Indonesians Human Leukocyte Antigen (HLA) Distributions and Correlations with Global Diseases.

In Human, Major Histocompatibility Complex known as Human Leukocyte Antigen (HLA). The HLA grouped into three subclasses regions: the class I region, the class II region, and the class III region. There are thousands of polymorphic HLAs, many of them are proven to have correlations with diseases. Indonesia consists of diverse ethnicity people and populations. It carries a unique genetic diversity between one and another geographical positions. This paper aims to extract Indonesians HLA allele data, mapping the data, and correlating them with global diseases. From the study, it is found that global diseases, like Crohn's disease, rheumatoid arthritis, Graves' disease, gelatin allergy, T1D, HIV, systemic lupus erythematosus, juvenile chronic arthritis, and Mycobacterial disease (tuberculosis and leprosy) suspected associated with the Indonesian HLA profiles.

Development of a dry, stable and inhalable acyl-homoserine-lactone-acylase powder formulation for the treatment of pulmonary Pseudomonas aeruginosa infections.

In the lungs of cystic fibrosis (CF) patients, Pseudomonas aeruginosa commonly causes chronic infections. It has been shown that the P. aeruginosa quorum sensing (QS) system controls the expression of virulence factors during invasion and infection to host cells. PvdQ is an acyl-homoserine lactone (AHL) acylase able to degrade the signal molecule of P. aeruginosa QS. The role of PvdQ in inhibiting the QS and its successive virulence determinants has been established in in vitro as well as in in vivo, the latter in a Caenorabdhitis elegans model. For the treatment of pulmonary P. aeruginosa infections, we propose that PvdQ can be best administered directly to the lungs of the patients as a dry powder because this is expected to give specific advantages in delivery as compared to nebulizing. Therefore in this study we investigated the production of a PvdQ powder by spray-freeze drying using mannitol, trehalose and inulin as excipient. The activity of PvdQ in the powder was determined immediately after production and after subsequent storage during 4 weeks at 20°C and 55°C. We found that the enzymatic activity of PvdQ is fully maintained during spray-freeze drying using mannitol, trehalose or inulin as excipient. However, mannitol was not able to stabilize the protein during storage, while PvdQ incorporated in trehalose or inulin was fully stabilized even during storage at 55°C for at least 4 weeks. The poor stabilizing capacities of mannitol during storage could be related to its crystalline nature while the excellent stabilizing capacities of trehalose and inulin during storage could be related to their amorphous nature. The trehalose and inulin-based particles consisted of porous spheres with a volume average aerodynamical diameter of ∼1.8 µm implying that they are suitable for pulmonary delivery. This is the first study in which an AHL-degrading enzyme is processed into spray-freeze-dried powder suitable for inhalation.

PA0305 of Pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily.

The Pseudomonas aeruginosa PAO1 genome has at least two genes, pvdQ and quiP, encoding acylhomoserine lactone (AHL) acylases. Two additional genes, pa1893 and pa0305, have been predicted to encode penicillin acylase proteins, but have not been characterized. Initial studies on a pa0305 transposon insertion mutant suggested that the gene is not related to the AHL growth phenotype of P. aeruginosa. The close similarity (67 %) of pa0305 to HacB, an AHL acylase of Pseudomonas syringae, prompted us to investigate whether the PA0305 protein might also function as an AHL acylase. The pa0305 gene has been cloned and the protein (PA0305) has been overproduced, purified and subjected to functional characterization. Analysis of the purified protein showed that, like ß-lactam acylases, PA0305 undergoes post-translational processing resulting in α- and ß-subunits, with the catalytic serine as the first amino acid of the ß-subunit, strongly suggesting that PA0305 is a member of the N-terminal nucleophile hydrolase superfamily. Using a biosensor assay, PA0305his was shown to degrade AHLs with acyl side chains ranging in length from 6 to 14 carbons. Kinetics studies using N-octanoyl-L-homoserine lactone (C(8)-HSL) and N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C(12)-HSL) as substrates showed that the enzyme has a robust activity towards these two AHLs, with apparent K(cat)/K(m) values of 0.14 × 10(4) M(-1) s(-1) towards C(8)-HSL and 7.8 × 10(4) M(-1 )s(-1) towards 3-oxo-C(12)-HSL. Overexpression of the pa0305 gene in P. aeruginosa showed significant reductions in both accumulation of 3-oxo-C(12)-HSL and expression of virulence factors. A mutant P. aeruginosa strain with a deleted pa0305 gene showed a slightly increased capacity to kill Caenorhabditis elegans compared with the P. aeruginosa PAO1 wild-type strain and the PAO1 strain carrying a plasmid overexpressing pa0305. The harmful effects of the Δpa0305 strain on the animals were most visible at 5 days post-exposure and the mortality rate of the animals fed on the Δpa0305 strain was faster than for the animals fed on either the wild-type strain or the strain overexpressing pa0305. In conclusion, the pa0305 gene encodes an efficient acylase with activity towards long-chain homoserine lactones, including 3-oxo-C(12)-HSL, the natural quorum sensing signal molecule in P. aeruginosa, and we propose to name this acylase HacB.

Role of PvdQ in Pseudomonas aeruginosa virulence under iron-limiting conditions.

PvdQ, an acylase from Pseudomonas aeruginosa PAO1, has been shown to have at least two functions. It can act as a quorum quencher due to its ability to degrade long-chain N-acylhomoserine lactones (AHLs), e.g. 3-oxo-C12-HSL, leading to a decrease in virulence factors. In addition, PvdQ is involved in iron homeostasis by playing a role in the biosynthesis of pyoverdine, the major siderophore of P. aeruginosa. In accordance with earlier studies on RNA level, we could show at the protein level that PvdQ is only expressed when iron is present at very low concentrations. We therefore set out to investigate the two functions of PvdQ under iron-limiting conditions. Gene deletion of pvdQ does not affect growth of P. aeruginosa but abrogates pyoverdine production, and results in an accumulation of 3-oxo-C12-HSL. Phenotypic analyses of our DeltapvdQ mutant at low iron concentrations revealed that this mutant is impaired in swarming motility and biofilm formation. Additionally, a plant and a Caenorhabditis elegans infection model demonstrated that the deletion of pvdQ resulted in reduced virulence. None of the phenotypes in the present study could be linked to the presence or absence of AHLs. These results clearly indicate that under iron-limiting conditions PvdQ plays a major role in swarming motility, in biofilm development and in infection that is more likely to be linked to the pyoverdine pathway rather than the LasI/LasR/3-oxo-C12-HSL quorum-sensing circuit.

Quorum-quenching acylase reduces the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model.

The Pseudomonas aeruginosa PAO1 gene pvdQ encodes an acyl-homoserine lactone (AHL) acylase capable of degrading N-(3-oxododecanoyl)-L-homoserine lactone by cleaving the AHL amide. PvdQ has been proven to function as a quorum quencher in vitro in a number of phenotypic assays. To address the question of whether PvdQ also shows quorum-quenching properties in vivo, an infection model based on the nematode Caenorhabditis elegans was explored. In a fast-acting paralysis assay, strain PAO1(pMEpvdQ), which overproduces PvdQ, was shown to be less virulent than the wild-type strain. More than 75% of the nematodes exposed to PAO1(pMEpvdQ) survived and continued to grow when using this strain as a food source. Interestingly, in a slow-killing assay monitoring the survival of the nematodes throughout a 4-day course, strain PAO1-Delta pvdQ was shown to be more virulent than the wild-type strain, confirming the role of PvdQ as a virulence-reducing agent. It was observed that larval stage 1 (L1) to L3-stage larvae benefit much more from protection by PvdQ than L4 worms. Finally, purified PvdQ protein was added to C. elegans worms infected with wild-type PAO1, and this resulted in reduced pathogenicity and increased the life span of the nematodes. From our observations we can conclude that PvdQ might be a strong candidate for antibacterial therapy against Pseudomonas infections.