Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns

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

Transcriptome analysis of Vibrio parahaemolyticus in type III secretion system 1 inducing conditions

Vibrio parahaemolyticus is an emerging bacterial pathogen capable of causing inflammatory gastroenteritis, wound infections, and septicemia. As a food-borne illness, infection is most frequently associated with the consumption of raw or undercooked seafood, particularly shellfish. It is the primary cause of Vibrio-associated food-borne illness in the United States and the leading cause of food-borne illness in Japan. The larger of its two chromosomes harbors a set of genes encoding type III section system 1 (T3SS1), a virulence factor present in all V. parahaemolyticus strains that is similar to the Yersinia ysc T3SS. T3SS1 translocates effector proteins into eukaryotic cells where they induce changes to cellular physiology and modulate host-pathogen interactions. T3SS1 is also responsible for cytotoxicity toward several different cultured cell lines as well as mortality in a mouse model. Herein we used RNA-seq to obtain global transcriptome patterns of V. parahaemolyticus under conditions that either induce [growth in Dulbecco's Modified Eagle Medium (DMEM) media, in trans expression of transcriptional regulator exsA] or repress T3SS1 expression (growth in LB-S media, in trans exsD expression) and during infection of HeLa cells over time. Comparative transcriptomic analysis demonstrated notable differences in the expression patterns under inducing conditions and was also used to generate an expression profile of V. parahaemolyticus during infection of HeLa cells. In addition, we identified several new genes that are associated with T3SS1 expression and may warrant further study

Generation of Antigenic Variants via Gene Conversion: Evidence for Recombination Fitness Selection at the Locus Level in Anaplasma marginale

Multiple bacterial and protozoal pathogens utilize gene conversion to generate antigenically variant surface proteins to evade immune clearance and establish persistent infection. Both the donor alleles that encode the variants following recombination into an expression site and the donor loci themselves are under evolutionary selection: the alleles that encode variants that are sufficiently antigenically unique yet retain growth fitness and the loci that allow efficient recombination. We examined allelic usage in generating Anaplasma marginale variants during in vivo infection in the mammalian reservoir host and identified preferential usage of specific alleles in the absence of immune selective pressure, consistent with certain individual alleles having a fitness advantage for in vivo growth. In contrast, the loci themselves appear to have been essentially equally selected for donor function in gene conversion with no significant effect of locus position relative to the expression site or origin of replication. This pattern of preferential allelic usage but lack of locus effect was observed independently for Msp2 and Msp3 variants, both generated by gene conversion. Furthermore, there was no locus effect observed when a single locus contained both msp2 and msp3 alleles in a tail-to-tail orientation flanked by a repeat. These experimental results support the hypothesis that predominance of specific variants reflects in vivo fitness as determined by the encoding allele, independent of locus structure and chromosomal position. Identification of highly fit variants provides targets for vaccines that will prevent the high-level bacteremia associated with acute disease

Identification of Potential Type III Secretion Proteins via Heterologous Expression of Vibrio parahaemolyticus DNA

We employed a heterologous secretion assay to identify proteins potentially secreted by type III secretion systems (T3SSs) in Vibrio parahaemolyticus . N-terminal sequences from 32 proteins within T3SS genomic islands and seven proteins from elsewhere in the chromosome included proteins that were recognized for export by the Yersinia enterocolitica flagellar T3SS

Modeling host-pathogen interactions in the context of the microenvironment : three-dimensional cell culture comes of agee

Tissues and organs provide the structural and biochemical landscapes upon which microbial pathogens and commensals function to regulate health and disease. While flat two-dimensional (2-D) monolayers composed of a single cell type have provided important insight into understanding host-pathogen interactions and infectious disease mechanisms, these reductionist models lack many essential features present in the native host microenvironment that are known to regulate infection, including three-dimensional (3-D) architecture, multicellular complexity, commensal microbiota, gas exchange and nutrient gradients, and physiologically relevant biomechanical forces (e.g., fluid shear, stretch, compression). A major challenge in tissue engineering for infectious disease research is recreating this dynamic 3-D microenvironment (biological, chemical, and physical/mechanical) to more accurately model the initiation and progression of host-pathogen interactions in the laboratory. Here we review selected 3-D models of human intestinal mucosa, which represent a major portal of entry for infectious pathogens and an important niche for commensal microbiota. We highlight seminal studies that have used these models to interrogate host-pathogen interactions and infectious disease mechanisms, and we present this literature in the appropriate historical context. Models discussed include 3-D organotypic cultures engineered in the rotating wall vessel (RWV) bioreactor, extracellular matrix (ECM)-embedded/organoid models, and organ-on-a-chip (OAC) models. Collectively, these technologies provide a more physiologically relevant and predictive framework for investigating infectious disease mechanisms and antimicrobial therapies at the intersection of the host, microbe, and their local microenvironments

Modeling Host-Pathogen Interactions in the Context of the Microenvironment: Three-Dimensional Cell Culture Comes of Age.

Tissues and organs provide the structural and biochemical landscapes upon which microbial pathogens and commensals function to regulate health and disease. While flat two-dimensional (2-D) monolayers composed of a single cell type have provided important insight into understanding host-pathogen interactions and infectious disease mechanisms, these reductionist models lack many essential features present in the native host microenvironment that are known to regulate infection, including three-dimensional (3-D) architecture, multicellular complexity, commensal microbiota, gas exchange and nutrient gradients, and physiologically relevant biomechanical forces (e.g., fluid shear, stretch, compression). A major challenge in tissue engineering for infectious disease research is recreating this dynamic 3-D microenvironment (biological, chemical, and physical/mechanical) to more accurately model the initiation and progression of host-pathogen interactions in the laboratory. Here we review selected 3-D models of human intestinal mucosa, which represent a major portal of entry for infectious pathogens and an important niche for commensal microbiota. We highlight seminal studies that have used these models to interrogate host-pathogen interactions and infectious disease mechanisms, and we present this literature in the appropriate historical context. Models discussed include 3-D organotypic cultures engineered in the rotating wall vessel (RWV) bioreactor, extracellular matrix (ECM)-embedded/organoid models, and organ-on-a-chip (OAC) models. Collectively, these technologies provide a more physiologically relevant and predictive framework for investigating infectious disease mechanisms and antimicrobial therapies at the intersection of the host, microbe, and their local microenvironments