Loss of Pde1 function acts as an evolutionary gateway to penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen and rising resistance to ß-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance.

Zn2+-binding in the glutamate-rich region of the intrinsically disordered protein prothymosin-alpha.

Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn2+ ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn2+ ions, suggesting that Zn2+ binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn2+-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn2+-prothymosin-α interactions and the protein's ability to discriminate Zn2+ over other divalent cations (e.g., Ca2+, Co2+, Mg2+) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn2+ ions with apparent, stepwise Kd values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn2+. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn2+ to induce a structural change; rather, Zn2+ binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.

Quinary structure modulates protein stability in cells.

Protein quinary interactions organize the cellular interior and its metabolism. Although the interactions stabilizing secondary, tertiary, and quaternary protein structure are well defined, details about the protein-matrix contacts that comprise quinary structure remain elusive. This gap exists because proteins function in the crowded cellular environment, but are traditionally studied in simple buffered solutions. We use NMR-detected H/D exchange to quantify quinary interactions between the B1 domain of protein G and the cytosol of Escherichia coli. We demonstrate that a surface mutation in this protein is 10-fold more destabilizing in cells than in buffer, a surprising result that firmly establishes the significance of quinary interactions. Remarkably, the energy involved in these interactions can be as large as the energies that stabilize specific protein complexes. These results will drive the critical task of implementing quinary structure into models for understanding the proteome.

Residue level quantification of protein stability in living cells.

The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed. This difference has led both experimentalists and theoreticians to tackle the challenging task of understanding how the intracellular environment affects the properties of biopolymers. Despite a growing number of in-cell studies, there is a lack of quantitative, residue-level information about equilibrium thermodynamic protein stability under nonperturbing conditions. We report the use of NMR-detected hydrogen-deuterium exchange of quenched cell lysates to measure individual opening free energies of the 56-aa B1 domain of protein G (GB1) in living Escherichia coli cells without adding destabilizing cosolutes or heat. Comparisons to dilute solution data (pH 7.6 and 37 °C) show that opening free energies increase by as much as 1.14 ± 0.05 kcal/mol in cells. Importantly, we also show that homogeneous protein crowders destabilize GB1, highlighting the challenge of recreating the cellular interior. We discuss our findings in terms of hard-core excluded volume effects, charge-charge GB1-crowder interactions, and other factors. The quenched lysate method identifies the residues most important for folding GB1 in cells, and should prove useful for quantifying the stability of other globular proteins in cells to gain a more complete understanding of the effects of the intracellular environment on protein chemistry.

Development and implementation of a core genome multilocus sequence typing scheme for Haemophilus influenzae.

Haemophilus influenzae is part of the human nasopharyngeal microbiota and a pathogen causing invasive disease. The extensive genetic diversity observed in H. influenzae necessitates discriminatory analytical approaches to evaluate its population structure. This study developed a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae using pangenome analysis tools and validated the cgMLST scheme using datasets consisting of complete reference genomes (N = 14) and high-quality draft H. influenzae genomes (N = 2297). The draft genome dataset was divided into a development dataset (N = 921) and a validation dataset (N = 1376). The development dataset was used to identify potential core genes, and the validation dataset was used to refine the final core gene list to ensure the reliability of the proposed cgMLST scheme. Functional classifications were made for all the resulting core genes. Phylogenetic analyses were performed using both allelic profiles and nucleotide sequence alignments of the core genome to test congruence, as assessed by Spearman's correlation and ordinary least square linear regression tests. Preliminary analyses using the development dataset identified 1067 core genes, which were refined to 1037 with the validation dataset. More than 70% of core genes were predicted to encode proteins essential for metabolism or genetic information processing. Phylogenetic and statistical analyses indicated that the core genome allelic profile accurately represented phylogenetic relatedness among the isolates (R 2 = 0.945). We used this cgMLST scheme to define a high-resolution population structure for H. influenzae, which enhances the genomic analysis of this clinically relevant human pathogen.

Global Variation in Escherichia coli mcr-1 Genes and Plasmids from Animal and Human Genomes Following Colistin Usage Restrictions in Livestock.

Antimicrobial resistance (AMR) is a significant global health threat, with multidrug-resistant (MDR) bacterial clones becoming a major concern. Polymyxins, especially colistin, have reemerged as last-resort treatments for MDR Gram-negative infections. However, colistin use in livestock has spread mobile colistin resistance (mcr) genes, notably mcr-1, impacting human health. In consequence, its livestock use was banned in 2017, originating a natural experiment to study bacterial adaptation. The aim of this work was to analyse the changes in the mcr-1 genetic background after colistin restriction across the world. This study analyses 3163 Escherichia coli genomes with the mcr-1 gene from human and livestock hosts, mainly from Asia (n = 2621) and Europe (n = 359). Genetic characterisation identifies IncI2 (40.4%), IncX4 (26.7%), and multidrug-resistant IncHI2 (18.8%) as the most common plasmids carrying mcr-1. There were differences in plasmids between continents, with IncX4 (56.6%) being the most common in Europe, while IncI2 (44.8%) was predominant in Asia. Promoter variants related to reduced fitness costs and ISApl1 showed a distinct pattern of association that appears to be associated with adaptation to colistin restriction, which differed between continents. Thus, after the colistin ban, Europe saw a shift to specialised mcr-1 plasmids as IncX4, while ISApl1 decreased in Asia due to changes in the prevalence of the distinct promoter variants. These analyses illustrate the evolution of mcr-1 adaptation following colistin use restrictions and the need for region-specific strategies against AMR following colistin restrictions.

Contrasting genes conferring short- and long-term biofilm adaptation in Listeria.

Listeria monocytogenes is an opportunistic food-borne bacterium that is capable of infecting humans with high rates of hospitalization and mortality. Natural populations are genotypically and phenotypically variable, with some lineages being responsible for most human infections. The success of L. monocytogenes is linked to its capacity to persist on food and in the environment. Biofilms are an important feature that allow these bacteria to persist and infect humans, so understanding the genetic basis of biofilm formation is key to understanding transmission. We sought to investigate the biofilm-forming ability of L. monocytogenes by identifying genetic variation that underlies biofilm formation in natural populations using genome-wide association studies (GWAS). Changes in gene expression of specific strains during biofilm formation were then investigated using RNA sequencing (RNA-seq). Genetic variation associated with enhanced biofilm formation was identified in 273 genes by GWAS and differential expression in 220 genes by RNA-seq. Statistical analyses show that the number of overlapping genes flagged by either type of experiment is less than expected by random sampling. This novel finding is consistent with an evolutionary scenario where rapid adaptation is driven by variation in gene expression of pioneer genes, and this is followed by slower adaptation driven by nucleotide changes within the core genome.

Characterisation of Salmonella enterica clones carrying mcr-1 plasmids in meat products and patients in Northern Thailand using long read sequencing.

Salmonella spp. is an important foodborne pathogen associated with consumption of contaminated food, especially food of livestock origin. Antimicrobial resistance (AMR) in Salmonella has been reported globally and increasing AMR in food production is a major public health issue worldwide. The objective of this study was to describe the genetic relatedness among Salmonella enterica isolates, which displayed identical DNA fingerprint profiles. Ten S. enterica isolates were selected from meat and human cases with an identical rep-PCR profile of serovars Rissen (n = 4), Weltevreden (n = 4), and Stanley (n = 2). We used long-read whole genome sequencing (WGS) on the MinION sequencing platform to type isolates and investigate in silico the presence of specific AMR genes. Antimicrobial susceptibility testing was tested by disk diffusion and gradient diffusion method to corroborate the AMR phenotype. Multidrug resistance and resistance to more than one antimicrobial agent were observed in eight and nine isolates, respectively. Resistance to colistin with an accompanying mcr-1 gene was observed among the Salmonella isolates. The analysis of core genome and whole genome MLST revealed that the Salmonella from meat and human salmonellosis were genetically related. Hence, it could be concluded that meat is one of the important sources for Salmonella infection in human.

Simple and inexpensive incorporation of 19F-tryptophan for protein NMR spectroscopy.

Fluorine-containing amino acids are valuable probes for the biophysical characterization of proteins. Current methods for (19)F-labeled protein production involve time-consuming genetic manipulation, compromised expression systems and expensive reagents. We show that Escherichia coli BL21, the workhorse of protein production, can utilise fluoroindole for the biosynthesis of proteins containing (19)F-tryptophan.

Zinc deposition during ESI-MS analysis of peptide-zinc complexes.

Electrospray ionization (ESI) mass spectrometry (MS) has proven to be an extremely powerful technique for studying the stoichiometry and binding strength of peptide-metal complexes. We have found a significant new problem in the ESI-MS of zinc-peptide systems involving the deposition of zinc in the ESI emitter. This deposition of zinc during the experiment removes a significant amount of zinc ions from the solution, impacting the resulting mass spectral intensities used to quantify the amount of the zinc-bound species. Analysis of infused zinc-peptide samples with atomic absorption spectrometry and with a custom-built nanoflow ESI source confirms the alteration of the analyte solutions with positive or negative or no potential applied to the emitter. Ultimately, the location of the zinc deposition was determined to be the stainless steel emitter. The use of a custom-built nanoESI interface using glass emitters was found to mitigate the zinc deposition problem. The phenomenon of metal deposition warrants further investigation as it may not be limited to just zinc and may represent a significant obstacle in the ESI-MS analysis of all protein-metal systems.

Purification and characterization of the central segment of prothymosin-alpha: methodology for handling highly acidic peptides.

Prothymosin-alpha is a highly acidic protein consisting of 110 amino acids. The central segment of this protein, residues 51-89, is thought to be involved in metal binding which may be necessary for its physiological function. To carry out studies of this peptide, this central segment was synthesized in a linear fashion using Fmoc-based methods on rink amide MBHA resin. However, this peptide could not be purified with the typical straightforward approach of RP HPLC followed by negative mode electrospray ionization mass spectrometry (ESI-MS). This was attributed to the high proportion of acidic residues: 26 out of the 39 residues are aspartic and glutamic acids. The acidity of the peptide prevented retention on the RP HPLC column. Additionally, the ability of the highly negatively charged peptide to retain sodium ions prevented molecular weight determination with ESI-MS. A systematic approach to the purification of this highly acidic peptide was undertaken. Ultimately, strong anion exchange chromatography was used to purify the peptide. Extensive desalting using dialysis was required prior to ESI-MS, and the choice of the buffer proved to be critical. In the end, a purification method was devised that yielded a highly purified peptide and is readily compatible with analysis by ESI-MS.