69 research outputs found
Antimicrobial Properties of Syringopeptin 25A and Rhamnolipids
The increasing bacterial resistance to available antibiotics requires the search for new antibacterial compounds to be broadened. This study investigated the antimicrobial properties of two secondary metabolites from fluorescent pseudo monads -- syringopeptin 25A, a lipodepsipeptide produced by Pseudomonas syringae pv. syringae, and a rhamnolipid mixture produced by Pseudomonas aeruginosa. The rate of antimicrobial action was determined by monitoring the rate of uptake of propidium iodide during exposure to the compounds. Inhibition was also confirmed by the microbroth dilution method to determine the MI Cs. Both the compounds inhibited growth of Gram-positive organisms, including Mycobacterium smegmatis, staphylococci, and listeria. Inhibition of spore germination was also notable. SP 25A inhibited two multiple antibiotic strains of Staphylococcus aureus subsp. aureus and Enterococcus faecalis, while RLs failed to do so, even at 60 μg/ml. Addition of the compounds together showed a synergistic activity against Listeria monocytogenes. Neither compound was toxic to human cells in vitro at 8 μg/ml.
It is postulated that both compounds exert their antimicrobial effect by forming pores in the bacterial cell membrane, but we did not observe a relation between membrane permeabilization and inhibition of growth in each case. At sub-MIC concentrations RLs did cause pores in the membrane of L. monocytogenes, while SP 25A did not. However, RLs did not inhibit cell growth, while SP 25A completely inhibited cell growth.
To investigate these effects gene expression was monitored just before treating the cells with the antimicrobials, 30 min after treatment and 120 min after treatment. The gene expression profile was distinct when cells were treated with both the antimicrobials. SP 25A repressed genes related to cell division, intermediary metabolism, transcription, translation, and virulence genes. These effects were not produced when cells were treated with RLs, hence giving indications that even though both the antimicrobials may act on the same site (i.e. the cell membrane), the cellular response was different, which led to different phenotypes for growth.
This work indicates that SP 25A holds promise for further development as a therapeutic agent and provides evidence that the proposed pore-forming model alone does not suffice to explain the mode of action of SP 25A
Whole Cell Cross-Linking to Discover Host–Microbe Protein Cognate Receptor/Ligand Pairs
Bacterial surface ligands mediate interactions with the host cell during association that determines the specific outcome for the host–microbe association. The association begins with receptors on the host cell binding ligands on the microbial cell to form a partnership that initiates responses in both cells. Methods to determine the specific cognate partnerships are lacking. Determining these molecular interactions between the host and microbial surfaces are difficult, yet crucial in defining biologically important events that are triggered during association of the microbiome, and critical in defining the initiating signal from the host membrane that results in pathology or commensal association. In this study, we designed an approach to discover cognate host–microbe receptor/ligand pairs using a covalent cross-linking strategy with whole cells. Protein/protein cross-linking occurred when the interacting molecules were within 9–12 Å, allowing for identification of specific pairs of proteins from the host and microbe that define the molecular interaction during association. To validate the method three different bacteria with three previously known protein/protein partnerships were examined. The exact interactions were confirmed and led to discovery of additional partnerships that were not recognized as cognate partners, but were previously reported to be involved in bacterial interactions. Additionally, three unknown receptor/ligand partners were discovered and validated with in vitro infection assays by blocking the putative host receptor and deleting the bacterial ligand. Subsequently, Salmonella enterica sv. Typhimurium was cross-linked to differentiated colonic epithelial cells (caco-2) to discover four previously unknown host receptors bound to three previously undefined host ligands for Salmonella. This approach resulted in a priori discovery of previously unknown and biologically important molecules for host/microbe association that were casually reported to mediate bacterial invasion. The whole cell cross-linking approach promises to enable discovery of possible targets to modulate interaction of the microbiome with the host that are important in infection and commensalism, both of with initiate a host response
Whole Cell Cross-Linking to Discover Host-Microbe Protein Cognate Receptor/Ligand Pairs
Bacterial surface ligands mediate interactions with the host cell during association that determines the specific outcome for the host–microbe association. The association begins with receptors on the host cell binding ligands on the microbial cell to form a partnership that initiates responses in both cells. Methods to determine the specific cognate partnerships are lacking. Determining these molecular interactions between the host and microbial surfaces are difficult, yet crucial in defining biologically important events that are triggered during association of the microbiome, and critical in defining the initiating signal from the host membrane that results in pathology or commensal association. In this study, we designed an approach to discover cognate host–microbe receptor/ligand pairs using a covalent cross-linking strategy with whole cells. Protein/protein cross-linking occurred when the interacting molecules were within 9–12 Å, allowing for identification of specific pairs of proteins from the host and microbe that define the molecular interaction during association. To validate the method three different bacteria with three previously known protein/protein partnerships were examined. The exact interactions were confirmed and led to discovery of additional partnerships that were not recognized as cognate partners, but were previously reported to be involved in bacterial interactions. Additionally, three unknown receptor/ligand partners were discovered and validated with in vitro infection assays by blocking the putative host receptor and deleting the bacterial ligand. Subsequently, Salmonella enterica sv. Typhimurium was cross-linked to differentiated colonic epithelial cells (caco-2) to discover four previously unknown host receptors bound to three previously undefined host ligands for Salmonella. This approach resulted in a priori discovery of previously unknown and biologically important molecules for host/microbe association that were casually reported to mediate bacterial invasion. The whole cell cross-linking approach promises to enable discovery of possible targets to modulate interaction of the microbiome with the host that are important in infection and commensalism, both of with initiate a host response
Virulence of 32 Salmonella Strains in Mice
Virulence and persistence in the BALB/c mouse gut was tested for 32 strains of Salmonella enterica for which genome sequencing is complete or underway, including 17 serovars within subspecies I (enterica), and two representatives of each of the other five subspecies. Only serovar Paratyphi C strain BAA1715 and serovar Typhimurium strain 14028 were fully virulent in mice. Three divergent atypical Enteritidis strains were not virulent in BALB/c, but two efficiently persisted. Most of the other strains in all six subspecies persisted in the mouse intestinal tract for several weeks in multiple repeat experiments although the frequency and level of persistence varied considerably. Strains with heavily degraded genomes persisted very poorly, if at all. None of the strains tested provided immunity to Typhimurium infection. These data greatly expand on the known significant strain-to-strain variation in mouse virulence and highlight the need for comparative genomic and phenotypic studies
Distinct Salmonella Enteritidis lineages associated with enterocolitis in high-income settings and invasive disease in low-income settings.
An epidemiological paradox surrounds Salmonella enterica serovar Enteritidis. In high-income settings, it has been responsible for an epidemic of poultry-associated, self-limiting enterocolitis, whereas in sub-Saharan Africa it is a major cause of invasive nontyphoidal Salmonella disease, associated with high case fatality. By whole-genome sequence analysis of 675 isolates of S. Enteritidis from 45 countries, we show the existence of a global epidemic clade and two new clades of S. Enteritidis that are geographically restricted to distinct regions of Africa. The African isolates display genomic degradation, a novel prophage repertoire, and an expanded multidrug resistance plasmid. S. Enteritidis is a further example of a Salmonella serotype that displays niche plasticity, with distinct clades that enable it to become a prominent cause of gastroenteritis in association with the industrial production of eggs and of multidrug-resistant, bloodstream-invasive infection in Africa.This work was supported by the Wellcome Trust. We would like to thank the members of the Pathogen Informatics Team and the core sequencing teams at the Wellcome Trust Sanger Institute (Cambridge, UK). We are grateful to D. Harris for work in managing the sequence data
Molecular Interactions of Salmonella with the Host Epithelium in Presence of Commensals
Food-borne infections are a major source of mortality and morbidity. Salmonella causes the highest number of Food-borne bacterial infections in the US. This work contributes towards developing strategies to control Salmonella by (a) defining receptors used by Salmonella to adhere to and invade the host epithelium; (b) developing a host receptor based rapid detection method for the pathogen in food matrix; (C) and defining mechanisms of how probiotics can help alleviate Salmonella-induced cell death in the host epithelium.
We developed a cell-cell crosslinking method to discover host-microbe receptors, and discovered three new receptor-ligand interactions. Interaction of Salmonella Ef-Tu with Hsp90 from epithelial cells mediated adhesion, while interaction of Salmonella Ef-Tu with two host proteins that negatively regulate membrane ruffling (myosin phosphatase and alpha catenin) mediated adhesion and invasion. We also showed the role of host ganglioside GM1 in mediating invasion of epithelial cells by Salmonella.
Further we exploited pathogen affinity for immobilized gangliosides to concentrate them out of solution and from complex food matrices for detection by qPCR. A sensitivity of 4 CFU/ml (3 hours) in samples without competing microflora was achieved. Samples with competing microflora had a sensitivity of 40,000 CFU/ml.
Next we screened several probiotic strains for pathogen exclusion potential and found that Bifidobacterium longum subspp. infantis showed the highest potential for Salmonella enterica subspp. enterica ser. Typhimurium exclusion in a caco-2 cell culture model. B. infantis shared its binding specificity to ganglioside GM1 with S. ser. Typhimurium. Further, B. infantis completely inhibited Salmonella-induced caspase 8 and caspase 9 activity in intestinal epithelial cells. B. infantis also reduced the basal caspase 9 and caspase 3/7 activity in epithelial cells in absence of the pathogen. Western blots and gene expression profiling of epithelial cells revealed that the decreased caspase activation was concomitant with increased phosphorylation of pro-survival protein kinase Akt, increased expression of caspase inhibiting protein cIAP, and decreased expression of genes involved in mitochondrion organization, biogenesis and reactive oxygen species metabolic processes. Hence, B. infantis exerted its protective effects by repression of mitochondrial cell death pathway which was induced in the presence of S. ser. Typhimurium
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A genome-wide collection of barcoded single-gene deletion mutants in Salmonella enterica serovar Typhimurium
Genetic screening of pools of mutants can reveal genetic determinants involved in complex biological interactions, processes, and systems. We previously constructed two single-gene deletion resources for Salmonella enterica serovar Typhimurium 14028s in which kanamycin (KanR) and chloramphenicol (CamR) cassettes were used to replace non-essential genes. We have now used lambda-red recombination to convert the antibiotic cassettes in these resources into a tetracycline-resistant (TetR) version where each mutant contains a different 21-base barcode flanked by Illumina Read1 and Read2 primer sequences. A motility assay of a pool of the entire library, followed by a single-tube processing of the bacterial pellet, PCR, and sequencing, was used to verify the performance of the barcoded TetR collection. The new resource is useful for experiments with defined subsets of barcoded mutant strains where biological bottlenecks preclude high numbers of founder bacteria, such as in animal infections. The TetR version of the library will also facilitate the construction of triple mutants by transduction. The resource of 6197 mutants covering 3490 genes is deposited at Biological and Emerging Infections Resources (beiresources.org)
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Whole Cell Cross-Linking to Discover Host-Microbe Protein Cognate Receptor/Ligand Pairs.
Bacterial surface ligands mediate interactions with the host cell during association that determines the specific outcome for the host-microbe association. The association begins with receptors on the host cell binding ligands on the microbial cell to form a partnership that initiates responses in both cells. Methods to determine the specific cognate partnerships are lacking. Determining these molecular interactions between the host and microbial surfaces are difficult, yet crucial in defining biologically important events that are triggered during association of the microbiome, and critical in defining the initiating signal from the host membrane that results in pathology or commensal association. In this study, we designed an approach to discover cognate host-microbe receptor/ligand pairs using a covalent cross-linking strategy with whole cells. Protein/protein cross-linking occurred when the interacting molecules were within 9-12 Å, allowing for identification of specific pairs of proteins from the host and microbe that define the molecular interaction during association. To validate the method three different bacteria with three previously known protein/protein partnerships were examined. The exact interactions were confirmed and led to discovery of additional partnerships that were not recognized as cognate partners, but were previously reported to be involved in bacterial interactions. Additionally, three unknown receptor/ligand partners were discovered and validated with in vitro infection assays by blocking the putative host receptor and deleting the bacterial ligand. Subsequently, Salmonella enterica sv. Typhimurium was cross-linked to differentiated colonic epithelial cells (caco-2) to discover four previously unknown host receptors bound to three previously undefined host ligands for Salmonella. This approach resulted in a priori discovery of previously unknown and biologically important molecules for host/microbe association that were casually reported to mediate bacterial invasion. The whole cell cross-linking approach promises to enable discovery of possible targets to modulate interaction of the microbiome with the host that are important in infection and commensalism, both of with initiate a host response
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Virulence of 32 Salmonella Strains in Mice
Virulence and persistence in the BALB/c mouse gut was tested for 32 strains of Salmonella enterica for which genome sequencing is complete or underway, including 17 serovars within subspecies I (enterica), and two representatives of each of the other five subspecies. Only serovar Paratyphi C strain BAA1715 and serovar Typhimurium strain 14028 were fully virulent in mice. Three divergent atypical Enteritidis strains were not virulent in BALB/c, but two efficiently persisted. Most of the other strains in all six subspecies persisted in the mouse intestinal tract for several weeks in multiple repeat experiments although the frequency and level of persistence varied considerably. Strains with heavily degraded genomes persisted very poorly, if at all. None of the strains tested provided immunity to Typhimurium infection. These data greatly expand on the known significant strain-to-strain variation in mouse virulence and highlight the need for comparative genomic and phenotypic studies
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