Publications by authors named "Victor Nizet"

450 Publications

Ticagrelor Increases Platelet-Mediated Staphylococcus aureus Killing Resulting in Clearance of Bacteremia.

J Infect Dis 2021 May 9. Epub 2021 May 9.

Department of Pediatrics, University of California San Diego School of Medicine, La Jolla, CA, USA.

Platelets are a critical immune defense against Staphylococcus aureus bloodstream infections. S. aureus alpha-toxin is a virulence factor that decreases platelet viability and accelerates platelet clearance. It was shown that ticagrelor blocks alpha-toxin-mediated platelet injury and resulting thrombocytopenia, protecting mice in a lethal S. aureus sepsis model. We now present the use of ticagrelor as adjunctive therapy in a patient with a S. aureus endovascular infection and thrombocytopenia, associated with restoration of platelet count and bacteremia clearance. Ticagrelor enhanced platelet killing of the S. aureus bloodstream isolate from the treated patient in vitro.
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http://dx.doi.org/10.1093/infdis/jiab146DOI Listing
May 2021

Streptococcus pyogenes upregulates arginine catabolism to exert its pathogenesis on the skin surface.

Cell Rep 2021 Mar;34(13):108924

Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japan. Electronic address:

The arginine deiminase (ADI) pathway has been found in many kinds of bacteria and functions to supplement energy production and provide protection against acid stress. The Streptococcus pyogenes ADI pathway is upregulated upon exposure to various environmental stresses, including glucose starvation. However, there are several unclear points about the advantages to the organism for upregulating arginine catabolism. We show that the ADI pathway contributes to bacterial viability and pathogenesis under low-glucose conditions. S. pyogenes changes global gene expression, including upregulation of virulence genes, by catabolizing arginine. In a murine model of epicutaneous infection, S. pyogenes uses the ADI pathway to augment its pathogenicity by increasing the expression of virulence genes, including those encoding the exotoxins. We also find that arginine from stratum-corneum-derived filaggrin is a key substrate for the ADI pathway. In summary, arginine is a nutrient source that promotes the pathogenicity of S. pyogenes on the skin.
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http://dx.doi.org/10.1016/j.celrep.2021.108924DOI Listing
March 2021

Repurposed drugs block toxin-driven platelet clearance by the hepatic Ashwell-Morell receptor to clear bacteremia.

Sci Transl Med 2021 Mar;13(586)

Biomedical Sciences Graduate Program, UC San Diego, La Jolla, CA 92093, USA.

(SA) bloodstream infections cause high morbidity and mortality (20 to 30%) despite modern supportive care. In a human bacteremia cohort, we found that development of thrombocytopenia was correlated to increased mortality and increased α-toxin expression by the pathogen. Platelet-derived antibacterial peptides are important in bloodstream defense against SA, but α-toxin decreased platelet viability, induced platelet sialidase to cause desialylation of platelet glycoproteins, and accelerated platelet clearance by the hepatic Ashwell-Morell receptor (AMR). Ticagrelor (Brilinta), a commonly prescribed P2Y12 receptor inhibitor used after myocardial infarction, blocked α-toxin-mediated platelet injury and resulting thrombocytopenia, thereby providing protection from lethal SA infection in a murine intravenous challenge model. Genetic deletion or pharmacological inhibition of AMR stabilized platelet counts and enhanced resistance to SA infection, and the anti-influenza sialidase inhibitor oseltamivir (Tamiflu) provided similar therapeutic benefit. Thus, a "toxin-platelet-AMR" regulatory pathway plays a critical role in the pathogenesis of SA bloodstream infection, and its elucidation provides proof of concept for repurposing two commonly prescribed drugs as adjunctive therapies to improve patient outcomes.
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http://dx.doi.org/10.1126/scitranslmed.abd6737DOI Listing
March 2021

Driving to Safety: CRISPR-Based Genetic Approaches to Reducing Antibiotic Resistance.

Trends Genet 2021 Mar 18. Epub 2021 Mar 18.

Tata Institute for Genetics and Society, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0349, USA; Collaborative to Halt Antibiotic-Resistant Microbes, Department of Pediatrics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA; Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA.

Bacterial resistance to antibiotics has reached critical levels, skyrocketing in hospitals and the environment and posing a major threat to global public health. The complex and challenging problem of reducing antibiotic resistance (AR) requires a network of both societal and science-based solutions to preserve the most lifesaving pharmaceutical intervention known to medicine. In addition to developing new classes of antibiotics, it is essential to safeguard the clinical efficacy of existing drugs. In this review, we examine the potential application of novel CRISPR-based genetic approaches to reducing AR in both environmental and clinical settings and prolonging the utility of vital antibiotics.
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http://dx.doi.org/10.1016/j.tig.2021.02.007DOI Listing
March 2021

The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection.

Nat Commun 2021 02 23;12(1):1230. Epub 2021 Feb 23.

Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway.

The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.
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http://dx.doi.org/10.1038/s41467-021-21473-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902821PMC
February 2021

More than a Pore: Nonlytic Antimicrobial Functions of Complement and Bacterial Strategies for Evasion.

Microbiol Mol Biol Rev 2021 02 27;85(1). Epub 2021 Jan 27.

Division of Host-Microbe Systems and Therapeutics, Department of Pediatrics, UC San Diego, La Jolla, California, USA

The complement system is an evolutionarily ancient defense mechanism against foreign substances. Consisting of three proteolytic activation pathways, complement converges on a common effector cascade terminating in the formation of a lytic pore on the target surface. The classical and lectin pathways are initiated by pattern recognition molecules binding to specific ligands, while the alternative pathway is constitutively active at low levels in circulation. Complement-mediated killing is essential for defense against many Gram-negative bacterial pathogens, and genetic deficiencies in complement can render individuals highly susceptible to infection, for example, invasive meningococcal disease. In contrast, Gram-positive bacteria are inherently resistant to the direct bactericidal activity of complement due to their thick layer of cell wall peptidoglycan. However, complement also serves diverse roles in immune defense against all bacteria by flagging them for opsonization and killing by professional phagocytes, synergizing with neutrophils, modulating inflammatory responses, regulating T cell development, and cross talk with coagulation cascades. In this review, we discuss newly appreciated roles for complement beyond direct membrane lysis, incorporate nonlytic roles of complement into immunological paradigms of host-pathogen interactions, and identify bacterial strategies for complement evasion.
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http://dx.doi.org/10.1128/MMBR.00177-20DOI Listing
February 2021

Exploring the Impact of Ketodeoxynonulosonic Acid in Host-Pathogen Interactions Using Uptake and Surface Display by Nontypeable Haemophilus influenzae.

mBio 2021 01 19;12(1). Epub 2021 Jan 19.

Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, USA

Surface expression of the common vertebrate sialic acid (Sia) -acetylneuraminic acid (Neu5Ac) by commensal and pathogenic microbes appears structurally to represent "molecular mimicry" of host sialoglycans, facilitating multiple mechanisms of host immune evasion. In contrast, ketodeoxynonulosonic acid (Kdn) is a more ancestral Sia also present in prokaryotic glycoconjugates that are structurally quite distinct from vertebrate sialoglycans. We detected human antibodies against Kdn-terminated glycans, and sialoglycan microarray studies found these anti-Kdn antibodies to be directed against Kdn-sialoglycans structurally similar to those on human cell surface Neu5Ac-sialoglycans. Anti-Kdn-glycan antibodies appear during infancy in a pattern similar to those generated following incorporation of the nonhuman Sia -glycolylneuraminic acid (Neu5Gc) onto the surface of nontypeable (NTHi), a human commensal and opportunistic pathogen. NTHi grown in the presence of free Kdn took up and incorporated the Sia into its lipooligosaccharide (LOS). Surface display of the Kdn within NTHi LOS blunted several virulence attributes of the pathogen, including Neu5Ac-mediated resistance to complement and whole blood killing, complement C3 deposition, IgM binding, and engagement of Siglec-9. Upper airway administration of Kdn reduced NTHi infection in human-like null (Neu5Gc-deficient) mice that express a Neu5Ac-rich sialome. We propose a mechanism for the induction of anti-Kdn antibodies in humans, suggesting that Kdn could be a natural and/or therapeutic "Trojan horse" that impairs colonization and virulence phenotypes of free Neu5Ac-assimilating human pathogens. All cells in vertebrates are coated with a dense array of glycans often capped with sugars called sialic acids. Sialic acids have many functions, including serving as a signal for recognition of "self" cells by the immune system, thereby guiding an appropriate immune response against foreign "nonself" and/or damaged cells. Several pathogenic bacteria have evolved mechanisms to cloak themselves with sialic acids and evade immune responses. Here we explore a type of sialic acid called "Kdn" (ketodeoxynonulosonic acid) that has not received much attention in the past and compare and contrast how it interacts with the immune system. Our results show potential for the use of Kdn as a natural intervention against pathogenic bacteria that take up and coat themselves with external sialic acid from the environment.
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http://dx.doi.org/10.1128/mBio.03226-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845648PMC
January 2021

Novel Models of Colonization and Disease Reveal Modest Contributions of M-Like (SCM) Protein.

Microorganisms 2021 Jan 16;9(1). Epub 2021 Jan 16.

Department of Molecular Virology and Microbiology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.

is a common colonizing bacterium of the urogenital tract of cats and dogs that can also cause invasive disease in these animal populations and in humans. Although the virulence mechanisms of are not well-characterized, an M-like protein, SCM, has recently identified been as a potential virulence factor. SCM is a surface-associated protein that binds to host plasminogen and IgGs suggesting its possible importance in host-pathogen interactions. In this study, we developed in vitro and ex vivo blood component models and murine models of vaginal colonization, systemic infection, and dermal infection to compare the virulence potential of the zoonotic vaginal isolate G361 and its isogenic SCM-deficient mutant (G361∆). We found that while establishes vaginal colonization and causes invasive disease in vivo, the contribution of the SCM protein to virulence phenotypes in these models is modest. We conclude that SCM is dispensable for invasive disease in murine models and for resistance to human blood components ex vivo, but may contribute to mucosal persistence, highlighting a potential contribution to the recently appreciated genetic diversity of SCM across strains and hosts.
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http://dx.doi.org/10.3390/microorganisms9010183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829700PMC
January 2021

A Novel N4-Like Bacteriophage Isolated from a Wastewater Source in South India with Activity against Several Multidrug-Resistant Clinical Pseudomonas aeruginosa Isolates.

mSphere 2021 01 13;6(1). Epub 2021 Jan 13.

School of Biotechnology, Amrita Vishwa Vidyapeetham, Amritapuri, Kerala, India

Multidrug-resistant community-acquired infections caused by the opportunistic human pathogen are increasingly reported in India and other locations globally. Since this organism is ubiquitous in the environment, samples such as sewage and wastewater are rich reservoirs of bacteriophages. In this study, we report the isolation and characterization of a novel N4-like lytic bacteriophage, vB_Pae_AM.P2 (AM.P2), from wastewater in Kerala, India. AM.P2 is a double-stranded DNA podovirus that efficiently lyses the model strain, PAO1, at a multiplicity of infection as low as 0.1 phage per bacterium and resistance frequency of 6.59 × 10 Synergy in bactericidal activity was observed between AM.P2 and subinhibitory concentrations of the antibiotic ciprofloxacin. Genome sequencing of AM.P2 revealed features similar to those of the N4-like phages LUZ7 and KPP21. As judged by two independent assay methods, spot tests and growth inhibition, AM.P2 successfully inhibited the growth of almost 30% of strains from a contemporary collection of multidrug-resistant clinical isolates from South India. Thus, AM.P2 may represent an intriguing candidate for inclusion in bacteriophage cocktails developed for various applications, including water decontamination and clinical bacteriophage therapy. In India, multidrug resistance determinants are much more abundant in community-associated bacterial pathogens due to the improper treatment of domestic and industrial effluents. In particular, a high bacterial load of the opportunistic pathogen in sewage and water bodies in India is well documented. The isolation and characterization of bacteriophages that could target emerging strains, representing possible epicenters for community-acquired infections, could serve as a useful alternative tool for various applications, such as phage therapy and environmental treatment. Continuing to supplement the repertoire of broad-spectrum bacteriophages is an essential tool in confronting this problem.
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http://dx.doi.org/10.1128/mSphere.01215-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845610PMC
January 2021

Identifying the effect of vancomycin on health care-associated methicillin-resistant Staphylococcus aureus strains using bacteriological and physiological media.

Gigascience 2021 Jan;10(1)

Department of Bioengineering, University of California, 9500 Gilman Dr, La Jolla, CA 92093, USA.

Background: The evolving antibiotic-resistant behavior of health care-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) USA100 strains are of major concern. They are resistant to a broad class of antibiotics such as macrolides, aminoglycosides, fluoroquinolones, and many more.

Findings: The selection of appropriate antibiotic susceptibility examination media is very important. Thus, we use bacteriological (cation-adjusted Mueller-Hinton broth) as well as physiological (R10LB) media to determine the effect of vancomycin on USA100 strains. The study includes the profiling behavior of HA-MRSA USA100 D592 and D712 strains in the presence of vancomycin through various high-throughput assays. The US100 D592 and D712 strains were characterized at sub-inhibitory concentrations through growth curves, RNA sequencing, bacterial cytological profiling, and exo-metabolomics high throughput experiments.

Conclusions: The study reveals the vancomycin resistance behavior of HA-MRSA USA100 strains in dual media conditions using wide-ranging experiments.
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http://dx.doi.org/10.1093/gigascience/giaa156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794652PMC
January 2021

Elongated neutrophil-derived structures are blood-borne microparticles formed by rolling neutrophils during sepsis.

J Exp Med 2021 Mar;218(3)

La Jolla Institute for Immunology, La Jolla, CA.

Rolling neutrophils form tethers with submicron diameters. Here, we report that these tethers detach, forming elongated neutrophil-derived structures (ENDS) in the vessel lumen. We studied ENDS formation in mice and humans in vitro and in vivo. ENDS do not contain mitochondria, endoplasmic reticulum, or DNA, but are enriched for S100A8, S100A9, and 57 other proteins. Within hours of formation, ENDS round up, and some of them begin to present phosphatidylserine on their surface (detected by annexin-5 binding) and release S100A8-S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. ENDS appear in blood plasma of mice upon induction of septic shock. Compared with healthy donors, ENDS are 10-100-fold elevated in blood plasma of septic patients. Unlike neutrophil-derived extracellular vesicles, most ENDS are negative for the tetraspanins CD9, CD63, and CD81. We conclude that ENDS are a new class of bloodborne submicron particles with a formation mechanism linked to neutrophil rolling on the vessel wall.
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http://dx.doi.org/10.1084/jem.20200551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721910PMC
March 2021

Exploration of Bacterial Bottlenecks and Streptococcus pneumoniae Pathogenesis by CRISPRi-Seq.

Cell Host Microbe 2021 01 28;29(1):107-120.e6. Epub 2020 Oct 28.

Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Biophore Building, Lausanne 1015, Switzerland. Electronic address:

Streptococcus pneumoniae is an opportunistic human pathogen that causes invasive diseases, including pneumonia, with greater health risks upon influenza A virus (IAV) co-infection. To facilitate pathogenesis studies in vivo, we developed an inducible CRISPR interference system that enables genome-wide fitness testing in one sequencing step (CRISPRi-seq). We applied CRISPRi-seq to assess bottlenecks and identify pneumococcal genes important in a murine pneumonia model. A critical bottleneck occurs at 48 h with few bacteria causing systemic infection. This bottleneck is not present during IAV superinfection, facilitating identification of pneumococcal pathogenesis-related genes. Top in vivo essential genes included purA, encoding adenylsuccinate synthetase, and the cps operon required for capsule production. Surprisingly, CRISPRi-seq indicated no fitness-related role for pneumolysin during superinfection. Interestingly, although metK (encoding S-adenosylmethionine synthetase) was essential in vitro, it was dispensable in vivo. This highlights advantages of CRISPRi-seq over transposon-based genetic screens, as all genes, including essential genes, can be tested for pathogenesis potential.
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http://dx.doi.org/10.1016/j.chom.2020.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855995PMC
January 2021

Evaluation of IL-17D in Host Immunity to Group A Infection.

J Immunol 2020 12 19;205(11):3122-3129. Epub 2020 Oct 19.

Department of Pathology, University of California, San Diego, La Jolla, CA 92093;

IL-17D is a cytokine that belongs to the IL-17 family and is conserved in vertebrates and invertebrates. In contrast to IL-17A and IL-17F, which are expressed in Th17 cells, IL-17D is expressed broadly in nonimmune cells. IL-17D can promote immune responses to cancer and viruses in part by inducing chemokines and recruiting innate immune cells such as NK cells. Although bacterial infection can induce IL-17D in fish and invertebrates, the role of mammalian IL-17D in antibacterial immunity has not been established. To determine whether IL-17D has a role in mediating host defense against bacterial infections, we studied i.p. infection by group A (GAS) in wild-type (WT) and mice. Compared with WT animals, mice deficient in IL-17D experienced decreased survival, had greater weight loss, and showed increased bacterial burden in the kidney and peritoneal cavity following GAS challenge. In WT animals, IL-17D transcript was induced by GAS infection and correlated to increased levels of chemokine CCL2 and greater neutrophil recruitment. Of note, GAS-mediated IL-17D induction in nonimmune cells required live bacteria, suggesting that processes beyond recognition of pathogen-associated molecular patterns were required for IL-17D induction. Based on our results, we propose a model in which nonimmune cells can discriminate between nonviable and viable GAS cells, responding only to the latter by inducing IL-17D.
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http://dx.doi.org/10.4049/jimmunol.1901482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686091PMC
December 2020

TLR4 signaling and macrophage inflammatory responses are dampened by GIV/Girdin.

Proc Natl Acad Sci U S A 2020 10 14;117(43):26895-26906. Epub 2020 Oct 14.

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093;

Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multimodular scaffold, GIV (a.k.a. Girdin), titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress proinflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene-depletion studies confirmed that the presence of GIV ameliorates dextran sodium sulfate-induced colitis and sepsis-induced death. The antiinflammatory actions of GIV are mediated via its C-terminally located TIR-like BB-loop (TILL) motif which binds the cytoplasmic TIR modules of TLR4 in a manner that precludes receptor dimerization; such dimerization is a prerequisite for proinflammatory signaling. Binding of GIV's TILL motif to TIR modules inhibits proinflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses.
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http://dx.doi.org/10.1073/pnas.2011667117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604444PMC
October 2020

Prophage exotoxins enhance colonization fitness in epidemic scarlet fever-causing Streptococcus pyogenes.

Nat Commun 2020 10 6;11(1):5018. Epub 2020 Oct 6.

Australian Infectious Diseases Research Centre and School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD, Australia.

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for resistance to neutrophil killing. Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the pathogenesis of scarlet fever-causing GAS mediated by prophage ΦHKU.vir exotoxins.
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http://dx.doi.org/10.1038/s41467-020-18700-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538557PMC
October 2020

Evaluating Organism-Wide Changes in the Metabolome and Microbiome following a Single Dose of Antibiotic.

mSystems 2020 Oct 6;5(5). Epub 2020 Oct 6.

Department of Pediatrics, UC San Diego, La Jolla, California, USA

Antibiotics are a mainstay of modern medicine, but as they kill their target pathogen(s), they often affect the commensal microbiota. Antibiotic-induced microbiome dysbiosis is a growing research focus and health concern, often assessed via analysis of fecal samples. However, such analysis does not inform how antibiotics influence the microbiome across the whole host or how such changes subsequently alter host chemistry. In this study, we investigated the acute (1 day postadministration) and delayed (6 days postadministration) effects of a single parenteral dose of two common antibiotics, ampicillin or vancomycin, on the global metabolome and microbiome of mice across 77 different body sites from 25 different organs. The broader-spectrum agent ampicillin had the greatest impact on the microbiota in the lower gastrointestinal tract (cecum and colon), where microbial diversity is highest. In the metabolome, the greatest effects were seen 1 day posttreatment, and changes in metabolite abundances were not confined to the gut. The local abundance of ampicillin and its metabolites correlated with increased metabolome effect size and a loss of alpha diversity versus control mice. Additionally, small peptides were elevated in the lower gastrointestinal tract of mice 1 day after antibiotic treatment. While a single parenteral dose of antibiotic did not drastically alter the microbiome, nevertheless, changes in the metabolome were observed both within and outside the gut. This study provides a framework for how whole-organism -omics approaches can be employed to understand the impact of antibiotics on the entire host. We are just beginning to understand the unintended effects of antibiotics on our microbiomes and health. In this study, we aimed to define an approach by which one could obtain a comprehensive picture of (i) how antibiotics spatiotemporally impact commensal microbes throughout the gut and (ii) how these changes influence host chemistry throughout the body. We found that just a single dose of antibiotic altered host chemistry in a variety of organs and that microbiome alterations were not uniform throughout the gut. As technological advances increase the feasibility of whole-organism studies, we argue that using these approaches can provide further insight on both the wide-ranging effects of antibiotics on health and how to restore microbial communities to mitigate these effects.
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http://dx.doi.org/10.1128/mSystems.00340-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542558PMC
October 2020

The Pseudomonas aeruginosa protease LasB directly activates IL-1β.

EBioMedicine 2020 Oct 23;60:102984. Epub 2020 Sep 23.

Department of Microbiology and Immunology, Emory School of Medicine, Atlanta GA, United States; Division of Infectious Diseases, Emory School of Medicine, Atlanta GA, United States; Antimicrobial Resistance Center, Emory University, Atlanta GA, United States. Electronic address:

Background: Pulmonary damage by Pseudomonas aeruginosa during cystic fibrosis lung infection and ventilator-associated pneumonia is mediated both by pathogen virulence factors and host inflammation. Impaired immune function due to tissue damage and inflammation, coupled with pathogen multidrug resistance, complicates the management of these deep-seated infections. Pathological inflammation during infection is driven by interleukin-1β (IL-1β), but the molecular processes involved are not fully understood.

Methods: We examined IL-1β activation in a pulmonary model infection of Pseudomonas aeruginosa and in vitro using genetics, specific inhibitors, recombinant proteins, and targeted reporters of protease activity and IL-1β bioactivity.

Findings: Caspase-family inflammasome proteases canonically regulate maturation of this proinflammatory cytokine, but we report that plasticity in IL-1β proteolytic activation allows for its direct maturation by the pseudomonal protease LasB. LasB promotes IL-1β activation, neutrophilic inflammation, and destruction of lung architecture characteristic of severe P. aeruginosa pulmonary infection.

Interpretation: Preservation of lung function and effective immune clearance may be enhanced by selectively controlling inflammation. Discovery of this IL-1β regulatory mechanism provides a distinct target for anti-inflammatory therapeutics, such as matrix metalloprotease inhibitors that inhibit LasB and limit inflammation and pathology during P. aeruginosa pulmonary infections.

Funding: Full details are provided in the Acknowledgements section.
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http://dx.doi.org/10.1016/j.ebiom.2020.102984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511813PMC
October 2020

Mortality Risk Profiling of Staphylococcus aureus Bacteremia by Multi-omic Serum Analysis Reveals Early Predictive and Pathogenic Signatures.

Cell 2020 09;182(5):1311-1327.e14

Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA; Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA; Collaborative to Halt Antibiotic-Resistant Microbes, University of California, San Diego, La Jolla, CA 92093, USA; Center for Microbiome Innovation, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Staphylococcus aureus bacteremia (SaB) causes significant disease in humans, carrying mortality rates of ∼25%. The ability to rapidly predict SaB patient responses and guide personalized treatment regimens could reduce mortality. Here, we present a resource of SaB prognostic biomarkers. Integrating proteomic and metabolomic techniques enabled the identification of >10,000 features from >200 serum samples collected upon clinical presentation. We interrogated the complexity of serum using multiple computational strategies, which provided a comprehensive view of the early host response to infection. Our biomarkers exceed the predictive capabilities of those previously reported, particularly when used in combination. Last, we validated the biological contribution of mortality-associated pathways using a murine model of SaB. Our findings represent a starting point for the development of a prognostic test for identifying high-risk patients at a time early enough to trigger intensive monitoring and interventions.
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http://dx.doi.org/10.1016/j.cell.2020.07.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494005PMC
September 2020

Multidimensional Proteome Profiling of Blood-Brain Barrier Perturbation by Group B .

mSystems 2020 Aug 25;5(4). Epub 2020 Aug 25.

Department of Pharmacology, University of California, San Diego, La Jolla, California, USA

Group B (GBS) remains the leading cause of neonatal meningitis, a disease associated with high rates of adverse neurological sequelae. The relationship between GBS and brain tissues remains poorly characterized, partly because past studies had focused on microbial rather than host processes. Additionally, the field has not capitalized on systems-level technologies to probe the host-pathogen relationship. Here, we use multiplexed quantitative proteomics to investigate the effect of GBS infection in the murine brain at various levels of tissue complexity, beginning with the whole organ and moving to brain vascular substructures. Infected whole brains showed classical signatures associated with the acute-phase response. In isolated brain microvessels, classical blood-brain barrier proteins were unaltered, but interferon signaling and leukocyte recruitment proteins were upregulated. The choroid plexus showed increases in peripheral immune cell proteins. Proteins that increased in abundance in the vasculature during GBS invasion were associated with major histocompatibility complex (MHC) class I antigen processing and endoplasmic reticulum dysfunction, a finding which correlated with altered host protein glycosylation profiles. Globally, there was low concordance between the infection proteome of whole brains and isolated vascular tissues. This report underscores the utility of unbiased, systems-scale analyses of functional tissue substructures for understanding disease. Group B (GBS) meningitis remains a major cause of poor health outcomes very early in life. Both the host-pathogen relationship leading to disease and the massive host response to infection contributing to these poor outcomes are orchestrated at the tissue and cell type levels. GBS meningitis is thought to result when bacteria present in the blood circumvent the selectively permeable vascular barriers that feed the brain. Additionally, tissue damage subsequent to bacterial invasion is mediated by inflammation and by immune cells from the periphery crossing the blood-brain barrier. Indeed, the vasculature plays a central role in disease processes occurring during GBS infection of the brain. Here, we employed quantitative proteomic analysis of brain vascular substructures during invasive GBS disease. We used the generated data to map molecular alterations associated with tissue perturbation, finding widespread intracellular dysfunction and punctuating the importance of investigations relegated to tissue type over the whole organ.
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http://dx.doi.org/10.1128/mSystems.00368-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449606PMC
August 2020

Antibiotics and Innate Immunity: A Cooperative Effort Toward the Successful Treatment of Infections.

Open Forum Infect Dis 2020 Aug 20;7(8):ofaa302. Epub 2020 Jul 20.

Collaborative to Halt Antimicrobial Resistant Microbes, University of California San Diego School of Medicine, La Jolla, California, USA.

Despite the common ancestry of antimicrobial and immunological science, a divergence driven by artificially construed paradigms in microbiology has placed limits on how we understand the mechanisms of antibiotics in vivo. We summarize recent updates on data that shed light on how antibiotics interact with components of innate immunity.
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http://dx.doi.org/10.1093/ofid/ofaa302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423293PMC
August 2020

Opportunistic Invasive Infection by Group A Streptococcus During Anti-Interleukin-6 Immunotherapy.

J Infect Dis 2021 Apr;223(7):1260-1264

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA.

Invasive group A Streptococcus (GAS) in immunocompetent individuals is largely linked to hypervirulent strains. Congenital immunodeficiencies and those acquired from chronic disease or immunosuppressant drugs also increase risk of severe illness. We recovered GAS from the blood of a patient receiving a biologic inhibitor of interleukin 6 (IL-6). Growth of this serotype M4 isolate in human blood or a murine bacteremia model was promoted by interleukin 1 or IL-6 inhibition. Hyperinvasive M1T1 GAS was unaffected by IL-6 in both models. These findings based on a natural experiment introduce IL-6 signaling deficiencies as a risk factor for invasive GAS.
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http://dx.doi.org/10.1093/infdis/jiaa511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8030709PMC
April 2021

T4 Pili Promote Colonization and Immune Evasion Phenotypes of Nonencapsulated M4 Streptococcus pyogenes.

mBio 2020 07 21;11(4). Epub 2020 Jul 21.

Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan

(group A [GAS]) is an important human pathogen causing a broad spectrum of diseases and associated with significant global morbidity and mortality. Almost all GAS isolates express a surface hyaluronic acid capsule, a virulence determinant that facilitates host colonization and impedes phagocyte killing. However, recent epidemiologic surveillance has reported a sustained increase in both mucosal and invasive infections caused by nonencapsulated GAS, which questions the indispensable role of hyaluronic acid capsule in GAS pathogenesis. In this study, we found that pilus of M4 GAS not only significantly promotes biofilm formation, adherence, and cytotoxicity to human upper respiratory tract epithelial cells and keratinocytes, but also promotes survival in human whole blood and increased virulence in murine models of invasive infection. T4 antigen, the pilus backbone protein of M4 GAS, binds haptoglobin, an abundant human acute-phase protein upregulated upon infection and inflammation, on the bacterial surface. Haptoglobin sequestration reduces the susceptibility of nonencapsulated M4 GAS to antimicrobial peptides released from activated neutrophils and platelets. Our results reveal a previously unappreciated virulence-promoting role of M4 GAS pili, in part mediated by co-opting the biology of haptoglobin to mitigate host antimicrobial defenses. Group A (GAS) is a strict human pathogen causing more than 700 million infections globally each year. The majority of the disease-causing GAS are encapsulated, which greatly guarantees survival and dissemination in the host. Emergence of the capsule-negative GAS, such as M4 GAS, in recent epidemiologic surveillance alarms the necessity to elucidate the virulence determinants of these pathogens. Here, we found that M4 pili play an important role in promoting M4 GAS adherence and cytotoxicity to human pharyngeal epithelial cells and keratinocytes. The same molecule also significantly enhanced M4 GAS survival and replication in human whole blood and experimental murine infection. T4 antigen, which composes the backbone of M4 pili, was able to sequester the very abundant serum protein haptoglobin to further confer M4 GAS resistance to antibacterial substances released by neutrophils and platelets.
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http://dx.doi.org/10.1128/mBio.01580-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374061PMC
July 2020

An Irreversible Inhibitor to Probe the Role of Cysteine Protease SpeB in Evasion of Host Complement Defenses.

ACS Chem Biol 2020 08 28;15(8):2060-2069. Epub 2020 Jul 28.

Members of the CA class of cysteine proteases have multifaceted roles in physiology and virulence for many bacteria. Streptococcal pyrogenic exotoxin B (SpeB) is secreted by and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we developed and characterized a clickable inhibitor, , based on X-ray crystallographic analysis and structure-activity relationships. Our SpeB probe showed irreversible enzyme inhibition in biochemical assays and labeled endogenous SpeB in cultured supernatants. Importantly, application of decreased survival in the presence of human neutrophils and supports the role of SpeB-mediated proteolysis as a mechanism to limit complement-mediated host defense. We posit that our SpeB inhibitor will be a useful chemical tool to regulate, label, and quantitate secreted cysteine proteases with SpeB-like activity in complex biological samples and a lead candidate for new therapeutics designed to sensitize to host immune clearance.
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http://dx.doi.org/10.1021/acschembio.0c00191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7755099PMC
August 2020

Revealing 29 sets of independently modulated genes in , their regulators, and role in key physiological response.

Proc Natl Acad Sci U S A 2020 07 2;117(29):17228-17239. Epub 2020 Jul 2.

Department of Bioengineering, University of California San Diego, La Jolla, CA 92093;

The ability of to infect many different tissue sites is enabled, in part, by its transcriptional regulatory network (TRN) that coordinates its gene expression to respond to different environments. We elucidated the organization and activity of this TRN by applying independent component analysis to a compendium of 108 RNA-sequencing expression profiles from two clinical strains (TCH1516 and LAC). ICA decomposed the transcriptome into 29 independently modulated sets of genes (i-modulons) that revealed: 1) High confidence associations between 21 i-modulons and known regulators; 2) an association between an i-modulon and σS, whose regulatory role was previously undefined; 3) the regulatory organization of 65 virulence factors in the form of three i-modulons associated with AgrR, SaeR, and Vim-3; 4) the roles of three key transcription factors (CodY, Fur, and CcpA) in coordinating the metabolic and regulatory networks; and 5) a low-dimensional representation, involving the function of few transcription factors of changes in gene expression between two laboratory media (RPMI, cation adjust Mueller Hinton broth) and two physiological media (blood and serum). This representation of the TRN covers 842 genes representing 76% of the variance in gene expression that provides a quantitative reconstruction of transcriptional modules in , and a platform enabling its full elucidation.
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http://dx.doi.org/10.1073/pnas.2008413117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382225PMC
July 2020

Developmental Immaturity of Siglec Receptor Expression on Neonatal Alveolar Macrophages Predisposes to Severe Group B Streptococcal Infection.

iScience 2020 Jun 28;23(6):101207. Epub 2020 May 28.

Department of Pediatrics, University of California, San Diego, Rady Children's Hospital, San Diego, 9500 Gilman Drive, Mail Code 0760, La Jolla, CA 92093-0760, USA. Electronic address:

Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common neonatal pathogen. However, the cellular and molecular mechanisms for neonatal susceptibility to GBS pneumonia and sepsis are incompletely understood. Here we optimized a mouse model of GBS pneumonia to test the role of alveolar macrophage (ΑΜΦ) maturation in host vulnerability to disease. Compared with juvenile and adult mice, neonatal mice infected with GBS had increased mortality and persistence of lung injury. In addition, neonatal mice were defective in GBS phagocytosis and killing. ΑΜΦ depletion and disruption of ΑΜΦ differentiation in Csf2 mice both impaired GBS clearance. AMΦ engage the heavily sialylated GBS capsule via the cell surface Siglec receptors Sn and Siglec-E. Although both newborn and adult ΑΜΦ expressed Siglec-E, newborn ΑΜΦ expressed significantly lower levels of Sn. We propose that a developmental delay in Sn expression on ΑΜΦ may prevent effective killing and clearing of GBS from the newborn lung.
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http://dx.doi.org/10.1016/j.isci.2020.101207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300150PMC
June 2020

All major cholesterol-dependent cytolysins use glycans as cellular receptors.

Sci Adv 2020 May 22;6(21):eaaz4926. Epub 2020 May 22.

Institute for Glycomics, Griffith University, Gold Coast, QLD 4222, Australia.

Cholesterol-dependent cytolysins (CDCs) form pores in cholesterol-rich membranes, but cholesterol alone is insufficient to explain their cell and host tropism. Here, we show that all eight major CDCs have high-affinity lectin activity that identifies glycans as candidate cellular receptors. Streptolysin O, vaginolysin, and perfringolysin O bind multiple glycans, while pneumolysin, lectinolysin, and listeriolysin O recognize a single glycan class. Addition of exogenous carbohydrate receptors for each CDC inhibits toxin activity. We present a structure for suilysin domain 4 in complex with two distinct glycan receptors, P antigen and αGal/Galili. We report a wide range of binding affinities for cholesterol and for the cholesterol analog pregnenolone sulfate and show that CDCs bind glycans and cholesterol independently. Intermedilysin binds to the sialyl-TF -glycan on its erythrocyte receptor, CD59. Removing sialyl-TF from CD59 reduces intermedilysin binding. Glycan-lectin interactions underpin the cellular tropism of CDCs and provide molecular targets to block their cytotoxic activity.
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http://dx.doi.org/10.1126/sciadv.aaz4926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244308PMC
May 2020

Sulfur(VI) Fluoride Exchange (SuFEx)-Enabled High-Throughput Medicinal Chemistry.

J Am Chem Soc 2020 06 10;142(25):10899-10904. Epub 2020 Jun 10.

Laboratory of Advanced Food Process Engineering, Osaka Prefecture University, 1-2, Gakuen-cho, Nakaku, Sakai, Osaka 599-8570, Japan.

Optimization of small-molecule probes or drugs is a synthetically lengthy, challenging, and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible sulfur(VI) fluoride exchange (SuFEx) click chemistry. A high-throughput screening hit benzyl (cyanomethyl)carbamate ( = 8 μM) against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN═S(O)F] motif, rapidly diversified into 460 analogs in overnight reactions, and the products were directly screened to yield drug-like inhibitors with 480-fold higher potency ( = 18 nM). We showed that the improved molecule is active in a bacteria-host coculture. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.
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http://dx.doi.org/10.1021/jacs.9b13652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751259PMC
June 2020

Tuning the Innate Immune Response to Cyclic Dinucleotides by Using Atomic Mutagenesis.

Chembiochem 2020 09 8;21(18):2595-2598. Epub 2020 Jun 8.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0358, USA.

Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues. By employing an "atomic mutagenesis" strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c-di-GMP analogues induce type-I interferon (IFN) production, with some being more potent than c-di-GMP, their native archetype. This study demonstrates that CDN analogues bearing modified nucleobases are able to tune the innate immune response in eukaryotic cells.
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http://dx.doi.org/10.1002/cbic.202000162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494572PMC
September 2020