The outer-membrane protein MafA of Neisseria meningitidis constitutes a novel protein secretion pathway specific for the fratricide protein MafB.

MafB proteins are toxins secreted by Neisseria spp. which are involved in interbacterial competition. Their secretion mechanism has so far not been elucidated. Each strain can produce several MafB variants. On the chromosome, the mafB genes are localized on genomic islands also containing mafA genes. MafA proteins have a role in virulence with reported activities in adhesion and transcytosis of pathogenic Neisseria, a priori unrelated to MafB activities. In this study, we investigated the possible involvement of MafA in the transport of MafB across the outer membrane of Neisseria meningitidis. In wild-type strains, proteolytic fragments of MafB proteins were detected in the extracellular medium. In the absence of MafA, secretion was abrogated, and, in the case of MafBI, full-length and truncated polypeptides were detected inside the cells and inside outer-membrane vesicles. MafBI secretion required its cognate MafA, whereas MafBIII could use any MafA. Heterologous expression in Escherichia coli showed that MafBIII is transported to a cell-surface-exposed, i.e. protease-accessible, location in a MafA-dependent way. MafA itself was found to be localized to the outer membrane, forming large oligomeric complexes. As homologs were found in diverse bacteria, the Maf system represents a new protein secretion system in Gram-negative bacteria.

Conditionally essential genes for survival during starvation in Enterococcus faecium E745.

BACKGROUND: 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. 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. RESULTS: 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 °C. A total of 24 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. The usp gene is present in 99% of a set of 1644 E. faecium genomes that collectively span the diversity of the species. CONCLUSIONS: We postulate that usp 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.

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. 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. faeciumß-galactosidase. Blue/white screening using 5-bromo-4-chloro-3-indolyl-ß-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.

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 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.

RNA-seq and Tn-seq reveal fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum.

BACKGROUND: The 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). RESULTS: We 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. CONCLUSIONS: Genes 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.

Fratricide activity of MafB protein of N. meningitidis strain B16B6.

BACKGROUND: Neisseria 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. RESULTS: Blast 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. CONCLUSIONS: The meningococcal MafB proteins are novel toxic proteins involved in interbacterial competition.