ABSTRACT
BACKGROUND: The COVID-19 pandemic has necessitated the need to rapidly make public health decisions. We systematically evaluated SARS-CoV-2 seropositivity to understand local COVID-19 epidemiology and support evidence-based public health decision making. METHODS: Residual blood samples were collected for SARS-CoV-2 receptor binding domain (RBD) IgG testing over a 1-5 day period monthly from 26 February 2021-9 July 2021 from six clinical laboratories across the province of Alberta, Canada. Monthly crude and adjusted (for age and gender) seropositivity were calculated. Results were linked to provincial administrative, laboratory, and vaccine databases. RESULTS: 60,632 individual blood samples were tested. Vaccination data were available for 98.8% of samples. Adjusted RBD IgG positivity rose from 11.9% (95% confidence interval [CI] 11.9-12.0%) in March 2021 to 70.2% (95% CI 70.2-70.3%) in July 2021 (p < .0001). Seropositivity rose from 9.4% (95% CI 9.3-9.4%) in March 2021 to 20.2% (95% CI 20.1-20.2%) in July 2021 in unvaccinated Albertans. Unvaccinated seropositive individuals were from geographic areas with significantly (p < .001) lower median household income, lower proportion of married/common-law relationships, larger average household size and higher proportions of visible minorities compared to seronegative unvaccinated individuals. In July 2021, the age groups with the lowest and highest seropositivity in unvaccinated Albertans were those ≥80 years (12.0%, 95% CI 5.3-18.6%) and 20-29 years (24.2%, 95% CI 19.6-28.8%), respectively. Of seropositive unvaccinated individuals, 50.2% (95% CI 45.9-54.5%) had no record of prior SARS-CoV-2 molecular testing. CONCLUSIONS: Longitudinal surveillance of SARS-CoV-2 seropositivity with data linkage is valuable for decision-making during the pandemic.
Subject(s)
COVID-19 , SARS-CoV-2 , Aged, 80 and over , Alberta/epidemiology , Antibodies, Viral , COVID-19/epidemiology , COVID-19/prevention & control , Humans , Immunoglobulin G , Pandemics , VaccinationABSTRACT
We systematically evaluated SARS-CoV-2 IgG positivity in a provincial cohort to understand the local epidemiology of COVID-19 and support evidence-based public health decisions. Residual blood samples were collected for serology testing over 5-day periods monthly from June 2020 to January 2021 from six clinical laboratories across the province of Alberta, Canada. A total of 93,993 individual patient samples were tested with a SARS-CoV-2 nucleocapsid antibody assay with positives confirmed using a spike antibody assay. Population-adjusted SARS-CoV-2 IgG seropositivity was 0.92% (95% confidence interval [CI]: 0.91 to 0.93%) shortly after the first COVID-19 wave in June 2020, increasing to 4.63% (95% CI: 4.61 to 4.65%) amid the second wave in January 2021. There was no significant difference in seropositivity between males and females (1.39% versus 1.27%; P = 0.11). Ages with highest seropositivity were 0 to 9 years (2.71%, 95% CI: 1.64 to 3.78%) followed by 20 to 29 years (1.58%, 95% CI: 1.12 to 2.04%), with the lowest rates seen in those aged 70 to 79 (0.79%, 95% CI: 0.65 to 0.93%) and >80 (0.78%, 95% CI: 0.60 to 0.97%). Compared to the seronegative group, seropositive patients inhabited geographic areas with lower household income ($87,500 versus $97,500; P < 0.001), larger household sizes, and higher proportions of people with education levels of secondary school or lower, as well as immigrants and visible minority groups (all P < 0.05). A total of 53.7% of seropositive individuals were potentially undetected cases with no prior positive COVID-19 nucleic acid test (NAAT). Antibodies were detectable in some patients up to 9 months post positive NAAT result. This seroprevalence study will continue to inform public health decisions by identifying at-risk demographics and geographical areas. IMPORTANCE Using SARS-CoV-2 serology testing, we assessed the proportion of people in Alberta, Canada (population 4.4 million) positive for COVID-19 antibodies, indicating previous infection, during the first two waves of the COVID-19 pandemic (prior to vaccination programs). Linking these results with sociodemographic population data provides valuable information as to which groups of the population are more likely to have been infected with the SARS-CoV-2 virus to help facilitate public health decision-making and interventions. We also compared seropositivity data with previous COVID-19 molecular testing results. Absence of antibody and molecular testing were highly correlated (95% negative concordance). Positive antibody correlation with a previous positive molecular test was low, suggesting the possibility of mild/asymptomatic infection or other reasons leading individuals from seeking medical attention. Our data highlight that the true estimate of population prevalence of COVID-19 is likely best informed by combining data from both serology and molecular testing.
Subject(s)
Antibodies, Viral/blood , COVID-19 Vaccines/immunology , COVID-19/epidemiology , COVID-19/immunology , Pandemics , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Alberta , Asymptomatic Infections/epidemiology , COVID-19/prevention & control , Child , Child, Preschool , Female , Humans , Immunoglobulin G/blood , Infant , Infant, Newborn , Male , Middle Aged , Molecular Diagnostic Techniques , Prevalence , Seroepidemiologic Studies , Social Class , Young AdultABSTRACT
BACKGROUND: Hyponatremia is a common and under-recognized adverse drug reaction of selective serotonin re-uptake inhibitor (SSRI) antidepressants. Despite its clinical importance, there are few large-scale studies on the factors associated with hyponatremia. OBJECTIVE: The aim of this study was to determine the incidence of hyponatremia and to identify patient factors associated with hyponatremia in a large, population-based cohort initiating new prescriptions for citalopram. METHODS: We included all patients with a new prescription for citalopram during 2010-2017, inclusive, with baseline and post-initiation serum sodium values available. Data were obtained from an Alberta Health Pharmacy database to identify new citalopram prescriptions. Laboratory values for patients with new prescriptions were obtained from linked Calgary Laboratory Services data. Incident hyponatremia was defined as serum sodium level < 135 mmol/L, following prescription initiation. Associations were determined by performing Cox regression with time-varying covariate analysis, with the development of hyponatremia as the dependent variable. RESULTS: A total of 19,679 patients with new prescriptions were identified; 12,842 females and 6837 males. The mean age was 55.48 years (SD 21.35). Of these patients, 3250 (16.5%) developed hyponatremia, 1996 (15.5% of) females and 1254 (18.3% of) males (p = 0.002). Cox regression showed significant associations of hyponatremia with lower baseline sodium (HR 0.788), older age (HR 1.029), thiazide diuretic use (HR 1.141), and male sex (HR 1.168). Pharmaceutical manufacturer or strength of citalopram did not have significant effects on the development of hyponatremia. CONCLUSION: This study provides additional data on the predictors of hyponatremia among patients initiating citalopram therapy. We report a 16.5% incidence of hyponatremia after starting citalopram treatment, and significant new findings include a higher incidence in males. This is the first published incidence of hyponatremia following the initiation of citalopram treatment across all ages in Canada.
ABSTRACT
Key problems for seniors are their exposure to "potentially inappropriate medications" and "potential medication omissions", which place them at risk for moderate, severe, or fatal adverse drug reactions. This study of 82,935 first admissions to acute care hospitals in Calgary during 2013-2018 identified 294,160 Screening Tool of Older People's Prescriptions (STOPP) potentially inappropriate medications (PIMs) (3.55/patient), 226,970 American Geriatric Society (AGS) Beers PIMs (2.74/patient), 59,396 START potential prescribing omissions (PPOs) (0.72/patient), and 85,288 STOPP PPOs (1.03/patient) for which a new prescription corrected the omission. This represents an overwhelming workload to prevent inappropriate prescriptions continuing during the hospitalisation and then deprescribe them judiciously. Limiting scrutiny to the most frequent PIMs and PPOs will identify many moderate, severe, or fatal risks of causing adverse drug reactions (ADRs) but to identify all PIMs or PPO involving moderate or severe risks of ADRs also involves searching lower in the frequency list of patients. Deciding whether to use the STOPP or AGS Beers PIM lists is an important issue in searching for ADRs, because the Pearson correlation coefficient for agreement between the STOPP and AGS Beers PIM totals in this study was 0.7051 (95% CI 0.7016 to 0.7085; p < 0.001). The combined lists include 289 individual PIM medications but STOPP and AGS have only 159 (55%) in common. The AGS Beers lists include medications used in the US and STOPP/START those used in Europe. The AGS authors recommend using both criteria. The ideal solution to the problem is to implement carefully constructed Clinical Decision Support Systems (CDSS) as in the SENATOR trial, then for an experienced pharmacist to focus on the key PIMs and PPOs likely to lead to moderate, severe, or fatal ADRs. The pharmacist and key decision makers on the services need to establish a collegial relationship to discuss frequently changing the medications that place the patients at risk. Then, the remaining PIMs and PPOs that relate to chronic disease management can be discussed by phone with the family physician using the discharge summary, which lists the medications for potential deprescribing.
ABSTRACT
Polypharmacy with "potentially inappropriate medications" (PIMs) and "potential prescribing omissions" (PPOs) are frequent among those 65 and older. We assessed PIMs and PPOs in a retrospective study of 82,935 patients ≥ 65 during their first admission in the period March 2013 through February 2018 to the four acute-care Calgary hospitals. We used the American Geriatric Society (AGS) and STOPP/START criteria to assess PIMs and PPOs. We computed odds ratios (ORs) for key outcomes of concern to patients, their families, and physicians, namely readmission and/or mortality within six months of discharge, and controlled for age, sex, numbers of medications, PIMs, and PPOs. For readmission, the adjusted OR for number of medications was 1.09 (1.09-1.09), for AGS PIMs 1.14 (1.13-1.14), for STOPP PIMs 1.15 (1.14-1.15), for START PPOs 1.04 (1.02-1.06), and for START PPOs correctly prescribed 1.16 (1.14-1.17). For mortality within 6 months of discharge, the adjusted OR for the number of medications was 1.02 (1.01-1.02), for STOPP PIMs 1.07 (1.06-1.08), for AGS PIMs 1.11 (1.10-1.12), for START PPOs 1.31 (1.27-1.34), and for START PPOs correctly prescribed 0.97 (0.94-0.99). Algorithm rule mining identified an 8.772 higher likelihood of mortality with the combination of STOPP medications of duplicate drugs from the same class, neuroleptics, and strong opioids compared to a random relationship, and a 2.358 higher likelihood of readmission for this same set of medications. Detailed discussions between patients, physicians, and pharmacists are needed to improve these outcomes.
ABSTRACT
This data incorporates 2016 testing volumes ordered by family physicians and performed at Calgary Laboratory Services (CLS), the sole supplier of clinical laboratory services for the catchment area of the City of Calgary, Alberta, Canada. For each test, the mean number of tests ordered per patient was calculated over ordering Calgary physicians, along with arithmetic coefficients of variation (CV's). The latter parameter is reflective of variance in ordering practice among family physicians practicing in Calgary and is proposed as a benchmark measure for laboratory utilization in our accompanying research article [1]. The data table encompasses 358 tests ordered by at least 3 family physicians at a minimum total frequency of 100 within the 2016 study period and is presented in ascending order of rank in CV.
ABSTRACT
BACKGROUND: There is widespread variation in testing practice among practitioners, however there has been no objective way to pinpoint target tests for utilization management. We propose to take advantage of inter-physician variance in clinical practice as a quantitative measure to generate lists of potentially misutilized tests. METHODS: Testing frequencies from a database of clinical testing volumes for outpatients in Calgary, Canada, were obtained for the study period of 2016. For each chemistry, microbiology or hematology test, an arithmetic coefficient of variation (CV) was calculated from family physicians' ordering frequencies. RESULTS: The mean CV for all 358 tests considered was 219% (95% CI 206-231%) with a range of 52-729%. The highest variance was observed for human T-lymphotropic virus antibody testing and several tests for heavy metal levels (mercury, copper, zinc and chromium). Among the 100 most commonly run tests, high variance was found for several endocrinology tests including cortisol. CONCLUSIONS: The utility of ranking clinical tests by ordering variance presents a practical approach to evaluate relative variation in physician practice strategy and to identify potential areas of misutilization.
Subject(s)
Clinical Laboratory Techniques , Physicians, Family , Antibodies, Viral/blood , Canada , Humans , Metals, Heavy/bloodABSTRACT
Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. Moreover, the MIP-3α protein has been shown to display direct antimicrobial, antiviral and antiprotozoal activities. Because of the potential therapeutic uses of this protein, the efficient production of MIP-3α is of great interest. However, bacterial recombinant production of the MIP-3α protein has been limited by the toxicity of this extremely basic protein (pI 9.7) toward prokaryotic cells, and by solubility problems during expression and purification. In an attempt to overcome these issues, we have investigated the bacterial recombinant expression of MIP-3α by using several common expression and fusion tags, including 6× histidine (His), small ubiquitin modifier protein (SUMO), thioredoxin (TRX), ketosteroid isomerase (KSI), and maltose binding protein (MBP). We have also evaluated a recently introduced calmodulin (CaM)-tag that has been used for the effective expression of many basic antimicrobial peptides (AMPs). Here, we show that the CaM fusion tag system effectively expressed soluble MIP-3α in the cytoplasm of Escherichia coli with good yields. Rapid purification was facilitated by the His-tag that was integrated in the CaM-fusion protein system. Multidimensional nuclear magnetic resonance (NMR) studies demonstrated that the recombinant protein was properly folded, with the correct formation of disulfide bonds. In addition, the recombinant MIP-3α had antibacterial activity, and was shown to inhibit the formation of Pseudomonas aeruginosa biofilms.
ABSTRACT
OBJECTIVE: Acute lymphocytic leukemia (ALL) is a rare malignant neoplasm that develops from abnormal lymphoid stem cells. ALL incidence is highest among children and declines towards adolescence. There is limited data on the epidemiology of ALL, especially in Canada. This retrospective cohort study used patient data from the Calgary Laboratory Services Cancer Cytogenetics Laboratory to report the incidence rate of ALL in Calgary, Alberta, Canada. New cases of ALL were identified for the 5-year period of January 1, 2011 until December 31, 2015. Reported incidence rates were categorized by sex and age groups, and age-standardized to the Canadian population. RESULTS: There were an average of 11.4 new cases of ALL diagnosed per year between 2011 and 2015. The total incidence rate per 100,000 person-years was 0.84. Incidence rates peaked in children aged 0-4 with 7.55 and 3.32 cases per 100,000 person-years for males and females, respectively. The median age of diagnosis was 8 years. Incidence rates were generally lowest for adults aged 20 and over. The ratio of males to females diagnosed with ALL was 1.59. Overall, the recent incidence of ALL in Calgary is comparatively low with a preference for males and children below 5 years of age.
Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma/epidemiology , Adolescent , Adult , Aged , Aged, 80 and over , Alberta/epidemiology , Child , Child, Preschool , Female , Humans , Incidence , Infant , Male , Middle Aged , Retrospective Studies , Young AdultABSTRACT
OBJECTIVES: Serum iron is an important clinical test to help identify cases of iron deficiency or overload. Fluctuations caused by diurnal variation and diet are thought to influence test results, which may affect clinical patient management. We examined the impact of these preanalytical factors on iron concentrations in a large community-based cohort. DESIGN AND METHODS: Serum iron concentration, blood collection time, fasting duration, patient age and sex were obtained for community-based clinical testing from the Laboratory Information Service at Calgary Laboratory Services for the period of January 2011 to December 2015. RESULTS: A total of 276,307 individual test results were obtained. Iron levels were relatively high over a long period from 8:00 to 15:00. Mean concentrations were highest at blood collection times of 11:00 for adult men and 12:00 for adult women and children, however iron levels peaked as late as 15:00 in teenagers. With regard to fasting, iron levels required approximately 5h post-prandial time to return to a baseline, except for children and teenage females where no significant variation was seen until after 11h fasting. After 10h fasting, iron concentrations in all patient groups gradually increased to higher levels compared to earlier fasting times. CONCLUSIONS: Serum iron concentrations remain reasonably stable during most daytime hours for testing purposes. In adults, blood collection after 5 to 9h fasting provides a representative estimate of a patient's iron levels. For patients who have fasted overnight, i.e. ≥12h fasting, clinicians should be aware that iron concentrations may be elevated beyond otherwise usual levels.
Subject(s)
Circadian Rhythm , Fasting/blood , Iron Deficiencies , Iron Overload/blood , Iron/blood , Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , MaleABSTRACT
OBJECTIVES: The increasing cost of clinical laboratory testing is a challenge in our health care system. This study aims to calculate the annual clinical laboratory test costs attributed to patients in a major Canadian city and to correlate them to their sociodemographic variables. METHODS: Retrospective cohort study involving patients who received clinical chemistry, hematology, and microbiology tests in 2011 in Calgary, Canada (n = 610,409). Test volumes were obtained from a laboratory informatics database. Total expenditures per patient were calculated using estimated test costs and then combined with the 2011 Canadian Census Household Survey results to infer sociodemographic correlates. RESULTS: While more women received laboratory testing (58.4%), men had slightly higher testing costs per capita. Except for Chinese, visible minority and Aboriginal populations had higher testing costs. There was an inverse correlation between testing cost and household income, and accordingly, higher costs were found in those without postsecondary education and the unemployed. Furthermore, hotspot mapping revealed the geographical distribution of patient test costs within the city. CONCLUSIONS: There is variation in testing costs for patients among different sociodemographic variables.
Subject(s)
Clinical Laboratory Services/economics , Health Expenditures , Canada , Educational Status , Employment , Female , Humans , Income , Male , Retrospective Studies , Sex Factors , Socioeconomic FactorsABSTRACT
Friulimicin is a cyclic lipodecapeptide antibiotic that is produced by Actinoplanes friuliensis. Similar to the related lipopeptide drug daptomycin, the peptide skeleton of friulimicin is synthesized by a large multienzyme nonribosomal peptide synthetase (NRPS) system. The LipD protein plays a major role in the acylation reaction of friulimicin. The attachment of the fatty acid group promotes its antibiotic activity. Phylogenetic analysis reveals that LipD is most closely related to other freestanding acyl carrier proteins (ACPs), for which the genes are located near to NRPS gene clusters. Here, we report that the solution NMR structure of apo-LipD is very similar to other four-helix bundle forming ACPs from fatty acid synthase (FAS), polyketide synthase, and NRPS systems. By recording NMR dynamics data, we found that the backbone motions in holo-LipD are more restricted than in apo-LipD due to the attachment of phosphopantetheine moiety. This enhanced stability of holo-LipD was also observed in differential scanning calorimetry experiments. Furthermore, we demonstrate that, unlike several other ACPs, the folding of LipD does not depend on the presence of divalent cations, although the presence of Mg2+ or Ca2+ can increase the protein stability. We propose that small structural rearrangements in the tertiary structure of holo-LipD which lead to the enhanced stability are important for the cognate enzyme recognition for the acylation reaction. Our results also highlight the different surface charges of LipD and FAS-ACP from A. friuliensis that would allow the acyl-CoA ligase to interact preferentially with the LipD instead of binding to the FAS-ACP.
Subject(s)
Acyl Carrier Protein/chemistry , Anti-Bacterial Agents/biosynthesis , Bacterial Proteins/chemistry , Lipopeptides/biosynthesis , Micromonosporaceae/chemistry , Acyl Carrier Protein/metabolism , Bacterial Proteins/metabolism , Magnetic Resonance Spectroscopy , Micromonosporaceae/metabolism , Peptide Synthases/chemistry , Peptide Synthases/metabolism , Protein DomainsABSTRACT
Staphylokinase (Sak) is a plasminogen activator protein that is secreted by many Staphylococcus aureus strains. Sak also offers protection by binding and inhibiting specific antimicrobial peptides (AMPs). Here, we evaluate Sak as a more general interaction partner for AMPs. Studies with melittin, mCRAMP, tritrpticin and bovine lactoferricin indicate that the truncation of the first ten residues of Sak (SakΔN10), which occurs in vivo and uncovers important residues in a bulge region, improves its affinity for AMPs. Melittin and mCRAMP have a lower affinity for SakΔN10, and in docking studies, they bind to the N-terminal segment and bulge region of SakΔN10. By comparison, lactoferricin and tritrpticin form moderately high affinity 1:1 complexes with SakΔN10 and their cationic residues form several electrostatic interactions with the protein's α-helix. Overall, our work identifies two distinct AMP binding surfaces on SakΔN10 whose occupation would lead to either inhibition or promotion of its plasminogen activating properties.
Subject(s)
Antimicrobial Cationic Peptides/metabolism , Metalloendopeptidases/metabolism , Plasminogen/metabolism , Staphylococcus aureus/enzymology , Animals , Binding Sites , Cathelicidins/metabolism , Cattle , Lactoferrin/metabolism , Melitten/metabolism , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , Models, Molecular , Molecular Docking Simulation , Oligopeptides/metabolism , Protein Structure, Secondary , Staphylococcus aureus/chemistry , Staphylococcus aureus/genetics , Substrate SpecificityABSTRACT
In recent years, the increasing number of antibiotic-resistant bacteria has become a serious health concern. Antimicrobial peptides (AMPs) are an important component of the innate immune system of most organisms. A better understanding of their structures and mechanisms of action would lead to the design of more potent and safer AMPs as alternatives for current antibiotics. For detailed investigations, effective recombinant production which allows the facile modification of the amino acid sequence, the introduction of unnatural amino acids, and labeling with stable isotopes for nuclear magnetic resonance (NMR) studies is desired. Several expression strategies have been introduced in previous reports; however, their effectiveness has been limited to a select few AMPs. Here, we have studied calmodulin (CaM) as a more universal carrier protein to express many types of AMPs in E. coli. We have discovered that the unique architecture of CaM, consisting of two independent target binding domains with malleable methionine-rich interaction surfaces, can accommodate numerous amino acid sequences containing basic and hydrophobic residues. This effectively masks the toxic antimicrobial activities of many amphipathic AMPs and protects them from degradation during expression and purification. Here, we demonstrate the expression of various AMPs using a CaM-fusion expression system, including melittin, fowlicidin-1, tritrpticin, indolicidin, puroindoline A peptide, magainin II F5W, lactoferrampin B, MIP3α51-70, and human ß-defensin 3 (HBD-3), the latter requiring three disulfide bonds for proper folding. In addition, our approach was extended to the transmembrane domain of the cell adhesion protein l-selectin. We propose the use of the CaM-fusion system as a universal approach to express many cationic amphipathic peptides that are normally toxic and would kill the bacterial host cells.
Subject(s)
Antimicrobial Cationic Peptides/genetics , Antineoplastic Agents/metabolism , Calmodulin/genetics , Escherichia coli/genetics , Genetic Engineering , Membrane Proteins/genetics , Recombinant Fusion Proteins/genetics , Amino Acid Sequence , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/metabolism , Calmodulin/metabolism , Gene Expression , Humans , Models, Molecular , Protein DomainsABSTRACT
The Escherichia coli Fec system, consisting of an outer membrane receptor (FecA), a periplasmic substrate binding protein (FecB) and an inner membrane permease-ATPase type transporter (FecC/D), plays an important role in the uptake and transport of Fe(3+)-citrate. Although several FecB sequences from various organisms have been reported, there are no biophysical or structural data available for this protein to date. In this work, using isothermal titration calorimetry (ITC), we report for the first time the ability of FecB to bind different species of Fe(3+)-citrate as well as other citrate complexes with trivalent (Ga(3+), Al(3+), Sc(3+) and In(3+)) and a representative divalent metal ion (Mg(2+)) with low µM affinity. Interestingly, ITC experiments with various iron-free di- and tricarboxylic acids show that FecB can bind tricarboxylates with µM affinity but not biologically relevant dicarboxylates. The ability of FecB to bind with metal-free citrate is also observed in (1)H,(15)N HSQC-NMR titration experiments reported here at two different pH values. Further, differential scanning calorimetry (DSC) experiments indicate that the ligand-bound form of FecB has greater thermal stability than ligand-free FecB under all pH and ligand conditions tested, which is consistent with the idea of domain closure subsequent to ligand binding for this type of periplasmic binding proteins.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Ferric Compounds/metabolism , Ion Pumps/metabolism , Metals/metabolism , Periplasm/metabolism , Tricarboxylic Acids/metabolism , Amino Acids/metabolism , Calorimetry , Calorimetry, Differential Scanning , Citric Acid/pharmacology , Escherichia coli/drug effects , Kinetics , Ligands , Magnetic Resonance Spectroscopy , Nitrogen Isotopes , Periplasm/drug effects , Protein Binding/drug effects , Protein Stability/drug effectsABSTRACT
Tritrpticin is an antimicrobial peptide with a strong microbicidal activity against Gram-positive and Gram-negative bacteria as well as fungi. The 13-residue peptide is essentially symmetrical and possesses a unique cluster of three Trp residues near the center of its amino acid sequence. The mechanism of action of tritrpticin is believed to involve permeabilization of the cytoplasmic membrane of susceptible bacteria. However it has been suggested that intracellular targets may also play a role in its antimicrobial activity. In this work the mechanism of action of several tritrpticin derivatives was studied through substitution of the three Trp residues with 5-hydroxy-tryptophan (5OHW), a naturally occurring non-ribosomal amino acid. Although it is more polar, 5OHW preserves many of the biophysical and biochemical properties of Trp, allowing the use of fluorescence spectroscopy and NMR techniques to study the interaction of the modified peptides with membrane mimetics. Single or triple 5OHW substitution did not have a large effect on the MIC of the parent peptide against Escherichia coli and Bacillus subtilis. However, the mechanism of action was altered by simultaneously replacing all three Trp with 5OHW. Our results suggest that the inner membrane of Gram-negative bacteria did not constitute the main target of this particular tritrpticin derivative. Since the addition of a hydroxyl group to the indole motif of the Trp residue was able to modify the mechanism of action of the peptides, our data confirm the importance of the Trp cluster in tritrpticin. This work also shows that 5OHW constitutes a new probe to modulate the antimicrobial activity and mechanism of action of other Trp-rich antimicrobial peptides.
Subject(s)
Anti-Infective Agents/pharmacology , Cell Membrane/drug effects , Oligopeptides/pharmacology , Peptides/pharmacology , 5-Hydroxytryptophan , Bacteria/drug effects , Calorimetry, Differential Scanning , Circular Dichroism , DNA/chemistry , Magnetic Resonance Spectroscopy , Peptides/chemistry , Spectrometry, Fluorescence , Structure-Activity RelationshipABSTRACT
Antimicrobial peptides (AMPs) constitute promising candidates for the development of new antibiotics. Among the ever-expanding family of AMPs, tritrpticin has strong antimicrobial activity against a broad range of pathogens. This 13-residue peptide has an unusual amino acid sequence that is almost symmetrical and features three central Trp residues with two Arg residues near each end of the peptide. In this work, the role of the three sequential Trp residues in tritrpticin was studied in a systematic fashion by making a series of synthetic peptides with single-, double- and triple-Trp substitutions to Tyr or Ala. ¹H NMR and fluorescence spectroscopy demonstrated the ability of all of the tritrpticin-analog peptides to interact with negatively-charged membranes. Consequently, most tritrpticin analogs exhibited the ability to permeabilize synthetic ePC:ePG (egg-yolk phosphatidylcholine (ePC), egg-yolk phosphatidylglycerol (ePG)) vesicles and live Escherichia coli bacteria. The membrane perturbation characteristics were highly dependent on the location of the Trp residue substitution, with Trp6 being the most important residue and Trp8 the least. The membrane permeabilization activity of the peptides in synthetic and biological membranes was directly correlated with the antimicrobial potency of the peptides against E. coli. These results contribute to the understanding of the role of each of the three Trp residues to the antimicrobial activity of tritrpticin.
ABSTRACT
A number of physicochemical characteristics have been described which contribute to the biological activity of antimicrobial peptides. This information was used to design a novel antimicrobial peptide sequence by using an intrinsically inactive membrane-associated peptide derived from the HIV glycoprotein, gp41, as a starting scaffold. This peptide corresponds to the tryptophan-rich membrane-proximal region of gp41, which is known to interact at the interfacial region of the viral membrane and adopts a helical structure in the presence of lipids. Three synthetic peptides were designed to increase the net positive charge and amphipathicity of this 19-residue peptide. Ultimately, the peptide with the greatest degree of amphipathicity and largest positive charge proved to be the most potent antimicrobial, and this peptide could be further modified to improve the antimicrobial activity. However, the other two peptides were relatively ineffective antimicrobials and instead proved to be extremely hemolytic. This work demonstrates a novel approach for the design of unexplored antimicrobial peptide sequences but it also reveals that the biological and cytotoxic activities of these polypeptides depend on a number of interrelated factors.
ABSTRACT
We have recently reported a series of synthetic anticancer heptapeptides (H-KKWß(2,2) WKK-NH(2) ) containing a central achiral and lipophilic ß(2,2) -amino acid that display low toxicity against non-malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising ß(2,2) -amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC-5). The results demonstrated a considerable increase in anticancer potency following head-to-tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High-resolution NMR studies and molecular dynamics simulations together with an annexin-V-FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents.
Subject(s)
Amino Acids/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Burkitt Lymphoma/drug therapy , Peptides, Cyclic/pharmacology , Burkitt Lymphoma/pathology , Cell Line, Tumor , Cyclization , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Erythrocytes/drug effects , Fibroblasts/drug effects , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Dynamics Simulation , Molecular Structure , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Structure-Activity RelationshipABSTRACT
Chemokines are best known as signaling proteins in the immune system. Recently however, a large number of human chemokines have been shown to exert direct antimicrobial activity. This moonlighting activity appears to be related to the net high positive charge of these immune signaling proteins. Chemokines can be divided into distinct structural elements and some of these have been studied as isolated peptide fragments that can have their own antimicrobial activity. Such peptides often encompass the α-helical region found at the C-terminal end of the parent chemokines, which, similar to other antimicrobial peptides, adopt a well-defined membrane-bound amphipathic structure. Because of their relatively small size, intact chemokines can be studied effectively by NMR spectroscopy to examine their structures in solution. In addition, NMR relaxation experiments of intact chemokines can provide detailed information about the intrinsic dynamic behavior; such analyses have helped for example to understand the activity of TC-1, an antimicrobial variant of CXCL7/NAP-2. With chemokine dimerization and oligomerization influencing their functional properties, the use of NMR diffusion experiments can provide information about monomer-dimer equilibria in solution. Furthermore, NMR chemical shift perturbation experiments can be used to map out the interface between self-associating subunits. Moreover, the unusual case of XCL1/lymphotactin presents a chemokine that can interconvert between two distinct folds in solution, both of which have been elucidated. Finally, recent advances have allowed for the determination of the structures of chemokines in complex with glycosaminoglycans, a process that could interfere with their antimicrobial activity. Taken together, these studies highlight several different structural facets that contribute to the way in which chemokines exert their direct microbicidal actions.