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

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

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

STATISTICAL ISSUES IN THE NORMALIZATIONOF MULTI-SPECIES MICROARRAY DATA

Several species of bacteria are involved in the production of cheese, including Lactobacillus brevis and Lactococcus lactis. A custom-designed Affymetrix microarray was recently developed to study gene expression in three organisms on a single chip. This array contains only perfect match features for the coding and non-coding regions in the genomes of all three sequences. The multi-species nature of this array version raises interesting questions regarding the preprocessing or normalization strategies for the analysis of gene expression data. We present and evaluate several possible strategies using both cDNA dilution data and experimental expression data from a repeated measures design. The statistical protocols highlighted in this work are applicable to other multi-species microarrays

Salmonella Degrades the Host Glycocalyx Leading to Altered Infection and Glycan Remodeling.

Complex glycans cover the gut epithelial surface to protect the cell from the environment. Invasive pathogens must breach the glycan layer before initiating infection. While glycan degradation is crucial for infection, this process is inadequately understood. Salmonella contains 47 glycosyl hydrolases (GHs) that may degrade the glycan. We hypothesized that keystone genes from the entire GH complement of Salmonella are required to degrade glycans to change infection. This study determined that GHs recognize the terminal monosaccharides (N-acetylneuraminic acid (Neu5Ac), galactose, mannose, and fucose) and significantly (p < 0.05) alter infection. During infection, Salmonella used its two GHs sialidase nanH and amylase malS for internalization by targeting different glycan structures. The host glycans were altered during Salmonella association via the induction of N-glycan biosynthesis pathways leading to modification of host glycans by increasing fucosylation and mannose content, while decreasing sialylation. Gene expression analysis indicated that the host cell responded by regulating more than 50 genes resulting in remodeled glycans in response to Salmonella treatment. This study established the glycan structures on colonic epithelial cells, determined that Salmonella required two keystone GHs for internalization, and left remodeled host glycans as a result of infection. These data indicate that microbial GHs are undiscovered virulence factors

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

Defined Single-Gene and Multi-Gene Deletion Mutant Collections in Salmonella enterica sv Typhimurium

Artículo de publicación ISIWe constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available

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