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.

The most common evolutionary events at the molecular level are single-base substitutions, as well as insertions and deletions (indels) of short DNA segments. A large body of research has been devoted to develop probabilistic substitution models and to infer their parameters using likelihood and Bayesian approaches. In contrast, relatively little has been done to model indel dynamics, probably due to the difficulty in writing explicit likelihood functions. Here, we contribute to the effort of modeling indel dynamics by presenting SpartaABC, an approximate Bayesian computation (ABC) approach to infer indel parameters from sequence data (either aligned or unaligned). SpartaABC circumvents the need to use an explicit likelihood function by extracting summary statistics from simulated sequences. First, summary statistics are extracted from the input sequence data. Second, SpartaABC samples indel parameters from a prior distribution and uses them to simulate sequences. Third, it computes summary statistics from the simulated sets of sequences. By computing a distance between the summary statistics extracted from the input and each simulation, SpartaABC can provide an approximation to the posterior distribution of indel parameters as well as point estimates. We study the performance of our methodology and show that it provides accurate estimates of indel parameters in simulations. We next demonstrate the utility of SpartaABC by studying the impact of alignment errors on the inference of positive selection. A C ++ program implementing SpartaABC is freely available in http://spartaabc.tau.ac.il.

Under models of isolation-by-distance, population structure is determined by the probability of identity-by-descent between pairs of genes according to the geographic distance between them. Well established analytical results indicate that the relationship between geographical and genetic distance depends mostly on the neighborhood size of the population which represents a standardized measure of gene flow. To test this prediction, we model local dispersal of haploid individuals on a two-dimensional landscape using seven dispersal kernels: Rayleigh, exponential, half-normal, triangular, gamma, Lomax and Pareto. When neighborhood size is held constant, the distributions produce similar patterns of isolation-by-distance, confirming predictions. Considering this, we propose that the triangular distribution is the appropriate null distribution for isolation-by-distance studies. Under the triangular distribution, dispersal is uniform over the neighborhood area which suggests that the common description of neighborhood size as a measure of an effective, local panmictic population is valid for popular families of dispersal distributions. We further show how to draw random variables from the triangular distribution efficiently and argue that it should be utilized in other studies in which computational efficiency is important.

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.

Mutation is the ultimate source of all genetic variation and is, therefore, central to evolutionary change. Previous work on Paramecium tetraurelia found an unusually low germline base-substitution mutation rate in this ciliate. Here, we tested the generality of this result among ciliates using Tetrahymena thermophila. We sequenced the genomes of 10 lines of T. thermophila that had each undergone approximately 1,000 generations of mutation accumulation (MA). We applied an existing mutation-calling pipeline and developed a new probabilistic mutation detection approach that directly models the design of an MA experiment and accommodates the noise introduced by mismapped reads. Our probabilistic mutation-calling method provides a straightforward way of estimating the number of sites at which a mutation could have been called if one was present, providing the denominator for our mutation rate calculations. From these methods, we find that T. thermophila has a germline base-substitution mutation rate of 7.61 × 10 ^-12 per-site, per cell division, which is consistent with the low base-substitution mutation rate in P. tetraurelia. Over the course of the evolution experiment, genomic exclusion lines derived from the MA lines experienced a fitness decline that cannot be accounted for by germline base-substitution mutations alone, suggesting that other genetic or epigenetic factors must be involved. Because selection can only operate to reduce mutation rates based upon the "visible" mutational load, asexual reproduction with a transcriptionally silent germline may allow ciliates to evolve extremely low germline mutation rates.

Inbreeding in hermaphroditic plants can occur through two different mechanisms: biparental inbreeding, when a plant mates with a related individual, or self-fertilization, when a plant mates with itself. To avoid inbreeding, many hermaphroditic plants have evolved self-incompatibility (SI) systems which prevent or limit self-fertilization. One particular SI system—homomorphic SI—can also reduce biparental inbreeding. Homomorphic SI is found in many angiosperm species, and it is often assumed that the additional benefit of reduced biparental inbreeding may be a factor in the success of this SI system. To test this assumption, we developed a spatially-explicit, individual-based simulation of plant populations that displayed three different types of homomorphic SI. We measured the total level of inbreeding avoidance by comparing each population to a self-compatible population (NSI), and we measured biparental inbreeding avoidance by comparing to a population of self-incompatible plants that were free to mate with any other individual (PSI).

Because biparental inbreeding is more common when offspring dispersal is limited, we examined the levels of biparental inbreeding over a range of dispersal distances. We also tested whether the introduction of inbreeding depression affected the level of biparental inbreeding avoidance. We found that there was a statistically significant decrease in autozygosity in each of the homomorphic SI populations compared to the PSI population and, as expected, this was more pronounced when seed and pollen dispersal was limited. However, levels of homozygosity and inbreeding depression were not reduced. At low dispersal, homomorphic SI populations also suffered reduced female fecundity and had smaller census population sizes. Overall, our simulations showed that the homomorphic SI systems had little impact on the amount of biparental inbreeding in the population especially when compared to the overall reduction in inbreeding compared to the NSI population. With further study, this observation may have important consequences for research into the origin and evolution of homomorphic self-incompatibility systems.

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.

Plasmodium vivax is the most prevalent malarial species in South America and exerts a substantial burden on the populations it affects. The control and eventual elimination of P. vivax are global health priorities. Genomic research contributes to this objective by improving our understanding of the biology of P. vivax and through the development of new genetic markers that can be used to monitor efforts to reduce malaria transmission. Here we analyze whole-genome data from eight field samples from a region in Cordóba, Colombia where malaria is endemic. We find considerable genetic diversity within this population, a result that contrasts with earlier studies suggesting that P. vivax had limited diversity in the Americas. We also identify a selective sweep around a substitution known to confer resistance to sulphadoxine-pyrimethamine (SP). This is the first observation of a selective sweep for SP resistance in this species. These results indicate that P. vivax has been exposed to SP pressure even when the drug is not in use as a first line treatment for patients afflicted by this parasite. We identify multiple non-synonymous substitutions in three other genes known to be involved with drug resistance in Plasmodium species. Finally, we found extensive microsatellite polymorphisms. Using this information we developed 18 polymorphic and easy to score microsatellite loci that can be used in epidemiological investigations in South America.

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.