A Fragment of 21 ORFs Around the Direct Repeat (DR) Region of Mycobacterium tuberculosis is Absent From the Other Sequenced Mycobacterial Genomes: Implications for the Evolution of the DR Region

The direct repeat (DR) region is a singular locus of the Mycobacterium tuberculosis complex genome. This region consists of 36 bp repetitive sequences separated by non-repetitive unique spacer sequences. Around this region there are several genes coding for proteins of unknown function. To determine whether the M. smegmatis, M. avium, M. marinum and M. leprae genomes contain sequences and ORFs similar to those of the DR locus of the M. tuberculosis complex, we analysed the corresponding regions in these species. As a first step, some conserved genes that flank the DR genes [Rv2785c (rpsO), Rv2786c (ribF), Rv2790c (ltp1 ), Rv2793c (truB), Rv2800, Rv2825, Rv2828, Rv2831 (echA16 ), Rv2838 (rbfA) and Rv2845 (proS )] were used as markers to locate the corresponding orthologues in M. smegmatis, M. avium, M. marinum and M. leprae in silico. Most of these M. tuberculosis marker genes have highly similar orthologues located in the same order and orientation in the other mycobacteria. In contrast, no orthologues were found for ORFs Rv2801–Rv2824, suggesting that these genes are unique to M. tuberculosis within the genus Mycobacterium.We observed that in M. smegmatis and M. avium, Rv2800 and Rv2825 are adjacent. This observation was experimentally confirmed by PCR. In conclusion, as the DR locus and the ORFs around it are absent in M. smegmatis and M. avium and, as it is possible that these species are older than M. tuberculosis, we postulated that the DR locus was acquired by the M. tuberculosis complex species or by an ancestor bacterium

In vitro anti-tuberculosis activity of azole drugs against Mycobacterium tuberculosis clinical isolates

Background: Latent tuberculosis has been associated with the persistence of dormant Mycobacterium tuberculosis in the organism of infected individuals, who are reservoirs of the bacilli and the source for spreading the disease in the community. New active anti-TB drugs exerting their metabolic action at different stages and on latent/dormant bacilli are urgently required to avoid endogenous reactivations and to be part of treatments of multi- and extensively-drug resistant tuberculosis (M/XDR-TB). It was previously reported that azole drugs are active against M. tuberculosis. For that reason, the aims of this study were to determine the in vitro activity of azole drugs, imidazole (clotrimazole, CLO and econazole, ECO) and nitroimidazole (metronidazole, MZ and ipronidazole, IPZ), against a collection of MDR M. tuberculosis clinical isolates; and to analyze their potential use in both the LTB and the active forms of M/XDR-TB treatments. Methods: A total of 55 MDR M. tuberculosis isolates and H37Rv were included. MZ and IPZ activity against M. tuberculosis isolates were tested using anaerobic culture conditions. The activity of ECO and CLO was measured by the minimal inhibitory concentration (MIC) using a microdilution colorimetric method.Inst. de BiotecnologíaFil: Imperiale, Belen Rocio. Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; ArgentinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Morcillo, Nora. Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; Argentin

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

Good Protection but Excessive Pulmonary Inflammation in Balb/C Mice Vaccinated with Mycobacterium Bovis Mce-2A Mutant after Challenge with Homologous Strains

Tuberculosis (TB) remains a major threat to public and veterinary health. Zoonotic TB (caused by Mycobacterium bovis) is present in wild animals and cattle in most developing countries, and M. bovis is also able to infect humans on a worldwide basis. Thus, the high incidence of bovine TB is a major economic problem and an additional risk to human health, being the development of new vaccines to prevent both human and bovine TB urgent and a major challenge. The aims of the present study were to characterize the pathogenicity and immunogenicity of M. bovis mce2A mutant in BALB/c mice, and then evaluate its potential as vaccine. Mutant M. bovis mce2A produced limited tissue damage (pneumonia) and lower bacilli burdens than its parental strain when administered in high dose by intratracheal inoculation, and showed limited dissemination when used as subcutaneous vaccine. Challenge experiments using low, middle and highly virulent M. tuberculosis or M. bovis strains showed similar protection conferred by mce-2 mutant than BCG. Interestingly, vaccinated animals showed low bacilli loads but high inflammatory response when were challenged with M. bovis strains, while vaccinated mice challenged with M. tuberculosis exhibited low bacilli burdens and scarce inflammation. Thus, in spite of the high genome homology between M. tuberculosis and M. bovis, it seems that there is higher antigenic recognition and in consequence extensive inflammatory response when the strain used as vaccine is homologous to the challenge strain, in this case M. bovis.Fil: Alfonseca Silva, Edgar. Universidad Nacional Autónoma de México; MéxicoFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Hernández Pando, Rogelio. Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán”; Méxic

Mycobacterium bovis in Swine: Spoligotyping of Isolates from Argentina

A total of 143 Mycobacterium bovis isolates of pigs, from the most productive swine area in Argentina, were typed by spoligotyping. Twenty-two different spoligotypes were identified, and 133 (93%) isolates were grouped into 12 clusters. One of them, designed SB0140, was the most frequent because it held 83 (58%) isolates. This spoligotype also grouped 362 (43%) out of 841 isolates from previously typed cattle and, thus, constitutes the most frequent in our country. In addition, 135 (94%) isolates revealed spoligotypes identical to those of cattle, showing an epidemiological link. On the other hand, there were seven novel spoligotypes, six of which were also unique since they had only one isolate each. This study aimed to identify the spoligotypes of M. bovis isolated from pigs to contribute to a better understanding of the distribution of bovine tuberculosis in the main productive area of Argentina

The Role of Glucose in the Pathology of EHEC O157: H7

The pathogen enterohemorrhagic Escherichia coli (EHEC) O157: H7 is responsible for hemorrhagic colitis and hemolytic uremic syndrome in humans [1]. During the colonization process in the gastrointestinal tract, EHEC needs to adapt to changes in nutrient availability [2]. The objective of this study was to evaluate the influence of glucose on physiology and processes involved in the pathogenesis of EHEC O157: H7 in order to improve our understanding of the mechanisms controlling EHEC growth and survival in the bovine gut.Fil: Marques Da Silva, Wanderson. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Taibo, Catalina Beatriz. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Sabio y Garcia, Julia Veronica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Larzabal, Mariano. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin

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

Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection

The confirmatory diagnosis of Mycobacterium bovis (M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis–positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.Instituto de BiotecnologíaFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Meikle, Virginia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Bernardelli, Amelia. Servicio Nacional de Sanidad y Calidad Agroalimentaria. Dirección de Laboratorios y Control Técnico; ArgentinaFil: Abdala, Alejandro Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Tarabla, Hector Dante. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

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