Human and Veterinary Vaccines against Pathogenic <em>Escherichia coli</em>

PathogenicEscherichia coli constitute an important current problem of public health and animal production. Efforts have been made to fight the infections caused by these bacteria, and in this chapter, we present the progress made up to date in the vaccines generated for this purpose. Different vaccines have been tested against the pathotypes responsible for human diseases such as diarrhea and urinary infections. Also, the poultry market has deserved the effort of the researchers to obtain a product that fights the E. coli strains that cause diseases in them. Finally, advances are also presented for the zoonotic enterohemorrhagic E. coli (EHEC), which are a different problem due to their low importance as a disease factor in cattle, but they are a very important pathogen in humans. In several of these fields, authorized products have been developed and are currently being marketed

Designed Coiled-Coil Peptides Inhibit the Type Three Secretion System of Enteropathogenic Escherichia coli

BACKGROUND: Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry. METHODS/PRINCIPAL FINDINGS: We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled-coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments. CONCLUSIONS: Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis

Identification of two proteins that interact with the Erp virulence factor from Mycobacterium tuberculosis by using the bacterial two-hybrid system

<p>Abstract</p> <p>Background</p> <p>The exported repetitive protein (<it>erp</it>) gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS) repeats. It has been demonstrated that <it>erp </it>is a virulence-associated factor since the disruption of this gene impairs the growth of <it>Mycobacterium bovis </it>and <it>Mycobacterium tuberculosis </it>in mice.</p> <p>Results</p> <p>In order to elucidate the function of Erp we searched for Erp-binding proteins from <it>M. tuberculosis </it>by using a bacterial two-hybrid system. Our results indicate that Erp interacts specifically with two putative membrane proteins, Rv1417 and Rv2617c. Further analysis revealed that the latter two interact with each other, indicating that Rv1417, Rv2617c and Erp are connected through multiple interactions. While Rv1417 is disseminated in several <it>Actinomycetales </it>genera, orthologues of Rv2617c are exclusively present in members of the <it>M. tuberculosis </it>complex (MTC). The central and amino-terminal regions of Erp were determined to be involved in the interaction with Rv1417 and Rv2627c. Erp forms from <it>Mycobacterium smegmatis </it>and <it>Mycobacterium leprae </it>were not able to interact with Rv2617c in two-hybrid assays. Immunolocalization experiments showed that Rv1417 and Rv2617c are found on the cell membrane and Erp on the bacterial cell wall. Finally, comparative genomics and expression studies revealed a possible role of Rv1417 in riboflavin metabolism.</p> <p>Conclusion</p> <p>We identified interactive partners of Erp, an <it>M. tuberculosis </it>protein involved in virulence, which will be the focus of future investigation to decipher the function of the Erp family protein.</p

Study of the role of Mce3R on the transcription of mce genes of Mycobacterium tuberculosis

<p>Abstract</p> <p>Background</p> <p><it>mce3 </it>is one of the four virulence-related <it>mce </it>operons of <it>Mycobacterium tuberculosis</it>. In a previous work we showed that the overexpression of Mce3R in <it>Mycobacterium smegmatis </it>and <it>M. tuberculosis </it>abolishes the expression of <it>lacZ </it>fused to the <it>mce3 </it>promoter, indicating that Mce3R represses <it>mce3 </it>transcription.</p> <p>Results</p> <p>We obtained a knockout mutant strain of <it>M. tuberculosis </it>H37Rv by inserting a hygromycin cassette into the <it>mce3R </it>gene. The mutation results in a significant increase in the expression of <it>mce3 </it>genes either <it>in vitro </it>or in a murine cell macrophages line as it was determined using promoter-<it>lacZ </it>fusions in <it>M. tuberculosis</it>. The abundance of <it>mce1</it>, <it>mce2 </it>and <it>mce4 </it>mRNAs was not affected by this mutation as it was demonstrated by quantitative RT-PCR. The <it>mce3R </it>promoter activity in the presence of Mce3R was significantly reduced compared with that in the absence of the regulator, during the <it>in vitro </it>culture of <it>M. tuberculosis</it>.</p> <p>Conclusion</p> <p>Mce3R repress the transcription of <it>mce3 </it>operon and self regulates its own expression but does not affect the transcription of <it>mce1</it>, <it>mce2 </it>and <it>mce4 </it>operons of <it>M. tuberculosis</it>.</p

Genomic analysis of shiga toxin-containing Escherichia coli O157:H7 isolated from Argentinean cattle

Cattle are the main reservoir of Enterohemorrhagic Escherichia coli (EHEC), with O157:H7 the distinctive serotype. EHEC is the main causative agent of a severe systemic disease, Hemolytic Uremic Syndrome (HUS). Argentina has the highest pediatric HUS incidence worldwide with 12–14 cases per 100,000 children. Herein, we assessed the genomes of EHEC O157:H7 isolates recovered from cattle in the humid Pampas of Argentina. According to phylogenetic studies, EHEC O157 can be divided into clades. Clade 8 strains that were classified as hypervirulent. Most of the strains of this clade have a Shiga toxin stx2a-stx2c genotype. To better understand the molecular bases related to virulence, pathogenicity and evolution of EHEC O157:H7, we performed a comparative genomic analysis of these isolates through whole genome sequencing. The isolates classified as clade 8 (four strains) and clade 6 (four strains) contained 13 to 16 lambdoid prophages per genome, and the observed variability of prophages was analysed. An inter strain comparison show that while some prophages are highly related and can be grouped into families, other are unique. Prophages encoding for stx2a were highly diverse, while those encoding for stx2c were conserved. A cluster of genes exclusively found in clade 8 contained 13 genes that mostly encoded for DNA binding proteins. In the studied strains, polymorphisms in Q antiterminator, the Q-stx2A intergenic region and the O and P γ alleles of prophage replication proteins are associated with different levels of Stx2a production. As expected, all strains had the pO157 plasmid that was highly conserved, although one strain displayed a transposon interruption in the protease EspP gene. This genomic analysis may contribute to the understanding of the genetic basis of the hypervirulence of EHEC O157:H7 strains circulating in Argentine cattle. This work aligns with other studies of O157 strain variation in other populations that shows key differences in Stx2a-encoding prophages

Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov.

A comparison of Mycobacterium tuberculosis complex isolates from seals (pinnipeds) in Australia, Argentina, Uruguay, Great Britain and New Zealand was undertaken to determine their relationships to each other and their taxonomic position within the complex. Isolates from 30 cases of tuberculosis in six species of pinniped and seven related isolates were compared to representative and standard strains of the M. tuberculosis complex. The seal isolates could be distinguished from other members of the M. tuberculosis complex, including the recently defined ‘Mycobacterium canettii’ and ‘Mycobacterium caprae’, on the basis of host preference and phenotypic and genetic tests. Pinnipeds appear to be the natural host for this ‘seal bacillus’, although the organism is also pathogenic in guinea pigs, rabbits, humans, Brazilian tapir (Tapirus terrestris) and, possibly, cattle. Infection caused by the seal bacillus is predominantly associated with granulomatous lesions in the peripheral lymph nodes, lungs, pleura, spleen and peritoneum. Cases of disseminated disease have been found. As with other members of the M. tuberculosis complex, aerosols are the most likely route of transmission. The name Mycobacterium pinnipedii sp. nov. is proposed for this novel member of the M. tuberculosis ..

Preparation of a working seed lot of BCG and quality control by PCR genotyping

The bacillus Calmette-Guérin (BCG) was obtained in 1920 after successive passages leading to the attenuation of a Mycobacterium bovis strain. For the following 40 years, BCG had been replicated, resulting in substrains with genotypic and phenotypic differences. Several genomic studies have compared two BCG strains, M. bovis and Mycobacterium tuberculosis, and observed that deleted regions in the different strains could be related to differences in antigenic properties. In this work, a working seed lot was obtained from a lyophilized secondary seed lot from the BCG Pasteur strain 1173 P2 and genetically characterized. The genome was analyzed by PCR directed to five regions (RD1, RD2, RD14, RD15, DU2), using the seed lot and different available strains as templates. No genetic differences were found in the fragments studied as compared to the Pasteur strain. A total of 20 passages were carried out and no differences were found in the size of the fragments amplified by PCR. In conclusion, this method allows to control a working seed lot genotypically and to assess the stability of the BCG genome.El bacilo de Calmette-Guérin (BCG) se obtuvo en 1920, después de sucesivos pasajes que llevaron a la atenuación de una cepa de Mycobacterium bovis. A lo largo de los 40 años subsiguientes la cepa BCG fue replicada y surgieron subcepas con diferencias fenotípicas y genotípicas. Se realizaron varios estudios de comparación genómica de diferentes cepas de BCG, M. bovis y Mycobacterium tuberculosis, y se observó que las deleciones de regiones en las diferentes cepas podrían estar relacionadas con diferencias en las propiedades antigénicas. En este trabajo se describe la preparación y caracterización genética de un lote semilla de trabajo obtenido a partir de un lote semilla secundaria liofilizado de la cepa BCG Pasteur 1173 P2. Se analizaron por PCR cinco regiones (RD1, RD2, RD14, RD15, DU2) en el lote semilla de trabajo utilizando como control las diferentes cepas disponibles. No se hallaron diferencias genéticas en los fragmentos estudiados al comparar el lote semilla de trabajo con la cepa BCG Pasteur 1173 P2. Asimismo, se efectuaron hasta 20 pasajes y no se encontraron diferencias en el tamaño de los fragmentos amplificados por PCR. En conclusión, se ha puesto a punto un método que permite controlar el genotipo de un lote semilla de trabajo y evaluar la estabilidad del genoma del BCG.Instituto de BiotecnologíaFil: Trovero, A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Argüelles, Claudia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Bacillus anthracis: una mirada molecular a un patógeno célebre Bacillus anthracis: a molecular look at a famous pathogen

Bacillus anthracis es un bacilo gram positivo del grupo Bacillus cereus, que posee un genoma extremadamente monomórfco y comparte gran similitud fsiológica y de estructura genética con B. cereus y Bacillus thuringiensis. En este artículo se describen nuevos métodos moleculares para la identifcación y tipifcación de B. anthracis, basados en repeticiones en tándem de número variable o en diferencias genéticas detectadas por secuenciación, desarrollados en los últimos años. Los aspectos moleculares de los factores de virulencia tradicionales, cápsula, antígeno protector, factor letal y factor edema se describen en profundidad, junto con factores de virulencia recientemente propuestos, como los sideróforos, petrobactina y bacilibactina, la adhesina de la capa S y la lipoproteína MntA. También se detalla la organización molecular de los megaplásmidos pXO1 y pXO2, incluyendo la isla de patogenicidad de pXO1. El esqueleto genético de estos plásmidos se ha encontrado en otras especies relacionadas, probablemente debido a eventos de transferencia lateral. Finalmente, se presentan los dos receptores celulares del antígeno protector, ANTXR1/TEM8 y ANTXR2/CMG2, esenciales en la interacción del patógeno con el hospedador. Los estudios moleculares realizados en los últimos años han permitido aumentar enormemente el conocimiento de los diferentes aspectos de este microorganismo y su relación con el hospedador, pero a la vez han abierto nuevos interrogantes sobre este notorio patógeno.Bacillus anthracis, a gram-positive rod belonging to the Bacillus cereus group, has an extremely monomorphic genome, and presents high structural and physiological similarity with B. cereus and Bacillus thuringiensis. In this work, the new molecular methods for the identifcation and typing of B. anthracis developed in the last years, based on variable number tandem repeats or on genetic differences detected through sequencing, are described. The molecular aspects of traditional virulence factors: capsule, protective antigen, lethal factor and edema factor are described in depth, together with virulence factors recently proposed, such as the siderophores petrobactin and bacillibactin, the S-layer adhesin and the MntA lipoprotein. It is detailed the molecular organization of megaplasmids pXO1 and pXO2, including the pathogenicity island of pXO1. The genetic skeleton of these plasmids has been observed in related species, and this could be attributed to lateral gene transfer. Finally, the two anthrax toxin protective antigen receptors, ANTXR1/TEM8 and ANTXR2/CMG2, essential for the interaction of the pathogen with the host, are presented. The molecular studies performed in recent years have greatly increased knowledge in different aspects of this microorganism and its relationship with the host, but at the same time they have raised new questions about this noted pathogen