A D-Alanine auxotrophic live vaccine is effective against lethal infection caused by Staphylococcus aureus.

Staphylococcus aureus infections are becoming a major global health issue due to the rapid emergence of multidrug-resistant strains. Therefore, there is an urgent need to develop an effective vaccine to prevent and control these infections. In order to develop a universal immunization strategy, we constructed a mutant derivative of S. aureus 132 which lacks the genes involved in D-alanine biosynthesis, a structural component of cell wall peptidoglycan. This unmarked deletion mutant requires the exogenous addition of D-alanine for in vitro growth. The aim of this study was to examine the ability of this D-alanine auxotroph to induce protective immunity against staphylococcal infection. Our findings demonstrate that this deletion mutant is highly attenuated, elicits a protective immune response in mice and generates cross-reactive antibodies. Moreover, the D-alanine auxotroph was completely eliminated from the blood of mice after its intravenous or intraperitoneal injection. We determined that the protective effect was dependent on antibody production since the adoptive transfer of immune serum into naïve mice resulted in effective protection against S. aureus bacteremia. In addition, splenocytes from mice immunized with the D-alanine auxotroph vaccine showed specific production of IL-17A after ex vivo stimulation. We conclude that this D-alanine auxotroph protects mice efficiently against virulent staphylococcal strains through the combined action of antibodies and IL-17A, and therefore constitutes a promising vaccine candidate against staphylococcal disease, for which no licensed vaccine is available yet.

Estudo do metaboloma salivar e sua associação com a doença periodontal em pacientes com síndrome de Down / Saliva metabolome in patients with Down syndrome and its association with periodontal disease

Os pacientes com Síndrome de Down (SD) possuem grande incidência de doença periodontal (DP), caracterizada por um curso precoce e com maior severidade. O estudo de metaboloma pode contribuir para o entendimento deste curso da doença, identificando possíveis metabólitos como biomarcadores nestes indivíduos. Para entender o perfil metabolômico dos indivíduos com síndrome de Down e a sua relação com a doença periodontal, realizamos a identificação de metabólitos salivares de adolescentes e adultos jovens, entre 12 e 21 anos, ambos os gêneros. Foram coletados dados sobre o estado geral de saúde e realizados exames clínicos bucais, como índice de higiene oral simplificado, sangramento e profundidade de sondagem. Para a análise do metaboloma foi coletada amostra de saliva não estimulada, analisadas por meio de cromatografia gasosa acoplada á espectrometria de massas. Saliva e fluido crevicular gengival também foram coletados para identificação microbiana através do MALDI-TOF. Os dados encontrados foram submetidos a análise estátisca por meio da Análise dos Componentes Principais (PCA) e quantificação relativa dos metabólitos foi avaliada por testes não paramétricos, Mann-Whitney

e Kruskal-Wallis. Foi possível observar através dos modelos de PCA separação dos indivíduos com SD e controles, independente da doença periodontal. A quantificação relativa revelou maiores níveis de glicina, lprolina, l-leucina, l-serina, ácido palmítico, ácido pentanóico, ácido tetradecanóico, tirosina e l-fenilalanina nos grupos SD quando comparados aos controles. Controles com DP também apresentaram níveis elevados de glicina, l-alanina, l-serina e manopiranose quando comparados com controles saudáveis. A microbiota de indivíduos com SD apresentous diferenças siginificantes em relação aos individuos controles, principalmente para Rothia dentocariosa, Staphylococcus epidermidis, Tannerella forsythia quando avaliado a saliva e A. Actinomycetemcomitans, Micrococcus luteus, Rothia aeria, Treponema denticola no fluido crevicular gengival. Em conclusão, o perfil metabolômico impresso nos indivíduos com SD difere significativamente dos indivíduos controles, independente da doença periodontal. Entretanto, os metabólitos que diferenciam indivíduos controles com e sem DP, apresentam-se elevados em todos indivíduos com SD, promovendo novos "insights" para o perfil metabólico relacionado a DP na SD.

Down Syndrome (DS) patients have a high incidence of periodontal disease (PD), characterized by an early course and greater severity. The metabolome study may contribute to the understanding of the disease course, identifying possible metabolites as biomarkers in these individuals. To understand the metabolomic profile of the DS and their relationship with PD, we conducted the identification of salivary metabolites of adolescents and young adults between 12 and 21 years, both genders. Data were collected on general health and was performed oral clinical examination, as the IHOS, bleeding index and probing depth. For metabolome analysis was collected unstimulated saliva sample, analyzed by gas chromatography coupled to mass spectrometry. Saliva and gingival crevicular fluid were also collected for microbial identification by MALDI-TOF. Data were submitted to analysis-statistic by PCA and relative quantification

of metabolites was evaluated by Mann-Whitney and Kruskal-Wallis tests. It can be observed through the PCA models separation of DS groups and controls groups, regardless of periodontal disease. Relative quantification showed higher levels of glycine, L-proline, L-leucine, L-serine, palmitic acid, pentanoic acid, tetradecanoic acid, tyrosine and L-phenylalanine in the SD groups when compared to controls groups. Controls with PD also showed high levels of glycine, L-alanine, L-serine and mannopyranose compared with healthy controls. The microbiota of individuals with DS groups show significant differences compared to control groups, especially for Rothia dentocariosa, Staphylococcus epidermidis, Tannerella forsythia when evaluated saliva and A. actinomycetemcomitans, Micrococcus luteus, Rothia aeria, Treponema denticola in gingival crevicular fluid. In conclusion, the printed metabolomic profile in individuals with Down syndrome differs significantly from control subjects, regardless of periodontal disease. However, the metabolites that distinguish controls group with and without PD, show up high in all DS individuals, promoting new "insights" to the metabolic profile related to PD in DS.

The alanine racemase of Mycobacterium smegmatis is essential for growth in the absence of D-alanine.

Alanine racemase, encoded by the gene alr, is an important enzyme in the synthesis of d-alanine for peptidoglycan biosynthesis. Strains of Mycobacterium smegmatis with a deletion mutation of the alr gene were found to require d-alanine for growth in both rich and minimal media. This indicates that alanine racemase is the only source of d-alanine for cell wall biosynthesis in M. smegmatis and confirms alanine racemase as a viable target gene for antimycobacterial drug development.

Late presentation and development of nephrocalcinosis in primary hyperoxaluria.

A case of primary hyperoxaluria type 1 with complete deficiency of alanine:glyoxalate aminotransferase that first manifested at the age of 59 with irreversible acute on chronic renal failure is reported. Nephrocalcinosis, initially absent, developed rapidly after renal failure evolved. The possible role of hypovolaemia and contrast nephrotoxicity in precipitating the clinical onset is discussed. Primary hyperoxaluria should be considered in patients of any age presenting with unexplained renal failure, and appropriate systemic pathology of oxalosis.

A deficiency in A alpha chain's N-terminal alanine residue as a major cause of the slow coagulation of fetal fibrinogen.

To clarify the mechanism which causes the coagulation time of fetal fibrinogen to be longer than that of adult fibrinogen, the N-terminal amino acid residues of fetal fibrinogen were analyzed. The results showed that the amount of A alpha chain's N-terminal alanine residues in fetal fibrinogen was only 54% of that in adult fibrinogen. When the amount of A alpha chain's N-terminal residual alanine in adult fibrinogen was decreased to 57% of the normal level by digestion with aminopeptidase M, the adult fibrinogen yielded a prolonged thrombin time and a retarded release rate for fibrino-peptides A and B, both values approximating to those of fetal fibrinogen. The results suggest that the deficiency in alanine residue at the N-terminus of the A alpha chain is a major cause of the slow coagulation of fetal fibrinogen.

Hemoglobin J-Chicago (beta76(E20) Ala yields Asp): a new hemoglobin variant resulting from substitution of an external residue.

An electrophoretically fast-moving hemoglobin variant was found in a 2-yr-old boy who was referred for evaluation with findings of iron deficiency anemia. The anemia was corrected, and no hematologic abnormality remained after treatment with iron. Oxygen affinity of the blood was normal, and no evidence was found of instability of the variant hemoglobin. Structural studies demonstrated a substitution of aspartic acid for alanine at beta76 (E20). This change did not appear to cause any functional disruption of the hemoglobin in this patient, as would be predicted by the position of the affected animo acid residue on the surface of the molecule.