Binding of HasA by its transmembrane receptor HasR follows a conformational funnel mechanism.

HasR in the outer membrane of Serratia marcescens binds secreted, heme-loaded HasA and translocates the heme to the periplasm to satisfy the cell's demand for iron. The previously published crystal structure of the wild-type complex showed HasA in a very specific binding arrangement with HasR, apt to relax the grasp on the heme and assure its directed transfer to the HasR-binding site. Here, we present a new crystal structure of the heme-loaded HasA arranged with a mutant of HasR, called double mutant (DM) in the following that seemed to mimic a precursor stage of the abovementioned final arrangement before heme transfer. To test this, we performed first molecular dynamics (MD) simulations starting at the crystal structure of the complex of HasA with the DM mutant and then targeted MD simulations of the entire binding process beginning with heme-loaded HasA in solution. When the simulation starts with the former complex, the two proteins in most simulations do not dissociate. When the mutations are reverted to the wild-type sequence, dissociation and development toward the wild-type complex occur in most simulations. This indicates that the mutations create or enhance a local energy minimum. In the targeted MD simulations, the first protein contacts depend upon the chosen starting position of HasA in solution. Subsequently, heme-loaded HasA slides on the external surface of HasR on paths that converge toward the specific arrangement apt for heme transfer. The targeted simulations end when HasR starts to relax the grasp on the heme, the subsequent events being in a time regime inaccessible to the available computing power. Interestingly, none of the ten independent simulation paths visits exactly the arrangement of HasA with HasR seen in the crystal structure of the mutant. Two factors which do not exclude each other could explain these observations: the double mutation creates a non-physiologic potential energy minimum between the two proteins and /or the target potential in the simulation pushes the system along paths deviating from the low-energy paths of the native binding processes. Our results support the former view, but do not exclude the latter possibility.

Structures of the Heme Acquisition Protein HasA with Iron(III)-5,15-Diphenylporphyrin and Derivatives Thereof as an Artificial Prosthetic Group.

Iron(III)-5,15-diphenylporphyrin and several derivatives were accommodated by HasA, a heme acquisition protein secreted by Pseudomonas aeruginosa, despite possessing bulky substituents at the meso position of the porphyrin. Crystal structure analysis revealed that the two phenyl groups at the meso positions of porphyrin extend outside HasA. It was shown that the growth of P. aeruginosa was inhibited in the presence of HasA coordinating the synthetic porphyrins under iron-limiting conditions, and that the structure of the synthetic porphyrins greatly affects the inhibition efficiency.

Inhibition of heme uptake in Pseudomonas aeruginosa by its hemophore (HasA(p)) bound to synthetic metal complexes.

The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasA(p)) can capture several synthetic metal complexes other than heme. The crystal structures of HasA(p) harboring synthetic metal complexes revealed only small perturbation of the overall HasA(p) structure. An inhibitory effect upon heme acquisition by HasA(p) bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasA(p) bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasA(p) as an iron source.

Health Literacy throughout adolescence: Invariance and validity study of three measurement scales in the general population.

OBJECTIVE: To simultaneously investigate the psychometric properties of three recently developed health literacy measurement scales throughout adolescence in the general population. METHODS: French versions of the Health Literacy for School-Aged Children (HLSAC, unidimensional) scale, the Health Literacy Assessment Scale for Adolescents (HAS-A, multidimensional) and the 16-item European Health Literacy Survey questionnaire (HLS-EU-Q16, unidimensional) were completed by 1 444 adolescents in 8th, 9th, 11th grade in general school and 11-12th grade in vocational school. Psychometric properties were studied using confirmatory factor analysis, McDonald's omega coefficient and hypothesis testing. RESULTS: Structural validity was acceptable (HLS-EU-Q16) to good (HAS-A and HLSAC), no measurement invariance issue was found and internal consistency was acceptable for the three scales (0.68-0.84). Convergent validity was low (Pearson correlation coefficients<0.5) and the only scale for which results were in agreement with a priori hypotheses was the HLSAC. CONCLUSIONS: Our results were supportive of the use of HLSAC to assess health literacy during adolescence but the HAS-A, with a slightly better structural validity, can also be promoted due to its three measured dimensions. PRACTICE IMPLICATIONS: The use of these scales in practice will help to focus on health literacy, a critical factor for prevention and health promotion in adolescence.

Infectious bronchitis virus from chickens in Al-Hasa, Saudi Arabia 2015-2016.

AIM: This study aimed to isolate some of the currently circulating infectious bronchitis virus (IBV) strains from some broiler chicken farms in Al-Hasa and to do some molecular characteristics of these strains. MATERIALS AND METHODS: We collected 300 tissue specimens, including the trachea, bronchi, lungs, and kidneys from some four commercial chicken farms showing respiratory manifestations. We tested these tissue specimens by the real-time polymerase chain reaction (RT-PCR) and gel-based PCR. We selected some PCR positive samples for isolation in the embryonated chicken eggs (ECE). We sequenced some PCR-positive samples and conducted phylogenetic analysis based on the obtained sequences. RESULTS: Our molecular surveillance revealed that 31.6% of the tested specimens were IBV positive by PCR. We selected some positive specimens showing low Ct values by the qRT-PCR for virus isolation by the ECE. The infected eggs showed hemorrhage, dwarfing, and death in some cases after three passages in the ECE. We sequenced some of the positive PCR specimens and used the obtained sequences to draw the phylogenetic tree based on the partial IBV-ORF-1a, N, and S1 gene sequences. The phylogenetic trees based on the IBV-N and S1 gene sequences showed that the circulating IBV strains in Al-Hasa during 2016 was showing a high degree of identity to some strains from Taiwan and Italy. Meanwhile, the grouping of these strains based on the IBV-S1 sequences revealed that the currently circulating IBV strains in Al-Hasa belonged to Gr.I.7 along with strains from Taiwan. CONCLUSION: Our results confirmed the continuous circulation of the IBV among the chicken population in Al-Hasa despite the intensive application of vaccines against this virus.

Evidence for involvement of the Fas BCAX regulon in capsule synthesis by Streptococcus equi.

The constitutively expressed hyaluronic acid capsule is an important virulence factor of Streptococcus equi, the cause of equine strangles. Study of the genomic sequence of CF22caps-, a non-encapsulated mutant of S. equi CF22 generated by gamma (Co60) irradiation revealed a non-sense mutation in fasC (SEQ_0302), a sensor kinase gene in FasBCAX an operon with an important regulatory role in expression of streptococcal secreted virulence and matrix binding proteins. The mutation was associated with a significant (p < .05) decrease in transcription of hasA, the synthase gene essential for hyaluronic acid synthesis and, conversely, with small increases in transcription of skc, covR and seM. The early growth phase of CF22caps- was also delayed compared to the CF22caps+ parent. In contrast to the human pathogen, S. pyogenes, capsule synthesis in S. equi therefore appears to be controlled by FasBCAX and not by CovRS.

Enhanced hyluronic acid production in Streptococcus zooepidemicus by over expressing HasA and molecular weight control with Niscin and glucose.

Hyaluronic acid (HA) is a high molecular weight linear polysaccharide, endowed with unique physiological and biological properties. Given its unique properties, HA have unprecedented applications in the fields of medicine and cosmetics. The ever growing demand for HA production is the driving force behind the need for finding and developing novel and amenable sources of the HA producers. Microbial fermentation of Streptococcus zooepidemicus deemed as one the most expeditious and pervasive methods of HA production. Herein, a wild type Streptococcus zooepidemicus, intrinsically expressing high levels of HA, was selected and optimized for HA production. HasA gene was amplified and introduced into the wild type Streptococcus zooepidemicus, under the control of Nisin promoter. The HasA over-expression increased the HA production, while the molecular weight was decreased. In order to compensate for molecular weight loss, the glucose concentration was increased to an optimum amount of 90 g/L. It is hypostatizes that excess glucose would rectify the distribution of the monomers and each HasA molecule would be provided with sufficient amount of substrates to lengthen the HA molecules. Arriving at an improved strain and optimized cultivating condition would pave the way for industrial grade HA production with high quality and quantity.

Continuous Reusability using Immobilized HasApf in Chemoenzymatic Deracemization: A New Heterogeneous Enzyme Catalysis.

This study found that the calibration curve of heme acquisition system A (HasA, a new reactive active species) immobilized by a porous ceramic particle (ImHApf; immobilized HasA from Pseudomonas fluorescens) can be constructed in the range of 1750-1450 cm-1 using Fourier transform infrared spectroscopy (FTIR) analysis, and evaluated its catalytic efficiency. In the asymmetric oxidation of rac-1-(6-methoxynaphthalen-2-yl)ethanol (rac-1: a naproxen precursor), a product ketone from the (R)-isomer is desymmetrized using NaBH4 and continuously reused even if treated with an organic solvent in 50 mM glycine-NaOH buffer at 40 °C in the absence of nicotinamide adenine dinucleotide (NAD(P)), leading to >99% enantiomeric excess and >90% chemical yield; the activity was calculated at 0.74 ± 0.03 mU/(mg·min) and the turnover number was determined to be approximately 2 × 106. It was confirmed that the other sec-alcohols such as rac-1-(2-naphthyl)ethanol (rac-2) and m- and p-substituted rac-1-phenyl ethanols (rac-3ab-6ab) using ImHApf can also yield a single stereoisomer from a racemate. Therefore, HasA immobilization can be expected to become an important tool for building an environmentally friendly system that promotes industrial sustainability.

Capsular hyaluronic acid of equine isolates of Streptococcus zooepidemicus is upregulated at temperatures below 35°C.

REASONS FOR PERFORMING STUDY: Streptococcus zooepidemicus causes opportunist respiratory and other infections in the horse. Capsule expression is highly variable and known to affect resistance to phagocytosis. Most clinical isolates producing small, dry colonies at 37°C produce mucoid colonies at temperatures below 35°C. OBJECTIVES: The aim was to understand the molecular basis of increased capsule expression by equine isolates of S. zooepidemicus at temperatures lower than 35°C. STUDY DESIGN: Cross-sectional observational study. METHODS: Capsule production by groups of equine S. zooepidemicus strains was determined at 23, 30, 35 and 37°C. Hyaluronidase (HylC) at 23 and 37°C was measured by quantitative enzyme-linked immunosorbent assay. Expressions of hasA and hylC at 23 and 37°C were measured by quantitative reverse transcriptase-PCR. The covRS genes in representative S. zooepidemicus were sequenced and checked for mutations. RESULTS: Colonies of randomly selected S. zooepidemicus strains became mucoid or showed marked increase in colony mucoidy following the change in temperature to 23°C. Expression of hasA at 23°C was 45- to 700-fold greater than at 37°C. Transcription of hylC at 23°C was 2.5- to 200-fold greater than at 37°C, yet enzyme concentrations in cultures were significantly higher at 37°C (P<0.05), suggesting that production of HylC is regulated post transcriptionally. The covRS gene in S. zooepidemicus was not mutated as seen in isolates of Streptococcus pyogenes with increased capsule production at 25°C. CONCLUSIONS: Sensitivity of capsule expression to temperature above 35°C but not HylC by the general population of equine S. zooepidemicus indicates that capsule is not required for extended colonisation nor for opportunist invasion. Instead, capsule production at lower than body temperature may reflect adaptation to life on skin and mucosal surfaces, where hyaluronic acid contributes to adhesion and resistance to desiccation. Pathogenicity of S. zooepidemicus following opportunist invasion is possibly dependent on factors other than capsule that may be co-regulated with HylC. The Summary is available in Chinese - see Supporting information.