The role of small molecule signalling in biofilm migration of Pseudomonas aeruginosa

University of Technology, Sydney. Faculty of Science.Pseudomonas aeruginosa is a Gram-negative pathogen which exploits damaged epithelium to cause acute and chronic infections in a range of immunocompromised individuals. The chronic nature of infections caused by P. aeruginosa is often associated with the formation of biofilms. Extension and retraction of type IV pili (tfp) mediates a form of surface translocation, termed twitching motility, which is involved in active biofilm expansion and sessile biofilm formation. In P. aeruginosa the biogenesis, assembly and twitching motility function of tfp is controlled by a number of complex regulatory systems, however the signals that these systems respond to are not well characterised. The aim of this Thesis was to understand how intracellular and extracellular signals control P. aeruginosa twitching motility-mediated biofilm expansion. In this Thesis five independent fimL mutants, that had presumably acquired extragenic suppressor mutations which restored twitching motility ability, were characterised. All fimL revertants were found to have increased levels of intracellular cyclic AMP (icAMP). While an extragenic suppressor mutation in the cAMP phosphodiesterase CpdA was shown to be responsible for the increase in icAMP levels and restoration of twitching motility in one fimL revertant, the site of suppressor mutation(s) in the remaining four revertants was not identified. These results suggest that twitching motility reversion in fimL mutants occurs via at least two mechanisms and that an increase in icAMP levels is correlated with twitching motility. Extracellular ATP (eATP) is released by damaged epithelial cells which acts as a “danger” signal to recruit host immune system factors to repair the damage. As P. aeruginosa has a propensity for damaged epithelia the effect of eATP on P. aeruginosa biofilm expansion and formation was investigated. The results presented in this Thesis demonstrate that eATP inhibits P. aeruginosa twitching motility-mediated biofilm expansion and stimulates sessile biofilm formation, which may provide a potential advantage for P. aeruginosa within an infection setting. Additionally, our results suggest that high levels of endogenously-produced bacterial eATP acts to coordinate P. aeruginosa multicellular behaviours. This Thesis also reports the identification of a novel extracellular signal N-acetylglucosamine, which stimulates P. aeruginosa twitching motility. Additionally, the twitching motility response of P. aeruginosa to the host derived signals serum albumin, mucin and oligopeptides was characterised in detail. These analyses implicated the CheW-homolog, ChpC which is a component of the Chp chemosensory system, in this response. Overall the results presented in this Thesis provide insight into the regulation P. aeruginosa twitching motility by a number of intracellular and extracellular signals. Our results suggest that the adaptive response of P. aeruginosa to these signals is likely to have significant implications in the success of this pathogen within an infection setting

Extracellular ATP inhibits twitching motility-mediated biofilm expansion by Pseudomonas aeruginosa

© 2015 Nolan et al.; licensee BioMed Central. Background: Pseudomonas aeruginosa is an opportunistic pathogen that exploits damaged epithelia to cause infection. Type IV pili (tfp) are polarly located filamentous structures which are the major adhesins for attachment of P. aeruginosa to epithelial cells. The extension and retraction of tfp powers a mode of surface translocation termed twitching motility that is involved in biofilm development and also mediates the active expansion of biofilms across surfaces. Extracellular adenosine triphosphate (eATP) is a key "danger" signalling molecule that is released by damaged epithelial cells to alert the immune system to the potential presence of pathogens. As P. aeruginosa has a propensity for infecting damaged epithelial tissues we have explored the influence of eATP on tfp biogenesis and twitching motility-mediated biofilm expansion by P. aeruginosa. Results: In this study we have found that eATP inhibits P. aeruginosa twitching motility-mediated expansion of interstitial biofilms at levels that are not inhibitory to growth. We have determined that eATP does not inhibit expression of the tfp major subunit, PilA, but reduces the levels of surface assembled tfp. We have also determined that the active twitching zone of expanding P. aeruginosa interstitial biofilms contain large quantities of eATP which may serve as a signalling molecule to co-ordinate cell movements in the expanding biofilm. The inhibition of twitching motility-mediated interstitial biofilm expansion requires eATP hydrolysis and does not appear to be mediated by the Chp chemosensory system. Conclusions: Endogenous eATP produced by P. aeruginosa serves as a signalling molecule to co-ordinate complex multicellular behaviours of this pathogen. Given the propensity for P. aeruginosa to infect damaged epithelial tissues, our observations suggest that eATP released by damaged cells may provide a cue to reduce twitching motility of P. aeruginosa in order to establish infection at the site of damage. Furthermore, eATP produced by P. aeruginosa biofilms and by damaged epithelial cells may play a role in P. aeruginosa pathogenesis by inducing inflammatory damage and fibrosis. Our findings have significant implications in the development and pathogenesis of P. aeruginosa biofilm infections

Recombinant factorVIII Fc fusion protein for the prevention and treatment of bleeding in children with severe hemophilia A

This work was supported by funding from Biogen, including funding for the editorial and writing support in the the development of this paper

Extragenic suppressor mutations that restore twitching motility to fimL mutants of Pseudomonas aeruginosa are associated with elevated intracellular cyclic AMP levels

Cyclic AMP (cAMP) is a signaling molecule that is involved in the regulation of multiple virulence systems of the opportunistic pathogen Pseudomonas aeruginosa. The intracellular concentration of cAMP in P. aeruginosa cells is tightly controlled at the levels of cAMP synthesis and degradation through regulation of the activity and/or expression of the adenylate cyclases CyaA and CyaB or the cAMP phosphodiesterase CpdA. Interestingly, mutants of fimL, which usually demonstrate defective twitching motility, frequently revert to a wild-type twitching-motility phenotype presumably via the acquisition of an extragenic suppressor mutation(s). In this study, we have characterized five independent fimL twitching-motility revertants and have determined that all have increased intracellular cAMP levels compared with the parent fimL mutant. Whole-genome sequencing revealed that only one of these fimL revertants has acquired a loss-of-function mutation in cpdA that accounts for the elevated levels of intracellular cAMP. As mutation of cpdA did not account for the restoration of twitching motility observed in the other four fimL revertants, these observations suggest that there is at least another, as yet unidentified, site of extragenic suppressor mutation that can cause phenotypic reversion in fimL mutants and modulation of intracellular cAMP levels of P. aeruginosa

Transcriptional variation of sensory-related genes in natural populations of Aedes albopictus

BACKGROUND: The Asian tiger mosquito, Aedes albopictus, is a highly dangerous invasive vector of numerous medically important arboviruses including dengue, chikungunya and Zika. In four decades it has spread from tropical Southeast Asia to many parts of the world in both tropical and temperate climes. The rapid invasion process of this mosquito is supported by its high ecological and genetic plasticity across different life history traits. Our aim was to investigate whether wild populations, both native and adventive, also display transcriptional genetic variability for functions that may impact their biology, behaviour and ability to transmit arboviruses, such as sensory perception. RESULTS: Antennal transcriptome data were derived from mosquitoes from a native population from Ban Rai, Thailand and from three adventive Mediterranean populations: Athens, Greece and Arco and Trento from Italy. Clear inter-population differential transcriptional activity was observed in different gene categories related to sound perception, olfaction and viral infection. The greatest differences were detected between the native Thai and the Mediterranean populations. The two Italian populations were the most similar. Nearly one million quality filtered SNP loci were identified. CONCLUSION: The ability to express this great inter-population transcriptional variability highlights, at the functional level, the remarkable genetic flexibility of this mosquito species. We can hypothesize that the differential expression of genes, including those involved in sensory perception, in different populations may enable Ae. albopictus to exploit different environments and hosts, thus contributing to its status as a global vector of arboviruses of public health importance. The large number of SNP loci present in these transcripts represents a useful addition to the arsenal of high-resolution molecular markers and a resource that can be used to detect selective pressure and adaptive changes that may have occurred during the colonization process

Device Design Modifications Informed by In Vitro Testing of Bacterial Attachment Reduce Infection Rates of Cochlear Implants in Clinical Practice.

Recalcitrant chronic infections of implanted medical devices are often linked to the presence of biofilms. The prevention and treatment of medical device-associated infections is a major source of antibiotic use and driver of antimicrobial resistance globally. Lowering the incidence of infection in patients that receive implanted medical devices could therefore significantly improve antibiotic stewardship and reduce patient morbidity. Here we determined if modifying the design of an implantable medical device to reduce bacterial attachment, impacted the incidence of device-associated infections in clinical practice. Since the 1980s cochlear implants have provided long-term treatment of sensorineural hearing deficiency in hundreds of thousands of patients world-wide. Nonetheless, a relatively small number of devices are surgically explanted each year due to unresolvable infections. Features associated with the accumulation of bacteria on the Cochlear™ Nucleus® CI24RE™ model of cochlear implant devices were identified using both in vitro bacterial attachment assays and examination of explanted devices. Macro-scale design modifications that reduced bacterial attachment in vitro were incorporated into the design of the CI500™ and Profile™ series of Nucleus implant. Analyses of mandatory post-market vigilance data of 198,757 CI24RE and 123,084 CI500/Profile series implantation surgeries revealed that these design modifications correlated with significantly reduced infection rates. This study demonstrates that a design-centric approach aimed at mitigating bacterial attachment was a simple, and effective means of reducing infections associated with Cochlear Nucleus devices. This approach is likely to be applicable to improving the designs of other implantable medical devices to reduce device-associated infections

Self-organization of bacterial biofilms is facilitated by extracellular DNA

Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa. We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze individual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the "bulldozer" aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA

Entangled-State Cycles of Atomic Collective-Spin States

We study quantum trajectories of collective atomic spin states of $N$ effective two-level atoms driven with laser and cavity fields. We show that interesting ``entangled-state cycles'' arise probabilistically when the (Raman) transition rates between the two atomic levels are set equal. For odd (even) $N$, there are $(N+1)/2$ ($N/2$) possible cycles. During each cycle the $N$-qubit state switches, with each cavity photon emission, between the states $(|N/2,m>\pm |N/2,-m>)/\sqrt{2}$, where $|N/2,m>$ is a Dicke state in a rotated collective basis. The quantum number $m$ ($>0$), which distinguishes the particular cycle, is determined by the photon counting record and varies randomly from one trajectory to the next. For even $N$ it is also possible, under the same conditions, to prepare probabilistically (but in steady state) the Dicke state $|N/2,0>$, i.e., an $N$-qubit state with $N/2$ excitations, which is of particular interest in the context of multipartite entanglement.Comment: 10 pages, 9 figure

Helminth infections and gut microbiota - a feline perspective

$\textbf{BACKGROUND}$: Investigations of the relationships between the gut microbiota and gastrointestinal parasitic nematodes are attracting growing interest by the scientific community, driven by the need to better understand the contribution of parasite-associated changes in the composition of the gut flora to both host malnutrition and immune modulation. These studies have however been carried out mainly in humans and experimental animals, while knowledge of the make-up of the gut commensal flora in presence or absence of infection by parasitic nematodes in domestic animals is limited. In this study, we investigate the qualitative and quantitative impact that infections by a widespread parasite of cats (i.e. $\textit{Toxocara cati}$) exert on the gut microbiota of feline hosts. $\textbf{METHODS}$: The faecal microbiota of cats with patent infection by $\textit{T. cati}$ (= $\textit{Tc+}$), as well as that of negative controls (= $\textit{Tc-}$) was examined via high-throughput sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene, followed by bioinformatics and biostatistical analyses of sequence data. $\textbf{RESULTS}$: A total of 2,325,366 useable high-quality sequences were generated from the faecal samples analysed in this study and subjected to further bioinformatics analyses, which led to the identification of 128 OTUs and nine bacterial phyla, respectively. The phylum Firmicutes was predominant in all samples analysed (mean of 53.0%), followed by the phyla Proteobacteria (13.8%), Actinobacteria (13.7%) and Bacteroidetes (10.1%). Among others, bacteria of the order Lactobacillales, the family $\textit{Enterococcaceae}$ and genera $\textit{Enterococcus}$ and $\textit{Dorea}$ showed a trend towards increased abundance in $\textit{Tc+}$ compared with $\textit{Tc-}$ samples, while no significant differences in OTU richness and diversity were recorded between $\textit{Tc+}$ and $\textit{Tc-}$ samples ($\textit{P}$=0.485 and $\textit{P}$=0.581, respectively). However, Canonical Correlation and Redundancy Analyses were able to separate samples by infection status ($\textit{P}$=0.030 and $\textit{P}$=0.015, respectively), which suggests a correlation between the latter and the composition of the feline faecal microbiota. $\textbf{CONCLUSIONS}$: In spite of the relatively small number of samples analysed, subtle differences in the composition of the gut microbiota of $\textit{Tc+}$ vs $\textit{Tc-}$ cats could be identified, some of which in accordance with current data from humans and laboratory animal hosts. Nevertheless, the findings from this study contribute valuable knowledge to the yet little explored area of parasite-microbiota interactions in domestic animals.This work was supported by Merial Pty Ltd (CC, AG, DO) and ESCCAP UK & Ireland (CC). CC is supported by grants from the Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge and the Royal Society. TPJ is supported by scholarships from the Biotechnology and Biological Sciences Research Council (BBSRC) Doctoral Training Partnerships program

Ears of the Armadillo: Global Health Research and Neglected Diseases in Texas

Neglected tropical diseases (NTDs) have\ud been recently identified as significant public\ud health problems in Texas and elsewhere in\ud the American South. A one-day forum on the\ud landscape of research and development and\ud the hidden burden of NTDs in Texas\ud explored the next steps to coordinate advocacy,\ud public health, and research into a\ud cogent health policy framework for the\ud American NTDs. It also highlighted how\ud U.S.-funded global health research can serve\ud to combat these health disparities in the\ud United States, in addition to benefiting\ud communities abroad