Laboratory and clinical Pseudomonas aeruginosa strains do not bind glycosphingolipids in vitro or during type IV pili-mediated initial host cell attachment

The glycosphingolipids (GSLs) gangliotriaosylceramide (Gg3) and gangliotetraosylceramide (Gg4) have been implicated as receptors for type IV pili (T4P)-mediated Pseudomonas aeruginosa epithelial cell attachment. Since P. aeruginosa T4P are divided into five groups, the authors determined whether GSLs in general, and Gg3 and Gg4 in particular, are specifically bound and required for host epithelial cell attachment of clinical and laboratory strains within these groups. An enterohaemorrhagic Escherichia coli strain, CL56, known to bind to both Gg3 and Gg4, provided a positive control. TLC overlay showed no binding of more than 12 P. aeruginosa strains to either Gg3 or Gg4 (or other GSLs), while CL56 Gg3/Gg4 binding was readily detectable. GSL ELISA similarly demonstrated no significant P. aeruginosa binding to Gg3 or Gg4, compared with CL56. Using a selective chemical inhibitor, epithelial cell GSL synthesis was abrogated, and Gg3 and Gg4 expression deleted, but P. aeruginosa attachment was not impaired. Target cell attachment was mediated by T4P, since non-piliated, but flagellated, mutants were unable to bind to the target cells. CFTR (cystic fibrosis transmembrane conductance regulator) has also been implicated as a receptor; however, in this work, overexpression of CFTR had no effect on P. aeruginosa binding. It is concluded that neither Gg3 nor Gg4 are specifically recognized by P. aeruginosa, and that endogenous GSLs do not have a role in the attachment of live intact P. aeruginosa to cultured lung epithelial cells. In contrast to whole piliated P. aeruginosa, T4P sheared from such bacteria showed significant Gg3 and Gg4 binding, which may explain the results of other studies.

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Type IV Secretion System Is Not Involved in Infection Process in Citrus

International Journal of Microbiology ◽

10.1155/2014/763575 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 10

Author(s):

Tiago Rinaldi Jacob ◽

Marcelo Luiz de Laia ◽

Leandro Marcio Moreira ◽

Janaína Fernandes Gonçalves ◽

Flavia Maria de Souza Carvalho ◽

...

Keyword(s):

Cell Envelope ◽

Positive Control ◽

Secretion System ◽

Infection Process ◽

Type Iv Secretion ◽

Target Cells ◽

Type Iv Secretion System ◽

In Planta ◽

Type Iv

The type IV secretion system (T4SS) is used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells.Xanthomonas citrisubsp.citricontains two copies of the T4SS, one in the chromosome and the other is plasmid-encoded. To understand the conditions that induce expression of the T4SS inXcc, we analyzed,in vitroandin planta, the expression of 18 ORFs from the T4SS and 7 hypothetical flanking genes by RT-qPCR. As a positive control, we also evaluated the expression of 29 ORFs from the type III secretion system (T3SS), since these genes are known to be expressed during plant infection condition, but not necessarily in standard culture medium. From the 29 T3SS genes analyzed by qPCR, onlyhrpAwas downregulated at 72 h after inoculation. All genes associated with the T4SS were downregulated onCitrusleaves 72 h after inoculation. Our results showed that unlike the T3SS, the T4SS is not induced during the infection process.

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Mucin Promotes Rapid Surface Motility in Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.00073-12 ◽

2012 ◽

Vol 3 (3) ◽

Cited By ~ 38

Author(s):

Amy T. Y. Yeung ◽

Alicia Parayno ◽

Robert E. W. Hancock

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iv Pili ◽

Transcriptional Analysis ◽

Bacterial Cells ◽

Content Type ◽

Type Iv ◽

Sensing Systems ◽

Bright Green ◽

Surface Motility

ABSTRACTAn important environmental factor that determines the mode of motility adopted byPseudomonas aeruginosais the viscosity of the medium, often provided by adjusting agar concentrationsin vitro. However, the viscous gel-like property of the mucus layer that overlays epithelial surfaces is largely due to the glycoprotein mucin.P. aeruginosais known to swim within 0.3% (wt/vol) agar and swarm on the surface at 0.5% (wt/vol) agar with amino acids as a weak nitrogen source. When physiological concentrations or as little as 0.05% (wt/vol) mucin was added to the swimming agar, in addition to swimming,P. aeruginosawas observed to undergo highly accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin appeared to be circular, with a bright green center surrounded by a thicker white edge. While intact flagella were required for the surface motility in the presence of mucin, type IV pili and rhamnolipid production were not. Replacement of mucin with other wetting agents indicated that the lubricant properties of mucin might contribute to the surface motility. Based on studies with mutants, the quorum-sensing systems (lasandrhl) and the orphan autoinducer receptor QscR played important roles in this form of surface motility. Transcriptional analysis of cells taken from the motility zone revealed the upregulation of genes involved in virulence and resistance. Based on these results, we suggest that mucin may be promoting a new or highly modified form of surface motility, which we propose should be termed “surfing.”IMPORTANCEAn important factor that dictates the mode of motility adopted byP. aeruginosais the viscosity of the medium, often provided by adjusting agar concentrationsin vitro. However, the gel-like properties of the mucous layers that overlay epithelial surfaces, such as those of the lung, a major site ofPseudomonasinfection, are contributed mostly by the production of the glycoprotein mucin. In this study, we added mucin to swimming media and found that it promoted the ability ofP. aeruginosato exhibit rapid surface motility. These motility colonies appeared in a circular form, with a bright green center surrounded by a thicker white edge. Interestingly, bacterial cells at the thick edge appeared piled up and lacked flagella, while cells at the motility center had flagella. Our data from various genetic and phenotypic studies suggest that mucin may be promoting a modified form of swarming or a novel form of surface motility inP. aeruginosa.

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The In Vitro Antibiofilm Activity of Water Leaf Extract of Papaya (Carica papaya L.) against Pseudomonas aeruginosa

Current Biochemistry ◽

10.29244/cb.2.3.150-163 ◽

2017 ◽

Vol 2 (3) ◽

pp. 150-163

Author(s):

Ekajayanti Kining ◽

Syamsul Falah ◽

Novik Nurhidayat

Keyword(s):

Pseudomonas Aeruginosa ◽

Contact Time ◽

Cell Attachment ◽

Water Extract ◽

Opportunistic Pathogen ◽

Bacterial Biofilm ◽

Antibiofilm Activity ◽

Bacterial Survival ◽

Optimum Conditions

Pseudomonas aeruginosa is one of opportunistic pathogen forming bacterial biofilm. The biofilm sustains the bacterial survival and infections. This study aimed to assess the activity of water extract of papaya leaves on inhibition of cells attachment, growth and degradation of the biofilm using crystal violet (CV) biofilm assay. Research results showed that water extract of papaya leaves contains alkaloids, tanins, flavonoids, and steroids/terpenoids and showed antibacterial activity and antibiofilm against P. aeruginosa. Addition of extract can inhibit the cell attachment and was able to degrade the biofilm of 40.92% and 48.058% respectively at optimum conditions: extract concentration of 25% (v/v), temperature 37.5 °C and contact time 45 minutes. With a concentration of 25% (v/v), temperature of 50 °C and the contact time of 3 days, extract of papaya leaves can inhibit the growth of biofilms of 39.837% v/v.

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Faculty Opinions recommendation of Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1011135.192160 ◽

2003 ◽

Author(s):

Leo Eberl

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Type Iv Pili ◽

Wild Type ◽

Type Iv

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Faculty Opinions recommendation of Distinct function of Pseudomonas aeruginosa type IV pili disclosed in the bacterial pass-through of membrane filter with smaller pore sizes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1081001.533960 ◽

2007 ◽

Author(s):

Esther Bullitt

Keyword(s):

Pseudomonas Aeruginosa ◽

Membrane Filter ◽

Type Iv Pili ◽

Pore Sizes ◽

Type Iv ◽

Pass Through

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The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

Cancers ◽

10.3390/cancers13123024 ◽

2021 ◽

Vol 13 (12) ◽

pp. 3024

Author(s):

Martin Fogtmann Berthelsen ◽

Maria Riedel ◽

Huiqiang Cai ◽

Søren H. Skaarup ◽

Aage K. O. Alstrup ◽

...

Keyword(s):

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Somatic Cells ◽

Genetic Alterations ◽

Lung Epithelial Cells ◽

Target Cells ◽

Multiple Gene ◽

Conditional Expression ◽

Gene Alterations

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

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Aztreonam Lysine Increases the Activity of Phages E79 and phiKZ against Pseudomonas aeruginosa PA01

Microorganisms ◽

10.3390/microorganisms9010152 ◽

2021 ◽

Vol 9 (1) ◽

pp. 152

Author(s):

Carly M. Davis ◽

Jaclyn G. McCutcheon ◽

Jonathan J. Dennis

Keyword(s):

Pseudomonas Aeruginosa ◽

Morphological Changes ◽

Burst Size ◽

Type Iv Pili ◽

Biofilm Growth ◽

Promising Alternative ◽

Type Iv ◽

Alternative Treatment Option ◽

One Step ◽

Step Growth

Pseudomonas aeruginosa is a pernicious bacterial pathogen that is difficult to treat because of high levels of antibiotic resistance. A promising alternative treatment option for such bacteria is the application of bacteriophages; the correct combination of phages plus antibiotics can produce synergistic inhibitory effects. In this study, we describe morphological changes induced by sub-MIC levels of the antibiotic aztreonam lysine (AzLys) on P. aeruginosa PA01, which may in part explain the observed phage–antibiotic synergy (PAS). One-step growth curves for phage E79 showed increased adsorption rates, decreased infection latency, accelerated time to lysis and a minor reduction in burst size. Phage E79 plus AzLys PAS was also able to significantly reduce P. aeruginosa biofilm growth over 3-fold as compared to phage treatment alone. Sub-inhibitory AzLys-induced filamentation of P. aeruginosa cells resulted in loss of twitching motility and a reduction in swimming motility, likely due to a reduction in the number of polar Type IV pili and flagella, respectively, on the filamented cell surfaces. Phage phiKZ, which uses Type IV pili as a receptor, did not exhibit increased activity with AzLys at lower sub-inhibitory levels, but still produced phage–antibiotic synergistic killing with sub-inhibitory AzLys. A one-step growth curve indicates that phiKZ in the presence of AzLys also exhibits a decreased infection latency and moderately undergoes accelerated time to lysis. In contrast to prior PAS studies demonstrating that phages undergo delayed time to lysis with cell filamentation, these PAS results show that phages undergo accelerated time to lysis, which therefore suggests that PAS is dependent upon multiple factors, including the type of phages and antibiotics used, and the bacterial host being tested.

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