scholarly journals Differential Regulation by Magnesium of the Two MsbB Paralogs of Shigella flexneri

2008 ◽  
Vol 190 (10) ◽  
pp. 3526-3537 ◽  
Author(s):  
Seth R. Goldman ◽  
Yupeng Tu ◽  
Marcia B. Goldberg
Keyword(s):  
Lipid A ◽  
Shigella Flexneri ◽  
Differential Regulation ◽  
Virulence Plasmid ◽  
Transferase Activity ◽  
Dependent Manner ◽  
Transcriptional Fusion ◽  
Environmental Signals ◽  
Stressful Environments ◽  
Insight Into

ABSTRACT Shigella flexneri, a gram-negative enteric pathogen, is unusual in that it contains two nonredundant paralogous genes that encode the myristoyl transferase MsbB (LpxM) that catalyzes the final step in the synthesis of the lipid A moiety of lipopolysaccharide. MsbB1 is encoded on the chromosome, and MsbB2 is encoded on the large virulence plasmid present in all pathogenic shigellae. We demonstrate that myristoyl transferase activity due to MsbB2 is detected in limited magnesium medium, but not in replete magnesium medium, whereas that due to MsbB1 is detected under both conditions. MsbB2 increases overall hexa-acylation of lipid A under limited magnesium conditions. Regulation of MsbB2 by magnesium occurs at the level of transcription and is dependent on the conserved magnesium-inducible PhoPQ two-component regulatory pathway. Direct hexanucleotide repeats within the promoter upstream of msbB2 were identified as a putative PhoP binding site, and mutations within the repeats led to diminished PhoP-dependent expression of a transcriptional fusion of lacZ to this promoter. Thus, the virulence plasmid-encoded paralog of msbB is induced under limited magnesium in a PhoPQ-dependent manner. PhoPQ regulates the response of many Enterobacteriaceae to environmental signals, which include modifications of lipid A that confer increased resistance of the organism to stressful environments and antimicrobial peptides. The findings reported here are the first example of gene duplication in which one paralog has selectively acquired the mechanism for differential regulation by PhoPQ. Our findings provide molecular insight into the mechanisms by which each of the two MsbB proteins of S. flexneri likely contributes to pathogenesis.

scholarly journals VirB, a key transcriptional regulator of virulence plasmid genes in Shigella flexneri, forms DNA-binding site dependent foci in the bacterial cytoplasm

2021 ◽  
Author(s):  
Jillian N. Soceaa ◽  
Grant R. Bowmanb ◽  
Helen J. Wing
Keyword(s):  
Dna Binding ◽  
Subcellular Localization ◽  
Binding Site ◽  
Shigella Flexneri ◽  
Transcriptional Regulator ◽  
Virulence Plasmid ◽  
Focus Formation ◽  
Dna Binding Site ◽  
Bacterial Cytoplasm ◽  
Insight Into

VirB is a key regulator of genes located on the large virulence plasmid (pINV) in the bacterial pathogen Shigella flexneri. VirB is unusual; it is not related to other transcriptional regulators, instead, it belongs to a family of proteins that primarily function in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer. To do this, a GFP-VirB fusion that retains the regulatory activity of VirB and yet, does not undergo significant protein degradation in S. flexneri, was used. Surprisingly, discrete fluorescent foci were observed in live wild-type S. flexneri cells and an isogenic virB mutant using fluorescence microscopy. In contrast, foci were rarely observed (<10%) in pINV-cured cells or in cells expressing a GFP-VirB fusion carrying amino acid substitutions in the VirB DNA binding domain. Finally, the 25 bp VirB-binding site was demonstrated to be sufficient and necessary for GFP-VirB focus formation using a set of small surrogate plasmids. Combined, these data demonstrate that the VirB:DNA interactions required for the transcriptional anti-silencing activity of VirB on pINV are a prerequisite for the subcellular localization of VirB in the bacterial cytoplasm. The significance of these findings, in light of the anti-silencing activity of VirB, is discussed. Importance This study reveals the subcellular localization of VirB, a key transcriptional regulator of virulence genes found on the large virulence plasmid (pINV) in Shigella. Fluorescent signals generated by an active GFP-VirB fusion form 2, 3, or 4 discrete foci in the bacterial cytoplasm, predominantly at the quarter cell position. These signals are completely dependent upon VirB interacting with its DNA binding site found either on the virulence plasmid or an engineered surrogate. Our findings: 1) provide novel insight into VirB:pINV interactions, 2) suggest that VirB may have utility as a DNA marker, and 3) raise questions about how and why this anti-silencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm.

scholarly journals Inactivation of the sfgtr4 Gene of Shigella flexneri Induces Biofilm Formation and Affects Bacterial Pathogenicity

2020 ◽  
Vol 8 (6) ◽  
pp. 841
Author(s):  
Abdelmoughit Kaoukab-Raji ◽  
Latéfa Biskri ◽  
Abdelmounaaïm Allaoui
Keyword(s):  
Foodborne Pathogens ◽  
Biofilm Formation ◽  
Lipid A ◽  
Shigella Flexneri ◽  
Polymorphonuclear Neutrophils ◽  
Virulence Plasmid ◽  
Environmental Persistence ◽  
Bacterial Killing ◽  
Type Iii Effectors

Biofilm formation is a significant cause for the environmental persistence of foodborne pathogens. This phenomenon remains misunderstood in Shigella flexneri whose pathogenicity is mainly associated with the virulence plasmid pWR100. Sequence analysis of the latter predicts a putative lipopolysaccharides (LPS) glycosyltransferase (Gtr) encoded by Sfgtr4, which is the second gene of the SfpgdA-orf186-virK-msbB2 locus. We demonstrated here that purified SfGtr4 exhibited a Gtr activity in vitro by transferring glucose to lipid A. To establish the role of SfGtr4 in virulence, we generated a Sfgtr4 mutant and assessed its phenotype in vitro. Sfgtr4 mutant significantly reduced HeLa cells invasion without impairing type III effectors secretion, increased susceptibility to lysozyme degradation, and enhanced bacterial killing by polymorphonuclear neutrophils (PMNs). SfGtr4 is related to proteins required in biofilm formation. We established conditions whereby wild-type Shigella formed biofilm and revealed that its appearance was accelerated by the Sfgtr4 mutant. Additional phenotypical analysis revealed that single SfpdgA and double SfpgdA-Sfgtr4 mutants behaved similarly to Sfgtr4 mutant. Furthermore, a molecular interaction between SfGtr4 and SfPgdA was identified. In summary, the dual contribution of SfGtr4 and SfPgdA to the pathogenicity and the regulation biofilm formation by S. flexneri was demonstrated here.

Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 951-962 ◽  
Author(s):  
Tony Le Gall ◽  
Maria Mavris ◽  
Maria Celeste Martino ◽  
Maria Lina Bernardini ◽  
Erick Denamur ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Expression Profiles ◽  
Shigella Flexneri ◽  
Transcriptional Regulators ◽  
Host Cells ◽  
Virulence Plasmid ◽  
Dependent Manner ◽  
Regulation Of Expression ◽  
Type Iii ◽  
Plasmid Genes

Proteins directly involved in entry and dissemination of Shigella flexneri into epithelial cells are encoded by a virulence plasmid of 200 kb. A 30-kb region (designated the entry region) of this plasmid encodes components of a type III secretion (TTS) apparatus, substrates of this apparatus and their dedicated chaperones. During growth of bacteria in broth, expression of these genes is induced at 37 °C and the TTS apparatus is assembled in the bacterial envelope but is not active. Secretion is activated upon contact of bacteria with host cells and is deregulated in an ipaB mutant. The plasmid encodes four transcriptional regulators, VirF, VirB, MxiE and Orf81. VirF controls transcription of virB, whose product is required for transcription of entry region genes. MxiE, with the chaperone IpgC acting as a co-activator, controls expression of several effectors that are induced under conditions of secretion. Genes under the control of Orf81 are not known. The aim of this study was to define further the repertoires of virulence plasmid genes that are under the control of (i) the growth temperature, (ii) each of the known virulence plasmid-encoded transcriptional regulators (VirF, VirB, MxiE and Orf81) and (iii) the activity of the TTS apparatus. Using a macroarray analysis, the expression profiles of 71 plasmid genes were compared in the wild-type strain grown at 37 and 30 °C and in virF, virB, mxiE, ipaB, ipaB mxiE and orf81 mutants grown at 37 °C. Many genes were found to be under the control of VirB and indirectly of VirF. No alteration of expression of any gene was detected in the orf81 mutant. Expression of 13 genes was increased in the secretion-deregulated ipaB mutant in an MxiE-dependent manner. On the basis of their expression profile, substrates of the TTS apparatus can be classified into three categories: (i) those that are controlled by VirB, (ii) those that are controlled by MxiE and (iii) those that are controlled by both VirB and MxiE. The differential regulation of expression of TTS effectors in response to the TTS apparatus activity suggests that different effectors might be required at different times following contact of bacteria with host cells.

scholarly journals VirB, a key transcriptional regulator of virulence plasmid genes in Shigella flexneri, forms DNA-binding site dependent foci in the bacterial cytoplasm

2020 ◽  
Author(s):  
Jillian N. Socea ◽  
Grant R. Bowman ◽  
Helen J. Wing
Keyword(s):  
Dna Binding ◽  
Subcellular Localization ◽  
Binding Site ◽  
Virulence Genes ◽  
Shigella Flexneri ◽  
Transcriptional Regulator ◽  
Virulence Plasmid ◽  
Dna Binding Site ◽  
Bacterial Cytoplasm ◽  
Insight Into

AbstractVirB is a key regulator of virulence genes located on the large virulence plasmid (pINV) of the bacterial pathogen Shigella flexneri. VirB is unusual in that it is not related to other transcriptional regulators, instead, it belongs to a protein family that primarily functions in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing of virulence genes mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer. To do this, a GFP-VirB fusion that retains the regulatory activity of VirB and yet, does not undergo significant protein degradation in S. flexneri, was used. Surprisingly, discrete fluorescent foci were observed in live wild-type S. flexneri cells and an isogenic virB mutant using fluorescence microscopy. In contrast, foci were rarely observed (<10%) in cells cured of pINV. Moreover, in the context of the fusion, amino acid substitutions in the DNA binding domain of VirB resulted in the fluorescent signal becoming entirely diffuse. Combined, these data demonstrate that the VirB:DNA interactions required for the transcriptional anti-silencing activity of VirB on pINV are a prerequisite for the subcellular localization of VirB in the bacterial cytoplasm. The significance of these findings, in light of the anti-silencing activity of VirB, is discussed.ImportanceThis study reveals the subcellular localization of VirB, a key transcriptional regulator of virulence genes found on the large virulence plasmid in Shigella. Fluorescent signals generated by an active GFP-VirB fusion form 2, 3, or 4 discrete foci in the bacterial cytoplasm, predominantly at the quarter cell position. These signals are completely dependent upon VirB interacting with its DNA binding site found either on the virulence plasmid or an engineered surrogate. Our findings: 1) provide novel insight into VirB:pINV interactions, 2) suggest that VirB may have utility as a DNA marker, and 3) raise questions about how and why this anti-silencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm.

scholarly journals Detection of Other Microbial Species by Salmonella: Expression of the SdiA Regulon

2003 ◽  
Vol 185 (4) ◽  
pp. 1357-1366 ◽  
Author(s):  
Jenée N. Smith ◽  
Brian M. M. Ahmer
Keyword(s):  
Sequence Analysis ◽  
Unknown Function ◽  
Bacterial Species ◽  
Virulence Plasmid ◽  
Dependent Manner ◽  
Transcriptional Fusion ◽  
Natural Position ◽  
Acylhomoserine Lactone ◽  
The Third ◽  
Antisense Orientation

ABSTRACT Salmonella, Escherichia, and Klebsiella do not encode any recognized type of N-acylhomoserine lactone (AHL) synthase, and consistent with this, they do not synthesize AHLs under any conditions tested. However, they do encode an AHL receptor of the LuxR family, named SdiA. MudJ fusions in four loci are known to respond to plasmid-encoded sdiA in Salmonella, but only the rck locus has been described. Here we report the location and sequence analysis of the remaining three loci. The srg-6::MudJ is within gtgA of the gifsy-2 prophage, and the srg-7::MudJ is within PSLT61 of the virulence plasmid. Both fusions are in the antisense orientation. The third fusion, srgE5::MudJ, is within a horizontally acquired gene of unknown function at 33.6 centisomes that we have named srgE. Previously, sdiA expressed from its natural position in the chromosome was demonstrated to activate a plasmid-based transcriptional fusion to the rck promoter in response to AHL production by other bacterial species. However, the MudJ fusions did not respond to chromosomal sdiA. Here we report that MudJ fusions to three of the four loci (not srg-6) are activated by AHL in an sdiA-dependent manner during growth in motility agar (0.25% agar) but not during growth in top agar (0.7% agar) or on agar plates (1.2% agar). In motility agar, the srgE promoter responds to sdiA at 30°C and higher while the rck and srg-7 promoters respond only at 37 or 42°C. Substantial AHL-independent SdiA activity was observed at 30°C but not at 37°C.

scholarly journals Control of microtubule dynamics using an optogenetic microtubule plus end–F-actin cross-linker

2017 ◽  
Vol 217 (2) ◽  
pp. 779-793 ◽  
Author(s):  
Rebecca C. Adikes ◽  
Ryan A. Hallett ◽  
Brian F. Saway ◽  
Brian Kuhlman ◽  
Kevin C. Slep
Keyword(s):  
Blue Light ◽  
Actin Binding ◽  
Cross Linking ◽  
Dependent Manner ◽  
Exclusion Zone ◽  
Actin Networks ◽  
Drosophila Melanogaster S2 Cells ◽  
Actin Binding Domain ◽  
Specific Factors ◽  
Insight Into

We developed a novel optogenetic tool, SxIP–improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein–dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes.

scholarly journals Antidiarrheal Activity of Dissotis multiflora (Sm) Triana (Melastomataceae) Leaf Extract in Wistar Rats and Subacute Toxicity Evaluation

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Alian Désiré Afagnigni ◽  
Maximilienne Ascension Nyegue ◽  
Chantal Florentine Ndoye Foe ◽  
Youchahou Njankouo Ndam ◽  
Frédéric Nico Njayou ◽  
...  
Keyword(s):  
Wistar Rats ◽  
Leaf Extract ◽  
Shigella Flexneri ◽  
Female Rats ◽  
Male Rats ◽  
Dependent Manner ◽  
Subacute Toxicity ◽  
Antidiarrheal Activity ◽  
Dose Dependent ◽  
Ethanolic Leaf Extract

The present work was undertaken to evaluate antidiarrheal activity of ethanolic leaf extract of Dissotis multiflora (Sm) Triana (D. multiflora) on Shigella flexneri-induced diarrhea in Wistar rats and its subacute toxicity. Diarrhea was induced by oral administration of 1.2 × 109 cells/mL S. flexneri to rats. Antidiarrheal activity was investigated in rats with the doses of 111.42 mg/kg, 222.84 mg/kg, and 445.68 mg/kg. The level of biochemical parameters was assessed and organs histology examined by 14 days’ subacute toxicity. S. flexneri stool load decreased significantly in dose-dependent manner. The level of ALT increased (p<0.05) in male rats treated with the dose of 445.68 mg/kg while creatinine level increased in rats treated with both doses. In female rats, a significant decrease (p<0.05) of the level of AST and creatinine was noted in rats treated with the dose of 222.84 mg/kg of D. multiflora. Histological exams of kidney and liver of treated rats showed architectural modifications at the dose of 445.68 mg/kg. This finding suggests that D. multiflora leaf extract is efficient against diarrhea caused by S. flexneri but the treatment with doses lower than 222.84 mg/kg is recommended while further study is required to define the exact efficient nontoxic dose.

scholarly journals Bicarbonate Evokes Reciprocal Changes in Intracellular Cyclic di-GMP and Cyclic AMP Levels in Pseudomonas aeruginosa

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 519
Author(s):  
Kasidid Ruksakiet ◽  
Balázs Stercz ◽  
Gergő Tóth ◽  
Pongsiri Jaikumpun ◽  
Ilona Gróf ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cyclic Amp ◽  
Biofilm Formation ◽  
Second Messengers ◽  
Brain Heart Infusion ◽  
Ambient Air ◽  
Dependent Manner ◽  
Environmental Signals ◽  
Common Causes ◽  
Camp Levels

The formation of Pseudomonas aeruginosa biofilms in cystic fibrosis (CF) is one of the most common causes of morbidity and mortality in CF patients. Cyclic di-GMP and cyclic AMP are second messengers regulating the bacterial lifestyle transition in response to environmental signals. We aimed to investigate the effects of extracellular pH and bicarbonate on intracellular c-di-GMP and cAMP levels, and on biofilm formation. P. aeruginosa was inoculated in a brain–heart infusion medium supplemented with 25 and 50 mM NaCl in ambient air (pH adjusted to 7.4 and 7.7 respectively), or with 25 and 50 mM NaHCO3 in 5% CO2 (pH 7.4 and 7.7). After 16 h incubation, c-di-GMP and cAMP were extracted and their concentrations determined. Biofilm formation was investigated using an xCelligence real-time cell analyzer and by crystal violet assay. Our results show that HCO3− exposure decreased c-di-GMP and increased cAMP levels in a dose-dependent manner. Biofilm formation was also reduced after 48 h exposure to HCO3−. The reciprocal changes in second messenger concentrations were not influenced by changes in medium pH or osmolality. These findings indicate that HCO3− per se modulates the levels of c-di-GMP and cAMP, thereby inhibiting biofilm formation and promoting the planktonic lifestyle of the bacteria.

scholarly journals Shigella flexneri Phagosomal Escape Is Independent of Invasion

2007 ◽  
Vol 75 (10) ◽  
pp. 4826-4830 ◽  
Author(s):  
Susanne Paetzold ◽  
Sebastian Lourido ◽  
Bärbel Raupach ◽  
Arturo Zychlinsky
Keyword(s):  
Cell Invasion ◽  
Type Iii Secretion ◽  
Shigella Flexneri ◽  
Mucosal Inflammation ◽  
Virulence Plasmid ◽  
Secretion Systems ◽  
Type Iii Secretion Systems ◽  
Macrophage Cytotoxicity ◽  
Serovar Typhimurium ◽  
Epithelial Cell Invasion

ABSTRACT Infections with Salmonella enterica serovar Typhimurium and Shigella flexneri result in mucosal inflammation in response to epithelial cell invasion and macrophage cytotoxicity. These processes are mediated by type III secretion systems encoded in homologous virulence loci in the two species, namely, Salmonella pathogenicity island 1 (SPI-1), carried in the genome, and the Shigella entry region (SER), carried in a large virulence plasmid. Here we show that SPI-1 can functionally complement a deletion of SER in S. flexneri, restoring invasion of epithelial cells, macrophage cytotoxicity, and phagosomal escape. Furthermore, S. flexneri phagosomal escape requires the SER and another gene(s) carried on the large virulence plasmid. We demonstrate that the processes of invasion and phagosomal escape can be uncoupled in S. flexneri.

scholarly journals Virulent Shigella flexneri subverts the host innate immune response through manipulation of antimicrobial peptide gene expression

2008 ◽  
Vol 205 (5) ◽  
pp. 1121-1132 ◽  
Author(s):  
Brice Sperandio ◽  
Béatrice Regnault ◽  
Jianhua Guo ◽  
Zhi Zhang ◽  
Samuel L. Stanley ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Shigella Flexneri ◽  
Host Cells ◽  
Virulence Plasmid ◽  
Cationic Peptides ◽  
Immunity Genes ◽  
Genes Encoding ◽  
Peptide Gene

Antimicrobial factors are efficient defense components of the innate immunity, playing a crucial role in the intestinal homeostasis and protection against pathogens. In this study, we report that upon infection of polarized human intestinal cells in vitro, virulent Shigella flexneri suppress transcription of several genes encoding antimicrobial cationic peptides, particularly the human β-defensin hBD-3, which we show to be especially active against S. flexneri. This is an example of targeted survival strategy. We also identify the MxiE bacterial regulator, which controls a regulon encompassing a set of virulence plasmid-encoded effectors injected into host cells and regulating innate signaling, as being responsible for this dedicated regulatory process. In vivo, in a model of human intestinal xenotransplant, we confirm at the transcriptional and translational level, the presence of a dedicated MxiE-dependent system allowing S. flexneri to suppress expression of antimicrobial cationic peptides and promoting its deeper progression toward intestinal crypts. We demonstrate that this system is also able to down-regulate additional innate immunity genes, such as the chemokine CCL20 gene, leading to compromised recruitment of dendritic cells to the lamina propria of infected tissues. Thus, S. flexneri has developed a dedicated strategy to weaken the innate immunity to manage its survival and colonization ability in the intestine.

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