25 research outputs found
Análisis proteómico de proteínas de membrana ancladas a glicosilfosfatidilinositol en Candida albicans
Evolution of a Bacterial Regulon Controlling Virulence and Mg2+ Homeostasis
Related organisms typically rely on orthologous regulatory proteins to respond to a given signal. However, the extent to which (or even if) the targets of shared regulatory proteins are maintained across species has remained largely unknown. This question is of particular significance in bacteria due to the widespread effects of horizontal gene transfer. Here, we address this question by investigating the regulons controlled by the DNA-binding PhoP protein, which governs virulence and Mg2+ homeostasis in several bacterial species. We establish that the ancestral PhoP protein directs largely different gene sets in ten analyzed species of the family Enterobacteriaceae, reflecting both regulation of species-specific targets and transcriptional rewiring of shared genes. The two targets directly activated by PhoP in all ten species (the most distant of which diverged >200 million years ago), and coding for the most conserved proteins are the phoPQ operon itself and the lipoprotein-encoding slyB gene, which decreases PhoP protein activity. The Mg2+-responsive PhoP protein dictates expression of Mg2+ transporters and of enzymes that modify Mg2+-binding sites in the cell envelope in most analyzed species. In contrast to the core PhoP regulon, which determines the amount of active PhoP and copes with the low Mg2+ stress, the variable members of the regulon contribute species-specific traits, a property shared with regulons controlled by dissimilar regulatory proteins and responding to different signals
Evolution of a Bacterial Regulon Controlling Virulence and Mg2+ Homeostasis
Related organisms typically rely on orthologous regulatory proteins to respond to a given signal. However, the extent to which (or even if) the targets of shared regulatory proteins are maintained across species has remained largely unknown. This question is of particular significance in bacteria due to the widespread effects of horizontal gene transfer. Here, we address this question by investigating the regulons controlled by the DNA-binding PhoP protein, which governs virulence and Mg2+ homeostasis in several bacterial species. We establish that the ancestral PhoP protein directs largely different gene sets in ten analyzed species of the family Enterobacteriaceae, reflecting both regulation of species-specific targets and transcriptional rewiring of shared genes. The two targets directly activated by PhoP in all ten species (the most distant of which diverged >200 million years ago), and coding for the most conserved proteins are the phoPQ operon itself and the lipoprotein-encoding slyB gene, which decreases PhoP protein activity. The Mg2+-responsive PhoP protein dictates expression of Mg2+ transporters and of enzymes that modify Mg2+-binding sites in the cell envelope in most analyzed species. In contrast to the core PhoP regulon, which determines the amount of active PhoP and copes with the low Mg2+ stress, the variable members of the regulon contribute species-specific traits, a property shared with regulons controlled by dissimilar regulatory proteins and responding to different signals
Feedback Inhibition in the PhoQ/PhoP Signaling System by a Membrane Peptide
The PhoQ/PhoP signaling system responds to low magnesium and the presence of certain cationic antimicrobial peptides. It regulates genes important for growth under these conditions, as well as additional genes important for virulence in many gram-negative pathogens. PhoQ is a sensor kinase that phosphorylates and activates the transcription factor PhoP. Since feedback inhibition is a common theme in stress-response circuits, we hypothesized that some members of the PhoP regulon may play such a role in the PhoQ/PhoP pathway. We therefore screened for PhoP-regulated genes that mediate feedback in this system. We found that deletion of mgrB (yobG), which encodes a 47 amino acid peptide, results in a potent increase in PhoP-regulated transcription. In addition, over-expression of mgrB decreased transcription at both high and low concentrations of magnesium. Localization and bacterial two-hybrid studies suggest that MgrB resides in the inner-membrane and interacts directly with PhoQ. We further show that MgrB homologs from Salmonella typhimurium and Yersinia pestis also repress PhoP-regulated transcription in these organisms. In cell regulatory circuits, feedback has been associated with modulating the induction kinetics and/or the cell-to-cell variability in response to stimulus. Interestingly, we found that elimination of MgrB-mediated feedback did not have a significant effect on the kinetics of reporter protein production and did not decrease the variability in expression among cells. Our results indicate MgrB is a broadly conserved membrane peptide that is a critical mediator of negative feedback in the PhoQ/PhoP circuit. This new regulator may function as a point of control that integrates additional input signals to modulate the activity of this important signaling system
Identification of differentially expressed genes of Xanthomonas axonopodis pv. citri by representational difference analysis of cDNA
Nitric Oxide Antagonizes the Acid Tolerance Response that Protects Salmonella against Innate Gastric Defenses
Reactive nitrogen species (RNS) derived from dietary and salivary inorganic nitrogen oxides foment innate host defenses associated with the acidity of the stomach. The mechanisms by which these reactive species exert antimicrobial activity in the gastric lumen are, however, poorly understood.The genetically tractable acid tolerance response (ATR) that enables enteropathogens to survive harsh acidity was screened for signaling pathways responsive to RNS. The nitric oxide (NO) donor spermine NONOate derepressed the Fur regulon that controls secondary lines of resistance against organic acids. Despite inducing a Fur-mediated adaptive response, acidified RNS largely repressed oral virulence as demonstrated by the fact that Salmonella bacteria exposed to NO donors during mildly acidic conditions were shed in low amounts in feces and exhibited ameliorated oral virulence. NO prevented Salmonella from mounting a de novo ATR, but was unable to suppress an already functional protective response, suggesting that RNS target regulatory cascades but not their effectors. Transcriptional and translational analyses revealed that the PhoPQ signaling cascade is a critical ATR target of NO in rapidly growing Salmonella. Inhibition of PhoPQ signaling appears to contribute to most of the NO-mediated abrogation of the ATR in log phase bacteria, because the augmented acid sensitivity of phoQ-deficient Salmonella was not further enhanced after RNS treatment.Since PhoPQ-regulated acid resistance is widespread in enteric pathogens, the RNS-mediated inhibition of the Salmonella ATR described herein may represent a common component of innate host defenses
The 3-Hydroxy-2-Butanone Pathway Is Required for Pectobacterium carotovorum Pathogenesis
Pectobacterium species are necrotrophic bacterial pathogens that cause soft rot diseases in potatoes and several other crops worldwide. Gene expression data identified Pectobacterium carotovorum subsp. carotovorum budB, which encodes the α-acetolactate synthase enzyme in the 2,3-butanediol pathway, as more highly expressed in potato tubers than potato stems. This pathway is of interest because volatiles produced by the 2,3-butanediol pathway have been shown to act as plant growth promoting molecules, insect attractants, and, in other bacterial species, affect virulence and fitness. Disruption of the 2,3-butanediol pathway reduced virulence of P. c. subsp. carotovorum WPP14 on potato tubers and impaired alkalinization of growth medium and potato tubers under anaerobic conditions. Alkalinization of the milieu via this pathway may aid in plant cell maceration since Pectobacterium pectate lyases are most active at alkaline pH
Comparative gene expression analysis of Porphyromonas gingivalis ATCC 33277 in planktonic and biofilms states.
BACKGROUND AND OBJECTIVE:Porphyromonas gingivalis is a keystone pathogen in the onset and progression of periodontitis. Its pathogenicity has been related to its presence and survival within the subgingival biofilm. The aim of the present study was to compare the genome-wide transcription activities of P. gingivalis in biofilm and in planktonic growth, using microarray technology. MATERIAL AND METHODS:P. gingivalis ATCC 33277 was incubated in multi-well culture plates at 37°C for 96 hours under anaerobic conditions using an in vitro static model to develop both the planktonic and biofilm states (the latter over sterile ceramic calcium hydroxyapatite discs). The biofilm development was monitored by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM). After incubation, the bacterial cells were harvested and total RNA was extracted and purified. Three biological replicates for each cell state were independently hybridized for transcriptomic comparisons. A linear model was used for determining differentially expressed genes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to confirm differential expression. The filtering criteria of ≥ ±2 change in gene expression and significance p-values of <0.05 were selected. RESULTS:A total of 92 out of 1,909 genes (4.8%) were differentially expressed by P. gingivalis growing in biofilm compared to planktonic. The 54 up-regulated genes in biofilm growth were mainly related to cell envelope, transport, and binding or outer membranes proteins. Thirty-eight showed decreased expression, mainly genes related to transposases or oxidative stress. CONCLUSION:The adaptive response of P. gingivalis in biofilm growth demonstrated a differential gene expression
In vitro biofilm formation on different ceramic biomaterial surfaces: Coating with two bactericidal glasses
[Objectives] To compare biofilm formation on the surface of different ceramic biomaterials to be used in implant dentistry.[Methods] In vitro biofilm formation was investigated from mixtures of standard reference strains of Streptococcus oralis, Veillonella parvula, Actinomyces naeslundii, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Sterile ceramic calcium hydroxyapatite discs (HA) as control, sterile Al2O3/Ce-TZP nanocomposite sandblasted discs (material A1) and sterile Al2O3/Ce-TZP nanocomposite sandblasted discs and coated with two types of antimicrobial glasses (materials A2 and A3) were used. Biofilms were grown on the four surfaces and evaluated after 12, 24, 48 and 72 h of incubation. Biofilms were examined by confocal laser scanning microscopy (CLSM). In addition, counts of live bacterial cells of the target species A. actinomycetemcomitans, F. nucleatum and P. gingivalis were calculated by quantitative polymerase chain reaction (qPCR) combined with propidium monoazide (PMA). For data analysis, bacterial counts were compared with a multivariate general lineal model.[Results] Using CLSM, cell vitality decreased in A2 and A3. With qPCR-PMA, significant differences in vitality were observed forA. actinomycetemcomitans in A3 after 48 and 72 h of incubation. With respect to the development of the biofilms, a significant increase in counts on HA and materials A1 and A2 was observed for A. actinomycetemcomitans and F. nucleatum. Conversely, for P. gingivalis, no differences were found for HA and materials A1 and A2.[Significance] Differences in biofilm formation were detected among the different tested materials. The ceramic material A3 has an effect on the vitality of A. actinomycetemcomitans growing in an in vitro biofilm model.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) under the project MAT2012-38645. A.L.-P. and M.C.S. received funding from Extraordinary Chair of DENTAID-UCM.Peer reviewe
Representative confocal (A) and scanning electron (B) micrographs representing <i>Porphyromonas gingivalis</i> ATCC 33277 biofilm after 96h of growth.
<p>BacLight Live/Dead strain was used to assess the viability of cells in CLSM distinguishing viable bacteria depicted as green and non-viable as red stained cells.</p