The type II secretion system and its ubiquitous lipoprotein substrate, SsIE are required for biofilm formation and virulence of enteropathogenic escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenic E. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease

MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices

La coscienza del futuro

Prefazion

The regulation of Saxitoxin production in Cyanobacteria

Aquatic microalgae produce a variety of toxic secondary metabolites, which are a concern for public health and seafood industries, while also presenting a source of pharmacologically valuable compounds. The present study deals with the physiology and molecular genetics of saxitoxin (STX), a cyanobacterial neurotoxic alkaloid. Ecological and chemical parameters have been investigated for their effects on growth and STX production in the cyanobacterium Cylindrospermopsis raciborskii T3, in order to better understand the physiological responses of this cyanobacterium to the anthropogenic eutrophication of water bodies. The results indicated that phosphate, in particular, had an incremental effect on STX production, as well as promoting the up-regulation of transcription of the STX biosynthetic gene cluster (sxt). High temperature was found to negatively affect growth and STX production in this organism. The effects of the plant hormone, jasmonic acid, were also tested, since it has previously been shown to affect plant alkaloid production. The hypothesised similarity between cyanobacterial and plant secondary metabolism in response to this plant hormone was confirmed in the neurotoxic cyanobacterium, C. raciborskii T3, as well as the non-toxic Anabaena sp. PCC7120. Furthermore, investigation of the sxt gene cluster transcriptional map in C. raciborskii T3 was carried out, with identification of three main polycistronic and one monocistronic transcripts. Promoter regions putatively involved in the regulation of STX production in C. raciborskii T3 were also identified. Transcription factor consensus motifs, the pho boxes, were identified in the main promoter region. These conserved motifs are the binding regions for the transcriptional regulator, PhoB, to the pho regulon genes, involved in phosphate uptake during conditions of its depletion in the environment. Moreover, a genomic region adjacent to the sxt gene cluster in C. raciborskii T3 was identified and characterised, putatively encoding a regulatory two-component system. This system appears to be involved in the sensing of environmental signals, in particular depleted phosphate, while activating the transcription of genes involved in its uptake and transport. The results of this study lead to a greater understanding of the complex factors associated with the regulation of STX biosynthesis and bloom-formation, by the cyanobacterium C. raciborskii T3

Fluorescence in situ hybridization and microbial community profiling analysis of explanted cochlear implants

Background: Cochlear implant (CI) infections affect a small, but significant number of patients. Unremitting infections can lead to explantation. Fluorescence in situ hybridization (FISH) and microbial community profiling (MCP) are methods of studying microbial environments of explanted devices that can provide information to reduce morbidity and costs of infected CIs. Aims/objectives: To describe the results and clinical significance of bacterial analyses conducted on explanted CIs. Material and methods: Between 2013 and 2017, 12 explanted devices underwent microbiological analysis in addition to the manufacturer’s device failure analysis. Patients’ clinical history, infection status and outcome were reviewed and correlated with microbial analysis results. Results: From 2013 to 2017, 12 Cochlear™ devices from 11 patients underwent additional MCP or FISH analysis. Five devices were explanted due to suspected implant associated infection, and seven were explanted for other reasons. FISH analysis revealed biofilm presence on all infected devices, only partial correlation of cultures with biofilm composition and confirmation that biofilm formation occurs preferentially at particular device interfaces and geometries. MCP analysis presented challenges in data analysis inherent to its technique but correlated with cultures of infected devices and suggested a diverse microbial composition of explanted devices. Conclusions and significance: Microbial analysis of explanted devices can aid in further elucidating treatment approaches to infected CIs

Combination of Silver Nanoparticles and Curcumin Nanoparticles for Enhanced Anti-biofilm Activities

Biofilm tolerance has become a serious clinical concern in the treatment of nosocomial pneumonia owing to the resistance to various antibiotics. There is an urgent need to develop alternative antimicrobial agents or combination drug therapies that are effective via different mechanisms. Silver nanoparticles (AgNPs) have been developed as an anti-biofilm agent for the treatment of infections associated with the use of mechanical ventilations, such as endotracheal intubation. Meanwhile curcumin, a phenolic plant extract, has displayed natural anti-biofilm properties through the inhibition of bacterial quorum sensing systems. The aim of this study was to investigate the possible synergistic/additive interactions of AgNPs and curcumin nanoparticles (Cur-NPs) against both Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) microorganisms. The combination of AgNPs and Cur-NPs (termed Cur-SNPs) at 100 μg/mL disrupted 50% of established bacterial biofilms (formed on microtiter plates). However, further increase in the concentration of Cur-SNPs failed to effectively eliminate the biofilms. To achieve the same effect, at least 500 μg/mL Cur-NP alone was needed. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) revealed that combination therapy (Cur-SNPs) was the most potent to eradicate preformed biofilm compared to monodrug therapy. These agents are also nontoxic to healthy human bronchial epithelial cells (BEAS2B)

Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms

Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs