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.

Interference with Lipoprotein Maturation Sensitizes Methicillin-Resistant Staphylococcus aureus to Human Group IIA-Secreted Phospholipase A2 and Daptomycin.

Methicillin-resistant Staphylococcus aureus (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA-secreted phospholipase A2 (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including S. aureus. To determine hGIIA-resistance mechanisms of MRSA, we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of lspA, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the lspA mutant was associated with enhanced activity of hGIIA on the cell membrane. Moreover, lspA deletion increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. MRSA wild type could be sensitized to hGIIA and daptomycin killing through exposure to LspA-specific inhibitors globomycin and myxovirescin A1. Analysis of >26,000 S. aureus genomes showed that LspA is highly sequence-conserved, suggesting universal application of LspA inhibition. The role of LspA in hGIIA resistance was not restricted to MRSA since Streptococcus mutans and Enterococcus faecalis were also more hGIIA-susceptible after lspA deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological interference with LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically applied antibiotics.

Photochemical internalization enhances cytosolic release of antibiotic and increases its efficacy against staphylococcal infection.

Bacterial pathogens such as Staphylococcus aureus and Staphylococcus epidermidis can survive in different types of cells including professional phagocytes, causing intracellular infections. Antibiotic treatment of intracellular infections is often unsuccessful due to the low efficacy of most antibiotics inside cells. Therefore, novel techniques which can improve intracellular activity of antibiotics are urgently needed. We aimed to use photochemical internalization (PCI) to enhance cytosolic release of antibiotics from endocytic vesicles after internalization. Our results show that PCI indeed caused cytosolic release of gentamicin and significantly increased its efficacy against S. epidermidis in vitro in mouse macrophages. Upon illumination for 15 min, the killing of intracellular S. epidermidis in RAW 264.7 cells by 10 or 30 µg/ml gentamicin was increased to 1 or 3 CFU log, respectively, owing to the use of PCI, whereas no killing by gentamicin only without PCI was observed. Moreover, survival of S. aureus-infected zebrafish embryos was significantly improved by treatment with PCI-gentamicin. PCI improved the therapeutic efficacy of gentamicin at a dose of 0.1 ng per embryo to a level similar to that of a dose of 0.4 ng per embryo, indicating that PCI can lower the antibiotic dose required for treating (intracellular) staphylococcal infection. Thus, the present study shows that PCI is a promising novel approach to enhance the intracellular efficacy of antibiotics via cytosolic release, allowing them to reach intracellular bacteria. This will expand their therapeutic window and will increase the numbers of antibiotics which can be used for treatment of intracellular infections.

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.

Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species.

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease (n = 101) and from carriage (n = 103), and on non-typeable pneumococci from asymptomatic individuals (n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae, targeting cpsA, lytA, piaB, ply, Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae, whereas assays targeting cpsA, ply, Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates.

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.

Meningococcal Two-Partner Secretion Systems and Their Association with Outcome in Patients with Meningitis.

Two-partner secretion (TPS) systems export large TpsA proteins to the surface and extracellular milieu. In meningococci, three different TPS systems exist, and of these, TPS system 2 (TPS2) and TPS3 can be detected by the host's immune system. We evaluated the distribution of TPS systems among clinical isolates from two prospective cohort studies comprising 373 patients with meningococcal meningitis. TPS system 1 was present in 91% of isolates, and system 2 and/or 3 was present in 67%. The TPS system distribution was related to clonal complexes. Infection with strains with TPS2 and/or TPS3 resulted in less severe disease and better outcomes than infection with strains without these systems. Using whole-blood stimulation experiments, we found no differences in the host cytokine response between patients infected with TPS system 2 and 3 knockout strains and patients infected with a wild-type strain. In conclusion, meningococcal TPS system 2 and/or 3 is associated with disease severity and outcome in patients with meningitis.

Disease Burden of Invasive Meningococcal Disease in the Netherlands Between June 1999 and June 2011: A Subjective Role for Serogroup and Clonal Complex.

BACKGROUND: Several countries consider the implementation of a meningococcal serogroup B vaccine for young children and/or serogroup C or ACWY conjugate vaccine for adolescents. Representative information on clinical course of invasive meningococcal disease (IMD) is useful to evaluate cost-effectiveness of vaccination. Information on the relation between infecting meningococcal clonal complex (CC), disease course and outcome of IMD is scarce. METHODS: A retrospective study using Dutch surveillance data on IMD from June 1999 to June 2011. Clinical information was retrieved from hospital records. The effect of age, comorbidity, clinical manifestation, serogroup, and CC on disease course and outcome was assessed in multivariable analyses. Meningococcal CCs were assessed by multilocus sequence typing. RESULTS: Clinical information was retrieved for 879 IMD cases: 48% of patients presented with meningitis, 17% with septic shock, and 22% with septic shock plus meningitis. Development of septic shock was not related to CC or serogroup. Median (interquartile range) duration of hospital admission was 10 (8-13) days. Intensive care unit admittance (38%) was higher for patients aged ≥10 years and patients with septic shock (P-values ≤.001). Case-fatality rate (8%) and development of sequelae (29%) was dependent on age and clinical manifestation (P-values ≤.001) and not affected by comorbidity, CC, or serogroup. CONCLUSIONS: IMD still coincides with a considerable disease burden and mortality. Disease course and outcome depend mainly on age and clinical manifestation and less on meningococcal CC or serogroup.

Group A Streptococcal meningitis in adults.

OBJECTIVES: We report on the incidence, clinical characteristics, and bacterial genotype of group A streptococcal (GAS) meningitis in the Netherlands. METHODS: We assessed the incidence, clinical characteristics, and outcome of patients with GAS meningitis from a nationwide cohort study of adults with community-acquired bacterial meningitis in the Netherlands from 2006 to 2013. RESULTS: GAS was identified in 26 of 1322 patients with community-acquired bacterial meningitis (2%); 9 cases (35%) occurred in the first four months of 2013. GAS meningitis was often preceded by otitis or sinusitis (24 of 26 [92%]) and a high proportion of patients developed complications during clinical course (19 of 26 [73%]). Subdural empyema occurred in 8 of 26 patients (35%). Nine patients underwent mastoidectomy and in 5 patients neurosurgical evacuation of the subdural empyema was performed. Five of 26 patients (19%) died and 11 of 21 surviving patient had neurologic sequelae (52%). Infection with the emm1 and cc28 GAS genotype was associated with subdural empyema (both 4 of 6 [67%] vs. 2 of 14 [14%]; P = 0.037). CONCLUSIONS: GAS meningitis is an uncommon but severe disease. Patients are at risk for empyema, which is associated with infection with the emm1 and cc28 genotype.

Listeria monocytogenes sequence type 6 and increased rate of unfavorable outcome in meningitis: epidemiologic cohort study.

BACKGROUND: We analyzed clinical characteristics, treatment, genetic diversity, and outcome of 92 adults with Listeria monocytogenes meningitis included in 2 prospective nationwide cohort studies. METHODS: Episodes of community-acquired listerial meningitis confirmed by cerebrospinal fluid culture were included from 1998 to 2002 and 2006 to 2012. We compared patients and pathogen characteristics between cohorts and identified predictors for an unfavorable outcome according to the Glasgow Outcome Scale. RESULTS: Thirty episodes were included from 1998 to 2002 and 62 from 2006 to 2012; clinical and laboratory characteristics on admission were similar between cohorts. However, the rate of unfavorable outcome increased from 27% in the 1998-2002 cohort to 61% in the 2006-2012 cohort (P = .002). Differences between cohorts were increased use of adjunctive dexamethasone therapy (0% in 1998-2002 vs 53% in 2006-2012; P < .001) and emergence of infection by L. monocytogenes genotype sequence type 6 (ST6; 4% in 1998-2002 vs 29% in 2006-2012; P = .009). Multivariate regression analysis identified infection with L. monocytogenes ST6 as the sole predictor of unfavorable outcome (odds ratio, 3.77; 95% confidence interval, 1.07-13.33). Patients infected with genotypes other than ST6 also had an increased rate of unfavorable outcome over time (P = .03). CONCLUSIONS: The rate of unfavorable outcome among adults with listerial meningitis has increased over a 14-year period, from 27% to 61%. The emerging L. monocytogenes genotype ST6 was identified as the main factor leading to poorer prognosis. Adjunctive dexamethasone may be discontinued if L. monocytogenes is identified, as there is no proven benefit in Listeria meningitis.