence, deaths, hospital length of stay, and health-care costs for specific pathogen-drug combinations in select locations. To our knowledge, this study presents the most comprehensive estimates of AMR burden to date. MethodsWe estimated deaths and disability-adjusted life-years (DALYs) attributable to and associated with bacterial AMR for 23 pathogens and 88 pathogen-drug combinations in 204 countries and territories in 2019. We obtained data from systematic literature reviews, hospital systems, surveillance systems, and other sources, covering 471 million individual records or isolates and 7585 study-location-years. We used predictive statistical modelling to produce estimates of AMR burden for all locations, including for locations with no data. Our approach can be divided into five broad components: number of deaths where infection played a role, proportion of infectious deaths attributable to a given infectious syndrome, proportion of infectious syndrome deaths attributable to a given pathogen, the percentage of a given pathogen resistant to an antibiotic of interest, and the excess risk of death or duration of an infection associated with this resistance. Using these components, we estimated disease burden based on two counterfactuals: deaths attributable to AMR (based on an alternative scenario in which all drugresistant infections were replaced by drug-susceptible infections), and deaths associated with AMR (based on an alternative scenario in which all drug-resistant infections were replaced by no infection). We generated 95% uncertainty intervals (UIs) for final estimates as the 25th and 975th ordered values across 1000 posterior draws, and models were cross-validated for out-of-sample predictive validity. We present final estimates aggregated to the global and regional level. FindingsOn the basis of our predictive statistical models, there were an estimated 4•95 million (3•62-6•57) deaths associated with bacterial AMR in 2019, including 1•27 million (95% UI 0•911-1•71) deaths attributable to bacterial AMR. At the regional level, we estimated the all-age death rate attributable to resistance to be highest in western sub-Saharan Africa, at 27•3 deaths per 100 000 (20•9-35•3), and lowest in Australasia, at 6•5 deaths (4•3-9•4) per 100 000. Lower respiratory infections accounted for more than 1•5 million deaths associated with resistance in 2019, making it the most burdensome infectious syndrome. The six leading pathogens for deaths associated with resistance (Escherichia coli, followed by Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) were responsible for 929 000 (660 000-1 270 000) deaths attributable to AMR and 3•57 million (2•62-4•78) deaths associated with AMR in 2019. One pathogen-drug combination, meticillinresistant S aureus, caused more than 100 000 deaths attributa...

The Aspergillus nidulans ambient pH signaling pathway involves two transmembrane domain (TMD)-containing proteins, PalH and PalI. We provide in silico and mutational evidence suggesting that PalI is a three TMD (3-TMD) protein with an N-terminal signal peptide, and we show that PalI localizes to the plasma membrane. PalI is not essential for the proteolytic conversion of the PacC translation product into the processed 27-kDa form, but its absence markedly reduces the accumulation of the 53-kDa intermediate after cells are shifted to an alkaline pH. PalI and its homologues contain a predicted luminal, conserved Gly-Cyscontaining motif that distantly resembles a Gly-rich dimerization domain. The Gly44Arg and Gly47Asp substitutions within this motif lead to loss of function. The Gly47Asp substitution prevents plasma membrane localization of PalI-green fluorescent protein (GFP) and leads to its missorting into the multivesicular body pathway. Overexpression of the likely ambient alkaline pH receptor, the 7-TMD protein PalH, partially suppresses the null palI32 mutation. Although some PalH-GFP localizes to the plasma membrane, it predominates in internal membranes. However, the coexpression of PalI to stoichiometrically similar levels results in the strong predominance of PalH-GFP in the plasma membrane. Thus, one role for PalI, but possibly not the only role, is to assist with plasma membrane localization of PalH. These data, considered along with previous reports for both Saccharomyces cerevisiae and A. nidulans, strongly support the prevailing model of pH signaling involving two spatially segregated complexes: a plasma membrane complex containing PalH, PalI, and the arrestin-like protein PalF and an endosomal membrane complex containing PalA and PalB, to which PacC is recruited for its proteolytic activation.

The Aspergillus nidulans pH-responsive transcription factor PacC is modulated by limited, two-step proteolysis. The first, pH-regulated cleavage occurs in the 24-residue highly conserved "signaling protease box" in response to the alkaline pH signal. This is transduced by the Pal signaling pathway, containing the predicted calpain-like cysteine protease and likely signaling protease, PalB. In this work, we carried out classical mutational analysis of the putative signaling protease PalB, and we describe 9 missense and 18 truncating loss-of-function (including null) mutations. Mutations in the region of and affecting directly the predicted catalytic cysteine strongly support the deduction that PalB is a cysteine protease. Truncating and missense mutations affecting the C terminus highlight the importance of this region. Analysis of three-hemagglutinintagged PalB in Western blots demonstrates that PalB levels are independent of pH and Pal signal transduction. We have followed the processing of MYC 3 -tagged PacC in Western blots. We show unequivocally that PalB is essential for signaling proteolysis and is definitely not the processing protease. In addition, we have replaced 15 residues of the signaling protease box of MYC 3 -tagged PacC (pacC900) with alanine. The majority of these substitutions are silent. Leu481Ala, Tyr493Ala, and Gln499Ala result in delayed PacC processing in response to shifting from acidic to alkaline medium, as determined by Western blot analysis. Leu498Ala reduces function much more markedly, as determined by plate tests and processing recalcitrance. Excepting Leu498, this demonstrates that PacC signaling proteolysis is largely independent of sequence in the cleavage region.The activities of an increasing number of proteins, including a number of transcription factors, have been shown to be modulated by limited and site-specific proteolysis. One important mechanism involving incomplete proteasomal degradation, known as regulated ubiquitin proteasome-dependent processing (RUP) (31), mediates the proteolytic activation of transcription factors NF-B1, NF-B2, and the yeast NF-Blike proteins Spt23p and Mga2p (5,23,49; reviewed in references 55 and 56). Other RUP substrates include Drosophila Cubitus interruptus (Ci) and one of its vertebrate homologs, glioma-associated protein (Gli3), which in the absence of Hedgehog (Hh) signaling are converted from the Ci155 and Gli3-190 activators of Hh target genes to the Ci75 and Gli3-83 respective repressors of Hh target genes (3,10,50,54,67). Regulated intramembrane proteolysis (RIP) is a process whereby certain proteins are released from a membranebound sequestered state by a process essentially involving an intramembrane cleaving protease. The intramembrane cleaving protease cleavage usually proceeds constitutively, following a regulated primary extramembrane cleavage which tailors its substrate (reviewed in references 7, 40, 72, and 76). Other modulatory proteolytic processes involve cysteine proteases acting independently of RUP and RIP and potential...

SummaryThe A spergillus nidulans PacC transcription factor mediates gene regulation in response to alkaline ambient pH which, signalled by the Pal pathway, results in the processing of PacC72 to PacC27 via PacC53. Here we investigate two levels at which the pH regulatory system is transcriptionally moderated by pH and identify and characterise a new component of the pH regulatory machinery, PacX. Transcript level analysis and overexpression studies demonstrate that repression of acid‐expressed pal F, specifying the Pal pathway arrestin, probably by PacC27 and/or PacC53, prevents an escalating alkaline pH response. Transcript analyses using a reporter and constitutively expressed pac C  trans‐alleles show that pac C preferential alkaline‐expression results from derepression by depletion of the acid‐prevalent PacC72 form. We additionally show that pac C repression requires PacX. pac X mutations suppress PacC processing recalcitrant mutations, in part, through derepressed PacC levels resulting in traces of PacC27 formed by pH‐independent proteolysis. pac X was cloned by impala transposon mutagenesis. PacX, with homologues within the Leotiomyceta, has an unusual structure with an amino‐terminal coiled‐coil and a carboxy‐terminal zinc binuclear cluster. pacX mutations indicate the importance of these regions. One mutation, an unprecedented finding in A . nidulans genetics, resulted from an insertion of an endogenous Fot1‐like transposon.

SummaryThe pal/RIM ambient pH signalling pathway is crucial for the ability of pathogenic fungi to infect hosts. The Aspergillus nidulans 7‐TMD receptor PalH senses alkaline pH, subsequently facilitating ubiquitination of the arrestin PalF. Ubiquitinated PalF triggers downstream signalling events. The mechanism(s) by which PalH transduces the alkaline pH signal to PalF is poorly understood. We show that PalH is phosphorylated in a signal dependent manner, resembling mammalian GPCRs, although PalH phosphorylation, in contrast to mammalian GPCRs, is arrestin dependent. A genetic screen revealed that an ambient‐exposed region comprising the extracellular loop connecting TM4‐TM5 and ambient‐proximal residues within TM5 is required for signalling. In contrast, substitution by alanines of four aromatic residues within TM6 and TM7 results in a weak ‘constitutive’ activation of the pathway. Our data support the hypothesis that PalH mechanistically resembles mammalian GPCRs that signal via arrestins, such that the relative positions of individual helices within the heptahelical bundle determines the Pro316‐dependent transition between inactive and active PalH conformations, governed by an ambient‐exposed region including critical Tyr259 that potentially represents an agonist binding site. These findings open the possibility of screening for agonist compounds stabilizing the inactive conformation of PalH, which might act as antifungal drugs against ascomycetes.