Engineering a new vaccine platform for heterologous antigen delivery in live-attenuated Mycobacterium tuberculosis

Live vaccines are attractive vehicles for antigen delivery as a strategy to immunize against heterologous pathogens. The live vaccine MTBVAC is based on rational attenuation of Mycobacterium tuberculosis with the objective of improving BCG protection against pulmonary tuberculosis. However, the development of recombinant mycobacteria as antigen-presenting microorganisms has been hindered due to their fastidious genetic manipulation. In this study, we used MTBVAC as a genetic platform to deliver diphtheria, tetanus, or pertussis toxoids, which are the immunogenic constituents of the DTP vaccine. When using nonoptimal genetic conditions, the expression of these immunogens was barely detectable. Accordingly, we pursued a rational, step-by-step optimization of the genetic components to achieve the expression and secretion of these toxoids. We explored variants of the L5 mycobacteriophage promoter to ensure balanced antigen expression and plasmid stability. Optimal signal sequences were identified by comparative proteomics of MTBVAC and its parental strain. It was determined that proteins secreted by the Twin Arginine Translocation pathway displayed higher secretion in MTBVAC, and the Ag85A secretion sequence was selected as the best candidate. Because the coding regions of diphtheria, tetanus, and pertussis toxoids significantly differ in G + C content relative to mycobacterial genes, their codon usage was optimized. We also placed a 3xFLAG epitope in frame with the C-terminus of these toxoids to facilitate protein detection. Altogether, these optimizations resulted in the secretion of DTP antigens by MTBVAC, as demonstrated by western blot and MRM-MS. Finally, we examined specific antibody responses in mice vaccinated with recombinant MTBVAC expressing DTP antigens.

Delineation of a novel subgroup 16SrXIII-J phytoplasma, a 'Candidatus Phytoplasma hispanicum'-related strain, based on computer-simulated RFLP and phylogenetic analysis.

Symptoms of fruit phyllody and slow growth, which are suggestive of phytoplasma infection, were observed in strawberry plants cultivated in commercial fields. In order to provide evidence of association of phytoplasma with affected plants, assays for detecting and identifying were performed through computer-simulated restriction fragment length polymorphism (RFLP) and phylogenetic analysis. Total DNA was extracted from symptomatic and asymptomatic samples and used as template in nested PCR primed by the primers P1/Tint followed by R16F2n/16R2. Amplified DNA fragments of 1.2 kb from the 16S rRNA gene revealed the presence of phytoplasma in all symptomatic samples. Molecular detection was confirmed by electron transmission microscopy, which evidenced pleomorphic bodies in the phloem vessels. Nucleotide sequence representative of the strawberry phytoplasma shared 97.2 to 99 % similarity with phytoplasmas currently classified as members of the distinct subgroups within the 16SrXIII group. Similarity coefficient (F) values ranged from 0.70 to 0.92, indicating that strawberry phytoplasma delineates a new strain in addition to 'Candidatus Phytoplasma hispanicum'-related strains. The evolutionary tree displayed that this strain emerges as a new branch in relation to those previously described. The novel strain, designated SFP (strawberry fruit phyllody) phytoplasma represents the new 16SrXIII-J subgroup and its sequence, denominated SFP-Br02, was deposited in the GenBank database (EU719108). These findings contribute for the knowledge of the genetic diversity existing among members of the group 16SrXIII and establishes strawberry as an additional host of representatives of this group in Brazil.

A Gardnerella vaginalis e as infecções do trato urinário / The Gardnerella vaginalis and the urinary tract infections

As infecções do trato urinário (ITUs) estão entre as mais frequentes nos seres humanos. São causadas por grande variedade de uropatógenos habituais, porém podem ser provocadas por alguns micro-organismos fastidiosos, como a Gardnerella vaginalis, que é uma bactéria anaeróbia facultativa, observada sob a forma de cocobacilos Gram-variáveis. Ela habita a mucosa vaginal e eventualmente pode ocasionar ITUs. O isolamento pode ser realizado em amostras de urina utilizando o ágar CNA (colistina e ácido nalidíxico), com incubação de 48 a 72 horas em atmosfera rica em CO2. O exame de Gram de urina não centrifugada pode auxiliar o microbiologista na identificação das amostras nas quais uma bactéria fastidiosa seja o agente causal, já que a visualização de inúmeras células epiteliais, a ausência de leucócitos e a presença de mais de um tipo morfológico sugerem contaminação da amostra. Como estudos comprovam a incidência de G. vaginalis entre os agentes causadores de ITUs, o isolamento em uroculturas não deve ser desprezado. A interpretação clínica do crescimento da G. vaginalis é de difícil avaliação, sendo imprescindível a troca de informações entre o laboratório de microbiologia clínica e a equipe médica, investigando a presença de sinais e sintomas que possam estar associados à ITU.

Urinary tract infections are among the most recurrent in human beings. They are caused by a wide variety of usual uropathogens, although they may be caused by some fastidious micro-organisms, such as Gardnerella vaginalis. It is a facultative anaerobe, found in the form of Gram-variable coccobacilli. It inhabits the vaginal mucosa and occasionally may cause urinary tract infections. The isolation may be performed on urine samples using CNA agar, incubated for 48-72 hours in atmosphere rich in CO2. The Gram examination of non-centrifuged urine may aid the microbiologist in identifying samples in which a fastidious bacterium is the causative agent, since the visualization of numerous epithelial cells, the absence of leukocytes and the presence of more than one morphological type suggest sample contamination. As studies show the Gardnerella vaginalis incidence among the causative agents of urinary tract infections, the isolation in urine cultures can not be overlooked. The clinical interpretation of the Gardnerella vaginalis growth is difficult to assess, thus being essential the information exchange between the clinical microbiology laboratory and medical staff in order to investigate the presence of signs and symptoms that may be associated with urinary tract infections.