The streptococcal phase-variable type I restriction modification system SsuCC20p dictates the methylome of Streptococcus suis impacting the transcriptome and virulence in a zebrafish larvae infection model.

IMPORTANCE: Phase variation allows a single strain to produce phenotypic diverse subpopulations. Phase-variable restriction modification (RM) systems are systems that allow for such phase variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis. We show that the phase variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and demonstrated the consequential impact on the transcriptome and virulence in a zebrafish infection model. Acquiring mobile genetic elements containing epigenetic regulatory systems, like phase-variable RM systems, enables bacterial pathogens to produce diverse phenotypic subpopulations that are better adapted to specific (host) environments encountered during infection.

Neisseria meningitidis Sibling Small Regulatory RNAs Connect Metabolism with Colonization by Controlling Propionate Use.

Neisseria meningitidis (meningococcus) colonizes the human nasopharynx, primarily as a commensal, but sporadically causing septicemia and meningitis. During colonization and invasion, it encounters different niches with specific nutrient compositions. Small noncoding RNAs (sRNAs) are used to fine-tune expression of genes, allowing adaptation to their physiological differences. We have previously characterized sRNAs (Neisseria metabolic switch regulators [NmsRs]) controlling switches between cataplerotic and anaplerotic metabolism. Here, we extend the NmsR regulon by studying methylcitrate lyase (PrpF) and propionate kinase (AckA-1) involved in the methylcitrate cycle and serine hydroxymethyltransferase (GlyA) and 3-hydroxyacid dehydrogenase (MmsB) involved in protein degradation. These proteins were previously shown to be dysregulated in a ΔnmsRs strain. Levels of transcription of target genes and NmsRs were assessed by reverse transcriptase quantitative PCR (RT-qPCR). We also used a novel gene reporter system in which the 5' untranslated region (5' UTR) of the target gene is fused to mcherry to study NmsRs-target gene interaction in the meningococcus. Under nutrient-rich conditions, NmsRs downregulate expression of PrpF and AckA-1 by direct interaction with the 5' UTR of their mRNA. Overexpression of NmsRs impaired growth under nutrient-limiting growth conditions with pyruvate and propionic acid as the only carbon sources. Our data strongly suggest that NmsRs downregulate propionate metabolism by lowering methylcitrate enzyme activity under nutrient-rich conditions. Under nutrient-poor conditions, NmsRs are downregulated, increasing propionate metabolism, resulting in higher tricarboxylic acid (TCA) activities. IMPORTANCE Neisseria meningitidis colonizes the human nasopharynx, forming a reservoir for the sporadic occurrence of epidemic invasive meningococcal disease like septicemia and meningitis. Propionic acid generated by other bacteria that coinhabit the human nasopharynx can be utilized by meningococci for replication in this environment. Here, we showed that sibling small RNAs, designated NmsRs, riboregulate propionic acid utilization by meningococci and, thus, colonization. Under conditions mimicking the nasopharyngeal environment, NmsRs are downregulated. This leads to the conversion of propionic acid to pyruvate and succinate, resulting in higher tricarboxylic acid cycle activity, allowing colonization of the nasopharynx. NmsRs link metabolic state with colonization, which is a crucial step on the trajectory to invasive meningococcal disease.

Meningococcal virulence in zebrafish embryos depends on capsule polysaccharide structure.

Neisseria meningitidis or the meningococcus, can cause devasting diseases such as sepsis and meningitis. Its polysaccharide capsule, on which serogrouping is based, is the most important virulence factor. Non-encapsulated meningococci only rarely cause disease, due to their sensitivity to the host complement system. How the capsular polysaccharide structure of N. meningitidis relates to virulence is largely unknown. Meningococcal virulence can be modeled in zebrafish embryos as the innate immune system of the zebrafish embryo resembles that of mammals and is fully functional two days post-fertilization. In contrast, the adaptive immune system does not develop before 4 weeks post-fertilization. We generated isogenic meningococcal serogroup variants to study how the chemical composition of the polysaccharide capsule affects N. meningitidis virulence in the zebrafish embryo model. H44/76 serogroup B killed zebrafish embryos in a dose-dependent manner, whereas the non-encapsulated variant was completely avirulent. Neutrophil depletion was observed after infection with encapsulated H44/76, but not with its non-encapsulated variant HB-1. The survival of embryos infected with isogenic capsule variants of H44/76 was capsule specific. The amount of neutrophil depletion differed accordingly. Both embryo killing capacity and neutrophil depletion after infection correlated with the number of carbons used per repeat unit of the capsule polysaccharide during its biosynthesis (indicative of metabolic cost). Conclusion: Meningococcal virulence in the zebrafish embryo largely depends on the presence of the polysaccharide capsule but the extent of the contribution is determined by its structure. The observed differences between the meningococcal isogenic capsule variants in zebrafish embryo virulence may depend on differences in metabolic cost.

Genetic diversity of Staphylococcus aureus wall teichoic acid glycosyltransferases affects immune recognition.

Staphylococcus aureus is a leading cause of skin and soft tissue infections and systemic infections. Wall teichoic acids (WTAs) are cell wall-anchored glycopolymers that are important for S. aureus nasal colonization, phage-mediated horizontal gene transfer, and antibiotic resistance. WTAs consist of a polymerized ribitol phosphate (RboP) chain that can be glycosylated with N-acetylglucosamine (GlcNAc) by three glycosyltransferases: TarS, TarM, and TarP. TarS and TarP modify WTA with ß-linked GlcNAc at the C-4 (ß1,4-GlcNAc) and the C-3 position (ß1,3-GlcNAc) of the RboP subunit, respectively, whereas TarM modifies WTA with α-linked GlcNAc at the C-4 position (α1,4-GlcNAc). Importantly, these WTA glycosylation patterns impact immune recognition and clearance of S. aureus. Previous studies suggest that tarS is near-universally present within the S. aureus population, whereas a smaller proportion co-contain either tarM or tarP. To gain more insight into the presence and genetic variation of tarS, tarM and tarP in the S. aureus population, we analysed a collection of 25 652 S. aureus genomes within the PubMLST database. Over 99 % of isolates contained tarS. Co-presence of tarS/tarM or tarS/tarP occurred in 37 and 7 % of isolates, respectively, and was associated with specific S. aureus clonal complexes. We also identified 26 isolates (0.1 %) that contained all three glycosyltransferase genes. At sequence level, we identified tar alleles with amino acid substitutions in critical enzymatic residues or with premature stop codons. Several tar variants were expressed in a S. aureus tar-negative strain. Analysis using specific monoclonal antibodies and human langerin showed that WTA glycosylation was severely attenuated or absent. Overall, our data provide a broad overview of the genetic diversity of the three WTA glycosyltransferases in the S. aureus population and the functional consequences for immune recognition.

Regulation of Neisseria meningitidis cytochrome bc1 components by NrrF, a Fur-controlled small noncoding RNA.

NrrF is a small regulatory RNA of the human pathogen Neisseria meningitidis. NrrF was previously shown to repress succinate dehydrogenase (sdhCDAB) under control of the ferric uptake regulator (Fur). Here, we provide evidence that cytochrome bc1 , encoded by the polycistronic mRNA petABC, is a NrrF target as well. We demonstrated differential expression of cytochrome bc1 comparing wild-type meningococci and meningococci expressing NrrF when sufficient iron is available. Using a gfp-reporter system monitoring translational control and target recognition of sRNA in Escherichia coli, we show that interaction between NrrF and the 5' untranslated region of the petABC mRNA results in its repression. The NrrF region essential for repression of petABC was identified by site-directed mutagenesis and is fully conserved among meningococci. Our results provide further insights into the mechanism by which Fur controls essential components of the N. meningitidis respiratory chain. Adaptation of cytochrome bc1 complex component levels upon iron limitation is post-transcriptionally regulated via the small regulatory RNA NrrF.

Neisseria meningitidis Uses Sibling Small Regulatory RNAs To Switch from Cataplerotic to Anaplerotic Metabolism.

Neisseria meningitidis (the meningococcus) is primarily a commensal of the human oropharynx that sporadically causes septicemia and meningitis. Meningococci adapt to diverse local host conditions differing in nutrient supply, like the nasopharynx, blood, and cerebrospinal fluid, by changing metabolism and protein repertoire. However, regulatory transcription factors and two-component systems in meningococci involved in adaptation to local nutrient variations are limited. We identified novel sibling small regulatory RNAs ( Neisseriametabolic switch regulators [NmsRs]) regulating switches between cataplerotic and anaplerotic metabolism in this pathogen. Overexpression of NmsRs was tolerated in blood but not in cerebrospinal fluid. Expression of six tricarboxylic acid cycle enzymes was downregulated by direct action of NmsRs. Expression of the NmsRs themselves was under the control of the stringent response through the action of RelA. Small sibling regulatory RNAs of meningococci, controlling general metabolic switches, add an exciting twist to their versatile repertoire in bacterial pathogens.IMPORTANCE Regulatory small RNAs (sRNAs) of pathogens are coming to be recognized as highly important components of riboregulatory networks, involved in the control of essential cellular processes. They play a prominent role in adaptation to physiological changes as represented by different host environments. They can function as posttranscriptional regulators of gene expression to orchestrate metabolic adaptation to nutrient stresses. Here, we identified highly conserved sibling sRNAs in Neisseria meningitidis which are functionally involved in the regulation of gene expression of components of the tricarboxylic acid cycle. These novel sibling sRNAs that function by antisense mechanisms extend the so-called stringent response which connects metabolic status to colonization and possibly virulence as well as pathogenesis in meningococci.

Meningitis caused by a lipopolysaccharide deficient Neisseria meningitidis.

OBJECTIVE: Lipopolysaccharide (LPS) is a major component of the Neisseria meningitidis outer membrane. Here we report a patient with meningococcal meningitis of which the causative isolate lacked LPS. Thus far, no naturally occurring LPS-deficient meningococcal isolate has been known to cause clinical disease. METHODS: We used SDS-PAGE, silver staining and LPS-specific antibodies in whole cell ELISA to determine LPS presence in the causative isolate. Meningococcal whole genome sequencing was performed using Roche 454-sequencing. The N. meningitidis strain MC58 was used to compare all LPS biosynthesis associated genes. We compared growth characteristics of Escherichia coli transformed with a plasmid containing 2 lpxH types. RESULTS: The patient presented with isolated thunderclap headache. Analysis of the causative N. meningitidis showed no LPS. Whole genome sequencing revealed a mutation located in lpxH explaining LPS-deficiency. Expression of this lpxH variant in E. coli resulted in growth impairment compared to E. coli expressing the meningococcal wild type lpxH variant. In addition, inactivating lpxH in N. meningitidis H44/76 by insertional inactivation with a kanamycin cassette resulted in a LPS-deficient phenotype. CONCLUSIONS: We describe invasive meningococcal disease caused by a naturally occurring LPS-deficient meningococcal isolate.

Meningococcal serogroup Y lpxL1 variants from South Africa are associated with clonal complex 23 among young adults.

OBJECTIVES: To determine the genotypes of serogroup Y meningococcus (MenY), and to determine the prevalence of and identify factors associated with MenY lpxL1 variants. METHODS: Isolates, collected from 2003 to 2007 through national surveillance for invasive meningococcal disease, were characterized by multilocus sequence typing and screened for interleukin-6 induction. LpxL1 genes were sequenced from low IL-6 inducers. RESULTS: MenY represented 13% (n = 219/1702) of meningococcal disease. Clonal complex (cc) 175, ST-23/Cluster A3 (cc23), cc11 and cc167 accounted for 82% (176/214), 11% (24/214), 3% (6/214) and 3% (7/214) respectively. Low cytokine induction was evident in 15% (32/218). Cc23 isolates (24/24) had an lpxL1 mutation, while among the remaining isolates the proportion of lpxL1 variants was 4% (8/189, p < 0.001), and these were all cc175. Compared to wild type isolates, lpxL1 variants were associated with patients aged 5-14 years [unadjusted OR (95% CI): 4.3 (1.5-12)] or 15-24 years [unadjusted OR (95% CI): 9.1 (2.8-29)] compared to children <5 years; and were more likely have been isolated from CSF than blood [unadjusted OR (95% CI): 3.5 (1-11.9)]. On multivariable analysis, age remained significant [adjusted OR (95% CI), 5-14 years: 4.2 (1.5-12); 15-24 years: 8.9 (2.7-29)]. CONCLUSION: LpxL1 variants were associated with cc23 among young adults.

Streptococcus pneumoniae arginine synthesis genes promote growth and virulence in pneumococcal meningitis.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing pneumonia, sepsis and bacterial meningitis. Using a clinical phenotype based approach with bacterial whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with pneumococcal meningitis. Pneumococci harboring these genes show increased growth in human blood and cerebrospinal fluid (CSF). Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in growth or cleared, from lung, blood and CSF. Thus, S. pneumoniae arginine synthesis genes promote growth and virulence in invasive pneumococcal disease.

Domain exchange at the 3' end of the gene encoding the fratricide meningococcal two-partner secretion protein A.

BACKGROUND: Two-partner secretion systems in Gram-negative bacteria consist of an outer membrane protein TpsB that mediates the secretion of a cognate TpsA protein into the extracellular milieu. TpsA proteins have diverse, often virulence-related functions, and some of them inhibit the growth of related bacteria. In Neisseria meningitidis, several functions have been attributed to the TpsA proteins. Downstream of the tpsB and tpsA genes, several shorter tpsA-related gene cassettes, called tpsC, are located interspersed with intervening open-reading frames (IORFs). It has been suggested that the tpsC cassettes may recombine with the tpsA gene as a mechanism of antigenic variation. Here, we investigated (i) whether TpsA of N. meningitidis also has growth-inhibitory properties, (ii) whether tpsC cassettes recombine with the tpsA gene, and (iii) what the consequences of such recombination events might be. RESULTS: We demonstrate that meningococcal TpsA has growth-inhibitory properties and that the IORF located immediately downstream of tpsA confers immunity to the producing strain. Although bioinformatics analysis suggests that recombination between tpsC cassettes and tpsA occurs, detailed analysis of the tpsA gene in a large collection of disease isolates of three clonal complexes revealed that the frequency is very low and cannot be a mechanism of antigenic variation. However, recombination affected growth inhibition. In vitro experiments revealed that recombination can be mediated through acquirement of tpsC cassettes from the environment and it identified the regions involved in the recombination. CONCLUSIONS: Meningococcal TpsA has growth-inhibitory properties. Recombination between tpsA and tpsC cassettes occurs in vivo but is rare and has consequences for growth inhibition. A recombination model is proposed and we propose that the main goal of recombination is the collection of new IORFs for protection against a variety of TpsA proteins.

Prevalence and clinical course in invasive infections with meningococcal endotoxin variants.

BACKGROUND: Meningococci produce a penta-acylated instead of hexa-acylated lipid A when their lpxL1 gene is inactivated. Meningococcal strains with such lipid A endotoxin variants have been found previously in adult meningitis patients, where they caused less blood coagulopathy because of decreased TLR4 activation. METHODS: A cohort of 448 isolates from patients with invasive meningococcal disease in the Netherlands were screened for the ability to induce IL-6 in monocytic cell Mono Mac 6 cells. The lpxL1 gene was sequenced of isolates, which show poor capacity to induce IL-6.. Clinical characteristics of patients were retrieved from hospital records. RESULTS: Of 448 patients, 29 (6.5%) were infected with meningococci expressing a lipid A variant strain. Lipid A variation was not associated with a specific serogroup or genotype. Infections with lipid A variants were associated with older age (19.3 vs. 5.9 (median) years, p = 0.007) and higher prevalence of underlying comorbidities (39% vs. 17%; p = 0.004) compared to wild-type strains. Patients infected with lipid A variant strains had less severe infections like meningitis or shock (OR 0.23; 95%CI 0.09-0.58) and were less often admitted to intensive care (OR 0.21; 95%CI 0.07-0.60) compared to wild-type strains, independent of age, underlying comorbidities or strain characteristics. CONCLUSIONS: In adults with meningococcal disease lipid A variation is rather common. Infection with penta-acylated lipid A variant meningococci is associated with a less severe disease course.

The influence of antibodies on Staphylococcus epidermidis adherence to polyvinylpyrrolidone-coated silicone elastomer in experimental biomaterial-associated infection in mice.

Biomaterial-associated infection (BAI) is a major problem in modern medicine, and is often caused by Staphylococcus epidermidis. We aimed to raise monoclonal antibodies (mAbs) against major surface protein antigens of S. epidermidis, and to assess their possible protective activity in experimental BAI. Mice were vaccinated with a cell wall protein preparation of S. epidermidis. A highly immunodominant antigen was identified as Accumulation-associated protein (Aap). mAbs against Aap and against surface-exposed lipoteichoic acid (LTA) were used for passive immunization of mice in experimental biomaterial-associated infection. Neither anti-Aap nor anti-LTA mAbs showed protection. Either with or without antibodies, tissue surrounding the implants was more often culture positive than the implants themselves, but bacterial adherence to the implants was significantly increased in mice injected with anti-LTA. In vitro, anti-Aap and anti-LTA did show binding to S. epidermidis, but no opsonic activity was observed. We conclude that antibodies against S. epidermidis LTA or Aap showed no opsonic activity and did not protect mice against BAI. Moreover, the increase in binding to implanted biomaterial suggests that passive immunization may increase the risk for BAI.

Staphylococcus epidermidis is cleared from biomaterial implants but persists in peri-implant tissue in mice despite rifampicin/vancomycin treatment.

Infections associated with implanted biomedical devices (BAI) are predominantly caused by Staphylococcus epidermidis. We previously observed in murine experimental BAI that S. epidermidis persists in peri-implant tissue rather than on the implanted biomaterial itself (Boelens et al., J Infect Dis 2000;181:1337-1349; Broekhuizen et al., Infect Immun 2007;75:1129-1136). To investigate the efficacy of rifampicin/vancomycin to clear S. epidermidis from implants and peri-implant tissues, mice with two implants were challenged with 10(7) cfu S. epidermidis per implant and received daily injections of rifampicin (25 mg/kg) and vancomycin (50 mg/kg). On the day of termination, implants and peri-implant tissue were collected and processed for culture and histology. After 1 and 8 days, implants of control mice were culture positive in 14/18 and 5/16 cases, respectively, and tissue biopsies were all culture positive. In the antibiotic-treated mice, bacteria were recovered from only 1/18 and 1/16 implants after 1 and 8 days, respectively, whereas the tissues were culture positive in 14/18 and 7/16 biopsies, respectively. In microscopy, bacteria were seen in the tissue at a distance of several cell layers from the tissue-implant interface, colocalized with host cells. Thus, although a regimen of rifampicin/vancomycin sterilized the implants, S. epidermidis persisted in peri-implant tissue, which might be an as yet unrecognized reservoir in the pathogenesis of BAI.

Peri-implant tissue is an important niche for Staphylococcus epidermidis in experimental biomaterial-associated infection in mice.

Biomaterial-associated infections (BAI), which are predominantly caused by Staphylococcus epidermidis, are a significant problem in modern medicine. Biofilm formation is considered the pivotal element in the pathogenesis, but in previous mouse studies we retrieved S. epidermidis from peri-implant tissue. To assess the kinetics and generality of tissue colonization, we investigated BAI using two S. epidermidis strains, two biomaterials, and two mouse strains. With small inocula all implants were culture negative, whereas surrounding tissues were positive. When higher doses were used, tissues were culture positive more often than implants, with higher numbers of CFU. This was true for the different biomaterials tested, for both S. epidermidis strains, at different times, and for both mouse strains. S. epidermidis colocalized with host cells at a distance that was >10 cell layers from the biomaterial-tissue interface. We concluded that in mouse experimental BAI S. epidermidis peri-implant tissue colonization is more important than biofilm formation.

Application of minimal sequence quality values prevents misidentification of the blaSHV type in single bacterial isolates carrying different SHV extended-spectrum beta-lactamase genes.

Nucleotide sequencing is the standard molecular method for determination of the beta-lactamase gene present in an isolate. Using minimal sequence quality values prevents misidentification of bla(SHV) genes, as illustrated by three strains of three different species that each contained two different bla(SHV) alleles, SHV-2 and SHV-12.

Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin.

BACKGROUND: Lactobacilli are probiotic bacteria that are frequently tested in the management of allergic diseases or gastroenteritis. It is hypothesized that these probiotics have immunoregulatory properties and promote mucosal tolerance, which is in part mediated by regulatory T cells (Treg cells). On the basis of pathogenic or tissue-specific priming, dendritic cells (DC) acquire different T cell-instructive signals and drive the differentiation of naive T H cells into either T H 1, T H 2, or regulatory effector T cells. OBJECTIVE: We studied in what way different species of lactobacilli prime human DCs for their ability to drive Treg cells. METHODS: Human monocyte-derived DCs were cultured in vitro with lactobacilli of different species. RESULTS: Two different species of lactobacilli, Lactobacillus reuteri and Lactobacillus casei , but not Lactobacillus plantarum, prime monocyte-derived DCs to drive the development of Treg cells. These Treg cells produced increased levels of IL-10 and were capable of inhibiting the proliferation of bystander T cells in an IL-10-dependent fashion. Strikingly, both L reuteri and L casei , but not L plantarum , bind the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). Blocking antibodies to DC-SIGN inhibited the induction of the Treg cells by these probiotic bacteria, stressing that ligation of DC-SIGN can actively prime DCs to induce Treg cells. CONCLUSIONS: The targeting of DC-SIGN by certain probiotic bacteria might explain their beneficial effect in the treatment of a number of inflammatory diseases, including atopic dermatitis and Crohn's disease.