NDP-rhamnose biosynthesis and rhamnosyltransferases: building diverse glycoconjugates in nature.

Rhamnose is an important 6-deoxy sugar present in many natural products, glycoproteins, and structural polysaccharides. Whilst predominantly found as the l-enantiomer, instances of d-rhamnose are also found in nature, particularly in the Pseudomonads bacteria. Interestingly, rhamnose is notably absent from humans and other animals, which poses unique opportunities for drug discovery targeted towards rhamnose utilizing enzymes from pathogenic bacteria. Whilst the biosynthesis of nucleotide-activated rhamnose (NDP-rhamnose) is well studied, the study of rhamnosyltransferases that synthesize rhamnose-containing glycoconjugates is the current focus amongst the scientific community. In this review, we describe where rhamnose has been found in nature, as well as what is known about TDP-ß-l-rhamnose, UDP-ß-l-rhamnose, and GDP-α-d-rhamnose biosynthesis. We then focus on examples of rhamnosyltransferases that have been characterized using both in vivo and in vitro approaches from plants and bacteria, highlighting enzymes where 3D structures have been obtained. The ongoing study of rhamnose and rhamnosyltransferases, in particular in pathogenic organisms, is important to inform future drug discovery projects and vaccine development.

Group A, B, C, and G Streptococcus Lancefield antigen biosynthesis is initiated by a conserved α-d-GlcNAc-ß-1,4-l-rhamnosyltransferase.

Group A carbohydrate (GAC) is a bacterial peptidoglycan-anchored surface rhamnose polysaccharide (RhaPS) that is essential for growth of Streptococcus pyogenes and contributes to its ability to infect the human host. In this study, using molecular and synthetic biology approaches, biochemistry, radiolabeling techniques, and NMR and MS analyses, we examined the role of GacB, encoded in the S. pyogenes GAC gene cluster, in the GAC biosynthesis pathway. We demonstrate that GacB is the first characterized α-d-GlcNAc-ß-1,4-l-rhamnosyltransferase that synthesizes the committed step in the biosynthesis of the GAC virulence determinant. Importantly, the substitution of S. pyogenes gacB with the homologous gene from Streptococcus agalactiae (Group B Streptococcus), Streptococcus equi subsp. zooepidemicus (Group C Streptococcus), Streptococcus dysgalactiae subsp. equisimilis (Group G Streptococcus), or Streptococcus mutans complemented the GAC biosynthesis pathway. These results, combined with those from extensive in silico studies, reveal a common phylogenetic origin of the genes required for this priming step in >40 pathogenic species of the Streptococcus genus, including members from the Lancefield Groups B, C, D, E, G, and H. Importantly, this priming step appears to be unique to streptococcal ABC transporter-dependent RhaPS biosynthesis, whereas the Wzx/Wzy-dependent streptococcal capsular polysaccharide pathways instead require an α-d-Glc-ß-1,4-l-rhamnosyltransferase. The insights into the RhaPS priming step obtained here open the door to targeting the early steps of the group carbohydrate biosynthesis pathways in species of the Streptococcus genus of high clinical and veterinary importance.

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.

Increased flexibility in the use of exogenous lipoic acid by Staphylococcus aureus.

Lipoic acid is a cofactor required for intermediary metabolism that is either synthesized de novo or acquired from environmental sources. The bacterial pathogen Staphylococcus aureus encodes enzymes required for de novo biosynthesis, but also encodes two ligases, LplA1 and LplA2, that are sufficient for lipoic acid salvage during infection. S. aureus also encodes two H proteins, GcvH of the glycine cleavage system and the homologous GcvH-L encoded in an operon with LplA2. GcvH is a recognized conduit for lipoyl transfer to α-ketoacid dehydrogenase E2 subunits, while the function of GcvH-L remains unclear. The potential to produce two ligases and two H proteins is an unusual characteristic of S. aureus that is unlike most other Gram positive Firmicutes and might allude to an expanded pathway of lipoic acid acquisition in this microorganism. Here, we demonstrate that LplA1 and LplA2 facilitate lipoic acid salvage by differentially targeting lipoyl domain-containing proteins; LplA1 targets H proteins and LplA2 targets α-ketoacid dehydrogenase E2 subunits. Furthermore, GcvH and GcvH-L both facilitate lipoyl relay to E2 subunits. Altogether, these studies identify an expanded mode of lipoic acid salvage used by S. aureus and more broadly underscore the importance of bacterial adaptations when faced with nutritional limitation.

Staphylococcus aureus Tissue Infection During Sepsis Is Supported by Differential Use of Bacterial or Host-Derived Lipoic Acid.

To thrive in diverse environments, bacteria must shift their metabolic output in response to nutrient bioavailability. In many bacterial species, such changes in metabolic flux depend upon lipoic acid, a cofactor required for the activity of enzyme complexes involved in glycolysis, the citric acid cycle, glycine catabolism, and branched chain fatty acid biosynthesis. The requirement of lipoic acid for metabolic enzyme activity necessitates that bacteria synthesize the cofactor and/or scavenge it from environmental sources. Although use of lipoic acid is a conserved phenomenon, the mechanisms behind its biosynthesis and salvage can differ considerably between bacterial species. Furthermore, low levels of circulating free lipoic acid in mammals underscore the importance of lipoic acid acquisition for pathogenic microbes during infection. In this study, we used a genetic approach to characterize the mechanisms of lipoic acid biosynthesis and salvage in the bacterial pathogen Staphylococcus aureus and evaluated the requirements for both pathways during murine sepsis. We determined that S. aureus lipoic acid biosynthesis and salvage genes exist in an arrangement that directly links redox stress response and acetate biosynthesis genes. In addition, we found that lipoic acid salvage is dictated by two ligases that facilitate growth and lipoylation in distinct environmental conditions in vitro, but that are fully compensatory for survival in vivo. Upon infection of mice, we found that de novo biosynthesis or salvage promotes S. aureus survival in a manner that depends upon the infectious site. In addition, when both lipoic acid biosynthesis and salvage are blocked S. aureus is rendered avirulent, implying an inability to induce lipoic acid-independent metabolic programs to promote survival. Together, our results define the major pathways of lipoic acid biosynthesis and salvage in S. aureus and support the notion that bacterial nutrient acquisition schemes are instrumental in dictating pathogen proclivity for an infectious niche.

Transitioning from Triton X-100 to Tergitol 15-S-9: impacts on diagnostic assays using viral PCR sample solution.

In 2019, the European Union banned Triton X-100, a detergent widely used in laboratory diagnostics, including the Viral PCR Sample Solution (VPSS), and urged manufacturers to find environmentally sustainable alternatives. Tergitol 15-S-9 (VPSS2) has been proposed as an alternative surfactant. This multicenter study evaluated the effectiveness of VPSS2, a Tergitol-based viral solution, as a replacement for VPSS. Our results show the equivalent performance of VPSS2 to VPSS for nucleic acid extraction and viral stability over time at different temperatures. The new VPSS formulation was also tested against external quality assurance panels and clinical samples. The results of this work support adopting this modified viral PCR sample solution to replace Triton X-100-containing viral transport solutions.

The European Union has banned Triton X-100. All reagents containing it should be replaced. Could a new Viral PCR Sample Solution (VPSS) containing Tergitol 15-S-9 be a suitable replacement?

Genomic diversity in Fructobacillus spp. isolated from fructose-rich niches.

The Fructobacillus genus is a group of obligately fructophilic lactic acid bacteria (FLAB) that requires the use of fructose or another electron acceptor for their growth. In this work, we performed a comparative genomic analysis within the genus Fructobacillus by using 24 available genomes to evaluate genomic and metabolic differences among these organisms. In the genome of these strains, which varies between 1.15- and 1.75-Mbp, nineteen intact prophage regions, and seven complete CRISPR-Cas type II systems were found. Phylogenetic analyses located the studied genomes in two different clades. A pangenome analysis and a functional classification of their genes revealed that genomes of the first clade presented fewer genes involved in the synthesis of amino acids and other nitrogen compounds. Moreover, the presence of genes strictly related to the use of fructose and electron acceptors was variable within the genus, although these variations were not always related to the phylogeny.

Impact of swab removal in the detection of SARS-CoV-2 weakly-positive specimens.

Removing the swab after collection can speed up diagnosis and improve the quality of laboratory procedures. This study investigates the impact of swab removal on SARS-CoV-2 detection in clinical specimens with a focus on high Cycle threshold (Ct) samples (Cts≥32). The method assessed pairs of SARS-CoV-2 samples mimicking combined throat and nose swabs and tested them on two real-time-PCR platforms; the Applied Biosystems 7500 and the Abbott Alinity. Swab removal did not significantly affect detection rates of SARS-CoV-2 samples with Ct values<32, regardless of the PCR platform. However, reduced reproducibility was seen at the endpoint limit of detection of the platforms, which meant that fewer samples with Ct values≥32 were detected in the swab removal group.

Evaluation of SARS-CoV-2 antibody point of care devices in the laboratory and clinical setting.

SARS-CoV-2 antibody tests have been marketed to diagnose previous SARS-CoV-2 infection and as a test of immune status. There is a lack of evidence on the performance and clinical utility of these tests. We aimed to carry out an evaluation of 14 point of care (POC) SARS-CoV-2 antibody tests. Serum from participants with previous RT-PCR (real-time polymerase chain reaction) confirmed SARS-CoV-2 infection and pre-pandemic serum controls were used to determine specificity and sensitivity of each POC device. Changes in sensitivity with increasing time from infection were determined on a cohort of study participants. Corresponding neutralising antibody status was measured to establish whether the detection of antibodies by the POC device correlated with immune status. Paired capillary and serum samples were collected to ascertain whether POC devices performed comparably on capillary samples. Sensitivity and specificity varied between the POC devices and in general did not meet the manufacturers' reported performance characteristics, which signifies the importance of independent evaluation of these tests. The sensitivity peaked at ≥20 days following onset of symptoms, however sensitivity of 3 of the POC devices evaluated at extended time points showed that sensitivity declined with time. This was particularly marked at >140 days post infection. This is relevant if the tests are to be used for sero-prevalence studies. Neutralising antibody data showed that positive antibody results on POC devices did not necessarily confer high neutralising antibody titres, and that these POC devices cannot be used to determine immune status to the SARS-CoV-2 virus. Comparison of paired serum and capillary results showed that there was a decline in sensitivity using capillary blood. This has implications in the utility of the tests as they are designed to be used on capillary blood by the general population.