1,721,025 research outputs found
Genetic background influences survival of infections with Salmonella enterica serovar Typhimurium in the Collaborative Cross
No full textData here is data underlying pre-print: https://www.biorxiv.org/content/10.1101/2022.02.07.479341v1
Manuscript submitted to PLOS Genetics and is in revisio
Taxonomic and metagenomic analyses define the development of the microbiota in the chick
No full textData underlying this manuscript: Taxonomic and metagenomic analyses define the development of the microbiota in the chick, by Bogomolnaya et al. Currently in biorXv but to be published in mBio can be found here
Esterases IroE and Fes Participate in the Protection of Salmonella Against Oxidative Stress
Full text linkSalmonella enterica serotype Typhimurium is the leading cause of foodborne illnesses in the United States causing nearly 1.4 million cases and causes hundreds of millions of cases worldwide each year. A multidrug efflux pump called MacAB is essential for the survival of S. Typhimurium in the presence of reactive oxygen species, including hydrogen peroxide (H2O2), in vitro. We know that the expression of macAB is induced upon exposure to H2O2 and is necessary for the survival of S. Typhimurium in the presence of H2O2.
Two esterases, Fes and IroE, produced by Salmonella, degrade enterobactin to linear products including 2,3-dihydroxybenzoylserine trimer (DHBS3), a linear trimer of enterobactin. We found that removing the genes encoding the pump (MacAB) or the iroE and fes esterases, reduces survival of the mutant strains when reactive oxygen species are present. Surprisingly, inserting a functional iroE gene back into the mutant strain lacking both esterases (��fes��iroE) allows S. Typhimurium to survive oxidative stress. From this data we conclude that IroE can restore peroxide resistance to a double mutant lacking both IroE and Fes. In the future, we will determine if Fes can also rescue this double mutant from oxidative stress. These genes and the processes in which they participate are potential targets for the development of novel therapeutics to combat this increasingly antibiotic resistant pathogen
Controlled human malaria infection: applications, advances, and challenges
Full text linkControlled human malaria infection (CHMI) entails deliberate infection with malaria parasites either by mosquito bite or by direct injection of sporozoites or parasitized erythrocytes. When required, the resulting blood-stage infection is curtailed by the administration of antimalarial drugs. Inducing a malaria infection via inoculation with infected blood was first used as a treatment (malariotherapy) for neurosyphilis in Europe and the United States in the early 1900s. More recently, CHMI has been applied to the fields of malaria vaccine and drug development, where it is used to evaluate products in well-controlled early-phase proof-of-concept clinical studies, thus facilitating progression of only the most promising candidates for further evaluation in areas where malaria is endemic. Controlled infections have also been used to immunize against malaria infection. Historically, CHMI studies have been restricted by the need for access to insectaries housing infected mosquitoes or suitable malaria-infected individuals. Evaluation of vaccine and drug candidates has been constrained in these studies by the availability of a limited number of Plasmodium falciparum isolates. Recent advances have included cryopreservation of sporozoites, the manufacture of well-characterized and genetically distinct cultured malaria cell banks for blood-stage infection, and the availability of Plasmodium vivax-specific reagents. These advances will help to accelerate malaria vaccine and drug development by making the reagents for CHMI more widely accessible and also enabling a more rigorous evaluation with multiple parasite strains and species. Here we discuss the different applications of CHMI, recent advances in the use of CHMI, and ongoing challenges for consideration
Novel Mechanisms Used by Salmonella Typhimurium to Colonize the Intestine
Full text linkNon-typhoidal salmonellae are zoonotic pathogens that cause the largest number of cases of bacterial foodborne gastroenteritis in the United States annually. Food products of animal origin contaminated with Salmonella are major sources for human infection in the United States with food products of bovine origin responsible for up to 30% of all outbreaks of human disease. Salmonellae are heavily studied pathogens due to the ease of genetic manipulation and rapid growth rate. However, despite thorough investigation of Salmonella virulence mechanisms over the past three decades, few studies have used relevant animal models to study the gastrointestinal phase of infection. In addition, the vast majority of inquiry has focused on few virulence loci, leaving approximately half of the genome poorly explored.
We used the calf, a natural host of Salmonella and model that most closely recapitulates early enteric infection, to identify new Salmonella genes needed for infection and further characterized a subset of these genes. We identified more than 20 genes never previously implicated in enteric infection and confirmed the necessity of two genes, STM3846 (retron reverse transcriptase) and STM3602 (transcriptional regulator), during infection of the calf. Additional exploration using both mouse models and in vitro experiments showed that the STM3846 reverse transcriptase produces a RNA-DNA hybrid molecule called multicopy single-stranded DNA. This molecule regulates protein abundance during anaerobiosis, leading to poor colonization of mutants unable to produce this
molecule. Further characterization of STM3602 showed that this putative transcriptional regulator is involved in regulating multiple processes that are necessary for Salmonella to thrive within the complex microbial community of the intestine. Thus, through the use of a carefully orchestrated genetic screen in a relevant animal host, novel genes were identified and their functions for
colonization characterized. These genes and the processes in which they participate are potential targets for development of novel therapeutics to combat this increasingly antibiotic resistant pathogen
Comparison of cecal colonization of Salmonella enterica serotype Typhimurium in white leghorn chicks and Salmonella-resistant mice
Full text linkSalmonellosis is one of the most important bacterial food borne illnesses
worldwide. Among the many Salmonella serotypes, Typhimurium is the most
commonly implicated serotype in human disease in the United States. A major source of
infection for humans is consumption of chicken or egg products that have been
contaminated with S. Typhimurium. The breadth of knowledge regarding colonization
and persistence factors in the chicken is small when compared to our knowledge of
factors that are important for these processes in other species used in Salmonella
research, such as cattle and mice. Defining the factors important for these processes in
the chick is the first step in decreasing the transmission of Salmonella between animal
and human hosts.
In this work, we developed a chicken model to identify and study intestinal
colonization and persistence factors of Salmonella enterica serovar Typhimurium. We
studied the degree of enteric and systemic colonization of wild type S. Typhimurium
ATCC14028, one of the most widely studied Typhimurium isolates, in White Leghorn chicks and in Salmonella-resistant CBA/J mice during infection. Furthermore, we
determined the distribution of wild type S. Typhimurium and a SPI-1 mutant (invA)
during competitive infection in the cecum of 1-week-old chicks and 8-week-old mice.
Cell associated, intracellular and luminal distributions of these strains in the cecum were
analyzed as total counts in each compartment and also as a competitive index.
Localization of S. Typhimurium ATCC14028 and the role of SPI-1 in colonization are
well studied in murine models of infection, but comparative infection in chicks with the
same strain has not been undertaken previously.
We show that the cecal contents are the major site for recovery of S.
Typhimurium in the cecum of 1-week-old chicks and Salmonella-resistant mice. We
also show that while SPI-1 is important for successful infection in the murine model, it is
important only for cell association in the cecum of 1-week-old chicks. Finally, we found
that in chicks infected at 1 week of age, bacterial counts in the feces do not reflect those
seen in the cecum as they do in mice
DIFFERENTIAL RESPONSES TO SALMONELLA TYPHIMURIUM INFECTION IN THE COLLABORATIVE CROSS MOUSE POPULATION
Full text linkSalmonella Typhimurium (STm) usually cause self-limiting gastroenteritis. In some individuals, these bacteria can spread systemically and cause disseminated disease. Rising incidence of antibiotic resistance in STm is a world-wide health concern. Exploration of disease phenotypes that differ between individuals may offer alternative pathways for treatment of infections that do not rely on traditional antibiotics. We screened 32 Collaborative Cross (CC) lines for their responses to oral infection with STm to discover new pathways involved in response to infection and host disease outcome. Eighteen CC lines survived to day 7, while fourteen required euthanasia before day 7. The surviving 18 lines were infected and monitored for three weeks. Five of these lines reduced the bacterial load and were called resistant, 6 lines maintained a bacterial load and were categorized as tolerant, and 7 lines succumbed to infection before three-weeks and were called delayed susceptible. The tolerant lines maintained bacterial burdens in Peyer���s patches (PP), mesenteric lymph node (MLN), spleen, and liver, while resistant lines had significantly lower colonization in multiple tissues. Surviving mice had lower baseline temperatures than susceptible mice at one week post-infection and tolerant lines had lower baseline temperatures than both delayed susceptible and resistant. Resistant lines reduced tissue damage in spleen and liver while tolerant lines did not repair tissue damage between one and three weeks post-infection. We identified four suggestive loci for one-week survival, and one significant and six suggestive associations for three-week phenotypes. CC045 was particularly unusual as it survived one-week infections with high bacterial loads and modest weight loss, unlike other lines carrying the Slc11a1 mutation. We generated an Slc11a1- F2 population between CC045 and CC061. F1s all survived infection as did the majority of the F2s, supporting a dominant phenotype. The F2s were highly colonized in spleen and liver while maintaining body weight better than CC061, the parental susceptible line. Genetic analysis was performed on the survival data and four significant associations, and one significant allele interaction were identified
Identification of Novel Salmonella Typhimurium Genes Required For Survival During Intestinal Inflammation
No full textHead of Department: James SamuelBovine ligated ileal loops provide the best model to examine Salmonella Typhimurium genes required for survival during the early stages of infection, as humans and cattle develop very similar intestinal pathogenesis in response to the organism. Utilizing pools of mutants, both single gene and multi-gene deletion mutants, much of the S. Typhimurium genome can be screened simultaneously. A library consisting of multi-gene deletion mutants was screened in ligated ileal loops in calves. Regions of the genome required for survival in this model were identified in mucus and tissue samples using microarray analysis. STM1187-90 was one such region identified as under selection in both mucus and tissue. A ASTM1188 mutant was confirmed to poorly colonize the intestinal mucus and tissue in the presence of inflammation in competitive infections with the wild type. Complementation in trans reversed the phenotype of the ASTM1188 mutant. The phenotype of the ASTM1188 mutant was replicated and complemented in trans in the murine colitis model. STM1188 is a Salmonella specific gene whose protein product we show to be located in the inner membrane. This gene is absent in host-adapted serovars S. Typhi and S. Paratyphi A. Mutation of the putative lipobox cysteine to alanine resulted in mis- localization of STM1188C24A to the cytoplasm, and the inability to complement ASTM1188 in trans in mice.
A second screen of a single gene deletion pool (SGD) identified many novel genes with potential roles during inflammation as well as many predicted genes with defined roles during the early stages of infection. Several novel gene phenotypes were confirmed and subsequently complemented in competitive infections with wild type. AhilE was identified during SGD screening and confirmed in bovine ligated ileal loops as being selected against during competitive infections with extensive inflammation, however, it was not selected against when the inflammatory immune response was limited. HilE has been previously shown to negatively regulate SPI-1 expression. Utilizing the murine colitis model, the AhilE phenotype was confirmed and complemented during competitive infection with wild type. By using (3-galactosidase assays, AhilE was confirmed to overexpress SPI-1, however, surprisingly AhilE also overexpressed SPI-2 during SPI-1 inducing conditions. In the current studies we performed Salmonella screens in bovine ligated ileal loops and confirmed novel virulence genes required for survival during inflammation
Identification of Novel Virulence Genes of Salmonella enterica Using an Array Based Analysis of Cistrons Under Selection
Full text linkPools of mutants of minimal complexity but maximal coverage of genes of
interest facilitate screening for genes under selection in a particular environment. Prior to
this work, mutants were generated by random transposon insertions, which yielded
highly complex pools for in vivo studies. Recent advances in polymerase chain reaction
(PCR)-based mutagenesis in bacteria using the lambda red recombinase, as well as
whole genome sequencing, enable a more directed approach for the generation of
mutants. The lambda red approach was used to construct individual mutants in 1,023
Salmonella enterica serovar Typhimurium genes, including almost all genes found in
Salmonella, but not in related genera. All the mutations were confirmed simultaneously
using a novel amplification strategy to produce labeled ribonucleic acid (RNA) from a
T7 RNA polymerase promoter, introduced during the construction of each mutant,
followed by hybridization of this labeled RNA to a Typhimurium genome tiling array.
To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the
pool was subjected to selection by intraperitoneal injection into BALB/c (Bagg Albino)
mice and was recovered from the spleen. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal
microarray. Among the top 120 statistically significant spleen colonization phenotypes,
51 were mutations in genes with no previously known role in this model. Fifteen
phenotypes were tested using individual mutants in competitive assays and eleven were
confirmed in individual mixed intraperitoneal infection in mice, including the first two
examples of attenuation for sRNA mutants in Salmonella. We refer to our method as
Array-Based Analysis of Cistrons Under Selection (ABACUS).
Among the confirmed mutants identified in the ABACUS screen was a
component of the twin arginine transport (Tat) system, tatC, required for transport of
folded proteins across the cellular membrane. TatC is the highly conserved component
necessary for recognition of the twin arginine containing signal sequence S/T-R-R-x-FL-
K. We confirmed [delta] tatC mutants are defective for colonization of the liver and spleen
in competitive infections with wild type ATCC14028 after intraperitoneal infection in
Salmonella- susceptible (BALB/c). We also found that [delta] tatC mutants were defective for
swimming motility, but not swarming motility, which was linked to the ability to
elaborate flagellins on the bacterial surface under different conditions
An In-depth Analysis of Iron and Pathogenicity Regulatory Pathways in Pseudomonas syringae pv. syringae B728a
Full text linkPseudomonas syringae pv. syringae strain B728a (P.s.s. B728a) is an economically significant plant pathogen that is capable of successful epiphytic colonization of leaf surfaces. Although the virulence factors associated with this pathogen���s ability to cause disease have been well studied, the transition from epiphyte to pathogen is not well understood.
The research described in this dissertation utilizes high throughput sequencing transcriptome analyses to define an iron regulatory network that is predicted to be utilized during the epiphytic portion of the P.s.s. B728a lifecycle. This dissertation also describes a collaborative microarray analysis that analyzes the P.s.s. B728a transcriptome at a global level.
An iron associated sigma factor, AcsS, encoded within a peptide synthesis rich region of the P.s.s. B728a genome is shown to regulate the citrate siderophore achromobactin. RNA-seq transcriptome analysis reveals that this sigma factor regulates expression of genes predicted to be involved in functions that are important during the epiphytic stage of P.s.s. B728a, including genes involved in iron response, secretion, extracellular polysaccharide production, and cell motility.
As part of a collaboration, the transcriptomes of the P.s.s. B728a genome and nine deletion mutants in regulatory genes were analyzed by microarray analayses using seven treatment conditions, including epiphytic and in planta conditions. As part of these microarray analyses, results are described for the global regulator, GacS, and a downstream transcription factor, SalA. This study confirms the role of GacS and SalA in the regulation of major virulence components of P.s.s. B728a such as phytotoxin production and Type III secretion. This study also elucidates a role for GacS and SalA regulation of genes important for epiphytic survival and function, including the Type VI secretion system, iron acquisition, and EPS production
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