A Mig-14-like protein (PA5003) affects antimicrobial peptide recognition in Pseudomonas aeruginosa

The evolution of antibiotic resistance in pathogenic bacteria is a growing global health problem which is gradually making the treatment of infectious diseases less efficient. Antimicrobial peptides are small charged molecules found in organisms from the complete phylogenetic spectrum. The peptides are attractive candidates for novel drug development due to their activity against bacteria that are resistant to conventional antibiotics, and reports of peptide resistance are rare in the clinical setting. Paradoxically, many clinically relevant bacteria have mechanisms that can recognize and respond to the presence of cationic antimicrobial peptides (CAMPs) in the environment by changing the properties of the microbial surface thereby increasing the tolerance of the microbes towards the peptides. In Pseudomonas aeruginosa an essential component of this inducible tolerance mechanism is the lipopolysaccharide modification operon arnBCADTEF–PA3559 which encodes enzymes required for LPS alterations leading to increased antimicrobial peptide tolerance. The expression of the operon is induced by the presence of CAMPs in the environment but the molecular mechanisms underlying the cellular recognition of the peptides are poorly elucidated. In this work, we investigate the factors influencing arnB expression by transposon mutagenesis and arnB promoter green fluorescent protein reporters. We have identified a novel gene encoding a Mig-14-like protein that is required for recognition of the CAMPs colistin and Novispirin G10 by P. aeruginosa. Moreover, we show that this gene is also required for the formation of CAMP-tolerant subpopulations in P. aeruginosa hydrodynamic flow chamber biofilms.

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Stratified Growth in Pseudomonas aeruginosa Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.70.10.6188-6196.2004 ◽

2004 ◽

Vol 70 (10) ◽

pp. 6188-6196 ◽

Cited By ~ 230

Author(s):

Erin Werner ◽

Frank Roe ◽

Amandine Bugnicourt ◽

Michael J. Franklin ◽

Arne Heydorn ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Protein Synthesis ◽

Fluorescent Protein ◽

Anaerobic Growth ◽

Synthetic Activity ◽

Gene Encoding ◽

Rate Dependent ◽

Air Interface ◽

Transmission Electron ◽

Green Fluorescent

ABSTRACT In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp 1 promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 μm wide in colony biofilms and 30 μm wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 μm into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.

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The PhlA Hemolysin from the Entomopathogenic Bacterium Photorhabdusluminescens Belongs to the Two-Partner Secretion Family of Hemolysins

Journal of Bacteriology ◽

10.1128/jb.184.14.3871-3878.2002 ◽

2002 ◽

Vol 184 (14) ◽

pp. 3871-3878 ◽

Cited By ~ 56

Author(s):

Julien Brillard ◽

Eric Duchaud ◽

Noël Boemare ◽

Frank Kunst ◽

Alain Givaudan

Keyword(s):

Hemolytic Activity ◽

Pathogenic Bacteria ◽

Fluorescent Protein ◽

Gene Encoding ◽

Rich Media ◽

The Family ◽

Insect Interactions ◽

Green Fluorescent

ABSTRACT Photorhabdus is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae. Bacterial hemolysins found in numerous pathogenic bacteria are often virulence factors. We describe here the nucleotide sequence and the molecular characterization of the Photorhabdus luminescens phlBA operon, a locus encoding a hemolysin which shows similarities to the Serratia type of hemolysins. It belongs to the two-partner secretion (TPS) family of proteins. In low-iron conditions, a transcriptional induction of the phlBA operon was observed by using the chloramphenicol acetyltransferase reporter gene, causing an increase in PhlA hemolytic activity compared to iron-rich media. A spontaneous phase variant of P. luminescens was deregulated in phlBA transcription. The phlA mutant constructed by allelic exchange remained highly pathogenic after injection in the lepidopteran Spodoptera littoralis, indicating that PhlA hemolysin is not a major virulence determinant. Using the gene encoding green fluorescent protein as a reporter, phlBA transcription was observed in hemolymph before insect death. We therefore discuss the possible role of PhlA hemolytic activity in the bacterium-nematode-insect interactions.

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Exploring resveratrol dimers as virulence blocking agents – Attenuation of type III secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa

Scientific Reports ◽

10.1038/s41598-020-58872-0 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Charlotta Sundin ◽

Caroline E. Zetterström ◽

Duc Duy Vo ◽

Robert Brkljača ◽

Sylvia Urban ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Bacterial Infections ◽

Pathogenic Bacteria ◽

Yersinia Pseudotuberculosis ◽

Fluorescent Protein ◽

Screening Method ◽

Resistant Bacteria ◽

Type Iii ◽

Green Fluorescent

AbstractBacterial infections continue to threaten humankind and the rapid spread of antibiotic resistant bacteria is alarming. Current antibiotics target essential bacterial processes and thereby apply a strong selective pressure on pathogenic and non-pathogenic bacteria alike. One alternative strategy is to block bacterial virulence systems that are essential for the ability to cause disease but not for general bacterial viability. We have previously show that the plant natural product (-)-hopeaphenol blocks the type III secretion system (T3SS) in the Gram-negative pathogens Yersinia pseudotuberculosis and Pseudomonas aeruginosa. (-)-Hopeaphenol is a resveratrol tetramer and in the present study we explore various resveratrol dimers, including partial structures of (-)-hopeaphenol, as T3SS inhibitors. To allow rapid and efficient assessment of T3SS inhibition in P. aeruginosa, we developed a new screening method by using a green fluorescent protein reporter under the control of the ExoS promoter. Using a panel of assays we showed that compounds with a benzofuran core structure i.e. viniferifuran, dehydroampelopsin B, anigopreissin A, dehydro-δ-viniferin and resveratrol-piceatannol hybrid displayed significant to moderate activities towards the T3SS in Y. pseudotuberculosis and P. aeruginosa.

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Synergy pattern of short cationic antimicrobial peptides against multidrug-resistant Pseudomonas aeruginosa

10.1101/639286 ◽

2019 ◽

Author(s):

Serge Ruden ◽

Annika Rieder ◽

Thomas Schwartz ◽

Ralf Mikut ◽

Kai Hilpert

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Peptides ◽

Pathogenic Bacteria ◽

Synergistic Effects ◽

Polymyxin B ◽

Multidrug Resistant ◽

Multi Drug Resistance ◽

Resistant Bacteria ◽

Drug Resistant ◽

Conventional Antibiotics

AbstractWith the rise of various multi-drug resistance pathogenic bacteria, worldwide health care is under pressure to respond. Conventional antibiotics are failing and the development of novel classes or alternative strategies is a major priority. Antimicrobial peptides (AMPs) can not only kill multi-drug resistant bacteria, but also can be used synergistically with conventional antibiotics. We selected 30 short AMPs from different origins and measured their synergy in combination with Polymyxin B, Piperacillin, Ceftazidime, Cefepime, Meropenem, Imipenem, Tetracycline, Erythromycin, Kanamycin, Tobramycin, Amikacin, Gentamycin, and Ciprofloxacin. In total 403 unique combinations were tested against a multi-drug resistant Pseudomonas aeruginosa isolate (PA910). As a measure of the synergistic effects, fractional inhibitory concentrations (FICs) were determined using microdilution assays with FICs ranges between 0.25 and 2. A high number of combinations between peptides and Polymyxin B, Erythromycin and Tetracycline were found to be synergistic. Novel variants of Indolicidin also showed a high frequency in synergist interaction.

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Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

Applied and Environmental Microbiology ◽

10.1128/aem.06644-11 ◽

2011 ◽

Vol 77 (23) ◽

pp. 8310-8317 ◽

Cited By ~ 27

Author(s):

Joshua D. Morris ◽

Jessica L. Hewitt ◽

Lawrence G. Wolfe ◽

Nachiket G. Kamatkar ◽

Sarah M. Chapman ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Fluorescent Protein ◽

Nile Red ◽

Temporal Distribution ◽

Time Lapse ◽

Content Type ◽

Rhamnolipid Production ◽

Serovar Typhimurium ◽

Surface Motility ◽

Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

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WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum

Physiological Genomics ◽

10.1152/physiolgenomics.00105.2005 ◽

2005 ◽

Vol 22 (3) ◽

pp. 412-421 ◽

Cited By ~ 19

Author(s):

Mira Wouters ◽

Karine Smans ◽

Jean-Marie Vanderwinden

Keyword(s):

Small Intestine ◽

Green Fluorescent Protein ◽

Gastrointestinal Tract ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Molecular Layer ◽

Transgenic Animals ◽

The Novel ◽

Suitable Alternative ◽

Green Fluorescent

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel green fluorescent protein ZsGreen into the first exon of the c-Kit gene, creating a null allele called WZsGreen. In the gastrointestinal tract of heterozygous WZsGreen/+ mice, tiny ZsGreen fluorescent dots were observed in all KIT-expressing ICC populations, with exception of ICC at the deep muscular plexus in small intestine. During development of the gastrointestinal tract, ZsGreen expression followed KIT expression in a spatiotemporal way. Stellate and basket KIT-expressing cells in the molecular layer of the cerebellum also exhibited ZsGreen dots, whereas no ZsGreen was detected in skin, testis, and bone marrow. ZsGreen dot-containing intestinal cells could be isolated from jejunum and maintained alive in culture for at least 3 days. ZsGreen is a suitable alternative to EGFP in transgenic animals. The novel WZsGreen/+ model reported here appears to be a promising tool for live studies of KIT-expressing cells in the gastrointestinal tract and cerebellum and for the further analysis of pacemaker mechanisms.

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Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

Applied and Environmental Microbiology ◽

10.1128/aem.00414-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5060-5069 ◽

Cited By ~ 124

Author(s):

Morten T. Rybtke ◽

Bradley R. Borlee ◽

Keiji Murakami ◽

Yasuhiko Irie ◽

Morten Hentzer ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Subsequent Development ◽

Cellular Level ◽

Host Immune System ◽

Chronic Infections ◽

Content Type ◽

Genes Encoding ◽

Green Fluorescent

ABSTRACTThe increased tolerance toward the host immune system and antibiotics displayed by biofilm-formingPseudomonas aeruginosaand other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP inP. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsivecdrApromoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP inP. aeruginosastrains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment ofP. aeruginosawith the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology ofP. aeruginosa.

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Recruitment of the LIM protein hic-5 to focal contacts of human platelets

Journal of Cell Science ◽

10.1242/jcs.111.15.2181 ◽

1998 ◽

Vol 111 (15) ◽

pp. 2181-2188 ◽

Cited By ~ 1

Author(s):

J. Hagmann ◽

M. Grob ◽

A. Welman ◽

G. van Willigen ◽

M.M. Burger

Keyword(s):

Fluorescent Protein ◽

Human Platelets ◽

Gene Encoding ◽

Chain Reactions ◽

Amino Terminal ◽

Culture Cells ◽

Lim Domains ◽

Focal Contacts ◽

Green Fluorescent ◽

Carboxy Terminal

Platelets are anuclear, membrane-bounded fragments derived from megakaryocytes which, upon stimulation, assemble an actin skeleton including stress fibres and focal contacts. The focal contacts resemble those of tissue culture cells. However, they lack paxillin, a conspicuous component of these organelles. We found that instead of paxillin, platelets contain a related protein with a molecular mass of 55 kDa that crossreacts with a monoclonal antibody against paxillin. The gene for the 55 kDa protein was cloned from a bone marrow cDNA library and turned out to be identical to a recently discovered gene encoding hic-5. Like paxillin, hic-5 is a cytoskeletal protein containing four carboxy-terminal LIM domains and LD motifs in the amino-terminal half. The LIM domains of both hic-5 and paxillin are capable of targetting green fluorescent protein to focal contacts. In addition, GST-hic-5 precipitates the focal adhesion kinase pp125(FAK) and talin from platelet extracts. Only trace amounts of hic-5 occur in DAMI cells, a megakaryocytic cell line, and in megakaryocytes cultured from CD34+ cells obtained from umbilical cord blood. However, RT-polymerase chain reactions performed with RNA obtained from platelets gave a positive result when primers specific for hic-5 were used, but were negative with paxillin-specific primers, indicating that a switch from paxillin expression to hic-5 expression must occur late in the maturation of megakaryocytes into platelets.

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Induced Expression of the Heat Shock Protein Genes uspA and grpE during Starvation at Low Temperatures and Their Influence on Thermal Resistance of Escherichia coli O157:H7

Journal of Food Protection ◽

10.4315/0362-028x-66.11.2045 ◽

2003 ◽

Vol 66 (11) ◽

pp. 2045-2050 ◽

Cited By ~ 25

Author(s):

YI ZHANG ◽

MANSEL W. GRIFFITHS

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

Stress Responses ◽

Thermal Tolerance ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Bacterial Cells ◽

Induced Expression ◽

Green Fluorescent ◽

Shock Proteins

Heat shock proteins play an important role in protecting bacterial cells against several stresses, including starvation. In this study, the promoters for two genes encoding heat shock proteins involved in many stress responses, UspA and GrpE, were fused with the green fluorescent protein (gfp) gene. Thus, the expression of the two genes could be quantified by measuring the fluorescence emitted by the cells under different environmental conditions. The heat resistance levels of starved and nonstarved cells during storage at 5, 10, and 37°C were compared with the levels of expression of the uspA and grpE genes. D52-values (times required for decimal reductions in count at 52°C) increased by 11.5, 14.6, and 18.5 min when cells were starved for 3 h at 37°C, for 24 h at 10°C, and for 2 days at 5°C, respectively. In all cases, these increases were significant (P < 0.01), indicating that the stress imposed by starvation altered the ability of E. coli O157:H7 to survive subsequent heat treatments. Thermal tolerance was correlative with the induction of UspA and GrpE. At 5°C, the change in the thermal tolerance of the pathogen was positively linked to the induced expression of the grpE gene but negatively related to the expression of the uspA gene. The results obtained in this study indicate that UspA plays an important role in starvation-induced thermal tolerance at 37°C but that GrpE may be more involved in regulating this response at lower temperatures. An improvement in our understanding of the molecular mechanisms involved in these cross-protection responses may make it possible to devise strategies to limit their effects.

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Combined Antibacterial and Anti-Inflammatory Activity of a Cationic Disubstituted Dexamethasone-Spermine Conjugate

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01682-09 ◽

2010 ◽

Vol 54 (6) ◽

pp. 2525-2533 ◽

Cited By ~ 10

Author(s):

Robert Bucki ◽

Katarzyna Leszczyńska ◽

Fitzroy J. Byfield ◽

David E. Fein ◽

Esther Won ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Infections ◽

Glucocorticoid Receptors ◽

Fluorescent Protein ◽

Lipoteichoic Acid ◽

Bacterial Strains ◽

Bacterial Killing ◽

Antibiotic Resistant ◽

Anti Inflammatory ◽

Green Fluorescent

ABSTRACT The rising number of antibiotic-resistant bacterial strains represents an emerging health problem that has motivated efforts to develop new antibacterial agents. Endogenous cationic antibacterial peptides (CAPs) that are produced in tissues exposed to the external environment are one model for the design of novel antibacterial compounds. Here, we report evidence that disubstituted dexamethasone-spermine (D2S), a cationic corticosteroid derivative initially identified as a by-product of synthesis of dexamethasone-spermine (DS) for the purpose of improving cellular gene delivery, functions as an antibacterial peptide-mimicking molecule. This moiety exhibits bacterial killing activity against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa present in cystic fibrosis (CF) sputa, and Pseudomonas aeruginosa biofilm. Although compromised in the presence of plasma, D2S antibacterial activity resists the proteolytic activity of pepsin and is maintained in ascites, cerebrospinal fluid, saliva, and bronchoalveolar lavage (BAL) fluid. D2S also enhances S. aureus susceptibility to antibiotics, such as amoxicillin (AMC), tetracycline (T), and amikacin (AN). Inhibition of interleukin-6 (IL-6) and IL-8 release from lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated neutrophils in the presence of D2S suggests that this molecule might also prevent systemic inflammation caused by bacterial wall products. D2S-mediated translocation of green fluorescent protein (GFP)-labeled glucocorticoid receptor (GR) in bovine aorta endothelial cells (BAECs) suggests that some of its anti-inflammatory activities involve engagement of glucocorticoid receptors. The combined antibacterial and anti-inflammatory activities of D2S suggest its potential as an alternative to natural CAPs in the prevention and treatment of some bacterial infections.

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