Rapid Evolution of the Sequences and Gene Repertoires

Proteins secreted to the extracellular environment or to the periphery of the cell envelope, the secretome, play essential roles in foraging, antagonistic and mutualistic interactions. We hypothesize that arms races, genetic conflicts and varying selective pressures should lead to the rapid change of sequences and gene repertoires of the secretome. The analysis of 42 bacterial pan-genomes shows that secreted, and especially extracellular proteins, are predominantly encoded in the accessory genome, i.e. among genes not ubiquitous within the clade. Genes encoding outer membrane proteins might engage more frequently in intra-chromosomal gene conversion because they are more often in multi-genic families. The gene sequences encoding the secretome evolve faster than the rest of the genome and in particular at non-synonymous positions. Cell wall proteins in Firmicutes evolve particularly fast when compared with outer membrane proteins of Proteobacteria. Virulence factors are over-represented in the secretome, notably in outer membrane proteins, but cell localization explains more of the variance in substitution rates and gene repertoires than sequence homology to known virulence factors. Accordingly, the repertoires and sequences of the genes encoding the secretome change fast in the clades of obligatory and facultative pathogens and also in the clades of mutualists and free-living bacteria. Our study shows that cell localization shapes genome evolution. In agreement with our hypothesis, the repertoires and the sequences of genes encoding secreted proteins evolve fast. The particularly rapid change of extracellular proteins suggests that these public goods are key players in bacterial adaptation.

Evolution of Drug Resistance: Insight on TEM β-Lactamases Structure and Activity and β-Lactam Antibiotics

Since the discovery of the first penicillin bacterial resistance to β-lactam antibiotics has spread and evolved promoting new resistances to pathogens. The most common mechanism of resistance is the production of β-lactamases that have spread thorough nature and evolve to complex phenotypes like CMT type enzymes. New antibiotics have been introduced in clinical practice, and therefore it becomes necessary a concise summary about their molecular targets, specific use and other properties. β-lactamases are still a major medical concern and they have been extensively studied and described in the scientific literature. Several authors agree that Glu166 should be the general base and Ser70 should perform the nucleophilic attack to the carbon of the carbonyl group of the β-lactam ring. Nevertheless there still is controversy on their catalytic mechanism. TEMs evolve at incredible pace presenting more complex phenotypes due to their tolerance to mutations. These mutations lead to an increasing need of novel, stronger and more specific and stable antibiotics. The present review summarizes key structural, molecular and functional aspects of ESBL, IRT and CMT TEM β-lactamases properties and up to date diagrams of the TEM variants with defined phenotype. The activity and structural characteristics of several available TEMs in the NCBI-PDB are presented, as well as the relation of the various mutated residues and their specific properties and some previously proposed catalytic mechanisms.

Computational design with flexible backbone sampling for protein remodeling and scaffolding of complex binding sites

Dissertation presented to obtain the Doutoramento (Ph.D.) degree in Biochemistry at the Instituto de Tecnologia Qu mica e Biol ogica da Universidade Nova de Lisboa

REPRODUCIBILITY OF ALKALINE ANTIGENS OF Leishmania major-LIKE AND Leishmania (V.) braziliensis EVALUATED BY IgG-ELISA. COMPARISON OF ANTIGENS ADDED OF A PROTEIN INHIBITOR (PMSF) OR NOT

This paper deals with the analysis of 10 batches of L.major-like and L.(V.) braziliensis antigens added or not of a proteases inhibitor evaluated by means of an IgG-ELISA on three consecutive days using positive standard sera from patients with diagnosis of American Leishmaniasis previously tested for the presence of IgG antibodies by means of ELISA. The statistical analysis showed that for L. (V.) braziliensis the PMSF-containing antigen did not show any difference among batches or days of testing; the L.(V.) braziliensis antigen without PMSF showed statistical significance for differences among batches and a two-way ANOVA showed significant differences between antigens. L.major-like antigen prepared with or without PMSF showed differences among batches; all 3 days of testing displayed differences for the PMSF antigen but only for days 1 and 2 for the antigen without inhibitor. A two-way ANOVA showed differences among batches of the antigens but not for antigens with and without the protein inhibitor. According to the statistical analysis the L.major-like antigen added or not of PMSF has shown that it is the choice antigen for mucocutaneous leishmaniasis serology.

The Crystal Structure of the Escherichia coli Autoinducer-2 Processing Protein LsrF

PLOS ONE, 4(8):ARTe6820

INFLUENCE OF DIETARY PROTEIN CONTENT ON Trypanosoma cruzi INFECTION IN GERMFREE AND CONVENTIONAL MICE

Germfree (GF) and conventional (CV) mice were fed on diets containing 4.4, 13.2 or 26.4% of protein (weight/weight). CV mice fed on low protein diet did not gain weight during four weeks, whereas the protein deficient diet did not affect the growth of GF mice. After four weeks on these diets, the mice were inoculated with 5x103 trypomastigotes of Trypanosoma cruzi. The protein deficiency affected less the GF than the CV mice, according to the following parameters: weight gain, hemoglobin, plasma protein and albumin levels and water and protein contents of the carcass. Infection with T. cruzi produced a significant decrease in hemoglobin levels, red blood cell count, and water and protein contents in the carcass. This decrease was more pronounced in the GF mice. Histopathologically, there was no difference between the treatments in animals with the same microbiological status (GF or CV). However, the disease was more severe in the GF than in the CV mice.

Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT<1), group B (EIA > or = 3, ALT>1), group C (1<=EIA<3, ALT<1), group D (1<=EIA<3, ALT>1) and group E (EIA<=0.7). HCV sequences were detected by RT-nested PCR, using primers for the most conserved region of viral genome. RIBA-2 was applied to the same samples. In group A (n=6), all samples were positive by RT-nested PCR and RIBA-2. Among 124 samples in group B, 120 (96.8%) were RIBA-2 positive and 4 (3.2%) were RIBA-2 indeterminate but were seropositive for antigen c22.3. In group B, 109 (87.9%) of the RIBA-2 positive samples were also RT-nested PCR positive, as well as were all RIBA-2 indeterminate samples. In group C, all samples (n=9) were RT-nested PCR negative: 4 (44.4%) were also RIBA-2 negative, 4 (44.4%) were RIBA-2 positive and 1 (11.1%) was RIBA-2 indeterminate. HCV-RNA was detected by RT-nested PCR in 3 (37.5%) out of 8 samples in group D. Only one of them was also RIBA-2 positive, all the others were RIBA-2 indeterminate. All of the group E samples (controls) were RT- nested PCR and RIBA-2 negative. Our study suggests a strong relation between anti-HCV EIA-2 ratio > or = 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.

The genetic basis of morphological change in convergent evolution of natural populations: identifying candidate genes behind convergent evolution in blind cavefish astyanax mexicanus

Dissertation presented to obtain the Ph.D. degree in Biology at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa.

Mitosis and protein Na-terminal acetylation

Dissertation presented to obtain the Ph.D degree in Developmental Biology

Evaluation of a rapid dipstick test, Malar-CheckTM, for the diagnosis of Plasmodium falciparum malaria in Brazil

The present study was carried out to evaluate the Malar-CheckTM Pf test, an immunochromatographic assay that detects Plasmodium falciparum Histidine Rich Protein II, does not require equipment, and is easy and rapid to perform. In dilution assays performed to test sensitivity against known parasite density, Malar-CheckTMwere compared with thick blood smear (TBS), the gold standard for diagnosis. Palo Alto isolate or P. falciparum blood from patients with different parasitemias was used. The average cut-off points for each technique in three independent experiments were 12 and 71 parasites/mm³ (TBS and Malar-CheckTM, respectively). In the field assays, samples were collected from patients with fever who visited endemic regions. Compared to TBS, Malar-CheckTMyielded true-positive results in 38 patients, false-positive results in 3, true-negative results in 23, and false-negative result in 1. Malar-CheckTMperformed with samples from falciparum-infected patients after treatment showed persistence of antigen up to 30 days. Malar-CheckTM should aid the diagnosis of P. falciparum in remote areas and improve routine diagnosis even when microscopy is available. Previous P. falciparum infection, which can determine a false-positive test in cured individuals, should be considered. The prompt results obtained with the Malar-CheckTM for early diagnosis could avoid disease evolution to severe cases.

NMR studies of transiente protein complexes

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica,especialidade Bioquímica Física,pela Universidade Nova de Lisboa,Faculdade de Ciências e Tecnologia

Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%.

Protein-responsive polymers for point-of-care detection of cardiac biomarker

This work describes a novel use for the polymeric film, poly(o-aminophenol) (PAP) that was made responsive to a specific protein. This was achieved through templated electropolymerization of aminophenol (AP) in the presence of protein. The procedure involved adsorbing protein on the electrode surface and thereafter electroploymerizing the aminophenol. Proteins embedded at the outer surface of the polymeric film were digested by proteinase K and then washed away thereby creating vacant sites. The capacity of the template film to specifically rebind protein was tested with myoglobin (Myo), a cardiac biomarker for ischemia. The films acted as biomimetic artificial antibodies and were produced on a gold (Au) screen printed electrode (SPE), as a step towards disposable sensors to enable point-of-care applications. Raman spectroscopy was used to follow the surface modification of the Au-SPE. The ability of the material to rebind Myo was measured by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The devices displayed linear responses to Myo in EIS and SWV assays down to 4.0 and 3.5 μg/mL, respectively, with detection limits of 1.5 and 0.8 μg/mL. Good selectivity was observed in the presence of troponin T (TnT) and creatine kinase (CKMB) in SWV assays, and accurate results were obtained in applications to spiked serum. The sensor described in this work is a potential tool for screening Myo in point-of-care due to the simplicity of fabrication, disposability, short time response, low cost, good sensitivity and selectivity.

Smart Plastic Antibody Material (SPAM) tailored on disposable screen printed electrodes for protein recognition: application to Myoglobin detection

This work introduces two major changes to the conventional protocol for designing plastic antibodies: (i) the imprinted sites were created with charged monomers while the surrounding environment was tailored using neutral material; and (ii) the protein was removed from its imprinted site by means of a protease, aiming at preserving the polymeric network of the plastic antibody. To our knowledge, these approaches were never presented before and the resulting material was named here as smart plastic antibody material (SPAM). As proof of concept, SPAM was tailored on top of disposable gold-screen printed electrodes (Au-SPE), following a bottom-up approach, for targeting myoglobin (Myo) in a point-of-care context. The existence of imprinted sites was checked by comparing a SPAM modified surface to a negative control, consisting of similar material where the template was omitted from the procedure and called non-imprinted materials (NIMs). All stages of the creation of the SPAM and NIM on the Au layer were followed by both electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). AFM imaging was also performed to characterize the topography of the surface. There are two major reasons supporting the fact that plastic antibodies were effectively designed by the above approach: (i) they were visualized for the first time by AFM, being present only in the SPAM network; and (ii) only the SPAM material was able to rebind to the target protein and produce a linear electrical response against EIS and square wave voltammetry (SWV) assays, with NIMs showing a similar-to-random behavior. The SPAM/Au-SPE devices displayed linear responses to Myo in EIS and SWV assays down to 3.5 μg/mL and 0.58 μg/mL, respectively, with detection limits of 1.5 and 0.28 μg/mL. SPAM materials also showed negligible interference from troponin T (TnT), bovine serum albumin (BSA) and urea under SWV assays, showing promising results for point-of-care applications when applied to spiked biological fluids.