Biology subjects

de Maat, V.

Publications and source records attributed to de Maat, V..

4 recordsLinked to original sources

bioRxiv2020

Conditionally essential genes for survival during starvation in Enterococcus faecium E745

The nosocomial pathogen Enterococcus faecium can survive for prolonged periods of time on surfaces in the absence of nutrients. This trait is thought to contribute to the ability of E. faecium to spread among patients in hospitals. Because there is currently a lack of data on the mechanisms that are responsible for the ability of E. faecium to survive in the absence of nutrients, we performed a high-throughput transposon mutant library screening (Tn-seq) to identify genes that have a role in long-term survival during incubation in phosphate-buffered saline (PBS) at 20{degrees}C. A total of 18 genes were identified by Tn-seq to contribute to survival in PBS, with functions associated with the general stress response, DNA repair, metabolism, and membrane homeostasis. The gene which was quantitatively most important for survival in PBS was usp (locus tag: EfmE745_02439), which is predicted to encode a 17.4 kDa universal stress protein. After generating a targeted deletion mutant in usp, we were able to confirm that usp significantly contributes to survival in PBS and this defect was restored by in trans complementation. As usp is present in 99% of 1644 E. faecium genomes that span the diversity of the species, we postulate that this gene is a key determinant for the remarkable environmental robustness of E. faecium. Further mechanistic studies into usp and other genes identified in this study may shed further light on the mechanisms by which E. faecium can survive in the absence of nutrients for prolonged periods of time.

microbiology↗

bioRxiv2019

CRISPR-Cas9 mediated genome editing in vancomycin resistant Enterococcus faecium

The Gram-positive bacterium Enterococcus faecium is becoming increasingly prevalent as a cause of hospital-acquired, antibiotic-resistant infections. There is thus an urgent need to mechanistically characterize the traits that contribute to the emergence of E. faecium as a multidrug resistant opportunistic pathogen. A fundamental part of research into E. faecium biology relies on the ability to generate targeted mutants, but this process is currently labour-intensive and time-consuming, taking 4 to 5 weeks per mutant. In this report we describe a method relying on the high recombination rates of E. faecium and the application of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 genome editing tool to more efficiently generate targeted mutants in the E. faecium chromosome. Using this tool and the multi-drug resistant clinical E. faecium strain E745, we generated a deletion mutant in the lacL gene, which encodes the large subunit of the E. faecium {beta}-galactosidase. Blue/white screening using 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal) could be used to distinguish between the wild-type and lacL deletion mutant. We also inserted two copies of gfp into the intrinsic E. faecium macrolide resistance gene msrC to generate stable green fluorescent cells. We conclude that CRISPR-Cas9 can be used to generate targeted genome modifications in E. faecium in 3 weeks, with limited hands-on time. This method can potentially be implemented in other Gram-positive bacteria with high intrinsic recombination rates.

microbiology↗

bioRxiv2018

Streptococcal Lancefield polysaccharides are critical cell wall determinants for human group IIA secreted phospholipase A2 to exert its bactericidal effects

Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant human hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the{Delta} gacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the human bactericidal enzyme hGIIA to exert its bactericidal function.\n\nAuthor summaryThe human immune system is capable of killing invading bacteria by secreting antimicrobial proteins. Cationic human Group IIA secreted phospholipase A2 (hGIIA) is especially effective against Gram-positive bacteria by degrading the bacterial membrane. HGIIA requires binding to negatively charged surface structures before it can penetrate through the thick peptidoglycan layer and reach the target phospholipid membrane. HGIIA is constitutively expressed at high concentrations at sites of possible bacterial entry, e.g. in tears, skin and small intestine. In serum, normal concentrations are low but can increase up to 1,000-fold upon inflammation or infection. In vitro, ex vivo and in vivo experiments suggest an important role for hGIIA in defense against two human pathogens, Group A and Group B Streptococcus (GAS, GBS). We demonstrate that the Lancefield cell wall polysaccharides that are expressed by these bacteria, the Group A Carbohydrate (GAC) for GAS and the Group B Carbohydrate (GBC) for GBS, are required for optimal hGIIA bactericidal efficacy by facilitating penetration through the peptidoglycan layer. Given the increased hGIIA resistance of antigen-modified or antigen-deficient streptococci, it will be of interest to determine potential regulatory mechanisms regarding expression of streptococcal Lancefield polysaccharides.

microbiology↗

bioRxiv2017

Fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum

Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. Here, we identify genes that contribute to growth of E. faecium in human serum. We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, to completion and then compared its transcriptome during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. A high-throughput transposon sequencing (Tn-seq) approach was used to identify conditionally essential genes in E. faecium E745 during growth in serum. Genes involved in de novo nucleotide biosysnthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants also exhibited significantly attenuated virulence in an intravenous zebrafish infection model. We conclude that genes involved in carbohydrate and nucleotide metabolism of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism.

microbiology↗