Draft genome sequence of Sphingomonas paucimobilis strain Sph5, isolated from tap water filtration membrane.

Sphingomonadaceae are common membrane colonizers and biofilm formers. As part of our studies on long-term genetic changes in drinking water biofilm species, we report the draft genome sequence of Sphingomonas strain Sph5, isolated from a tap water filtration membrane. The isolate was determined as Sphingomonas paucimobilis through whole genome sequencing and de novo assembly.

Toward sustainable space exploration: a roadmap for harnessing the power of microorganisms.

Finding sustainable approaches to achieve independence from terrestrial resources is of pivotal importance for the future of space exploration. This is relevant not only to establish viable space exploration beyond low Earth-orbit, but also for ethical considerations associated with the generation of space waste and the preservation of extra-terrestrial environments. Here we propose and highlight a series of microbial biotechnologies uniquely suited to establish sustainable processes for in situ resource utilization and loop-closure. Microbial biotechnologies research and development for space sustainability will be translatable to Earth applications, tackling terrestrial environmental issues, thereby supporting the United Nations Sustainable Development Goals.

Mitigation and use of biofilms in space for the benefit of human space exploration.

Biofilms are self-organized communities of microorganisms that are encased in an extracellular polymeric matrix and often found attached to surfaces. Biofilms are widely present on Earth, often found in diverse and sometimes extreme environments. These microbial communities have been described as recalcitrant or protective when facing adversity and environmental exposures. On the International Space Station, biofilms were found in human-inhabited environments on a multitude of hardware surfaces. Moreover, studies have identified phenotypic and genetic changes in the microorganisms under microgravity conditions including changes in microbe surface colonization and pathogenicity traits. Lack of consistent research in microgravity-grown biofilms can lead to deficient understanding of altered microbial behavior in space. This could subsequently create problems in engineered systems or negatively impact human health on crewed spaceflights. It is especially relevant to long-term and remote space missions that will lack resupply and service. Conversely, biofilms are also known to benefit plant growth and are essential for human health (i.e., gut microbiome). Eventually, biofilms may be used to supply metabolic pathways that produce organic and inorganic components useful to sustaining life on celestial bodies beyond Earth. This article will explore what is currently known about biofilms in space and will identify gaps in the aerospace industry's knowledge that should be filled in order to mitigate or to leverage biofilms to the advantage of spaceflight.

Rotation-Time Symmetry Breaking in Frustrated Chiral Nematic Driven by a Pulse-Train Waveform.

We report waveform-induced rotation-time symmetry breaking in liquid crystal director motion. Homeotropic cells filled with a negative dielectric anisotropy chiral nematic exhibit persistent and visually observable waves of director orientation with a time period of at least 30 driving field cycles. Their existence in the space of driving waveform parameters is explored. The possibility of utilizing this system, which exhibits both spatial and temporal long-range order, as a modeling tool for experimental studies on discrete time crystals is discussed.

Role of RpoS in Regulating Stationary Phase Salmonella Typhimurium Pathogenesis-Related Stress Responses under Physiological Low Fluid Shear Force Conditions.

The discovery that biomechanical forces regulate microbial virulence was established with the finding that physiological low fluid shear (LFS) forces altered gene expression, stress responses, and virulence of the enteric pathogen Salmonella enterica serovar Typhimurium during the log phase. These log phase LFS-induced phenotypes were independent of the master stress response regulator, RpoS (σS). Given the central importance of RpoS in regulating stationary-phase stress responses of S. Typhimurium cultured under conventional shake flask and static conditions, we examined its role in stationary-phase cultures grown under physiological LFS. We constructed an isogenic rpoS mutant derivative of wild-type S. Typhimurium and compared the ability of these strains to survive in vitro pathogenesis-related stresses that mimic those encountered in the infected host and environment. We also compared the ability of these strains to colonize (adhere, invade, and survive within) human intestinal epithelial cell cultures. Unexpectedly, LFS-induced resistance of stationary-phase S. Typhimurium cultures to acid and bile salts stresses did not rely on RpoS. Likewise, RpoS was dispensable for stationary-phase LFS cultures to adhere to and survive within intestinal epithelial cells. In contrast, the resistance of these cultures to challenges of oxidative and thermal stresses, and their invasion into intestinal epithelial cells was influenced by RpoS. These findings expand our mechanistic understanding of how physiological fluid shear forces modulate stationary-phase S. Typhimurium physiology in unexpected ways and provide clues into microbial mechanobiology and nuances of Salmonella responses to microenvironmental niches in the infected host. IMPORTANCE Bacterial pathogens respond dynamically to a variety of stresses in the infected host, including physical forces of fluid flow (fluid shear) across their surfaces. While pathogens experience wide fluctuations in fluid shear during infection, little is known about how these forces regulate microbial pathogenesis. This is especially important for stationary-phase bacterial growth, which is a critical period to understand microbial resistance, survival, and infection potential, and is regulated in many bacteria by the general stationary-phase stress response protein RpoS. Here, we showed that, unlike conventional culture conditions, several stationary-phase Salmonella pathogenic stress responses were not impacted by RpoS when bacteria were cultured under fluid shear conditions relevant to those encountered in the intestine of the infected host. These findings offer new insight into how physiological fluid shear forces encountered by Salmonella during infection might impact pathogenic responses in unexpected ways that are relevant to their disease-causing ability.

Spaceflight Analogue Culture Enhances the Host-Pathogen Interaction Between Salmonella and a 3-D Biomimetic Intestinal Co-Culture Model.

Physical forces associated with spaceflight and spaceflight analogue culture regulate a wide range of physiological responses by both bacterial and mammalian cells that can impact infection. However, our mechanistic understanding of how these environments regulate host-pathogen interactions in humans is poorly understood. Using a spaceflight analogue low fluid shear culture system, we investigated the effect of Low Shear Modeled Microgravity (LSMMG) culture on the colonization of Salmonella Typhimurium in a 3-D biomimetic model of human colonic epithelium containing macrophages. RNA-seq profiling of stationary phase wild type and Δhfq mutant bacteria alone indicated that LSMMG culture induced global changes in gene expression in both strains and that the RNA binding protein Hfq played a significant role in regulating the transcriptional response of the pathogen to LSMMG culture. However, a core set of genes important for adhesion, invasion, and motility were commonly induced in both strains. LSMMG culture enhanced the colonization (adherence, invasion and intracellular survival) of Salmonella in this advanced model of intestinal epithelium using a mechanism that was independent of Hfq. Although S. Typhimurium Δhfq mutants are normally defective for invasion when grown as conventional shaking cultures, LSMMG conditions unexpectedly enabled high levels of colonization by an isogenic Δhfq mutant. In response to infection with either the wild type or mutant, host cells upregulated transcripts involved in inflammation, tissue remodeling, and wound healing during intracellular survival. Interestingly, infection by the Δhfq mutant led to fewer transcriptional differences between LSMMG- and control-infected host cells relative to infection with the wild type strain. This is the first study to investigate the effect of LSMMG culture on the interaction between S. Typhimurium and a 3-D model of human intestinal tissue. These findings advance our understanding of how physical forces can impact the early stages of human enteric salmonellosis.

Longitudinal characterization of multispecies microbial populations recovered from spaceflight potable water.

While sequencing technologies have revolutionized our knowledge of microbial diversity, little is known about the dynamic emergent phenotypes that arise within the context of mixed-species populations, which are not fully predicted using sequencing technologies alone. The International Space Station (ISS) is an isolated, closed human habitat that can be harnessed for cross-sectional and longitudinal functional microbiome studies. Using NASA-archived microbial isolates collected from the ISS potable water system over several years, we profiled five phenotypes: antibiotic resistance, metabolism, hemolysis, and biofilm structure/composition of individual or multispecies communities, which represent characteristics that could negatively impact astronaut health and life-support systems. Data revealed a temporal dependence on interactive behaviors, suggesting possible microbial adaptation over time within the ecosystem. This study represents one of the most extensive phenotypic characterization of ISS potable water microbiota with implications for microbial risk assessments of water systems in built environments in space and on Earth.

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.

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.

Physiological fluid shear alters the virulence potential of invasive multidrug-resistant non-typhoidal Salmonella Typhimurium D23580.

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.

Characterization of the Invasive, Multidrug Resistant Non-typhoidal Salmonella Strain D23580 in a Murine Model of Infection.

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 10(5) 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.

Comparative genome analysis of the high pathogenicity Salmonella Typhimurium strain UK-1.

Salmonella enterica serovar Typhimurium, a gram-negative facultative rod-shaped bacterium causing salmonellosis and foodborne disease, is one of the most common isolated Salmonella serovars in both developed and developing nations. Several S. Typhimurium genomes have been completed and many more genome-sequencing projects are underway. Comparative genome analysis of the multiple strains leads to a better understanding of the evolution of S. Typhimurium and its pathogenesis. S. Typhimurium strain UK-1 (belongs to phage type 1) is highly virulent when orally administered to mice and chickens and efficiently colonizes lymphoid tissues of these species. These characteristics make this strain a good choice for use in vaccine development. In fact, UK-1 has been used as the parent strain for a number of nonrecombinant and recombinant vaccine strains, including several commercial vaccines for poultry. In this study, we conducted a thorough comparative genome analysis of the UK-1 strain with other S. Typhimurium strains and examined the phenotypic impact of several genomic differences. Whole genomic comparison highlights an extremely close relationship between the UK-1 strain and other S. Typhimurium strains; however, many interesting genetic and genomic variations specific to UK-1 were explored. In particular, the deletion of a UK-1-specific gene that is highly similar to the gene encoding the T3SS effector protein NleC exhibited a significant decrease in oral virulence in BALB/c mice. The complete genetic complements in UK-1, especially those elements that contribute to virulence or aid in determining the diversity within bacterial species, provide key information in evaluating the functional characterization of important genetic determinants and for development of vaccines.

Live attenuated Salmonella vaccines displaying regulated delayed lysis and delayed antigen synthesis to confer protection against Mycobacterium tuberculosis.

Live recombinant attenuated Salmonella vaccine (RASV) strains have great potential to induce protective immunity against Mycobacterium tuberculosis by delivering M. tuberculosis antigens. Recently, we reported that, in orally immunized mice, RASV strains delivering the M. tuberculosis early secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10) antigens via the Salmonella type III secretion system (SopE amino-terminal region residues 1 to 80 with two copies of ESAT-6 and one copy of CFP-10 [SopE(Nt80)-E2C]) afforded protection against aerosol challenge with M. tuberculosis. Here, we constructed and evaluated an improved Salmonella vaccine against M. tuberculosis. We constructed translational fusions for the synthesis of two copies of ESAT-6 plus CFP-10 fused to the OmpC signal sequence (OmpC(SS)-E2C) and amino acids 44 to 338 of antigen 85A (Ag85A(294)) flanked by the signal sequence (SS) and C-terminal peptide (CT) of ß-lactamase (Bla(SS)-Ag85A(294)-Bla(CT)) to enable delivery via the Salmonella type II secretion system. The genes expressing these proteins were cloned as an operon transcribed from P(trc) into isogenic Asd(+)/MurA(+) pYA3681 lysis vector derivatives with different replication origins (pBR, p15A, pSC101), resulting in pYA4890, pYA4891, and pYA4892 for SopE(Nt80)-E2C/Ag85A(294) synthesis and pYA4893 and pYA4894 for OmpC(SS)-E2C/Ag85A(294) synthesis. Mice orally immunized with the RASV χ11021 strain engineered to display regulated delayed lysis and regulated delayed antigen synthesis in vivo and harboring pYA4891, pYA4893, or pYA4894 elicited significantly greater humoral and cellular immune responses, and the RASV χ11021 strain afforded a greater degree of protection against M. tuberculosis aerosol challenge in mice than RASVs harboring any other Asd(+)/MurA(+) lysis plasmid and immunization with M. bovis BCG, demonstrating that RASV strains displaying regulated delayed lysis with delayed antigen synthesis resulted in highly immunogenic delivery vectors for oral vaccination against M. tuberculosis infection.

Effect of deletion of genes involved in lipopolysaccharide core and O-antigen synthesis on virulence and immunogenicity of Salmonella enterica serovar typhimurium.

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 ΔwaaG and ΔwaaI mutations, in particular, were deficient in colonization of Peyer's patches and liver. This deficiency could be partially overcome in the ΔwaaI 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.

Complete genome sequence of the universal killer Salmonella enterica Serovar Typhimurium UK-1 (ATCC 68169).

The Salmonella enterica serovar Typhimurium strain UK-1 exhibits the highest invasion and virulence attributes among the most frequently studied strains. S. Typhimurium UK-1 has been used as the foundation for developing recombinant vaccines and has been used extensively on virulence and colonization studies in chickens and mice. We describe here the complete genome sequence of S. Typhimurium UK-1. Comparative genomics of Salmonella Typhimurium will provide insight into factors that determine virulence and invasion.

Protective cellular responses elicited by vaccination with influenza nucleoprotein delivered by a live recombinant attenuated Salmonella vaccine.

Orally administered recombinant attenuated Salmonella vaccines (RASVs) 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.