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Supplementary Table S2 from A Critical Role for the Inflammatory Response in a Mouse Model of Preneoplastic Progression

Supplementary Figure S1 from A Critical Role for the Inflammatory Response in a Mouse Model of Preneoplastic Progression

The tumor microenvironment, which includes inflammatory cells, vasculature, extracellular matrix, and fibroblasts, is a critical mediator of neoplastic progression and metastasis. Using an inducible transgenic mouse model of preneoplastic progression in the mammary gland, we discovered that activation of inducible fibroblast growth factor receptor-1 (iFGFR1) in the mammary epithelium rapidly increased the expression of several genes involved in the inflammatory response. Further analysis revealed that iFGFR1 activation induced recruitment of macrophages to the epithelium and continued association with the alveolar hyperplasias that developed following long-term activation. Studies using HC-11 mammary epithelial cells showed that iFGFR1-induced expression of the macrophage chemoattractant osteopontin was required for macrophage recruitment in vitro. Finally, conditional depletion of macrophages inhibited iFGFR1-mediated epithelial cell proliferation and lateral budding. These findings show that inflammatory cells, specifically macrophages, are critical for mediating early events in an inducible transgenic mouse model of preneoplastic progression. (Cancer Res 2006; 66(11): 5676-85)

This article reports the results of studying three novel bacteriophages, JL, Shanette, and Basilisk, which infect the pathogen Bacillus cereus and carry genes that may contribute to its pathogenesis. We analyzed host range and superinfection ability, mapped their genomes, and characterized phage structure by mass spectrometry and transmission electron microscopy (TEM). The JL and Shanette genomes were 96% similar and contained 217 open reading frames (ORFs) and 220 ORFs, respectively, while Basilisk has an unrelated genome containing 138 ORFs. Mass spectrometry revealed 23 phage particle proteins for JL and 15 for Basilisk, while only 11 and 4, respectively, were predicted to be present by sequence analysis. Structural protein homology to well-characterized phages suggested that JL and Shanette were members of the family Myoviridae , which was confirmed by TEM. The third phage, Basilisk, was similar only to uncharacterized phages and is an unrelated siphovirus. Cryogenic electron microscopy of this novel phage revealed a T=9 icosahedral capsid structure with the major capsid protein (MCP) likely having the same fold as bacteriophage HK97 MCP despite the lack of sequence similarity. Several putative virulence factors were encoded by these phage genomes, including TerC and TerD involved in tellurium resistance. Host range analysis of all three phages supports genetic transfer of such factors within the B. cereus group, including B. cereus , B. anthracis , and B. thuringiensis . This study provides a basis for understanding these three phages and other related phages as well as their contributions to the pathogenicity of B. cereus group bacteria. IMPORTANCE The Bacillus cereus group of bacteria contains several human and plant pathogens, including B. cereus , B. anthracis , and B. thuringiensis . Phages are intimately linked to the evolution of their bacterial hosts and often provide virulence factors, making the study of B. cereus phages important to understanding the evolution of pathogenic strains. Herein we provide the results of detailed study of three novel B. cereus phages, two highly related myoviruses (JL and Shanette) and an unrelated siphovirus (Basilisk). The detailed characterization of host range and superinfection, together with results of genomic, proteomic, and structural analyses, reveal several putative virulence factors as well as the ability of these phages to infect different pathogenic species.

This paper investigates the accumulation and release of deoxyribonucleic acid (DNA) relative to a surface micromachined silicon lance. The lance is a critical element of nanoinjection, a proposed approach for injecting foreign DNA into living cells. The quantity of DNA accumulated on the nanoinjector lance and the speed at which it can be moved on and off the lance are essential to the proposed system's function. Prototype nanoinjector lances were fabricated using a multilayer surface micromachining process. DNA stained with the fluorescent dye 4', 6-diamidino-2-phenylidole dihydrochloride was visualized using fluorescent illumination as the DNA was accumulated on and released from the tips of microelectromechanical systems (MEMS) microlances using a 1.5-V dc source. In 5 min 46 s, the lance accumulated over 32 000 DNA molecules from a dilute DNA solution. The lance then released over 6200 DNA molecules within 6 s. Finally, the nanoinjector lance was used to inject a reporter gene encoding a red fluorescent protein into a mouse embryo, resulting in expression of the gene. The nanoinjector lance represents an important and significant step in the development of a self-contained MEMS-based DNA injection system.

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Brevibacillus laterosporus has been isolated from many different environments, including beehives, and produces compounds that are toxic to many organisms. Five B. laterosporus phages have been isolated previously. Here, we announce five additional phages that infect this bacterium, including the first B. laterosporus siphoviruses to be discovered.

Paenibacillus larvae is a pathogen of honeybees that causes American foulbrood (AFB). We isolated bacteriophages from soil containing bee debris collected near beehives in Utah. We announce five high-quality complete genome sequences, which represent the first completed genome sequences submitted to GenBank for any P. larvae bacteriophage.

Transgenic mice expressing an inducible suicide gene, which allows systemic and reversible elimination of macrophages, were developed. A macrophage-specific c-fms promoter was used to express enhanced green fluorescent protein and a drug-inducible suicide gene that leads to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage. Transgenic mice were fertile, of normal weight, and showed no abnormal phenotype before drug exposure. The transgene was expressed constitutively in macrophages and dendritic cells (DC) but not significantly in T cells or B cells. Induction of the suicide gene led to depletion of 70–95% of macrophages and DC in nearly all tissues examined. Depletion reduced the ability to clear bacteria from the blood and led to increased bacterial growth in the liver. Depleted mice displayed several abnormalities, including splenomegaly, lymphadenopathy, thymic atrophy, extramedullary hematopoiesis, and development of peritoneal adhesions. This new, transgenic line will be useful in investigating the role of macrophages and DC.

Mycobacteriophages – viruses of mycobacterial hosts – are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages – Corndog, Catdawg, Dylan, Firecracker, and YungJamal – designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8–9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.

Background The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains. Results Whole genome nucleotide and proteome comparison of the 93 extant Bacillus phages revealed 12 distinct clusters, 28 subclusters and 14 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member of the group. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,922 protein families (phams) of which only 951 (19%) had a predicted function. In addition, 3,058 (62%) of phams were orphams (phams containing a gene product from a single phage). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains. Conclusions This analysis provides a basis for understanding and characterizing Bacillus phages and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.

Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students’ interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training., IMPORTANCE Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.

The Bacillus cereus group is an assemblage of highly related firmicute bacteria that cause a variety of diseases in animals, including insects and humans. We announce three high-quality, complete genome sequences of bacteriophages we isolated from soil samples taken at the bases of fruit trees in Utah County, Utah. While two of the phages (Shanette and JL) are highly related myoviruses, the bacteriophage Basilisk is a siphovirus.

Paenibacillus larvae is a pathogen of honeybees that causes American foulbrood (AFB). We isolated bacteriophages from soil containing bee debris collected near beehives in Utah. We announce five high-quality complete genome sequences, which represent the first completed genome sequences submitted to GenBank for any P. larvae bacteriophage.

Mycobacteriophages infect members of the Mycobacterium genus in the phylum Actinobacteria and exhibit remarkable diversity. Genome analysis groups the thousands of known mycobacteriophages into clusters, of which the B1 subcluster is currently the third most populous. We report the complete genome sequences of five additional members of the B1 subcluster.

Background Phylogenetic comparison of bacteriophages requires whole genome approaches such as dotplot analysis, genome pairwise maps, and gene content analysis. Currently mycobacteriophages, a highly studied phage group, are categorized into related clusters based on the comparative analysis of whole genome sequences. With the recent explosion of phage isolation, a simple method for phage cluster prediction would facilitate analysis of crude or complex samples without whole genome isolation and sequencing. The hypothesis of this study was that mycobacteriophage-cluster prediction is possible using comparison of a single, ubiquitous, semi-conserved gene. Tape Measure Protein (TMP) was selected to test the hypothesis because it is typically the longest gene in mycobacteriophage genomes and because regions within the TMP gene are conserved. Results A single gene, TMP, identified the known Mycobacteriophage clusters and subclusters using a Gepard dotplot comparison or a phylogenetic tree constructed from global alignment and maximum likelihood comparisons. Gepard analysis of 247 mycobacteriophage TMP sequences appropriately recovered 98.8% of the subcluster assignments that were made by whole-genome comparison. Subcluster-specific primers within TMP allow for PCR determination of the mycobacteriophage subcluster from DNA samples. Using the single-gene comparison approach for siphovirus coliphages, phage groupings by TMP comparison reflected relationships observed in a whole genome dotplot comparison and confirm the potential utility of this approach to another widely studied group of phages. Conclusions TMP sequence comparison and PCR results support the hypothesis that a single gene can be used for distinguishing phage cluster and subcluster assignments. TMP single-gene analysis can quickly and accurately aid in mycobacteriophage classification.

Intranasal application of vesicular stomatitis virus (VSV) induces acute encephalitis characterized by a pronounced myeloid and T cell infiltrate. The role of distinct phagocytic populations on VSV encephalitis was therefore examined in this study. Ablation of peripheral macrophages did not impair VSV encephalitis or viral clearance from the brain, whereas, depletion of splenic marginal dendritic cells impaired this response and enhanced morbidity/mortality. Selective depletion of brain perivascular macrophages also suppressed this response without altering viral clearance. Thus, two anatomically distinct phagocytic populations regulate VSV encephalitis in a non-redundant fashion although neither population is essential for viral clearance in the CNS.

The tumor microenvironment, which includes inflammatory cells, vasculature, extracellular matrix, and fibroblasts, is a critical mediator of neoplastic progression and metastasis. Using an inducible transgenic mouse model of preneoplastic progression in the mammary gland, we discovered that activation of inducible fibroblast growth factor receptor-1 (iFGFR1) in the mammary epithelium rapidly increased the expression of several genes involved in the inflammatory response. Further analysis revealed that iFGFR1 activation induced recruitment of macrophages to the epithelium and continued association with the alveolar hyperplasias that developed following long-term activation. Studies using HC-11 mammary epithelial cells showed that iFGFR1-induced expression of the macrophage chemoattractant osteopontin was required for macrophage recruitment in vitro. Finally, conditional depletion of macrophages inhibited iFGFR1-mediated epithelial cell proliferation and lateral budding. These findings show that inflammatory cells, specifically macrophages, are critical for mediating early events in an inducible transgenic mouse model of preneoplastic progression. (Cancer Res 2006; 66(11): 5676-85)

The V antigen (LcrV) of the plague bacteriumYersinia pestisis a potent protective antigen that is under development as a vaccine component for humans. LcrV is multifunctional. On the bacterial surface it mediates delivery of a set of toxins called Yops into host cells, and as a released protein it can cause production of the immunosuppressive cytokine interleukin-10 (IL-10) and can inhibit chemotaxis of polymorphonuclear neutrophils. It is not known how these mechanisms of LcrV operate, what their relative importance is, when they function during plague, and which are critical to protection by antibody. This study investigated several of these issues. C57BL/6 mice, mice unable to express IL-10, or mice with the macrophage lineage eliminated were treated with a protective anti-LcrV antibody or a nonprotective antibody against YopM and infected intravenously byY. pestisKIM5 or a strain that lacked the genes encoding all six effector Yops. Viable bacterial numbers were determined at various times. The data indicated that Yops were necessary forYersiniagrowth after the bacteria had seeded liver and spleen. Anti-LcrV antibody prevented this growth, even in IL-10−/−mice, demonstrating that one protective mechanism for anti-LcrV antibody is independent of IL-10. Anti-LcrV antibody had no effect on persistence in organs ofY. pestislacking effector Yops, even though the yersiniae could strongly express LcrV, suggesting that Yops are necessary for building sufficient bacterial numbers to produce enough LcrV for its immunosuppressive effects. In vitro assays showed that anti-LcrV antibody could partially block delivery of Yops and downstream effects of Yops in infected macrophage-like J774A.1 cells. However, cells of the macrophage lineage were found to be dispensable for protection by anti-LcrV antibody in spleen, although they contributed to protection in liver. Taken together, the data support the hypothesis that one protective effect of the antibody is to block delivery of Yops to host cells and prevent early bacterial growth. The findings also identified the macrophage lineage as one host cell type that mediates protection.

The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains. Whole genome nucleotide and proteome comparison of the 83 extant, fully sequenced Bacillus phages revealed 10 distinct clusters, 24 subclusters and 15 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,442 protein families (phams) of which only 894 (20%) had a predicted function. In addition, 2,583 (58%) of phams were orphams (phams containing a single member). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains. This analysis provides a basis for understanding and characterizing Bacillus and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.

Background Paenibacillus larvae is a Firmicute bacterium that causes American Foulbrood, a lethal disease in honeybees and is a major source of global agricultural losses. Although P. larvae phages were isolated prior to 2013, no full genome sequences of P. larvae bacteriophages were published or analyzed. This report includes an in-depth analysis of the structure, genomes, and relatedness of P. larvae myoviruses Abouo, Davis, Emery, Jimmer1, Jimmer2, and siphovirus phiIBB_Pl23 to each other and to other known phages. Results P. larvae phages Abouo, Davies, Emery, Jimmer1, and Jimmer2 are myoviruses with ~50 kbp genomes. The six P. larvae phages form three distinct groups by dotplot analysis. An annotated linear genome map of these six phages displays important identifiable genes and demonstrates the relationship between phages. Sixty phage assembly or structural protein genes and 133 regulatory or other non-structural protein genes were identifiable among the six P. larvae phages. Jimmer1, Jimmer2, and Davies formed stable lysogens resistant to superinfection by genetically similar phages. The correlation between tape measure protein gene length and phage tail length allowed identification of co-isolated phages Emery and Abouo in electron micrographs. A Phamerator database was assembled with the P. larvae phage genomes and 107 genomes of Firmicute-infecting phages, including 71 Bacillus phages. Phamerator identified conserved domains in 1,501 of 6,181 phamilies (only 24.3%) encoded by genes in the database and revealed that P. larvae phage genomes shared at least one phamily with 72 of the 107 other phages. The phamily relationship of large terminase proteins was used to indicate putative DNA packaging strategies. Analyses from CoreGenes, Phamerator, and electron micrograph measurements indicated Jimmer1, Jimmer2, Abouo and Davies were related to phages phiC2, EJ-1, KC5a, and AQ113, which are small-genome myoviruses that infect Streptococcus, Lactobacillus, and Clostridium, respectively. Conclusions This paper represents the first comparison of phage genomes in the Paenibacillus genus and the first organization of P. larvae phages based on sequence and structure. This analysis provides an important contribution to the field of bacteriophage genomics by serving as a foundation on which to build an understanding of the natural predators of P. larvae. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-745) contains supplementary material, which is available to authorized users.