Pseudomonas aeruginosa is a ubiquitous environmental bacterium capable of causing a variety of life-threatening human infections. The genetic basis for preferential infection of certain immunocompromised patients or individuals with cystic fibrosis by P. aeruginosa is not understood. To establish whether variation in the genomic repertoire of P. aeruginosa strains can be associated with a particular type of infection, we used a whole-genome DNA microarray to determine the genome content of 18 strains isolated from the most common human infections and environmental sources. A remarkable conservation of genes including those encoding nearly all known virulence factors was observed. Phylogenetic analysis of strain-specific genes revealed no correlation between genome content and infection type. Clusters of strainspecific genes in the P. aeruginosa genome, termed variable segments, appear to be preferential sites for the integration of novel genetic material. A specialized cloning vector was developed for capture and analysis of these genomic segments. With this capture system a site associated with the strain-specific ExoU cytotoxin-encoding gene was interrogated and an 80-kb genomic island carrying exoU was identified. These studies demonstrate that P. aeruginosa strains possess a highly conserved genome that encodes genes important for survival in numerous environments and allows it to cause a variety of human infections. The acquisition of novel genetic material, such as the exoU genomic island, through horizontal gene transfer may enhance colonization and survival in different host environments.

A novel porcine deltacoronavirus (PdCV) was first discovered in Ohio and Indiana in February 2014, rapidly spread to other states in the United States and Canada, and caused significant economic loss in the swine industry. The origin and virulence of this novel porcine coronavirus are not known. Here, we characterized U.S. PdCV isolates and determined their virulence in gnotobiotic and conventional piglets. Genome analyses revealed that U.S. PdCV isolates possess unique genetic characteristics and share a close relationship with Hong Kong and South Korean PdCV strains and coronaviruses (CoVs) of Asian leopard cats and Chinese ferret-badgers. The PdCV-positive intestinal content (Ohio CVM1) and the cell culture-adapted PdCV Michigan (MI) strain were orally inoculated into gnotobiotic and/or conventional piglets. Within 1 to 3 days postinfection, profuse watery diarrhea, vomiting, and dehydration were observed. Clinical signs were associated with epithelial necrosis in the gastric pits and small intestine, the latter resulting in severe villous atrophy. Mild interstitial pneumonia was identified in the lungs of PdCV-infected piglets. High levels of viral RNA (8 to 11 log RNA copies/g) were detected in intestinal tissues/luminal contents and feces of infected piglets, whereas moderate RNA levels (2 to 5 log RNA copies/g) were detected in blood, lung, liver, and kidney, indicating multisystemic dissemination of the virus. Polyclonal immune serum against PdCV but not immune serum against porcine epidemic diarrhea virus (PEDV) reacted with PdCV-infected small-intestinal epithelial cells, indicating that PdCV is antigenically distinct from PEDV. Collectively, we demonstrate for the first time that PdCV caused severe gastrointestinal diseases in swine.

e Porcine epidemic diarrhea coronavirus (PEDV) has significantly damaged America's pork industry. Here we investigate the receptor usage and cell entry of PEDV. PEDV recognizes protein receptor aminopeptidase N from pig and human and sugar coreceptor N-acetylneuraminic acid. Moreover, PEDV infects cells from pig, human, monkey, and bat. These results support the idea of bats as an evolutionary origin for PEDV, implicate PEDV as a potential threat to other species, and suggest antiviral strategies to control its spread. P orcine epidemic diarrhea coronavirus (PEDV) causes largescale outbreaks of diarrhea in pigs and an 80 to 100% fatality rate in suckling piglets (1-3). Since 2013, PEDV has swept throughout the United States, wiped out more than 10% of America's pig population in less than a year, and significantly damaged the U.S. pork industry (4-6). No vaccine or antiviral drug is currently available to keep the spread of PEDV in check. PEDV belongs to the ␣ genus of the coronavirus family (7,8), which also includes porcine transmissible gastroenteritis coronavirus (TGEV), bat coronavirus 512/2005 (BtCoV/512/2005), and human NL63 coronavirus (HCoV-NL63). Although both PEDV and TGEV infect pigs, PEDV is genetically more closely related to BtCoV/512/ 2005 than to TGEV, leading to the hypothesis that PEDV originated from bats (9).Receptor binding and cell entry are essential steps in viral infection cycles, critical determinants of viral host range and tropism, and important targets for antiviral interventions. An envelope-anchored spike protein mediates coronavirus entry into cells. The spike ectodomain consists of a receptor-binding subunit, S1, and a membrane fusion subunit, S2. S1 contains two domains, an N-terminal domain (S1-NTD) and a C-terminal domain (S1-CTD), both of which can potentially function as receptor-binding domains (RBDs) (Fig. 1A) (10, 11). The ability of coronavirus RBDs to recognize receptor orthologs from different species is one of the most important determinants of coronavirus host range and tropism (8,(12)(13)(14). HCoV-NL63 S1-CTD recognizes human angiotensin-converting enzyme 2 (ACE2), whereas TGEV S1-CTD recognizes porcine aminopeptidase N (APN), and its S1-NTD recognizes two sugar coreceptors, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) (15-18). Usage of sugar coreceptors is linked to the enteric tropism of coronaviruses (18,19). It has been shown that PEDV uses porcine APN as its receptor (20). However, it is not known whether PEDV recognizes APN from other species or whether it uses sugar coreceptors. Addressing these questions will be critical for understanding the host range, tropism, and evolutionary origin of PEDV, for evaluating its potential risk to other species, particularly humans, and for developing effective vaccines and antiviral drugs to curb the spread of PEDV in pigs and to other species.To characterize the receptor usage of PEDV, here we identified the two S1 domains of PEDV based on the sequence similarity between PEDV and TGEV S1 subun...

Internal N 6 -methyladenosine (m 6 A) modification is one of the most common and abundant modifications of RNA. However, the biological role(s) of viral RNA m 6 A remains elusive. Using human metapneumovirus (hMPV) as a model, we demonstrate that m 6 A serves as a molecular marker for innate immune discrimination of self from nonself RNAs. We show that hMPV RNAs are m 6 A methylated and that viral m 6 A methylation promotes hMPV replication and gene expression. Inactivating m 6 A addition sites with synonymous mutations or demethylase resulted in m 6 A deficient recombinant hMPVs and virion RNAs that induced significantly higher expression of type I interferon (IFN) which was dependent on the cytoplasmic RNA sensor RIG-I, not MDA5. Mechanistically, m 6 A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I, and facilitates the conformational change of RIG-I, leading to enhanced IFN expression. Furthermore, m 6 A-deficient rhMPVs triggered higher IFN in vivo and were significantly attenuated in cotton rats yet retained high immunogenicity. Collectively, our results highlight that (i) virus acquires m 6 A in their RNAs as a means of mimicking cellular RNA to avoid detection by innate immunity; and (ii) viral RNA m 6 A can serve as a target to attenuate hMPV for vaccine purposes.

Vertebrates vary in resistance and resilience to infectious diseases, and the mechanisms regulating the trade-off between these two often opposing protective processes are not well understood. Variability in the sensitivity of species to induction of damaging inflammation in response to equivalent pathogen loads (resilience) complicates the use of animal models that reflect human disease. We found that induction of pro-inflammatory cytokines from macrophages in response to inflammatory stimuli in vitro is regulated by proteins in the sera of species in inverse proportion to their in vivo resilience to lethal doses of bacterial lipopolysaccharide over a range of 10,000-fold. This finding suggests that proteins in serum rather than intrinsic cellular differences may play a role in regulating variations in resilience to microbe-associated molecular patterns between species. Involvement of circulating proteins as key molecules raises hope that the process might be manipulated to create better animal models and potentially new drug targets.

Agrobacterium tumefaciens transfers a piece of its Ti plasmid DNA (transferred DNA or T-DNA) into plant cells during crown gall tumorigenesis. A. tumefaciens can transfer its T-DNA to a wide variety of hosts, including both dicotyledonous and monocotyledonous plants. We show that the host range of A. tumefaciens can be extended to include Saccharomyces cerevisiae. Additionally, we demonstrate that while T-DNA transfer into S. cerevisiae is very similar to T-DNA transfer into plants, the requirements are not entirely conserved. The Ti plasmid-encoded vir genes ofA. tumefaciens that are required for T-DNA transfer into plants are also required for T-DNA transfer into S. cerevisiae, as is vir gene induction. However, mutations in the chromosomal virulence genes of A. tumefaciens involved in attachment to plant cells have no effect on the efficiency of T-DNA transfer into S. cerevisiae. We also demonstrate that transformation efficiency is improved 500-fold by the addition of yeast telomeric sequences within the T-DNA sequence.Agrobacterium tumefaciens causes crown gall tumors in plants by transferring a segment of DNA (transferred DNA or T-DNA) from its tumor-inducing (Ti) plasmid to the nucleus of plant cells. The T-DNA becomes integrated into the plant nuclear genome where it functions to give rise to the characteristic tumor (reviewed in refs. 1 and 2). This process depends on the induction of a set of Ti plasmid-encoded virulence (vir) genes. vir genes are induced via the virA/virG two-component regulatory system which senses monosaccharides and phenolic compounds from wounded plants (reviewed in ref.3). The T-DNA is a single-stranded DNA molecule produced by a virDl/D2-encoded site-specific endonuclease that nicks within two 24-bp direct repeat sequences on the Ti plasmid (4). These repeats, termed border sequences, flank the T-DNA. Following cleavage and excision, the T-DNA is coated by the singlestranded DNA binding protein VirE2 (5), and the resulting T-DNA complex is transferred to the plant cell.The mechanism by which the T-DNA complex is transported through the inner and outer bacterial membranes and into the plant cell is not well understood. It is believed on the basis of several lines of evidence that the VirB proteins and VirD4 are involved in T-DNA transport (reviewed in ref. 6). Once the T-DNA complex enters the plant cell, it is targeted to the nucleus via nuclear localization sequences in the VirD2 and VirE2 proteins (7,8). Upon entering the nucleus, the T-DNA is integrated into the plant genome by illegitimate recombination, a process likely mediated by host factors (9).The study of host factors involved in T-DNA transfer has been difficult and would be greatly facilitated by the availability of a host model amenable to genetic manipulation. Given the similarities between T-DNA transfer and conjugative transfer of broad-host-range plasmids (reviewed in refs. 1, 6, and 10), we set out to determine if A. tumefaciens can transfer T-DNA to the yeast Saccharomyces cerevisiae. It has been demo...

Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium, is capable of colonizing a wide range of environmental niches and can also cause serious infections in humans. In order to understand the genetic makeup of pathogenic P. aeruginosa strains, a method of differential hybridization of arrayed libraries of cloned DNA fragments was developed. An M13 library of DNA from strain X24509, isolated from a patient with a urinary tract infection, was screened using a DNA probe from P. aeruginosa strain PAO1. The genome of PAO1 has been recently sequenced and can be used as a reference for comparisons of genetic organization in different strains. M13 clones that did not react with a DNA probe from PAO1 carried X24509-specific inserts. When a similar array hybridization analysis with DNA probes from different strains was used, a set of M13 clones which carried sequences present in the majority of human P. aeruginosa isolates from a wide range of clinical sources was identified. The inserts of these clones were used to identify cosmids encompassing a contiguous 48.9-kb region of the X24509 chromosome called PAGI-1 (for "P. aeruginosa genomic island 1"). PAGI-1 is incorporated in the X24509 chromosome at a locus that shows a deletion of a 6,729-bp region present in strain PAO1. Survey of the incidence of PAGI-1 revealed that this island is present in 85% of the strains from clinical sources. Approximately half of the PAGI-1-carrying strains show the same deletion as X24509, while the remaining strains contain both the PAGI-1 sequences and the 6,729-bp PAO1 segment. Sequence analysis of PAGI-1 revealed that it contains 51 predicted open reading frames. Several of these genes encoded products with predictable function based on their sequence similarities to known genes, including insertion sequences, determinants of regulatory proteins, a number of dehydrogenase gene homologs, and two for proteins of implicated in detoxification of reactive oxygen species. It is very likely that PAGI-1 was acquired by a large number of P. aeruginosa isolates through horizontal gene transfer. The selection for its maintenance may be the consequence of expression of any one of the genes of unknown function or the genes which allow P. aeruginosa to survive under the conditions that generate reactive oxygen species. Alternatively, one or both of the transcriptional regulators encoded in PAGI-1 may control the expression of genes in the P. aeruginosa chromosome, which provides a selective advantage for strains that have acquired this genomic island.

Detection of LPS in tissues is an integral component of innate immunity that acts to protect against invasion by Gram-negative bacteria. Plasma down-regulates LPS-induced cytokine production from macrophages, thereby limiting systemic inflammation in blood and distant tissues. To identify the protein(s) involved in this process, we used classical biochemical chromatographic techniques to identify fractions of mouse sera that suppress LPS-induced TNF from bone marrow-derived macrophages (BMDMs). Fractionation yielded microgram quantities of a protein that was identified by MS to be hemopexin (Hx). Mouse Hx purified on hemin-agarose beads and rhHx decreased the production of cytokines from BMDMs and peritoneal macrophages induced by LPS. Preincubation of LPS with Hx did not affect the activity of LPS on LAL, whereas preincubation of Hx with macrophages followed by washing resulted in decreased activity of these cells in response to LPS, suggesting that Hx acts on macrophages rather than LPS. Heme-free Hx did not stimulate HO-1 in the macrophages. Purified Hx also decreased TNF and IL-6 from macrophages induced by the synthetic TLR2 agonist Pam3Cys. Our data suggest that Hx, which is an acute-phase protein that increases during inflammation, limits TLR4 and TLR2 agonist-induced macrophage cytokine production directly through a mechanism distinct from HO-1.