Publications by authors named "Steffen Porwollik"

111 Publications

Eradication of Intracellular Salmonella Typhimurium by Polyplexes of Acid-Transforming Chitosan and Fragment DNA.

Macromol Biosci 2021 Apr 18:e2000408. Epub 2021 Apr 18.

132 Sprague Hall, Irvine, CA, 92697, USA.

Antibiotics are highly successful against microbial infections. However, current challenges include rising antibiotic resistance rates and limited efficacy against intracellular pathogens. A novel form of a nanomaterial-based antimicrobial agent is investigated for efficient treatment of an intracellular Salmonella enterica sv Typhimurium infection. A known antimicrobial polysaccharide, chitosan, is engineered to be readily soluble under neutral aqueous conditions for systemic administration. The modified biologic, named acid-transforming chitosan (ATC), transforms into an insoluble, antimicrobial compound in the mildly acidic intracellular compartment. In cell culture experiments, ATC is confirmed to have antimicrobial activity against intracellular S. Typhimurium in a concentration- and pH-dependent manner, without affecting the host cells, RAW264.7 macrophages. For improved cellular uptake and pharmacokinetic/pharmacodynamic properties, ATC is further complexed with fragment DNA (fDNA), to form nano-sized spherical polyplexes. The resulting ATC/fDNA polyplexes efficiently eradicated S. Typhimurium from RAW264.7 macrophages. ATC/fDNA polyplexes may bind with microbial wall and membrane components. Consistent with this expectation, transposon insertion sequencing of a complex random mutant S. Typhimurium library incubated with ATC does not reveal specific genomic target regions of the antimicrobial. This study demonstrates the utility of a molecularly engineered nanomaterial as an efficient and safe antimicrobial agent, particularly against an intracellular pathogen.
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http://dx.doi.org/10.1002/mabi.202000408DOI Listing
April 2021

The multi-drug efflux system AcrABZ-TolC is essential for infection of Typhimurium by the flagellum-dependent bacteriophage Chi.

J Virol 2021 Mar 17. Epub 2021 Mar 17.

Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA

Bacteriophages are the most abundant biological entities in the biosphere. Due to their host specificity and ability to kill bacteria rapidly, bacteriophages have many potential healthcare applications, including therapy against antibiotic-resistant bacteria. Infection by flagellotropic bacteriophages requires a properly rotating bacterial flagellar filament. The flagella-dependent phage χ (Chi) infects serovars of the pathogenic enterobacterium However, cell surface receptors and proteins involved in other stages of χ infection have not been discovered to date. We screened a multi-gene deletion library of serovar Typhimurium by spotting mutants on soft agar plates seeded with bacteriophage χ and monitoring their ability to grow and form a swim ring, a characteristic of bacteriophage-resistant motile mutants. Those multi-gene deletion regions identified to be important for χ infectivity were further investigated by characterizing the phenotypes of corresponding single-gene deletion mutants. This way, we identified motile mutants with varying degrees of resistance to χ. Deletions in individual genes encoding the AcrABZ-TolC multi-drug efflux system drastically reduced infection by bacteriophage χ. Furthermore, an triple deletion strain was fully resistant to χ. Infection was severely reduced but not entirely blocked by the deletion of the gene encoding the molecular chaperone trigger factor. Finally, deletion in genes encoding enzymes involved in the synthesis of the antioxidants glutathione (GSH) and uric acid resulted in reduced infectivity. Our findings begin to elucidate poorly understood processes involved in later stages of flagellotropic bacteriophage infection and informs research aimed at the use of bacteriophages to combat antibiotic-resistant bacterial infections.Antimicrobial resistance is a large concern in the healthcare field. With more multi-drug resistant bacterial pathogens emerging, other techniques for eliminating bacterial infections are being explored. Among these is phage therapy, where combinations of specific phages are used to treat infections. Generally, phages utilize cell appendages and surface receptors for the initial attachment to their host. Phages that are flagellotropic are of particular interest because flagella are often important in bacterial virulence, making resistance to attachment of these phages harder to achieve without reducing virulence. This study discovered the importance of a multi-drug efflux pump for the infection of by a flagellotropic phage. In theory, if a bacterial pathogen develops phage resistance by altering expression of the efflux pump then the pathogen would simultaneously become more susceptible to the antibiotic substrates of the pump. Thus, co-administering antibiotics and flagellotropic phage may be a particularly potent antibacterial therapy.
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http://dx.doi.org/10.1128/JVI.00394-21DOI Listing
March 2021

Mechanisms of Attachment and Survival on In-Shell Black Peppercorns, Almonds, and Hazelnuts.

Front Microbiol 2020 23;11:582202. Epub 2020 Oct 23.

Department of Food Science and Nutrition, Illinois Institute of Technology, Bedford Park, IL, United States.

subspecies I (ssp 1) is the leading cause of hospitalizations and deaths due to known bacterial foodborne pathogens in the United States and is frequently implicated in foodborne disease outbreaks associated with spices and nuts. However, the underlying mechanisms of this association have not been fully elucidated. In this study, we evaluated the influence of storage temperature (4 or 25°C), relative humidity (20 or 60%), and food surface characteristics on the attachment and survival of five individual strains representing ssp 1 serovars Typhimurium, Montevideo, Braenderup, Mbandaka, and Enteritidis on raw in-shell black peppercorns, almonds, and hazelnuts. We observed a direct correlation between the food surface roughness and ssp 1 attachment, and detected significant inter-strain difference in survival on the shell surface under various storage conditions. A combination of low relative humidity (20%) and ambient storage temperature (25°C) resulted in the most significant reduction of on shell surfaces ( < 0.05). To identify genes potentially associated with attachment and survival on shell surfaces, we inoculated a library of 120,000 random transposon insertion mutants of an Enteritidis strain on almond shells, and screened for mutant survival after 1, 3, 7, and 14 days of storage at 20% relative humidity and 25°C. Mutants in 155 . Enteritidis genes which are involved in carbohydrate metabolic pathways, aerobic and anaerobic respiration, inner membrane transport, and glutamine synthesis displayed significant selection on almond shells ( < 0.05). Findings of this study suggest that various food attributes, environmental factors, and an unexpectedly complex metabolic and regulatory network in ssp 1 collectively contribute to the bacterial attachment and survival on low moisture shell surface, providing new data for the future development of knowledge-based intervention strategies.
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http://dx.doi.org/10.3389/fmicb.2020.582202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644838PMC
October 2020

Identification of Novel Genes Mediating Survival of on Low-Moisture Foods via Transposon Sequencing Analysis.

Front Microbiol 2020 15;11:726. Epub 2020 May 15.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, United States.

is the leading foodborne pathogen associated with outbreaks involving low-moisture foods (LMFs). However, the genes involved in s long-term survival on LMFs remain poorly characterized. In this study, in-shell pistachios were inoculated with Tnbased mutant libraries of . Enteritidis P125109, . Typhimurium 14028s, and . Newport C4.2 at approximate 10 CFU/g and stored at 25°C. Transposon sequencing analysis (Tn-seq) was then employed to determine the relative abundance of each Tn insertion site immediately after inoculation (T), after drying (T), and at 120 days (T). In . Enteritidis, . Typhimurium, and . Newport mutant libraries, the relative abundance of 51, 80, and 101 Tn insertion sites, respectively, was significantly lower at T compared to T, while in libraries of . Enteritidis and . Typhimurium the relative abundance of 42 and 68 Tn insertion sites, respectively, was significantly lower at T compared to T. Tn insertion sites with reduced relative abundance in this competition assay were localized in DNA repair, lipopolysaccharide biosynthesis and stringent response genes. Twelve genes among those under strong negative selection in the competition assay were selected for further study. Whole gene deletion mutants in ten of these genes, , , , , , , , , and , were impaired for individual survival on pistachios. The findings highlight the value of combined mutagenesis and sequencing to identify novel genes important for the survival of in low-moisture foods.
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http://dx.doi.org/10.3389/fmicb.2020.00726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242855PMC
May 2020

Import of Aspartate and Malate by DcuABC Drives H/Fumarate Respiration to Promote Initial Salmonella Gut-Lumen Colonization in Mice.

Cell Host Microbe 2020 06 15;27(6):922-936.e6. Epub 2020 May 15.

Institute of Microbiology, D-BIOL, ETH Zürich, 8093 Zürich, Switzerland. Electronic address:

Initial enteropathogen growth in the microbiota-colonized gut is poorly understood. Salmonella Typhimurium is metabolically adaptable and can harvest energy by anaerobic respiration using microbiota-derived hydrogen (H) as an electron donor and fumarate as an electron acceptor. As fumarate is scarce in the gut, the source of this electron acceptor is unclear. Here, transposon sequencing analysis along the colonization trajectory of S. Typhimurium implicates the C4-dicarboxylate antiporter DcuABC in early murine gut colonization. In competitive colonization assays, DcuABC and enzymes that convert the C4-dicarboxylates aspartate and malate into fumarate (AspA, FumABC), are required for fumarate/H-dependent initial growth. Thus, S. Typhimurium obtains fumarate by DcuABC-mediated import and conversion of L-malate and L-aspartate. Fumarate reduction yields succinate, which is exported by DcuABC in exchange for L-aspartate and L-malate. This cycle allows S. Typhimurium to harvest energy by H/fumarate respiration in the microbiota-colonized gut. This strategy may also be relevant for commensal E. coli diminishing the S. Typhimurium infection.
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http://dx.doi.org/10.1016/j.chom.2020.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292772PMC
June 2020

Glycolytic reprograming in Salmonella counters NOX2-mediated dissipation of ΔpH.

Nat Commun 2020 04 14;11(1):1783. Epub 2020 Apr 14.

Department of Immunology & Microbiology, University of Colorado School of Medicine, 12800 E. 19th Ave, Mail Box 8333, Aurora, CO, 80045, USA.

The microbial adaptations to the respiratory burst remain poorly understood, and establishing how the NADPH oxidase (NOX2) kills microbes has proven elusive. Here we demonstrate that NOX2 collapses the ΔpH of intracellular Salmonella Typhimurium. The depolarization experienced by Salmonella undergoing oxidative stress impairs folding of periplasmic proteins. Depolarization in respiring Salmonella mediates intense bactericidal activity of reactive oxygen species (ROS). Salmonella adapts to the challenges oxidative stress imposes on membrane bioenergetics by shifting redox balance to glycolysis and fermentation, thereby diminishing electron flow through the membrane, meeting energetic requirements and anaplerotically generating tricarboxylic acid intermediates. By diverting electrons away from the respiratory chain, glycolysis also enables thiol/disulfide exchange-mediated folding of bacterial cell envelope proteins during periods of oxidative stress. Thus, primordial metabolic pathways, already present in bacteria before aerobic respiration evolved, offer a solution to the stress ROS exert on molecular targets at the bacterial cell envelope.
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http://dx.doi.org/10.1038/s41467-020-15604-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156505PMC
April 2020

SpoT Induces Intracellular Virulence Programs in the Phagosome.

mBio 2020 02 25;11(1). Epub 2020 Feb 25.

University of Colorado School of Medicine, Department of Immunology and Microbiology, Aurora, Colorado, USA

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In , these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as , because the hydrolytic activity of this elA and poT omologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a -Δ mutant strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." expressing the -Δ variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. -Δ mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, -Δ mutant replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA. Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in , this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.
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http://dx.doi.org/10.1128/mBio.03397-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042702PMC
February 2020

Serovar Typhimurium 14028s Genomic Regions Required for Colonization of Lettuce Leaves.

Front Microbiol 2020 24;11. Epub 2020 Jan 24.

Department of Plant Sciences, University of California, Davis, Davis, CA, United States.

Contamination of edible produce leaves with human bacterial pathogens has been associated with serious disease outbreaks and has become a major public health concern affecting all aspects of the market, from farmers to consumers. While pathogen populations residing on the surface of ready-to-eat produce can be potentially removed through thorough washing, there is no disinfection technology available that effectively eliminates internal bacterial populations. By screening 303 multi-gene deletion (MGD) mutants of serovar Typhimurium (STm) 14028s, we were able to identify ten genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the bacterial membrane, and biosynthesis of surface appendages. Interestingly, at 21 days post inoculation, seven of these mutants showed increased population titers inside the leaf, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not Mut3, was significantly impaired in the leaf apoplastic wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. The genetic basis of STm colonization of leaves investigated in this study provides a foundation from which to develop mitigation tactics to enhance food safety.
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http://dx.doi.org/10.3389/fmicb.2020.00006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993584PMC
January 2020

Discovery of trehalose phospholipids reveals functional convergence with mycobacteria.

J Exp Med 2019 04 25;216(4):757-771. Epub 2019 Feb 25.

Department of Infectious Diseases and Immunology, School of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

species are among the world's most prevalent pathogens. Because the cell wall interfaces with the host, we designed a lipidomics approach to reveal pathogen-specific cell wall compounds. Among the molecules differentially expressed between Paratyphi and Typhi, we focused on lipids that are enriched in Typhi, because it causes typhoid fever. We discovered a previously unknown family of trehalose phospholipids, 6,6'-diphosphatidyltrehalose (diPT) and 6-phosphatidyltrehalose (PT). Cardiolipin synthase B (ClsB) is essential for PT and diPT but not for cardiolipin biosynthesis. Chemotyping outperformed homology analysis in evaluating synthesis of diPT. DiPT is restricted to a subset of Gram-negative bacteria: large amounts are produced by Typhi, lower amounts by other pathogens, and variable amounts by strains. DiPT activates Mincle, a macrophage activating receptor that also recognizes mycobacterial cord factor (6,6'-trehalose dimycolate). Thus, Gram-negative bacteria show convergent function with mycobacteria. Overall, we discovered a previously unknown immunostimulant that is selectively expressed among medically important bacterial species.
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http://dx.doi.org/10.1084/jem.20181812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446866PMC
April 2019

Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium.

PLoS One 2019 4;14(2):e0211584. Epub 2019 Feb 4.

Department of Microbial Physiology, Humboldt Universität zu Berlin, Berlin, Germany.

The Cpx-envelope stress system regulates the expression of virulence factors in many Gram-negative pathogens. In Salmonella enterica serovar Typhimurium deletion of the sensor kinase CpxA but not of the response regulator CpxR results in the down regulation of the key regulator for invasion, HilA encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we provide evidence that cpxA deletion interferes with dephosphorylation of CpxR resulting in increased levels of active CpxR and consequently in misregulation of target genes. 14 potential operons were identified to be under direct control of CpxR. These include the virulence determinants ecotin, the omptin PgtE, and the SPI-2 regulator SsrB. The Tat-system and the PocR regulator that together promote anaerobic respiration of tetrathionate on 1,2-propanediol are also under direct CpxR control. Notably, 1,2-propanediol represses hilA expression. Thus, our work demonstrates for the first time the involvement of the Cpx system in a complex network mediating metabolism and virulence function.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211584PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361445PMC
November 2019

A macrophage-based screen identifies antibacterial compounds selective for intracellular Salmonella Typhimurium.

Nat Commun 2019 01 14;10(1):197. Epub 2019 Jan 14.

Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St W, Hamilton, ON, L8S 4K1, Canada.

Salmonella Typhimurium (S. Tm) establishes systemic infection in susceptible hosts by evading the innate immune response and replicating within host phagocytes. Here, we sought to identify inhibitors of intracellular S. Tm replication by conducting parallel chemical screens against S. Tm growing in macrophage-mimicking media and within macrophages. We identify several compounds that inhibit Salmonella growth in the intracellular environment and in acidic, ion-limited media. We report on the antimicrobial activity of the psychoactive drug metergoline, which is specific against intracellular S. Tm. Screening an S. Tm deletion library in the presence of metergoline reveals hypersensitization of outer membrane mutants to metergoline activity. Metergoline disrupts the proton motive force at the bacterial cytoplasmic membrane and extends animal survival during a systemic S. Tm infection. This work highlights the predictive nature of intracellular screens for in vivo efficacy, and identifies metergoline as a novel antimicrobial active against Salmonella.
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http://dx.doi.org/10.1038/s41467-018-08190-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331611PMC
January 2019

Zinc-dependent substrate-level phosphorylation powers Salmonella growth under nitrosative stress of the innate host response.

PLoS Pathog 2018 10 26;14(10):e1007388. Epub 2018 Oct 26.

Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO, United States of America.

The metabolic processes that enable the replication of intracellular Salmonella under nitrosative stress conditions engendered in the innate response of macrophages are poorly understood. A screen of Salmonella transposon mutants identified the ABC-type high-affinity zinc uptake system ZnuABC as a critical determinant of the adaptation of Salmonella to the nitrosative stress generated by the enzymatic activity of inducible nitric oxide (NO) synthase of mononuclear phagocytic cells. NO limits the virulence of a znuB mutant in an acute murine model of salmonellosis. The ZnuABC transporter is crucial for the glycolytic function of fructose bisphosphate aldolase, thereby fueling growth of Salmonella during nitrosative stress produced in the innate response of macrophages. Our investigations demonstrate that glycolysis mediates resistance of Salmonella to the antimicrobial activity of NO produced in an acute model of infection. The ATP synthesized by substrate-level phosphorylation at the payoff phase of glycolysis and acetate fermentation powers the replication of Salmonella experiencing high levels of nitrosative stress. In contrast, despite its high potential for ATP synthesis, oxidative phosphorylation is a major target of inhibition by NO and contributes little to the antinitrosative defenses of intracellular Salmonella. Our investigations have uncovered a previously unsuspected conjunction between zinc homeostasis, glucose metabolism and cellular energetics in the adaptation of intracellular Salmonella to the reactive nitrogen species synthesized in the innate host response.
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http://dx.doi.org/10.1371/journal.ppat.1007388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221366PMC
October 2018

Neutral barcoding of genomes reveals the dynamics of Salmonella colonization in cattle and their peripheral lymph nodes.

Vet Microbiol 2018 Jul 16;220:97-106. Epub 2018 May 16.

Dept. of Microbiology and Molecular Genetics, UC Irvine, B240 Med Sci Bldg., Irvine, CA 92697, United States. Electronic address:

Feedlot cattle often contain Salmonella. The number of bacteria that initiate colonization of different cattle organs and the bacterial migration within these large animals are poorly understood. To investigate these questions, we constructed wild-type isogenic tagged strains (WITS) of Salmonella by inserting 21-base barcodes flanked by Illumina sequencing primers into a neutral genome location. We then delivered several different pools of uniquely barcoded clones orally and into multiple intradermal sites, in individual Holstein steers, and subsequently performed Salmonella-directed sequence tag-based analysis of microbial populations (STAMP). Using high-throughput sequencing of the barcodes of Salmonella grown from steer lymph nodes, organs and feces, we monitored how individual barcoded clones travel from different entry sites within animals. Data showed that gastrointestinal colonization was established by up to hundreds of Salmonella founder cells, whereas peripheral lymph nodes were usually colonized by very low numbers of founding bacteria, often originating from the nearest draining intradermal delivery site. Transmission of Salmonella from the gastrointestinal tract to the lymphatic system was frequently observed, whereas entry of intradermally delivered bacteria into the gut was rare. Bacteria undergo limited extraintestinal proliferation within or prior to arrival at peripheral lymph nodes. Overall, the application of the STAMP technique facilitated characterization of the migration routes and founder population size of Salmonella within feedlot cattle and their organs and lymph nodes in unprecedented detail.
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http://dx.doi.org/10.1016/j.vetmic.2018.05.007DOI Listing
July 2018

Genome-Wide Comparative Functional Analyses Reveal Adaptations of sv. Newport to a Plant Colonization Lifestyle.

Front Microbiol 2018 18;9:877. Epub 2018 May 18.

Soil and Water Sciences Department, University of Florida, Gainesville, FL, United States.

Outbreaks of salmonellosis linked to the consumption of vegetables have been disproportionately associated with strains of serovar Newport. We tested the hypothesis that strains of sv. Newport have evolved unique adaptations to persistence in plants that are not shared by strains of other serovars. We used a genome-wide mutant screen to compare growth in tomato fruit of a sv. Newport strain from an outbreak traced to tomatoes, and a sv. Typhimurium strain from animals. Most genes in the sv. Newport strain that were selected during persistence in tomatoes were shared with, and similarly selected in, the sv. Typhimurium strain. Many of their functions are linked to central metabolism, including amino acid biosynthetic pathways, iron acquisition, and maintenance of cell structure. One exception was a greater need for the core genes involved in purine metabolism in sv. Typhimurium than in sv. Newport. We discovered a gene, , that was unique to sv. Newport and contributed to the strain's fitness in tomatoes. The gene was present in about 25% of sv. Newport Group III genomes and generally absent from other genomes. Homologs of were detected in the genomes of , and , members of the Enterobacteriacea family that can colonize both plants and animals.
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http://dx.doi.org/10.3389/fmicb.2018.00877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5968271PMC
May 2018

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens.

Mol Microbiol 2018 05 30;108(3):288-305. Epub 2018 Mar 30.

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full-length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.
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http://dx.doi.org/10.1111/mmi.13936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912970PMC
May 2018

A simplified multiplex PCR-based typing method for common serovars supported by online server-based detection system.

Indian J Med Res 2017 Aug;146(2):272-280

Deparment of Microbiology & Molecular Genetics, University of California, Irvine, USA.

Background & Objectives: A rapid and simple alternative method is needed to replace the laborious, time-consuming Salmonella serotyping. The objective of the present study was to improve and simplify a previously reported multiplex polymerase chain reaction (PCR)-based method and to create an online server to enable rapid determination of serovars.

Methods: A method of multiplex PCR-based genome typing (MPGT) was standardized using 59 Salmonella isolates of 31 serovars. Several previously reported primers were modified to obtain a more accurate performance. The screen was separated into four different multiplex reactions distinguishable on standard electrophoresis. A blind study was subsequently performed with 81 isolates of 10 serovars most prevalent in India. Whole genome information from 440 Salmonella isolates was used to confirm the usefulness of this method and concurrence of in silico predictions and PCR results were investigated. A public server (http://www.mpgt-salmonella.res.in) was established for data storage and determination of closest previously observed Salmonella isolates based on obtained MPGT patterns.

Results: The 16 target genes amplified showed variability in their presence in strains from different serotypes. Hence, identical amplification patterns suggested genetic relatedness of strains and usually identical serological behaviour. The observed absence/presence patterns of genes were converted to an MPGT code. Altogether, 83 different codes were predicted in silico based on the whole genome information of 440 strains. Results confirmed that major serovars usually displayed unique MPGT codes.

Interpretation & Conclusions: The multiplex PCR assay resulted in specific binary codes for isolates from each of the 31 Salmonella serovars tested. The online server allowed the user to compare obtained PCR results with stored previous patterns. Simplicity, speed and cost-effectiveness make this tool useful for quick outbreak management.
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http://dx.doi.org/10.4103/ijmr.IJMR_1258_15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761039PMC
August 2017

Interactions of Salmonella enterica Serovar Typhimurium and Pectobacterium carotovorum within a Tomato Soft Rot.

Appl Environ Microbiol 2018 03 14;84(5). Epub 2018 Feb 14.

Soil and Water Science Department, Genetics Institute, University of Florida-IFAS, Gainesville, Florida, USA.

spp. are remarkably adaptable pathogens, and this adaptability allows these bacteria to thrive in a variety of environments and hosts. The mechanisms with which these pathogens establish within a niche amid the native microbiota remain poorly understood. Here, we aimed to uncover the mechanisms that enable serovar Typhimurium strain ATCC 14028 to benefit from the degradation of plant tissue by a soft rot plant pathogen, The hypothesis that in the soft rot, the liberation of starch (not utilized by ) makes this polymer available to spp., thus allowing it to colonize soft rots, was tested first and proven null. To identify the functions involved in soft rot colonization, we carried out transposon insertion sequencing coupled with the phenotypic characterization of the mutants. The data indicate that spp. experience a metabolic shift in response to the changes in the environment brought on by spp. and likely coordinated by the small regulatory RNA. While and appear to be of importance in the soft rot, the global two-component system encoded by (which controls and under laboratory conditions) does not appear to be necessary for the observed phenotype. Motility and the synthesis of nucleotides and amino acids play critical roles in the growth of spp. in the soft rot. Outbreaks of produce-associated illness continue to be a food safety concern. Earlier studies demonstrated that the presence of phytopathogens on produce was a significant risk factor associated with increased carriage on fruits and vegetables. Here, we genetically characterize some of the requirements for interactions between and phytobacteria that allow spp. to establish a niche within an alternate host (tomato). Pathways necessary for nucleotide synthesis, amino acid synthesis, and motility are identified as contributors to the persistence of spp. in soft rots.
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http://dx.doi.org/10.1128/AEM.01913-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812938PMC
March 2018

Gene Expression Response of Serotype Enteritidis Phage Type 8 to Subinhibitory Concentrations of the Plant-Derived Compounds -Cinnamaldehyde and Eugenol.

Front Microbiol 2017 26;8:1828. Epub 2017 Sep 26.

Department of Animal Science, University of Connecticut, Storrs, CT, United States.

Enteritidis phage type 8 (PT8) is a major poultry-associated strain implicated in foodborne outbreaks in the United States. We previously reported that two plant-derived compounds generally recognized as safe (GRAS), -cinnamaldehyde (TC), and eugenol (EG), significantly reduced . Enteritidis colonization in broiler and layer chickens. To elucidate potential PT8 genes affected by TC and EG during colonization, a whole-genome microarray analysis of the bacterium treated with TC and EG was conducted. . Enteritidis PT8 was grown in Luria-Bertani broth at 37°C to an OD of ~0.5. Subinhibitory concentrations (SICs; concentration that does not inhibit bacterial growth) of TC (0.01%; 0.75 mM) or EG (0.04%; 2.46 mM) were then added to the culture. . Enteritidis PT8 RNA was extracted before and 30 min after TC or EG addition. Labeled cDNA from three replicate experiments was subsequently hybridized to a microarray of over 99% of . Enteritidis PT4 genes, and the hybridization signals were quantified. The plant-derived compounds down-regulated ( < 0.005) expression of . Enteritidis PT8 genes involved in flagellar motility, regulation of the Pathogenicity Island 1, and invasion of intestinal epithelial cells. TC and EG also suppressed transcription of genes encoding multiple transport systems and outer membrane proteins. Moreover, several metabolic and biosynthetic pathways in the pathogen were down-regulated during exposure to the plant-derived compounds. Both TC and EG stimulated the transcription of heat shock genes, such as , and in . Enteritidis PT8 ( < 0.005). The results obtained from microarray were validated using a quantitative real-time PCR. The plant-derived compounds TC and EG exert antimicrobial effects on . Enteritidis PT8 by affecting multiple genes, including those associated with virulence, colonization, cell membrane composition, and transport systems.
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http://dx.doi.org/10.3389/fmicb.2017.01828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623010PMC
September 2017

Novel DNA Binding and Regulatory Activities for σ (RpoN) in Salmonella enterica Serovar Typhimurium 14028s.

J Bacteriol 2017 06 25;199(12). Epub 2017 May 25.

Department of Microbiology, University of Georgia, Athens, Georgia, USA

The variable sigma (σ) subunit of the bacterial RNA polymerase (RNAP) holoenzyme, which is responsible for promoter specificity and open complex formation, plays a strategic role in the response to environmental changes. serovar Typhimurium utilizes the housekeeping σ and five alternative sigma factors, including σ The σ-RNAP differs from other σ-RNAP holoenzymes in that it forms a stable closed complex with the promoter and requires ATP hydrolysis by an activated cognate bacterial enhancer binding protein (bEBP) to transition to an open complex and initiate transcription. In Typhimurium, σ-dependent promoters normally respond to one of 13 different bEBPs, each of which is activated under a specific growth condition. Here, we utilized a constitutively active, promiscuous bEBP to perform a genome-wide identification of σ-RNAP DNA binding sites and the transcriptome of the σ regulon of Typhimurium. The position and context of many of the identified σ RNAP DNA binding sites suggest regulatory roles for σ-RNAP that connect the σ regulon to regulons of other σ factors to provide a dynamic response to rapidly changing environmental conditions. The alternative sigma factor σ (RpoN) is required for expression of genes involved in processes with significance in agriculture, bioenergy production, bioremediation, and host-microbe interactions. The characterization of the σ regulon of the versatile pathogen Typhimurium has expanded our understanding of the scope of the σ regulon and how it links to other σ regulons within the complex regulatory network for gene expression in bacteria.
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http://dx.doi.org/10.1128/JB.00816-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446619PMC
June 2017

Salmonella Persistence in Tomatoes Requires a Distinct Set of Metabolic Functions Identified by Transposon Insertion Sequencing.

Appl Environ Microbiol 2017 03 15;83(5). Epub 2017 Feb 15.

Soil and Water Science Department, Genetics Institute, University of Florida-IFAS, Gainesville, Florida, USA.

Human enteric pathogens, such as spp. and verotoxigenic , are increasingly recognized as causes of gastroenteritis outbreaks associated with the consumption of fruits and vegetables. Persistence in plants represents an important part of the life cycle of these pathogens. The identification of the full complement of genes involved in the colonization of the model plant (tomato) was carried out using transposon insertion sequencing analysis. With this approach, 230,000 transposon insertions were screened in tomato pericarps to identify loci with reduction in fitness, followed by validation of the screen results using competition assays of the isogenic mutants against the wild type. A comparison with studies in animals revealed a distinct plant-associated set of genes, which only partially overlaps with the genes required to elicit disease in animals. biosynthesis of amino acids was critical to persistence within tomatoes, while amino acid scavenging was prevalent in animal infections. Fitness reduction of the amino acid synthesis mutants was generally more severe in the tomato mutant, which hyperaccumulates certain amino acids, suggesting that these nutrients remain unavailable to spp. within plants. lipopolysaccharide (LPS) was required for persistence in both animals and plants, exemplifying some shared pathogenesis-related mechanisms in animal and plant hosts. Similarly to phytopathogens, spp. required biosynthesis of amino acids, LPS, and nucleotides to colonize tomatoes. Overall, however, it appears that while shares some strategies with phytopathogens and taps into its animal virulence-related functions, colonization of tomatoes represents a distinct strategy, highlighting this pathogen's flexible metabolism. Outbreaks of gastroenteritis caused by human pathogens have been increasingly associated with foods of plant origin, with tomatoes being one of the common culprits. Recent studies also suggest that these human pathogens can use plants as alternate hosts as a part of their life cycle. While dual (animal/plant) lifestyles of other members of the family are well known, the strategies with which colonizes plants are only partially understood. Therefore, we undertook a high-throughput characterization of the functions required for persistence within tomatoes. The results of this study were compared with what is known about genes required for virulence in animals and interactions of plant pathogens with their hosts to determine whether repurposes its virulence repertoire inside plants or whether it behaves more as a phytopathogen during plant colonization. Even though utilized some of its virulence-related genes in tomatoes, plant colonization required a distinct set of functions.
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http://dx.doi.org/10.1128/AEM.03028-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311394PMC
March 2017

Draft Genome Sequence of Salmonella enterica subsp. diarizonae Serovar 61:k:1,5,(7) Strain CRJJGF_00165 (Phylum Gammaproteobacteria).

Genome Announc 2016 Nov 23;4(6). Epub 2016 Nov 23.

Bacterial Epidemiology and Antimicrobial Resistance Research Unit, USDA-ARS, Athens, Georgia, USA

Here, we report a 4.78-Mb draft genome sequence of the Salmonella enterica subsp. diarizonae serovar 61:k:1,5,(7) strain CRJJGF_00165 [also called S. enterica subsp. IIIb serovar 61:k:1,5,(7) strain CRJJGF_00165], isolated from ground beef in 2007.
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http://dx.doi.org/10.1128/genomeA.01322-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122689PMC
November 2016

Contribution of Asparagine Catabolism to Salmonella Virulence.

Infect Immun 2017 02 26;85(2). Epub 2017 Jan 26.

Department of Molecular Genetics and Microbiology and Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, USA

Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that l-asparaginase II produced by Salmonella enterica serovar Typhimurium (S Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by l-asparaginase II of S Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for l-asparaginase II stability and function. Furthermore, we report that l-asparaginase II localizes primarily to the periplasm and acts together with l-asparaginase I to provide S Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S Typhimurium lacking both l-asparaginase I and II genes competes poorly with wild-type S Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S Typhimurium virulence, providing new insights into the competition for nutrients at the host-pathogen interface.
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http://dx.doi.org/10.1128/IAI.00740-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278173PMC
February 2017

Genetic Determinants of Salmonella enterica Serovar Typhimurium Proliferation in the Cytosol of Epithelial Cells.

Infect Immun 2016 Dec 18;84(12):3517-3526. Epub 2016 Nov 18.

Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA

Intestinal epithelial cells provide an important colonization niche for Salmonella enterica serovar Typhimurium during gastrointestinal infections. In infected epithelial cells, a subpopulation of S Typhimurium bacteria damage their internalization vacuole, leading to escape from the Salmonella-containing vacuole (SCV) and extensive proliferation in the cytosol. Little is known about the bacterial determinants of nascent SCV lysis and subsequent survival and replication of Salmonella in the cytosol. To pinpoint S Typhimurium virulence factors responsible for these steps in the intracellular infectious cycle, we screened a S Typhimurium multigene deletion library in Caco-2 C2Bbe1 and HeLa epithelial cells for mutants that had an altered proportion of cytosolic bacteria compared to the wild type. We used a gentamicin protection assay in combination with a chloroquine resistance assay to quantify total and cytosolic bacteria, respectively, for each strain. Mutants of three S Typhimurium genes, STM1461 (ydgT), STM2829 (recA), and STM3952 (corA), had reduced cytosolic proliferation compared to wild-type bacteria, and one gene, STM2120 (asmA), displayed increased cytosolic replication. None of the mutants were affected for lysis of the nascent SCV or vacuolar replication in epithelial cells, indicating that these genes are specifically required for survival and proliferation of S Typhimurium in the epithelial cell cytosol. These are the first genes identified to contribute to this step of the S Typhimurium infectious cycle.
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http://dx.doi.org/10.1128/IAI.00734-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116728PMC
December 2016

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Orion Strain CRJJGF_00093 (Phylum Gammaproteobacteria).

Genome Announc 2016 Sep 29;4(5). Epub 2016 Sep 29.

Bacterial Epidemiology and Antimicrobial Resistance Research Unit, USDA-ARS, Athens, Georgia, USA

Here, we report a 4.70-Mbp draft genome sequence of Salmonella enterica subsp. enterica serovar Orion strain CRJJGF_00093, isolated from a dog in 2005.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043568PMC
http://dx.doi.org/10.1128/genomeA.01063-16DOI Listing
September 2016

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Bardo Strain CRJJGF_00099 (Phylum Gammaproteobacteria).

Genome Announc 2016 Sep 15;4(5). Epub 2016 Sep 15.

Bacterial Epidemiology and Antimicrobial Resistance Research Unit, USDA-ARS, Athens, Georgia, USA

Here, we report a 4.87-Mbp draft genome sequence of the multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar Bardo strain CRJJGF_00099, isolated from dairy cattle in 2005.
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http://dx.doi.org/10.1128/genomeA.00964-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026435PMC
September 2016

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Blockley Strain CRJJGF_00147 (Phylum Gammaproteobacteria).

Genome Announc 2016 Sep 8;4(5). Epub 2016 Sep 8.

Bacterial Epidemiology and Antimicrobial Resistance Research Unit, USDA-ARS, Athens, Georgia, USA

Here, we report a 4.72-Mbp draft genome sequence of Salmonella enterica subsp. enterica serovar Blockley strain CRJJGF_00147, isolated from chicken rinse in 2009.
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http://dx.doi.org/10.1128/genomeA.00954-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017228PMC
September 2016

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Putten Strain CRJJGF_00159 (Phylum Gammaproteobacteria).

Genome Announc 2016 Sep 1;4(5). Epub 2016 Sep 1.

Bacterial Epidemiology and Antimicrobial Resistance Research Unit, USDA-ARS, Athens, Georgia, USA

Here, we report a 4.90 Mbp draft genome sequence of Salmonella enterica subsp. enterica serovar Putten strain CRJJGF_00159 isolated from food animal in 2004.
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http://dx.doi.org/10.1128/genomeA.00895-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009971PMC
September 2016

Involvement of the Rcs regulon in the persistence of Salmonella Typhimurium in tomatoes.

Environ Microbiol Rep 2016 Oct 9;8(5):928-935. Epub 2016 Sep 9.

Soil and Water Science Department, Genetics Institute Rm330E, University of Florida-IFAS, Gainesville, FL, 32611, USA.

It is becoming clear that human enteric pathogens, like Salmonella, can efficiently colonize vegetative and reproductive organs of plants. Even though the bacterium's ability to proliferate within plant tissues has been linked to outbreaks of salmonellosis, little is known about regulatory and physiological adaptations of Salmonella, or other human pathogens, to their persistence in plants. A screen of Salmonella deletion mutants in tomatoes identified rcsA and rcsB genes as those under positive selection. In tomato fruits, populations of Salmonella rcsB mutants were as much as 100-fold lower than those of the wild type. In the follow-up experiments, competitive fitness of rcsA and rcsB mutants was strongly reduced in tomatoes. Bioinformatics predictions identified a putative Salmonella RcsAB binding box (TTMGGAWWAABCTYA) and revealed an extensive putative RcsAB regulon, of which many members were differentially fit within tomatoes.
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http://dx.doi.org/10.1111/1758-2229.12457DOI Listing
October 2016