Altered transcriptional responses in the lungs of aged mice after influenza infection.

BACKGROUND: Influenza causes a serious infection in older individuals who are at the highest risk for mortality from this virus. Changes in the immune system with age are well known. This study used transcriptomic analysis to evaluate how aging specifically affects the functional host response to influenza in the lung. Adult (12-16 weeks) and aged (72-76 weeks) mice were infected with influenza and lungs were processed for RNA analysis. RESULTS: Older mice demonstrated a delayed anti-viral response on the level of transcription compared to adults, similar to the immunologic responses measured in prior work. The transcriptional differences, however, were evident days before observable differences in the protein responses described previously. The transcriptome response to influenza in aged mice was dominated by immunoglobulin genes and B cell markers compared to adult animals, suggesting immune dysregulation. Despite these differences, both groups of mice had highly similar transcriptional responses involving non-immune genes one day after inoculation and T cell genes during resolution. CONCLUSIONS: These results define a delayed and dysregulated immune response in the lungs of aged mice infected with influenza. The findings implicate B cells and immunoglobulins as markers or mechanisms of immune aging. In addition to discovering new therapeutic targets, the findings underscore the value of transcription studies and network analysis to characterize complex biological processes, and serve as a model to analyze the susceptibility of the elderly to infectious agents.

Hyperammonemia syndrome due to Ureaplasma urealyticum in a kidney transplant recipient: A case of disseminated disease from a fluoroquinolone-resistant isolate.

Ureaplasma species (spp.) are common colonizers of the urogenital tract but may cause systemic infection in immunocompromised patients. They release significant amounts of ammonia via urea hydrolysis and have been recently implicated in the pathogenesis of hyperammonemia syndrome after organ transplantation. We describe a unique case of hyperammonemia syndrome after kidney transplant caused by U urealyticum infection, and the first, to our knowledge, case of a fluoroquinolone-resistant Ureaplasma strain causing hyperammonemia syndrome. A 17-year-old female developed intermittent fevers, rising creatinine, sterile pyuria and debilitating polyarthritis approximately 1 year after kidney transplant. Serum ammonia level was elevated, and urine PCR was positive for U urealyticum. Near the end of treatment with levofloxacin, she had rebound hyperammonemia, which preceded clinical relapse of polyarthritis and encephalopathy. Blood and urine PCR and synovial fluid culture were positive for U urealyticum. Susceptibility testing showed fluoroquinolone resistance, but she responded well to azithromycin and doxycycline. The frequency of Ureaplasma spp. infection in immunocompromised patients is probably underestimated due to diagnostic challenges. Ammonia levels were helpful biomarkers of response to antimicrobial therapy in our case. Susceptibility testing of clinical isolates should be pursued. In serious Ureaplasma spp. infections, particularly in immunocompromised patients, two empiric antibiotics may be indicated given the potential for antimicrobial resistance.

Androgen sensitivity gateway to COVID-19 disease severity.

In this communication, we present arguments for androgen sensitivity as a likely determinant of COVID-19 disease severity. The androgen sensitivity model explains why males are more likely to develop severe symptoms while children are ostensibly resistant to infection. Further, the model explains the difference in COVID-19 mortality rates among different ethnicities. Androgen sensitivity is determined by genetic variants of the androgen receptor. The androgen receptor regulates transcription of the transmembrane protease, serine 2 (TMPRSS2), which is required for SARS-CoV-2 infectivity. TMPRSS2 primes the Spike protein of the virus, which has two consequences: diminishing viral recognition by neutralizing antibodies and activating SARS-CoV-2 for virus-cell fusion. Genetic variants that have been associated with androgenetic alopecia, prostate cancer, benign prostatic hyperplasia and polycystic ovary syndrome could be associated with host susceptibility. In addition to theoretical epidemiological and molecular mechanisms, there are reports of high rates of androgenetic alopecia of from hospitalized COVID-19 patients due to severe symptoms. Androgen sensitivity is a likely determinant of COVID-19 disease severity. We believe that the evidence presented in this communication warrants the initiation of trials using anti-androgen agents.

The Infectious Diseases Society of America's 10 × '20 Initiative (10 New Systemic Antibacterial Agents US Food and Drug Administration Approved by 2020): Is 20 × '20 a Possibility?

BACKGROUND: Infections caused by antibiotic-resistant bacteria, including carbapenem-resistant Enterobacteriaceae, have increased in frequency, resulting in significant patient morbidity and mortality. The Infectious Diseases Society of America continues to propose legislative, regulatory, and funding solutions to address this escalating crisis. This report updates the status of development and approval of systemic antibiotics in the United States as of late 2018. METHODS: We performed a review of the published literature and on-line clinical trials registry at www.clinicaltrials.gov to identify new systemically acting orally and/or intravenously administered antibiotic drug candidates in the development pipeline, as well as agents approved by the US Food and Drug Administration since 2012. RESULTS: Since our 2013 pipeline status report, the number of new antibiotics annually approved for marketing in the United States has reversed its previous decline, likely influenced by new financial incentives and increased regulatory flexibility. Although our survey demonstrates progress in development of new antibacterial drugs that target infections caused by resistant bacterial pathogens, the majority of recently approved agents have been modifications of existing chemical classes of antibiotics, rather than new chemical classes. Furthermore, larger pharmaceutical companies continue to abandon the field, and smaller companies face financial difficulties as a consequence. CONCLUSIONS: Unfortunately, if 20 × '20 is achieved due to efforts embarked upon in decades past, it could mark the apex of antibiotic drug development for years to come. Without increased regulatory, governmental, industry, and scientific support and collaboration, durable solutions to the clinical, regulatory, and economic problems posed by bacterial multidrug resistance will not be found.

Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds.

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z' factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 µg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

Lactobacillus-derived extracellular vesicles enhance host immune responses against vancomycin-resistant enterococci.

BACKGROUND: Probiotic bacteria are known to modulate host immune responses against various pathogens. Recently, extracellular vesicles (EVs) have emerged as potentially important mediators of host-pathogen interactions. In this study, we explored the role of L. plantarum derived EVs in modulating host responses to vancomycin-resistant Enterococcus faecium (VRE) using both Caenorhabditis elegans and human cells. RESULTS: Our previous work has shown that probiotic conditioning C. elegans with L. acidophilus NCFM prolongs the survival of nematodes exposed to VRE. Similarly, L. plantarum WCFS1 derived extracellular vesicles (LDEVs) also significantly protected the worms against VRE infection. To dissect the molecular mechanisms of this EV-induced protection, we found that treatment of C. elegans with LDEVs significantly increased the transcription of host defense genes, cpr-1 and clec-60. Both cpr-1 and clec-60 have been previously reported to have protective roles against bacterial infections. Incubating human colon-derived Caco-2 cells with fluorescent dye-labeled LDEVs confirmed that LDEVs could be transported into the mammalian cells. Furthermore, LDEV uptake was associated with significant upregulation of CTSB, a human homologous gene of cpr-1, and REG3G, a human gene that has similar functions to clec-60. CONCLUSIONS: We have found that EVs produced from L. plantarum WCFS1 up-regulate the expression of host defense genes and provide protective effects on hosts. Using probiotic-derived EVs instead of probiotic bacteria themselves, this study provides a new direction to treat antimicrobial resistant pathogens, such as VRE.

ModuleBlast: identifying activated sub-networks within and across species.

Identifying conserved and divergent response patterns in gene networks is becoming increasingly important. A common approach is integrating expression information with gene association networks in order to find groups of connected genes that are activated or repressed. In many cases, researchers are also interested in comparisons across species (or conditions). Finding an active sub-network is a hard problem and applying it across species requires further considerations (e.g. orthology information, expression data and networks from different sources). To address these challenges we devised ModuleBlast, which uses both expression and network topology to search for highly relevant sub-networks. We have applied ModuleBlast to expression and interaction data from mouse, macaque and human to study immune response and aging. The immune response analysis identified several relevant modules, consistent with recent findings on apoptosis and NFκB activation following infection. Temporal analysis of these data revealed cascades of modules that are dynamically activated within and across species. We have experimentally validated some of the novel hypotheses resulting from the analysis of the ModuleBlast results leading to new insights into the mechanisms used by a key mammalian aging protein.

Ebola Virus Persistence in Semen of Male Survivors.

We investigated the duration of Ebola virus (EBOV) RNA and infectious EBOV in semen specimens of 5 Ebola virus disease (EVD) survivors. EBOV RNA and infectious EBOV was detected by real-time RT-PCR and virus culture out to 290 days and 70 days, respectively, after EVD onset.

MyD88-dependent signaling prolongs survival and reduces bacterial burden during pulmonary infection with virulent Francisella tularensis.

Francisella tularensis is the causative agent of the debilitating febrile illness tularemia. The severe morbidity associated with F. tularensis infections is attributed to its ability to evade the host immune response. Innate immune activation is undetectable until more than 48 hours after infection. The ensuing inflammatory response is considered pathological, eliciting a septic-like state characterized by hypercytokinemia and cell death. To investigate potential pathological consequences of the innate immune response, mice deficient in a key innate immune signaling molecule, MyD88, were studied. MyD88 knockout (KO) mice were infected with the prototypical virulent F. tularensis strain, Schu S4. MyD88 KO mice succumbed to infection more rapidly than wild-type mice. The enhanced pathogenicity of Schu S4 in MyD88 KO mice was associated with greater bacterial burdens in lungs and distal organs, and the absence of IFN-γ in the lungs, spleens, and sera. Cellular infiltrates were not observed on histological evaluation of the lungs, livers, or spleens of MyD88 KO mice, the first KO mouse described with this phenotype to our knowledge. Despite the absence of cellular infiltration, there was more cell death in the lungs of MyD88 KO mice. Thus, the host proinflammatory response is beneficial, and MyD88 signaling is required to limit bacterial burden and prolong survival during pulmonary infection by virulent F. tularensis.

Rapid optical determination of ß-lactamase and antibiotic activity.

BACKGROUND: The absence of rapid tests evaluating antibiotic susceptibility results in the empirical prescription of antibiotics. This can lead to treatment failures due to escalating antibiotic resistance, and also furthers the emergence of drug-resistant bacteria. This study reports a rapid optical method to detect ß-lactamase and thereby assess activity of ß-lactam antibiotics, which could provide an approach for targeted prescription of antibiotics. The methodology is centred on a fluorescence quenching based probe (ß-LEAF--ß-Lactamase Enzyme Activated Fluorophore) that mimics the structure of ß-lactam antibiotics. RESULTS: The ß-LEAF assay was performed for rapid determination of ß-lactamase production and activity of ß-lactam antibiotic (cefazolin) on a panel of Staphylococcus aureus ATCC strains and clinical isolates. Four of the clinical isolates were determined to be lactamase producers, with the capacity to inactivate cefazolin, out of the twenty-five isolates tested. These results were compared against gold standard methods, nitrocefin disk test for ß-lactamase detection and disk diffusion for antibiotic susceptibility, showing results to be largely consistent. Furthermore, in the sub-set of ß-lactamase producers, it was demonstrated and validated that multiple antibiotics (cefazolin, cefoxitin, cefepime) could be assessed simultaneously to predict the antibiotic that would be most active for a given bacterial isolate. CONCLUSIONS: The study establishes the rapid ß-LEAF assay for ß-lactamase detection and prediction of antibiotic activity using S. aureus clinical isolates. Although the focus in the current study is ß-lactamase-based resistance, the overall approach represents a broad diagnostic platform. In the long-term, these studies form the basis for the development of assays utilizing a broader variety of targets, pathogens and drugs.

Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity.

In innate immune sensing, the detection of pathogen-associated molecular patterns by recognition receptors typically involve leucine-rich repeats (LRRs). We provide a categorization of 375 human LRR-containing proteins, almost half of which lack other identifiable functional domains. We clustered human LRR proteins by first assigning LRRs to LRR classes and then grouping the proteins based on these class assignments, revealing several of the resulting protein groups containing a large number of proteins with certain non-LRR functional domains. In particular, a statistically significant number of LRR proteins in the typical (T) and bacterial + typical (S+T) categories have transmembrane domains, whereas most of the LRR proteins in the cysteine-containing (CC) category contain an F-box domain (which mediates interactions with the E3 ubiquitin ligase complex). Furthermore, by examining the evolutionary profiles of the LRR proteins, we identified a subset of LRR proteins exhibiting strong conservation in fungi and an enrichment for "nucleic acid-binding" function. Expression analysis of LRR genes identifies a subset of pathogen-responsive genes in human primary macrophages infected with pathogenic bacteria. Using functional RNAi, we show that MFHAS1 regulates Toll-like receptor (TLR)-dependent signaling. By using protein interaction network analysis followed by functional RNAi, we identified LRSAM1 as a component of the antibacterial autophagic response.

Interferon-γ, tumor necrosis factor, and interleukin-18 cooperate to control growth of Mycobacterium tuberculosis in human macrophages.

Mycobacterium tuberculosis (MTB) remains a leading infectious threat to human health. Macrophages are the cells targeted for infection by the bacterium as well as key effector cells for clearance of the pathogen. Interleukin (IL)-27 opposes macrophage-mediated control of MTB because supplying IL-12 and blocking the activity of IL-27 limits bacterial growth in primary human macrophages. The purpose of this study was to determine the immunological regulators of this macrophage mechanism to restrict MTB growth. Interferon (IFN)-γ, TNF-α, and IL-18 were all demonstrated to be important to the environment that limits bacterial growth when IL-12 is supplied and IL-27 is neutralized. We find IL-18 works in conjunction with IL-12 to achieve optimal IFN-γ production in this system. We also demonstrate novel interactions between these cytokines to influence the expression or responsiveness to one another. Quantitative assays show that IFN-γ enhances expression of the IL-18 receptor signaling chain, as well as TNF expression and secretion. In turn, TNF-α augments expression of the receptor for IFN-γ, the amount at the cell surface, and the extent of IFN-γ -induced signaling. We further define how the cytokine environment supports an enhanced state of classical macrophage activation. Collectively, these results describe novel immunological mechanisms that provide additional insights into the effects of IL-12 and IL-27 on macrophage regulation during MTB infection.

Invasion of erythrocytes by Francisella tularensis.

Francisella tularensis is the causative agent of tularemia and is classified as a category A biodefense agent by the Centers for Disease Control and Prevention because of its highly infectious nature. F. tularensis infects leukocytes and exhibits an extracellular phase in the blood of the host. It is unknown, however, whether F. tularensis can infect erythrocytes; thus, we examined this possibility in vivo and in vitro. In the murine model of pulmonary type A tularemia, we showed the presence of intraerythrocytic bacteria by double-immunofluorescence microscopy and ex vivo gentamicin protection of the purified erythrocyte fraction. In vitro, F. tularensis invaded human erythrocytes, as shown in the gentamicin protection assays, double-immunofluorescence microscopy, flow cytometry, scanning electron microscopy, and transmission electron microscopy with immunogold labeling of the bacteria. Additional in vitro tests indicated that serum complement-dependent and complement-independent mechanisms contribute to erythrocyte invasion. Our results reveal a novel intraerythrocytic phase during F. tularensis infection.

Identification and mechanistic basis of non-ACE2 blocking neutralizing antibodies from COVID-19 patients with deep RNA sequencing and molecular dynamics simulations.

Variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continue to cause disease and impair the effectiveness of treatments. The therapeutic potential of convergent neutralizing antibodies (NAbs) from fully recovered patients has been explored in several early stages of novel drugs. Here, we identified initially elicited NAbs (Ig Heavy, Ig lambda, Ig kappa) in response to COVID-19 infection in patients admitted to the intensive care unit at a single center with deep RNA sequencing (>100 million reads) of peripheral blood as a diagnostic tool for predicting the severity of the disease and as a means to pinpoint specific compensatory NAb treatments. Clinical data were prospectively collected at multiple time points during ICU admission, and amino acid sequences for the NAb CDR3 segments were identified. Patients who survived severe COVID-19 had significantly more of a Class 3 antibody (C135) to SARS-CoV-2 compared to non-survivors (15059.4 vs. 1412.7, p = 0.016). In addition to highlighting the utility of RNA sequencing in revealing unique NAb profiles in COVID-19 patients with different outcomes, we provided a physical basis for our findings via atomistic modeling combined with molecular dynamics simulations. We established the interactions of the Class 3 NAb C135 with the SARS-CoV-2 spike protein, proposing a mechanistic basis for inhibition via multiple conformations that can effectively prevent ACE2 from binding to the spike protein, despite C135 not directly blocking the ACE2 binding motif. Overall, we demonstrate that deep RNA sequencing combined with structural modeling offers the new potential to identify and understand novel therapeutic(s) NAbs in individuals lacking certain immune responses due to their poor endogenous production. Our results suggest a possible window of opportunity for administration of such NAbs when their full sequence becomes available. A method involving rapid deep RNA sequencing of patients infected with SARS-CoV-2 or its variants at the earliest infection time could help to develop personalized treatments using the identified specific NAbs.

Deep RNA sequencing of intensive care unit patients with COVID-19.

COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host's response in COVID-19. Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (> 100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies.

A Francisella tularensis locus required for spermine responsiveness is necessary for virulence.

Tularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensis senses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensis with host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensis subsp. holarctica live vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensis subsp. tularensis Schu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensis gene important for virulence and evasion of the host immune response.

Deep RNA Sequencing of Intensive Care Unit Patients with COVID-19.

PURPOSE: COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host's response in COVID-19. METHODS: Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (>100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. RESULTS: We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. CONCLUSIONS: Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies. TAKE HOME MESSAGE: Deep RNA sequencing provides a novel diagnostic tool for critically ill patients. Among ICU patients with COVID-19, RNA sequencings can identify gene expression, pathogens (including SARS-CoV-2), and can predict mortality. TWEET: Deep RNA sequencing is a novel technology that can assist in the care of critically ill COVID-19 patients & can be applied to other disease.

Francisella tularensis DeltapyrF mutants show that replication in nonmacrophages is sufficient for pathogenesis in vivo.

The pathogenesis of Francisella tularensis has been associated with this bacterium's ability to replicate within macrophages. F. tularensis can also invade and replicate in a variety of nonphagocytic host cells, including lung and kidney epithelial cells and hepatocytes. As uracil biosynthesis is a central metabolic pathway usually necessary for pathogens, we characterized DeltapyrF mutants of both F. tularensis LVS and Schu S4 to investigate the role of these mutants in intracellular growth. As expected, these mutant strains were deficient in de novo pyrimidine biosynthesis and were resistant to 5-fluoroorotic acid, which is converted to a toxic product by functional PyrF. The F. tularensis DeltapyrF mutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensis DeltapyrF strains would be attenuated in animal infection models. Surprisingly, these mutants retained virulence during infection of chicken embryos and in the murine model of pneumonic tularemia. We hypothesized that the F. tularensis DeltapyrF strains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensis DeltapyrF mutants replicated in HEK-293 cells and normal human fibroblasts in vitro. Moreover, immunofluorescence microscopy showed abundant staining of wild-type and mutant bacteria in nonmacrophage cells in the lungs of infected mice. These findings indicate that replication in nonmacrophages contributes to the pathogenesis of F. tularensis.

A probabilistic generative model for GO enrichment analysis.

The Gene Ontology (GO) is extensively used to analyze all types of high-throughput experiments. However, researchers still face several challenges when using GO and other functional annotation databases. One problem is the large number of multiple hypotheses that are being tested for each study. In addition, categories often overlap with both direct parents/descendents and other distant categories in the hierarchical structure. This makes it hard to determine if the identified significant categories represent different functional outcomes or rather a redundant view of the same biological processes. To overcome these problems we developed a generative probabilistic model which identifies a (small) subset of categories that, together, explain the selected gene set. Our model accommodates noise and errors in the selected gene set and GO. Using controlled GO data our method correctly recovered most of the selected categories, leading to dramatic improvements over current methods for GO analysis. When used with microarray expression data and ChIP-chip data from yeast and human our method was able to correctly identify both general and specific enriched categories which were overlooked by other methods.

Global transcriptional response to spermine, a component of the intramacrophage environment, reveals regulation of Francisella gene expression through insertion sequence elements.

Tularemia is caused by the category A biodefense agent Francisella tularensis. This bacterium is associated with diverse environments and a plethora of arthropod and mammalian hosts. How F. tularensis adapts to these different conditions, particularly the eukaryotic intracellular environment in which it replicates, is poorly understood. Here, we demonstrate that the polyamines spermine and spermidine are environmental signals that alter bacterial stimulation of host cells. Genomewide analysis showed that F. tularensis LVS undergoes considerable changes in gene expression in response to spermine. Unexpectedly, analysis of gene expression showed that multiple members of two classes of Francisella insertion sequence (IS) elements, ISFtu1 and ISFtu2, and the genes adjacent to these elements were induced by spermine. Spermine was sufficient to activate transcription of these IS elements and of nearby genes in broth culture and in macrophages. Importantly, the virulent strain of F. tularensis, Schu S4, exhibited similar phenotypes of cytokine induction and gene regulation in response to spermine. Distinctions in gene expression changes between Schu S4 and LVS at one orthologous locus, however, correlated with differences in IS element location. Our results indicate that spermine and spermidine are novel triggers to alert F. tularensis of its eukaryotic host environment. The results reported here also identify an unexpected mechanism of gene regulation controlled by a spermine-responsive promoter contained within IS elements. Different arrangements of these mobile genetic elements among Francisella strains may contribute to virulence by conveying new expression patterns for genes from different strains.