Biomedical subjects

V L Miller

Publications and source records attributed to V L Miller.

At least 37 records · Page 2Linked to original sources

PubMed2000

Identification and characterization of Yersinia enterocolitica genes induced during systemic infection.

Yersinia enterocolitica is one of three pathogenic Yersinia species that share a tropism for lymphoid tissues. However, infection of an immunocompromised host is likely to result in a systemic infection, which is often fatal. Little is known about the bacterial proteins needed to establish such an infection. The genes that encode these virulence factors are likely to be active only during systemic infection. A library of random cat fusions was used to inoculate BALB/c mice. Fusions expressed during a systemic infection were enriched by the administration of chloramphenicol-succinate. Y. enterocolitica isolates recovered from the mice were tested for chloramphenicol resistance in vitro. Fusions that were inactive in vitro were analyzed further and found to represent 31 allelic groups. Each was given a sif (for systemic infection factor) designation. Based on homology to known proteins, the sif genes are likely to encode proteins important for general physiology, transcription regulation, and other functions. During systemic infections, 13 of the sif-cat fusions were able to outcompete the wild type in the presence of chloramphenicol-succinate, confirming that the fusions were active. The in vitro expression of several sif genes was determined, showing modest changes in response to various growth conditions. A mutation in sif15, which encodes a putative outer membrane protein, caused attenuation during systemic infection but not during colonization of the Peyer's patches. Comparisons between the Y. enterocolitica sif genes and the previously identified hre genes imply that very different groups of genes are active during a systemic infection and during colonization of the Peyer's patches.

Animals↗

PubMed2000

Motility is required to initiate host cell invasion by Yersinia enterocolitica.

Invasin-mediated invasion of host cells by the pathogen Yersinia enterocolitica was shown to be affected by flagellar-dependent motility. Motility appears to be required to ensure the bacterium migrates to and contacts the host cell. Nonmotile strains of Y. enterocolitica were less invasive than motile strains, but the reduction in invasion could be overcome by artificially bringing the bacteria into host cell contact by centrifugation. Mutations in known regulatory genes of the flagellar regulon, flhDC and fliA, resulted in less inv expression but did not have a significant effect on invasin levels. However, invasin levels were reduced for strains that harbored flhDC on a multicopy plasmid, apparently as a result of increased proteolysis of invasin.

Adhesins, Bacterial↗

PubMed2000

Yersinia enterocolitica ClpB affects levels of invasin and motility.

Expression of the Yersinia enterocolitica inv gene is dependent on growth phase and temperature. inv is maximally expressed at 23 degrees C in late-exponential- to early-stationary-phase cultures. We previously reported the isolation of a Y. enterocolitica mutant (JB1A8v) that shows a decrease in invasin levels yet is hypermotile when grown at 23 degrees C. JB1A8v has a transposon insertion within uvrC. Described here is the isolation and characterization of a clone that suppresses these mutant phenotypes of the uvrC mutant JB1A8v. This suppressing clone encodes ClpB (a Clp ATPase homologue). The Y. enterocolitica ClpB homologue is 30 to 40% identical to the ClpB proteins from various bacteria but is 80% identical to one of the two ClpB homologues of Yersinia pestis. A clpB::TnMax2 insertion mutant (JB69Qv) was constructed and determined to be deficient in invasin production and nonmotile when grown at 23 degrees C. Analysis of inv and fleB (flagellin gene) transcript levels in JB69Qv suggested that ClpB has both transcriptional and posttranscriptional effects. In contrast, a clpB null mutant, BY1v, had no effect on invasin levels or motility. A model accounting for these observations is presented.

Adhesins, Bacterial↗

PubMed2000

The Yersinia enterocolitica phospholipase gene yplA is part of the flagellar regulon.

Yersinia enterocolitica yplA encodes a phospholipase required for virulence. Virulence genes are often regulated in response to environmental signals; therefore, yplA expression was examined using a yplA::lacZY transcriptional fusion. Maximal yplA expression occurred between pH 6.5 and pH 7.5 and was induced in the mid-logarithmic growth phase. Potential Fnr, cyclic AMP (cAMP)-cAMP receptor protein (Crp), and sigma(F) regulatory sites were identified in the nucleotide sequence. Reduction of yplA expression by aeration, addition of glucose and sucrose, and application of high temperature and salt is consistent with Fnr-, cAMP-Crp-, and sigma(F)-mediated regulation, respectively. Expression of yplA was reduced in flhDC and fliA null strains, indicating that yplA is part of the flagellar regulon.

Bacterial Proteins↗

PubMed1999

A new pathway for the secretion of virulence factors by bacteria: the flagellar export apparatus functions as a protein-secretion system.

Biogenesis of the flagellum, a motive organelle of many bacterial species, is best understood for members of the Enterobacteriaceae. The flagellum is a heterooligomeric structure that protrudes from the surface of the cell. Its assembly initially involves the synthesis of a dedicated protein export apparatus that subsequently transports other flagellar proteins by a type III mechanism from the cytoplasm to the outer surface of the cell, where oligomerization occurs. In this study, the flagellum export apparatus was shown to function also as a secretion system for the transport of several extracellular proteins in the pathogenic bacterium Yersinia enterocolitica. One of the proteins exported by the flagellar secretion system was the virulence-associated phospholipase, YplA. These results suggest type III protein secretion by the flagellar system may be a general mechanism for the transport of proteins that influence bacterial-host interactions.

Anti-Bacterial Agents↗

PubMed1999

Bacterial phospholipases and pathogenesis.

Phospholipases are produced from a diverse group of bacterial pathogens causing very different diseases. In some cases, secreted phospholipases appear to be the major cause of pathophysiological effects. Yet in other cases, phospholipases are key virulence factors, contributing to bacterial survival or dissemination without causing tissue destruction. Perhaps the most intriguing aspect of phospholipases as virulence factors is their potential to interfere with cellular signaling cascades and to modulate the host immune response.

Animals↗

PubMed1999

Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis.

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature-tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild-type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O-antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.

Animals↗

PubMed1999

Molecular basis of the interaction of Salmonella with the intestinal mucosa.

Salmonella is one of the most extensively characterized bacterial pathogens and is a leading cause of bacterial gastroenteritis. Despite this, we are only just beginning to understand at a molecular level how Salmonella interacts with its mammalian hosts to cause disease. Studies during the past decade on the genetic basis of virulence of Salmonella have significantly advanced our understanding of the molecular basis of the host-pathogen interaction, yet many questions remain. In this review, we focus on the interaction of enterocolitis-causing salmonellae with the intestinal mucosa, since this is the initiating step for most infections caused by Salmonella. Animal and in vitro cell culture models for the interaction of these bacteria with the intestinal epithelium are reviewed, along with the bacterial genes that are thought to affect this interaction. Lastly, recent studies on the response of epithelial cells to Salmonella infection and how this might promote diarrhea are discussed.

Animals↗

PubMed1999

InvF is required for expression of genes encoding proteins secreted by the SPI1 type III secretion apparatus in Salmonella typhimurium.

The expression of genes encoding proteins secreted by the SPI1 (Salmonella pathogenicity island) type III secretion apparatus is known to require the transcriptional activators SirA and HilA. However, neither SirA nor HilA is believed to directly activate the promoters of these genes. invF, the first gene of the inv-spa gene cluster, is predicted to encode an AraC-type transcriptional activator and is required for invasion into cultured epithelial cells. However, the genes which are regulated by InvF have not been identified. In this work, an in-frame deletion in invF was constructed and tested for the expression of Phi(sigD-lacZYA), sipC::Tn5lacZY, and a plasmid-encoded Phi(sicA-lacZYA). SigD (Salmonella invasion gene) is a secreted protein required for the efficient invasion of Salmonella typhimurium into cultured eucaryotic cells. sicA (Salmonella invasion chaperone) is the first gene of a putative operon encoding the Sip/Ssp (Salmonella invasion/Salmonella secreted proteins) invasion proteins secreted by the SPI1 type III export apparatus. invF was required for the expression of the sigD, sicA, and sipC fusions. This is the first demonstration that there is a functional promoter in the intergenic sequence between spaS and sicA. In addition, several proteins were either absent from or found in reduced amounts in the culture supernatants of the invF mutant. Therefore, invF is required for the optimal expression of several genes encoding SPI1-secreted proteins. Genetic evidence is also presented suggesting there is HilA-dependent readthrough transcription from the invF promoter at least through sipC.

AraC Transcription Factor↗

PubMed1999

The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility.

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen Yersinia enterocolitica can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as flgEF, flgMN, flhBA, and fliA, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by Y. enterocolitica to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon, flhDC, was cloned. Mutations within flhDC completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of flhDC indicated that it is required for the production of flagella. When flhDC was expressed from an inducible ptac promoter, flagellin production was shown to be dependent on levels of flhDC expression. Phenotypically, induction of the ptac-flhDC fusion also corresponded to increased levels of both swimming and swarming motility.

Bacterial Proteins↗

PubMed1998

An analysis of program and family costs of case managed care for technology-dependent infants with bronchopulmonary dysplasia.

Caring for technology-dependent infants with bronchopulmonary dysplasia requires a wide range and intense level of services. Case management programs can offer comprehensive services to patients with complex needs. The Neonatal Pulmonary Program at Tulane University Medical Center is a case management program for infants with chronic pulmonary problems. The purpose of this study was to describe the costs of providing care for technology-dependent infants with bronchopulmonary dysplasia and to include the direct and indirect costs to families as well as the Program costs. The study population included 89 infants enrolled in the program from September 1987 through February 1992. Outpatient, inpatient, and professional staff costs were derived from hospital and clinic billing information; all other costs were determined through family interviews. Total costs for outpatient services were $59,627 (89), professional team members time $185,539 (89); inpatient services $1,144,930 (89), professional costs for inpatient services $88,946; direct health costs $32,543 (37) for home health care, equipment, medications, special diets; direct non-health costs $30,670 (37) for transportation, meals, child care, lodging during clinic visits and rehospitalizations and household expenses to accommodate equipment needs; and indirect costs $26,173 (37) for missed work days and employment changes.

Bronchopulmonary Dysplasia↗

PubMed1998

Occupational upper extremity disorders in the federal workforce. Prevalence, health care expenditures, and patterns of work disability.

Upper extremity disorders (UEDs) account for a significant number of work-related illnesses in the US workforce. Little information exists on the distribution of UEDs, their associated health care and indemnity costs, or patterns of work disability. The study presented is an analysis of upper extremity claims within the federal workforce. In this study, the universe consisted of all claims accepted by the US Department of Labor, Office of Workers' Compensation Programs (OWCP), from October 1, 1993, through September 30, 1994. A total of 185,927 claims of notices of injury were processed during the study period, and of these, 8,147 or 4.4% had an UED diagnosis coded according to the International Classification of Diseases, Clinical Modification (ICD-9-CM). 5,844 claims involved a single UED diagnosis and were the only claims field by these employees between October 1, 1990, and September 30, 1994. These single claims with single diagnoses comprised the sample for further analysis. Mononeuritis and enthesopathies of the upper limb were the most common diagnoses, accounting for 43% and 31% of the claims, respectively. Women had a higher proportion of carpal tunnel syndrome, "unspecified" mononeuritis, and "unspecified" enthesopathies. The majority of claimants for both the mononeuritis- and enthesopathy-related diagnoses were between 31 and 50 years of age, received only health care benefits, and did not incur wage loss. Health care costs for mononeuritis and enthesopathy claims were $12,228,755 (M = $2,849). Carpal tunnel syndrome (CTS) and enthesopathy of the elbow were the most costly diagnoses, accounting for 57% and 16% of the total, respectively. Surgical services represented the highest expenditures in CTS claims. Physical therapy accounted for the majority of health care costs for enthesopathy cases. The mean number of workdays lost for CTS and enthesopathy claims were 84 and 79, and the average indemnity costs were $4,941 and $4,477, respectively. These findings indicate that while UEDs represent a relatively small percentage of all workers' compensation cases, the health care and indemnity costs are considerable. Also mean duration and pattern of work disability revealed that these disorders can result in chronic work disability similar to that observed in low back pain. The results highlight the need to determine whether interventions that account for the majority of costs significantly impact long-term outcomes. There is also a need to identify risk factors for prolonged disability in those who experience problems with delayed recovery.

Adolescent↗

PubMed1998

Phospholipase A of Yersinia enterocolitica contributes to pathogenesis in a mouse model.

Some isolates of Yersinia enterocolitica exhibit phospholipase activity, which has been linked to lecithin-dependent hemolysis (M. Tsubokura, K. Otsoki, I. Shimohira, and H. Yamamoto, Infect. Immun. 25:939-942, 1979). A gene encoding Y. enterocolitica phospholipase was identified, and analysis of the nucleotide sequence revealed two tandemly transcribed open reading frames. The first, yplA, has 74% identity and 85% similarity to the phospholipase A found in Serratia liquefaciens. Though the other, yplB, was less similar to the downstream accessory protein found in S. liquefaciens, the organization in both species is similar. Subsequently, a yplA-null Y. enterocolitica strain, YEDS10, was constructed and demonstrated to be phospholipase negative by plate and spectrophotometric assays. To ascertain whether the phospholipase has a role in pathogenesis, YEDS10 was tested in the mouse model. In experiments with perorally infected BALB/c mice, fewer YEDS10 organisms were recovered from the mesenteric lymph nodes and Peyer's patches (PP) than the parental strain at 3 or 5 days postinfection. Furthermore, bowel tissue and PP infected with YEDS10 appeared to be less inflamed than those infected with the parental strain. When extremely high doses of both the parental and YEDS10 strains were given, similar numbers of viable bacteria were recovered from the PP and mesenteric lymph nodes on day 3. However, the numbers of foci and the extent of inflammation and necrosis within them were noticeably less for YEDS10 compared to the parental strain. Together these findings suggest that Y. enterocolitica produces a phospholipase A which has a role in pathogenesis.

Amino Acid Sequence↗

PubMed1998

Expression of invasin and motility are coordinately regulated in Yersinia enterocolitica.

The Yersinia enterocolitica inv gene encodes the primary invasion factor invasin, which has been previously shown to be critical in the initial stages of infection. The expression of inv is influenced by growth phase and temperature and is maximal during late exponential-early stationary phase at 23 degrees C. In addition, motility of Y. enterocolitica is regulated by temperature. Y. enterocolitica cells are motile when grown at lower temperatures (30 degrees C or below), while bacteria grown at 37 degrees C are nonmotile. This study was initiated to determine the molecular basis for the temperature regulation of inv expression. Two mutants were isolated that both showed a significant decrease in invasin expression but are hypermotile when grown at 23 degrees C. The first mutant (JB1A8v) was a result of a random mTn5Km insertion into the uvrC gene. The uvrC mutant JB1A8v demonstrated a significant decrease in inv and an increase in fleB (encodes flagellin) expression. These results suggest that expression of inv and flagellin genes is coordinated at the level of transcription. The second regulatory mutant, JB16v, was a result of a targeted insertion into a locus similar to sspA which in E. coli encodes a stationary-phase regulator. The E. coli sspA gene was cloned and assayed for complementation in both of the regulatory mutants. It was determined that E. coli sspA restored invasin expression in both the uvrC mutant and the sspA mutant. In addition, the complementing clone decreased flagellin levels in these mutants.

Adhesins, Bacterial↗

PubMed1998

Identification of a novel Salmonella invasion locus homologous to Shigella ipgDE.

Genes essential for Salmonella typhimurium invasion have been localized to Salmonella pathogenicity island 1 (SPI1) on the chromosome. However, it is clear that other genes are required for the invasion process. Mutations that abolish the SPI1 invasion type III secretion system do not significantly reduce invasion into Chinese hamster ovary tissue culture cells. Two invasion defective mutants were isolated by screening 2,500 Tn10dTc insertion mutants of S. typhimurium in the tissue culture invasion assay. One of the invasion mutants, SVM167, has an insertion between centisomes 24.5 and 25.5 in an operon homologous to the ipgDEF operon of the Shigella flexneri and Shigella sonnei virulence plasmid. A second mutant, SVM168, has an insertion in an IS3-type element with homology to the Salmonella enteritidis IS1351 element and Yersinia enterocolitica IS1400 element from a high-pathogenicity island. Further characterization of SVM167 showed that culture supernatants from this mutant lack a previously uncharacterized protein that is also missing from culture supernatants of a SPI1 mutant, suggesting it can be secreted by the SPI1 type III secretion system. In addition, transcription of this operon, sigDE (Salmonella invasion gene), is dependent on the presence of sirA, an activator of hilA expression. HilA activates transcription of several of the SPI1 genes but does not appear to have a major role in activation of transcription from the sigDE promoter.

Amino Acid Sequence↗

PubMed1997

Identification of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis.

Pathogenic species of the genus Yersinia have a marked tropism for lymphoid tissue during the early stages of infection. Bacterial survival at this site determines whether the disease is localized or progresses systemically, leading to a high rate of mortality. Several plasmid-encoded virulence genes are known to be required for survival and pathogenesis, but the contribution of chromosomal genes has been largely unexplored. This study represents the first intensive effort to characterize and determine the function of Yersinia chromosomal genes expressed in lymphoid tissue after intragastric infection. Strains harbouring cat fusions expressed in the host were isolated from Peyer's patch tissue of mice intragastrically infected and treated with chloramphenicol (Cm); genes identified in this manner were designated hre for host responsive element. The hre::cat strains that were Cm resistant in vivo (in mouse tissue) and Cm sensitive in vitro (on laboratory media at 26 degrees C) were identified and shown to consist of 61 different allelic groups. The hre::cat fusions from 48 of the allelic groups were cloned and characterized by DNA sequence analysis. The results identified genes necessary for iron acquisition, protection from environmental stresses, biosynthesis of cell envelope components and other diverse metabolic activities. However, the DNA sequence of many clones had no homology to other known genes. Insertion mutations were constructed for four hre genes and the resulting Y. enterocolitica mutants were tested in the mouse model for effects on pathogenesis. All of the mutant strains were affected for virulence when assayed for survival in host tissues and LD50 analysis.

Animals↗

PubMed1997

Reevaluation of the virulence phenotype of the inv yadA double mutants of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis and Yersinia enterocolitica are closely related human pathogens causing gastroenteritis. Invasin and YadA are two of the most extensively studied virulence factors of the Yersinia genus. Invasin is the primary invasion factor encoded by the inv gene on the chromosome and is required for the penetration of the epithelial cells. YadA is encoded by the yadA gene on the 70-kb virulence plasmid and has multiple functions. Previous studies indicate that an inv yadA double mutant of Y. enterocolitica is avirulent while an inv yadA mutant of Y. pseudotuberculosis is hypervirulent. In this study, we investigated this unexpected difference. New constructs of the inv yadA mutants of Y. pseudotuberculosis were made and tested in mice. These new constructs were not hypervirulent; rather, they maintained the same virulence as the wild-type strain. Further examination of the inv mutant used for the previous study revealed that it carries an aberrant inv phenotype and has an altered outer membrane profile and an altered colony morphology. Therefore, the mutants used previously were not isogenic to the parental wild-type strain, which may in part account for the difference in the results obtained.

Adhesins, Bacterial↗

PubMed1996

Temperature-dependent regulation of Yersinia enterocolitica Class III flagellar genes.

Temperature is a key environmental cue for Yersinia enterocolitica as well as for the two other closely related pathogens, Yersinia pestis and Yersinia pseudotuberculosis. Between the range of 30 degrees C and 37 degrees C, Y. enterocolitica phase-varies between motility and plasmid-encoded virulence gene expression. To determine how temperature regulates Y. enterocolitica motility, we have been dissecting the flagellar regulatory hierarchy to determine at which level motility is blocked by elevated temperature (37 degrees C). Here we report the cloning, DNA sequences, and regulation of the two main regulators of Class III flagellar genes, fliA (sigma F) and flgM (anti-sigma F), and a third gene, flgN, which we show is required for filament assembly. Identification of the Y. enterocolitica fliA and flgM genes was accomplished by functional complementation of both S. typhimurium and Y. enterocolitica mutations and by DNA sequence analysis. The Y. enterocolitica fliA gene, encoding the flagellar-specific sigma-factor, sigma F, maps immediately downstream of the three flagellin structural genes. The flgM and flgN genes, encoding anti-sigma F and a gene product required for filament assembly, respectively, map downstream of the invasin (inv) gene but are transcribed in the opposite (convergent) direction. By using Northern blot analyses we show that transcription of both fliA and flgM is immediately arrested when cells are exposed to 37 degrees C, coincident with the timing of virulence gene induction. Unlike S. typhimurium flgM mutants, Y. enterocolitica flgM mutants are fully virulent.

Amino Acid Sequence↗