Predicting Essential Metabolic Genome Content of Niche-Specific Enterobacterial Human Pathogens during Simulation of Host Environments

Microorganisms have evolved to occupy certain environmental niches, and the metabolic genes essential for growth in these locations are retained in the genomes. Many microorganisms inhabit niches located in the human body, sometimes causing disease, and may retain genes essential for growth in locations such as the bloodstream and urinary tract, or growth during intracellular invasion of the hosts’ macrophage cells. Strains of Escherichia coli (E. coli) and Salmonella spp. are thought to have evolved over 100 million years from a common ancestor, and now cause disease in specific niches within humans. Here we have used a genome scale metabolic model representing the pangenome of E. coli which contains all metabolic reactions encoded by genes from 16 E. coli genomes, and have simulated environmental conditions found in the human bloodstream, urinary tract, and macrophage to determine essential metabolic genes needed for growth in each location. We compared the predicted essential genes for three E. coli strains and one Salmonella strain that cause disease in each host environment, and determined that essential gene retention could be accurately predicted using this approach. This project demonstrated that simulating human body environments such as the bloodstream can successfully lead to accurate computational predictions of essential/important genes.     

Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence

Anti-tumor CD8⁺ T cells are a key determinant for overall survival in patients following surgical resection for solid malignancies. Using a mouse model of cancer vaccination (adenovirus expressing melanoma tumor-associated antigen (TAA)—dopachrome tautomerase (AdDCT) and resection resulting in major surgical stress (abdominal nephrectomy), we demonstrate that surgical stress results in a reduction in the number of CD8⁺ T cell that produce cytokines (IFNγ, TNFα, Granzyme B) in response to TAA. This effect is secondary to both reduced proliferation and impaired T cell function following antigen binding. In a prophylactic model, surgical stress completely abrogates tumor protection conferred by vaccination in the immediate postoperative period. In a clinically relevant surgical resection model, vaccinated mice undergoing a positive margin resection with surgical stress had decreased survival compared to mice with positive margin resection alone. Preoperative immunotherapy with IFNα significantly extends survival in surgically stressed mice. Importantly, myeloid derived suppressor cell (MDSC) population numbers and functional impairment of TAA-specific CD8⁺ T cell were altered in surgically stressed mice. Our observations suggest that cancer progression may result from surgery-induced suppression of tumor-specific CD8⁺ T cells. Preoperative immunotherapies aimed at targeting the prometastatic effects of cancer surgery will reduce recurrence and improve survival in cancer surgery patients.     

Seasonal Dynamics of the Airborne Bacterial Community and Selected Viruses in a Children’s Daycare Center

Children’s daycare centers appear to be hubs of respiratory infectious disease transmission, yet there is only limited information about the airborne microbial communities that are present in daycare centers. We have investigated the microbial community of the air in a daycare center, including seasonal dynamics in the bacterial community and the presence of specific viral pathogens. We collected filters from the heating, ventilation, and air conditioning (HVAC) system of a daycare center every two weeks over the course of a year. Amplifying and sequencing the 16S rRNA gene revealed that the air was dominated by Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes that are commonly associated with the human skin flora. Clear seasonal differences in the microbial community were not evident; however, the community structure differed when the daycare center was closed and unoccupied for a 13-day period. These results suggest that human occupancy, rather than the environment, is the major driver in shaping the microbial community structure in the air of the daycare center. Using PCR for targeted viruses, we detected a seasonal pattern in the presence of respiratory syncytial virus that included the period of typical occurrence of the disease related to the virus; however, we did not detect the presence of adenovirus or rotavirus at any time.     

Early Indicators of Fatal Leptospirosis during the 2010 Epidemic in Puerto Rico

Background

Leptospirosis is a potentially fatal bacterial zoonosis that is endemic throughout the tropics and may be misdiagnosed as dengue. Delayed hospital admission of leptospirosis patients is associated with increased mortality.

Methodology/Principal Findings

During a concurrent dengue/leptospirosis epidemic in Puerto Rico in 2010, suspected dengue patients that tested dengue-negative were tested for leptospirosis. Fatal and non-fatal hospitalized leptospirosis patients were matched 1:1–3 by age. Records from all medical visits were evaluated for factors associated with fatal outcome. Among 175 leptospirosis patients identified (4.7 per 100,000 residents), 26 (15%) were fatal. Most patients were older males and had illness onset during the rainy season. Fatal case patients first sought medical care earlier than non-fatal control patients (2.5 vs. 5 days post-illness onset [DPO], p < 0.01), but less frequently first sought care at a hospital (52.4% vs. 92.2%, p < 0.01). Although fatal cases were more often diagnosed with leptospirosis at first medical visit (43.9% vs. 9.6%, p = 0.01), they were admitted to the hospital no earlier than non-fatal controls (4.5 vs. 6 DPO, p = 0.31). Cases less often developed fever (p = 0.03), but more often developed jaundice, edema, leg pain, hemoptysis, and had a seizure (p ≤ 0.03). Multivariable analysis of laboratory values from first medical visit associated with fatal outcome included increased white blood cell (WBC) count with increased creatinine (p = 0.001), and decreased bicarbonate with either increased WBC count, increased creatinine, or decreased platelet count (p < 0.001).

Conclusions/Significance

Patients with fatal leptospirosis sought care earlier, but were not admitted for care any earlier than non-fatal patients. Combinations of routine laboratory values predictive of fatal outcome should be considered in admission decision-making for patients with suspected leptospirosis.     

Inertia weight control strategies for particle swarm optimization

Particle swarm optimization (PSO) is a population-based, stochastic optimization technique inspired by the social dynamics of birds. The PSO algorithm is rather sensitive to the control parameters, and thus, there has been a significant amount of research effort devoted to the dynamic adaptation of these parameters. The focus of the adaptive approaches has largely revolved around adapting the inertia weight as it exhibits the clearest relationship with the exploration/exploitation balance of the PSO algorithm. However, despite the significant amount of research efforts, many inertia weight control strategies have not been thoroughly examined analytically nor empirically. Thus, there are a plethora of choices when selecting an inertia weight control strategy, but no study has been comprehensive enough to definitively guide the selection. This paper addresses these issues by first providing an overview of 18 inertia weight control strategies. Secondly, conditions required for the strategies to exhibit convergent behaviour are derived. Finally, the inertia weight control strategies are empirically examined on a suite of 60 benchmark problems. Results of the empirical investigation show that none of the examined strategies, with the exception of a randomly selected inertia weight, even perform on par with a constant inertia weight.     

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B₁, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B₁ and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network.Sphingolipids play critical roles in plant growth and development as essential components of endomembranes, including the plasma membrane, where they constitute more than 40% of the total lipid (Sperling et al., 2005; Cacas et al., 2016). Sphingolipids also are highly enriched in detergent-insoluble membrane fractions of the plasma membrane that form microdomains for proteins with important cell surface activities, including cell wall biosynthesis and hormone transport (Cacas et al., 2012, 2016; Perraki et al., 2012; Bayer et al., 2014). In addition, sphingolipids, particularly those with very-long-chain fatty acids (VLCFAs), are integrally associated with Golgi-mediated protein trafficking that underlies processes related to the growth of plant cells (Bach et al., 2008, 2011; Markham et al., 2011; Melser et al., 2011). Furthermore, sphingolipids function through their bioactive long-chain base (LCB) and ceramide metabolites to initiate programmed cell death (PCD), important for mediating plant pathogen resistance through the hypersensitive response (Greenberg et al., 2000; Liang et al., 2003; Shi et al., 2007; Bi et al., 2014; Simanshu et al., 2014).Sphingolipid biosynthesis is highly regulated in all eukaryotes. In plants, the maintenance of sphingolipid homeostasis is vital to ensure sufficient sphingolipids for growth (Chen et al., 2006; Kimberlin et al., 2013) while restricting the accumulation of PCD-inducing ceramides and LCBs until required for processes such as the pathogen-triggered hypersensitive response. Serine palmitoyltransferase (SPT), which catalyzes the first step in LCB synthesis, is generally believed to be the primary control point for sphingolipid homeostasis (Hanada, 2003). SPT synthesizes LCBs, unique components of sphingolipids, by catalyzing a pyridoxal phosphate-dependent condensation of Ser and palmitoyl (16:0)-CoA in plants (Markham et al., 2013). Similar to other eukaryotes, the Arabidopsis (Arabidopsis thaliana) SPT is a heterodimer consisting of LCB1 and LCB2 subunits (Chen et al., 2006; Dietrich et al., 2008; Teng et al., 2008). Research to date has shown that SPT is regulated primarily by posttranslational mechanisms involving physical interactions with noncatalytic, membrane-associated proteins that confer positive and negative regulation of SPT activity (Han et al., 2009, 2010; Breslow et al., 2010). These proteins include a 56-amino acid small subunit of SPT (ssSPT) in Arabidopsis, which was recently shown to stimulate SPT activity and to be essential for generating sufficient amounts of sphingolipids for pollen and sporophytic cell viability (Kimberlin et al., 2013).Evidence from yeast and mammalian research points to a more critical role for proteins termed ORMs (for orosomucoid-like proteins) in sphingolipid homeostatic regulation (Breslow et al., 2010; Han et al., 2010). The Saccharomyces cerevisiae Orm1p and Orm2p negatively regulate SPT through reversible phosphorylation of these polypeptides in response to intracellular sphingolipid levels (Breslow et al., 2010; Han et al., 2010; Roelants et al., 2011; Gururaj et al., 2013; Muir et al., 2014). Phosphorylation/dephosphorylation of ORMs in S. cerevisiae presumably affects the higher order assembly of SPT to mediate flux through this enzyme for LCB synthesis (Breslow, 2013). In this sphingolipid homeostatic regulatory mechanism, the S. cerevisiae Orm1p and Orm2p are phosphorylated at their N termini by Ypk1, a TORC2-dependent protein kinase (Han et al., 2010; Roelants et al., 2011). The absence of this phosphorylation domain in mammalian and plant ORM homologs brings into question the nature of SPT reversible regulation by ORMs in other eukaryotic systems (Hjelmqvist et al., 2002).Sphingolipid synthesis also is mediated by the N-acylation of LCBs by ceramide synthases to form ceramides, the hydrophobic backbone of the major plant glycosphingolipids, glucosylceramide (GlcCer) and glycosyl inositolphosphoceramide (GIPC). Two functionally distinct classes of ceramide synthases occur in Arabidopsis, designated class I and class II (Chen et al., 2008). Class I ceramide synthase activity resulting from the Longevity Assurance Gene One Homolog2 (LOH2)-encoded ceramide synthase acylates, almost exclusively, LCBs containing two hydroxyl groups (dihydroxy LCBs) with 16:0-CoA to form C16 ceramides, which are used primarily for GlcCer synthesis (Markham et al., 2011; Ternes et al., 2011; Luttgeharm et al., 2016). Class II ceramide synthase activities resulting from the LOH1- and LOH3-encoded ceramide synthases are most active in the acylation of LCBs containing three hydroxyl groups (trihydroxy LCBs) with VLCFA-CoAs, including primarily C₂₄ and C₂₆ acyl-CoAs (Markham et al., 2011; Ternes et al., 2011; Luttgeharm et al., 2016). Class II (LOH1 and LOH3) ceramide synthase activity is essential for producing VLCFA-containing glycosphingolipids to support the growth of plant cells, whereas class I (LOH2) ceramide synthase activity is nonessential under normal growth conditions (Markham et al., 2011; Luttgeharm et al., 2015b). It was speculated recently that LOH2 ceramide synthase functions, in part, as a safety valve to acylate excess LCBs for glycosylation, resulting in a less cytotoxic form (Luttgeharm et al., 2015b; Msanne et al., 2015). Recent studies have shown that the Lag1/Lac1 components of the S. cerevisiae ceramide synthase are phosphorylated by Ypk1, and this phosphorylation stimulates ceramide synthase activity in response to heat and reduced intracellular sphingolipid levels (Muir et al., 2014). This finding points to possible coordinated regulation of ORM-mediated SPT and ceramide synthase activities to regulate sphingolipid homeostasis, which is likely more complicated in plants and mammals due to the occurrence of functionally distinct ceramide synthases in these systems (Stiban et al., 2010; Markham et al., 2011; Ternes et al., 2011; Luttgeharm et al., 2016).RNA interference (RNAi) suppression of ORM genes in rice (Oryza sativa) has been shown to affect pollen viability (Chueasiri et al., 2014), but no mechanistic characterization of ORM proteins in plants has yet to be reported. Here, we describe two Arabidopsis ORMs, AtORM1 and AtORM2, that suppress SPT activity through direct interaction with the LCB1/LCB2 heterodimer. We also show that strong up-regulation of AtORM expression impairs growth. In addition, up- or down-regulation of ORMs is shown to differentially affect the sensitivity of Arabidopsis to the PCD-inducing mycotoxin fumonisin B₁ (FB₁), a ceramide synthase inhibitor, and to differentially affect the activities of class I and II ceramide synthases as a possible additional mechanism for regulating sphingolipid homeostasis.