Publications by authors named "Samuel I Miller"

156 Publications

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Maintenance tobramycin primarily affects untargeted bacteria in the CF sputum microbiome.

Thorax 2020 09 6;75(9):780-790. Epub 2020 Jul 6.

Pediatrics, University of Washington School of Medicine, Seattle, Washington, USA

Rationale: The most common antibiotic used to treat people with cystic fibrosis (PWCF) is inhaled tobramycin, administered as maintenance therapy for chronic lung infections. While the effects of inhaled tobramycin on abundance and lung function diminish with continued therapy, this maintenance treatment is known to improve long-term outcomes, underscoring how little is known about why antibiotics work in CF infections, what their effects are on complex CF sputum microbiomes and how to improve these treatments.

Objectives: To rigorously define the effect of maintenance tobramycin on CF sputum microbiome characteristics.

Methods And Measurements: We collected sputum from 30 PWCF at standardised times before, during and after a single month-long course of maintenance inhaled tobramycin. We used traditional culture, quantitative PCR and metagenomic sequencing to define the dynamic effects of this treatment on sputum microbiomes, including abundance changes in both clinically targeted and untargeted bacteria, as well as functional gene categories.

Main Results: CF sputum microbiota changed most markedly by 1 week of antibiotic therapy and plateaued thereafter, and this shift was largely driven by changes in non-dominant taxa. The genetically conferred functional capacities (ie, metagenomes) of subjects' sputum communities changed little with antibiotic perturbation, despite taxonomic shifts, suggesting functional redundancy within the CF sputum microbiome.

Conclusions: Maintenance treatment with inhaled tobramycin, an antibiotic with demonstrated long-term mortality benefit, primarily impacted clinically untargeted bacteria in CF sputum, highlighting the importance of monitoring the non-canonical effects of antibiotics and other treatments to accurately define and improve their clinical impact.
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http://dx.doi.org/10.1136/thoraxjnl-2019-214187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875198PMC
September 2020

Toxin Glycan Binding: Lectin Keys Unlocking Host and Tissue Specificity.

Cell Host Microbe 2020 06;27(6):851-853

Department of Microbiology, University of Washington, Seattle, WA 98195, USA; Department of Medicine, University of Washington, Seattle, WA 98195, USA; Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA. Electronic address:

In this issue of Cell Host and Microbe, Lee et al. define the glycan binding specificity of a variant of typhoid toxin produced by a non-typhoidal Salmonellae serotype. The authors elegantly demonstrate that tissue and host specificity of the toxin are related to specific glycan binding characteristics of the toxin.
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http://dx.doi.org/10.1016/j.chom.2020.05.019DOI Listing
June 2020

Structure of an Inner Membrane Protein Required for PhoPQ-Regulated Increases in Outer Membrane Cardiolipin.

mBio 2020 02 11;11(1). Epub 2020 Feb 11.

Department of Microbiology, University of Washington, Seattle, Washington, USA

The subsp. serovar Typhimurium PhoPQ two-component system is activated within the intracellular phagosome environment, where it promotes remodeling of the outer membrane and resistance to innate immune antimicrobial peptides. Maintenance of the PhoPQ-regulated outer membrane barrier requires PbgA, an inner membrane protein with a transmembrane domain essential for growth, and a periplasmic domain required for PhoPQ-activated increases in outer membrane cardiolipin. Here, we report the crystal structure of cardiolipin-bound PbgA, adopting a novel transmembrane fold that features a cardiolipin binding site in close proximity to a long and deep cleft spanning the lipid bilayer. The end of the cleft extends into the periplasmic domain of the protein, which is structurally coupled to the transmembrane domain via a functionally critical C-terminal helix. In conjunction with a conserved putative catalytic dyad situated at the middle of the cleft, our structural and mutational analyses suggest that PbgA is a multifunction membrane protein that mediates cardiolipin transport, a function essential for growth, and perhaps catalysis of an unknown enzymatic reaction. Gram-negative bacteria cause many types of infections and have become increasingly resistant to available antibiotic drugs. The outer membrane serves as an important barrier that protects bacteria against antibiotics and other toxic compounds. This outer membrane barrier function is regulated when bacteria are in host environments, and the protein PbgA contributes significantly to this increased barrier function by transporting cardiolipin to the outer membrane. We determined the crystal structure of PbgA in complex with cardiolipin and propose a model for its function. Knowledge of the mechanisms of outer membrane assembly and integrity can greatly contribute to the development of new and effective antibiotics, and this structural information may be useful in this regard.
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http://dx.doi.org/10.1128/mBio.03277-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018646PMC
February 2020

CFTR dysregulation drives active selection of the gut microbiome.

PLoS Pathog 2020 01 21;16(1):e1008251. Epub 2020 Jan 21.

Department of Comparative Medicine, University of Washington, Seattle, WA, United States of America.

Patients with cystic fibrosis (CF) have altered fecal microbiomes compared to those of healthy controls. The magnitude of this dysbiosis correlates with measures of CF gastrointestinal (GI) disease, including GI inflammation and nutrient malabsorption. However, whether this dysbiosis is caused by mutations in the CFTR gene, the underlying defect in CF, or whether CF-associated dysbiosis augments GI disease was not clear. To test the relationships between CFTR dysfunction, microbes, and intestinal health, we established a germ-free (GF) CF mouse model and demonstrated that CFTR gene mutations are sufficient to alter the GI microbiome. Furthermore, flow cytometric analysis demonstrated that colonized CF mice have increased mesenteric lymph node and spleen TH17+ cells compared with non-CF mice, suggesting that CFTR defects alter adaptive immune responses. Our findings demonstrate that CFTR mutations modulate both the host adaptive immune response and the intestinal microbiome.
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http://dx.doi.org/10.1371/journal.ppat.1008251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994172PMC
January 2020

Fecal dysbiosis in infants with cystic fibrosis is associated with early linear growth failure.

Nat Med 2020 02 20;26(2):215-221. Epub 2020 Jan 20.

Department of Microbiology, University of Washington, Seattle, WA, USA.

Most infants with cystic fibrosis (CF) have pancreatic exocrine insufficiency that results in nutrient malabsorption and requires oral pancreatic enzyme replacement. Newborn screening for CF has enabled earlier diagnosis, nutritional intervention and enzyme replacement for these infants, allowing most infants with CF to achieve their weight goals by 12 months of age. Nevertheless, most infants with CF continue to have poor linear growth during their first year of life. Although this early linear growth failure is associated with worse long-term respiratory function and survival, the determinants of body length in infants with CF have not been defined. Several characteristics of the CF gastrointestinal (GI) tract, including inflammation, maldigestion and malabsorption, may promote intestinal dysbiosis. As GI microbiome activities are known to affect endocrine functions, the intestinal microbiome of infants with CF may also impact growth. We identified an early, progressive fecal dysbiosis that distinguished infants with CF and low length from infants with CF and normal length. This dysbiosis included altered abundances of taxa that perform functions that are important for GI health, nutrient harvest and growth hormone signaling, including decreased abundance of Bacteroidetes and increased abundance of Proteobacteria. Thus, the GI microbiota represent a potential therapeutic target for the correction of low linear growth in infants with CF.
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http://dx.doi.org/10.1038/s41591-019-0714-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018602PMC
February 2020

Xenophagy: Pathogen-Containing Vacuoles Are Hard to Digest.

Curr Biol 2019 10;29(20):R1086-R1088

Department of Microbiology, University of Washington, Seattle, WA 98195, USA; Department of Medicine, University of Washington, Seattle, WA 98195, USA; Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA. Electronic address:

Many intracellular pathogens reside in host-membrane-encased vacuoles, but the mechanism initiating xenophagic targeting of these vacuoles was unknown. A recent study identifies the host vacuolar-ATPase as essential to xenophagic clearance and the Salmonellae effector SopF that inhibits bacterial clearance by its ADP-ribosylation.
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http://dx.doi.org/10.1016/j.cub.2019.08.069DOI Listing
October 2019

The cellular microbiology of Salmonellae interactions with macrophages.

Cell Microbiol 2019 11 23;21(11):e13116. Epub 2019 Oct 23.

Department of Microbiology, University of Washington, Seattle, WA, USA.

Salmonellae are important enteric pathogens that cause gastroenteritis and systemic illnesses. Macrophages are important components of both the innate and acquired immune system, acting as phagocytes with significant antimicrobial killing activities that present antigen to the adaptive immune system. Macrophages can also be cultured from a variety of sites as primary cells, and the study of the survival and interactions of Salmonellae with these cells is a very early model of infection and cellular microbiology. This review traces the history of discoveries made using Salmonellae infection of macrophages and addresses the possibility of future research in this area, in particular with regards to understanding the complexity of individual bacteria and macrophage cell variability and how such heterogeneity may alter the outcome of infection.
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http://dx.doi.org/10.1111/cmi.13116DOI Listing
November 2019

Salmonella Translocated Effectors Recruit OSBP1 to the Phagosome to Promote Vacuolar Membrane Integrity.

Cell Rep 2019 05;27(7):2147-2156.e5

Department of Microbiology, University of Washington, Seattle, WA 98195, USA; Department of Immunology, University of Washington, Seattle, WA 98195, USA; Department of Medicine, University of Washington, Seattle, WA 98195, USA; Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA. Electronic address:

Intracellular Salmonella use a type III secretion system (TTSS) to translocate effector proteins across the phagosome membrane and thus promote vacuole membrane tubulation, resulting in intracellular survival. This work demonstrates that the effector SseJ binds the eukaryotic lipid transporter oxysterol binding protein 1 (OSBP1). SseJ directs OSBP1 to the endosomal compartment in a manner dependent on the TTSS located on Salmonella pathogenicity island 2 (SPI2). OSBP1 localization is mediated by both SseJ and another OSBP1-binding SPI2 translocated effector, the deubiquitinase SseL. Deletion of both SseJ and SseL reduced vacuolar integrity with increased bacteria released into the eukaryotic cytoplasm of epithelial cells, indicating that their combined activities are necessary for vacuole membrane stability. Cells knocked down for OSBP1 or deleted for the OSBP1-binding proteins VAPA/B also demonstrate loss of vacuole integrity, consistent with the hypothesis that OSBP1 recruitment is required for SPI2-mediated alterations that promote vacuolar integrity of salmonellae.
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http://dx.doi.org/10.1016/j.celrep.2019.04.021DOI Listing
May 2019

Cyclic-di-GMP regulation promotes survival of a slow-replicating subpopulation of intracellular Typhimurium.

Proc Natl Acad Sci U S A 2019 03 12;116(13):6335-6340. Epub 2019 Mar 12.

Department of Microbiology, University of Washington, Seattle, WA 98195;

Typhimurium can invade and survive within macrophages where the bacterium encounters a range of host environmental conditions. Like many bacteria, Typhimurium rapidly responds to changing environments by the use of second messengers such as cyclic di-GMP (c-di-GMP). Here, we generate a fluorescent biosensor to measure c-di-GMP concentrations in thousands of individual bacteria during macrophage infection and to define the sensor enzymes important to c-di-GMP regulation. Three sensor phosphodiesterases were identified as critical to maintaining low c-di-GMP concentrations generated after initial phagocytosis by macrophages. Maintenance of low c-di-GMP concentrations by these phosphodiesterases was required to promote survival within macrophages and virulence for mice. Attenuation of Typhimurium virulence was due to overproduction of c-di-GMP-regulated cellulose, as deletion of the cellulose synthase machinery restored virulence to a strain lacking enzymatic activity of the three phosphodiesterases. We further identified that the cellulose-mediated reduction in survival was constrained to a slow-replicating persister population of Typhimurium induced within the macrophage intracellular environment. As utilization of glucose has been shown to be required for Typhimurium macrophage survival, one possible hypothesis is that this persister population requires the glucose redirected to the synthesis of cellulose to maintain a slow-replicating, metabolically active state.
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http://dx.doi.org/10.1073/pnas.1901051116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442585PMC
March 2019

Human and Extracellular DNA Depletion for Metagenomic Analysis of Complex Clinical Infection Samples Yields Optimized Viable Microbiome Profiles.

Cell Rep 2019 02;26(8):2227-2240.e5

Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98105, USA; Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98105, USA; Pulmonary and Sleep Medicine, Seattle Children's Hospital, Seattle, WA 98105, USA. Electronic address:

Metagenomic sequencing is a promising approach for identifying and characterizing organisms and their functional characteristics in complex, polymicrobial infections, such as airway infections in people with cystic fibrosis. These analyses are often hampered, however, by overwhelming quantities of human DNA, yielding only a small proportion of microbial reads for analysis. In addition, many abundant microbes in respiratory samples can produce large quantities of extracellular bacterial DNA originating either from biofilms or dead cells. We describe a method for simultaneously depleting DNA from intact human cells and extracellular DNA (human and bacterial) in sputum, using selective lysis of eukaryotic cells and endonuclease digestion. We show that this method increases microbial sequencing depth and, consequently, both the number of taxa detected and coverage of individual genes such as those involved in antibiotic resistance. This finding underscores the substantial impact of DNA from sources other than live bacteria in microbiological analyses of complex, chronic infection specimens.
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http://dx.doi.org/10.1016/j.celrep.2019.01.091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435281PMC
February 2019

The Mla system and glycerophospholipid transport to the outer membrane.

Elife 2019 01 14;8. Epub 2019 Jan 14.

Department of Microbiology, University of Washington, Seattle, United States.

The outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screened transposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system in We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system to OM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.
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http://dx.doi.org/10.7554/eLife.40171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365058PMC
January 2019

β-Barrel outer membrane proteins suppress mTORC2 activation and induce autophagic responses.

Sci Signal 2018 11 27;11(558). Epub 2018 Nov 27.

Department of Microbiology, University of Washington, Seattle, WA 98195, USA.

The outer membranes of Gram-negative bacteria and mitochondria contain proteins with a distinct β-barrel tertiary structure that could function as a molecular pattern recognized by the innate immune system. Here, we report that purified outer membrane proteins (OMPs) from different bacterial and mitochondrial sources triggered the induction of autophagy-related endosomal acidification, LC3B lipidation, and p62 degradation. Furthermore, OMPs reduced the phosphorylation and therefore activation of the multiprotein complex mTORC2 and its substrate Akt in macrophages and epithelial cells. The cell surface receptor SlamF8 and the DNA-protein kinase subunit XRCC6 were required for these OMP-specific responses in macrophages and epithelial cells, respectively. The addition of OMPs to mouse bone marrow-derived macrophages infected with Typhimurium facilitated bacterial clearance. These data identify a specific cellular response mediated by bacterial and mitochondrial OMPs that can alter inflammatory responses and influence the killing of pathogens.
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http://dx.doi.org/10.1126/scisignal.aat7493DOI Listing
November 2018

Inhibiting the Evolution of Antibiotic Resistance.

Mol Cell 2019 01 15;73(1):157-165.e5. Epub 2018 Nov 15.

Department of Microbiology, University of Washington, Seattle, WA, USA; Department of Genome Sciences, University of Washington, Seattle, WA, USA. Electronic address:

Efforts to battle antimicrobial resistance (AMR) are generally focused on developing novel antibiotics. However, history shows that resistance arises regardless of the nature or potency of new drugs. Here, we propose and provide evidence for an alternate strategy to resolve this problem: inhibiting evolution. We determined that the DNA translocase Mfd is an "evolvability factor" that promotes mutagenesis and is required for rapid resistance development to all antibiotics tested across highly divergent bacterial species. Importantly, hypermutator alleles that accelerate AMR development did not arise without Mfd, at least during evolution of trimethoprim resistance. We also show that Mfd's role in AMR development depends on its interactions with the RNA polymerase subunit RpoB and the nucleotide excision repair protein UvrA. Our findings suggest that AMR development can be inhibited through inactivation of evolvability factors (potentially with "anti-evolution" drugs)-in particular, Mfd-providing an unexplored route toward battling the AMR crisis.
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http://dx.doi.org/10.1016/j.molcel.2018.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320318PMC
January 2019

Identification of Small-Molecule Modulators of Diguanylate Cyclase by FRET-Based High-Throughput Screening.

Chembiochem 2019 02 27;20(3):394-407. Epub 2018 Dec 27.

University of Washington, Department of Microbiology, 1959 NE Pacific St., Box 357710, Seattle, WA, 98195, USA.

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low-micromolar range. Subsequent structure-activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP-associated phenotypes. In sum, our studies underline the importance of detailed mechanism-of-action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.
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http://dx.doi.org/10.1002/cbic.201800593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509406PMC
February 2019

Multidrug-Resistant Acinetobacter baumannii Chloramphenicol Resistance Requires an Inner Membrane Permease.

Antimicrob Agents Chemother 2018 08 27;62(8). Epub 2018 Jul 27.

Department of Microbiology, University of Washington, Seattle, Washington, USA

is a Gram-negative organism that is a cause of hospital-acquired multidrug-resistant (MDR) infections. has a unique cell surface compared to those of many other Gram-negative pathogens in that it can live without lipopolysaccharide (LPS) and it has a high content of cardiolipin in the outer membrane. Therefore, to better understand the cell envelope and mechanisms of MDR , we screened a transposon library for mutants with defective permeability barrier function, defined as a deficiency in the ability to exclude the phosphatase chromogenic substrate 5-bromo-4-chloro-3-indolylphosphate (XP). We identified multiple mutants with mutations in the ABUW_0982 gene, predicted to encode a permease broadly present in isolates with increased susceptibility to the ribosome-targeting antibiotic chloramphenicol (CHL). Moreover, compared to other known CHL resistance genes, such as chloramphenicol acyltransferase genes, we found that ABUW_0982 is the primary determinant of intrinsic CHL resistance in strain 5075 (Ab5075), an important isolate responsible for severe MDR infections in humans. Finally, studies measuring the efflux of chloramphenicol and expression of ABUW_0982 in CHL-susceptible support the conclusion that ABUW_0982 encodes a single-component efflux protein with specificity for small, hydrophobic molecules, including CHL.
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http://dx.doi.org/10.1128/AAC.00513-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105846PMC
August 2018

Adaptation of commensal proliferating to the intestinal tract of young children with cystic fibrosis.

Proc Natl Acad Sci U S A 2018 02 29;115(7):1605-1610. Epub 2018 Jan 29.

Department of Microbiology, University of Washington, Seattle, WA 98195;

The mature human gut microbiota is established during the first years of life, and altered intestinal microbiomes have been associated with several human health disorders. usually represents less than 1% of the human intestinal microbiome, whereas in cystic fibrosis (CF), greater than 50% relative abundance is common and correlates with intestinal inflammation and fecal fat malabsorption. Despite the proliferation of and other Proteobacteria in conditions involving chronic gastrointestinal tract inflammation, little is known about adaptation of specific characteristics associated with microbiota clonal expansion. We show that isolated from fecal samples of young children with CF has adapted to growth on glycerol, a major component of fecal fat. isolates from different CF patients demonstrate an increased growth rate in the presence of glycerol compared with from healthy controls, and unrelated CF strains have independently acquired this growth trait. Furthermore, CF and control isolates have differential gene expression when grown in minimal media with glycerol as the sole carbon source. While CF isolates display a growth-promoting transcriptional profile, control isolates engage stress and stationary-phase programs, which likely results in slower growth rates. Our results indicate that there is selection of unique characteristics within the microbiome of individuals with CF, which could contribute to individual disease outcomes.
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http://dx.doi.org/10.1073/pnas.1714373115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816161PMC
February 2018

The gram-negative bacterial periplasm: Size matters.

PLoS Biol 2018 01 17;16(1):e2004935. Epub 2018 Jan 17.

Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

Gram-negative bacteria are surrounded by two membrane bilayers separated by a space termed the periplasm. The periplasm is a multipurpose compartment separate from the cytoplasm whose distinct reducing environment allows more efficient and diverse mechanisms of protein oxidation, folding, and quality control. The periplasm also contains structural elements and important environmental sensing modules, and it allows complex nanomachines to span the cell envelope. Recent work indicates that the size or intermembrane distance of the periplasm is controlled by periplasmic lipoproteins that anchor the outer membrane to the periplasmic peptidoglycan polymer. This periplasm intermembrane distance is critical for sensing outer membrane damage and dictates length of the flagellar periplasmic rotor, which controls motility. These exciting results resolve longstanding debates about whether the periplasmic distance has a biological function and raise the possibility that the mechanisms for maintenance of periplasmic size could be exploited for antibiotic development.
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http://dx.doi.org/10.1371/journal.pbio.2004935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771553PMC
January 2018

Multi-drug resistant non-typhoidal Salmonella associated with invasive disease in western Kenya.

PLoS Negl Trop Dis 2018 01 12;12(1):e0006156. Epub 2018 Jan 12.

Department of Global Health, University of Washington, Seattle, WA, United States of America.

Non-typhoidal Salmonella (NTS) is a leading cause of bloodstream infections in Africa, but the various contributions of host susceptibility versus unique pathogen virulence factors are unclear. We used data from a population-based surveillance platform (population ~25,000) between 2007-2014 and NTS genome-sequencing to compare host and pathogen-specific factors between individuals presenting with NTS bacteremia and those presenting with NTS diarrhea. Salmonella Typhimurium ST313 and Salmonella Enteritidis ST11 were the most common isolates. Multi-drug resistant strains of NTS were more commonly isolated from patients presenting with NTS bacteremia compared to NTS diarrhea. This relationship was observed in patients under age five [aOR = 15.16, 95% CI (2.84-81.05), P = 0.001], in patients five years and older, [aOR = 6.70 95% CI (2.25-19.89), P = 0.001], in HIV-uninfected patients, [aOR = 21.61, 95% CI (2.53-185.0), P = 0.005], and in patients infected with Salmonella serogroup B [aOR = 5.96, 95% CI (2.28-15.56), P < 0.001] and serogroup D [aOR = 14.15, 95% CI (1.10-182.7), P = 0.042]. Thus, multi-drug-resistant NTS was strongly associated with bacteremia compared to diarrhea among children and adults. This association was seen in HIV-uninfected individuals infected with either S. Typhimurium or S. Enteritidis. Risk of developing bacteremia from NTS infection may be driven by virulence properties of the Salmonella pathogen.
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http://dx.doi.org/10.1371/journal.pntd.0006156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785031PMC
January 2018

Expression level of human TLR4 rather than sequence is the key determinant of LPS responsiveness.

PLoS One 2017 11;12(10):e0186308. Epub 2017 Oct 11.

Departments of Medicine, Microbiology and Genome Sciences, University of Washington, Seattle, Washington, United States of America.

To address the role of Toll-like receptor 4 (TLR4) single nucleotide polymorphisms (SNP) in lipopolysaccharide (LPS) recognition, we generated mice that differed only in the sequence of TLR4. We used a bacterial artificial chromosome (BAC) transgenic approach and TLR4/MD-2 knockout mice to specifically examine the role of human TLR4 variants in recognition of LPS. Using in vitro and in vivo assays we found that the expression level rather than the sequence of TLR4 played a larger role in recognition of LPS, especially hypoacylated LPS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186308PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636155PMC
October 2017

Editorial overview: Host-microbe interactions: Bacteria.

Curr Opin Microbiol 2017 02;35:v-viii

Department of Medical Microbiology and Immunology, University of California, Davis, United States.

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http://dx.doi.org/10.1016/j.mib.2017.06.002DOI Listing
February 2017

Clinical and Fecal Microbial Changes With Diet Therapy in Active Inflammatory Bowel Disease.

J Clin Gastroenterol 2018 02;52(2):155-163

Departments of Microbiology.

Goal: To determine the effect of the specific carbohydrate diet (SCD) on active inflammatory bowel disease (IBD).

Background: IBD is a chronic idiopathic inflammatory intestinal disorder associated with fecal dysbiosis. Diet is a potential therapeutic option for IBD based on the hypothesis that changing the fecal dysbiosis could decrease intestinal inflammation.

Study: Pediatric patients with mild to moderate IBD defined by pediatric Crohn's disease activity index (PCDAI 10-45) or pediatric ulcerative colitis activity index (PUCAI 10-65) were enrolled into a prospective study of the SCD. Patients started SCD with follow-up evaluations at 2, 4, 8, and 12 weeks. PCDAI/PUCAI, laboratory studies were assessed.

Results: Twelve patients, ages 10 to 17 years, were enrolled. Mean PCDAI decreased from 28.1±8.8 to 4.6±10.3 at 12 weeks. Mean PUCAI decreased from 28.3±23.1 to 6.7±11.6 at 12 weeks. Dietary therapy was ineffective for 2 patients while 2 individuals were unable to maintain the diet. Mean C-reactive protein decreased from 24.1±22.3 to 7.1±0.4 mg/L at 12 weeks in Seattle Cohort (nL<8.0 mg/L) and decreased from 20.7±10.9 to 4.8±4.5 mg/L at 12 weeks in Atlanta Cohort (nL<4.9 mg/L). Stool microbiome analysis showed a distinctive dysbiosis for each individual in most prediet microbiomes with significant changes in microbial composition after dietary change.

Conclusions: SCD therapy in IBD is associated with clinical and laboratory improvements as well as concomitant changes in the fecal microbiome. Further prospective studies are required to fully assess the safety and efficacy of dietary therapy in patients with IBD.
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http://dx.doi.org/10.1097/MCG.0000000000000772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484760PMC
February 2018

In vivo protein interaction network analysis reveals porin-localized antibiotic inactivation in Acinetobacter baumannii strain AB5075.

Nat Commun 2016 11 11;7:13414. Epub 2016 Nov 11.

Department of Genome Sciences, University of Washington, 850 Republican Street, Brotman Building Room 154, Seattle, Washington 98109, USA.

The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital-acquired infections worldwide and is a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. Here, to gain insight on A. baumannii antibiotic resistance mechanisms, we analyse the protein interaction network of a multidrug-resistant A. baumannii clinical strain (AB5075). Using in vivo chemical cross-linking and mass spectrometry, we identify 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter-molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 are identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO are verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrates changes in meropenem or imipenem sensitivity in strain AB5075. These results provide a view of porin-localized antibiotic inactivation and increase understanding of bacterial antibiotic resistance mechanisms.
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http://dx.doi.org/10.1038/ncomms13414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114622PMC
November 2016

Next-Generation High-Throughput Functional Annotation of Microbial Genomes.

mBio 2016 10 4;7(5). Epub 2016 Oct 4.

Department of Immunology and Infectious Disease, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.

Host infection by microbial pathogens cues global changes in microbial and host cell biology that facilitate microbial replication and disease. The complete maps of thousands of bacterial and viral genomes have recently been defined; however, the rate at which physiological or biochemical functions have been assigned to genes has greatly lagged. The National Institute of Allergy and Infectious Diseases (NIAID) addressed this gap by creating functional genomics centers dedicated to developing high-throughput approaches to assign gene function. These centers require broad-based and collaborative research programs to generate and integrate diverse data to achieve a comprehensive understanding of microbial pathogenesis. High-throughput functional genomics can lead to new therapeutics and better understanding of the next generation of emerging pathogens by rapidly defining new general mechanisms by which organisms cause disease and replicate in host tissues and by facilitating the rate at which functional data reach the scientific community.
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http://dx.doi.org/10.1128/mBio.01245-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050336PMC
October 2016

Antibiotic Resistance and Regulation of the Gram-Negative Bacterial Outer Membrane Barrier by Host Innate Immune Molecules.

Authors:
Samuel I Miller

mBio 2016 Sep 27;7(5). Epub 2016 Sep 27.

Departments of Microbiology, Medicine & Genome Sciences, University of Washington, Seattle, Washington, USA

The Gram-negative outer membrane is an important barrier that provides protection against toxic compounds, which include antibiotics and host innate immune molecules such as cationic antimicrobial peptides. Recently, significant research progress has been made in understanding the biogenesis, regulation, and functioning of the outer membrane, including a recent paper from the laboratory of Dr. Brett Finlay at the University of British Columbia (J. van der Heijden et al., mBio 7:e01238-16, 2016, http://dx.doi.org/10.1128/mBio.01541-16). These investigators demonstrate that toxic oxygen radicals, such as those found in host tissues, regulate outer membrane permeability by altering the outer membrane porin protein channels to regulate the influx of oxygen radicals as well as β-lactam antibiotics. This commentary provides context about this interesting paper and discusses the prospects of utilizing increased knowledge of outer membrane biology to develop new antibiotics for antibiotic-resistant Gram-negative bacteria.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040116PMC
http://dx.doi.org/10.1128/mBio.01541-16DOI Listing
September 2016

GUTSS: An Alignment-Free Sequence Comparison Method for Use in Human Intestinal Microbiome and Fecal Microbiota Transplantation Analysis.

PLoS One 2016 8;11(7):e0158897. Epub 2016 Jul 8.

Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

Background: Comparative analysis of gut microbiomes in clinical studies of human diseases typically rely on identification and quantification of species or genes. In addition to exploring specific functional characteristics of the microbiome and potential significance of species diversity or expansion, microbiome similarity is also calculated to study change in response to therapies directed at altering the microbiome. Established ecological measures of similarity can be constructed from species abundances, however methods for calculating these commonly used ecological measures of similarity directly from whole genome shotgun (WGS) metagenomic sequence are lacking.

Results: We present an alignment-free method for calculating similarity of WGS metagenomic sequences that is analogous to the Bray-Curtis index for species, implemented by the General Utility for Testing Sequence Similarity (GUTSS) software application. This method was applied to intestinal microbiomes of healthy young children to measure developmental changes toward an adult microbiome during the first 3 years of life. We also calculate similarity of donor and recipient microbiomes to measure establishment, or engraftment, of donor microbiota in fecal microbiota transplantation (FMT) studies focused on mild to moderate Crohn's disease. We show how a relative index of similarity to donor can be calculated as a measure of change in a patient's microbiome toward that of the donor in response to FMT.

Conclusion: Because clinical efficacy of the transplant procedure cannot be fully evaluated without analysis methods to quantify actual FMT engraftment, we developed a method for detecting change in the gut microbiome that is independent of species identification and database bias, sensitive to changes in relative abundance of the microbial constituents, and can be formulated as an index for correlating engraftment success with clinical measures of disease. More generally, this method may be applied to clinical evaluation of human microbiomes and provide potential diagnostic determination of individuals who may be candidates for specific therapies directed at alteration of the microbiome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158897PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938407PMC
July 2017

Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

Curr Biol 2016 07 23;26(14):1791-801. Epub 2016 Jun 23.

Department of Microbiology, University of Washington, Seattle, WA 98195, USA; Department of Immunology, University of Washington, Seattle, WA 98195, USA; Department of Medicine, University of Washington, Seattle, WA 98195, USA; Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA. Electronic address:

Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes.
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http://dx.doi.org/10.1016/j.cub.2016.05.003DOI Listing
July 2016

A Cellular GWAS Approach to Define Human Variation in Cellular Pathways Important to Inflammation.

Pathogens 2016 Apr 26;5(2). Epub 2016 Apr 26.

Department of Microbiology, University of Washington, Seattle, WA 98195, USA.

An understanding of common human diversity in innate immune pathways should be beneficial in understanding autoimmune diseases, susceptibility to infection, and choices of anti-inflammatory treatment. Such understanding could also result in definition of currently unknown components of human inflammation pathways. A cellular genome-wide association studies (GWAS) platform, termed Hi-HOST (High-throughput human in vitro susceptibility testing), was developed to assay in vitro cellular phenotypes of infection in genotyped lymphoblastoid cells from genetically diverse human populations. Hi-HOST allows for measurement of multiple host and pathogen parameters of infection/inflammation including: bacterial invasion and intracellular replication, host cell death, and cytokine production. Hi-HOST has been used to successfully define a significant portion of the heritable human diversity in inflammatory cell death in response to Salmonella typhimurium. It also led to the discovery of genetic variants important to protection against systemic inflammatory response syndrome (SIRS) and protection against death and bacteremia in individuals with SIRS. Our laboratory is currently using this platform to define human diversity in autophagy and the NLPR3 inflammasome pathways, and to define new components that can impact the expression of phenotypes related to these pathways.
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http://dx.doi.org/10.3390/pathogens5020039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931390PMC
April 2016

Pyomelanin-producing Pseudomonas aeruginosa selected during chronic infections have a large chromosomal deletion which confers resistance to pyocins.

Environ Microbiol 2016 10 2;18(10):3482-3493. Epub 2016 Jun 2.

Department of Microbiology, University of Washington, Seattle, WA, USA.

When bacterial lineages make the transition from free-living to permanent association with hosts, they can undergo massive gene losses, for which the selective forces within host tissues are unknown. We identified here melanogenic clinical isolates of Pseudomonas aeruginosa with large chromosomal deletions (66 to 270 kbp) and characterized them to investigate how they were selected. When compared with their wild-type parents, melanogenic mutants (i) exhibited a lower fitness in growth conditions found in human tissues, such as hyperosmolarity and presence of aminoglycoside antibiotics, (ii) narrowed their metabolic spectrum with a growth disadvantage with particular carbon sources, including aromatic amino acids and acyclic terpenes, suggesting a reduction of metabolic flexibility. Despite an impaired fitness in rich media, melanogenic mutants can inhibit their wild-type parents and compete with them in coculture. Surprisingly, melanogenic mutants became highly resistant to two intraspecific toxins, the S-pyocins AP41 and S1. Our results suggest that pyocins produced within a population of infecting P. aeruginosa may have selected for bacterial mutants that underwent massive gene losses and that were adapted to the life in diverse bacterial communities in the human host. Intraspecific interactions may therefore be an important factor driving the continuing evolution of pathogens during host infections.
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http://dx.doi.org/10.1111/1462-2920.13336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295658PMC
October 2016

Genomic Analysis of Salmonella enterica Serovar Typhimurium Characterizes Strain Diversity for Recent U.S. Salmonellosis Cases and Identifies Mutations Linked to Loss of Fitness under Nitrosative and Oxidative Stress.

mBio 2016 Mar 8;7(2):e00154. Epub 2016 Mar 8.

Department of Microbiology, University of Washington, Seattle, Washington, USA Department of Medicine, University of Washington, Seattle, Washington, USA Department of Genome Sciences, University of Washington, Seattle, Washington, USA

Unlabelled: Salmonella enterica serovar Typhimurium is one of the most common S. enterica serovars associated with U.S. foodborne outbreaks. S. Typhimurium bacteria isolated from humans exhibit wide-ranging virulence phenotypes in inbred mice, leading to speculation that some strains are more virulent in nature. However, it is unclear whether increased virulence in humans is related to organism characteristics or initial treatment failure due to antibiotic resistance. Strain diversity and genetic factors contributing to differential human pathogenicity remain poorly understood. We reconstructed phylogeny, resolved genetic population structure, determined gene content and nucleotide variants, and conducted targeted phenotyping assays for S. Typhimurium strains collected between 1946 and 2012 from humans and animals in the United States and abroad. Strains from recent U.S. salmonellosis cases were associated with five S. Typhimurium lineages distributed within three phylogenetic clades, which are not restricted by geography, year of acquisition, or host. Notably, two U.S. strains and four Mexican strains are more closely related to strains associated with human immunodeficiency virus (HIV)-infected individuals in sub-Saharan Africa than to other North American strains. Phenotyping studies linked variants specific to these strains in hmpA and katE to loss of fitness under nitrosative and oxidative stress, respectively. These results suggest that U.S. salmonellosis is caused by diverse S. Typhimurium strains circulating worldwide. One lineage has mutations in genes affecting fitness related to innate immune system strategies for fighting pathogens and may be adapting to immunocompromised humans by a reduction in virulence capability, possibly due to a lack of selection for its maintenance as a result of the worldwide HIV epidemic.

Importance: Nontyphoidal Salmonella bacteria cause an estimated 1.2 million illnesses annually in the United States, 80 million globally, due to ingestion of contaminated food or water. Salmonella Typhimurium is one of the most common serovars associated with foodborne illness, causing self-limiting gastroenteritis and, in approximately 5% of infected patients, systemic infection. Although some S. Typhimurium strains are speculated to be more virulent than others, it is unknown how strain diversity and genetic factors contribute to differential human pathogenicity. Ours is the first study to examine the diversity of S. Typhimurium associated with recent cases of U.S. salmonellosis and to provide some initial correlation between observed genotypes and phenotypes. Definition of specific S. Typhimurium lineages based on such phenotype/genotype correlations may identify strains with greater capability of associating with specific food sources, allowing outbreaks to be more quickly identified. Additionally, defining simple correlates of pathogenesis may have predictive value for patient outcome.
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http://dx.doi.org/10.1128/mBio.00154-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810482PMC
March 2016