RESUMEN

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. KEY POINTS: • Several T- and B-cell epitopes were identified in all the three proteins. • Four different adjuvants were used in vaccine formulations. • Immunization stimulated significant levels of IgG2/3 in vaccinated animals.

Asunto(s)

Anticuerpos Antibacterianos , Vacunas Bacterianas , Leptospirosis , Lipoproteínas , Animales , Leptospirosis/prevención & control , Leptospirosis/inmunología , Lipoproteínas/inmunología , Lipoproteínas/genética , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/genética , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Cricetinae , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Adyuvantes Inmunológicos/administración & dosificación , Inmunoglobulina G/sangre , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/genética , Leptospira interrogans/inmunología , Leptospira interrogans/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Vacunación , Inmunidad Humoral , Leptospira/inmunología , Leptospira/genética , Inmunogenicidad Vacunal

RESUMEN

Helicobacter pylori is the most common cause of gastroduodenal diseases. The concept that cagA-positive H. pylori is a risk factor for gastric cancer appears to be true only for H. pylori strains from Western countries. Other virulent genes may have a synergistic interaction with cagA during pathogenesis. This study aims to investigate H. pylori cagA, vacA, and iceA prevalence, genotypes, and their association to clinical outcomes in Vietnamese patients. The cagA status and vacA and iceA genotypes were determined using the PCR technique on DNA extracted from gastric biopsies of 141 patients with gastroduodenal diseases. After performing molecular analysis for cagA, vacA, and iceA genes, samples with mixed H. pylori strains, positivity, or negativity for both cagA and cagPAI-empty site, or unidentified genotypes were excluded. Finally, 107 samples were examined. The presence of the cagA, vacA, and iceA genes were detected in 77.6%, 100%, and 80.4% of cases, respectively. Notably, cagA( +) with EPIYA-ABD, vacA s1i1m1, vacA s1i1m2, iceA1, and iceA2 accounted for 73.8%, 44.9%, 33.6%, 48.6%, and 31.8% of cases, respectively. Four iceA2 subtypes (24-aa, 59-aa, 94-aa, and 129-aa variants) were found, with the 59-aa variant the most prevalent (70.6%). The cagA( +)/vacAs1i1m1/iceA1 and cagA( +)/vacAs1i1m2/iceA1 combinations were found in 26.2% and 25.1% of cases, respectively. A multivariable logistic regression analysis was performed, after adjusting for age and gender, with the gastritis group was used as a reference control. Statistically significant associations were found between the vacA s1i1m2 genotype, the iceA1 variant, and the cagA( +)/vacAs1i1m2/iceA1 combination and gastric cancer; the adjusted ORs were estimated as 18.02 (95% CI: 3.39-95.81), 4.09 (95% CI: 1.1-15.08), and 16.19 (95% CI: 3.42-76.66), respectively. Interestingly, for the first time, our study found that vacA s1i1m2, but not vacA s1i1m1, was a risk factor for gastric cancer. This study illustrates the genetic diversity of the H. pylori cagA, vacA, and iceA genes across geographical regions and contributes to understanding the importance of these genotypes for clinical outcomes.

Asunto(s)

Antígenos Bacterianos , Proteínas Bacterianas , Genotipo , Infecciones por Helicobacter , Helicobacter pylori , Humanos , Proteínas Bacterianas/genética , Helicobacter pylori/genética , Helicobacter pylori/aislamiento & purificación , Helicobacter pylori/clasificación , Helicobacter pylori/patogenicidad , Vietnam/epidemiología , Antígenos Bacterianos/genética , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/epidemiología , Estudios Transversales , Masculino , Femenino , Persona de Mediana Edad , Adulto , Proteínas de la Membrana Bacteriana Externa/genética , Anciano , Adulto Joven , Prevalencia , Factores de Virulencia/genética

RESUMEN

Acinetobacter baumannii is an opportunistic bacterium that causes infection in several sites. Carbapenem-resistant A. baumannii strains (CRAb) lead the World Health Organization's list of 12 pathogens considered a priority for developing new antimicrobials. The pathogenicity of A. baumannii is related to the different virulence factors employed in the colonization of biotic and abiotic surfaces, biofilm formation and multidrug resistance. We analyze the outer membrane protein FilF from A. baumannii in silico and produce it in recombinant form (rFilF). rFilF protein was successfully expressed in Escherichia coli BL21 Star in an insoluble form. Immunization with rFilF induced significant anti-rFilF IgG antibody production in mice, detected by indirect enzyme-linked immunosorbent assay, since the first evaluation until 49th. On the last experimentation day, the predominant immunoglobulin found was IgG1 followed by IgG2a, IgG2b, IgM, IgG3, and IgA. We observe that interleukins 4 and 10 show significant production after the 28th day of experimentation in mice immunized with rFilF. Anti-rFilF pAbs were able to inhibit biofilm formation in nine CRAb strains evaluated, and in the standard strain ATCC® 19606. These results demonstrate the anti-biofilm activity of anti-rFilF antibodies, promising in the development of a non-antibiotic approach based on the control of CRAb strains.

Asunto(s)

Acinetobacter baumannii , Anticuerpos Antibacterianos , Biopelículas , Carbapenémicos , Biopelículas/efectos de los fármacos , Acinetobacter baumannii/inmunología , Acinetobacter baumannii/efectos de los fármacos , Animales , Anticuerpos Antibacterianos/inmunología , Carbapenémicos/farmacología , Ratones , Inmunoglobulina G/inmunología , Antibacterianos/farmacología , Infecciones por Acinetobacter/inmunología , Infecciones por Acinetobacter/microbiología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/genética , Ratones Endogámicos BALB C , Femenino , Escherichia coli/genética , Escherichia coli/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/genética

RESUMEN

BACKGROUND: Helicobacter pylori is an etiologic agent of gastroduodenal diseases. The microorganism, considered a type I carcinogen, affects about 50% of the global population. H. pylori virulence factors are determinant for the clinical outcome of the infection. The outer inflammatory protein A (oipA) gene encodes an outer membrane adhesin and is related to severe gastropathies, such as gastric cancer. OBJECTIVE: The aim of this study was to evaluate the association of the oipA gene with the severity of gastroduodenal diseases in dyspeptic patients in region Central Brazil. METHODS: The polymerase chain reaction (PCR) was used to determine the presence of H. pylori. Samples positives were used for molecular screening of the oipA gene. Gastropathies were categorized as non-severe and severe diseases. RESULTS: Approximately 68% of patients had H. pylori and 36% were infected with H. pylori oipA+ strains. Infection was significantly associated in patients aged over 44 years (P=0.004). However, there was no association between oipA and patients' age (P=0.89). Approximately 46% of patients infected with oipA+ strains had some severe illness. Gastric adenocarcinoma was the most frequent severe gastropathy. The H. pylori oipA genotype was inversely associated with the severity of gastroduodenal diseases (OR=0.247, 95%CI: 0.0804-0.7149 and P=0.007). CONCLUSION: The characterization of possible molecular markers will contribute to personalized medicine, impacting the prognosis of patients. BACKGROUND: • Evidence points to an association between the H. pylori oipA gene and gastropathies. BACKGROUND: • There is a high prevalence of H. pylori infection with a relevant percentage of oipA+ strains. BACKGROUND: • More severe gastropathies were observed in those infected with H. pylori oipA+ strains.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa , Helicobacter pylori , Neoplasias Gástricas , Anciano , Humanos , Biomarcadores , Genotipo , Helicobacter pylori/genética , Virulencia/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo

RESUMEN

Escherichia coli cells rapidly respond to changes in the environment. Such response must be anticipated upon development of fermentation strategy for commercial purposes. The response may signal changes in cell physiology, which is critical for the cell growth and the level of the target protein production. One of the responses is the elevated expression of membrane proteins to tightly control the trafficking of molecules into and out from the cells. Normally, the expression level of the membrane protein is basal as the fermentation is carried out in physiological conditions. Here, we reported an elevated expression of the outer membrane protein A (OmpA) during a series of fermentation conduct, starting from the shake flask, 1-L to finally 10-L fermentor. The incidence led to a lower expression of the target protein and thereby resulting in lower process efficiency. OmpA expression was concomitant to the bacterial growth and already observed in the early exponential phase. Despite the drawback, this phenomenon actually inspires the observation of OmpA expression as one of the indicators for the E. coli cells response to the fermentation conditions. This auxiliary check would prevent the higher OmpA expression that led to the low expression of the target protein.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo

RESUMEN

BACKGROUND: The most prevalent stomach infection in the world is caused by Helicobacter pylori (H. pylori). Several pathogenicity genes, including cagA, vacA, babA2, dupA, iceA, and oipA, are associated with an increased risk of gastrointestinal disease such as peptic ulcer and stomach cancer. This research aims to determine the prevalence of different H. pylori genotypes and correlate their risk in the development of gastrointestinal diseases in the Ecuadorian population. METHODS: A cross-sectional research of 225 patients at the Calderón Hospital in Quito, Ecuador, was conducted. End point PCRs were run to determine the presence of 16S rRNA, cagA, vacA (m1), vacA (s1), babA2, dupA, iceA1, and oipA virulence genes. Chi-square test, odds ratios (OR) and 95% confidence intervals (CI) were utilized for the statistical analysis. RESULTS: H. pylori infection was present in 62.7% of people. Peptic ulcers were seen in 22.2% and malignant lesions in 3.6% of patients. Genes oipA (93.6%), vacA (s1) (70.9%), and babA2 (70.2%) were the most prevalent. cagA/vacA (s1m1) and cagA/oipA (s1m1) combinations were found in 31.2% and 22.7% of the cases, respectively. Acute inflammation has a significant correlation with the genes cagA (OR = 4.96 95% CI: 1.1-22.41), babA2 (OR = 2.78 95% CI: 1.06-7.3), and the cagA/oipA combination (OR = 4.78, 95% CI: 1.06-21.62). Follicular hyperplasia was associated with iceA1 (OR = 3.13; 95% CI: 1.2-8.16), babA2 (OR = 2.56; 95% CI: 1.14-5.77), cagA (OR = 2.19; 95% CI: 1.06-4.52), and the cagA/oipA combination (OR = 2.32, 95% CI: 1.12-4.84). The vacA (m1) and vacA (s1m1) genes were associated with gastric intestinal metaplasia (OR = 2.71 95% CI: 1.17-6.29) (OR = 2.33 95% CI: 1.03-5.24). Finally, we showed that cagA/vacA (s1m1) gene combination increased the risk of duodenal ulcer development (OR = 2.89, 95% CI 1.10-7.58). CONCLUSION: This study makes a significant contribution by offering genotypic information regarding H. pylori infection. The presence of several H. pylori genes was associated with the onset of gastrointestinal illness in the Ecuadorian population.

Asunto(s)

Infecciones por Helicobacter , Helicobacter pylori , Úlcera Péptica , Neoplasias Gástricas , Humanos , Proteínas Bacterianas/genética , Adhesinas Bacterianas/genética , Antígenos Bacterianos/genética , Helicobacter pylori/genética , Estudios Transversales , Proteínas de la Membrana Bacteriana Externa/genética , Ecuador/epidemiología , Prevalencia , ARN Ribosómico 16S , Úlcera Péptica/epidemiología , Úlcera Péptica/complicaciones , Genotipo , Neoplasias Gástricas/complicaciones , Infecciones por Helicobacter/complicaciones

RESUMEN

Renibacterium salmoninarum is one of the oldest known fish bacterial pathogens. This Gram-positive bacterium is the causative agent of Bacterial Kidney Disease (BKD), a chronic infection that primarily infects salmonids at low temperatures. Externally, infected fish may show exophthalmos, skin blisters, ulcerations, and hemorrhages at the base of the fins and along the lateral line. Internally, the kidney, heart, spleen, and liver may show signs of inflammation. The best characterized virulence factor of R. salmoninarum is p57, a 57 kDa protein located on the bacterial cell surface and secreted into surrounding fish tissue. The p57 protein in fish is the main mediator in suppressing the immune system, reducing antibody production, and intervening in cytokine activity. In this review, we will discuss aspects such as single nucleotide polymorphisms (SNPs) that modify the DNA sequence, variants in the number of copies of MSA genes, physical-chemical properties of the signal peptides, and the limited iron conditions that can modify p57 expression and increase the virulence of R. salmoninarum.

Asunto(s)

Enfermedades de los Peces , Infecciones por Bacterias Grampositivas , Animales , Proteómica , Virulencia/genética , Proteínas de la Membrana Bacteriana Externa/genética , Genómica , Enfermedades de los Peces/microbiología , Infecciones por Bacterias Grampositivas/veterinaria , Infecciones por Bacterias Grampositivas/microbiología

RESUMEN

Adherent-invasive E. coli (AIEC) is a pathotype associated with the etiopathogenesis of Crohn's disease (CD), albeit with an as-yet unclear role. The main pathogenic mechanisms described for AIEC are adherence to epithelial cells, invasion of epithelial cells, and survival and replication within macrophages. A few virulence factors have been described as participating directly in these phenotypes, most of which have been evaluated only in AIEC reference strains. To date, no molecular markers have been identified that can differentiate AIEC from other E. coli pathotypes, so these strains are currently identified based on the phenotypic characterization of their pathogenic mechanisms. The identification of putative AIEC molecular markers could be beneficial not only from the diagnostic point of view but could also help in better understanding the determinants of AIEC pathogenicity. The objective of this study was to identify molecular markers that contribute to the screening of AIEC strains. For this, we characterized outer membrane protein (OMP) profiles in a group of AIEC strains and compared them with the commensal E. coli HS strain. Notably, we found a set of OMPs that were present in the AIEC strains but absent in the HS strain. Moreover, we developed a PCR assay and performed phylogenomic analyses to determine the frequency and distribution of the genes coding for these OMPs in a larger collection of AIEC and other E. coli strains. As result, it was found that three genes (chuA, eefC, and fitA) are widely distributed and significantly correlated with AIEC strains, whereas they are infrequent in commensal and diarrheagenic E. coli strains (DEC). Additional studies are needed to validate these markers in diverse strain collections from different geographical regions, as well as investigate their possible role in AIEC pathogenicity.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Biomarcadores/metabolismo , Escherichia coli/metabolismo , Infecciones por Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/metabolismo

RESUMEN

OBJECTIVE.: To evaluate in silico and at the serological level the antigenic potential of the recombinant extracellular domain of the lipopolysaccharide assembly protein - D (LptD) of Bartonella bacilliformis (dexr_LptD). MATERIALS AND METHODS.: Through in silico analysis, we selected a B. bacilliformis protein with antigenic and immunogenic potential. The selected protein gene was cloned into Escherichia coli TOP10 and expressed in Escherichia coli BL21 (DE3) pLysS. Recombinant protein was expressed using isopropyl-ß-D-1-thiogalactopyranoside (IPTG) and induction conditions were optimized. Finally, it was purified with Ni-IDA resin (His60 Ni Superflow) and a Western Blot assay was conducted. RESULTS.: In silico, the selected protein was LptD because it is located in the outer membrane and is antigenic and immunogenic. Optimized conditions for dexr_LptD induction were 0.5 mM IPTG, 16 hours, TB (Terrific Broth) medium, 3% (v/v) ethanol, 28 ºC, OD600: 1-1.5 and 200 rpm. Purification was carried out under denaturating conditions on a small scale and we obtained 2.6 µg/mL of partially purified dexr_LptD. The Western Blot assay showed a positive reaction between the sera from patients with Carrión's Disease and dexr_LptD, which shows the antigenicity of dexr_LptD. CONCLUSIONS.: The dexr_LptD shows antigenicity both in silico and at the serological level, these results are the basis for further studies on vaccine candidates against Carrion's Disease.

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OBJETIVO.: Evaluar in silico y a nivel serológico el potencial antigénico del dominio extracelular recombinante de la proteína de ensamblaje de lipopolisacáridos - D (LptD) de Bartonella bacilliformis (dexr_LptD). MATERIALES Y MÉTODOS.: Mediante el análisis in silico se realizó la selección de una proteína de B. bacilliformis con potencial antigénico e inmunogénico. El gen de la proteína seleccionada se clonó en Escherichia coli TOP10 y se expresó en Escherichia coli BL21 (DE3) pLysS. La proteína recombinante fue expresada usando isopropil-ß-D-1-tiogalactopiranósido (IPTG) y se optimizaron las condiciones de inducción. Por último, se purificó con resina Ni-IDA (His60 Ni Superflow) y se realizó un ensayo de Western Blot. RESULTADOS.: In silico, la proteína seleccionada fue LptD por estar localizada en la membrana externa y ser antigénica e inmunogénica. Las condiciones optimizadas para la inducción del dexr_LptD fueron 0,5 mM IPTG, 16 h, medio TB (Terrific Broth), etanol al 3% (v/v), 28 ºC, OD600: 1-1,5 y 200 r.p.m. La purificación se realizó en condiciones denaturantes a pequeña escala y se obtuvo 2,6 µg/mL de dexr_LptD parcialmente purificada. El ensayo de Western Blot mostró una reacción positiva entre los sueros provenientes de pacientes con la enfermedad de Carrión y dexr_LptD, ello evidencia la antigenicidad del dexr_LptD. CONCLUSIONES.: El dexr_LptD muestra antigenicidad in silico y a nivel serológico, estos resultados son base para posteriores estudios sobre candidatos vacunales contra la enfermedad de Carrión.

Asunto(s)

Infecciones por Bartonella , Bartonella bacilliformis , Proteínas de Escherichia coli , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bartonella bacilliformis/genética , Clonación Molecular , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Isopropil Tiogalactósido/metabolismo , Lipopolisacáridos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

BACKGROUND: Salmonella Typhimurium is a Gram-negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella-containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin-encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post-transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post-transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S. Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild-type strain, suggesting that ompX mRNA is also regulated at a post-transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2-induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/genética , Porinas , Salmonella typhimurium , Animales , Regulación Bacteriana de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Ratones , Porinas/genética , Porinas/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo

RESUMEN

Anaplasma marginale is an obligate intracellular Gram-negative bacterium that is parasitic to erythrocytes and is the main agent of bovine anaplasmosis. This disease causes severe anemia and reduces weight gain and milk production, thus giving rise to major economic losses relating to livestock worldwide. The genetic diversity of this bacterium has been characterized based on sequences of major surface proteins (MSPs), especially MSP1α. This has enabled identification of several geographical strains, according to different amino acid sequences. The aim of this study was to investigate the genetic diversity of A. marginale in naturally infected Angus beef cattle during a disease outbreak in southeastern Brazil. Four blood samples were collected over a four-month period from each of 20 animals on a cattle farm in Itú, São Paulo, Brazil. Serum samples were subjected to indirect ELISA to detect anti-A. marginale IgG antibodies. The 80 whole-blood samples obtained were subjected to DNA extraction, quantitative real-time PCR (qPCR) for the msp1ß gene, semi-nested PCR (snPCR) for the msp1α gene, cloning of the target fragment and sequencing using the Sanger method. The sequences obtained were analyzed for genetic diversity using the RepeatAnalyzer software. Both iELISA tests, using recombinant MSP5 and the Anaplasma antibody test kit (VMRD), revealed high seroprevalence: 91.25% and 97.5%, respectively. In qPCR, 100% of the samples were positive, with between 103 and 107 DNA copies/µL. In the snPCR based on the msp1α gene, 57.5% (46/80) of the samples were positive. Microsatellite analysis on the 36 sequences obtained showed the presence of genotypes H (58.3%), F (25%), E (19.4%), C (2.7%) and G (2.7%). The RepeatAnalyzer software identified 36 strains in the study region, among which some had not previously been described in the literature (13 27 13 27 13 F; 16 FF; τ 27; 63 29 104 29; LJ1 13 LJ1 13; 16 F 17; 16 F 91). High genetic diversity of A. marginale bacteria was found on this farm in Itú, São Paulo.

Asunto(s)

Anaplasma marginale , Anaplasmosis , Enfermedades de los Bovinos , Sobreinfección , Anaplasma marginale/genética , Anaplasmosis/microbiología , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Brasil/epidemiología , Bovinos , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/microbiología , Brotes de Enfermedades/veterinaria , Variación Genética , Filogenia , Sobreinfección/epidemiología

RESUMEN

BACKGROUND: Salmonella Typhimurium is a Gram negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S . Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild type strain, suggesting that ompX mRNA is also regulated at a post transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2 induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

Asunto(s)

Animales , Ratones , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Porinas/genética , Porinas/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología

RESUMEN

Outer membrane vesicles (OMV) derived from Bordetella pertussis-the etiologic agent of the resurgent disease called pertussis-are safe and effective in preventing bacterial colonization in the lungs of immunized mice. Vaccine formulations containing those OMV are capable of inducing a mixed Th1/Th2/Th17 profile, but even more interestingly, they may induce a tissue-resident memory immune response. This immune response is recommended for the new generation of pertussis-vaccines that must be developed to overcome the weaknesses of current commercial acellular vaccines (second-generation of pertussis vaccine). The third-generation of pertussis vaccine should also deal with infections caused by bacteria that currently circulate in the population and are phenotypically and genotypically different [in particular those deficient in the expression of pertactin antigen, PRN(-)] from those that circulated in the past. Here we evaluated the protective capacity of OMV derived from bacteria grown in biofilm, since it was observed that, by difference with older culture collection vaccine strains, circulating clinical B. pertussis isolates possess higher capacity for this lifestyle. Therefore, we performed studies with a clinical isolate with good biofilm-forming capacity. Biofilm lifestyle was confirmed by both scanning electron microscopy and proteomics. While scanning electron microscopy revealed typical biofilm structures in these cultures, BipA, fimbria, and other adhesins described as typical of the biofilm lifestyle were overexpressed in the biofilm culture in comparison with planktonic culture. OMV derived from biofilm (OMVbiof) or planktonic lifestyle (OMVplank) were used to formulate vaccines to compare their immunogenicity and protective capacities against infection with PRN(+) or PRN(-) B. pertussis clinical isolates. Using the mouse protection model, we detected that OMVbiof-vaccine was more immunogenic than OMVplank-vaccine in terms of both specific antibody titers and quality, since OMVbiof-vaccine induced antibodies with higher avidity. Moreover, when OMV were administered at suboptimal quantity for protection, OMVbiof-vaccine exhibited a significantly adequate and higher protective capacity against PRN(+) or PRN(-) than OMVplank-vaccine. Our findings indicate that the vaccine based on B. pertussis biofilm-derived OMV induces high protection also against pertactin-deficient strains, with a robust immune response.

Asunto(s)

Membrana Externa Bacteriana/metabolismo , Biopelículas , Bordetella pertussis/metabolismo , Vesículas Extracelulares/metabolismo , Vacuna contra la Tos Ferina/administración & dosificación , Tos Ferina/prevención & control , Animales , Membrana Externa Bacteriana/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Biopelículas/crecimiento & desarrollo , Bordetella pertussis/genética , Bordetella pertussis/crecimiento & desarrollo , Bordetella pertussis/inmunología , Modelos Animales de Enfermedad , Vesículas Extracelulares/inmunología , Femenino , Inmunización , Inmunogenicidad Vacunal , Ratones Endogámicos BALB C , Vacuna contra la Tos Ferina/inmunología , Vacuna contra la Tos Ferina/metabolismo , Desarrollo de Vacunas , Factores de Virulencia de Bordetella/genética , Factores de Virulencia de Bordetella/metabolismo , Tos Ferina/inmunología , Tos Ferina/metabolismo , Tos Ferina/microbiología

RESUMEN

The genus Amblyomma is the most representative tick genus in Brazil and some species act as vectors of pathogenic organisms to animals and humans. Information on the seasonal dynamics of Amblyomma spp. as well as on rickettsial organisms infecting these ticks in some regions in Brazil is still fragmentary. Herein, we investigated the seasonal dynamics and rickettsial infections in Amblyomma dubitatum ticks collected in the Atlantic forest biome in north-eastern Brazil. Using carbon dioxide traps, ticks were collected monthly for two consecutive years. In total, 15,789 ticks were collected: 69 females (0.4%), 116 males (0.7%), 1,067 nymphs (6.8%), and 14,537 larvae (92.1%). All nymphs, females and males were identified as A. dubitatum, whereas larvae were identified as Amblyomma spp. Larvae were more frequent in summer (77% of the larvae collected), whereas nymphs were collected with similar frequency in summer (32.8%), autumn (30.0%) and spring (28.4%). Adults were more frequent in spring (47.6%). A total of 648 ticks (485 nymphs, 60 females, and 103 males) were tested by PCR for the gltA gene of Rickettsia spp. and 87 (13.4%; 95% CI: 10.9-16.3%) were positive. A consensus sequence (size, 350 bp) of 66 gltA gene sequences indicate that the organism detected herein is similar to Rickettsia tamurae, Rickettsia monacencis and Rickettsia sp. strain Pampulha. One of these positive samples was also positive for the ompA gene of spotted fever group rickettsiae, but attempts to sequence the amplicon were not successful. We also tested this sample by a PCR targeting the rickettsial htrA gene, but no amplification product could be detected. This study indicates that A. dubitatum may be a common tick in areas where capybaras are present in north-eastern Brazil, occurring during the whole year. It also suggests the circulation of a spotted fever group rickettsia in this A. dubitatum population, whose identity has yet to be determined.

Asunto(s)

Amblyomma/microbiología , Infecciones por Rickettsia/epidemiología , Infecciones por Rickettsia/veterinaria , Rickettsia/clasificación , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Brasil/epidemiología , Citrato (si)-Sintasa/genética , ADN Bacteriano/genética , Vectores de Enfermedades , Ecosistema , Femenino , Bosques , Larva/microbiología , Masculino , Ninfa/microbiología , Reacción en Cadena de la Polimerasa , Rickettsia/genética , Estaciones del Año , Análisis de Secuencia de ADN

RESUMEN

The most common presentation of animal leptospirosis is the subclinical and silent chronic form, that can lead to important reproductive disorders. The diagnosis of this chronic form remains a challenge. The aim of the present study is to gather and critically analyse the current information about molecular tools applied to animal leptospirosis diagnosis, particularly the silent chronic presentation of the infection. Regarding clinical specimens, samples from urinary tract were the most used (69/102, 67·7%), while few studies (12/102, 11·8%) investigated samples from reproductive tract. Concerning the molecular methods applied, the most used is still the conventional polymerase chain reaction (PCR) (46/102, 45%), followed by real-time PCR (38/102, 37·2%). The lipL32 gene is currently the most common target used for Leptospira detection, with 48% of studies applying this genetic marker. From all the studies, only few (21/102, 20·5%) performed gene sequencing. According to the majority of authors, current evidence suggests that lipL32-PCR is useful for an initial screening for Leptospira DNA detection in animal clinical samples. Posteriorly, if DNA sequencing could be performed on positive lipL32-PCR samples, we encourage the use of secY gene as a genetic marker. The molecular methods appear as the most important tools for the diagnosis of the chronic silent leptospirosis on domestic animals, reinforcing its evident impact not only on animal reproduction but also on a One Health context.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/genética , Leptospira/genética , Leptospirosis/diagnóstico , Leptospirosis/veterinaria , Lipoproteínas/genética , Canales de Translocación SEC/genética , Animales , Animales Domésticos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Análisis de Secuencia de ADN

RESUMEN

Borrelia burgdorferi s.s. is a Gram-negative spirochaete, the aetiological agent of Lyme disease, the most common vector-borne disease in the Northern hemisphere. Reports on the presence of B. burgdorferi in central Mexico have been strongly criticized, since these were based only on unspecific serological methods. Furthermore, the worldwide genetic diversity of B. burgdorferi s.s. has not been evaluated. For this reason, the aim of the present study was to confirm the presence of B. burgdorferi in the central area of Mexico and to evaluate its relationship with regard to the global genetic diversity of B. burgdorferi s.s. To achieve this, fragments of the flagellin and the outer surface protein A genes were amplified from ear biopsies of the arboreal wild endemic mice Habromys schmidlyi. With these sequences, a concatenated Bayesian analysis was performed to confirm the identity of B. burgdorferi s.s. Afterwards, the global genetic diversity of this bacterial species was evaluated using our sequences and those available in GenBank. A prevalence of 10.4% (5/48) of H. schmidlyi infected with Borrelia sp. was detected, and the phylogenetic analyses confirmed the identity of B. burgdorferi s.s. Using both genes, the genetic diversity was low. However, genetic structuring analyses revealed that populations of western United States and those from Mexico formed slightly different genetic groups, separated from the populations of the rest of the world. Our study not only confirms the presence of this bacterium in central Mexico, but also shows the most southern record of this bacterium so far. It also highlights the importance of H. schmidlyi as a new potential host of this bacterial species. Our study also provides first genetic data on an incipient process of divergence in B. burgdorferi s.s. populations of eastern United States and central Mexico.

Asunto(s)

Antígenos de Superficie/genética , Proteínas de la Membrana Bacteriana Externa/genética , Vacunas Bacterianas/genética , Borrelia burgdorferi/genética , Flagelina/genética , Variación Genética , Lipoproteínas/genética , Enfermedad de Lyme/veterinaria , Enfermedades de los Roedores/epidemiología , Sigmodontinae , Animales , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/microbiología , México , Enfermedades de los Roedores/microbiología

RESUMEN

Oxidative stress is the main mechanism behind efficient disinfectants, causing damage in bacterial macromolecules. Importantly, bacteria activate resistance mechanisms in response to damage generated by oxidative stress. Strategies allowing pathogens to survive oxidative stress are highly conserved among microorganisms. Many of these strategies entail genomic responses triggered by signals transduced through Two Component Systems (TCS). Recently, we demonstrated that the TCS ArcAB (specifically ArcA) participates in bacterial responses to hypochlorite, regulating the uptake of this toxic compound and being involved in resistance and survival inside neutrophils, where hypochlorous acid abounds. Here, we demonstrated that ArcA is required in the response to oxidative stress generated by hypochlorite, independent of its cognate sensor ArcB or the Asp54 of ArcA, the only phosphorylable residue in ArcA, which is required to function as a gene regulator. Our results suggest that ArcA could have additional functions to respond to oxidative stress, independent of its regulatory activity, which might require interaction with other unknown relevant proteins.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Ácido Hipocloroso/farmacología , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Asparagina/química , Proteínas de la Membrana Bacteriana Externa/química , Biopelículas/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Introduction: Molecular biology diagnostic methods such as real-time PCR should be used in Nicaragua to improve the diagnosis of leptospirosis in humans and animals. Objective: To evaluate three qPCR methods for pathogenic Leptospira detection in domestic animals. Materials and methods: Real-time PCR primers were designed for the amplification of specific regions from the Lip 32 gene of Leptospira in SYBER Green (SYBER Green-A) and TaqMan, as well in SYBER Green-B as previously published. The sequences of 12 strains obtained from the database of the National Center for Biotechnology Information (NCBI) were aligned to select probes and primers. The analytical sensitivity was determined by calculating the detectable genomic equivalent while 18 pathogenic references strains and 28 negative controls were used to evaluate the sensitivity and specificity of each one of the three sets in 129 urine samples of domestic animals. Results: The detection limit of four genomic equivalents per reaction was obtained from SYBR Green-A. The specificities were 94.4% (95% CI: 81.1-100.0) for TaqMan, 77.8% (95% CI: 55.8-99.8) for SYBR Green-A, while for SYBR Green-B it was 61.1% (95% CI: 35.8-86.4). In the three tests, we obtained a specificity of 100% (95% CI: 98.2-100.0). In the field samples, 26.4% were positive with SYBR Green-A and 6.1% with SYBR Green-B. Conclusion: SYBR Green-A presented the lowest detection limit while the three techniques under evaluation showed high specificity while TaqMan was the most sensitive.

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Introducción. En Nicaragua es necesario estandarizar pruebas moleculares como la PCR en tiempo real (quantitative Polymerase Chain Reaction, qPCR) que mejoren el diagnóstico de leptospirosis en humanos y animales. Objetivo. Evaluar tres qPCR para la detección de leptospiras patógenas en animales domésticos de Nicaragua. Materiales y métodos. Se diseñaron cebadores para la amplificación del gen LipL32 en SYBR Green (SYBR Green-A) y TaqMan, y en otros descritos previamente (SYBR Green-B). Las secuencias de 12 cepas obtenidas de la base de datos del National Center for Biotechnology Information (NCBI) se alinearon para la búsqueda de sondas y cebadores. La sensibilidad analítica se determinó calculando el equivalente genómico detectable, se utilizaron 18 cepas de referencia para la sensibilidad diagnóstica y 28 controles negativos para la especificidad. Los métodos se aplicaron en 129 muestras de orina de animales domésticos. Resultados. En SYBR Green-A se obtuvo un límite de detección de cuatro equivalentes genómicos; en TaqMan, la sensibilidad fue del 94,4 % (IC95% 81,1-100,0). Con SYBR Green-A, se obtuvo una sensibilidad del 77,8 % (IC95% 55,8-99,8), en tanto que con SYBR Green-B fue del 61,1 % (IC95% 35,8-86,4). En las tres pruebas se logró una especificidad del 100 % (IC95% 98,2-100,0). El 26,4 % de las muestras de animales domésticos fueron positivas con SYBR Green-A y el 6,2 % con SYBR Green-B. Conclusiones. El SYBR Green-A presentó un límite de detección bajo, en tanto que las tres técnicas evaluadas mostraron alta especificidad, en tanto que la TaqMan tuvo la mayor sensibilidad.

Asunto(s)

Animales Domésticos/microbiología , Proteínas de la Membrana Bacteriana Externa/genética , Genes Bacterianos , Leptospira/genética , Lipoproteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Animales , Animales Domésticos/orina , Bovinos , Sondas de ADN/genética , Perros , Amplificación de Genes , Caballos , Leptospira/aislamiento & purificación , Nicaragua , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Ovinos , Sus scrofa

RESUMEN

Leptospirosis is a zoonotic disease of worldwide distribution, affecting both humans and animals. The development of an effective vaccine against leptospirosis has long been pursued but without success. Humans are contaminated after direct contact with the urine of infected animals or indirectly by contaminated water or soil. The vaccines available consist of inactivated whole-bacterial cells, and the active immunoprotective antigen is the lipopolysaccharide moiety, which is also the basis for serovar classification. However, these vaccines are short-lasting, and protection is only against serovars contained in the preparation. The search for prevalent antigens, present in pathogenic species of Leptospira, represents the most cost-effective strategy for prevention of leptospirosis. Thus, the identification of these antigens is a priority. In this study, we examined the immunoprotective effect of eight leptospiral recombinant proteins using hamster as the challenge model. Animals received subcutaneously two doses of vaccine containing 50 µg of each recombinant protein adsorbed on alum adjuvant. Two weeks after the booster, animals were challenged with virulent leptospires and monitored for 21 days. All proteins were able to induce a specific immune response, although significant protective effects on survival rate were observed only for the proteins Lsa14, rLIC13259, and rLIC11711. Of these, only rLIC13259 and rLIC11711 were found to be highly prospective in promoting renal clearance. The sterilizing potential of both proteins will be further investigated to elucidate the immunoprotective mechanisms involved in leptospirosis control. These are the first proteins involved with human complement components with the capacity to protect against virulent challenge and to eliminate the bacteria from the host.

Asunto(s)

Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/farmacología , Leptospira/inmunología , Leptospirosis/prevención & control , Enfermedad Aguda , Adyuvantes Inmunológicos/farmacología , Compuestos de Alumbre/farmacología , Animales , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Cricetinae , Modelos Animales de Enfermedad , Masculino , Proteínas Recombinantes/farmacología

RESUMEN

Leptospirosis is an acute infection caused by pathogenic species of the genus Leptospira, which affects humans and animals in all world. In severe forms of the disease, kidneys, liver and lungs are the main affected organs, resulting in acute kidney injury, jaundice and pulmonary hemorrhage. Previous post-mortem studies have shown that lesions are not limited to these organs. Cardiac and striated muscle injuries have already been reported, but the pathophysiology of cardiac and skeletal lesions in leptospirosis is not fully understood. It has been suggested that the tissue damage observed in leptospirosis could be directly mediated by leptospires or by their toxic cellular components. LipL32 and Lp25 are leptospira membrane proteins with unknown functions, that are present only in pathogenic strains of Leptospira spp. Both proteins induce skeletal muscle lesions similar to those observed when normal guinea pigs are inoculated with leptospires. Through immunohistochemistry, this study showed the presence of LipL32 and Lp25 proteins on muscle cell membranes and in the underlying cytoplasm of skeletal muscles, as well as focal lesions in cardiac tissues of fatal cases of leptospirosis. Altogether, these results reinforce that both proteins can be important factors in the pathogenesis of leptospirosis.

Asunto(s)

Lesión Renal Aguda/patología , Proteínas de la Membrana Bacteriana Externa/genética , Riñón/patología , Leptospira/genética , Leptospirosis/complicaciones , Lipoproteínas/genética , Miocardio/patología , Lesión Renal Aguda/microbiología , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Femenino , Genes Bacterianos , Cobayas , Humanos , Leptospira/metabolismo , Leptospirosis/metabolismo , Lipoproteínas/metabolismo , Masculino , Persona de Mediana Edad , Músculos/patología