As plantas possuem um arsenal de substâncias utilizadas como defesa contra patógenos e predadores. A possibilidade de utilizar tais substâncias como biopesticidas revolucionou o estudo das proteínas tóxicas. Proteínas inativadoras de ribossomos (RIPs) e as ureases estão entre as proteínas que são abundantes em plantas. RIPs tipo 2 como a ricina, são muito tóxicas, e podem despurinar ribossomos de várias espécies e induzir lesão em DNA, levando à interrupção da síntese protéica e morte de células. Menos tóxicas que a ricina, a maior parte das RIPs conhecidas são do tipo 1 com apenas uma cadeia polipeptídica de 25 - 32 kDa. As ureases (EC 3.5.1.5) são metaloenzimas dependentes de níquel, que catalisam a hidrólise da uréia para formar amônia e dióxido de carbono. A semente do feijão-de-porco, Canavalia ensiformis, é fonte rica de isoformas de urease, entre elas, a canatoxina (CNTX). A proteína CNTX apresenta atividade inseticida contra diferentes espécies de insetos, e sua toxicidade depende da liberação de um peptídeo interno de 10kDa (pepcanatox), que ocorre por ação das catepsinas do sistema digestivo dos insetos suscetíveis. Um peptídeo equivalente ao pepcanatox foi obtido por expressão heteróloga em Escherichia coli - Jaburetox-2Ec, o qual apresentou atividade tóxica contra Dysdercus peruvianus, Rhodnius prolixus e Blatella germanica. Nesse trabalho demonstramos a atividade inseticida de cinco RIPs tipo 1 (PAP-S, gelonina, momordina, saporina-S6 e licnina) em Spodoptera frugiperda e Anticarsia gemmatalis. As RIPs mostraram um efeito entomotóxico espécie-específico para as lagartas, sendo que momordina foi a menos tóxica nos bioensaios. Perda de peso mais pronunciada foi observada em S. frugiperda no 4° dia após o início dos ensaios e para a A. gemmatalis, no 10° dia. A indução da mortalidade (larval e/ou pupal) foi de 57,13% para os tratamentos em A. gemmatalis e 29,45% para S. frugiperda. Para investigar o efeito deterrente de RIPs tipo 1 em insetos, verificou-se, através do teste cometa, o nível de danos ao DNA em tecidos de S. frugiperda e A. gemmatalis que ingeriram um total de 40 μg de RIPs. Os insetos tratados com RIPs mostraram um valor 2 a 3 vezes maior de células com sinais de dano de DNA do que o controle. O dano de DNA poderia ser conseqüência do estresse oxidativo, assim analisou-se atividade de enzimas antioxidantes CAT e SOD e níveis de peroxidação lipídica (TBARS) nos extratos celulares dos insetos, mas não houve uma correlação entre dano de DNA e marcadores de estresse oxidativo. O peptídeo recombinante derivado de urease, jaburetox-2Ec, induziu uma mortalidade de 100% de S. frugiperda após ingestão de 47μg do peptídeo. Em contraste com os dados obtidos com as RIPs, o jaburetox-2Ec não causou lesões no DNA ou alterações em marcadores do balanço redox em S. frugiperda evidenciando um mecanismo de ação distinto. Em linhagens de células de insetos em cultura (Tn5B e UFL-AG-286), a análise citomorfológica sugeriu a ocorrência de citotoxicidade e lise celular com exposição a 80 e 10 μg do jaburetox-2Ec, após 4 e 7 dias de incubação, respectivamente. Para compreender a ação do peptídeo entomotóxico em células de insetos, realizou-se testes de citotoxicidade utilizando o kit CyTotox-GloP TM P incubando-se células UFL-AG-286 e Sf21 com jaburetox-2Ec. Os resultados com esse kit não foram conclusivos, sugerindo que o peptídeo recombinante seria capaz de inibir as proteases intracelulares liberadas na lise celular. Nossos resultados mostraram que RIPs tipo 1 e o jaburetox-2Ec têm efeito inseticida em lepidópteros por mecanismos distintos, sendo que as RIPs tipo 1 induzem lesão de DNA em A. gemmatalis e S. frugiperda, enquanto que jaburetox-2Ec induz alterações ainda não identificadas, que resultam em morte do inseto. / The plants have an arsenal of substances used as a defense against pathogens and predators. The possibility of using these substances as biopesticides revolutionized the study of toxic proteins. Ribosomes-inactivating proteins (RIPs) and ureases are among the proteins that are abundant in plants. Type 2 RIPS, such as ricin, are highly toxic and depurinate ribosomes of different species and induce DNA damage, arresting protein synthesis and leading to cell death. Less toxic, most of the known RIPs are type 1, composed of a single polypeptide chain of 25-32 kDa. Ureases (EC 3.5.1.5) are nickel dependent metalloenzymes that catalyze the hydrolysis of urea into ammonia and carbon dioxide. The seed of jackbean (Canavalia ensiformis) is a rich source of ureases isoforms, one of which is canatoxin (CNTX). The protein CNTX presents insecticidal activity and its toxicity relies on an internal ~10 kDa peptide (pepcanatox) released upon hydrolysis of CNTX by digestive cathepsins of susceptible insects. A peptide equivalent to pepcanatox obtained by heterologous expression in Escherichia coli - Jaburetox-2EC, showed insecticidal activity against Dysdercus peruvianus, Rhodnius prolixus and Blatella germanica. In this study we demonstrated the insecticidal activity of five type 1 RIPs (PAP-S, gelonin, momordin, saporin-S6 and lychnin) in Spodoptera frugiperda and Anticarsia gemmatalis. The entomotoxic effect of RIPs was species-specific and momordin was shown to be the less toxic in the bioassays. S. frugiperda had a more pronounced weight loss on the 4th day of treatment and A. gemmatalis on the 10th day. Mortality (larval and/or pupal) rate reached 57.13% for A. gemmatalis and 29.45% for S. frugiperda. To investigate the deterrent effect of type 1 RIPs in insects, the levels of DNA damage were evaluated using the comet test in tissues homogenates of S. frugiperda and A. gemmatalis fed a total of 40μg of RIPs. The RIPs-treated insects showed 2 to 3 times more cells with DNA damage, as compared to controls. To test whether DNA damage could be consequent to oxidative stress, the activity of the antioxidant enzymes SOD and CAT and levels of lipid peroxidation (TBARS) were assayed in cellular extracts of S. frugiperda and A. gemmatalis fed 40 μg RIPs, but no correlations were found between DNA damage and stress markers. The urease-derived recombinant peptide, jaburetox-2Ec, induced 100% mortality of S. frugiperda fed 47 μg peptide. In contrast to the results observed for type 1 RIPs, treatment of S. frugiperda with jaburetox-2Ec did not cause damage in DNA nor modifications in redox balance markers, indicating a distinct mechanism of action. Microscopic analysis of lepidopteran insect cells in culture (lines Tn5B and UFL-AG-286) suggested cytotoxicity and cell lysis induced by incubation with 80 and 10 μg jaburetox- 2Ec, after 4 and 7 days, respectively. Aiming to understand the mode of action of the entomotoxic peptide in insects cells, the CyTotox-GloP TM Pkit was used to detect cytotoxicity in UFL-AG-286 and Sf21 cells incubated with jaburetox-2Ec. Unexpectedly, the results were not conclusive since it appears that the recombinant peptide is able to inhibit the intracellular proteases released upon cell lysis, preventing the hydrolysis of fluorogenic substrate used in the kit. In conclusion, our results demonstrated that type 1 RIPs and jaburetox-2Ec are insecticidal to lepidopterans acting through distinct mechanisms. Thus type 1 RIPs induce DNA damage in A. gemmatalis and S. frugiperda, while jaburetox-2Ec induce yet to be identified physiological changes that lead to insect death.

Termination of protein synthesis involves the recognition of one of three stop codons (UAG, UAA or UGA) and hydrolysis of the nascent polypeptide chain from the peptidyl-tRNA on the ribosome. Unlike sense codons, which are decoded by aminoacyl-tRNAs, stop codons are decoded by proteins known as release factors. The decoding release factors occupy the same site as aminoacyl-tRNA, interacting directly with the stop codon at the decoding centre and inducing peptidyl-tRNA hydrolysis at the peptidyl transferase centre. Eubacteria have two codon-specific decoding release factors - RF1, which recognizes UAG and UAA, and RF2, which recognizes UGA and UAA. Biochemical studies identified two tripeptide 'anticodon' motifs, PXT in RF1 and SPF in RF2, which structural studies have shown occur in exposed loops (anticodon loops) on the surface of the proteins. Structures of isolated release factors show a compact 'closed' conformation whereas structures of release factors bound to the ribosome show them to be in a highly extended 'open' conformation. This suggests that a large conformational change in the release factor must take place upon or before binding to the ribosome. This transition has been invoked as a mechanism for how translational fidelity is maintained (Rawat et al, 2003), however, small angle X-ray scattering data from E. coli RF1 suggest the decoding release factors are also in the open conformation in solution challenging this mechanism. Mora et al. (2003a) presented evidence that swapping the anticodon loop of RF2 with that of RF1 switched the stop codon specificity of the release factor. Recent structures of the decoding release factors bound to the ribosome showed that there was a second structural element of the release factor, the tip of helix α5, involved in recognition of the first base of the stop codon. The objectives of this thesis were to investigate both the anticodon loop and the helix α5 region for their roles in stop codon recognition, and to investigate whether there is a conformational change in the release factors on binding to the ribosome. The anticodon loop was investigated using chimeras of E. coli RF1/RF2 and E. coli RF1/C. elegans mitochondrial RF1 (MRF1) within the anticodon loop. An RF1 variant containing the RF2-specific SPF tripeptide motif did not switch stop codon specificity showing that the tripeptide motifs are not sufficient determinants for the codon specificity of RF1 and RF2 as was originally proposed. Surprisingly repeating the complete swap of the RF1 anticodon loop to that of RF2 did not switch the stop codon specificity as claimed in Mora et al. (2003a). The studies in this thesis identified additional regions of the anticodon loop of the release factor that are important for stop codon recognition. Two of the RF1/RF2 anticodon loop variants produced showed altered codon specificity recognizing all three standard stop codons and the sense codon UGG. These variants provided unexpected insights into the mechanism of stop codon recognition and can explain why there are two release factors in eubacteria. The C. elegans MRF1 contains a novel anticodon loop that is shorter and lacks the classical PXT motif. E. coli RF1/C. elegans MRF1 chimeras showed that this anticodon loop could function in E. coli RF1 and maintain the same codon specificity. While size and sequence within the loop together are important for recognition clearly there is more than one way RF1-type release factors can recognize the UAG and UAA stop codons. Vertebrate mitochondria use four stop codons, two of the standard stop codons, UAA and UAG, and the reassigned arginine codons AGA and AGG. Two vertebrate mitochondrial release factors have been identified, mtRF1a and mtRF1 (renamed here mRF1[Canonical] and mRF1[Noncanonical]). Bioinformatic studies showed mRF1[C] had similar helix α5 and anticodon loop regions to classical RF1s. mRF1[NC] had different helix α5 and anticodon loop regions and was hypothesized to recognize the non-standard stop codons AGA and AGG. E. coli RF1/Human mRF1[NC] chimeras were constructed that showed that the helix α5 and anticodon loop regions are important for stop codon recognition. Nevertheless the chimeras showed poor activity at the AGA and AGG stop codons on E. coli 70S ribosomes suggesting that mRF1[NC] has evolved to function exclusively on 55S mitoribosomes. A release factor variant of RF2 was designed that had the potential to trap this E. coli factor in the closed conformation in solution by disulphide bond formation. The RF2 double cysteine variant was successfully expressed and purified. The disulphide bond between the two cysteines was detected directly by mass spectrometry in a high proportion of molecules, showing the closed form of RF2 exists in solution. The RF2 closed form variant was shown to have release activity concomitant with the proportion of the open form in the RF preparation showing that the conformational change is required for normal release factor function. Preliminary binding studies have suggested that the RF2 closed form variant can bind to the ribosome. The ability of the closed form of RF2 to bind to the ribosome allowed a mechanism of translational fidelity to be proposed from the studies in this thesis; the release factor would recognize the stop codon in the decoding centre and, if cognate, the conformational change would occur allowing peptidyl-tRNA hydrolysis.

"March, 2005" / Bibliography: leaves 144-155. / ix, 155 p. : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / "The aims of this thesis were to investigate potentially novel aspects of the regulation of B and morphogenetic gene expression in coliphage 186, in order to understand more fully how late gene expression is controlled in this phage, and how gene expression may be regulated in general. Three specific aims were pursued in this project: 1. to characterize E. coli mutants that appear to abolish 186 B protein activity; 2. to determine the role of replication for the provision of late functions during the phage lytic cycle; and 3. to determine the role of CI repression of the 186 B promoter." --p. 41. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Sciences, Discipline of Biochemistry, 2005

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Diane S. Herson, Dept. of Biological Sciences. Includes bibliographical references.

Translation initiation factor IF1 is a small, essential and ubiquitous protein factor encoded by a single infA gene in bacteria. Although several important functions have been attributed to IF1, the precise reason for its indispensability is yet to be defined. It is known that IF1 binds to the ribosomal A-site during initiation, where it primarily contacts ribosomal RNA (rRNA) and induces large scale conformational changes in the small ribosomal subunit. To shed more light on the function of IF1 and its interaction with the ribosome, we have employed a genetic approach to elucidate structure-function interactions between IF1 and rRNA. A selection has been used to isolate second site suppressor mutations in rRNA that restore the growth of a cold sensitive mutant IF1 with an arginine to leucine substitution in position 69 (R69L).  This yielded two classes of suppressors – one class that mapped to the processing stem of 23S rRNA – a transient structure important for proper maturation of 23S rRNA; and the other class to the functional sequence of 16S rRNA. Suppressor mutations in the processing stem of 23S rRNA were shown to disrupt efficient processing of 23S rRNA. In addition, we report that at least one of the manifestations of cold sensitivity associated with the mutant IF1 is at the level of ribosomal subunit association. These results led to a model whereby the cold sensitive R69L mutant IF1 results in aberrant ribosomal subunit association properties, while the 23S processing stem mutations indirectly suppress this effect by decreasing the pool of mature 50S subunits available for association.  Spontaneous suppressor mutations in 16S rRNA were diverse in position and phenotypic properties, but all mutations affected ribosomal subunit association, in most cases by directly decreasing the affinity of the 30S for 50S subunits. Site directed mutagenesis of select positions in 16S rRNA yielded additional suppressor mutations that were localized to the mRNA and streptomycin binding sites on the small ribosomal subunit. We suggest that the 16S rRNA suppressors occur in positions that affect the conformational dynamics brought about by IF1. Taken together, this work indicates that the major function of IF1 is the modulation of ribosomal subunit association brought about through conformational changes of the 30S subunit. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Many studies have attempted to link antimicrobial use in food animal agriculture with an increased risk of antimicrobial-resistant (AR) bacterial levels in humans. Our data arise from longitudinal aggregated fecal samples in a 3-year cohort study of vertically integrated populations of human workers and consumers, and swine. Human and swine E. coli isolates (N = 2130 and 3485, respectively) were tested for antimicrobial susceptibility using the SensititreTM broth microdilution system. The associations between AR prevalence for each antimicrobial agent, multi-drug resistant E. coli, or multivariate AR E. coli, and the risk factors (host species, production type (swine), vocation (human swine worker versus non-worker), and season) in the study were assessed using generalized estimating equations (GEE), GLM with multinomial distribution, or GEE in a multivariate model using a SAS® macro to adjust for the correlated AR phenotypes. There were significant (p < 0.05) differences in AR isolates: 1) between host-species with swine at higher risk for ceftiofur, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. The prevalence of ciprofloxacin, nalidixic acid, and trimethoprim/sulfamethoxazole resistance were higher among human isolates, 2) swine production group was significantly associated with AR with purchased boars, nursery piglets, and breeding boars at a higher risk of resistance to streptomycin and tetracycline, and 3) human swine worker cohorts exhibited an elevated tetracycline prevalence, but lowered sulfisoxazole prevalence when compared to nonworkers. High variability among seasonal samples over the 3-year period was observed. There were significant differences in multiple resistance isolates between host species, with swine at higher risk than humans of carrying multi-resistant strains; however, no significant differences in multiple resistance isolates within humans by vocation or within swine by production group. The odds-ratios, adjusted for multivariate dependence of individual AR phenotypes, were increased relative to unadjusted oddsratios among 1) swine as compared to human for tetracycline (OR = 21.8 vs. 19.6), and 2) increased significantly among swine-workers as compared to non-workers only for tetracycline (OR = 1.4 vs. 1.3). Occupational exposure to swine-rearing facilities appears to be associated with an increased relative odds for the prevalence of tetracycline resistance compared to non-workers.

Fecal contamination is the pollution caused by the microorganisms residing in the intestine of warm blooded animals and humans. Bacteria are the prime cause of contamination of surface waters in the US. The transport of microorganisms into waterways can have detrimental effects on water quality and human health especially if the pathogenic strains are ingested. E. coli is used as an indicator of fecal contamination. Detection of these bacteria in a water body above set limits poses a potential health hazard. Various sources contribute to the bacterial contamination of a water body. The sources need to be identified and quantified for their E. coli content to measure bacteria loads in the waterbody accurately. In many cases, in-situ re-growth is also believed to be a considerable source of E. coli. Also re-growth of E. coli in landscapes due to favorable environmental conditions (e.g., rainfall after dry weather conditions) is one of the major phenomena affecting E. coli concentration in streams. Thus the environmental factors like temperature and soil moisture that influence transport, persistence, re-growth, and survival of E. coli in landscapes were studied. The objective of this study was to identify, characterize and quantify E. coli loads from feces of four different animals and monitor survival, growth and re-growth at four different temperatures and moisture contents over a period of seven days. Findings of this research will aid in Watershed Protection Plan (WPP) development and Total Maximum Daily Load (TMDL) development to address impairment from point and non-point source pollution of E. coli. Wildlife and range cattle manure samples responsible for fecal contamination of Cedar Creek were identified and four fecal sources out of those were quantified for the E. coli concentrations. No significant difference was found upon comparing the E. coli concentration for each species between the genders. Sub-adult cattle demonstrated significantly higher E. coli concentrations than adult cattle. Growth and die-off rates were measured at different temperatures (0degreesC, 10degreesC, 25degreesC, and 50degreesC) and moisture conditions (1%, 25% 56.5% and 83%). E. coli concentrations in cattle and raccoons feces showed highest survivability and growth at 20degreesC out of all the temperatures studied. There was no survival of E. coli from either species at 50degreesC after 24 h. E. coli in cattle and raccoons samples exhibited greater growth at lower, nearly aerobic soil moisture content (25%) for all days compared to nearly anaerobic soil moisture content (83%).

Free-ranging wildlife are an important contributor of fecal pollution in the form of Escherichia coli (E. coli) to water bodies. Currently, details of this contribution are nebulous and understudied. Much of the related research has not focused on freeranging wildlife; instead investigations examine entire systems while estimating wildlife contribution indirectly or with data of inconsistent quality and source. I began my research by conducting a meta-analysis of existing research to determine the current state of knowledge of wildlife’s specific contribution. Data were sparse, fragmented, of variable quality, and difficult to access. Researchers relied on a variety of outside sources (e.g., state natural resource agencies). Making comparison between studies was nearly impossible because methodologies differed greatly or were described inconsistently. I then calculated wildlife population densities, undertook fecal collection, and conducted spatial analyses of fecal deposition to gather accurate and relevant data of the study area. I augmented field data collection with data derived from my meta-analysis (i.e., fecal deposition rates). I was able to estimate the relative role of individual species (e.g., raccoons [Procyon lotor], white-tailed deer [Odocoileus virginianus], and feral hogs [Sus scrofa]). Finally, I created a model using these data to determine important parameters for future research (e.g., fecal deposition rates) and simulate various management strategies. Although all parameters need more research focus, I found defecation rates were especially important but little researched. I found raccoons were the greatest determiner of potential E. coli load in the floodplain though adjustment of other parameters would greatly impact these findings.

This study was conducted to determine if a relationship exists between temperament, stress response, and the shedding of Escerhichia coli O157:H7. Cattle (n = 150) were evaluated for disposition and stress response before shipping to the feeding operation, upon arrival at the feedlot, at approximately 70d on feed, and prior to transport to the harvesting facility. Chute and pen scores, as well as serum cortisol concentrations, were measured in order to assess individual temperament and stress response. A temperament index was created to classify cattle as Excitable, Intermediate, or Calm. The presence of E. coli O157:H7 was determined by rectal swabs on the live cattle and swabs of colons collected postmortem at the processing facility. As expected, variables for pre-shipment temperament index, exit velocity, pen score, arrival and midpoint exit velocity, and mid-point cortisol concentrations differed (P < 0.05) greatly between temperament groups. However, pre-shipment chute scores and cortisol concentration, as well as arrival and final cortisol concentrations differed (P < 0.05) only for Excitable cattle compared to both Calm and Intermediate groups. The percentage of cattle shedding the pathogen at arrival was approximately equal between temperament groups. When sampled before shipment to the processing facility, a higher proportion (P = 0.03) of cattle displaying Calm temperaments shed E. coli O157:H7 than the other groups. Results from postmortem colon samples exhibited a similar trend. When the results from all four sampling periods were pooled, the Calm cattle had a greater numerical percentage test positive for E. coli O157:H7. However, the pooled frequency distribution is largely dictated by the results of the final sampling time. Based on these results, it appears that Excitable cattle are not more likely to shed E. coli O157:H7. In fact, it seems that Calm cattle may be equally or more susceptible to shed at later points in the feeding period. However, it is important to note that a relatively small number of the samples tested positive for E. coli O157:H7, thus, potentially causing dramatic changes in the distributions.

Escherichia coli enteropatogéna (EPEC) se encuentra entre una de las principales causantes de diarrea en niños de países en vías de desarrollo, estas bacterias pueden ser calsificadas en típicas (tEPEC) y atípicas (aEPEC) dependiendo de la presencia o ausencia de un plásmido llamado EAF (E. coli adherence factor), respectivamente. El objetivo de este estudio fue describir la diversidad alélica de genes de virulencia de EPEC, el fenotipo, y su relación con las características clínicas. Se analizaron 120 cepas de EPEC aisladas de un estudio de cohorte en niños menores de un año en Lima. Utilizando la técnica de PCR-RFLP se caracterizaron los alelos de los genes eae (intimina), bfpA (proteína bundlina del bundle-forming pilus) y perA (plasmid encoded regulator). El fenotipo de EPEC fue evaluado mediante la precipitacion de proteínas secretadas (EspA, EspB, EspD y EspC), el ensayo de adherencia y el ensayo de polimerización de actina (FAS). Las cepas aEPEC (eae+, bfp-) fueron más comunes, tanto en casos de diarrea (54/74, 73%) como en controles (40/46, 87%). Del total de cepas evaluadas se encontraron 13 alelos del gen eae; los más frecuentes fueron: beta (34/120, 28%), theta (24/120, 20%), kappa (14/120, 12%) y mu (8/120, 7%). Se encontraron 5 alelos de bfpA; los más frecuentes fueron: beta1/7 (10/26), alpha (7/26) y beta5 (3/26). El alelo gamma del gen eae fue el más frecuente entre los episodios de diarrea de larga duración (> 7 días) que en los de menos duración (3/26, 12% vs. 0/48, 0%, p menor 0.05). Por otro lado, el alelo kappa del gen eae está relacionado con un puntaje cliníco más severos en comparación a otros alelos (p menor 0.05). De los cuatro patrones de adherencia encontrados LA (adherencia localizada); LAL (similar a la LA); DA (adherencia difusa) y IS/NA (bacterias aisladas/no adherencia), el patrón LA se encontró muy frecuente en las tEPEC (eae+, bfpA+) versus las aEPEC dentro de los casos de diarrea (6/8, 75% vs. 2/38, 5%, p menor 0.05), mientras el patrón IS/NA estuvo relacionado a las aEPEC versus las tEPEC en los casos de diarrea (26/38, 68% vs. 1/8, 13%, p menor 0.05). Se encontró que la polimerización de actina estuvo más relacionada a las tEPEC que a las cepas aEPEC en los casos de diarrea (6/8, 75% vs. 1/38, 3%, p menor 0.05). / Enteropathogenic Escherichia coli (EPEC) strains are amongst the major causes of infantile diarrhea in developing countries and can be classified as typical (tEPEC) atypical (aEPEC), depending on the presence or absence of the E. coli adherence factor plasmid (pEAF), respectively. The aim of this study was to describe the allelic diversity of critical virulence EPEC genes, phenotype; and the association of these results with clinical characteristics. 120 EPEC strains isolated from a cohort diarrhea study in Peruvian children were characterized for the allele of eae (intimin), bfpA (bundling pilin protein of bundle-forming pilus) and perA (plasmid encoded regulator) genes by PCR-restriction fragment length polymorphism. The EPEC phenotype was evaluated using, the protein precipitation assay (EspA, EspB, EspD y EspC), the adherence and the actin polymerization assay (FAS). aEPEC strains (eae+, bfp-) were the most common pathotype in diarrhea (54/74, 73%) and control samples from children without diarrhea (40/46, 87%). Overall there were 13 eae alleles; the most common were beta (34/120, 28%), theta (24/120, 20%), kappa (14/120, 12%) and mu (8/120, 7%). There were 5 bfpA alleles; the most common were beta1/7 (10/26), alpha3 (7/26) and beta5 (3/26). There were 3 perA alleles: beta (8/16), alpha (7/16) and gamma (1/16). The gamma-intimin allele was more frequently found in diarrhea episodes of longer duration (>7 days) than shorter (3/26, 12% vs. 0/48, 0%, p less than 0.05). The kappa-intimin allele had the highest clinical severity score in comparison to other alleles (p less than 0.05). Of the four adherence patterns found: LA (localized adherence); LAL (LA-like); DA (diffuse adherence) and IS/NA (isolated strain/ not adherence); LA pattern was more related to tEPEC (eae+, bfpA+) than aEPEC in diarrhea cases (6/8, 75% vs. 2/38, 5%, p less than 0.05). The IS/NA pattern was related to aEPEC compared to tEPEC in diarrhea cases (26/38, 68% vs. 1/8, 13%, p less than 0.05). We found that the actin polimerization (P) was significantly associated to tEPEC in both, diarrhea (6/8, 75% vs. 1/38, 3%, p less than0.05).