scholarly journals Growth Phase Regulation of Vibrio cholerae RTX Toxin Export

2006 ◽  
Vol 189 (5) ◽  
pp. 1827-1835 ◽  
Author(s):  
Bethany Kay Boardman ◽  
Brian M. Meehan ◽  
Karla J. Fullner Satchell
Keyword(s):  
Stationary Phase ◽  
Vibrio Cholerae ◽  
Growth Phase ◽  
Diarrheal Disease ◽  
Outer Membrane Vesicles ◽  
Transcriptional Level ◽  
Type I ◽  
Bacterial Membranes ◽  
Rtx Toxin ◽  
Phase Regulation

ABSTRACT Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, secretes several “accessory” toxins, including RTX toxin, which causes the cross-linking of the actin cytoskeleton. RTX toxin is exported to the extracellular milieu by an atypical type I secretion system (T1SS), and we previously noted that RTX-associated activity is detectable only in supernatant fluids from log phase cultures. Here, we investigate the mechanisms for regulating RTX toxin activity in supernatant fluids. We find that exported proteases are capable of destroying RTX activity and may therefore play a role in the growth phase regulation of toxin activity. We determined that the absence of RTX toxin in stationary-phase culture supernatant fluids is also due to a lack of toxin secretion and not attributable to solely proteolytic degradation. We ascertained that the T1SS apparatus is regulated at the transcriptional level by growth phase control that is independent of quorum sensing, unlike other virulence factors of V. cholerae. Additionally, in stationary-phase cultures, all RTX toxin activity is associated with bacterial membranes or outer membrane vesicles.

scholarly journals Vibrio cholerae Strains with Mutations in an Atypical Type I Secretion System Accumulate RTX Toxin Intracellularly

2004 ◽  
Vol 186 (23) ◽  
pp. 8137-8143 ◽  
Author(s):  
Bethany Kay Boardman ◽  
Karla J. Fullner Satchell
Keyword(s):  
Binding Site ◽  
Vibrio Cholerae ◽  
Secretion System ◽  
Atp Binding ◽  
Type I ◽  
Atp Binding Site ◽  
Rtx Toxin ◽  
Component Type ◽  
Transport Atpases

ABSTRACT This study shows that the Vibrio cholerae RTX toxin is secreted by a four-component type I secretion system (TISS) encoded by rtxB, rtxD, rtxE, and tolC. ATP-binding site mutations in both RtxB and RtxE blocked secretion, demonstrating that this atypical TISS requires two transport ATPases that may function as a heterodimer.

scholarly journals Mucosal Immunization with Vibrio cholerae Outer Membrane Vesicles Provides Maternal Protection Mediated by Antilipopolysaccharide Antibodies That Inhibit Bacterial Motility

2010 ◽  
Vol 78 (10) ◽  
pp. 4402-4420 ◽  
Author(s):  
Anne L. Bishop ◽  
Stefan Schild ◽  
Bharathi Patimalla ◽  
Brian Klein ◽  
Andrew Camilli
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Protective Antigen ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Clean Water ◽  
Mucosal Immunization ◽  
Maternal Protection

ABSTRACTVibrio choleraeis the causative agent of cholera, a severe diarrheal disease that remains endemic in many parts of the world and can cause outbreaks wherever sanitation and clean water systems break down. Prevention of disease could be achieved through improved sanitation and clean water provision supported by vaccination.V. choleraeserogroup O1 is the major cause of cholera; O1 serotypes Inaba and Ogawa have similar disease burdens, while O139 is the only non-O1 serogroup to cause epidemics. We showed previously that immunization of adult female mice with purifiedV. choleraeouter membrane vesicles (OMVs) elicits an antibody response that protect neonates from oralV. choleraechallenge and that suckling from an immunized dam accounts for the majority of protection fromV. choleraecolonization. Here we report that lipopolysaccharide (LPS) is the major OMV protective antigen. Mucosal immunization with OMVs from Inaba or Ogawa provides significant cross-serotype protection fromV. choleraecolonization, although serotype-specific antigens are dominant. OMVs from O1 or O139 do not provide cross-serogroup protection, but by immunization with a mixture of O1 and O139 OMVs, cross-serogroup protection was achieved. Neonatal protection is not associated with significant bacterial death but may involve inhibition of motility, as antibodies from OMV-immunized mice inhibitV. choleraemotilityin vitro, with trends that parallelin vivoprotection. Motility assays also reveal that a higher antibody titer is required to immobilize O139 compared to O1, a phenotype that is O139 capsule dependent.

scholarly journals A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

2015 ◽  
Vol 197 (6) ◽  
pp. 1051-1064 ◽  
Author(s):  
Bo R. Park ◽  
Ryszard A. Zielke ◽  
Igor H. Wierzbicki ◽  
Kristie C. Mitchell ◽  
Jeffrey H. Withey ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Type I Collagen ◽  
Diarrheal Disease ◽  
Type Ii Secretion ◽  
Type I ◽  
Type Ii ◽  
Marine Animals ◽  
Natural Habitats ◽  
Content Type ◽  
Functional Components

Vibrio choleraeis autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence ofV. choleraein natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studiedV. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues andV. choleraehas been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of theV. choleraeputative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose thatV. choleraeis a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of theV. choleraecollagen utilization program.

scholarly journals Role of mRNA Stability in Growth Phase Regulation of Gene Expression in the Group A Streptococcus

2006 ◽  
Vol 189 (5) ◽  
pp. 1866-1873 ◽  
Author(s):  
Timothy C. Barnett ◽  
Julia V. Bugrysheva ◽  
June R. Scott
Keyword(s):  
Gene Expression ◽  
Stationary Phase ◽  
Growth Phase ◽  
Group A Streptococcus ◽  
Exponential Phase ◽  
Northern Hybridization ◽  
Late Exponential Phase ◽  
Group A ◽  
Phase Regulation ◽  
The Stability

ABSTRACT The impressive disease spectrum of Streptococcus pyogenes (the group A streptococcus [GAS]) is believed to be determined by its ability to modify gene expression in response to environmental stimuli. Virulence gene expression is controlled tightly by several different transcriptional regulators in this organism. In addition, expression of most, if not all, GAS genes is determined by a global mechanism dependent on growth phase. To begin an analysis of growth-phase regulation, we compared the transcriptome 2 h into stationary phase to that in late exponential phase of a serotype M3 GAS strain. We identified the arc transcript as more abundant in stationary phase in addition to the sag and sda transcripts that had been previously identified. We found that in stationary phase, the stability of sagA, sda, and arcT transcripts increased dramatically. We found that polynucleotide phosphorylase (PNPase [encoded by pnpA]) is rate limiting for decay of sagA and sda transcripts in late exponential phase, since the stability of these mRNAs was greater in a pnpA mutant, while stability of control mRNAs was unaffected by this mutation. Complementation restored the wild-type decay rate. Furthermore, in a pnpA mutant, the sagA mRNA appeared to be full length, as determined by Northern hybridization. It seems likely that mRNAs abundant in stationary phase are insensitive to the normal decay enzyme(s) and instead require PNPase for this process. It is possible that PNPase activity is limited in stationary phase, allowing persistence of these important virulence factor transcripts at this phase of growth.

scholarly journals The Actin Cross-linking Domain of the Vibrio cholerae RTX Toxin Directly Catalyzes the Covalent Cross-linking of Actin

2006 ◽  
Vol 281 (43) ◽  
pp. 32366-32374 ◽  
Author(s):  
Christina L. Cordero ◽  
Dmitry S. Kudryashov ◽  
Emil Reisler ◽  
Karla J. Fullner Satchell
Keyword(s):  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Tissue Transglutaminase ◽  
Lethal Factor ◽  
Host Cells ◽  
Cross Linking ◽  
Cell Extracts ◽  
Rtx Toxin

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins to alter host cells and to elicit diarrheal disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization by covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we further investigated the role of the ACD in the actin cross-linking reaction. We show that the RTX toxin cross-links actin independently of tissue transglutaminase, thus eliminating an indirect model of ACD activity. We demonstrate that a fusion protein of the ACD and the N-terminal portion of lethal factor from Bacillus anthracis (LFNACD) has cross-linking activity in vivo and in crude cell extracts. Furthermore, we determined that LFNACD directly catalyzes the formation of covalent linkages between actin molecules in vitro and that Mg2+ and ATP are essential cofactors for the cross-linking reaction. In addition, G-actin is proposed as a cytoskeletal substrate of the RTX toxin in vivo. Future studies of the in vitro cross-linking reaction will facilitate characterization of the enzymatic properties of the ACD and contribute to our knowledge of the novel mechanism of covalent actin cross-linking.

scholarly journals Regulation of the type I protein secretion system by the MisR/MisS two-component system in Neisseria meningitidis

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1588-1601 ◽  
Author(s):  
Soma Sannigrahi ◽  
Xinjian Zhang ◽  
Yih-Ling Tzeng
Keyword(s):  
Neisseria Meningitidis ◽  
Growth Phase ◽  
Sequence Similarity ◽  
Secretion System ◽  
Type I ◽  
Two Component System ◽  
Promoter Elements ◽  
Component System ◽  
Rtx Toxin ◽  
Two Component

Neisseria meningitidis, an obligate human pathogen, remains a leading cause of meningitis and fatal sepsis. Meningococci are known to secrete a family of proteins, such as FrpC, with sequence similarity to the repeat-in-toxin (RTX) proteins via the type I secretion system. The meningococcal type I secretion proteins are encoded at two distant genetic loci, NMB1400 (hlyB) and NMB1738/1737 (hlyD/tolC), and are separated from the RTX toxin-like substrates. We have characterized the promoter elements of both hlyB and hlyD by primer extension and lacZ reporter fusions and revealed the growth phase-dependent upregulation of both genes. In addition, we showed that the MisR/MisS two-component system negatively regulates the expression of hlyB and hlyD/tolC. Direct binding of MisR to hlyB and hlyD promoters was demonstrated by electrophoretic mobility shift assay (EMSA), and DNase I protection assays identified MisR binding sites overlapping the promoter elements. Direct repression of hlyB transcription by MisR was supported by in vitro transcription assays. Mutations in the MisR/S system affected, but did not eliminate, the growth phase-dependent upregulation of hlyB, suggesting additional regulatory mechanisms. Increased secretion of RTX toxin-like proteins was detected in the cell-free media from misS mutant cultures, indicating that the amounts of extracellular RTX toxin-like proteins are, in part, controlled by the abundance of the type I secretion apparatus. This is, to our knowledge, the first example of a two-component system mediating secretion of cytotoxin family proteins by controlling expression of the type I secretion proteins.

Morphological Characterization of Mycobacteriophage R1

1974 ◽  
Vol 32 ◽  
pp. 254-255
Author(s):  
B. L. Soloff ◽  
T. A. Rado
Keyword(s):  
Carbon Source ◽  
Stationary Phase ◽  
Growth Phase ◽  
Minimal Medium ◽  
Morphological Characterization ◽  
Logarithmic Growth Phase ◽  
Complete Lysis ◽  
Lysogenic Culture ◽  
Logarithmic Growth

Mycobacteriophage R1 was originally isolated from a lysogenic culture of M. butyricum. The virus was propagated on a leucine-requiring derivative of M. smegmatis, 607 leu−, isolated by nitrosoguanidine mutagenesis of typestrain ATCC 607. Growth was accomplished in a minimal medium containing glycerol and glucose as carbon source and enriched by the addition of 80 μg/ ml L-leucine. Bacteria in early logarithmic growth phase were infected with virus at a multiplicity of 5, and incubated with aeration for 8 hours. The partially lysed suspension was diluted 1:10 in growth medium and incubated for a further 8 hours. This permitted stationary phase cells to re-enter logarithmic growth and resulted in complete lysis of the culture.

scholarly journals Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine

2008 ◽  
Vol 74 (15) ◽  
pp. 4847-4852 ◽  
Author(s):  
Anastasia Matthies ◽  
Thomas Clavel ◽  
Michael Gütschow ◽  
Wolfram Engst ◽  
Dirk Haller ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Enzyme Induction ◽  
Growth Phase ◽  
Anaerobic Bacterium ◽  
Stationary Growth Phase ◽  
Gut Bacteria ◽  
Soy Isoflavones ◽  
Strictly Anaerobic ◽  
Stationary Growth ◽  
Mouse Intestine

ABSTRACT The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.

scholarly journals Function of the Pyruvate Oxidase-Lactate Oxidase Cascade in Interspecies Competition between Streptococcus oligofermentans and Streptococcus mutans

2012 ◽  
Vol 78 (7) ◽  
pp. 2120-2127 ◽  
Author(s):  
Lei Liu ◽  
Huichun Tong ◽  
Xiuzhu Dong
Keyword(s):  
Stationary Phase ◽  
Streptococcus Mutans ◽  
Growth Phase ◽  
Large Degree ◽  
Interspecies Interactions ◽  
Lactate Oxidase ◽  
Interspecies Competition ◽  
Pyruvate Oxidase ◽  
Content Type

ABSTRACTComplex interspecies interactions occur constantly between oral commensals and the opportunistic pathogenStreptococcus mutansin dental plaque. Previously, we showed that oral commensalStreptococcus oligofermentanspossesses multiple enzymes for H2O2production, especially lactate oxidase (Lox), allowing it to out-competeS. mutans. In this study, through extensive biochemical and genetic studies, we identified a pyruvate oxidase (pox) gene inS. oligofermentans. Apoxdeletion mutant completely lost Pox activity, while ectopically expressedpoxrestored activity. Pox was determined to produce most of the H2O2in the earlier growth phase and log phase, while Lox mainly contributed to H2O2production in stationary phase. Bothpoxandloxwere expressed throughout the growth phase, while expression of theloxgene increased by about 2.5-fold when cells entered stationary phase. Since lactate accumulation occurred to a large degree in stationary phase, the differential Pox- and Lox-generated H2O2can be attributed to differential gene expression and substrate availability. Interestingly, inactivation ofpoxcauses a dramatic reduction in H2O2production from lactate, suggesting a synergistic action of the two oxidases in converting lactate into H2O2. In anin vitrotwo-species biofilm experiment, thepoxmutant ofS. oligofermentansfailed to inhibitS. mutanseven thoughloxwas active. In summary,S. oligofermentansdevelops a Pox-Lox synergy strategy to maximize its H2O2formation so as to win the interspecies competition.

scholarly journals Aggregatibacter actinomycetemcomitans Leukotoxin Is Delivered to Host Cells in an LFA-1-Indepdendent Manner When Associated with Outer Membrane Vesicles

Toxins ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 414 ◽  
Author(s):  
Justin Nice ◽  
Nataliya Balashova ◽  
Scott Kachlany ◽  
Evan Koufos ◽  
Eric Krueger ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Aggressive Periodontitis ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Type I ◽  
Outer Membranes ◽  
Independent Mechanism ◽  
Bacterial Outer Membrane

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.

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