BackgroundThe Gram-positive bacterium Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. The mechanisms by which E. faecium can survive and grow in blood during an infection have not yet been characterized. Here, we identify genes that contribute to growth of E. faecium in human serum through transcriptome profiling (RNA-seq) and a high-throughput transposon mutant library sequencing approach (Tn-seq).ResultsWe first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, using a combination of short- and long read sequencing, revealing a 2,765,010 nt chromosome and 6 plasmids, with sizes ranging between 9.3 kbp and 223.7 kbp. We then compared the transcriptome of E. faecium E745 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. The E. faecium E745 transposon mutant library was then used to identify genes that were specifically required for growth of E. faecium in serum. Genes involved in de novo nucleotide biosynthesis (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 were tested for their virulence in an intravenous zebrafish infection model and exhibited significantly attenuated virulence compared to E. faecium E745.ConclusionsGenes involved in carbohydrate metabolism and nucleotide biosynthesis of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism. These genes may serve as targets for the development of novel anti-infectives for the treatment of E. faecium bloodstream infections.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4299-9) contains supplementary material, which is available to authorized users.

BackgroundNeisseria meningitidis is an inhabitant of the mucosal surfaces of the human nasopharynx. We recently demonstrated that the secreted meningococcal Two-partner secretion protein A (TpsA) is involved in interbacterial competition. The C-terminal end of the large TpsA protein contains a small toxic domain that inhibits the growth of target bacteria. The producing cells are protected from this toxic activity by a small immunity protein that is encoded by the gene immediately downstream of the tpsA gene. Further downstream on the chromosome, a repertoire of toxic modules, designated tpsC cassettes, is encoded that could replace the toxic module of TpsA by recombination. Each tpsC cassette is associated with a gene encoding a cognate immunity protein.ResultsBlast searchers using the toxic domains of TpsA and TpsC proteins as queries identified homologies with the C-terminal part of neisserial MafB proteins, which, for the rest, showed no sequence similarity to TpsA proteins. On the chromosome, mafB genes are part of genomic islands, which include cassettes for additional toxic modules as well as genes putatively encoding immunity proteins. We demonstrate that a MafB protein of strain B16B6 inhibits the growth of a strain that does not produce the corresponding immunity protein. Assays in E. coli confirmed that the C-terminal region of MafB is responsible for toxicity, which is inhibited by the cognate immunity protein. Pull-down assays revealed direct interaction between MafB toxic domains and the cognate immunity proteins.ConclusionsThe meningococcal MafB proteins are novel toxic proteins involved in interbacterial competition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0493-6) contains supplementary material, which is available to authorized users.

The Gram-positive bacterium Enterococcus faecium is becoming increasingly prevalent as a cause of hospital-acquired, antibiotic-resistant infections. 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. faeciumβ-galactosidase. Blue/white screening using 5-bromo-4-chloro-3-indolyl-β-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.

23Human Group IIA secreted phospholipase A 2 (hGIIA) is an acute phase protein with 24 bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic 25 mice have highlighted the importance of hGIIA as an innate defense mechanism against the 26 human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). 27Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To 28 identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon 29 library to recombinant human hGIIA and compared relative mutant abundance with library 30 input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the 31 output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA 32 susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included 33 two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene 34 cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented 35 strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in 36 the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is 37 conserved across different GAS serotypes. Increased resistance is associated with delayed 38 penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B 39Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. 40This suggests that the streptococcal Lancefield antigens, which are critical determinants for 41 streptococcal physiology and virulence, are required for the human bactericidal enzyme 42 hGIIA to exert its bactericidal function.

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 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 ΔgacI 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 bactericidal enzyme hGIIA to exert its bactericidal function.