Development of two alternative models of campylobacter jejuni infection that more closely mimic the in vivo environment

Abstract Campylobacter JeJum IS the leading cause of foodborne gastroenteritis worldwide, yet despite the organism's prevalence, relatively little is known about the mechanisms of pathogenesis. This is mainly due to the lack of a convenient small animal model of infection combined with certain inherent weaknesses with widely used in vitro models. The aim was to develop two improved models to study C. jejuni interactions with intestinal epithelial cells. The Ex Vivo Organ Culture (EVOC) model involves co-culturing C. jejuni with human intestinal biopsies. C. jejuni 11168H and 81-176 wild-type strains were demonstrated to induce the secretion of human beta-defensins 2 and 3 (hBD-2 and hBD-3). Furthermore, the supernatants of infected biopsies were demonstrated to contain significantly higher levels of IL-l~, IL-6, IL-12 and IL-23 compared to uninfected controls. Experiments using 11168H flaA and neuBl mutants demonstrated that the induced defensin response was not due to host recognition of either flagellin or the terminal sialic acid residue of C. jejuni illS. The Vertical Diffusion Chamber (VDC) model involves co-culturing C. jejuni with polarised human intestinal epithelial cells (lECs) with micro aerobic conditions at the apical surface and aerobic conditions at the basolateral surface. Survival and integrity of the IECs under these conditions over 24 hours was demonstrated. Co- culture experiments under these conditions resulted in an increase in both C. jejuni interaction with and invasion of IECs. This was mirrored by an increased, polarised host innate immune response. Transcriptional analysis of aerobically and microaerobically co-cultured C. jejuni 11168H identified several genes that may playa role in these increased interactions. The levels of interaction and invasion of defined C. jejuni 11168H mutants with Caco-2 cells were analysed to identify bacterial factors that contribute to these increased interactions. Both 11168HflaA and rpoN mutants exhibited lower levels of interaction and invasion than the wild-type strain, suggesting the involvement of bacterial motility in the increased interactions under micro aerobic conditions. The reduction in this increased interaction phenotype was more pronounced at shorter co-incubation times, suggesting that motility is particularly important during the early phases of interaction. The development of these two model systems should allow· future 3 experiments to more accurately investigate host-pathogen interactions during C. jejuni infection of the human intestinal trac

The Engineering of a Microscale Niche to Test a Novel Anti-Cancer Agent

Cutting-edge biomedical research often relies on innovative tools from the field of engineering. Some of these tools are designed to investigate or probe a niche, analyzing its characteristics and affordances. Other devices are engineered to create a niche in which a specific, unique interaction can take place, while the outcomes are carefully monitored. This paper will focus of the latter and, more specifically, the production of a niche for inducing promising interaction between cancer cells and certain types of fungi. Preliminary research suggests that when maintained in close proximity to breast cancer cells, mushroom mycelia secrete compounds which trigger the programed cell death of the cancer cells. Experiments are currently being conducted to assist in the development of an appropriate scaffold to facilitate this interaction. Since niche construction can be viewed as the perception, utilization, destruction and creation of affordances, it is proposed that affordance-based design and reverse engineering techniques will prove advantageous in this work. The specific design question being asked is: “What set of affordances is necessarily to achieve a high success in the organism interactions, as well as ease of experimentation and repeatability?” As with all new creations, there has been much trial and error. The process for engineering this device was to first create a setup that would allow for the most basic affordances, i.e. allows for both the mushroom and cancer cells to survive, and then to add on more unique affordances. At this point, a prototype has been fully constructed and the physical parameters are being adjusted to allow for ease and accuracy of analysis. Affordance-based design allows for a more holistic understanding of the process and the created niche. The utilization of affordances in the understanding of any niche or system would allow for a greater interchange of information between disciplines, including Christian theology. While an individual affordance does not necessarily serve as an indicator of purpose or teleology, layers of interdependent affordances in both space and time point to the work of an engineer. Thus, this concept of affordances can be used to understand such things as a biblical creation, or can potentially be helpful in describing how a Christian worldview is consistent with the fields of science and engineering

Tool Use by Four Species of Indo-Pacific Sea Urchins

We compared the covering behavior of four sea urchin species, Tripneustes gratilla, Pseudoboletia maculata, Toxopneustes pileolus, and Salmacis sphaeroides found in the waters of Malapascua Island, Cebu Province and Bolinao, Panagsinan Province, Philippines. Specifically, we measured the amount and type of covering material on each sea urchin, and in several cases, the recovery of debris material after stripping the animal of its cover. We found that Tripneustes gratilla and Salmacis sphaeroides have a higher affinity for plant material, especially seagrass, compared to Pseudoboletia maculata and Toxopneustes pileolus, which prefer to cover themselves with coral rubble and other calcified material. Only in Toxopneustes pileolus did we find a significant corresponding depth-dependent decrease in total cover area, confirming previous work that covering behavior serves as a protection mechanism against UV radiation. We found no dependence of particle size on either species or size of sea urchin, but we observed that larger sea urchins generally carried more and heavier debris. We observed a transport mechanism of debris onto the echinoid body surface utilizing a combination of tube feet and spines. We compare our results to previous studies, comment on the phylogeny of sea urchin covering behavior, and discuss the interpretation of this behavior as animal tool use

Vibrio cholerae accessory colonisation factor AcfC: a chemotactic protein with a role in hyperinfectivity.

Vibrio cholerae O1 El Tor is an aquatic Gram-negative bacterium responsible for the current seventh pandemic of the diarrheal disease, cholera. A previous whole-genome analysis on V. cholerae O1 El Tor strains from the 2010 epidemic in Pakistan showed that all strains contained the V. cholerae pathogenicity island-1 and the accessory colonisation gene acfC (VC_0841). Here we show that acfC possess an open reading frame of 770 bp encoding a protein with a predicted size of 28 kDa, which shares high amino acid similarity with two adhesion proteins found in other enteropathogens, including Paa in serotype O45 porcine enteropathogenic Escherichia coli and PEB3 in Campylobacter jejuni. Using a defined acfC deletion mutant, we studied the specific role of AcfC in V. cholerae O1 El Tor environmental survival, colonisation and virulence in two infection model systems (Galleria mellonella and infant rabbits). Our results indicate that AcfC might be a periplasmic sulfate-binding protein that affects chemotaxis towards mucin and bacterial infectivity in the infant rabbit model of cholera. Overall, our findings suggest that AcfC contributes to the chemotactic response of WT V. cholerae and plays an important role in defining the overall distribution of the organism within the intestine

The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses.

The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide) respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2) stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA) expression. However, a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wild-type strain. Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2) stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the G mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment

Regulation of nitrogen metabolism in the marine diazotroph Trichodesmium IMS101 under varying temperatures and atmospheric CO2 concentrations

We examined the influence of forecasted changes in global temperatures and pCO2 on N2 fixation and assimilation in the ecologically important cyanobacterium Trichodesmium spp. Changes of mRNA transcripts (nifH, glnA, hetR, psbA, psaB), protein (nitrogenase, glutamine synthetase) pools and enzymatic activity (nitrogenase) were measured under varying pCO2 and temperatures. High pCO2 shifted transcript patterns of all genes, resulting in a more synchronized diel expression. Under the same conditions, we did not observe any significant changes in the protein pools or in total cellular allocations of carbon and nitrogen (i.e. C : N ratio remained stable). Independently of temperature, high pCO2 (900 µatm) elevated N2 fixation rates. Levels of the key enzymes, nitrogenase and glutamine synthetase that mediate nitrogen assimilation did not increase, implying that the high pCO2 allowed higher reaction turnover rates through these key enzymes. Moreover, increased temperatures and high pCO2 resulted in higher C : P ratios. The plasticity in phosphorous stoichiometry combined with higher enzymatic efficiencies lead to higher growth rates. In cyanobacteria photosynthesis, carbon uptake, respiration, N2 fixation and nitrogen assimilation share cellular components. We propose that shifted cellular resource and energy allocation among those components will enable Trichodesmium grown at elevated temperatures and pCO2 to extend its niche in the future ocean, through both tolerance of a broader temperature range and higher P plasticity