scholarly journals Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae

2015 ◽  
Vol 83 (11) ◽  
pp. 4335-4348 ◽  
Author(s):  
Amable J. Rivas ◽  
Gisela von Hoven ◽  
Claudia Neukirch ◽  
Martina Meyenburg ◽  
Qianqian Qin ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Epithelial Cells ◽  
Necrotizing Fasciitis ◽  
Vibrio Cholerae ◽  
Gene Product ◽  
Morphological Changes ◽  
Marine Animals ◽  
Content Type ◽  
Membrane Pores ◽  
Photobacterium Damselae

ABSTRACTPhotobacterium damselaesubsp.damselae, an important pathogen of marine animals, may also cause septicemia or hyperaggressive necrotizing fasciitis in humans. We previously showed that hemolysin genes are critical for virulence of this organism in mice and fish. In the present study, we characterized thehlyAgene product, a putative small β-pore-forming toxin, and termed it phobalysin P (PhlyP), for “photobacterial lysin encoded on a plasmid.” PhlyP formed stable oligomers and small membrane pores, causing efflux of K+, with no significant leakage of lactate dehydrogenase but entry of vital dyes. The latter feature distinguished PhlyP from the relatedVibrio choleraecytolysin. Attack by PhlyP provoked a loss of cellular ATP, attenuated translation, and caused profound morphological changes in epithelial cells. In coculture experiments with epithelial cells,Photobacterium damselaesubsp.damselaeled to rapid hemolysin-dependent membrane permeabilization. Unexpectedly, hemolysins also promoted the association ofP. damselaesubsp.damselaewith epithelial cells. The collective observations of this study suggest that membrane-damaging toxins commonly enhance bacterial adherence.

scholarly journals Synergistic and Additive Effects of Chromosomal and Plasmid-Encoded Hemolysins Contribute to Hemolysis and Virulence in Photobacterium damselae subsp. damselae

2013 ◽  
Vol 81 (9) ◽  
pp. 3287-3299 ◽  
Author(s):  
Amable J. Rivas ◽  
Miguel Balado ◽  
Manuel L. Lemos ◽  
Carlos R. Osorio
Keyword(s):  
Animal Species ◽  
Synergistic Effects ◽  
Additive Effects ◽  
Iron Starvation ◽  
Triple Mutant ◽  
Marine Animals ◽  
Content Type ◽  
Photobacterium Damselae ◽  
The One ◽  
The Individual

ABSTRACTPhotobacterium damselaesubsp.damselaecauses infections and fatal disease in marine animals and in humans. Highly hemolytic strains produce damselysin (Dly) and plasmid-encoded HlyA (HlyApl). These hemolysins are encoded by plasmid pPHDD1 and contribute to hemolysis and virulence for fish and mice. In this study, we report that all the hemolytic strains produce a hitherto uncharacterized chromosome-encoded HlyA (HlyAch). Hemolysis was completely abolished in a singlehlyAchmutant of a plasmidless strain and in adly hlyAplhlyAchtriple mutant. We found that Dly, HlyApl, and HlyAchare needed for full hemolytic values in strains harboring pPHDD1, and these values are the result of the additive effects between HlyApland HlyAch, on the one hand, and of the synergistic effect of Dly with HlyApland HlyAch, on the other hand. Interestingly, Dly-producing strains produced synergistic effects with strains lacking Dly production but secreting HlyA, constituting a case of the CAMP (Christie,Atkins, andMunch-Petersen) reaction. Environmental factors such as iron starvation and salt concentration were found to regulate the expression of the three hemolysins. We found that the contributions, in terms of the individual and combined effects, of the three hemolysins to hemolysis and virulence varied depending on the animal species tested. While Dly and HlyAplwere found to be main contributors in the virulence for mice, we observed that the contribution of hemolysins to virulence for fish was mainly based on the synergistic effects between Dly and either of the two HlyA hemolysins rather than on their individual effects.

scholarly journals Biofilms Comprise a Component of the Annual Cycle ofVibrio choleraein the Bay of Bengal Estuary

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e00483-18 ◽  
Author(s):  
Marzia Sultana ◽  
Suraia Nusrin ◽  
Nur A. Hasan ◽  
Abdus Sadique ◽  
Kabir U. Ahmed ◽  
...  
Keyword(s):  
Life Cycle ◽  
Vibrio Cholerae ◽  
Causative Agent ◽  
Aquatic Environment ◽  
Morphological Changes ◽  
Living Cells ◽  
Estuarine Environment ◽  
Annual Life Cycle ◽  
Free Living ◽  
Content Type

ABSTRACTVibrio cholerae, an estuarine bacterium, is the causative agent of cholera, a severe diarrheal disease that demonstrates seasonal incidence in Bangladesh. In an extensive study ofV. choleraeoccurrence in a natural aquatic environment, water and plankton samples were collected biweekly between December 2005 and November 2006 from Mathbaria, an estuarine village of Bangladesh near the mangrove forests of the Sundarbans. ToxigenicV. choleraeexhibited two seasonal growth peaks, one in spring (March to May) and another in autumn (September to November), corresponding to the two annual seasonal outbreaks of cholera in this region. The total numbers of bacteria determined by heterotrophic plate count (HPC), representing culturable bacteria, accounted for 1% to 2.7% of the total numbers obtained using acridine orange direct counting (AODC). The highest bacterial culture counts, including toxigenicV. cholerae, were recorded in the spring. The direct fluorescent antibody (DFA) assay was used to detectV. choleraeO1 cells throughout the year, as free-living cells, within clusters, or in association with plankton.V. choleraeO1 varied significantly in morphology, appearing as distinctly rod-shaped cells in the spring months, while small coccoid cells within thick clusters of biofilm were observed during interepidemic periods of the year, notably during the winter months. ToxigenicV. choleraeO1 was culturable in natural water during the spring when the temperature rose sharply. The results of this study confirmed biofilms to be a means of persistence for bacteria and an integral component of the annual life cycle of toxigenicV. choleraein the estuarine environment of Bangladesh.IMPORTANCEVibrio cholerae, the causative agent of cholera, is autochthonous in the estuarine aquatic environment. This study describes morphological changes in naturally occurringV. choleraeO1 in the estuarine environment of Mathbaria, where the bacterium is culturable when the water temperature rises and is observable predominantly as distinct rods and dividing cells. In the spring and fall, these morphological changes coincide with the two seasonal peaks of endemic cholera in Bangladesh.V. choleraeO1 cells are predominantly coccoid within biofilms but are rod shaped as free-living cells and when attached to plankton or to particulate matter in interepidemic periods of the year. It is concluded that biofilms represent a stage of the annual life cycle ofV. choleraeO1, the causative agent of cholera in Bangladesh.

scholarly journals Diaphorina citri Nymphs Are Resistant to Morphological Changes Induced by “ Candidatus Liberibacter asiaticus” in Midgut Epithelial Cells

2018 ◽  
Vol 86 (4) ◽  
Author(s):  
Marina Mann ◽  
Somayeh Fattah-Hosseini ◽  
El-Desouky Ammar ◽  
Richard Stange ◽  
EricaRose Warrick ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Morphological Changes ◽  
Diaphorina Citri ◽  
Content Type ◽  
Citrus Greening Disease ◽  
Mitochondrial Superoxide ◽  
Liberibacter Asiaticus

ABSTRACT “ Candidatus Liberibacter asiaticus” is the causative bacterium associated with citrus greening disease. “ Ca . Liberibacter asiaticus” is transmitted by Diaphorina citri more efficiently when it is acquired by nymphs rather than adults. Why this occurs is not known. We compared midguts of D. citri insects reared on healthy or “ Ca . Liberibacter asiaticus”-infected citrus trees using quantitative PCR, confocal microscopy, and mitochondrial superoxide staining for evidence of oxidative stress. Consistent with its classification as propagative, “ Ca . Liberibacter asiaticus” titers were higher in adults than in nymphs. Our previous work showed that adult D. citri insects have basal levels of karyorrhexis (fragmentation of the nucleus) in midgut epithelial cells, which is increased in severity and frequency in response to “ Ca . Liberibacter asiaticus.” Here, we show that nymphs exhibit lower levels of early-stage karyorrhexis than adults and are refractory to the induction of advanced karyorrhexis by “ Ca . Liberibacter asiaticus” in the midgut epithelium. MitoSox Red staining showed that guts of infected adults, particularly males, experienced oxidative stress in response to “ Ca . Liberibacter asiaticus.” A positive correlation between the titers of “ Ca . Liberibacter asiaticus” and the Wolbachia endosymbiont was observed in adult and nymph midguts, suggesting an interplay between these bacteria during development. We hypothesize that the resistance of the nymph midgut to late-stage karyorrhexis through as yet unknown molecular mechanisms benefits “ Ca . Liberibacter asiaticus” for efficient invasion of midgut epithelial cells, which may be a factor explaining the developmental dependency of “ Ca . Liberibacter asiaticus” acquisition by the vector.

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 Identification of a Novel Virulence Factor in Clostridium Difficile That Modulates Toxin Sensitivity of Cultured Epithelial Cells

2011 ◽  
Vol 79 (9) ◽  
pp. 3810-3820 ◽  
Author(s):  
Masashi Miura ◽  
Haru Kato ◽  
Osamu Matsushita
Keyword(s):  
Clostridium Difficile ◽  
Epithelial Cells ◽  
Culture Filtrate ◽  
Morphological Changes ◽  
Hypothetical Protein ◽  
Intestinal Epithelial ◽  
Content Type ◽  
Cultured Epithelial Cells ◽  
Toxin Sensitivity ◽  
In Cells

ABSTRACTTwo glucosylating toxins named toxins A and B play a role in the pathogenesis ofClostridium Difficileinfection. The interaction of the toxins with host cell factors proceeds to downstream stages of cytotoxic effects in cells, in which involvement of otherC. difficilefactors remains unknown. We utilized culture filtrate ofC. difficilewith a low dilution to characterize the influence of putative minor proteins on the organization of the actin cytoskeleton in cultured epithelial cells and found a previously uncharacterized F-actin aggregated structure, termed “actin aggregate,” at the juxtanuclear region. We reasoned that formation of actin aggregate was due to an additional factor(s) in the culture filtrate rather than the glucosylating toxins, because treatment of purified toxins rarely caused actin aggregate in cells. We focused on a previously uncharacterized hypothetical protein harboring a KDEL-like sequence as a candidate. The product of the candidate gene was detected in culture filtrate ofC. difficileATCC 9689 and was renamed Srl. Purified glutathioneS-transferase-tagged Srl triggered formation of actin aggregate in the cells in the presence of either toxin A or B and enhanced cytotoxicity of each of the two toxins, including decreases in both cell viability and transepithelial resistance of cultured epithelial monolayer, although the recombinant Srl alone did not show detectable cytotoxicity. Srl-neutralized culture filtrate partially inhibited morphological changes of the cells in parallel with decreased actin aggregate formation in the cells. Thus, Srl might contribute to the modulation of toxin sensitivity of intestinal epithelial cells by enhancing cytotoxicity ofC. difficiletoxins.

scholarly journals Photobacterium damselae subsp. damselae Major Virulence Factors Dly, Plasmid-Encoded HlyA, and Chromosome-Encoded HlyA Are Secreted via the Type II Secretion System

2015 ◽  
Vol 83 (4) ◽  
pp. 1246-1256 ◽  
Author(s):  
Amable J. Rivas ◽  
Ana Vences ◽  
Matthias Husmann ◽  
Manuel L. Lemos ◽  
Carlos R. Osorio
Keyword(s):  
Secretion System ◽  
Transcriptional Level ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Marine Animals ◽  
Content Type ◽  
Type Ii Secretion System ◽  
Photobacterium Damselae ◽  
Prepilin Peptidase

Photobacterium damselaesubsp.damselaeis a marine bacterium that causes septicemia in marine animals and in humans. Previously, we had determined a major role of pPHDD1 plasmid-encoded Dly (damselysin) and HlyA (HlyApl) and the chromosome-encoded HlyA (HlyAch) hemolysins in virulence. However, the mechanisms by which these toxins are secreted remain unknown. In this study, we found that a mini-Tn10transposon mutant in a plasmidless strain showing an impaired hemolytic phenotype contained an insertion inepsL, a component of a type II secretion system (T2SS). Reconstruction of the mutant by allelic exchange confirmed the specific involvement ofepsLin HlyAchsecretion. In addition, mutation ofepsLin a pPHDD1-harboring strain caused an almost complete abolition of hemolytic activity against sheep erythrocytes, indicating thatepsLplays a major role in secretion of the plasmid-encoded HlyApland Dly. This was further demonstrated by analysis of different combinations of hemolysin gene mutants and by strain-strain complementation assays. We also found that mutation of the putative prepilin peptidase genepilDseverely affected hemolysis, which dropped at levels inferior to those ofepsLmutants. Promoter expression analyses suggested that impairment of hemolysin secretion inepsLandpilDmutants might constitute a signal that affects hemolysin and T2SS gene expression at the transcriptional level. In addition, singleepsLandpilDmutations caused a drastic decrease in virulence for mice, demonstrating a major role of T2SS andpilDinP. damselaesubsp.damselaevirulence.

scholarly journals The Photobacterium damselae subsp. damselae Hemolysins Damselysin and HlyA Are Encoded within a New Virulence Plasmid

2011 ◽  
Vol 79 (11) ◽  
pp. 4617-4627 ◽  
Author(s):  
Amable J. Rivas ◽  
Miguel Balado ◽  
Manuel L. Lemos ◽  
Carlos R. Osorio
Keyword(s):  
Marine Bacterium ◽  
Conjugative Plasmid ◽  
Virulence Plasmid ◽  
Marine Animals ◽  
Fatal Disease ◽  
Content Type ◽  
Wide Range ◽  
Photobacterium Damselae ◽  
Different Levels

ABSTRACTPhotobacterium damselaesubsp.damselae(formerlyVibrio damsela) is a marine bacterium that causes infections and fatal disease in a wide range of marine animals and in humans. Highly hemolytic strains produce damselysin (Dly), a cytolysin encoded by thedlygene that is lethal for mice and has hemolytic activity. We found that Dly is encoded in the highly hemolytic strain RM-71 within a 153,429-bp conjugative plasmid that we dubbed pPHDD1. In addition to Dly, pPHDD1 also encodes a homologue of the pore-forming toxin HlyA. We found a direct correlation between presence of pPHDD1 and a strong hemolytic phenotype in a collection ofP. damselaesubsp.damselaeisolates. Hemolysis was strongly reduced in a doubledly hlyAmutant, demonstrating the role of the two pPHDD1-encoded genes in hemolysis. Interestingly, although singlehlyAanddlymutants showed different levels of hemolysis reduction depending on the erythrocyte source, hemolysis was not abolished in any of the single mutants, suggesting that the hemolytic phenotype is the result of the additive effect of Dly and HlyA. We found that pPHDD1-encodeddlyandhlyAgenes are necessary for full virulence for mice and fish. Our results suggest that pPHDD1 can be considered as a driving force for the emergence of a highly hemolytic lineage ofP. damselaesubsp.damselae.

scholarly journals Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Ana Vences ◽  
Amable J. Rivas ◽  
Manuel L. Lemos ◽  
Matthias Husmann ◽  
Carlos R. Osorio
Keyword(s):  
Sea Bass ◽  
Virulence Plasmid ◽  
Type Ii Secretion ◽  
Cell Toxicity ◽  
Marine Animals ◽  
Marine Aquaculture ◽  
Content Type ◽  
Type Ii Secretion System ◽  
Photobacterium Damselae ◽  
Chromosome I

ABSTRACT Photobacterium damselae subsp. damselae is a pathogen of marine animals, including fish of importance in aquaculture. The virulence plasmid pPHDD1, characteristic of highly hemolytic isolates, encodes the hemolysins damselysin (Dly) and phobalysin (PhlyP). Strains lacking pPHDD1 constitute the vast majority of the isolates from fish outbreaks, but genetic studies to identify virulence factors in plasmidless strains are scarce. Here, we show that the chromosome I-encoded hemolysin PhlyC plays roles in virulence and cell toxicity in pPHDD1-negative isolates of this pathogen. By combining the analyses of whole genomes and of gene deletion mutants, we identified two hitherto uncharacterized chromosomal loci encoding a phospholipase (PlpV) and a collagenase (ColP). PlpV was ubiquitous in the subspecies and exerted hemolytic activity against fish erythrocytes, which was enhanced in the presence of lecithin. ColP was restricted to a fraction of the isolates and was responsible for the collagen-degrading activity in this subspecies. Consistent with the presence of signal peptides in PlpV and ColP sequences, mutants for the type II secretion system (T2SS) genes epsL and pilD exhibited impairments in phospholipase and collagenase activities. Sea bass virulence experiments and cell culture assays demonstrated major contributions of PhlyC and PlpV to virulence and toxicity. IMPORTANCE This study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture, Photobacterium damselae subsp. damselae. To date, studies on the genetic basis of virulence were restricted to the pPHDD1 plasmid-encoded toxins Dly and PhlyP. However, the vast majority of the recent isolates of this pathogen from fish farm outbreaks lack this plasmid. Here we demonstrate that the plasmidless strains produce two hitherto uncharacterized ubiquitous toxins encoded in chromosome I, namely, the hemolysin PhlyC and the phospholipase PlpV. We report the main roles of these two toxins in fish virulence and in cell toxicity. Our results constitute the basis for a better understanding of the virulence of a widespread marine pathogen.

scholarly journals Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

2015 ◽  
Vol 83 (7) ◽  
pp. 2614-2626 ◽  
Author(s):  
Rohitashw Kumar ◽  
Darpan Saraswat ◽  
Swetha Tati ◽  
Mira Edgerton
Keyword(s):  
Candida Albicans ◽  
Epithelial Cells ◽  
Oral Candidiasis ◽  
Oral Microbiome ◽  
Proteinase Activity ◽  
Adhesion Properties ◽  
Content Type ◽  
Oral Epithelial Cells ◽  
Oral Epithelial ◽  
Cell Cell

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually withC. albicanscells overexpressing Sap6 (SAP6OE and a Δsap8strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6strain was attenuated. These hypervirulent strains had highly aggregative colony structurein vitroand higher secreted proteinase activity; however, the levels of proteinase activity ofC. albicansSaps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6OE and Δsap8cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increasedC. albicansadhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

Modulation effects of hexamethylene bisacetamide on growth and differentiation of cultured human malignant glioma cells

1996 ◽  
Vol 84 (5) ◽  
pp. 831-838 ◽  
Author(s):  
Xiao-Nan Li ◽  
Zi-Wei Du ◽  
Qiang Huang
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Malignant Glioma ◽  
Culture Media ◽  
Morphological Changes ◽  
Control Group ◽  
Cell Cycle Distribution ◽  
Content Type ◽  
Hexamethylene Bisacetamide ◽  
Human Malignant Glioma

✓ The modulation effects of hexamethylene bisacetamide (HMBA), a differentiation-inducing agent, on growth and differentiation of cells from human malignant glioma cell line SHG-44 were studied. At cytostatic doses (2.5 mM, 5 mM, 7.5 mM, and 10 mM for 15 days), HMBA exerted a marked inhibitory effect on cell proliferation. Exposure to HMBA (5 mM and 10 mM for 12 days) also resulted in an accumulation of cells in G0/G1 phase and a decrease of cells in S phase as analyzed by flow cytometry. The reversible effects of 7.5 mM HMBA and 10 mM HMBA on cell proliferation and 10 mM HMBA on disruption of cell cycle distribution were observed when HMBA was removed from culture media on Day 6 and replaced with HMBA-free media. Colony-forming efficiency (CFE) in soft agar was remarkably decreased by HMBA (2.5 mM, 5 mM, 7.5 mM, and 10 mM for 14 days), and in 7.5 mM HMBA— and 10 mM HMBA—treated cells, the CFEs were reduced to 25% and 12.5%, respectively, of that in untreated cells. Cells treated with HMBA (5 mM and 10 mM for 15 days) remained tumorigenic in athymic nude mice, but the growth rates of the xenografts were much slower than those in the control group. The effects of HMBA on cell proliferation, cell cycle distribution, CFE, and growth of xenografts were dose dependent. A more mature phenotype was confirmed by the morphological changes from spindle shape to large polygonal stellate shape and remarkably elevated expression of glial fibrillary acidic protein in cells exposed to HMBA (5 mM, 10 mM for 15 days). Our results showed that a more differentiated phenotype with marked growth arrest was induced in SHG-44 cells by HMBA.

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