Search PubMed⌕ Search

Biomedical subjects

S I Miller

Publications and source records attributed to S I Miller.

At least 37 records · Page 2Linked to original sources

Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa.

Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and hexa-acylated lipid A structures under different environmental conditions. CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment. CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.

Acylation↗

Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo.

Systemically administered tumor-targeted Salmonella has been developed as an anticancer agent, although its use could be limited by the potential induction of tumor necrosis factor alpha (TNFalpha)-mediated septic shock stimulated by lipid A. Genetic modifications of tumor-targeting Salmonella that alter lipid A and increase safety must, however, retain the useful properties of this bacteria. We report here that disruption of the Salmonella msbB gene reduces TNFalpha induction and increases the LD50 of this pathogenic bacteria by 10,000-fold. Notwithstanding this enormous difference, Salmonella retains its tumor-targeting properties, exhibiting tumor accumulation ratios in excess of 1000:1 compared with normal tissues. Administration of this bacteria to mice bearing melanoma results in tumors that are less than 6% the size of tumors in untreated controls at day 18. Thus, the antitumor activity previously demonstrated using tumor-targeting Salmonella with normal lipid A is retained. Lipid modification of tumor-specific bacterial vectors provides a means for reducing septic shock and further suggests that the antitumor activity of these bacteria may be independent of TNFalpha.

Acyltransferases↗

Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems.

Salmonellae encode two virulence-associated type III secretion systems (TTSS) within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2). Two Salmonella typhimurium genes, sspH1 and sspH2, that encode proteins similar to the Shigella flexneri and Yersinia species TTSS substrates, IpaH and YopM, were identified. SspH1 and SspH2 are proteins containing leucine-rich repeats that are differentially targeted to the SPI1 and SPI2 TTSS. sspH2 transcription was induced within RAW264.7 macrophages, and was dependent upon the SPI2-encoded regulator ssrA/ssrB. In contrast, sspH1 transcription is independent of SPI2, and is not induced after bacterial phagocytosis by eukaryotic cells. Infection of eukaryotic cells with strains expressing a SspH2-CyaA fusion protein resulted in SPI2 TTSS-dependent cAMP increases. In contrast, SspH1-CyaA-mediated cAMP increases were both SPI1 and SPI2 TTSS dependent. sspH2-like sequences were found in most Salmonella serotypes examined, whereas sspH1 was detected in only one S. typhimurium isolate, indicating that the copy number of sspH genes can be variable within Salmonella serotypes. S. typhimurium deleted for both sspH1 and sspH2 was not able to cause a lethal infection in calves, indicating that these genes participate in S. typhimurium virulence for animals.

Animals↗

How intracellular bacteria survive: surface modifications that promote resistance to host innate immune responses.

Bacterial pathogens regulate the expression of virulence factors in response to environmental signals. In the case of salmonellae, many virulence factors are regulated via PhoP/PhoQ, a two-component signal transduction system that is repressed by magnesium and calcium in vitro. PhoP/PhoQ-activated genes promote intracellular survival within macrophages, whereas PhoP-repressed genes promote entrance into epithelial cells and macrophages by macropinocytosis and stimulate epithelial cell cytokine production. PhoP-activated genes include those that alter the cell envelope through structural alterations of lipopolysaccharide and lipid A, the bioactive component of lipopolysaccharide. PhoP-activated changes in the bacterial envelope likely promote intracellular survival by increasing resistance to host cationic antimicrobial peptides and decreasing host cell cytokine production.

Animals↗

Identification of a putative Salmonella enterica serotype typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis.

The genetic basis for the host adaptation of Salmonella serotypes is currently unknown. We have explored a new strategy to identify Salmonella enterica serotype Typhimurium (S. typhimurium) genes involved in host adaptation, by comparing the virulence of 260 randomly generated signature-tagged mutants during the oral infection of mice and calves. This screen identified four mutants, which were defective for colonization of only one of the two host species tested. One mutant, which only displayed a colonization defect during the infection of mice, was further characterized. During competitive infection experiments performed with the S. typhimurium wild type, the mutant was defective for colonization of murine Peyer's patches but colonized bovine Peyer's patches at the wild-type level. No difference in virulence between wild type and mutant was observed when calves were infected orally with 10(10) CFU/animal. In contrast, the mutant possessed a sixfold increase in 50% lethal morbidity dose when mice were infected orally. The transposon in this mutant was inserted in a 2.9-kb pathogenicity islet, which is located between uvrB and yphK on the S. typhimurium chromosome. This pathogenicity islet contained a single gene, termed slrP, with homology to ipaH of Shigella flexneri and yopM of Yersinia pestis. These data show that comparative screening of signature-tagged mutants in two animal species can be used for scanning the S. typhimurium genome for genes involved in host adaptation.

Amino Acid Sequence↗

A HilA-independent pathway to Salmonella typhimurium invasion gene transcription.

Salmonella typhimurium invasion of nonphagocytic cells requires the expression of a type III secretion system (TTSS) encoded within Salmonella pathogenicity island 1 (SPI1). TTSS gene transcription is activated in response to environmental signals and requires transcriptional regulators encoded within (HilA) and outside (SirA) SPI1. Two unique loci, sirB and sirC, which contribute to SPI1 gene transcription were defined. sirC is an SPI1-encoded transcription factor of the AraC family that contributes to the invasive phenotype. sirB is required for maximal expression of sirC and consists of two open reading frames located near kdsA, a gene involved in lipopolysaccharide biosynthesis. sirC expression, unlike expression of other SPI1 genes, does not require HilA. Overexpression of sirC or sirA restores expression of a subset of SPI1 genes, including invF and sspC, in the absence of HilA. These data define roles for SirC and SirA as part of a HilA-independent pathway to SPI1 gene expression. We postulate that HilA-independent activation of inv expression is important for efficient assembly and function of the SPI1 TTSS.

Bacterial Proteins↗

Genomic subtraction identifies Salmonella typhimurium prophages, F-related plasmid sequences, and a novel fimbrial operon, stf, which are absent in Salmonella typhi.

Salmonella typhimurium causes systemic and fatal infection in inbred mice, while the related serotype Salmonella typhi is avirulent for mammals other than humans. In order to identify genes from the virulent strain S. typhimurium ATCC 14028 that are absent in S. typhi Ty2, and therefore might be involved in S. typhimurium mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon, stfACDEFG, located at centisome 5 of the S. typhimurium chromosome, which is absent in S. typhi, Salmonella arizonae, and Salmonella bongori but was detected in several other Salmonella serotypes. The fimbrial genes represent a genomic insertion in S. typhimurium compared to the respective region between fhuB and hemL in Escherichia coli K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the S. typhimurium virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57.

Animals↗

Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides.

The Salmonellae PhoP-PhoQ virulence regulators induce resistance to host cationic antimicrobial peptides (CAMP) after infection of vertebrate tissues, and Mg2+ or Ca2+ limitation. The PhoP-PhoQ activated gene, pagP, was identified as important to inducible CAMP resistance and increased acylation of lipid A, the major component of the outer leaflet of the outer membrane. pagP mutants demonstrated increased outer membrane permeability in response to CAMP, supporting the hypothesis that increased lipid A acylation is a CAMP resistance mechanism. Similarly, in response to Mg2+ limited growth, other enteric Gram-negative bacteria demonstrated increased lipid A acylation. Compounds that inhibit the ability to increase lipid A acylation may have utility as new antimicrobial agents.

Acylation↗

Identification of PhoP-PhoQ activated genes within a duplicated region of the Salmonella typhimurium chromosome.

Salmonellae virulence requires the PhoP-PhoQ two-component regulatory system. PhoP-PhoQ activate the transcription of genes following phagocytosis by macrophages which are necessary for survival within the phagosome environment. Thirteen previously undefined PhoP-activated gene fusions generated by MudJ and TnphoA (pag A, and E-P, respectively) were cloned and sequenced. Most pag products show no similarity to proteins in the database, while others are predicted to encode: a UDP-glucose dehydrogenase (pagA); a protein with similarity to the product of an E. coli aluminium-induced gene (pagH); a protein encoded within a Salmonella-unique region adjacent to the sinR gene (pagN); a protein similar to a product of the Yersinia virulence plasmid (pagO); and a protein with similarity to CrcA which is necessary for resistance of E. coli to camphor (pagP). Of the pag characterized, only pagK, M and O were closely linked. pagJ and pagK were shown to be unlinked but nearly identical in DNA sequence, as each was located within a 1.6 kb DNA duplication. The translations of sequences surrounding pagJ and pagK show similarity to proteins from extrachromosomal elements as well as those involved in DNA transposition and rearrangement, suggesting that this region may have been or is a mobile element. The transcriptional start sites of pagK, M, and J were determined; however, comparison to other known pag gene promoters failed to reveal a consensus sequence for PhoP-regulated activation. DNA sequences hybridizing to a Salmonella typhimurium pagK specific probe were found in S. enteritidis but absent in other Salmonella serotypes and Enterobacteriaceae tested, suggesting that these genes are specific for broad host range Salmonellae that cause diarrhoea in humans. Cumulatively, these data further demonstrate: (1) that PhoP-PhoQ is a global regulator of the production of diverse envelope or secreted proteins; (2) that PhoP-PhoQ regulate the production of proteins of redundant function; and (3) that pag are often located in regions of horizontally acquired DNA that are absent in other Enterobacteriaceae.

Artificial Gene Fusion↗

Characteristics of short-stay admissions to a psychiatric inpatient service.

Since the rapid expansion of managed care coupled with dramatic reductions in lengths of inpatient stay, there is widespread concern that the emphasis on cost containment is eclipsing attention to patient care. The present study was undertaken to evaluate speculations that the majority of short-stay (less than 48 hours) admissions to a psychiatric inpatient service at a large teaching hospital in the midwestern United States consisted of public pay patients who were rapidly transferred to area state hospitals. Using two cases mix measures, severity of illness and changes in acuity and clinical outcomes of a sample of short-stay (n = 77) and longer stay (n = 145) admissions were compared. Short-stay admissions, although similar to longer stay patients in terms of demographics, Axis I diagnosis, payer status, and appropriateness of admission, are clinically distinct. The use of nonhospital alternatives in treating a subsample of suicidal patients and the implications for improved mental health services delivery are discussed.

Adult↗

Immunosuppression and nitric oxide production induced by parenteral live Salmonella vaccines do not correlate with protective capacity: a phoP::Tn10 mutant does not suppress but does protect.

Previous work from our laboratory showed that an aroA mutant strain of S. typhimurium, SL3235, induces profound immunosuppression 7 days post-parenteral inoculation, and that the suppression is mediated by nitric oxide. Suppression was measured by the capacity of spleen cells to mount a primary in vitro plaque-forming cell response to sheep red blood cells in Mishell-Dutton cultures. In the present studies, the capacity of a panel of strains of attenuated Salmonella with various genetic lesions was tested. Most of the strains were S. typhimurium, but several were S. dublin. It was found that a variety of Salmonella strains induced suppression, demonstrating that suppressive capacity is not unique to SL3235 or to S. typhimurium. A strong correlation was obtained between the log10 of the microbial burden (cfu spleen-1) on the seventh day post-vaccine inoculation and the degree of immunosuppression. Strains that gave high spleen counts gave greater suppression. Microbial burden also correlated with the size of the spleen and the amount of nitrite produced by spleen-cell cultures, a measure of nitric oxide. Finally, the degree of immunosuppression was found to be linearly related to the log10 of the amount of nitrite produced. The capacity of the various strains of Salmonella to protect against challenge with virulent S. typhimurium, strain W118-2, was also tested. No correlation was found between suppressive and protective capacities of the various strains. Two strains suppressed, but did not protect. While most strains that protected grew or persisted in vivo, a phoP::Tn10 mutant of S. typhimurium did not grow or persist; this phoP mutant did not cause immunosuppression, but gave 100% protection against challenge with wild type S. typhimurium, suggesting that such mutants have advantageous properties as live vaccines.

Animals↗

PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance.

Antimicrobial peptides are distributed throughout the animal kingdom and are a key component of innate immunity. Salmonella typhimurium regulates mechanisms of resistance to cationic antimicrobial peptides through the two-component systems PhoP-PhoQ and PmrA-PmrB. Polymyxin resistance is encoded by the PmrA-PmrB regulon, whose products modify the lipopolysaccharide (LPS) core and lipid A regions with ethanolamine and add aminoarabinose to the 4' phosphate of lipid A. Two PmrA-PmrB-regulated S. typhimurium loci (pmrE and pmrF) have been identified that are necessary for resistance to polymyxin and for the addition of aminoarabinose to lipid A. One locus, pmrE, contains a single gene previously identified as pagA (or ugd) that is predicted to encode a UDP-glucose dehydrogenase. The second locus, pmrF, is the second gene of a putative operon predicted to encode seven proteins, some with similarity to glycosyltransferases and other complex carbohydrate biosynthetic enzymes. Genes immediately flanking this putative operon are also regulated by PmrA-PmrB and/or have been associated with S. typhimurium polymyxin resistance. This work represents the first identification of non-regulatory genes necessary for modification of lipid A and subsequent antimicrobial peptide resistance, and provides support for the hypothesis that lipid A aminoarabinose modification promotes resistance to cationic antimicrobial peptides.

Anti-Bacterial Agents↗

Cell-contact-stimulated formation of filamentous appendages by Salmonella typhimurium does not depend on the type III secretion system encoded by Salmonella pathogenicity island 1.

The formation of filamentous appendages on Salmonella typhimurium has been implicated in the triggering of bacterial entry into host cells (C. C. Ginocchio, S. B. Olmsted, C. L. Wells, and J. E. Galán, Cell 76:717-724, 1994). We have examined the roles of cell contact and Salmonella pathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of S. typhimurium strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine. Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type S. typhimurium bacteria adhering to both cultured epithelial cells and to murine Peyer's patch follicle-associated epithelia. Wild-type S. typhimurium adhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages. Invasion-deficient S. typhimurium strains carrying mutations in components of SPI1 (invA, invG, sspC, and prgH) exhibited filamentous appendages similar to those on wild-type S. typhimurium when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion. When adhering to cell-free culture inserts, an S. typhimurium invG mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia. Our data on S. typhimurium strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages. However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.

Animals↗

Hearing discordant voices: some notes on using Bayes's theorem in interpretive inquiry.

The focus of these brief comments is to argue that Bayes's theorem may have conceptual and pedagogical applications for qualitative analysis. We suggest that the theorem is a salient alternative voice for the development of sensitizing concepts. It is also proposed that the theorem is a useful heuristic for raising deep-structure issues such as the nature of qualitative evidence and how such evidence becomes confirming for a given claim.

Bayes Theorem↗

Psychiatric hospital outcomes of dual diagnosis patients under managed care.

The authors compared patterns of psychiatric hospitalization utilization and outcomes between persons with and without co-existing substance-related disorders in a managed care environment by means of a prospective follow-along study of persons hospitalized for psychiatric reasons under the auspices of a large regional managed care firm. Forty-two psychiatric inpatients with comorbid substance disorders and 121 inpatients without coexisting substance disorders were compared across measures of service use and psychiatric acuity. Readmission to the hospital was assessed at 30 days and after 6 months. Patients with coexisting substance disorders spent fewer days in the hospital, but were rehospitalized at a higher rate both within 30 days and (significantly) after 6 months. These results suggest that the revolving-door pattern of service utilization is also present in managed care environments.

Adolescent↗

Regulation of lipid A modifications by Salmonella typhimurium virulence genes phoP-phoQ.

Bacterial pathogenesis requires proteins that sense host microenvironments and respond by regulating virulence gene transcription. For Salmonellae, one such regulatory system is PhoP-PhoQ, which regulates genes required for intracellular survival and resistance to cationic peptides. Analysis by mass spectrometry revealed that Salmonella typhimurium PhoP-PhoQ regulated structural modifications of lipid A, the host signaling portion of lipopolysaccharide (LPS), by the addition of aminoarabinose and 2-hydroxymyristate. Structurally modified lipid A altered LPS-mediated expression of the adhesion molecule E-selectin by endothelial cells and tumor necrosis factor-alpha expression by adherent monocytes. Thus, altered responses to environmentally induced lipid A structural modifications may represent a mechanism for bacteria to gain advantage within host tissues.

Acylation↗