Adaptation and Preadaptation of Salmonella enterica to Bile

Bile possesses antibacterial activity because bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile. Sublethal concentrations of the bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to bile is supported by the observation that RpoS− mutants are bile-sensitive. While adaptation to bile involves a response by the bacterial population, individual cells can become bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of bile yields bile-resistant colonies at frequencies between 10−6 and 10−7 per cell and generation. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that bile resistance can be acquired by mutation. Full genome sequencing of bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational bile resistance. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations

Duckweed (Lemna minor) as a Model Plant System for the Study of Human Microbial Pathogenesis

BACKGROUND: Plant infection models provide certain advantages over animal models in the study of pathogenesis. However, current plant models face some limitations, e.g., plant and pathogen cannot co-culture in a contained environment. Development of such a plant model is needed to better illustrate host-pathogen interactions. METHODOLOGY/PRINCIPAL FINDINGS: We describe a novel model plant system for the study of human pathogenic bacterial infection on a large scale. This system was initiated by co-cultivation of axenic duckweed (Lemna minor) plants with pathogenic bacteria in 24-well polystyrene cell culture plate. Pathogenesis of bacteria to duckweed was demonstrated with Pseudomonas aeruginosa and Staphylococcus aureus as two model pathogens. P. aeruginosa PAO1 caused severe detriment to duckweed as judged from inhibition to frond multiplication and chlorophyll formation. Using a GFP-marked PAO1 strain, we demonstrated that bacteria colonized on both fronds and roots and formed biofilms. Virulence of PAO1 to duckweed was attenuated in its quorum sensing (QS) mutants and in recombinant strains overexpressing the QS quenching enzymes. RN4220, a virulent strain of S. aureus, caused severe toxicity to duckweed while an avirulent strain showed little effect. Using this system for antimicrobial chemical selection, green tea polyphenols exhibited inhibitory activity against S. aureus virulence. This system was further confirmed to be effective as a pathogenesis model using a number of pathogenic bacterial species. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that duckweed can be used as a fast, inexpensive and reproducible model plant system for the study of host-pathogen interactions, could serve as an alternative choice for the study of some virulence factors, and could also potentially be used in large-scale screening for the discovery of antimicrobial chemicals

Mitochondrial Structure, Function and Dynamics Are Temporally Controlled by c-Myc

Although the c-Myc (Myc) oncoprotein controls mitochondrial biogenesis and multiple enzymes involved in oxidative phosphorylation (OXPHOS), the coordination of these events and the mechanistic underpinnings of their regulation remain largely unexplored. We show here that re-expression of Myc in myc−/− fibroblasts is accompanied by a gradual accumulation of mitochondrial biomass and by increases in membrane polarization and mitochondrial fusion. A correction of OXPHOS deficiency is also seen, although structural abnormalities in electron transport chain complexes (ETC) are not entirely normalized. Conversely, the down-regulation of Myc leads to a gradual decrease in mitochondrial mass and a more rapid loss of fusion and membrane potential. Increases in the levels of proteins specifically involved in mitochondrial fission and fusion support the idea that Myc affects mitochondrial mass by influencing both of these processes, albeit favoring the latter. The ETC defects that persist following Myc restoration may represent metabolic adaptations, as mitochondrial function is re-directed away from producing ATP to providing a source of metabolic precursors demanded by the transformed cell

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.Fondo Especial de la Educación Superior/[0500-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0504-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0505-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0248-13]/FEES/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí