Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Disease risk factors and humoral antibody response to periodontal-associated pathogens: A model to evaluate periodontal disease in older adults

Periodontal diseases (PD) are infectious, inflammatory, and tissue destructive events which affect the periodontal ligament that surround and support the teeth. Periodontal diseases are the major cause of tooth loss after age 35, with gingivitis and periodontitis affecting 75% of the adult population. A select group of bacterial organisms are associated with periodontal pathogenesis. There is a direct association between oral hygiene and prevention of PD. The importance of genetic differences and host immune response capabilities in determining host, susceptibility or resistance to PD has not been established. This study examined the risk factors and serum (humoral) immune response to periodontal diseased-associated pathogens in a 55 to 80+ year old South Texas study sample with PD. This study sample was described by: age, sex, ethnicity, the socioeconomic factors marital status, income and occupation, IgG, IgA, IgM immunoglobulin status, and the autoimmune response markers rheumatoid factor (RF) and antinuclear antibody (ANA). These variables were used to determine the risk factors associated with development of PD. Serum IgG, IgA, IgM antibodies to bacterial antigens provided evidence for disease exposure.^ A causal model for PD was constructed from associations for risk factors (ethnicity, marital status, income, and occupation) with dental exam and periodontitis. The multiple correlation between PD and ethnicity, income and dental exam was significant. Hispanics of low income were least likely to have had a dental exam in the last year and most likely to have PD. The etiologic agents for PD, as evidenced by elevated humoral antibody responses, were the Gram negative microorganisms Bacteroides gingivalis, serotypes FDC381 and SUNYaBA7A1-28, and Wolinella recta. Recommendation for a PD prevention and control program are provided. ^

Transcriptional regulation of the {\it neu\/} oncogene and its negative regulation by the c-{\it myc\/} oncogene

The first part of my research involved the characterization of the neu gene promoter. I subcloned a 2.2-kb sequence located upstream to the extreme 5$\sp\prime$ end of the neu gene, in front of the bacterial reporter gene, chloramphenicol acetyltransferase (CAT). Transfection of this construct into different cell lines and subsequent CAT assays demonstrated that this 2.2-kb fragment was functional as a promoter. A series of deletion constructs was engineered to study the contribution of different fragments to transcription. Subcloning of individual fragments was followed by a cotransfection competition experiment, which demonstrated the involvement of protein factors interacting with the promoter. A gel retardation assay was also performed to show the physical binding of protein factors to the promoter. The combined results suggested that both positively and negatively acting protein factors are involved in interacting with different regions of the promoter, contributing to the overall transcription activity. My findings provide an insight into the regulation of neu gene expression, which in turn provides the tools to understand the molecular mechanisms of overexpression of the neu gene in some breast cancer and ovarian cancer cell lines.^ In the second part of my research, I discovered that another oncogene, c-myc, was able to reverse the transformed morphology that was induced by the neu oncogene. Utilizing the promoter constructs that I made, I was able to show that the c-myc oncogene has a negative regulatory effect on the expression of the neu oncogene. Further studies suggested that c-myc is able to lower the effective concentration of a positive factor(s) that interact with a 139-bp fragment of the neu gene promoter. These findings may provide a direct evidence of the long suspected role of the c-myc gene in transcriptional regulation. The neu gene may very well be the first identified mammalian target gene that is regulated by the c-myc oncogene. Since c-myc is known to be stimulated by various mitogenic signals and the neu gene is likely to be a growth factor receptor, it is possible that c-myc, when stimulated by the signal transduction pathway of the neu gene, would function as a negative feedback regulator on the neu gene receptor. (Abstract shortened with permission of author.) ^

Proliferation index in ductal carcinoma in situ of the breast: Relation to estrogen, progesterone and HER2/neu receptors

Background. Ductal carcinoma in situ (DCIS) is the most prevalent precursor to invasive breast cancer (IBC), the second leading cause of death in women in the United States. The three most important prognostic markers for IBC are Estrogen receptor (ER), Progesterone receptor (PR) and HER2/neu. The four groups (IBC) defined as (1) ER and/or PR positive and HER2/neu negative, (2) ER and/or PR positive and HER2/neu positive (3) ER and/or PR negative and HER2/neu positive and (4) negative for all three of these receptors (Triple negative). However, they have not been well studied in DCIS. This is an exploratory study with a primary objective to examine the prevalence of ER, PR, and HER2/neu in DCIS, to explore if the defined groups of IBC occur in DCIS and to consider the biological relationship between these four groups and the proliferative activity of the tumor. A secondary goal of this study is to examine the relationship between grade and proliferative activity. Methods. Using immunohistochemistry, I have measured Ki-67, ER, PR and HER2/neu positivity for a series of cases of DCIS. Results. 20 ER and/or PR positive and HER2/neu negative (50%) with average PI of 0.05, 7 ER and/or PR positive and HER2/neu positive (17.5%) with average PI of 0.14, 10 ER and/or PR negative and HER2/neu positive (25%) with average PI of 0.18, and three triple negative (7.5%) with average PI of 0.18. ER and/or PR positive and HER2/neu positive group has the highest PI (p<0.001). Further, the ER and/or PR positive and HER2/neu positive group show a linear relationship between PI and average ER/PR positivity (R=0.6). PI increases with higher grades. Conclusion. PI appears to depend upon the average fraction of positive ER/PR tumor cells, possibly with a synergistic dependence when HER2/neu is positive. If ER/PR is negative, then both HER2/neu positive and the triple negative cases appear to cluster around an average PI that is higher than the average PI in HER2/neu negative ER/PR positive negative cases. In the triple negative tumors there must be another driver of proliferation.^

False -positives and false-negatives in tests for trend

False-positive and false-negative values were calculated for five different designs of the trend test and it was demonstrated that a design suggested by Portier and Hoel in 1984 for a different problem produced the lowest false-positive and false-negative rates when applied to historical spontaneous tumor rate data for Fischer Rats. ^

Assessing pediatric nasal carriage of S. aureus & MRSA

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) has become an increasing problem in the community. Nasal carriage of these bacteria has been shown to be a predisposing factor for infection and environmental contamination. This serious public health concern prompted an investigation to assess the pediatric nasal carriage of these bacteria in an effort to better understand the populations at risk and prevention of infection.^ This prospective study surveys 30 children from the Northwest Assistance Ministries (NAM) pediatric clinic from October 2008 to the present. Two nasal swabs were taken in 2-4 week intervals to determine S. aureus carrier status. Microbiologic tests were conducted to isolate and identify S. aureus from nasal cultures. Children with 2 cultures positive for S. aureus were classified as persistent carriers, those with 1 positive and 1 negative culture were classified as intermittent carriers, and those with 2 negative cultures were classified as noncarriers. This information was related to patient records and statistical analyses (X 2 and t-tests) were performed.^ Distribution of S. aureus carriage related to patient demographics (age, sex, & race) was showed no significant differences between S. aureus positive and S. aureus negative patient populations (p = 0.8). Additionally, the distribution of carrier status related to demographics also showed no significant difference (p = 0.8). Finally, the distribution of carrier status related to relevant medical history (immunizations current, past infection, & antibiotic use at time of swabbing) showed no significant difference (p = 0.4).^ This study is a snapshot of an ongoing study to assess the pediatric nasal carriage of S. aureus and MRSA. The inability to draw any reliable conclusion from the distribution of data is likely a result of an inadequate samples size. This is one of few studies assessing pediatric nasal carriage of S. aureus and targeting an underrepresented, Hispanic population is especially unique. Continuing this study allows for a better understanding of the epidemiology of this bacterium which will hopefully lead to appropriate interventions thus preventing future S. aureus infections.^

A retrospective analysis of patients referred for tuberculosis testing at Parkland Hospital, Dallas, TX

Tuberculosis (TB) is an infectious disease of great public health importance, particularly to institutions that provide health care to large numbers of TB patients such as Parkland Hospital in Dallas, TX. The purpose of this retrospective chart review was to analyze differences in TB positive and TB negative patients to better understand whether or not there were variables that could be utilized to develop a predictive model for use in the emergency department to reduce the overall number of suspected TB patients being sent to respiratory isolation for TB testing. This study included patients who presented to the Parkland Hospital emergency department between November 2006 and December 2007 and were isolated and tested for TB. Outcome of TB was defined as a positive sputum AFB test or a positive M. tuberculosis culture result. Data were collected utilizing the UT Southwestern Medical Center computerized database OACIS and included demographic information, TB risk factors, physical symptoms, and clinical results. Only two variables were significantly (P<0.05) related to TB outcome: dyspnea (shortness of breath) (P<0.001) and abnormal x-ray (P<0.001). Marginally significant variables included hemoptysis (P=0.06), weight loss (P=0.11), night sweats (P=0.20), history of homelessness or incarceration (P=0.15), and history of positive skin PPD (P=0.19). Using a combination of significant and marginally significant variables, a predictive model was designed which demonstrated a specificity of 24% and a sensitivity of 70%. In conclusion, a predictive model for TB outcome based on patients who presented to the Parkland Hospital emergency department between November 2006 and December 2007 was unsuccessful given the limited number of variables that differed significantly between TB positive and TB negative patients. It is suggested that a future prospective cohort study should be implemented to collect data on TB positive and TB negative patients. It may be possible that a more thorough prospective collection of data may lead to clearer comparisons between TB positive and TB negative patients and ultimately to the design of a more sensitive predictive model for TB outcome. ^

Outcomes associated with chemotherapy in elderly patients with early stage operable breast cancer

Background. Various clinical trials have proved the efficacy of adjuvant chemotherapy in women with breast cancer. Chemotherapy efficacy and guidelines for its use differ by stage of tumor and age of the patient with no clear recommendations for patients aged 70 and above. Objective. To examine the clinical and economic outcomes associated with chemotherapy use in and to examine the disparities in treatment and survival in elderly patients with early stage operable breast cancer by age and axillary node status. Methods. We studied a cohort of 23,110 node positive and 31,572 node negative women aged 65 and over diagnosed with incident American Joint Committee on Cancer (AJCC) stage I, II or IIIa breast cancer between January 1, 1991 and December 31, 2002 using SEER-Medicare data. Total patient costs were estimated using the phase of care approach and adjusted cost estimates were obtained from regression analysis using a 3% discount rate. Cox proportional hazard ratio of mortality was used to determine the effectiveness of chemotherapy. Propensity score approach was also used to minimize the bias associated with receipt of chemotherapy. To assess disparity in treatment, multivariate logistic regression analyses were performed to assess the relative odds of receiving surgery, chemotherapy and radiation after BCS for African Americans compared to Whites. Results. Regression adjusted cost estimates for all node positive patients receiving chemotherapy was approximately $2,300 and was significantly higher (p<0.05) than for patients not receiving chemotherapy. Mortality was significantly lower in node positive and node negative women aged 65-74 years receiving chemotherapy. There was a significant difference between African American and White women in receiving BCS and radiation after BCS; however this difference was explained by patient demographics, tumor characteristics and socioeconomic status (SES). African American node positive women were 21% less likely to receive chemotherapy than White women (OR, 0.79; CI, 0.68-0.92) in multivariate analysis. Conclusion. Chemotherapy is associated with increased survival in patients aged 65-74 and total costs attributable to chemotherapy differ by phase and age of the patient. Underutilization of systemic adjuvant chemotherapy in African American women requires attention and may serve as potential areas for appropriate intervention.^

A STUDY TO INVESTIGATE DIET DIVERSITY AS A NUTRITION ASSESSMENT INSTRUMENT

Nutrient intake and specific food item data from 24-hour dietary recalls were utilized to study the relationship between measures of diet diversity and dietary adequacy in a population of white females of child-bearing age and socioeconomic subgroups of that population. As the basis of the diet diversity measures, twelve food groups were constructed from the 24-hour recall data and the number of unique foods per food group counted and weighted according to specified weighting schemes. Utilizing these food groups, nine diet diversity indices were developed.^ Sensitivity/specificity analysis was used to determine the ability of varying levels of selected diet diversity indices to identify individuals above and below preselected intakes of different nutrients. The true prevalence proportions, sensitivity and specificity, false positive and false negative rates, and positive predictive values observed at the selected levels of diet diversity indices were investigated in relation to the objectives and resources of a variety of nutrition improvement programs. Diet diversity indices constructed from the total population data were evaluated as screening tools for respondent nutrient intakes in each of the socioeconomic subgroups as well.^ The results of the sensitivity/specificity analysis demonstrated that the false positive rate, the false negative rate, or both were too high at each diversity cut-off level to validate the widespread use of any of the diversity indices in the dietary assessment of the study population. Although diet diversity has been shown to be highly correlated with the intakes of a number of nutrients, the diet diversity indices constructed in this study did not adequately represent nutrient intakes in the diet as reported, in this study, intakes as reported in the 24-hour dietary recall. Specific cut-off levels of selected diversity indices might have limited application in some nutrition programs. The results were applicable to the sensitivity/specificity analyses in the socioeconomic subgroups as well as in the total population. ^

THE USE OF ARM CIRCUMFERENCE FOR SIMPLIFIED SCREENING FOR MALNUTRITION BY MINIMALLY TRAINED HEALTH WORKERS

Because of its simplicity and low cost, arm circumference (AC) is being used increasingly in screening for protein energy malnutrition among pre-school children in many parts of the developing world, especially where minimally trained health workers are employed. The objectives of this study were as follows: (1) To determine the relationship of the AC measure with weight for age and weight for height in the detection of malnutrition among pre-school children in a Guatemalan Indian village. (2) To determine the performance of minimally trained promoters under field conditions in measuring AC, weight and height. (3) To describe the practical aspects of taking AC measures versus weight, age and height.^ The study was conducted in San Pablo La Laguna, one of four villages situated on the shores of Lake Atitlan, Guatemala, in which a program of simplified medical care was implemented by the Institute for Nutrition for Central America and Panama (INCAP). Weight, height, AC and age data were collected for 144 chronically malnourished children. The measurements obtained by the trained investigator under the controlled conditions of the health post were correlated against one another and AC was found to have a correlation with weight for age of 0.7127 and with weight for height of 0.7911, both well within the 0.65 to 0.80 range reported in the literature. False positive and false negative analysis showed that AC was more sensitive when compared with weight for height than with weight for age. This was fortunate since, especially in areas with widespread chronic malnutrition, weight for height detects those acute cases in immediate danger of complicating illness or death. Moreover, most of the cases identified as malnourished by AC, but not by weight for height (false positives), were either young or very stunted which made their selection by AC better than weight for height. The large number of cases detected by weight for age, but not by AC (false negative rate--40%) were, however, mostly beyond the critical age period and had normal weight for heights.^ The performance of AC, weight for height and weight for age under field conditions in the hands of minimally trained health workers was also analyzed by correlating these measurements against the same criterion measurements taken under ideally controlled conditions of the health post. AC had the highest correlation with itself indicating that it deteriorated the least in the move to the field. Moreover, there was a high correlation between AC in the field and criterion weight for height (0.7509); this correlation was almost as high as that for field weight for height versus the same measure in the health post (0.7588). The implication is that field errors are so great for the compounded weight for height variable that, in the field, AC is about as good a predictor of the ideal weight for height measure.^ Minimally trained health workers made more errors than the investigator as exemplified by their lower intra-observer correlation coefficients. They consistently measured larger than the investigator for all measures. Also there was a great deal of variability between these minimally trained workers indicating that careful training and followup is necessary for the success of the AC measure.^ AC has many practical advantages compared to the other anthropometric tools. It does not require age data, which are often unreliable in these settings, and does not require sophisticated subtraction and two dimensional table-handling skills that weight for age and weight for height require. The measure is also more easily applied with less disturbance to the child and the community. The AC tape is cheap and not easily damaged or jarred out of calibration while being transported in rugged settings, as is often the case with weight scales. Moreover, it can be kept in a health worker's pocket at all times for continual use in a widespread range of settings. ^

Is H. pylori infection associated with diarrhea in a cohort of 3- to 8-year-old Colombian children?

The Estudio Comunitario sobre la Salud del Niño cohort study followed 326 3- to 8-year-old Colombian children for 4 years to observe the natural history of Helicobacter pylori infection and identify risk factors for acquisition, recurrence and persistence. Acute H. pylori infection during childhood may predispose to other enteric infections and therefore increase the risk of diarrheal disease. This dissertation aimed to estimate the effect of H. pylori infection on the occurrence of diarrhea and parasitic co-infections. The analysis used Generalized Estimating Equations to obtain odds ratios to estimate relative risks for diarrhea and the Zhang-Yu algorithm to estimate relative risks for on parasitic infections. Andersen-Gill models were used to estimate rate ratios for the effect of H. pylori status on the recurrence of parasitic infections. H. pylori status was classified for the entire follow-up duration in 1 of 3 categories: persistently positive, intermittently positive, and persistently negative. Multivariable models included child’s sex, age, symptoms, medication use, and socio-environmental factors. H. pylori infection was weakly and imprecisely associated with diarrheal disease, which occurred at an unexpectedly low frequency in this study. Persistently H. pylori-positive children had a somewhat higher incidence of reported diarrhea than intermittently positive or persistently negative children. Stratified analysis revealed that the presence of specific helminthes modified the effect of persistent H. pylori infection on diarrhea. The incidence of any parasitic infections was higher in children with persistent H. pylori infection relative to those with intermittent or persistently negative status, but this association did not hold when adjusted for the full set of selected covariates. The effects of H. pylori persistent status were similar for the occurrence or recurrence of Giardia duodenalis, Entamoeba histolytica, and Ascaris lumbricoides. These results show that H. pylori frequently co-exists with other parasites in Andean children and suggest that intermittently H. pylori–positive children might be at a lower risk of parasitic infections than persistently positive children. The relationship of H. pylori infection, helminthic infection and diarrheal disease should be further explored in studies that devote more intensive resources to accurate ascertainment of diarrhea.^

Clinical, microbiological, and immunological markers of gingival and periodontal disease induction and progression

The potential impact of periodontal disease, a suspected risk factor for systemic diseases, presents challenges for health promotion and disease prevention strategies. This study examined clinical, microbiological, and immunological factors in a disease model to identify potential biomarkers that may be useful in predicting the onset and severity of both inflammatory and destructive periodontal disease. This project used an historical cohort design based on data obtained from 47 adult, female nonhuman primates followed over a 6-year period for 5 unique projects where the ligature-induced model of periodontitis was utilized. Standardization of protocols for sample collection allowed for comparison over time. Bleeding and pocket depth measures were selected as the dependent variables of relevance to humans based upon the literature and historical observations. Exposure variables included supragingival plaque, attachment level, total bacteria, black-pigmented bacteria, Gram-negative and Gram-positive bacteria, total IgG and IgA in crevicular fluid, specific IgG antibody in both crevicular fluid and serum, and IgG antibody to four select pathogenic microorganisms. Three approaches were used to analyze the data from this study. The first approach tested for differences in the means of the response variables within the group and among longitudinal observations within the group at each time point. The second approach examined the relationship among the clinical, microbiological, and immunological variables using correlation coefficients and stratified analyses. Multivariable models using GEE for repeated measures were produced as a predictive description of the induction and progression of gingivitis and periodontal disease. The multivariable models for bleeding (gingivitis) include supragingival plaque, total bacteria and total IgG while the second also contains supragingival plaque, Gram-positive bacteria, and total IgG. Two multivariable models emerged for periodontal disease. One multivariable model contains plaque, total bacteria, total IgG and attachment level. The second model includes black-pigmented bacteria, total bacteria, antibody to Campylobacter rectus, and attachment level. Utilization of the nonhuman primate model to prospectively examine causal hypotheses can provide a focus for human research on the mechanisms of progression from health to gingivitis to periodontitis. Ultimately, causal theories can guide strategies to prevent disease initiation and reduce disease severity. ^

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.