The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis (CF) and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals; pigment production has been reported to enable Bcc strains to overcome the host cell oxidative burst. In this work, we investigated the role of pyomelanin in resistance to oxidative stress and virulence in strains J2315 and K56-2, two epidemic CF isolates belonging to the Burkholderia cenocepacia ET-12 lineage. We previously reported that a single amino acid change from glycine to arginine at residue 378 in homogentisate 1,2-dioxygenase (HmgA) affects the pigment production phenotype: pigmented J2315 has an arginine at position 378, while non-pigmented K56-2 has a glycine at this position. Herein, we performed allelic exchange to generate isogenic non-pigmented and pigmented strains of J2315 and K56-2, respectively, and tested these to determine whether pyomelanin contributes to the protection against oxidative stress in vitro as well as in a respiratory infection in CGD mice in vivo. Our results indicate that the altered pigment phenotype does not significantly impact these strains\' ability to resist oxidative stress with H2O2 and NO in vitro and did not change the virulence and infection outcome in CGD mice in vivo suggesting that other factors besides pyomelanin are contributing to the pathophysiology of these strains.IMPORTANCEThe Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria that are often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals and overcoming the host cell oxidative burst. We investigated the role of pyomelanin in Burkholderia cenocepacia strains J2315 (pigmented) and K56-2 (non-pigmented) and performed allelic exchange to generate isogenic non-pigmented and pigmented strains, respectively. Our results indicate that the altered pigment phenotype does not significantly impact these strains\' ability to resist H2O2 or NO in vitro and did not alter the outcome of a respiratory infection in CGD mice in vivo. These results suggest that pyomelanin may not always constitute a virulence factor and suggest that other features are contributing to the pathophysiology of these strains.

White-rot fungi, such as Phanerochaete chrysosporium, are the most efficient degraders of lignin, a major component of plant biomass. Enzymes produced by these fungi, such as lignin peroxidases and manganese peroxidases, break down lignin polymers into various aromatic compounds based on guaiacyl, syringyl, and hydroxyphenyl units. These intermediates are further degraded, and the aromatic ring is cleaved by 1,2,4-trihydroxybenzene dioxygenases. This study aimed to characterize homogentisate dioxygenase (HGD)-like proteins from P. chrysosporium that are strongly induced by the G-unit fragment of vanillin. We overexpressed two homologous recombinant HGDs, PcHGD1 and PcHGD2, in Escherichia coli. Both PcHGD1 and PcHGD2 catalyzed the ring cleavage in methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ). The two enzymes had the highest catalytic efficiency (kcat/Km) for MHQ, and therefore, we named PcHGD1 and PcHGD2 as MHQ dioxygenases 1 and 2 (PcMHQD1 and PcMHQD2), respectively, from P. chrysosporium. This is the first study to identify and characterize MHQ and DMHQ dioxygenase activities in members of the HGD superfamily. These findings highlight the unique and broad substrate spectra of PcHGDs, rendering them attractive candidates for biotechnological applications.IMPORTANCEThis study aimed to elucidate the properties of enzymes responsible for degrading lignin, a dominant natural polymer in terrestrial lignocellulosic biomass. We focused on two homogentisate dioxygenase (HGD) homologs from the white-rot fungus, P. chrysosporium, and investigated their roles in the degradation of lignin-derived aromatic compounds. In the P. chrysosporium genome database, PcMHQD1 and PcMHQD2 were annotated as HGDs that could cleave the aromatic rings of methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ) with a preference for MHQ. These findings suggest that MHQD1 and/or MHQD2 play important roles in the degradation of lignin-derived aromatic compounds by P. chrysosporium. The preference of PcMHQDs for MHQ and DMHQ not only highlights their potential for biotechnological applications but also underscores their critical role in understanding lignin degradation by a representative of white-rot fungus, P. chrysosporium.

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by pathogenic variants in the homogentisate 1,2-dioxygenase (HGD) gene. This leads to a deficient HGD enzyme with the consequent accumulation of homogentisic acid (HGA) in different tissues causing complications in various organs, particularly in joints, heart valves and kidneys. The genetic basis of AKU in Egypt is completely unknown. We evaluated the clinical and genetic spectrum of six pediatric and adolescents AKU patients from four unrelated Egyptian families. All probands had a high level of HGA in urine by qualitative GC/MS before genetic confirmation by Sanger sequencing. Recruited AKU patients were four females and two males (median age 13 years). We identified four different pathogenic missense variants within HGD gene. Detected variants included a novel variant c.1079G > T;p.(Gly360Val) and three recurrent variants; c.1078G > C;p.(Gly360Arg), c.808G > A;p.(Gly270Arg) and c.473C > T;p.(Pro158Leu). All identified variants were properly segregating in the four families consistent with autosomal recessive inheritance. In this study, we reported the phenotypic and genotypic spectrum of alkaptonuria for the first time in Egypt. We further enriched the HGD-variant database with another novel pathogenic variant. The recent availability of nitisinone may promote the need for genetic confirmation at younger ages to start therapy earlier and prevent serious complications.

Until now, only a few studies have focused on the early onset of symptoms of alkaptonuria (AKU) in the pediatric population. This prospective, longitudinal study is a comprehensive approach to the assessment of children with recognized AKU during childhood. The study includes data from 32 visits of 13 patients (five males, eight females; age 4-17 years) with AKU. A clinical evaluation was performed with particular attention to eye, ear, and skin pigmentation, musculoskeletal complaints, magnetic resonance imaging (MRI), and ultrasound (US) imaging abnormalities. The cognitive functioning and adaptive abilities were examined. Molecular genetic analyses were performed. The most common symptoms observed were dark urine (13/13), followed by joint pain (6/13), and dark ear wax (6/13). In 4 of 13 patients the values obtained in the KOOS-child questionnaire were below the reference values. MRI and US did not show degenerative changes in knee cartilages. One child had nephrolithiasis. Almost half of the children with AKU (5/13) presented deficits in cognitive functioning and/or adaptive abilities. The most frequent HGD variants observed in the patients were c.481G>A (p.Gly161Arg) mutation and the c.240A>T (p.His80Gln) polymorphism. The newly described allele of the HGD gene (c.948G>T, p.Val316Phe) which is potentially pathogenic was identified.

A 6-yr-old female orangutan presented with a history of dark urine that turned brown upon standing since birth. Repeated routine urinalysis and urine culture were unremarkable. Urine organic acid analysis showed elevation in homogentisic acid consistent with alkaptonuria. Sequence analysis identified a homozygous missense variant, c.1081G>A (p.Gly361Arg), of the homogentisate 1,2-dioxygenase (HGD) gene. Familial studies, molecular modeling, and comparison to human variant databases support this variant as the underlying cause of alkaptonuria in this orangutan. This is the first report of molecular confirmation of alkaptonuria in a nonhuman primate.

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs, which occurs because the homogentisate 1,2-dioxygenase (HGD) enzyme is not functional due to gene variants. Over time, HGA oxidation and accumulation cause the formation of the ochronotic pigment, a deposit that provokes tissue degeneration and organ malfunction. Here, we report a comprehensive review of the variants so far reported, the structural studies on the molecular consequences of protein stability and interaction, and molecular simulations for pharmacological chaperones as protein rescuers. Moreover, evidence accumulated so far in alkaptonuria research will be re-proposed as the bases for a precision medicine approach in a rare disease.

Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defective homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called ochronosis, which results on the long term in an early-onset and severe osteoarthropathy. HGD\'s quaternary structure is known to be easily disrupted by missense mutations, which makes them an interesting target for novel treatment strategies that aim to rescue enzyme activity. However, only prediction models are available providing information on a structural basis. Therefore, an E. coli based whole-cell screening was developed to evaluate HGD missense variants in 96-well microtiter plates. The screening principle is based on HGD\'s ability to convert the oxidation sensitive HGA into maleylacetoacetate. More precisely, catalytic activity could be deduced from pyomelanin absorbance measurements, derived from the auto-oxidation of remaining HGA. Optimized screening conditions comprised several E. coli expression strains, varied expression temperatures and varied substrate concentrations. In addition, plate uniformity, signal variability and spatial uniformity were investigated and optimized. Finally, eight HGD missense variants were generated via site-directed mutagenesis and evaluated with the developed high-throughput screening (HTS) assay. For the HTS assay, quality parameters passed the minimum acceptance criterion for Z\' values > 0.4 and single window values > 2. We found that activity percentages versus wildtype HGD were 70.37 ± 3.08% (for M368V), 68.78 ± 6.40% (for E42A), 58.15 ± 1.16% (for A122V), 69.07 ± 2.26% (for Y62C), 35.26 ± 1.90% (for G161R), 35.86 ± 1.14% (for P230S), 23.43 ± 4.63% (for G115R) and 19.57 ± 11.00% (for G361R). To conclude, a robust, simple, and cost-effective HTS system was developed to reliably evaluate and distinguish human HGD missense variants by their HGA consumption ability. This HGA quantification assay may lay the foundation for the development of novel treatment strategies for missense variants in AKU.

The emergence and spread of antibiotic resistance pose serious environmental and health challenges. Attention has been drawn to phage therapy as an alternative approach to combat antibiotic resistance with immense potential. However, one of the obstacles to phage therapy is phage resistance, and it can be acquired through genetic mutations, followed by consequences of phenotypic variations. Therefore, understanding the mechanisms underlying phage-host interactions will provide us with greater detail on how to optimize phage therapy. In this study, three lytic phages (phipa2, phipa4, and phipa10) were isolated to investigate phage resistance and the potential fitness trade-offs in Pseudomonas aeruginosa. Specifically, in phage-resistant mutants phipa2-R and phipa4-R, mutations in conferring resistance occurred in genes pilT and pilB, both essential for type IV pili (T4P) biosynthesis. In the phage-resistant mutant phipa10-R, a large chromosomal deletion of ~294 kb, including the hmgA (homogentisate 1,2-dioxygenase) and galU (UTP-glucose-1-phosphate uridylyltransferase) genes, was observed and conferred phage phipa10 resistance. Further, we show examples of associated trade-offs in these phage-resistant mutations, e.g., impaired motility, reduced biofilm formation, and increased antibiotic susceptibility. Collectively, our study sheds light on resistance-mediated genetic mutations and their pleiotropic phenotypes, further emphasizing the impressive complexity and diversity of phage-host interactions and the challenges they pose when controlling bacterial diseases in this important pathogen. IMPORTANCE Battling phage resistance is one of the main challenges faced by phage therapy. To overcome this challenge, detailed information about the mechanisms of phage-host interactions is required to understand the bacterial evolutionary processes. In this study, we identified mutations in key steps of type IV pili (T4P) and O-antigen biosynthesis leading to phage resistance and provided new evidence on how phage predation contributed toward host phenotypes and fitness variations. Together, our results add further fundamental knowledge on phage-host interactions and how they regulate different aspects of Pseudomonas cell behaviors.

OBJECTIVE: Alkaptonuria is a rare autosomal recessive genetic disorder resulting from the deficiency of homogentisate 1,2 dioxygenase (HGD), the third enzyme in the tyrosine degradation pathway. Homogentisic acid produced in excess oxidizes into ochronotic pigment polymer. Accumulation of this pigment in various tissues leads to systemic disease.
METHODS: Clinical, laboratory, molecular findings and treatment characteristics of 35 patients followed up in Ege University Pediatric Nutrition, and Metabolism Department with the diagnosis of alkaptonuria were evaluated retrospectively.
RESULTS: Twenty-four males (68.57%) and 11 females (31.42%) with a confirmed diagnosis of alkaptonuria from 32 different families were included in the study. We identified 11 different genetic variants; six of these were novel. c.1033C>T, c.676G>A, c.664G>A, c.731_734del, c.1009G>T, c.859_862delins ATAC were not previously reported in the literature. 24 (68.57%) patients only adhered to a low-protein diet in our study group. Seven (20%) patients initiated a low protein diet and NTBC therapy. Mean urinary HGA decreased by 88.7% with nitisinone. No statistical changes were detected in urinary HGA excretion with the low protein diet group.
CONCLUSIONS: In our study, alkaptonuria patients were diagnosed at different ages, from infancy to adulthood, and progressed with other systemic involvement in the follow-up. Since the initial period is asymptomatic, giving potentially effective treatment from an early age is under discussion. Raising disease awareness is very important in reducing disease mortality and morbidity rates.

Kidney renal clear cell carcinoma (KIRC) with poor prognosis is the main histological subtype of renal cell carcinoma, accounting for more than 80% of patients. Most patients are diagnosed at an advanced stage due to being asymptomatic early on. Advanced KIRC has an extremely poor prognosis due to its inherent resistance to radiotherapy and chemotherapy. Therefore, a comprehensive understanding of the molecular mechanisms of KIRC and the development of effective early diagnostic and therapeutic strategies is urgently needed. In this study, we aimed to identify the prognosis-related biomarker and analyzed its relationship with tumor progression. Metabolic changes are an important feature of kidney cancer, where the reduction of fumarate allows us to target the tyrosine metabolic pathway. The homogentisate 1,2-dioxygenase (HGD) and glutathione S-transferase zeta 1 (GSTZ1) related with prognosis of KIRC was identified through bioinformatics analysis based on The Cancer Genome Atlas (TCGA) databases. Mechanistically, we found that decreased HGD and GSTZ1 promote aerobic glycolysis in KIRC, coordinate the balance of amino acid metabolism and energy metabolism in tumor cells, and ultimately activate the tumor cell cycle and tumor progression. In summary, we identified the tyrosine metabolizing enzymes HGD and GSTZ1 as biomarkers of KIRC, which will further the understanding of the tumor metabolism profile, provide novel strategies and theoretical support for diagnosing and treating KIRC and as referential for future clinical research.