NAA10 dysfunction with normal NatA-complex activity in a girl with non-syndromic ID and a de novo NAA10 p.(V111G) variant - a case report

Background: The NAA10-NAA15 (NatA) protein complex is an N-terminal acetyltransferase responsible for acetylating ~ 40% of eukaryotic proteins. In recent years, NAA10 variants have been found in patients with an X-linked developmental disorder called Ogden syndrome in its most severe form and, in other familial or de novo cases, with variable degrees of syndromic intellectual disability (ID) affecting both sexes. Case presentation: Here we report and functionally characterize a novel and de novo NAA10 (NM_003491.3) c.332 T > G p.(V111G) missense variant, that was detected by trio-based whole exome sequencing in an 11 year old girl with mild/moderate non-syndromic intellectual disability. She had delayed motor and language development, but normal behavior without autistic traits. Her blood leukocyte X-inactivation pattern was within normal range (80/20). Functional characterization of NAA10-V111G by cycloheximide chase experiments suggests that NAA10-V111G has a reduced stability compared to NAA10-WT, and in vitro acetylation assays revealed a reduced enzymatic activity of monomeric NAA10-V111G but not for NAA10-V111G in complex with NAA15 (NatA enzymatic activity). Conclusions: We show that NAA10-V111G has a reduced stability and monomeric catalytic activity, while NatA function remains unaltered. This is the first example of isolated NAA10 dysfunction in a case of ID, suggesting that the syndromic cases may also require a degree of compromised NatA function.publishedVersio

Using CRISPR interference to study novel biofilm-associated genes in Staphylococcus aureus

The opportunistic pathogen S. aureus causes biofilm-associated infections which are often chronic and difficult to eradicate. As S. aureus is often resistant to multiple antibiotics, understanding the underlying processes of biofilm formation and regulation is crucial to identify novel treatment strategies. Although many genes have already been implicated to be involved in S. aureus biofilm formation, much is still unknown. Functional studies of genes by deletion or inactivation is time-consuming and restricted to non-essential genes. In this work, knockdown of gene expression by CRISPR interference (CRISPRi) was explored as a method to identify new biofilm-associated genes in S. aureus. Initially it was shown that CRISPRi can be used as a fast and simple method to study biofilm-associated genes in S. aureus in two different biofilm model systems, namely the crystal violet microtiter plate assay and the macrocolony formation assay. Thereafter, several new putative biofilm-associated genes were identified. By CRISPRi-based knockdown of gene expression for a selection of genes, we identified three WalRK regulated genes involved in biofilm formation in the crystal violet microtiter plate assay. While two of the genes, atl and sle1 have already been associated with biofilm formation, we also discovered an uncharacterized gene SAOUHSC_00671 whose role in biofilm formation is still unknown. SAOUHSC_00671 depleted cells showed a reduction in biofilm formation and increased clustering when analyzed by confocal microscopy. SAOUHSC_00671 harbors LysM domains and a CHAP domain, suggesting that this protein might be a cell wall hydrolase, whose role in S. aureus biofilm needs to be further investigated. A genome wide CRISPRi library was used to screen for novel biofilm genes using a macrocolony formation assay. With this approach, several genes of the central metabolic pathways were found to affect the wrinkling and structuring of S. aureus macrocolonies. These include PckA, involved in gluconeogenesis, and FumC and SucA, both involved in the TCA cycle. Furthermore, depletion of ubiE and hemE, encoding enzymes involved in the synthesis of menaquinone and heme, respectively, resulted in macrocolonies with a loss of structure and surface wrinkling. Based on these data, we propose a link between macrocolony formation and respiratory metabolism in S. aureus. Finally, in the same screen, a putative methyltransferase was also shown to be important for S. aureus macrocolony structuring, and the molecular function of this gene needs to be further investigated.S. aureus er en opportunistisk bakterie som forårsaker biofilm-relaterte infeksjoner som ofte er kroniske og vanskelig å behandle. Siden S. aureus også ofte er resistent mot flere antibiotika, er det viktig å finne nye behandlingsmetoder. For å kunne gjøre dette er det nødvendig å forstå prosessene som ligger til grunn for biofilm dannelse- og regulering. Selv om mange gener allerede har blitt beskrevet i S. aureus biofilm, er det fortsatt mye som er ukjent. Det å studere gen-funksjonalitet i S. aureus er en tidkrevende prosess som også er begrenset til å studere ikke-essensielle gener. I dette arbeidet har vi undersøkt om nedregulering av genekspresjon ved CRISPR interferens (CRISPRi) kan brukes som en metode for å identifisere nye biofilm-relaterte gener i S. aureus. Det ble innledningsvis vist at CRISPRi kan brukes som en rask og enkel metode for å studere gener involvert i biofilm dannelse i S. aureus ved hjelp av to ulike modell systemer, krystallfiolett-mikrotiterassayet og et makrokoloniassay. Deretter ble det identifisert flere nye gener som potensielt er involvert i biofilm dannelse. Ved å slå ned genekspresjon ved bruk av CRISPRi for en rekke gener, identifiserte vi tre gener under regulering av WalRK som var involvert i biofilmdannelse i krystallfiolett-mikrotiterassayet. Selv om to av disse genene, atl og sle1, allerede har blitt assosiert med biofilmdannelse, identifiserte vi et gen, SAOUHSC_00671, hvis rolle i biofilmdannelse fortsatt er ukjent. Celler hvor SAOUHSC_00671 ekspresjon var slått ned viste en reduksjon av biofilm. Uvanlige klynger av celler ble observert ved bruk av konfokal mikroskopi. SAOUHSC_00671 har flere LysM domener i tillegg til et CHAP domene, noe som indikerer at dette proteinet potensielt er involvert i hydrolyse av cellevegg. Rollen til dette proteinet i biofilmdannelse krever videre undersøkelser. Ved bruk av et CRISPRi bibliotek ble det gjennomført et genomskala makrokoloniassayeksperiment for å lete etter nye gener involvert i biofilmdannelse. Genene pckA, som er involvert i glukoneogenesen, samt fumC og sucA som er involvert i TCA syklusen viste seg å påvirke struktureringen av makrokolonier. Videre viste det seg at mangelen på UbiE og HemE, enzymer som er involvert i henholdsvis syntese av menaquinone (vitamin K2) og heme, resulterte i makrokolonier med tap av struktur og rynker. Basert på disse resultatene foreslår vi en forbindelse mellom makrokolonidannelse og respirasjon i S. aureus. Avslutningsvis fant vi, i den samme screeningen, en potensiell metyltransferase som også viste seg å være viktig for strukturering av makrokolonier i S. aureus. De molekylære funksjonene til dette genet krever videre studier.submittedVersionM-BIOTE

Staphylococcal Biofilms: Challenges and Novel Therapeutic Perspectives

Staphylococci, like Staphylococcus aureus and S. epidermidis, are common colonizers of the human microbiota. While being harmless in many cases, many virulence factors result in them being opportunistic pathogens and one of the major causes of hospital-acquired infections worldwide. One of these virulence factors is the ability to form biofilms—three-dimensional communities of microorganisms embedded in an extracellular polymeric matrix (EPS). The EPS is composed of polysaccharides, proteins and extracellular DNA, and is finely regulated in response to environmental conditions. This structured environment protects the embedded bacteria from the human immune system and decreases their susceptibility to antimicrobials, making infections caused by staphylococci particularly difficult to treat. With the rise of antibiotic-resistant staphylococci, together with difficulty in removing biofilms, there is a great need for new treatment strategies. The purpose of this review is to provide an overview of our current knowledge of the stages of biofilm development and what difficulties may arise when trying to eradicate staphylococcal biofilms. Furthermore, we look into promising targets and therapeutic methods, including bacteriocins and phage-derived antibiofilm approaches

NAA10 dysfunction with normal NatA-complex activity in a girl with non-syndromic ID and a de novo NAA10 p.(V111G) variant - a case report

Background: The NAA10-NAA15 (NatA) protein complex is an N-terminal acetyltransferase responsible for acetylating ~ 40% of eukaryotic proteins. In recent years, NAA10 variants have been found in patients with an X-linked developmental disorder called Ogden syndrome in its most severe form and, in other familial or de novo cases, with variable degrees of syndromic intellectual disability (ID) affecting both sexes. Case presentation: Here we report and functionally characterize a novel and de novo NAA10 (NM_003491.3) c.332 T > G p.(V111G) missense variant, that was detected by trio-based whole exome sequencing in an 11 year old girl with mild/moderate non-syndromic intellectual disability. She had delayed motor and language development, but normal behavior without autistic traits. Her blood leukocyte X-inactivation pattern was within normal range (80/20). Functional characterization of NAA10-V111G by cycloheximide chase experiments suggests that NAA10-V111G has a reduced stability compared to NAA10-WT, and in vitro acetylation assays revealed a reduced enzymatic activity of monomeric NAA10-V111G but not for NAA10-V111G in complex with NAA15 (NatA enzymatic activity). Conclusions: We show that NAA10-V111G has a reduced stability and monomeric catalytic activity, while NatA function remains unaltered. This is the first example of isolated NAA10 dysfunction in a case of ID, suggesting that the syndromic cases may also require a degree of compromised NatA function

Systematic analysis of drug combinations against Gram-positive bacteria

Drug combinations can expand options for antibacterial therapies but have not been systematically tested in Gram-positive species. We profiled similar to 8,000 combinations of 65 antibacterial drugs against the model species Bacillus subtilis and two prominent pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Thereby, we recapitulated previously known drug interactions, but also identified ten times more novel interactions in the pathogen S. aureus, including 150 synergies. We showed that two synergies were equally effective against multidrug-resistant S. aureus clinical isolates in vitro and in vivo. Interactions were largely species-specific and synergies were distinct from those of Gram-negative species, owing to cell surface and drug uptake differences. We also tested 2,728 combinations of 44 commonly prescribed non-antibiotic drugs with 62 drugs with antibacterial activity against S. aureus and identified numerous antagonisms that might compromise the efficacy of antimicrobial therapies. We identified even more synergies and showed that the anti-aggregant ticagrelor synergized with cationic antibiotics by modifying the surface charge of S. aureus. All data can be browsed in an interactive interface (https://apps.embl.de/combact/).ISSN:2058-527