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... Consensus sequences were assembled and edited using the EditSeq and SeqMan programs, and multiple sequence alignment analysis was done using MegAlign (DNASTAR Inc., Madison, WI) or Clustal W (v1.81) =-=(31)-=-. Establishment of stable cell lines expressing CD163. Cell culture wells (35 mm) containing approximately 1 106 cells each were transfected with 2 to 4 g of CD163 expression vector or a negative c...

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...nicol selection marker (cat). The marker was inserted by electroporation and lambda red-directed recombination of PCR products containing cat flanked by 40-bp sequences homologous to the target locus =-=(11)-=-. Selected deletion mutants were complemented by single-copy gene insertion into the chromosomal attTn7 locus. A modification of the method of Bao et al. (3) was used. Plasmid pCIITn7K-a was a gift of...

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...nd incubation overnight at 37°C. A disk-diffusion assay (Kirby-Bauer testing) was performed by diluting overnight broth cultures with PBS to an OD 600 of 0.13, or approximately 0.5 McFarland standard =-=(4)-=-. A cotton swab was used to spread the bacteria onto an LB agar plate. Paper disks 6 mm in diameter (0.25 in.; Schleicher & Schuell, Keene, NH) were saturated with 20 �l test reagent and placed atop t...

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...20% glycerol, 10% SDS, and 5% 2-�-mercaptoethanol. Proteinase K was added (0.2 mg), and samples were incubated overnight at 37°C. Samples were boiled for 5 min and run on a 10% SDS-polyacrylamide gel =-=(15)-=-. A silver stain kit was used for visualization (Bio-Rad, Hercules, CA). Statistical analysis. Raw colony counts were adjusted to CFU per gram of kidney or bladder. The minimum threshold of detection ...

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...(ATCC 47076) is a laboratory K-12 strain. CFT073 (ATCC 700928) is a clinical strain isolated from a woman with pyelonephritis (28). The genomic sequences of both MG1655 and CFT073 have been published =-=(5, 45)-=-. Cultures were grown at 37°C in Luria-Bertani (LB) broth or 1.5% LB agar unless otherwise noted. The following antibiotics (Sigma, St. Louis, MO) were added, as appropriate: ampicillin at 100 �g/ml, ...

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...has since spread to become endemic in the major swine-producing nations of Europe, Asia, and the Americas. The etiologic agent of PRRS is a member of the family Arteriviridae in the order Nidovirales =-=(1)-=-. Two distinct genotypes of PRRS virus (PRRSV), designated “European” (type I) and “North American” (type II), have been described. These two genotypes share only about 60% identity at the nucleotide ...

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...omolecular biosynthesis towards the de novo production of amino acids. Consequently, among the first manifestations of the SR is the shutting down of stable RNA (comprised of rRNA and tRNA) synthesis =-=(9)-=-. This, in turn, leads to a reduction in the growth rate of the organism (transcription of the rrn operons is tightly coupled to the growth rate) (10, 11). The stringent response has been implicated i...

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...s of DegP function inside the mouse, nor does it seem to have an independent role in infection. DegP is at present the only known periplasmic protein that functions as both a protease and a chaperone =-=(39)-=-. We modified the genetic complementation method used in this study to see if the protease function is necessary for virulence. Fulllength degP with its sigma E promoter was inserted into the CFT073 c...

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... minutes and separated by sodium dodecyl sulfate (SDS)–polyacrylamide (10%) gel electrophoresis. After transfer to nitrocellulose, blots were exposed to anti-DegP/MBP antibody, a gift of Jon Beckwith =-=(40)-=-. Anti-rabbit immunoglobulin G–horseradish peroxidase was used as secondary antibody, and its binding was visualized by using the LumiGLO chemiluminescence kit (Kierkegaard & Perry Laboratories, Gaith...

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...sistance in E. coli K-12 in 1988 (25). Johnson et al. found it during a screen of transposon-generated mutants of Salmonella enterica serovar Typhimurium for avirulence after oral inoculation of mice =-=(19)-=-. Klebsiella pneumoniae, Brucella abortus, Brucella melitensis, and Yersinia enterocolitica mutants of degP (htrA) were subsequently also found to have reduced virulence in infection models, and a liv...

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... coli virulence in an animal infection model, though it has been wellstudied as a virulence gene in other pathogens. It was described as “htrA” for high temperature resistance in E. coli K-12 in 1988 =-=(25)-=-. Johnson et al. found it during a screen of transposon-generated mutants of Salmonella enterica serovar Typhimurium for avirulence after oral inoculation of mice (19). Klebsiella pneumoniae, Brucella...

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... the E. coli homolog over its entire length (745 residues). Like the E. coli protein, the E. carotovora subsp. atroseptica homolog lacks a catalytically active N-terminal phosphohydrolase (HD) domain =-=(3)-=-. Instead, the H and D residues required for phosphohydrolase activity are replaced with F and P (respectively), suggesting that the E. carotovora subsp. atroseptica protein functions exclusively as a...

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... sequences homologous to the target locus (11). Selected deletion mutants were complemented by single-copy gene insertion into the chromosomal attTn7 locus. A modification of the method of Bao et al. =-=(3)-=- was used. Plasmid pCIITn7K-a was a gift of Eric Stabb. It features a mini-Tn7 transposon with a kanamycin resistance marker. For complementation, the fulllength gene and its promoter were amplified f...

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... stable RNA (comprised of rRNA and tRNA) synthesis (9). This, in turn, leads to a reduction in the growth rate of the organism (transcription of the rrn operons is tightly coupled to the growth rate) =-=(10, 11)-=-. The stringent response has been implicated in controlling virulence factor production in a number of pathogens (17, 20, 23, 31, 50). The SR is brought about as a result of the rapid accumulation of ...

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...Apart from primary PAM, the only other cell type known to be fully permissive for the growth of PRRSV in vitro is the immortalized monkey kidney cell line MA-104 and its derivatives, such as MARC-145 =-=(12)-=-. In MARC-145 cells, the internalization of the virus by endocytosis and subsequent uncoating in low-pH vesicles seem to mimic similar events in PAM (14). However, MAbs that bind to porcine sialoadhes...

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...bsp. atroseptica are primarily involved in degrading plant cell walls and include a range of pectate lyases (Pels), polygalacturonases, pectin methylesterases, proteases (Prts), and cellulases (Cels) =-=(4)-=-. Over the last decade, it has become increasingly clear that production of many of these secreted virulence factors are under the control of an N-acyl-homoserine lactone (AHL)-dependent quorum-sensin...

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...o have no apparent in vivo survival defect, though they appear important in other pathogens. For example, fkpA, like surA, encodes a periplasmic prolyl isomerase, although it is in a different family =-=(27)-=-. FkpA also has chaperone activity. In Salmonella enterica serovar Typhimurium, an fkpA inactivation mutant shows decreased intracellular survival in a macrophage-like cell line (16). Mutants of fkpA ...

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...odstream and exhibits cytokine-like functions (9, 19). One well-characterized function of CD163 involves scavenging of hemoglobin, which is mediated by endocytosis of haptoglobin-hemoglobin complexes =-=(15)-=-. SusCD163v1 is missing the entire first and second SRCR repeats, yet it still confers permissivity to PRRSV infection, indicating that these two domains are not required for binding of PRRSV. Of six ...

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...ors. We have used a mouse model of UTI both as a screening tool for genes specifically expressed during infection and for assessment of the relative virulence of mutants compared to wild-type E. coli =-=(32, 37, 38)-=-. Previously, we found that degS is essential for infection by the uropathogenic E. coli strain CFT073 (32). DegS is an indirect regulator of the alternate sigma factor, sigma E. DegS is an inner memb...

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... media. Table 1 shows Escherichia coli strains used in this study. MG1655 (ATCC 47076) is a laboratory K-12 strain. CFT073 (ATCC 700928) is a clinical strain isolated from a woman with pyelonephritis =-=(28)-=-. The genomic sequences of both MG1655 and CFT073 have been published (5, 45). Cultures were grown at 37°C in Luria-Bertani (LB) broth or 1.5% LB agar unless otherwise noted. The following antibiotics...

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... stable RNA (comprised of rRNA and tRNA) synthesis (9). This, in turn, leads to a reduction in the growth rate of the organism (transcription of the rrn operons is tightly coupled to the growth rate) =-=(10, 11)-=-. The stringent response has been implicated in controlling virulence factor production in a number of pathogens (17, 20, 23, 31, 50). The SR is brought about as a result of the rapid accumulation of ...

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... (14, 52), an enteric pathogen (Salmonella enterica serovar Typhimurium) (43, 45, 48), gram-positive and gram-negative intracellular pathogens (Mycobacterium tuberculosis [44], Legionella pneumophila =-=[22]-=-, and Listeria monocytogenes [50]), a mucosal pathogen (Helicobacter pylori [37]), Neisseria gonorrhoeae (16), the principal 7650 WANG ET AL. J. BACTERIOL.so nsM arch 4, 2014 by PENN STATE UNIV http:/...

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...E. coli K-12 the products of these three genes, YaeT, YfiO, and NlpB, have been shown to associate, along with YfgL, to form a multicomponent complex important in the biogenesis of the outer membrane =-=(47)-=-. One mutant, CFT073 surA, shows minimal guinea pig red blood cell agglutination. This suggests diminished type 1 pilus expression. The association of SurA and type 1 pilus expression has recently bee...

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...nished type 1 pilus expression. The association of SurA and type 1 pilus expression has recently been reported (22). The type 1 pilus adhesin is important for E. coli persistence in the mouse bladder =-=(2)-=-. Therefore, the nonagglutinating CFT073 surA mutant was not assayed for virulence in the mouse UTI model. Of the 11 mutants inoculated into mice, 5 have deletions of genes mostly uncharacterized (gen...

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...of sigma E. Freed from RseA repression, sigma E mediates the recognition by RNA polymerase of the promoters of the genes in its regulon (1). The sigma E regulon has been characterized in E. coli K-12 =-=(10, 33)-=-. It consists of at least 40 genes, and those with known function can be divided into categories of periplasmic folding factors and degradases, lipopolysaccharide (LPS) biogenesis, sensor proteins, an...

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...hrocytes (Hema Resource & Supply, Aurora, OR) or human erythrocytes (University of Wisconsin Hospital) on a glass slide (23). Mannose was added to agglutinated cells to a final concentration of 50 mM =-=(29)-=-. Hemolysis was assessed by measurement of zones of clearing around colonies grown overnight on 5% sheep blood agar supplemented with 10 mM calcium chloride. Motility was evaluated by inoculation into...

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...nes. Fluorescent-antibody (FA) assays. Cell monolayers were washed once with PBS, fixed in 80% acetone for 5 to 10 min, and air dried. For detection of the PRRSV nucleocapsid (N) protein, MAb SDOW-17 =-=(22)-=- conjugated with fluorescein isothiocyanate (FITC) was added to each well. After 30 to 60 min of incubation, the antibody was aspirated and monolayers were washed three times with PBS prior to viewing...

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... and a live Salmonella enterica serovar Typhi vaccine strain with inactivation mutations in aroC and aroD was associated with no bacteremia in human volunteers when a third mutation in degP was added =-=(8, 24, 31, 41, 42)-=-. More recently, degP orthologs have been shown to contribute to the virulence of the gram-positive pathogens Streptococcus pyogenes and Streptococcus pneumoniae (18, 20). Interestingly, the uropathog...

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...n growing plants, and soft rot in stored tubers. Due to the significant economic impact of E. carotovora subsp. atroseptica, the genome of E. carotovora subsp. atroseptica SCRI1043 has been sequenced =-=(5)-=-. E. carotovora subsp. atroseptica produces numerous secreted enzymes that macerate plant tissues, releasing nutrients that enable rapid bacterial growth. The exoenzymes produced by E. carotovora subs...

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...that are associated with RelA and SpoT from most other organisms (Fig. 1A). The ACT domain is predicted to function in regulation, while the TGS domain is thought to play a role in nucleotide binding =-=(18, 54, 55)-=-. E. carotovora subsp. atroseptica SCRI1043 also contains a spoT homolog (ECA038). In E. carotovora subsp. atroseptica SCRI1043, relA is flanked by mazG and rumA (Fig. 1B). The mazG gene encodes a nuc...

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...s) or intracelDownloaded from http://iai.asm.org/ on February 21, 2013 by PENN STATE UNIVVOL. 74, 2006 SIGMA E-REGULATED GENES IN E. COLI UROPATHOGENESIS 4037 lular survival (Haemophilus influenzae) =-=(9, 17, 46, 48)-=-. It may be that some sigma E-regulated genes important for virulence in other bacteria are less necessary in E. coli uropathogenesis. Furthermore, the in vitro phenotype of the degP mutant of E. coli...

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...ed at least 60 times, yielding in excess of 105 50% tissue culture infective doses/ml. Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine =-=(23)-=-. The syndrome appeared almost simultaneously in Western Europe and North America in the late 1980s and has since spread to become endemic in the major swine-producing nations of Europe, Asia, and the...

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... quorum sensing) were significantly modulated, so the two signaling systems clearly do communicate, although more work needs to be done to elucidate the details of this. In E. coli and other bacteria =-=(6, 19, 26, 48)-=-, elevated (p)ppGpp levels lead to an increase in RpoS (sigma 38) production. This sigma factor controls the expression of a wide range of genes during stationary-phase growth and during adaptation to...

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..., it is unclear whether skp per se is involved in virulence. Skp has been described as a periplasmic chaperone important in the maturation of outer membrane porins, such as OmpA, OmpC, OmpF, and LamB =-=(7)-=-. It may have functional redundance with DegP and SurA, but these “parallel pathway” chaperones (34) may not be able to make up for its loss during infection. The members of the sigma E regulon play i...

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...c chaperone important in the maturation of outer membrane porins, such as OmpA, OmpC, OmpF, and LamB (7). It may have functional redundance with DegP and SurA, but these “parallel pathway” chaperones =-=(34)-=- may not be able to make up for its loss during infection. The members of the sigma E regulon play important roles in periplasmic and outer membrane homeostasis. It is notable, therefore, that so few ...

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...s) or intracelDownloaded from http://iai.asm.org/ on February 21, 2013 by PENN STATE UNIVVOL. 74, 2006 SIGMA E-REGULATED GENES IN E. COLI UROPATHOGENESIS 4037 lular survival (Haemophilus influenzae) =-=(9, 17, 46, 48)-=-. It may be that some sigma E-regulated genes important for virulence in other bacteria are less necessary in E. coli uropathogenesis. Furthermore, the in vitro phenotype of the degP mutant of E. coli...

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... domain structures similar to that reported for human CD163 (16) (Fig. 3). Transient transfection of various cell lines with susCD163v1 confers permissivity to PRRSV. Because the nonpermissive BHK-21 =-=(18)-=-, PK032495, NLFK, NLST, and RL cell lines (unpublished data) have the ability to replicate PRRSV and produce progeny virus after transfection with infectious cDNA clones, these cell lines are assumed ...

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...ws and gilts (late-term abortions, stillbirths, and mummies), high mortality rates among nursery pigs, and respiratory disease in swine of all ages. The disease has been the subject of recent reviews =-=(17, 20, 25, 26)-=-. Fully differentiated primary porcine alveolar macrophage (PAM) constitute the predominant cell target for viral replication (4, 5). A published example of an immortalized PAM cell line, however, is ...

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...ors. We have used a mouse model of UTI both as a screening tool for genes specifically expressed during infection and for assessment of the relative virulence of mutants compared to wild-type E. coli =-=(32, 37, 38)-=-. Previously, we found that degS is essential for infection by the uropathogenic E. coli strain CFT073 (32). DegS is an indirect regulator of the alternate sigma factor, sigma E. DegS is an inner memb...

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...on the CFT073 chromosome (45). An inactivation mutant of degQ has no apparent phenotype in E. coli K-12, but when overexpressed it can rescue the temperature-sensitive phenotype of a K-12 degP mutant =-=(44)-=-. We wondered if degQ plays a role in E. coli virulence and made a deletion mutant of it in CFT073. In competition with the wild type in the mouse UTI model, no significant difference was seen in thei...

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...irions is mediated by interaction of the viral matrix protein with heparin sulfate proteoglycans (3). Virus particles are internalized in clathrin-coated vesicles and released following acidification =-=(21)-=-. Internalization may be facilitated by sialoadhesin, a 210or 220-kDa membrane glycoprotein in the siglec family of sialic acid binding immunoglobulin-like lectins (32), since incubation of PAM cells ...

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...gS is an inner membrane protein that degrades RseA, a repressor of sigma E. Freed from RseA repression, sigma E mediates the recognition by RNA polymerase of the promoters of the genes in its regulon =-=(1)-=-. The sigma E regulon has been characterized in E. coli K-12 (10, 33). It consists of at least 40 genes, and those with known function can be divided into categories of periplasmic folding factors and...

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... in degP was added (8, 24, 31, 41, 42). More recently, degP orthologs have been shown to contribute to the virulence of the gram-positive pathogens Streptococcus pyogenes and Streptococcus pneumoniae =-=(18, 20)-=-. Interestingly, the uropathogenic E. coli CFT073 mutant shows few of the defects described for degP mutants in other pathogens. Most of the above-named bacteria, including the streptococci, have been...

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... ages. The disease has been the subject of recent reviews (17, 20, 25, 26). Fully differentiated primary porcine alveolar macrophage (PAM) constitute the predominant cell target for viral replication =-=(4, 5)-=-. A published example of an immortalized PAM cell line, however, is not permissive for PRRSV infection (33). In addition, monocytes from peripheral blood are largely refractory to PRRSV infection. A p...

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...pond to nutrient downshift by producing (p)ppGpp. The relA-mediated SR to nutrient limitation has been shown to affect virulence and quorum-sensingrelated phenotypes in several gram-negative bacteria =-=(14, 16, 17, 23)-=-. PCR screening of the arrayed SCRI1043 mutant bank failed to identify any mutants carrying insertions in the relA gene. Therefore, to investigate whether relA affects virulence in E. carotovora subsp...

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... and a live Salmonella enterica serovar Typhi vaccine strain with inactivation mutations in aroC and aroD was associated with no bacteremia in human volunteers when a third mutation in degP was added =-=(8, 24, 31, 41, 42)-=-. More recently, degP orthologs have been shown to contribute to the virulence of the gram-positive pathogens Streptococcus pyogenes and Streptococcus pneumoniae (18, 20). Interestingly, the uropathog...

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... in degP was added (8, 24, 31, 41, 42). More recently, degP orthologs have been shown to contribute to the virulence of the gram-positive pathogens Streptococcus pyogenes and Streptococcus pneumoniae =-=(18, 20)-=-. Interestingly, the uropathogenic E. coli CFT073 mutant shows few of the defects described for degP mutants in other pathogens. Most of the above-named bacteria, including the streptococci, have been...

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...anism (transcription of the rrn operons is tightly coupled to the growth rate) (10, 11). The stringent response has been implicated in controlling virulence factor production in a number of pathogens =-=(17, 20, 23, 31, 50)-=-. The SR is brought about as a result of the rapid accumulation of two small molecule effectors (known as alarmones), guanosine 3-diphosphate 5-triphosphate (pppGpp) and guanosine 35-bispyrophosph...

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...eleased following acidification (21). Internalization may be facilitated by sialoadhesin, a 210or 220-kDa membrane glycoprotein in the siglec family of sialic acid binding immunoglobulin-like lectins =-=(32)-=-, since incubation of PAM cells with monoclonal antibody (MAb) to this polypeptide blocks PRRSV infection (6, 34). Transfection of the nonpermissive PK-15 (porcine kidney) cell line with porcine sialo...

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...stimulate the expression of other global regulators, which in Escherichia coli include the stationary-phase factor (RpoS), the leucine-responsive regulatory protein (Lrp), and integration host factor =-=(26)-=-. However, in the absence of (p)ppGpp, expression of RpoS alone is insufficient to activate expression of the rpoS regulon (30). Therefore, it seems that the ppGpp-dependent conformational change in R...

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...nella pneumophila and Chlamydia trachomatis Downloaded from http://iai.asm.org/ on February 21, 2013 by PENN STATE UNIV4036 REDFORD AND WELCH INFECT. IMMUN. also show impaired survival in host cells =-=(12, 26)-=-. It is notable that disease with these three pathogens features a well-characterized and necessary intracellular replication phase, while E. coli uropathogenesis does not. This is the first report of...

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...ors. We have used a mouse model of UTI both as a screening tool for genes specifically expressed during infection and for assessment of the relative virulence of mutants compared to wild-type E. coli =-=(32, 37, 38)-=-. Previously, we found that degS is essential for infection by the uropathogenic E. coli strain CFT073 (32). DegS is an indirect regulator of the alternate sigma factor, sigma E. DegS is an inner memb...

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... ages. The disease has been the subject of recent reviews (17, 20, 25, 26). Fully differentiated primary porcine alveolar macrophage (PAM) constitute the predominant cell target for viral replication =-=(4, 5)-=-. A published example of an immortalized PAM cell line, however, is not permissive for PRRSV infection (33). In addition, monocytes from peripheral blood are largely refractory to PRRSV infection. A p...

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...e MA-104 and its derivatives, such as MARC-145 (12). In MARC-145 cells, the internalization of the virus by endocytosis and subsequent uncoating in low-pH vesicles seem to mimic similar events in PAM =-=(14)-=-. However, MAbs that bind to porcine sialoadhesin fail to detect a homologous protein on the surfaces of MARC-145 cells (6, 34), suggesting that MARC-145 cells may use a divergent member of the same p...

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... The SR proteins comprise a large number of cell surface and soluble glycoproteins involved in the recognition of various ligands, including proteins, polyribonucleotides, polysaccharides, and lipids =-=(28)-=-. These proteins are therefore capable of binding a wide range of host molecules and pathogens. Sequencing revealed that SR genes could be further divided into different types based on the commonality...

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...rR is a repressor, so up-regulation of this transcript might be expected to decrease exoenzyme production (7). As outlined in the introduction, ExpI generates OHHL, which (according to current models =-=[7]-=-) should compensate for the repression brought about by VirR overexpression. The function of ExpR in strain SCRI1043 is not clear, although it is known to be convergently transcribed (and overlapping)...

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...t, CFT073 surA, shows minimal guinea pig red blood cell agglutination. This suggests diminished type 1 pilus expression. The association of SurA and type 1 pilus expression has recently been reported =-=(22)-=-. The type 1 pilus adhesin is important for E. coli persistence in the mouse bladder (2). Therefore, the nonagglutinating CFT073 surA mutant was not assayed for virulence in the mouse UTI model. Of th...

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...rane glycoprotein in the siglec family of sialic acid binding immunoglobulin-like lectins (32), since incubation of PAM cells with monoclonal antibody (MAb) to this polypeptide blocks PRRSV infection =-=(6, 34)-=-. Transfection of the nonpermissive PK-15 (porcine kidney) cell line with porcine sialoadhesin conferred the ability to internalize PRRSV particles, but there remained an apparent block at the uncoati...

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...anism (transcription of the rrn operons is tightly coupled to the growth rate) (10, 11). The stringent response has been implicated in controlling virulence factor production in a number of pathogens =-=(17, 20, 23, 31, 50)-=-. The SR is brought about as a result of the rapid accumulation of two small molecule effectors (known as alarmones), guanosine 3-diphosphate 5-triphosphate (pppGpp) and guanosine 35-bispyrophosph...

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... different family (27). FkpA also has chaperone activity. In Salmonella enterica serovar Typhimurium, an fkpA inactivation mutant shows decreased intracellular survival in a macrophage-like cell line =-=(16)-=-. Mutants of fkpA in Legionella pneumophila and Chlamydia trachomatis Downloaded from http://iai.asm.org/ on February 21, 2013 by PENN STATE UNIV4036 REDFORD AND WELCH INFECT. IMMUN. also show impair...

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...ly refractory to PRRSV infection. A previous study demonstrated that heparin sulfate serves as an attachment factor in the binding and internalization of PRRSV but is not required for internalization =-=(2)-=-. In PAM cells, initial binding of virions is mediated by interaction of the viral matrix protein with heparin sulfate proteoglycans (3). Virus particles are internalized in clathrin-coated vesicles a...

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...eir relative recovery. This is similar to results recently reported for a degQ mutant of S. enterica serovar Typhimurium which was not attenuated in mice either by oral or intraperitoneal inoculation =-=(13)-=-. Compensation by DegQ may be responsible for the relative resilience to stress seen here of the CFT073 degP mutant in vitro, but DegQ does not appear to make up for the loss of DegP function inside t...

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... and a live Salmonella enterica serovar Typhi vaccine strain with inactivation mutations in aroC and aroD was associated with no bacteremia in human volunteers when a third mutation in degP was added =-=(8, 24, 31, 41, 42)-=-. More recently, degP orthologs have been shown to contribute to the virulence of the gram-positive pathogens Streptococcus pyogenes and Streptococcus pneumoniae (18, 20). Interestingly, the uropathog...

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...essed by mixing suspensions of bacteria in PBS with 3% (vol/vol) guinea pig erythrocytes (Hema Resource & Supply, Aurora, OR) or human erythrocytes (University of Wisconsin Hospital) on a glass slide =-=(23)-=-. Mannose was added to agglutinated cells to a final concentration of 50 mM (29). Hemolysis was assessed by measurement of zones of clearing around colonies grown overnight on 5% sheep blood agar supp...

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...ed cells (10) and generally increases following stimulation and activation of macrophages. Human CD163 lacking the transmembrane domain sheds into the bloodstream and exhibits cytokine-like functions =-=(9, 19)-=-. One well-characterized function of CD163 involves scavenging of hemoglobin, which is mediated by endocytosis of haptoglobin-hemoglobin complexes (15). SusCD163v1 is missing the entire first and seco...

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...ologous protein on the surfaces of MARC-145 cells (6, 34), suggesting that MARC-145 cells may use a divergent member of the same protein family or a different receptor altogether. Recently Kim et al. =-=(13)-=- reported that simian vimentin is a part of the PRRSV receptor complex. The following data support this hypothesis: (i) vimentin is expressed on the surface of MARC-145; (ii) MAb to vimentin blocked P...

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... the open reading frame to an upstream ATG initiation codon as observed with susCD163v1. SusCD163v1 and susCD163v2 encode proteins with SRCR domain structures similar to that reported for human CD163 =-=(16)-=- (Fig. 3). Transient transfection of various cell lines with susCD163v1 confers permissivity to PRRSV. Because the nonpermissive BHK-21 (18), PK032495, NLFK, NLST, and RL cell lines (unpublished data)...

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...ed cells (10) and generally increases following stimulation and activation of macrophages. Human CD163 lacking the transmembrane domain sheds into the bloodstream and exhibits cytokine-like functions =-=(9, 19)-=-. One well-characterized function of CD163 involves scavenging of hemoglobin, which is mediated by endocytosis of haptoglobin-hemoglobin complexes (15). SusCD163v1 is missing the entire first and seco...

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...e been described. These two genotypes share only about 60% identity at the nucleotide level and appear to have evolved independently prior to appearing almost simultaneously in commercial swine herds =-=(8, 11, 26)-=-. Both European and North American PRRSV cause reproductive failure in sows and gilts (late-term abortions, stillbirths, and mummies), high mortality rates among nursery pigs, and respiratory disease ...

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...e been described. These two genotypes share only about 60% identity at the nucleotide level and appear to have evolved independently prior to appearing almost simultaneously in commercial swine herds =-=(8, 11, 26)-=-. Both European and North American PRRSV cause reproductive failure in sows and gilts (late-term abortions, stillbirths, and mummies), high mortality rates among nursery pigs, and respiratory disease ...

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...ws and gilts (late-term abortions, stillbirths, and mummies), high mortality rates among nursery pigs, and respiratory disease in swine of all ages. The disease has been the subject of recent reviews =-=(17, 20, 25, 26)-=-. Fully differentiated primary porcine alveolar macrophage (PAM) constitute the predominant cell target for viral replication (4, 5). A published example of an immortalized PAM cell line, however, is ...

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...(GenBank accession no. BC051281), and mouse (GenBank accession no. AF274883). Total cellular RNA was isolated from PAM, U937, MARC-145, Vero, dexamethasone-induced primary mouse peritoneal macrophage =-=(29)-=-, and DH82 cells using the RNeasy minikit (QIAGEN, Santa Clarita, CA). U937 cells were treated with phorbol 12–myristate 13–acetate (100 ng/ml) for 3 days before harvesting of total cellular RNA in or...

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... inocula for further propagation in either PAM cells or stable cell lines expressing CD163. Preparation of recombinant P129-GFP plasmid and generation of P129-GFP virus have been described previously =-=(35)-=-. Briefly, green fluorescent protein (GFP) was inserted between ORF1b and ORF2a/b and was expressed as an additional subgenomic RNA from the ORF2 transcriptional regulation sequence. A second copy of ...

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...The rotted matter was scooped out of each tuber and weighed. Rotted matter from seven tubers were measured for each time point. OHHL assay. OHHL was assayed in liquid cultures as previously described =-=(8)-=-. Briefly, 100-l aliquots of culture supernatant, diluted 1/100, were dispensed into black optiplate 96-well plates. The E. coli sensor strain was grown in LB containing 10 g/ml Tet to an optical de...

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... in this study are listed in Table 1. Unless otherwise indicated, the E. carotovora subsp. atroseptica strains were grown in baffled flasks at 25°C in Pel minimal medium (PMM) as described previously =-=(12)-=-. When required, antibiotics were used at the following concentrations: kanamycin (Kn) at 50 g/ml, ampicillin (Ap) at 100 g/ml, streptomycin (Sm) at 100 g/ml, tetracycline (Tet) at 10 g/ml, and ch...

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...brane domain and functioned as PRRSV receptors. Among these are alternative splice patterns that encode cytoplasmic tails in different reading frames, as has been reported for human CD163 transcripts =-=(24)-=-. Therefore, sequence variations within the cytoplasmic domain do not appear to determine PRRSV receptor function. Sánchez-Torres et al. (27) reported that CD163 is involved in the uptake of another ...

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...rane glycoprotein in the siglec family of sialic acid binding immunoglobulin-like lectins (32), since incubation of PAM cells with monoclonal antibody (MAb) to this polypeptide blocks PRRSV infection =-=(6, 34)-=-. Transfection of the nonpermissive PK-15 (porcine kidney) cell line with porcine sialoadhesin conferred the ability to internalize PRRSV particles, but there remained an apparent block at the uncoati...

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...anism (transcription of the rrn operons is tightly coupled to the growth rate) (10, 11). The stringent response has been implicated in controlling virulence factor production in a number of pathogens =-=(17, 20, 23, 31, 50)-=-. The SR is brought about as a result of the rapid accumulation of two small molecule effectors (known as alarmones), guanosine 3-diphosphate 5-triphosphate (pppGpp) and guanosine 35-bispyrophosph...

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...ws and gilts (late-term abortions, stillbirths, and mummies), high mortality rates among nursery pigs, and respiratory disease in swine of all ages. The disease has been the subject of recent reviews =-=(17, 20, 25, 26)-=-. Fully differentiated primary porcine alveolar macrophage (PAM) constitute the predominant cell target for viral replication (4, 5). A published example of an immortalized PAM cell line, however, is ...

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...a factor controls the expression of a wide range of genes during stationary-phase growth and during adaptation to certain environmental stresses. Mukherjee et al. (38, 39) and later, Andersson et al. =-=(2)-=-, independently examined how disruption of rpoS in Erwinia carotovora subsp. carotovora affects exoenzyme production and virulence. The former workers found that in E. carotovora subsp. carotovora str...

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... CFT073, and three candidates, yaeT, yfiO, and nlpB, could not be deleted. The latter two have subsequently been described as encoding lipoproteins, and yfiO was found to be essential in E. coli K-12 =-=(30)-=-. Also, in E. coli K-12 the products of these three genes, YaeT, YfiO, and NlpB, have been shown to associate, along with YfgL, to form a multicomponent complex important in the biogenesis of the oute...

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...ues. CD163, which was originally identified as a specific differentiation protein of macrophages and monocytes, is now known to be a group B SRCR protein containing eight cysteine residues per domain =-=(7)-=-. CD163 is a type 1 membrane protein. The extracellular domain of CD163 consists of nine SRCR tandem repeats, followed by a transmembrane segment and an intracellular cytoplasmic tail. Several variant...

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...ropism for kidney or bladder infection has been observed in uropathogenic E. coli (pyelonephritis versus cystitis strains), and this tropism has been associated with differences in adhesin expression =-=(35)-=-. It may be that the skp mutant has an adhesin expression defect. P pilus and type 1 fimbria expression did appear normal in agglutination testing of CFT073 skp prior to infection. However, in vivo ex...

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...e been described. These two genotypes share only about 60% identity at the nucleotide level and appear to have evolved independently prior to appearing almost simultaneously in commercial swine herds =-=(8, 11, 26)-=-. Both European and North American PRRSV cause reproductive failure in sows and gilts (late-term abortions, stillbirths, and mummies), high mortality rates among nursery pigs, and respiratory disease ...

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...nding monolayer. A similar (but typically shorter) period of adaptation is required when primary PRRSV field isolates are grown on MARC-145 cells, and this corresponds to genetic changes in the virus =-=(30)-=-. Some field isolates (e.g., P3412) could not be adapted to MARC-145 cells even after several attempts but could be adapted to growth on both FK-D4 and PK-9 cells. After adaptation to stable CD-163-ex...

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...lA is probably driven from a conserved putatives70 promoter located between nucleotides 950 and 980 of the rumA gene. In E. coli, RumA methylates U1939 on the 23S rRNA molecule (33). Agarwalla et al. =-=(1)-=- have suggested that U1939 may be strategically positioned for sensing the presence of uncharged tRNA in the A site, thereby potentially linking the function of RumA with that of RelA. To investigate ...

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... were selected on minimal agar medium (12) containing Cm. The colonies were then patched onto Cm-containing plates, pooled for DNA preparation, and screened, essentially as described by Holeva et al. =-=(27)-=-. Assay of secreted exoenzymes. Pel assays were performed using a method adapted from the procedure of Starr et al. (49). The rates of polygalacturonic acid breakdown were measured spectrophotometrica...

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...s) or intracelDownloaded from http://iai.asm.org/ on February 21, 2013 by PENN STATE UNIVVOL. 74, 2006 SIGMA E-REGULATED GENES IN E. COLI UROPATHOGENESIS 4037 lular survival (Haemophilus influenzae) =-=(9, 17, 46, 48)-=-. It may be that some sigma E-regulated genes important for virulence in other bacteria are less necessary in E. coli uropathogenesis. Furthermore, the in vitro phenotype of the degP mutant of E. coli...

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...cholate; Difco, Sparks, MD). Complement resistance was observed by survival of bacteria in 50% serum. Normal or heat-inactivated human serum was added to bacteria suspended in Veronal-buffered saline =-=(43)-=-. Tubes were incubated at 37°C, and quantitative cultures were performed at 0, 1, 2, and 3 h. Lipopolysaccharide was examined by silver staining. Bacteria were grown to mid- logarithmic phase, pellete...

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...sy minikit (QIAGEN, Santa Clarita, CA). U937 cells were treated with phorbol 12–myristate 13–acetate (100 ng/ml) for 3 days before harvesting of total cellular RNA in order to induce CD163 expression =-=(10)-=-. CD163 transcripts were amplified in a single-tube RT-PCR using the SuperScript One-Step RT-PCR System for Long Templates kit (Invitrogen) according to the manufacturer’s instructions. The primer pai...

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... frames, as has been reported for human CD163 transcripts (24). Therefore, sequence variations within the cytoplasmic domain do not appear to determine PRRSV receptor function. Sánchez-Torres et al. =-=(27)-=- reported that CD163 is involved in the uptake of another porcine virus, African swine fever virus (ASFV), and that expression of CD163 on porcine macrophages and/or monocytes correlates with suscepti...

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... nloaded from by both strains was similar (data not shown). However, not all virulence factors were affected by the relA mutation; siderophores play a central role in the virulence of Erwinia species =-=(15)-=-, yet production of these molecules by JWC7 was essentially normal (Fig. 4B). The relA mutation can be complemented by relA in trans. The ability of plasmid-borne relA to complement a range of virulen...

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... and a live Salmonella enterica serovar Typhi vaccine strain with inactivation mutations in aroC and aroD was associated with no bacteremia in human volunteers when a third mutation in degP was added =-=(8, 24, 31, 41, 42)-=-. More recently, degP orthologs have been shown to contribute to the virulence of the gram-positive pathogens Streptococcus pyogenes and Streptococcus pneumoniae (18, 20). Interestingly, the uropathog...

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...pond to nutrient downshift by producing (p)ppGpp. The relA-mediated SR to nutrient limitation has been shown to affect virulence and quorum-sensingrelated phenotypes in several gram-negative bacteria =-=(14, 16, 17, 23)-=-. PCR screening of the arrayed SCRI1043 mutant bank failed to identify any mutants carrying insertions in the relA gene. Therefore, to investigate whether relA affects virulence in E. carotovora subsp...