Sex-specific newborn screening for X-linked adrenoleukodystrophy.

Males with X-linked adrenoleukodystrophy (ALD) are at high risk for developing adrenal insufficiency and/or progressive leukodystrophy (cerebral ALD) at an early age. Pathogenic variants in ABCD1 result in elevated levels of very long-chain fatty acids (VLCFA), including C26:0-lysophosphatidylcholine (C26:0-LPC). Newborn screening for ALD enables prospective monitoring and timely therapeutic intervention, thereby preventing irreversible damage and saving lives. The Dutch Health Council recommended to screen only male newborns for ALD without identifying untreatable conditions associated with elevated C26:0-LPC, like Zellweger spectrum disorders and single peroxisomal enzyme defects. Here, we present the results of the SCAN (Screening for ALD in the Netherlands) study which is the first sex-specific newborn screening program worldwide. Males with ALD are identified based on elevated C26:0-LPC levels, the presence of one X-chromosome and a variant in ABCD1, in heel prick dried bloodspots. Screening of 71 208 newborns resulted in the identification of four boys with ALD who, following referral to the pediatric neurologist and confirmation of the diagnosis, enrolled in a long-term follow-up program. The results of this pilot show the feasibility of employing a boys-only screening algorithm that identifies males with ALD without identifying untreatable conditions. This approach will be of interest to countries that are considering ALD newborn screening but are reluctant to identify girls with ALD because for girls there is no direct health benefit. We also analyzed whether gestational age, sex, birth weight and age at heel prick blood sampling affect C26:0-LPC concentrations and demonstrate that these covariates have a minimal effect.

Disruption of protein rhamnosylation affects the Sporothrix schenckii-host interaction.

Sporotrichosis is a fungal disease caused by the members of the Sporothrix pathogenic clade, and one of the etiological agents is Sporothrix schenckii. The cell wall of this organism has been previously analyzed and thus far is known to contain an inner layer composed of chitin and ß -glucans, and an outer layer of glycoproteins, which are decorated with mannose and rhamnose-containing oligosaccharides. The L-rhamnose biosynthesis pathway is common in bacteria but rare in members of the Fungi kingdom. Therefore, in this study, we aimed to disrupt this metabolic route to assess the contribution of rhamnose during the S. schenckii-host interaction. We identified and silenced in S. schenckii a functional ortholog of the bacterial rmlD gene, which encodes for an essential reductase for the synthesis of nucleotide-activated L-rhamnose. RmlD silencing did not affect fungal growth or morphology but decreased cell wall rhamnose content. Compensatory, the ß-1,3-glucan levels increased and were more exposed at the cell surface. Moreover, when incubated with human peripheral blood mononuclear cells, the RmlD silenced mutants differentially stimulated cytokine production when compared with the wild-type strain, reducing TNFα and IL-6 levels and increasing IL-1 ß and IL-10 production. Upon incubation with human monocyte-derived macrophages, the silenced strains were more efficiently phagocytosed than the wild-type strain. In both cases, our data suggest that rhamnose-based oligosaccharides are ligands that interact with TLR4. Finally, our findings showed that cell wall rhamnose is required for the S. schenckii virulence in the G. mellonella model of infection.

Streptococcal dTDP-L-rhamnose biosynthesis enzymes: functional characterization and lead compound identification.

Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP-L-rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio-layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose-dependent streptococcal pathogens as well as M. tuberculosis with an IC50 of 120-410 µM. Importantly, we confirmed that Ri03 inhibited dTDP-L-rhamnose formation in a concentration-dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP-L-rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.

Highly conserved nucleotide phosphatase essential for membrane lipid homeostasis in Streptococcus pneumoniae.

Proteins belonging to the DHH family, a member of the phosphoesterase superfamily, are produced by most bacterial species. While some of these proteins are well studied in Bacillus subtilis and Escherichia coli, their functions in Streptococcus pneumoniae remain unclear. Recently, the highly conserved DHH subfamily 1 protein PapP (SP1298) has been reported to play an important role in virulence. Here, we provide a plausible explanation for the attenuated virulence of the papP mutant. Recombinant PapP specifically hydrolyzed nucleotides 3'-phosphoadenosine-5'-phosphate (pAp) and 5'-phosphoadenylyl-(3'->5')-adenosine (pApA). Deletion of papP, potentially leading to pAp/pApA accumulation, resulted in morphological defects and mis-localization of several cell division proteins. Incubation with both polar solvent and detergent led to robust killing of the papP mutant, indicating that membrane integrity is strongly affected. This is in line with previous studies showing that pAp inhibits the ACP synthase, an essential enzyme involved in lipid precursor production. Remarkably, partial inactivation of the lipid biosynthesis pathway, by inhibition of FabF or depletion of FabH, phenocopied the papP mutant. We conclude that pAp and pApA phosphatase activity of PapP is required for maintenance of membrane lipid homeostasis providing an explanation how inactivation of this protein may attenuate pneumococcal virulence.

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP-4-dehydrorhamnose reductases (RmlD).

The sugar nucleotide dTDP-L-rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four-step dTDP-L-rhamnose biosynthesis pathway is catalyzed by dTDP-4-dehydrorhamnose reductases (RmlD). RmlD from the Gram-negative bacterium Salmonella is the only structurally characterized family member and requires metal-dependent homo-dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram-negative and Gram-positive RmlD homologues predicts that enzymes from all Gram-positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn-sequencing and generation of a conditional-expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram-positive bacteria and a subset of Gram-negative bacteria. These results will help future screens for novel inhibitors of dTDP-L-rhamnose biosynthesis.

Aptasensors for viral diagnostics.

Novel viral diagnostic tools need to be affordable, fast, accurate and easy to use with sensitivity and specificity equivalent or superior to current standards. At present, viral diagnostics are based on direct detection of viral components or indirect detection by measuring antibodies generated in response to viral infection. While sensitivity of detection and quantification are still important challenges, we expect major advances from new assay formats and synthetic binding molecules, such as aptamers. Compared to traditional antibody-based detection, aptamers could provide faster adaptation to continuously evolving virus strains and higher discriminating capacity between specific virus serotypes. Aptamers are very stable and easily modifiable, so are ideal molecules for detection and chemical sensing applications. Here, we review the use of aptasensors for detection of viral pathogens and consider the feasibility of aptasensors to become standard devices for point-of-care diagnostics of viruses.