Protein quality control in the mammalian endoplasmic reticulum

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.Cataloged from PDF version of thesis. "February, 2011."Includes bibliographical references.Quality control is an important part of protein biogenesis. Aberrant proteins must be destroyed before they aggregate and cause deleterious effects. Failure to do so can result in cell death or malfunction and, ultimately, disease. Quality control involves the recognition of misfolded proteins and their degradation. For secretory and membrane proteins, folding occurs in the endoplasmic reticulum (ER), but degradation is performed by the cytosolic proteasome. Thus, quality control in the secretory system includes the dislocation of misfolded proteins from the ER to the cytoplasm. ER proteins that fail to fold correctly are identified and directed to the membrane-associated ER quality control machinery. They are then moved across the ER membrane, tagged with ubiquitin, and extracted from the membrane and complex. Finally, the dislocation substrate is taken to the proteasome where it is degraded. Many proteins constituting the mammalian quality control machinery have been identified. Several different dislocation complexes work in parallel to clear misfolded proteins from the ER and they are distinguished by their E3 ubiquitin ligase. This thesis describes the identification and characterization of several previously unknown members of the HRD1-associated ER quality control complex. The newly-identified proteins are osteosarcoma amplified-9 (OS9), UBX domain containing-8 (UBXD8), Ubiquitinconjugating enzyme-6e (UBC6e), and ancient ubiquitous protein-i (AUP1). Each of these proteins participates in different steps of ER quality control. OS9 directs misfolded soluble glycoproteins to the dislocation complex. The mannose-6- phosphate homology domain of OS9 is involved in the recognition of glycans on the misfolded protein. UBXD8 recruits p97, the AAA+ ATPase responsible for membrane extraction of dislocated proteins, to the ER using its UBX domain. UBC6e is a membrane-anchored E2 ubiquitin conjugating enzyme. AUP1 recruits a second E2, soluble UBE2G2. Additionally, AUPI regulates substrate mono- and poly-ubiquitylation. AUPI is also necessary for lipid droplet formation. Lipid droplets are cytoplasmic organelles that store neutral lipids. Based on the data that AUPI depletion affects both ER quality control and lipid droplet formation and that pharmacological inhibition of lipid droplet formation perturbs dislocation, we propose that lipid droplet formation may also play a role in ER protein quality control.by Elizabeth J. Klemm.Ph.D

Antibiotic Resistance and Typhoid.

Multiple drug (antibiotic) resistance (MDR) has become a major threat to the treatment of typhoid and other infectious diseases. Since the 1970s, this threat has increased in Salmonella enterica serovar Typhi, driven in part by the emergence of successful genetic clades, such as haplotype H58, associated with the MDR phenotype. H58 S. Typhi can express multiple antibiotic resistance determinants while retaining the ability to efficiently transmit and persist within the human population. The recent identification of extensively drug resistant S. Typhi only highlights the dangers of ignoring this threat. Here we discuss the evolution of the S. Typhi MDR phenotype and consider options for management

PlasmidTron: assembling the cause of phenotypes and genotypes from NGS data.

Increasingly rich metadata are now being linked to samples that have been whole-genome sequenced. However, much of this information is ignored. This is because linking this metadata to genes, or regions of the genome, usually relies on knowing the gene sequence(s) responsible for the particular trait being measured and looking for its presence or absence in that genome. Examples of this would be the spread of antimicrobial resistance genes carried on mobile genetic elements (MGEs). However, although it is possible to routinely identify the resistance gene, identifying the unknown MGE upon which it is carried can be much more difficult if the starting point is short-read whole-genome sequence data. The reason for this is that MGEs are often full of repeats and so assemble poorly, leading to fragmented consensus sequences. Since mobile DNA, which can carry many clinically and ecologically important genes, has a different evolutionary history from the host, its distribution across the host population will, by definition, be independent of the host phylogeny. It is possible to use this phenomenon in a genome-wide association study to identify both the genes associated with the specific trait and also the DNA linked to that gene, for example the flanking sequence of the plasmid vector on which it is encoded, which follows the same patterns of distribution as the marker gene/sequence itself. We present PlasmidTron, which utilizes the phenotypic data normally available in bacterial population studies, such as antibiograms, virulence factors, or geographical information, to identify traits that are likely to be present on DNA that can randomly reassort across defined bacterial populations. It is also possible to use this methodology to associate unknown genes/sequences (e.g. plasmid backbones) with a specific molecular signature or marker (e.g. resistance gene presence or absence) using PlasmidTron. PlasmidTron uses a k-mer-based approach to identify reads associated with a phylogenetically unlinked phenotype. These reads are then assembled de novo to produce contigs in a fast and scalable-to-large manner. PlasmidTron is written in Python 3 and is available under the open source licence GNU GPL3 from https://github.com/sanger-pathogens/plasmidtron

Population structure and antimicrobial resistance patterns of Salmonella Typhi isolates in urban Dhaka, Bangladesh from 2004 to 2016.

BACKGROUND: Multi-drug resistant typhoid fever remains an enormous public health threat in low and middle-income countries. However, we still lack a detailed understanding of the epidemiology and genomics of S. Typhi in many regions. Here we have undertaken a detailed genomic analysis of typhoid in urban Dhaka, Bangladesh to unravel the population structure and antimicrobial resistance patterns in S. Typhi isolated between 2004-2016. PRINCIPAL FINDINGS: Whole genome sequencing of 202 S. Typhi isolates obtained from three study locations in urban Dhaka revealed a diverse range of S. Typhi genotypes and AMR profiles. The bacterial population within Dhaka were relatively homogenous with little stratification between different healthcare facilities or age groups. We also observed evidence of exchange of Bangladeshi genotypes with neighboring South Asian countries (India, Pakistan and Nepal) suggesting these are circulating throughout the region. This analysis revealed a decline in H58 (genotype 4.3.1) isolates from 2011 onwards, coinciding with a rise in a diverse range of non-H58 genotypes and a simultaneous rise in isolates with reduced susceptibility to fluoroquinolones, potentially reflecting a change in treatment practices. We identified a novel S. Typhi genotype, subclade 3.3.2 (previously defined only to clade level, 3.3), which formed two localized clusters (3.3.2.Bd1 and 3.3.2.Bd2) associated with different mutations in the Quinolone Resistance Determining Region (QRDR) of gene gyrA. SIGNIFICANCE: Our analysis of S. Typhi isolates from urban Dhaka, Bangladesh isolated over a twelve year period identified a diverse range of AMR profiles and genotypes. The observed increase in non-H58 genotypes associated with reduced fluoroquinolone susceptibility may reflect a change in treatment practice in this region and highlights the importance of continued molecular surveillance to monitor the ongoing evolution of AMR in Dhaka. We have defined new genotypes and lineages of Bangladeshi S. Typhi which will facilitate the identification of these emerging AMR clones in future surveillance efforts

PlasmidTron: assembling the cause of phenotypes from NGS data

AbstractWhen defining bacterial populations through whole genome sequencing (WGS) the samples often have detailed associated metadata that relate to disease severity, antimicrobial resistance, or even rare biochemical traits. When comparing these bacterial populations, it is apparent that some of these phenotypes do not follow the phylogeny of the host i.e. they are genetically unlinked to the evolutionary history of the host bacterium. One possible explanation for this phenomenon is that the genes are moving independently between hosts and are likely associated with mobile genetic elements (MGE). However, identifying the element that is associated with these traits can be complex if the starting point is short read WGS data. With the increased use of next generation WGS in routine diagnostics, surveillance and epidemiology a vast amount of short read data is available and these types of associations are relatively unexplored. One way to address this would be to perform assembly de novo of the whole genome read data, including its MGEs. However, MGEs are often full of repeats and can lead to fragmented consensus sequences. Deciding which sequence is part of the chromosome, and which is part of a MGE can be ambiguous. We present PlasmidTron, which utilises the phenotypic data normally available in bacterial population studies, such as antibiograms, virulence factors, or geographic information, to identify sequences that are likely to represent MGEs linked to the phenotype. Given a set of reads, categorised into cases (showing the phenotype) and controls (phylogenetically related but phenotypically negative), PlasmidTron can be used to assemble de novo reads from each sample linked by a phenotype. A k-mer based analysis is performed to identify reads associated with a phylogenetically unlinked phenotype. These reads are then assembled de novo to produce contigs. By utilising k-mers and only assembling a fraction of the raw reads, the method is fast and scalable to large datasets. This approach has been tested on plasmids, because of their contribution to important pathogen associated traits, such as AMR, hence the name, but there is no reason why this approach cannot be utilized for any MGE that can move independently through a bacterial population. PlasmidTron is written in Python 3 and available under the open source licence GNU GPL3 from https://github.com/sanger-pathogens/plasmidtron.DATA SUMMARYSource code for PlasmidTron is available from Github under the open source licence GNU GPL 3; (url - https://goo.gl/ot6rT5)Simulated raw reads files have been deposited in Figshare; (url - https://doi.org/10.6084/m9.figshare.5406355.vl)Salmonella enterica serovar Weltevreden strain VNS10259 is available from GenBank; accession number GCA_001409135.Salmonella enterica serovar Typhi strain BL60006 is available from GenBank; accession number GCA_900185485.Accession numbers for all of the Illumina datasets used in this paper are listed in the supplementary tables.I/We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. ⊠IMPACT STATEMENTPlasmidTron utilises the phenotypic data normally available in bacterial population studies, such as antibiograms, virulence factors, or geographic information, to identify sequences that are likely to represent MGEs linked to the phenotype.</jats:sec

Emergence of an Extensively Drug-Resistant Salmonella enterica Serovar Typhi Clone Harboring a Promiscuous Plasmid Encoding Resistance to Fluoroquinolones and Third-Generation Cephalosporins.

Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel S Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR S Typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the blaCTX-M-15 extended-spectrum β-lactamase, and carrying the qnrS fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of S Typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally.IMPORTANCE Typhoid fever is a severe disease caused by the Gram-negative bacterium Salmonella enterica serovar Typhi. Antibiotic-resistant S Typhi strains have become increasingly common. Here, we report the first large-scale emergence and spread of a novel extensively drug-resistant (XDR) S Typhi clone in Sindh, Pakistan. The XDR S Typhi is resistant to the majority of drugs available for the treatment of typhoid fever. This study highlights the evolving threat of antibiotic resistance in S Typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases. We genetically characterized the XDR S Typhi to investigate the phylogenetic relationship between these isolates and a global collection of S Typhi isolates and to identify multiple genes linked to antibiotic resistance. This S Typhi clone harbored a promiscuous antibiotic resistance plasmid previously identified in other enteric bacteria. The increasing antibiotic resistance in S Typhi observed here adds urgency to the need for typhoid prevention measures

Mathematical modelling for antibiotic resistance control policy: do we know enough?

Background: Antibiotics remain the cornerstone of modern medicine. Yet there exists an inherent dilemma in their use: we are able to prevent harm by administering antibiotic treatment as necessary to both humans and animals, but we must be mindful of limiting the spread of resistance and safeguarding the efficacy of antibiotics for current and future generations. Policies that strike the right balance must be informed by a transparent rationale that relies on a robust evidence base. Main text: One way to generate the evidence base needed to inform policies for managing antibiotic resistance is by using mathematical models. These models can distil the key drivers of the dynamics of resistance transmission from complex infection and evolutionary processes, as well as predict likely responses to policy change in silico. Here, we ask whether we know enough about antibiotic resistance for mathematical modelling to robustly and effectively inform policy. We consider in turn the challenges associated with capturing antibiotic resistance evolution using mathematical models, and with translating mathematical modelling evidence into policy. Conclusions: We suggest that in spite of promising advances, we lack a complete understanding of key principles. From this we advocate for priority areas of future empirical and theoretical research

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation

Abstract: Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum β-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA

Rapid Probing of Biological Surfaces with a Sparse-Matrix Peptide Library

Finding unique peptides to target specific biological surfaces is crucial to basic research and technology development, though methods based on biological arrays or large libraries limit the speed and ease with which these necessary compounds can be found. We reasoned that because biological surfaces, such as cell surfaces, mineralized tissues, and various extracellular matrices have unique molecular compositions, they present unique physicochemical signatures to the surrounding medium which could be probed by peptides with appropriately corresponding physicochemical properties. To test this hypothesis, a naïve pilot library of 36 peptides, varying in their hydrophobicity and charge, was arranged in a two-dimensional matrix and screened against various biological surfaces. While the number of peptides in the matrix library was very small, we obtained “hits” against all biological surfaces probed. Sequence refinement of the “hits” led to peptides with markedly higher specificity and binding activity against screened biological surfaces. Genetic studies revealed that peptide binding to bacteria was mediated, at least in some cases, by specific cell-surface molecules, while examination of human tooth sections showed that this method can be used to derive peptides with highly specific binding to human tissue

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events.

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species