ASU Scholarship Showcase

Salmonella enterica serovar Typhimurium strains belonging to sequence type ST313 are a major cause of fatal bacteremia among HIV-infected adults and children in sub-Saharan Africa. Unlike “classical” non-typhoidal Salmonella (NTS), gastroenteritis is often absent during ST313 infections and isolates are most commonly recovered from blood, rather than from stool. This is consistent with observations in animals, in which ST313 strains displayed lower levels of intestinal colonization and higher recovery from deeper tissues relative to classic NTS isolates. A better understanding of the key environmental factors regulating these systemic infections is urgently needed. Our previous studies using dynamic Rotating Wall Vessel (RWV) bioreactor technology demonstrated that physiological levels of fluid shear regulate virulence, gene expression, and stress response profiles of classic S. Typhimurium. Here we provide the first demonstration that fluid shear alters the virulence potential and pathogenesis-related stress responses of ST313 strain D23580 in a manner that differs from classic NTS.

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection.

Variation in behaviour among group members often impacts collective outcomes. Individuals may vary both in the task that they perform and in the persistence with which they perform each task. Although both the distribution of individuals among tasks and differences among individuals in behavioural persistence can each impact collective behaviour, we do not know if and how they jointly affect collective outcomes. Here, we use a detailed computational model to examine the joint impact of colony-level distribution among tasks and behavioural persistence of individuals, specifically their fidelity to particular resource sites, on the collective trade-off between exploring for new resources and exploiting familiar ones. We developed an agent-based model of foraging honeybees, parametrized by data from five colonies, in which we simulated scouts, who search the environment for new resources, and individuals who are recruited by the scouts to the newly found resources, i.e. recruits. We varied the persistence of returning to a particular food source of both scouts and recruits and found that, for each value of persistence, there is a different optimal ratio of scouts to recruits that maximizes resource collection by the colony. Furthermore, changes to the persistence of scouts induced opposite effects from changes to the persistence of recruits on the collective foraging of the colony. The proportion of scouts that resulted in the most resources collected by the colony decreased as the persistence of recruits increased. However, this optimal proportion of scouts increased as the persistence of scouts increased. Thus, behavioural persistence and task participation can interact to impact a colony's collective behaviour in orthogonal directions. Our work provides new insights and generates new hypotheses into how variations in behaviour at both the individual and colony levels jointly impact the trade-off between exploring for new resources and exploiting familiar ones.

The molecular mechanisms that allow generalist parasitoids to exploit many, often very distinct hosts are practically unknown. The wasp Aphidius ervi, a generalist koinobiont parasitoid of aphids, was introduced from Europe into Chile in the late 1970s to control agriculturally important aphid species. A recent study showed significant differences in host preference and host acceptance (infectivity) depending on the host A. ervi were reared on. In contrast, no genetic differentiation between A. ervi populations parasitizing different aphid species and aphids of the same species reared on different host plants was found in Chile. Additionally, the same study did not find any fitness effects in A. ervi if offspring were reared on a different host as their mothers. Here, we determined the effect of aphid host species (Sitobion avenae versus Acyrthosiphon pisum reared on two different host plants alfalfa and pea) on the transcriptome of adult A. ervi females.

We found a large number of differentially expressed genes (between host species: head: 2,765; body: 1,216; within the same aphid host species reared on different host plants: alfalfa versus pea: head 593; body 222). As expected, the transcriptomes from parasitoids reared on the same host species (pea aphid) but originating from different host plants (pea versus alfalfa) were more similar to each other than the transcriptomes of parasitoids reared on a different aphid host and host plant (head: 648 and 1,524 transcripts; body: 566 and 428 transcripts). We found several differentially expressed odorant binding proteins and olfactory receptor proteins in particular, when we compared parasitoids from different host species. Additionally, we found differentially expressed genes involved in neuronal growth and development as well as signaling pathways.

These results point towards a significant rewiring of the transcriptome of A. ervi depending on aphid-plant complex where parasitoids develop, even if different biotypes of a certain aphid host species (A. pisum) are reared on the same host plant. This difference seems to persist even after the different wasp populations were reared on the same aphid host in the laboratory for more than 50 generations. This indicates that either the imprinting process is very persistent or there is enough genetic/allelic variation between A. ervi populations. The role of distinct molecular mechanisms is discussed in terms of the formation of host fidelity.

Nasonia, a genus of four closely related parasitoid insect species, is a model system for genetic research. Their haplodiploid genetics (haploid males and diploid females) and interfertile species are advantageous for the genetic analysis of complex traits and the genetic basis of species differences. A fine-scale genomic map is an important tool for advancing genetic studies in this system. We developed and used a hybrid genotyping microarray to generate a high-resolution genetic map that covers 79% of the sequenced genome of Nasonia vitripennis. The microarray is based on differential hybridization of species-specific oligos between N. vitripennis and Nasonia giraulti at more than 20,000 markers spanning the Nasonia genome. The map places 729 scaffolds onto the five linkage groups of Nasonia, including locating many smaller scaffolds that would be difficult to map by other means. The microarray was used to characterize 26 segmental introgression lines containing chromosomal regions from one species in the genetic background of another. These segmental introgression lines have been used for rapid screening and mapping of quantitative trait loci involved in species differences. Finally, the microarray is extended to bulk-segregant analysis and genotyping of other Nasonia species combinations. These resources should further expand the usefulness of Nasonia for studies of the genetic basis and architecture of complex traits and speciation.

Low fluid shear force, including that encountered in microgravity models, induces bacterial responses, but the range of bacteria capable of responding to this signal remains poorly characterized. We systematically analyzed a range of Gram negative Enterobacteriaceae for conservation of the low-shear modeled microgravity (LSMMG) response using phenotypic assays, qPCR, and targeted mutations. Our results indicate LSMMG response conservation across Enterobacteriacae with potential variance in up- or down-regulation of a given response depending on genus. Based on the data, we analyzed the role of the trp operon genes and the TrpR regulator in the LSMMG response using targeted mutations in these genes in S. Typhimurium and E. coli. We found no alteration of the LSMMG response compared to WT in these mutant strains under the conditions tested here. To our knowledge, this study is first-of-kind for Citrobacter, Enterobacter, and Serratia, presents novel data for Escherichia, and provides the first analysis of trp genes in LSMMG responses. This impacts our understanding of how LSMMG affects bacteria and our ability to modify bacteria with this condition in the future.

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3′untranslated region (3′UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3′UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3′UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis.

Biomarkers encompass a wide range of different measurable indicators, representing a tangible link to physiological changes occurring within the body. Accessibility, sensitivity, and specificity are significant factors in biomarker suitability. New biomarkers continue to be discovered, and questions over appropriate selection and assessment of their usefulness remain. If traditional markers of inflammation are not sufficiently robust in their specificity, then perhaps alternative means of detection may provide more information. Epigenetic drift (epigenetic modifications as they occur as a direct function with age), and its ancillary elements, including platelets, secreted microvesicles (MVs), and microRNA (miRNA), may hold enormous predictive potential. The majority of epigenetic drift observed in blood is independent of variations in blood cell composition, addressing concerns affecting traditional blood-based biomarker efficacy. MVs are found in plasma and other biological fluids in healthy individuals. Altered MV/miRNA profiles may also be found in individuals with various diseases. Platelets are also highly reflective of physiological and lifestyle changes, making them extremely sensitive biomarkers of human health. Platelets release increased levels of MVs in response to various stimuli and under a plethora of disease states, which demonstrate a functional effect on other cell types.

A distinct pathovar of Salmonella enterica serovar Typhimurium, ST313, has emerged in sub-Saharan Africa as a major cause of fatal bacteremia in young children and HIV-infected adults. D23580, a multidrug resistant clinical isolate of ST313, was previously shown to have undergone genome reduction in a manner that resembles that of the more human-restricted pathogen, Salmonella enterica serovar Typhi. It has since been shown through tissue distribution studies that D23580 is able to establish an invasive infection in chickens. However, it remains unclear whether ST313 can cause lethal disease in a non-human host following a natural course of infection. Herein we report that D23580 causes lethal and invasive disease in a murine model of infection following peroral challenge. The LD⁵⁰ of D23580 in female BALB/c mice was 4.7 x 10⁵ CFU. Tissue distribution studies performed 3 and 5 days post-infection confirmed that D23580 was able to more rapidly colonize the spleen, mesenteric lymph nodes and gall bladder in mice when compared to the well-characterized S. Typhimurium strain SL1344. D23580 exhibited enhanced resistance to acid stress relative to SL1344, which may lend towards increased capability to survive passage through the gastrointestinal tract as well as during its intracellular lifecycle. Interestingly, D23580 also displayed higher swimming motility relative to SL1344, S. Typhi strain Ty2, and the ST313 strain A130. Biochemical tests revealed that D23580 shares many similar metabolic features with SL1344, with several notable differences in the Voges-Proskauer and catalase tests, as well alterations in melibiose, and inositol utilization. These results represent the first full duration infection study using an ST313 strain following the entire natural course of disease progression, and serve as a benchmark for ongoing and future studies into the pathogenesis of D23580.

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS.