Lipopolysaccharide (LPS) is a major virulence factor of Salmonella enterica serovar Typhimurium and is composed of lipid A, core oligosaccharide (C-OS), and O-antigen polysaccharide (O-PS). While the functions of the gene products involved in synthesis of core and O-antigen have been elucidated, the effect of removing O-antigen and core sugars on the virulence and immunogenicity of Salmonella enterica serovar Typhimurium has not been systematically studied. We introduced nonpolar, defined deletion mutations in waaG (rfaG), waaI (rfaI), rfaH, waaJ (rfaJ), wbaP (rfbP), waaL (rfaL), or wzy (rfc) into wild-type S. Typhimurium. The LPS structure was confirmed, and a number of in vitro and in vivo properties of each mutant were analyzed. All mutants were significantly attenuated compared to the wild-type parent when administered orally to BALB/c mice and were less invasive in host tissues. Strains with ⌬waaG and ⌬waaI mutations, in particular, were deficient in colonization of Peyer's patches and liver. This deficiency could be partially overcome in the ⌬waaI mutant when it was administered intranasally. In the context of an attenuated vaccine strain delivering the pneumococcal antigen PspA, all of the mutations tested resulted in reduced immune responses against PspA and Salmonella antigens. Our results indicate that nonreversible truncation of the outer core is not a viable option for developing a live oral Salmonella vaccine, while a wzy mutant that retains one O-antigen unit is adequate for stimulating the optimal protective immunity to homologous or heterologous antigens by oral, intranasal, or intraperitoneal routes of administration.

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.

Orally administered recombinant attenuated Salmonella vaccines (RASV) elicit humoral and mucosal immune responses against the immunizing antigen. The challenge in developing an effective vaccine against a virus or an intracellular bacterium delivered by RASVs is to introduce the protective antigen inside the host cell cytoplasm for presentation to MHC-I molecules for an efficient cell mediated immune response. To target the influenza nucleoprotein (NP) into the host cell cytosol, we constructed a regulated delayed lysis in vivo RASV strain χ11246(pYA4858) encoding influenza NP with a chromosomal deletion of the sifA gene to enable it to escape from the endosome prior to lysis. Oral immunization of mice with χ11246(pYA4858) (SifA−) with 3 booster immunizations resulted in complete protection (100%) against a lethal influenza virus (rWSN) challenge (100 LD50) compared to 25% survival of mice immunized with the isogenic χ11017(pYA4858) (SifA+) strain. Reducing the number of booster immunizations with χ11246(pYA4858) from 3 to 2 resulted in 66% survival of mice challenged with rWSN (100 LD50). Immunization with χ11246(pYA4858) via different routes provided protection in 80% orally, 100% intranasally and 100% intraperitoneally immunized mice against rWSN (100 LD50). A Th1 type immune response was elicited against influenza NP in all experiments. IFN-γ secreting NP147–155 specific T cells were not found to be correlated with protection. The role of antigen-specific CD8+ T cells remains to be determined. To conclude, we showed that Salmonella can be designed to deliver antigen(s) to the host cell cytosol for presumably class I presentation for the induction of protective immune responses.

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.

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.