Epigenetic acquisition of inducibility of type III cytotoxicity in P. aeruginosa

BACKGROUND: Pseudomonas aeruginosa, an opportunistic pathogen, is often encountered in chronic lung diseases such as cystic fibrosis or chronic obstructive pneumonia, as well as acute settings like mechanical ventilation acquired pneumonia or neutropenic patients. It is a major cause of mortality and morbidity in these diseases. In lungs, P. aeruginosa settles in a biofilm mode of growth with the secretion of exopolysaccharides in which it is encapsulated, enhancing its antibiotic resistance and contributing to the respiratory deficiency of patients. However, bacteria must first multiply to a high density and display a cytotoxic phenotype to avoid the host's defences. A virulence determinant implicated in this step of infection is the type III secretion system (TTSS), allowing toxin injection directly into host cells. At the beginning of the infection, most strains isolated from patients' lungs possess an inducible TTSS allowing toxins injection or secretion upon in vivo or in vitro activation signals. As the infection persists most of the bacteria permanently loose this capacity, although no mutations have been evidenced. We name "non inducible" this phenotype. As suggested by the presence of a positive feedback circuit in the regulatory network controlling TTSS expression, it may be due to an epigenetic switch allowing heritable phenotypic modifications without genotype's mutations. RESULTS: Using the generalised logical method, we designed a minimal model of the TTSS regulatory network that could support the epigenetic hypothesis, and studied its dynamics which helped to define a discriminating experimental scenario sufficient to validate the epigenetic hypothesis. A mathematical framework based on formal methods from computer science allowed a rigorous validation and certification of parameters of this model leading to epigenetic behaviour. Then, we demonstrated that a non inducible strain of P. aeruginosa can stably acquire the capacity to be induced by calcium depletion for the TTSS after a short pulse of a regulatory protein. Finally, the increased cytotoxicity of a strain after this epigenetic switch was demonstrated in vivo in an acute pulmonary infection model. CONCLUSION: These results may offer new perspectives for therapeutic strategies to prevent lethal infections by P. aeruginosa by reverting the epigenetic inducibility of type III cytotoxicity

Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas fluorescens </it>is a ubiquitous Gram-negative bacterium frequently encountered in hospitals as a contaminant of injectable material and surfaces. This psychrotrophic bacterium, commonly described as unable to grow at temperatures above 32°C, is now considered non pathogenic. We studied a recently identified clinical strain of <it>P. fluorescens </it>biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37°C in laboratory conditions.</p> <p>Results</p> <p>We found that MFN1032 secreted extracellular factors with a lytic potential at least as high as that of MF37, a psychrotrophic strain of <it>P. fluorescens </it>or the mesophilic opportunistic pathogen, <it>Pseudomonas aeruginosa </it>PAO1. We demonstrated the direct, and indirect – through increases in biosurfactant release – involvement of a phospholipase C in the hemolytic activity of this bacterium. Sequence analysis assigned this phospholipase C to a new group of phospholipases C different from those produced by <it>P. aeruginosa</it>. We show that changes in PlcC production have pleiotropic effects and that <it>plcC </it>overexpression and <it>plcC </it>extinction increase MFN1032 toxicity and colonization, respectively.</p> <p>Conclusion</p> <p>This study provides the first demonstration that a PLC is involved in the secreted hemolytic activity of a clinical strain of <it>Pseudomonas fluorescens</it>. Moreover, this phospholipase C seems to belong to a complex biological network associated with the biosurfactant production.</p

Boolean Models of Biosurfactants Production in Pseudomonas fluorescens

Cyclolipopeptides (CLPs) are biosurfactants produced by numerous Pseudomonas fluorescens strains. CLP production is known to be regulated at least by the GacA/GacS two-component pathway, but the full regulatory network is yet largely unknown. In the clinical strain MFN1032, CLP production is abolished by a mutation in the phospholipase C gene () and not restored by complementation. Their production is also subject to phenotypic variation. We used a modelling approach with Boolean networks, which takes into account all these observations concerning CLP production without any assumption on the topology of the considered network. Intensive computation yielded numerous models that satisfy these properties. All models minimizing the number of components point to a bistability in CLP production, which requires the presence of a yet unknown key self-inducible regulator. Furthermore, all suggest that a set of yet unexplained phenotypic variants might also be due to this epigenetic switch. The simplest of these Boolean networks was used to propose a biological regulatory network for CLP production. This modelling approach has allowed a possible regulation to be unravelled and an unusual behaviour of CLP production in P. fluorescens to be explained

Cell-associated hemolysis activity in the clinical strain of Pseudomonas fluorescens MFN1032

<p>Abstract</p> <p>Background</p> <p>MFN1032 is a clinical <it>Pseudomonas fluorescens </it>strain able to grow at 37°C. MFN1032 cells induce necrosis and apoptosis in rat glial cells at this temperature. This strain displays secretion-mediated hemolytic activity involving phospholipase C and cyclolipopeptides. Under laboratory conditions, this activity is not expressed at 37°C. This activity is tightly regulated and is subject to phase variation.</p> <p>Results</p> <p>We found that MFN1032 displays a cell-associated hemolytic activity distinct from the secreted hemolytic activity. Cell-associated hemolysis was expressed at 37°C and was only detected <it>in vitro </it>in mid log growth phase in the presence of erythrocytes. We studied the regulation of this activity in the wild-type strain and in a mutant defective in the Gac two-component pathway. GacS/GacA is a negative regulator of this activity. In contrast to the <it>Pseudomonas fluorescens </it>strains PfO-1 and Pf5, whose genomes have been sequenced, the MFN1032 strain has the type III secretion-like genes <it>hrc</it>RST belonging to the <it>hrpU </it>operon. We showed that disruption of this operon abolished cell-associated hemolytic activity. This activity was not detected in <it>P.fluorescens </it>strains carrying similar <it>hrc </it>genes, as for the <it>P. fluorescens </it>psychrotrophic strain MF37.</p> <p>Conclusions</p> <p>To our knowledge this the first demonstration of cell-associated hemolytic activity of a clinical strain of <it>Pseudomonas fluorescens</it>. Moreover, this activity seems to be related to a functional <it>hrpU </it>operon and is independent of biosurfactant production. Precise link between a functional <it>hrpU </it>operon and cell-associated hemolytic activity remains to be elucidated.</p

HISTONE DEACETYLASES IN INFLAMMATORY BOWEL DISEASES - INFLUENCE ON THE ANTIMICROBIAL BARRIER

The development and clinical behavior of the two major inflammatory bowel diseases (IBD) subgroups Crohn’s disease (CD) and ulcerative colitis (UC), are determined by multiple underlying factors leading to an impaired antimicrobial barrier and chronic inflammation. Contributing environmental influences on the onset of the diseases such as the intestinal microbiota, antibiotics use, smoking, or nutrition have more and more entered the limelight of the field. The increasing incidence of IBD, the big variance in disease progression, and the discrepancy between monocygotic twins urgently impose the question how exactly environmental factors might impact on IBD risk and progression. The emerging field of epigenetics offers a mechanistic framework to dissect the present interplay between environment and genome in the context of IBD and will allow a better understanding of disease pathology. Histone deacetylases (HDACs) are important epigenetic factors implicated in intestinal tissue homeostasis. They deacetylate histones but also a vast number of non-histone proteins, e.g. the transcription factor NF-κB, thereby impacting on transcriptional regulation on different levels. A role for HDACs in the epigenetically-mediated modulation of hBDs has been demonstrated in a number of studies, underscoring a potential involvement of HDACs in the β-defensin-related defects found in colonic IBD. In addition, inhibiting HDACs has been proposed as an IBD intervention, making more detailed studies on the role of HDACs in gut antimicrobial barrier function indespensible. Firstly, in this work, a systematic overview of class I HDAC mRNA intestinal expression has been performed in a large cohort of IBD patients. First insights on the expression pattern on the protein level are also given. Secondly, this study aimed to contribute to the small body of knowledge on HDAC-mediated epigenetic control of antimicrobial peptide (AMP) expression; focusing on human β-defensin 2 (hBD2). Herein, emphasis has been laid on the therapeutically relevant probiotic E. coli Nissle 1917 (EcN) as a potent hBD2-inducing factor in addition to the pro-inflammatory cytokine IL1β and the bacterial membrane component LPS. In vitro HDAC inhibition (HDACi) in colonic epithelial cells generated a strong, NF-κB-dependent enhancement of EcN- and LPS-induced hBD2 expression, but also of the pro-inflammatory cytokine IL8. For IL1β-induced hBD2, the observed augmentation seems to be mediated by additional transducing factors and conditions depending on which HDACs are inhibited. In the case of IL1β, the enhancing effect of HDACi on hBD2 could be evinced in a second colonic epithelial cell line. In an effort to closer mimic the in vivo situation, an ex vivo human colonic biopsy culture has been established with almost identical culture conditions using IBD and healthy control tissue. This was aimed to allow a comparison to the in vitro results, since it is of substantial importance to learn how a more complex, non-tumorous human tissue compound reacts to HDACi. Strikingly, in this context, an opposing impact of HDACi on EcN-stimulated hBD2 expression was observed. Inhibiting HDAC function using pan-HDAC inhibitors hindered hBD2 expression instead of enhancing it, but did not impede IL8 expression. Whether these contrary results could be due to the malignant nature of the in vitro cell lines was investigated using ex vivo treated human colorectal cancer tissue showing the same response as non-cancerous intestinal biopsies. In addition, first insights into the reactivity of a primary, non-transformed gingival epithelial cell line towards HDACi showed a comparable hBD2 response upon IL1β-stimulation as did the cancerous intestinal cell lines. Furthermore, ex vivo fold induction levels of hBD2 in CD patients have been reduced hinting towards a disturbance in hBD2 inducibility in response to EcN. In this study, differential intestinal mRNA expression patterns have been unveiled for class I HDACs. Importantly, a strong regulatory influence of HDACs on the expression of hBD2 could be demonstrated. The simultaneous upregulation of IL8 in epithelial cells and the missing downregulation of the same in biopsy culture under HDACi, advises caution in considering HDACi as therapeutic in IBD. Furthermore, a dependency of the HDACi-induced hBD2 enhancement on NF-κB could be found. Utilizing different culture approaches, the obtained results argue for a cellular context-dependent modulation of the epigenetic regulation of hBD2 expression by HDACs. Overall, this work promotes the understanding of epigenetics as the conjoining integrative mechanism between genome and environment, bridging the way to answering many yet elusive questions in the pathogenesis of IBD

Nanotheranostics: A Possible Solution for Drug-Resistant Staphylococcus aureus and their Biofilms?

Staphylococcus aureus is a notorious pathogen that colonizes implants (orthopedic and breast implants) and wounds with a vicious resistance to antibiotic therapy. Methicillin-resistant S. aureus (MRSA) is a catastrophe mainly restricted to hospitals and emerged to community reservoirs, acquiring resistance and forming biofilms. Treating biofilms is problematic except via implant removal or wound debridement. Nanoparticles (NPs) and nanofibers could combat superbugs and biofilms and rapidly diagnose MRSA. Nanotheranostics combine diagnostics and therapeutics into a single agent. This comprehensive review is interpretative, utilizing mainly recent literature (since 2016) besides the older remarkable studies sourced via Google Scholar and PubMed. We unravel the molecular S. aureus resistance and complex biofilm. The diagnostic properties and detailed antibacterial and antibiofilm NP mechanisms are elucidated in exciting stories. We highlight the challenges of bacterial infections nanotheranostics. Finally, we discuss the literature and provide “three action appraisals”. (i) The first appraisal consists of preventive actions (two wings), avoiding unnecessary hospital visits, hand hygiene, and legislations against over-the-counter antibiotics as the general preventive wing. Our second recommended preventive wing includes preventing the adverse side effects of the NPs from resistance and toxicity by establishing standard testing procedures. These standard procedures should provide breakpoints of bacteria’s susceptibility to NPs and a thorough toxicological examination of every single batch of synthesized NPs. (ii) The second appraisal includes theranostic actions, using nanotheranostics to diagnose and treat MRSA, such as what we call “multifunctional theranostic nanofibers. (iii) The third action appraisal consists of collaborative actions

Bioproducts for health II

In order to build a promising future, health and sustainability must be interelated. Marine, forestry, agriculture, and food systems are important sources of bioproducts used in health applications. To explore the potential of such sources for the development of natural products capable of biological activities, it is necessary to develop new technologically sustainable strategies. Despite the range of natural compounds already available, there is a need to identify bioactive molecules (e.g., polysaccharides, proteins and peptides, polyunsaturated fatty acids, and polyphenols) from different natural sources with positive health properties, including antihypertensive, antidiabetic, anti-obesity, antimicrobial, anti-atherosclerotic, antioxidant, antithrombotic, immune-modulatory, relaxing, and satiety-inducing effects. The Second Edition of this Special Issue aimed to identify and gather works on the latest varied sources of bioproducts, the biological and functional activities of these bioactive compounds, their mechanisms of action, and the methods used for extraction and purification, without losing our focus on alignment with the concept of green technology.info:eu-repo/semantics/publishedVersio

Nanotheranostics: A Possible Solution for Drug-Resistant Staphylococcus aureus and their Biofilms?

Staphylococcus aureus is a notorious pathogen that colonizes implants (orthopedic and breast implants) and wounds with a vicious resistance to antibiotic therapy. Methicillin-resistant S. aureus (MRSA) is a catastrophe mainly restricted to hospitals and emerged to community reservoirs, acquiring resistance and forming biofilms. Treating biofilms is problematic except via implant removal or wound debridement. Nanoparticles (NPs) and nanofibers could combat superbugs and biofilms and rapidly diagnose MRSA. Nanotheranostics combine diagnostics and therapeutics into a single agent. This comprehensive review is interpretative, utilizing mainly recent literature (since 2016) besides the older remarkable studies sourced via Google Scholar and PubMed. We unravel the molecular S. aureus resistance and complex biofilm. The diagnostic properties and detailed antibacterial and antibiofilm NP mechanisms are elucidated in exciting stories. We highlight the challenges of bacterial infections nanotheranostics. Finally, we discuss the literature and provide “three action appraisals”. (i) The first appraisal consists of preventive actions (two wings), avoiding unnecessary hospital visits, hand hygiene, and legislations against over-the-counter antibiotics as the general preventive wing. Our second recommended preventive wing includes preventing the adverse side effects of the NPs from resistance and toxicity by establishing standard testing procedures. These standard procedures should provide breakpoints of bacteria’s susceptibility to NPs and a thorough toxicological examination of every single batch of synthesized NPs. (ii) The second appraisal includes theranostic actions, using nanotheranostics to diagnose and treat MRSA, such as what we call “multifunctional theranostic nanofibers. (iii) The third action appraisal consists of collaborative actions

Complex regulation of BpeEF-OprC mediated drug efflux in Burkholderia pseudomallei

2016 Spring.Includes bibliographical references.Burkholderia pseudomallei (Bp) is a Gram-negative bacillus and the etiologic agent of melioidosis, a multifaceted syndrome causing high mortality in tropical regions of the world. The bacteria is classified as a Tier-1 Select Agent due to the seriousness of infection, low infectious dose, lack of effective vaccine, and difficulty of treatment. Bp’s many acquired and intrinsic antimicrobial resistance determinants make the study of these factors vital to improving the efficacy of bi-phasic treatment currently used to treat melioidosis. This study examines one factor in particular: the BpeEF-OprC efflux pump, a member of the resistance-nodulation and cell division family of efflux proteins, and capable of extruding both trimethoprim and sulfamethoxazole. A combination of these compounds (co-trimoxazole) is the first line of eradication phase therapy, making BpeEF-OprC the most clinically important efflux pump encoded by Bp. In spite of this, little is understood of the regulation of bpeEF-oprC, other than it is controlled in part by two LysR family proteins, BpeS, and BpeT. We hypothesized that these regulatory proteins 1) exert their action(s) by interacting with bpeEF-oprC at a specific site within the bpeT-llpE-bpeEF-oprC intergenic region, 2) are capable of influencing transcription of additional operons, and 3) that mutations to these proteins altered ability to form multimers, thereby influencing their function as observed by increased co-trimoxazole resistance and bpeF transcript levels. In Aim I of the study, we identified the cis regulatory regions by which these proteins interact within the bpeT-llpE-bpeE intergenic region using a combination of 5’ deletion assays, S1 nuclease protection, fluorescent-linked oligo extension and electrophoretic mobility assays. With this information we were able to locate bpeT transcriptional start sites and promoter regions as well as binding sites for both BpeT and BpeS. In Aim II, we examined the function of BpeT and BpeS as trans regulatory factors of BpeEF-OprC through mutation and deletion of both genes in part I, and as global regulatory factors in part II. Through overexpression and qRT-PCR or MIC analysis of wild type and mutant forms of both genes, we observed that while BpeT is a direct transcriptional activator of bpeEF-oprC, BpeS is not. Additionally, mutation position in BpeS seems to play a role in the expression phenotype of bpeEF-oprC. However, these mutations do not influence the ability of BpeS or BpeT to form multimers, as we observed no change between wild type and mutant protein oligomer formation through low-pressure gel chromatography and native gel electrophoresis. These same mutations also appeared to have no deleterious effect on the ability of the protein to bind their consensus region within the IR. Additionally, the loss of both genes did not interrupt the ability of bpeEF-oprC to be induced by substrates of BpeEF-OprC, suggesting an additional regulatory factor is at play. In Part II, RNA sequencing analysis and confirmation of select transcriptionally altered operons by RT-qPCR revealed that BpeS might influence expression of the Bsa Type 3 Secretion System (T3SS), while BpeT seems only to target bpeEF-oprC. This may have implication in the pathogenesis of Bp, and must be confirmed in in-vivo cell models using Select Agent excluded strain Bp82 in order to solidify the link between efflux and T3SS during infection. Ultimately, more work is needed to identify the missing regulatory factors in play during expression of bpeEF-oprC, understand how mutations to BpeT and BpeS alter their function, and confirm the relevance of a putative link between co-regulation of efflux and virulence during Bp infection

The impact of a single nucleotide polymorphism in fusA1 on biofilm formation and virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that is now the leading cause of morbidity and mortality in immunocompromised individuals. Those suffering with the genetic disease cystic fibrosis (CF) commonly encounter P. aeruginosa infections. P. aeruginosa infection can present itself as an acute infection, which is characterised by highly virulent, “free-swimming” bacteria, or as a chronic infection associated with the formation of surface-adhered bacterial communities known as biofilms. The labyrinth of interconnecting signalling networks has meant that the regulatory mechanisms behind biofilm formation and virulence are largely undefined. In this dissertation, a single nucleotide polymorphism was identified within the gene, fusA1, encoding elongation factor G (EF-G). The mutation introduced minor structural changes to the protein which were likely to have functional repercussions in its involvement in protein synthesis. Phenotypic analysis revealed that the mutation conferred changes in both resistance and sensitivity to various antibiotics, as well as changes in motility, exoenzyme production, quorum sensing, metabolism, synthesis of biofilm-associated proteins and exopolysaccharide production. Most notably was the up-regulation of a major virulence determinant, the type three secretion system, typically characteristic of cells comprising an acute infection. Proteomic and transcriptomic profiling of the mutant strain provided an insight into the genetic basis behind these phenotypes, identifying the up-regulation of multidrug efflux systems and modulations to the chemotactic systems. This study also found links between several biological processes that were modulated in the mutant strain, such as crosstalk between sulfur metabolism, iron uptake and the oxidative stress response. In summary, the work presented in this dissertation highlights the susceptibility of fusA1 to spontaneous mutation and identifies a novel role for EF-G in bacterial virulence and antibiotic sensitivity, both of which have worrying implications for infection within the CF lung.Medical Research Council King's College Cambridge Cambridge Philosophical Societ