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Due to its attractive mechanical properties and biocompatibility, poly(dimethyl) siloxane (PDMS) is widely used in the fabrication of biomedical materials. On the other hand, PDMS is also prone to adsorption of both protein and bacteria, making PDMS implants susceptible to infection. Herein, we examine the use of durably crosslinked zwitterionic coatings for PDMS surfaces to mitigate bacterial adhesion. Using a single step photografting technique, poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA) thin films were covalently attached to PDMS substrates. The abilities of these coatings to resist adhesion of S. aureus and S. epidermidis were tested in vitro under both wet and droplet conditions, as well as in subcutaneous and transcutaneous implantation models using Sprague-Dawley rats. Zwitterionic thin films effectively reduced bacterial adhesion in both in vitro and in vivo conditions. This was particularly true for pCBMA coated materials which exhibited significant reductions in bacterial adhesion and growth with respect to S. aureus and S. epidermidis for all in vitro conditions as well as the ability to resist bacterial growth on PDMS implants. The results of this study suggest that a simple and durable photografting process can be used to produce polymer thin films capable of preventing infection of implantable medical devices.

In vitro conjugate vaccine expression technology enables rapid and portable biosynthesis of protective antibacterial vaccines., Conjugate vaccines are among the most effective methods for preventing bacterial infections. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro conjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of conjugate vaccines against diverse bacterial pathogens in 1 hour. We show that iVAX-synthesized vaccines against Francisella tularensis subsp. tularensis (type A) strain Schu S4 protected mice from lethal intranasal F. tularensis challenge. The iVAX platform promises to accelerate development of new conjugate vaccines with increased access through refrigeration-independent distribution and portable production.

Background Many CpGs become hyper or hypo-methylated with age. Multiple methods have been developed by Horvath et al. to estimate DNA methylation (DNAm) age including Pan-tissue, Skin & Blood, PhenoAge, and GrimAge. Pan-tissue and Skin & Blood try to estimate chronological age in the normal population whereas PhenoAge and GrimAge use surrogate markers associated with mortality to estimate biological age and its departure from chronological age. Here, we applied Horvath’s four methods to calculate and compare DNAm age in 499 subjects with type 1 diabetes (T1D) from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study using DNAm data measured by Illumina EPIC array in the whole blood. Association of the four DNAm ages with development of diabetic complications including cardiovascular diseases (CVD), nephropathy, retinopathy, and neuropathy, and their risk factors were investigated. Results Pan-tissue and GrimAge were higher whereas Skin & Blood and PhenoAge were lower than chronological age (p < 0.0001). DNAm age was not associated with the risk of CVD or retinopathy over 18–20 years after DNAm measurement. However, higher PhenoAge (β = 0.023, p = 0.007) and GrimAge (β = 0.029, p = 0.002) were associated with higher albumin excretion rate (AER), an indicator of diabetic renal disease, measured over time. GrimAge was also associated with development of both diabetic peripheral neuropathy (OR = 1.07, p = 9.24E−3) and cardiovascular autonomic neuropathy (OR = 1.06, p = 0.011). Both HbA1c (β = 0.38, p = 0.026) and T1D duration (β = 0.01, p = 0.043) were associated with higher PhenoAge. Employment (β = − 1.99, p = 0.045) and leisure time (β = − 0.81, p = 0.022) physical activity were associated with lower Pan-tissue and Skin & Blood, respectively. BMI (β = 0.09, p = 0.048) and current smoking (β = 7.13, p = 9.03E−50) were positively associated with Skin & Blood and GrimAge, respectively. Blood pressure, lipid levels, pulse rate, and alcohol consumption were not associated with DNAm age regardless of the method used. Conclusions Various methods of measuring DNAm age are sub-optimal in detecting people at higher risk of developing diabetic complications although some work better than the others.

T cells are the immunological cornerstone in host defense against infections by intracellular bacterial pathogens, such as virulent Francisella tularensis spp. tularensis (Ftt). The general paucity of novel vaccines for Ftt during the past 60 y can, in part, be attributed to the poor understanding of immune parameters required to survive infection. Thus, we developed a strategy utilizing classical immunological tools to elucidate requirements for effective adaptive immune responses directed against Ftt. Following generation of various Francisella strains expressing well-characterized lymphocytic choriomeningitis virus epitopes, we found that survival correlated with persistence of Ag-specific CD4+ T cells. Function of these cells was confirmed in their ability to more effectively control Ftt replication in vitro. The importance of understanding the Ag-specific response was underscored by our observation that inclusion of an epitope that elicits high-avidity CD4+ T cells converted a poorly protective vaccine to one that engenders 100% protection. Taken together, these data suggest that improved efficacy of current tularemia vaccine platforms will require targeting appropriate Ag-specific CD4+ T cell responses and that elucidation of Francisella epitopes that elicit high-avidity CD4+ T cell responses, specifically in humans, will be required for successful vaccine development.

A shift in macrophage metabolism from oxidative phosphorylation to aerobic glycolysis is a requirement for activation to effectively combat invading pathogens. Francisella tularensis is a facultative intracellular bacterium that causes an acute, fatal disease called tularemia. Its primary mechanism of virulence is its ability to evade and suppress inflammatory responses while replicating in the cytosol of macrophages. The means by which F. tularensis modulates macrophage activation are not fully elucidated. In this study, we demonstrate that virulent F. tularensis impairs production of inflammatory cytokines in primary macrophages by preventing their shift to aerobic glycolysis, as evidenced by the downregulation of hypoxia inducible factor 1α and failure to upregulate pfkfb3. We also show that Francisella capsule is required for this process. In addition to modulating inflammatory responses, inhibition of glycolysis in host cells is also required for early replication of virulent Francisella. Taken together, our data demonstrate that metabolic reprogramming of host cells by F. tularensis is a key component of both inhibition of host defense mechanisms and replication of the bacterium.

Many microbial pathogens produce a β-(1→6)-linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule, including bacterial, fungal, and protozoan cells. Broadly protective immune responses to this single conserved polysaccharide antigen in animals are possible but only when a deacetylated poly-N-acetyl-d-glucosamine (dPNAG

Francisella tularensis is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies., IMPORTANCE The low infectious dose, the high potential mortality/morbidity rates, and the ability to be disseminated as an aerosol make Francisella tularensis a potential agent for bioterrorism. These characteristics led the Centers for Disease Control (CDC) to classify F. tularensis as a Tier 1 pathogen. Currently, there is no vaccine approved for general use in the United States.

Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI , iglJ , or pdpC , three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10 8 CFU iglJ- or pdpC -deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype.

The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS-specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

The mechanisms by which the intracellular pathogen Francisella tularensis evades innate immunity are not well defined. We have identified a gene with homology to Escherichia coli mviN, a putative lipid II flippase, which F. tularensis uses to evade activation of innate immune pathways. Infection of mice with a F. tularensis mviN mutant resulted in improved survival and decreased bacterial burdens compared to infection with wild-type F. tularensis. The mviN mutant also induced increased absent in melanoma 2 inflammasome-dependent IL-1β secretion and cytotoxicity in macrophages. The compromised in vivo virulence of the mviN mutant depended upon inflammasome activation, as caspase 1- and apoptosis-associated speck-like protein containing a caspase recruitment domain-deficient mice did not exhibit preferential survival following infection. This study demonstrates that mviN limits F. tularensis-induced absent in melanoma 2 inflammasome activation, which is critical for its virulence in vivo.

Francisella tularensis uses both pre- and post-assembly mechanisms to inhibit NADPH oxidase activity at its own phagosome and throughout infected human neutrophils. Ft is a facultative intracellular pathogen that infects many cell types, including neutrophils. In previous work, we demonstrated that the type B Ft strain LVS disrupts NADPH oxidase activity throughout human neutrophils, but how this is achieved is incompletely defined. Here, we used several type A and type B strains to demonstrate that Ft-mediated NADPH oxidase inhibition is more complex than appreciated previously. We confirm that phagosomes containing Ft opsonized with AS exclude flavocytochrome b558 and extend previous results to show that soluble phox proteins were also affected, as indicated by diminished phosphorylation of p47phox and other PKC substrates. However, a different mechanism accounts for the ability of Ft to inhibit neutrophil activation by formyl peptides, Staphylococcus aureus, OpZ, and phorbol esters. In this case, enzyme targeting and assembly were normal, and impaired superoxide production was characterized by sustained membrane accumulation of dysfunctional NADPH oxidase complexes. A similar post-assembly inhibition mechanism also diminished the ability of anti-Ft IS to confer neutrophil activation and bacterial killing, consistent with the limited role for antibodies in host defense during tularemia. Studies of mutants that we generated in the type A Ft strain Schu S4 demonstrate that the regulatory factor fevR is essential for NADPH oxidase inhibition, whereas iglI and iglJ, candidate secretion system effectors, and the acid phosphatase acpA are not. As Ft uses multiple mechanisms to block neutrophil NADPH oxidase activity, our data strongly suggest that this is a central aspect of virulence.

Francisella tularensis is the etiologic agent of an intracellular systemic infection of the lymphatic system in humans called tularemia. The organism has become the subject of considerable research interest due to its classification as a category A select agent by the CDC. To aid genetic analysis of this pathogen, we have constructed a temperature-sensitive Tn 5 -based transposon delivery system that is capable of generating chromosomal reporter fusions with lacZ or luxCDABE , enabling us to monitor gene expression. Transposition is catalyzed by the hyperactive Tn 5 transposase, whose expression is driven by the Francisella groES promoter. When high-temperature selection (42°C) is applied to a bacterial culture carrying the transposon delivery plasmid, ∼0.1% of the population is recovered with Tn 5 insertions in the chromosome. Nucleotide sequence analysis of a sample of mutants revealed that the insertions occur randomly throughout the chromosome. The kanamycin-selectable marker of the transposon is also flanked by FLP recombination target sequences that allow deletion of the antibiotic resistance gene when desired. This system has been used to generate transposon mutant libraries for the F. tularensis live vaccine strain as well as two different virulent F. tularensis strains. Chromosomal reporters delivered with the transposon were used to identify genes upregulated by growth in Chamberlain's defined medium. Genes in the fsl operon, reported to be involved in iron acquisition, as well as genes in the igl gene cluster were among those identified by the screen. Further experiments implicate the ferric uptake regulator (Fur) protein in the negative regulation of fsl but not igl reporters, which occurs in an iron-dependent manner. Our results indicate that we have created a valuable new transposon that can be used to identify and characterize virulence genes in F. tularensis strains.

Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections.

The lipopolysaccharide (LPS) and O-antigen polysaccharide capsule structures of Francisella tularensis play significant roles in helping these highly virulent bacteria avoid detection within a host. We previously created pools of F. tularensis mutants that we screened to identify strains that were not reactive to a monoclonal antibody to the O-antigen capsule. To follow up previously published work, we characterize further seven of the F. tularensis Schu S4 mutant strains identified by our screen. These F. tularensis strains carry the following transposon mutations: FTT0846::Tn5, hemH::Tn5, wbtA::Tn5, wzy::Tn5, FTT0673p/prsA::Tn5, manB::Tn5, or dnaJ::Tn5. Each of these strains displayed sensitivity to human serum, to varying degrees, when compared to wild-type F. tularensis Schu S4. By Western blot, only FTT0846::Tn5, wbtA::Tn5, wzy::Tn5, and manB::Tn5 strains did not react to the capsule and LPS O-antigen antibody 11B7, although the wzy::Tn5 strain did have a single O-antigen reactive band that was detected by the FB11 monoclonal antibody. Of these strains, manB::Tn5 and FTT0846 appear to have LPS core truncations, whereas wbtA::Tn5 and wzy::Tn5 had LPS core structures that are similar to the parent F. tularensis Schu S4. These strains were also shown to have poor growth within human monocyte derived macrophages (MDMs) and bone marrow derived macrophages (BMDMs). We examined the virulence of these strains in mice, following intranasal challenge, and found that each was attenuated compared to wild type Schu S4. Our results provide additional strong evidence that LPS and/or capsule are F. tularensis virulence factors that most likely function by providing a stealth shield that prevents the host immune system from detecting this potent pathogen.

Recent work has demonstrated that Salmonella enterica serovar Typhimurium forms biofilms on HEp-2 tissue culture cells in a type 1 fimbria-dependent manner. To investigate how biofilm growth of HEp-2 tissue culture cells affects gene expression in Salmonella , we compared global gene expression during planktonic growth and biofilm growth. Microarray results indicated that the transcription of ∼100 genes was substantially altered by growth in a biofilm. These genes encode proteins with a wide range of functions, including antibiotic resistance, central metabolism, conjugation, intracellular survival, membrane transport, regulation, and fimbrial biosynthesis. The identification of five fimbrial gene clusters was of particular interest, as we have demonstrated that type 1 fimbriae are required for biofilm formation on HEp-2 cells and murine intestinal epithelium. Mutations in each of these fimbriae were constructed in S. enterica serovar Typhimurium strain BJ2710, and the mutants were found to have various biofilm phenotypes on plastic, HEp-2 cells, and chicken intestinal tissue. The pef and csg mutants were defective for biofilm formation on each of the three surfaces tested, while the lpf mutant exhibited a complete loss of the ability to form a biofilm on chicken intestinal tissue but only an intermediate loss of the ability to form a biofilm on tissue culture cells and plastic surfaces. The bcf mutant displayed increased biofilm formation on both HEp-2 cells and chicken intestinal epithelium, while the sth mutant had no detectable biofilm defects. In all instances, the mutants could be restored to a wild-type phenotype by a plasmid carrying the functional genes. This is the first work to identify the genomic responses of Salmonella to biofilm formation on host cells, and this work highlights the importance of fimbriae in adhering to and adapting to a eukaryotic cell surface. An understanding of these interactions is likely to provide new insights for intervention strategies in Salmonella colonization and infection.

Recently, we demonstrated that Salmonella enterica serovar Typhimurium can form biofilm on HEp-2 cells in a type 1 fimbria-dependent manner. Previous work on Salmonella exopolysaccharide (EPS) in biofilm indicated that the EPS composition can vary based upon the substratum on which the bacterial biofilm forms. We have investigated the role of genes important in the production of colanic acid and cellulose, common components of EPS. A mutation in the colanic acid biosynthetic gene, wcaM , was introduced into S. enterica serovar Typhimurium strain BJ2710 and was found to disrupt biofilm formation on HEp-2 cells and chicken intestinal tissue, although biofilm formation on a plastic surface was unaffected. Complementation of the wcaM mutant with the functional gene restored the biofilm phenotype observed in the parent strain. A mutation in the putative cellulose biosynthetic gene, yhjN , was found to disrupt biofilm formation on HEp-2 cells and chicken intestinal epithelium, as well as on a plastic surface. Our data indicate that Salmonella attachment to, and growth on, eukaryotic cells represent complex interactions that are facilitated by species of EPS.

Salmonella enterica serovar Typhimurium causes self-limiting gastroenteritis in humans and a typhoid-like disease in mice that serves as a model for typhoid infections in humans. A critical step in Salmonella pathogenesis is the invasion of enterocytes and M cells of the small intestine via expression of a type III secretion system, encoded on Salmonella pathogenicity island 1 (SPI-1), that secretes effector proteins into host cells, leading to engulfment of the bacteria within large membrane ruffles. The in vitro regulation of invasion genes has been the subject of much scientific investigation. Transcription of the hilA gene, which encodes an OmpR/ToxR-type transcriptional activator of downstream invasion genes, is increased during growth under high-osmolarity and low-oxygen conditions, which presumably mimic the environment found within the small intestine. Several negative regulators of invasion gene expression have been identified, including HilE, Hha, and Lon protease. Mutations within the respective genes increase the expression of hilA when the bacteria are grown under environmental conditions that are not favorable for hilA expression and invasion. In this study, the intracellular expression of invasion genes was examined, after bacterial invasion of HEp-2 epithelial cells, using Salmonella strains containing plasmid-encoded short-half-life green fluorescent protein reporters of hilA , hilD , hilC , or sicA expression. Interestingly, the expression of SPI-1 genes was down-regulated after invasion, and this was important for the intracellular survival of the bacteria. In addition, the effects of mutations in genes encoding negative regulators of invasion on intracellular hilA expression were examined. Our results indicate that Lon protease is important for down-regulation of hilA expression and intracellular survival after the invasion of epithelial cells.

The ability of Salmonella enterica serovar Typhimurium to traverse the intestinal mucosa of a host is an important step in its ability to initiate gastrointestinal disease. The majority of the genes required for this invasive characteristic are encoded on Salmonella pathogenicity island 1 (SPI1), and their expression is controlled by the transcriptional activator HilA, a member of the OmpR/ToxR family of proteins. A variety of genes ( hilC , hilD , fis , sirA / barA , csrAB , phoB , fadD , envZ / ompR , fliZ , hilE , ams , lon , pag , and hha ) have been identified that exert positive or negative effects on hilA expression, although the mechanisms by which these gene products function remain relatively unclear. Recent work indicates that the small DNA-binding protein, Hha, has a significant role in repressing hilA transcription and the invasive phenotype, particularly in response to osmolarity signals. We have characterized the Salmonella -specific gene, hilE , and found that it plays an important regulatory role in hilA transcription and invasion gene expression. Mutation of hilE causes derepression of hilA transcription, and overexpression of hilE superrepresses hilA expression and the invasive phenotype. Bacterial two-hybrid experiments indicate that the HilE protein interacts with HilD, suggesting a possible mechanism for HilE negative regulation of hilA gene expression and the Salmonella invasive phenotype. Finally, we have found that the hilE gene resides on a region of the serovar Typhimurium chromosome that has many characteristics of a pathogenicity island.

Implementation of multicenter and/or longitudinal studies requires an effective quality assurance program to identify trends, data inconsistencies and process variability of results over time. The Diabetes Control and Complications Trial (DCCT) and the follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) study represent over 30 years of data collection among a cohort of participants across 27 clinical centers. The quality assurance plan is overseen by the Data Coordinating Center and is implemented across the clinical centers and central reading units. Each central unit incorporates specific DCCT/EDIC quality monitoring activities into their routine quality assurance plan. The results are reviewed by a data quality assurance committee whose function is to identify variances in quality that may impact study results from the central units as well as within and across clinical centers, and to recommend implementation of corrective procedures when necessary. Over the 30-year period, changes to the methods, equipment, or clinical procedures have been required to keep procedures current and ensure continued collection of scientifically valid and clinically relevant results. Pilot testing to compare historic processes with contemporary alternatives is performed and comparability is validated prior to incorporation of new procedures into the study. Details of the quality assurance plan across and within the clinical and central reading units are described, and quality outcomes for core measures analyzed by the central reading units (e.g. biochemical samples, fundus photographs, ECGs) are presented.

Type 1 fimbria-mediated adherence to HEp-2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose-sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp-2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp-2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp-2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp-2 cells using once-flow-through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process.

Pathogenic Salmonella species initiate infection of a mammalian host by inducing their own uptake into intestinal M-cells. During the uptake process, the bacteria utilize an intrinsic secretion system to release proteins that enter host cells. The secreted invasion-mediating proteins subsequently interact with host cell components that induce alterations in the actin cytoskeleton. To identify potential cellular determinants of invasion, we employed a yeast two-hybrid system using the secreted Salmonella invasion protein (SipC) as the bait protein. This system identified cytokeratins, supportive components of the cytoskeletal matrix, as proteins that may physically interact with SipC. Transfection-based studies revealed an inhibition of Salmonella invasion when a dominant negative cytokeratin-18 was expressed. Immunofluorescent confocal microscopy studies revealed that Salmonella did not enter HEp-2 cells expressing the dominant negative cytokeratin-18. These results suggest that an interaction between SipC and cytokeratin-18 may occur as part of Salmonella invasion.

Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14C] acetate‐labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose‐dependent neutrophil apoptosis inhibition via a TLR2‐dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function.

Salmonellae initiate disease through the invasion of host cells within the intestine. This ability to invade requires the coordinated action of numerous genes, many of which are found within Salmonella pathogenicity island 1 (SPI-1). The key to this process is the ability of the bacteria to respond to the environment, thereby upregulating the necessary genes under optimal conditions. Central to the control of SPI-1 is the transcriptional activator hilA . Work has identified at least 10 different activators and 8 different repressors responsible for the control of hilA . We have previously shown that hilE is a Salmonella -specific negative regulator that is able to repress hilA expression and invasion. Additionally, fimZ , a transcriptional activator responsible for the expression of type I fimbriae as well as flagellar genes, has also been implicated in this process. fimZ is homologous to response regulators from other two-component regulatory systems, although a sensor for the system has not been identified. The phoPQ and phoBR regulons are both two-component systems that negatively affect hilA expression, although the mechanism of action has not been determined. Our results show that PhoBR is capable of inducing fimZ expression, whereas PhoPQ does not affect fimZ expression but does upregulate hilE in an FimZ-dependent manner. Therefore, phosphate (sensed by PhoBR) and magnesium (sensed by PhoPQ) levels are important in controlling hilA expression levels when Salmonella is in the intestinal environment.

Refractive error (RE) is a complex, multifactorial disorder characterized by a mismatch between the optical power of the eye and its axial length that causes object images to be focused off the retina. The two major subtypes of RE are myopia (nearsightedness) and hyperopia (farsightedness), which represent opposite ends of the distribution of the quantitative measure of spherical refraction. We performed a fixed effects meta-analysis of genome-wide association results of myopia and hyperopia from 9 studies of European-derived populations: AREDS, KORA, FES, OGP-Talana, MESA, RSI, RSII, RSIII and ERF. One genome-wide significant region was observed for myopia, corresponding to a previously identified myopia locus on 8q12 (p = 1.25×10-8), which has been reported by Kiefer et al. as significantly associated with myopia age at onset and Verhoeven et al. as significantly associated to mean spherical-equivalent (MSE) refractive error. We observed two genome-wide significant associations with hyperopia. These regions overlapped with loci on 15q14 (minimum p value = 9.11×10-11) and 8q12 (minimum p value 1.82×10-11) previously reported for MSE and myopia age at onset. We also used an intermarker linkage- disequilibrium-based method for calculating the effective number of tests in targeted regional replication analyses. We analyzed myopia (which represents the closest phenotype in our data to the one used by Kiefer et al.) and showed replication of 10 additional loci associated with myopia previously reported by Kiefer et al. This is the first replication of these loci using myopia as the trait under analysis. "Replication-level" association was also seen between hyperopia and 12 of Kiefer et al.'s published loci. For the loci that show evidence of association to both myopia and hyperopia, the estimated effect of the risk alleles were in opposite directions for the two traits. This suggests that these loci are important contributors to variation of refractive error across the distribution.

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing foreign (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a metabolite in the 2-C-methyl-d-erythritol-4-phosphate pathway used by most eubacteria and apicomplexan parasites, and self isopentenyl pyrophosphate, a metabolite in the mevalonate pathway used by humans. Whereas microbial infections elicit prolonged expansion of memory Vγ2Vδ2 T cells, immunization with prenyl pyrophosphates or aminobisphosphonates elicit short-term Vγ2Vδ2 expansion with rapid anergy and deletion upon subsequent immunizations. We hypothesized that a live, attenuated bacterial vaccine that overproduces HMBPP would elicit long-lasting Vγ2Vδ2 T cell immunity by mimicking a natural infection. Therefore, we metabolically engineered the avirulent aroA− Salmonella enterica serovar Typhimurium SL7207 strain by deleting the gene for LytB (the downstream enzyme from HMBPP) and functionally complementing for this loss with genes encoding mevalonate pathway enzymes. LytB− Salmonella SL7207 had high HMBPP levels, infected human cells as efficiently as did the wild-type bacteria, and stimulated large ex vivo expansions of Vγ2Vδ2 T cells from human donors. Importantly, vaccination of a rhesus monkey with live lytB− Salmonella SL7207 stimulated a prolonged expansion of Vγ2Vδ2 T cells without significant side effects or anergy induction. These studies provide proof-of-principle that metabolic engineering can be used to derive live bacterial vaccines that boost Vγ2Vδ2 T cell immunity. Similar engineering of metabolic pathways to produce lipid Ags or B vitamin metabolite Ags could be used to derive live bacterial vaccine for other unconventional T cells that recognize nonpeptide Ags.

The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes ( FTT1236 and FTT1238c ) revealed similarity to those for waa ( rfa ) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD 50 ) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensis challenge.

Over the last decade, studies on the virulence of the highly pathogenic intracellular bacterial pathogen Francisella tularensis have increased dramatically. The organism produces an inert LPS, a capsule, escapes the phagosome to grow in the cytosol (FPI genes mediate phagosomal escape) of a variety of host cell types that include epithelial, endothelial, dendritic, macrophage, and neutrophil. This review focuses on the work that has identified and characterized individual virulence factors of this organism and we hope to highlight how these factors collectively function to produce the pathogenic strategy of this pathogen. In addition, several recent studies have been published characterizing F. tularensis mutants that induce host immune responses not observed in wild type F. tularensis strains that can induce protection against challenge with virulent F. tularensis. As more detailed studies with attenuated strains are performed, it will be possible to see how host models develop acquired immunity to Francisella. Collectively, detailed insights into the mechanisms of virulence of this pathogen are emerging that will allow the design of anti-infective strategies.

An early step in the establishment of Salmonella enterica serovar Typhimurium murine infection is the penetration of the intestinal mucosa of the small intestine. The majority of the genes responsible for the Salmonella invasive phenotype are encoded on Salmonella pathogenicity island 1, and their transcription is controlled by the hilA transcriptional activator. The expression of hilA is regulated by environmental signals including oxygen, osmolarity, pH, and growth phase such that the presence of any one suboptimal condition results in repression of hilA expression and the invasive phenotype. We have conducted a search for negative regulators of hilA by introduction of a Salmonella enterica serovar Typhimurium chromosomal DNA gene bank into a Salmonella enterica serovar Typhimurium hilA ::Tn 5lacZY reporter strain. This screen has identified the hha gene as a regulator that exerts a negative influence on hilA expression. Plasmid-encoded hha significantly reduces hilA ::Tn 5lacZY chromosomal expression, as well as expression of the invasion genes invF , prgH , and sipC . An hha null mutation results in substantial derepression of both chromosomally encoded and plasmid-encoded hilA ::Tn 5lacZY expression. Introduction of plasmid-encoded hha into strain SL1344 results in attenuation of invasion using in vitro and in vivo assays. Importantly, purified Hha protein was found to bind to a hilA DNA promoter fragment, suggesting that the regulatory activity of the Hha protein occurs at the hilA promoter. These data add detail to the developing model of the regulation of Salmonella invasion genes.

Salmonella typhimurium requires a type III secretion system encoded by pathogenicity island (SPI)-2 to survive and proliferate within macrophages. This survival implies that S. typhimurium avoids or withstands bactericidal events targeted to the microbe-containing vacuole, which include intraphagosomal production of reactive oxygen species (ROS), phagosomal acidification, and delivery of hydrolytic enzymes to the phagosome via fusion with lysosomes. Recent evidence suggests that S. typhimurium alters ROS production by murine macrophages in an SPI-2-dependent manner. To gain insights into the mechanism by which S. typhimurium inhibits intraphagosomal ROS production, we analyzed the subcellular distribution of NADPH oxidase components during infection of human monocyte-derived macrophages by wild-type (WT) or several SPI-2 mutant strains of S. typhimurium. We found that the membrane component of the NADPH oxidase, flavocytochrome b558, was actively excluded or rapidly removed from the phagosomal membrane of WT-infected monocyte-derived macrophages, thereby preventing assembly of the NADPH oxidase complex and intraphagosomal production of superoxide anion. In contrast, the NADPH oxidase assembled on and generated ROS in phagosomes containing SPI-2 mutant S. typhimurium. Subversion of NADPH oxidase assembly by S. typhimurium was accompanied by increased bacterial replication relative to that of SPI-2 mutant strains, suggesting that the ability of WT S. typhimurium to prevent NADPH oxidase assembly at the phagosomal membrane represents an important virulence factor influencing its intracellular survival.

Salmonella enterica serovars Gallinarum and Pullorum are S. enterica biotypes that exhibit host specificity for poultry and aquatic birds and are not normally capable of causing disease in mammalian hosts. During their evolution toward host restriction serovars Gallinarum and Pullorum lost their ability to mediate mannose-sensitive hemagglutination (MSHA), a phenotype correlated with adherence to certain cell types. Because adherence is an essential requirement for invasion of cells by bacterial pathogens, we examined whether MHSA type 1 fimbriae would increase the ability of serovars Pullorum and Gallinarum to invade normally restrictive cells. Serovars Gallinarum and Pullorum expressing S. enterica serovar Typhimurium strain LT2 type 1 fimbriae exhibited a 10- to 20-fold increased ability to adhere to and a 20- to 60-fold increased invasion efficiency of the human epithelial HEp-2 cell line. Invasion was accompanied by extensive ruffling of the membranes of the HEp-2 cells. In a murine ligated ileal loop model, a 32% increase in the number of M-cell ruffles was seen when serovar Gallinarum expressed serovar Typhimurium type 1 fimbriae.

Salmonella enterica serovar Typhimurium initiates infection of a host by inducing its own uptake into specialized M cells which reside within the epithelium overlaying Peyer's patches. Entry of Salmonella into intestinal epithelial cells is dependent upon invasion genes that are clustered together in Salmonella pathogenicity island 1 (SPI-1). Upon contact between serovar Typhimurium and epithelial cells targeted for bacterial internalization, bacterial proteins are injected into the host cell through a type III secretion system that leads to internalization of the bacteria. Previous work has established that the prgH , - I , - J , and - K and orgA genes reside in SPI-1, and the products of these genes are predicted to be components of the invasion secretion apparatus. We report that an error in the published orgA DNA sequence has been identified so that this region encodes two small genes rather than a single large open reading frame. These genes have been designated orgA and orgB . Additionally, an opening reading frame downstream of orgB , which we have designated orgC , has been identified and partially characterized. Previously published work has indicated that the prgH , - I , - J , and - K genes are transcribed from a promoter distinct from that used by the gene immediately downstream, orgA . Here, we present experiments indicating that orgA expression is driven by the prgH promoter. In addition, using reverse transcriptase PCR analysis, we have found that this polycistronic message extends downstream of prgH to include a total of 10 genes. To more fully characterize this invasion operon, we demonstrate that the prgH , prgI , prgJ , prgK , orgA , and orgB genes are each required for invasion and secretion, while orgC is not essential for the invasive phenotype.

Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Salmonella typhimurium DT104 further poses a major health concern due to its acquisition of resistance to multiple antibiotics. The rapid detection of multiresistant S. typhimurium DT104 would facilitate strategies aimed at controlling this pathogen. We developed a specific and sensitive polymerase chain reaction (PCR) assay that amplifies a segment of DNA that is conserved in multiresistant S. typhimurium DT104. To provide further specificity for this PCR-based diagnostic test, we amplified two other gene fragments that are present in S. typhimurium DT104. A multiplex PCR containing primers for targeted sequences resulted in the amplification of predicted size fragments from S. typhimurium DT104 exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline) or ASSuT resistance phenotypes. A minor modification of the multiplex PCR enabled the detection of other related multiresistant Salmonella such as S. typhimurium U302. To augment the detection process, we also designed a fluorogenic PCR assay that can detect the DNA of multiresistant S. typhimurium DT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistant S. typhimurium DT104, or potentially the next emerging multiresistant Salmonella, can be accurately detected in only 3-4 h.

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A beta-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV(A), which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a well-defined murine typhoid model of animal pathogenesis. To this end, an r- m+ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter- and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.

The Francisella tularensis pathogenicity island (FPI) encodes many proteins that are required for virulence. Expression of these genes depends upon the FevR (PigR) regulator and its interactions with the MglA/SspA and RNA polymerase transcriptional complex. Experiments to identify how transcription of the FPI genes is activated have led to identification of mutations within the migR , trmE , and cphA genes that decrease FPI expression. Recent data demonstrated that the small alarmone ppGpp, produced by RelA and SpoT, is important for stabilizing MglA/SspA and FevR (PigR) interactions in Francisella . Production of ppGpp is commonly known to be activated by cellular and nutritional stress in bacteria, which indicates that cellular and nutritional stresses act as important signals for FPI activation. In this work, we demonstrate that mutations in migR , trmE , or cphA significantly reduce ppGpp accumulation. The reduction in ppGpp levels was similar for each of the mutants and correlated with a corresponding reduction in iglA reporter expression. In addition, we observed that there were differences in the ability of each of these mutants to replicate within various mammalian cells, indicating that the migR , trmE , and cphA genes are likely parts of different cellular stress response pathways in Francisella . These results also indicate that different nutritional and cellular stresses exist in different mammalian cells. This work provides new information to help understand how Francisella regulates its virulence genes in response to host cell environments, and it contributes to our growing knowledge of this highly successful bacterial pathogen.

▪ Abstract The lifestyle of bacterial pathogens requires them to establish infection in the face of host immunity. Upon entering a potential host, a variety of interactions are initiated, the outcome of which depends upon a myriad of attributes of each of the participants. In this review we discuss the interactions that occur between pathogenic Salmonella species and the host immune systems, but when appropriate to broaden perspective, we have provided a general overview of the interactions between bacterial pathogens and animal hosts. Pathogenic Salmonella species possess an array of invasion genes that produce proteins secreted by a specialized type III secretion apparatus. These proteins are used by the bacteria to penetrate the intestinal mucosa by invading and destroying specialized epithelial M cells of the Peyer's patches. This manuever deposits the bacteria directly within the confines of the reticuloendothelial system. The host responds to these actions with nonspecific phagocytic cells and an imflammatory response as well as by activating specific cellular and humoral immune responses. Salmonella responds to this show of force directly. It appears that the bacteria invade and establish a niche within the very cells that have been sent to destroy them. Efforts are underway to characterize the factors that allow these intracellular bacteria to customize intracellular vacuoles for their own purposes. It is the constant play between these interactions that determines the outcome of the host infection, and clearly they will also shape the evolution of new survival strategies for both the bacterium and the host.

Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI, iglJ, or pdpC, three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10(8) CFU iglJ- or pdpC-deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype.

Francisella tularensis is a Gram-negative bacterium and the causative agent of the disease tularemia. Escape of F. tularensis from the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses to F. tularensis . We have identified the 5-formyltetrahydrofolate cycloligase gene ( FTL _ 0724 ) as being important for F. tularensis live vaccine strain (LVS) virulence. Infection of mice in vivo with a F. tularensis LVS FTL _ 0724 mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. The FTL _ 0724 mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophages in vitro . In contrast, infection of macrophages with a F. tularensis LVS rluD pseudouridine synthase ( FTL _ 0699 ) mutant resulted in diminished IL-1β and IL-18 secretion from macrophages in vitro compared to infection of macrophages with wild-type LVS. In addition, the FTL _ 0699 mutant was not attenuated in vivo . These findings further illustrate that F. tularensis LVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogen in vivo .

The use of phacoemulsification by ophthalmic surgeons has increased markedly over the past five years. Previous studies have reported relatively high rates of vitreous loss by residents learning phacoemulsification. We retrospectively analyzed the complications and results in 300 cases of phacoemulsification with intraocular lens implantation performed by residents. The first 40 cases done by four residents in their second year of training were compared with approximately the last 40 cases done by each resident at the end of the third year. The overall rate of surgical complications was 6.3%, and the total rate of vitreous loss was 3.3%. The rate of surgical complications during the initial surgeries in the second year of residency was 9.3%; it was 3.3% by the end of the third year. The rate of vitreous loss was 5.3% in the second year and 1.3% during the third year. Postoperatively, 90.6% of all eyes had a final best corrected visual acuity of 20/40 or better (95% excluding patients with pre-existing ocular disease). With proper training and supervision, the rate of surgical complications for residents learning phacoemulsification is acceptably low when compared with the rate for extracapsular cataract extraction.

Francisella tularensis subsp. tularensis strain SchuS4 (Ftt) is a highly virulent bacterial pathogen and the causative agent of tularemia. There are no licensed tularemia vaccines and experimental live vaccine strains (LVS) vary in their protective efficacy and are poorly defined. Development of novel vaccines will require improvement of protection over the existing LVS and preferably incorporation of well-defined epitopes. However, protective epitopes for tularemia have not been identified. Surviving tularemia requires a strong polyfunctional CD4+ T cell response. Due to the importance of the CD4+ T cell response, we hypothesized ectopic expression of an epitope recognized by high avidity CD4+ T cells would enhance existing vaccine efficacy, regardless of the epitope’s origin. gp61 and LLO are well characterized CD4+ T cell epitopes derived from LCMV and Listeria, respectively, and are recognized by C57Bl/6, but not Balb/c mice. Expression of gp61 or LLO during primary infection with LVS or Ftt improved survival of C57Bl/6, but not Balb/c mice, indicating the presence of low numbers of high avidity CD4+ precursor T cells alters survival during Francisella infection. Furthermore, mice vaccinated with LVS gp61 had significantly improved survival during low and high dose secondary Ftt infection and decreased bacterial burdens compared to controls. Together, this demonstrates that improved efficacy of current tularemia vaccine platforms is achieved by properly targeting appropriate antigen-specific cellular responses. Moreover, the elucidation of Francisella epitopes that elicit high-avidity CD4+ T cell responses, specifically in humans, will be required for successful vaccine development.

Growth of Salmonella typhimurium in a low-oxygen environment induces the ability of these bacteria to enter mammalian cells. We have carried out a search for invasion genes that are expressed under low-oxygen conditions by using Tn5lacZY transcriptional fusions. Several noninvasive oxygen-regulated lacZY insertion strains have been identified. The invasion defect in one of these noninvasive S. typhimurium strains, BJ66, has been complemented by introduction of a cosmid (pBDJ125) from an S. typhimurium SL1344 gene bank. A 1.9-kb EcoRV DNA fragment subcloned from this cosmid, containing a single open reading frame (orgA), restores the ability of BJ66 to invade mammalian cells. Comparative searches of the GenBank and EMBL sequence data banks with the nucleotide sequence of the gene and deduced amino acid sequence of the protein reveal no significant similarities. Interestingly, hybridization of an orgA gene probe with a P22 chromosomal mapping library demonstrated that the orgA gene maps to a region on the chromosome between 57.5 and 60 min where other Salmonella invasion genes have been mapped. Other enteroinvasive bacteria (Shigella flexneri, Escherichia coli, Yersinia spp., and Listeria monocytogenes) lack sequences which cross hybridize to the probe. We have compared the virulence of S. typhimurium SL1344 and an isogenic orgA mutant in a mouse model of typhoid fever. The orgA mutant was as virulent as the wild-type strain was when inoculated intraperitoneally but is significantly reduced (> 60-fold) in its ability to cause disease by an oral route of infection.

Salmonella species are known to initiate infection of mammalian hosts by penetrating the intestinal epithelium of the small bowel. These bacteria preferentially interact with Peyer's patches which are collections of lymphoid follicles making up the gut-associated lymphoid tissue. We infected murine ligated intestinal loops with invasive and noninvasive Salmonella typhimurium strains for 30, 60, 120, and 180 min and examined the infected tissue by transmission electron microscopy. Within 30 min, we found that invasive S. typhimurium exclusively entered M cells found within the follicle-associated epithelium (FAE) of the Peyer's patches. Initially, interactions between invasive bacteria and enterocytes adjacent to the M cells were not found. Invasion of M cells was associated with the ability of the bacteria to invade tissue culture cells. S. typhimurium mutants, which were noninvasive for tissue culture cells, could not be found in ligated loops associated with M cells or enterocytes after incubations of 30, 60, 120, or 180 min. At 60 min, internalized invasive S. typhimurium were cytotoxic for the M cells. Destruction of an M cell formed a gap in the FAE which allowed organisms to invade enterocytes adjacent to the dead cell. Later in the infection process (120 and 180 min), the presence of bacteria beneath the FAE correlated with changes in the cytoarchitecture of the lymphoid follicle. In addition, replicating Salmonella began to enter both the apical and basolateral surfaces of enterocytes adjacent to infected M cells.

Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella , relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236 , FTT1237 , and FTT1238 . Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236 , FTT1237 , and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.

Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDS-PAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)-alpha-D-GalNAcAN-(1->4)-alpha-D-GalNAcAN-(1->3)-beta-D-QuiNAc-(1->2)-beta-D-Qui4NFm-(1-, which is identical to the previously described F. tularensis O-antigen subunit. This indicates that the F. tularensis capsule can be classified as an O-antigen capsular polysaccharide. Our studies indicate that F. tularensis O-antigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an O-antigen polymerase mutant had no evidence of an O-antigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 microg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 microg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an O-antigen capsule that may be the basis of a future vaccine.

When grown aerobically, Salmonella typhimurium exhibits a low level of entry into tissue culture cells. We have isolated an S. typhimurium Tn10 mutant which, when grown under aerobic conditions, efficiently invades HEp-2 cells. Sequencing of S. typhimurium DNA adjacent to the site of the Tn10 element showed that the insertion disrupted transcription of the aspartate receptor gene, tar. Polar effects of the transposon on downstream genes also eliminated chemotaxis. Isogenic nonchemotactic (Che-), as well as nonmotile (Mot-) and nonflagellated (Fla-), S. typhimurium strains were examined for their ability to invade HEp-2 cells. "Smooth" swimming Che- mutants (cheA, cheW, cheR, and cheY) were found to possess increased invasiveness for cultured mammalian cells. In contrast, a "tumbly" cheB mutant and Mot- (flagellated) strain were found to have decreased levels of tissue culture invasiveness. A Fla- strain was found to be as invasive as the wild-type strain if centrifugation was used to facilitate contact with the monolayer surface. In addition, the observed hyperinvasiveness of the smooth swimming tar::Tn10 mutant was suppressed when the strain was paralyzed by the introduction of a mot or fla mutation. A murine infection model was used to demonstrate that the mutant invasive phenotypes were also observed in vivo. These data are most consistent with the idea that the rotation and physical orientation of flagella around the bacteria affect the ability of salmonellae to enter host cells.