Meningococcal disease in North America: Updates from the Global Meningococcal Initiative

This review summarizes the recent Global Meningococcal Initiative (GMI) regional meeting, which explored meningococcal disease in North America. Invasive meningococcal disease (IMD) cases are documented through both passive and active surveillance networks. IMD appears to be decreasing in many areas, such as the Dominican Republic (2016: 18 cases; 2021: 2 cases) and Panama (2008: 1 case/100,000; 2021: <0.1 cases/100,000); however, there is notable regional and temporal variation. Outbreaks persist in at-risk subpopulations, such as people experiencing homelessness in the US and migrants in Mexico. The recent emergence of β-lactamase-positive and ciprofloxacin-resistant meningococci in the US is a major concern. While vaccination practices vary across North America, vaccine uptake remains relatively high. Monovalent and multivalent conjugate vaccines (which many countries in North America primarily use) can provide herd protection. However, there is no evidence that group B vaccines reduce meningococcal carriage. The coronavirus pandemic illustrates that following public health crises, enhanced surveillance of disease epidemiology and catch-up vaccine schedules is key. Whole genome sequencing is a key epidemiological tool for identifying IMD strain emergence and the evaluation of vaccine strain coverage. The Global Roadmap on Defeating Meningitis by 2030 remains a focus of the GMI.Medical writing support for the development of this manuscript, under the direction of the authors, was provided Matthew Gunther of Ashfield MedComms, an Inizio company. Medical writing support was funded by Sanofi Pasteur. All authors discussed and agreed to the objectives of this manuscript and con- tributed throughout its production. All authors read and approved the final manuscript.S

dGTP Starvation in <i>Escherichia coli</i> Provides New Insights into the Thymineless-Death Phenomenon

<div><p>Starvation of cells for the DNA building block dTTP is strikingly lethal (thymineless death, TLD), and this effect is observed in all organisms. The phenomenon, discovered some 60 years ago, is widely used to kill cells in anticancer therapies, but many questions regarding the precise underlying mechanisms have remained. Here, we show for the first time that starvation for the DNA precursor dGTP can kill <i>E. coli</i> cells in a manner sharing many features with TLD. dGTP starvation is accomplished by combining up-regulation of a cellular dGTPase with a deficiency of the guanine salvage enzyme guanine-(hypoxanthine)-phosphoribosyltransferase. These cells, when grown in medium without an exogenous purine source like hypoxanthine or adenine, display a specific collapse of the dGTP pool, slow-down of chromosomal replication, the generation of multi-branched nucleoids, induction of the SOS system, and cell death. We conclude that starvation for a single DNA building block is sufficient to bring about cell death.</p></div

Partial Restoration of Antibacterial Activity of the Protein Encoded by a Cryptic Open Reading Frame (cyt1Ca) from Bacillus thuringiensis subsp. israelensis by Site-Directed Mutagenesis

Insecticidal crystal proteins of Bacillus thuringiensis belong to two unrelated toxin families: receptor-specific Cry toxins against insects and Cyt toxins that lyse a broad range of cells, including bacteria, via direct binding to phospholipids. A new cyt-like open reading frame (cyt1Ca) encoding a 60-kDa protein, has recently been discovered (C. Berry et al., Appl. Environ. Microbiol. 68:5082-5095, 2002). Cyt1Ca displays the structure of a two-domain fusion protein: the N-terminal moiety resembles the full-length Cyt toxins, and the C-terminal moiety is similar to the receptor-binding domains of several ricin-like toxins, such as Mtx1. Neither the larvicidal activity of cyt1Ca expressed in Escherichia coli nor the hemolytic effect of His-tagged purified Cyt1Ca has been observed (R. Manasherob et al., unpublished). This was attributed to five amino acid differences between the sequences of its N-terminal moiety and Cyt1Aa. The 3′ end of cyt1Ca was truncated (removing the ricin-binding domain of Cyt1Ca), and six single bases were appropriately changed by site-directed mutagenesis, sequentially replacing the noncharged amino acids by charged ones, according to Cyt1Aa, to form several versions. Expression of these mutated cyt1Ca versions caused loss of the colony-forming ability of the corresponding E. coli cells to different extents compared with the original gene. In some mutants this antibacterial effect was associated by significant distortion of cell morphology and in others by generation of multiple inclusion bodies spread along the cell envelope. The described deleterious effects of mutated cyt1Ca versions against E. coli may reflect an evolutionary relationship between Cyt1Aa and Cyt1Ca

Expression in Escherichia coli of the Native cyt1Aa from Bacillus thuringiensis subsp. israelensis▿

The gene cyt1Aa is one of the genes in the complex determining the mosquito larvicidity of Bacillus thuringiensis subsp. israelensis. Previous cloning in Escherichia coli resulted in a 48-bp addition upstream, encoding a chimera. Here, cyt1Aa was recloned without the artifact, and its toxicity against Aedes aegypti larvae and host E. coli cells was retested

Primers specific to the origin (ori) and the terminus (ter) regions of the <i>E. coli</i> chromosome [49] used for ori/ter ratio determination using Quantitative PCR.

<p>Primers specific to the origin (ori) and the terminus (ter) regions of the <i>E. coli</i> chromosome <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004310#pgen.1004310-Stokke1" target="_blank">[49]</a> used for ori/ter ratio determination using Quantitative PCR.</p

The outcome of dGTP starvation experiments for cultures initiated at decreasing cell densities.

<p>A stationary phase <i>optA1 gpt</i> culture was diluted to the indicated extents into minimal medium with casamino acids with hypoxanthine (Hx) (open symbols) or without Hx (closed symbols). The resulting cultures were subjected to overnight growth at 37°C, and samples of the resulting cultures were analyzed for the presence of suppressors resistant to subsequent repeat starvation using the tests described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004310#pgen-1004310-g006" target="_blank">Fig. 6C</a>. The results are presented as the % suppressors observed. Assuming, based on the data of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004310#pgen-1004310-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004310#pgen-1004310-g003" target="_blank">3</a>, that an about 100-fold increase in OD is needed from the beginning of starvation to reach lethal status, the critical biomass density (midpoint value) for cellular adaptation can be roughly estimated. At an inoculate value of 5×10⁵ cells for the midpoint and an experimentally determined correlation between OD_{630 nm} and cell titer (3.3×10⁸ cells/ml per OD_{630 nm}), a 100-fold OD_{630 nm} increase would correspond to 100×(5×10⁵)/(3.3×10⁸) = 0.15.</p

Defective growth upon purine starvation.

<p>(<b>A</b>) Growth defect of an <i>optA1 gpt</i> strain upon culturing in minimal glucose medium enriched with casamino acids without added purine source. Several controls are also shown (see text for details). Error bars are from three different measurements. (<b>B</b>) Complementation of the growth defect of an <i>optA1 gpt</i> strain by hypoxanthine (Hx), adenine (Ade), but not guanine (Gua), added at 50 µg/ml each. The cultures were started from a 5,000-fold dilution of an overnight stationary culture grown in the presence of hypoxanthine. (<b>C</b>) Relevant metabolic pathways for <i>de novo</i> synthesis and salvage of guanine and guanine nucleotides, illustrating how the <i>optA1 gpt</i> combination may become starved for dGTP (see text for details). One alternative pathway for synthesis of GMP from Gua that is not indicated is the conversion of Gua to guanosine by the DeoD purine nucleoside phosphorylase by condensation with Rib-1-P followed by conversion of guanosine to GMP by guanosine kinase (<i>gsk</i> gene product). However, this pathway for GMP synthesis is not very efficient <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004310#pgen.1004310-HoveJensen1" target="_blank">[52]</a>. The gene symbols are: <i>dgt</i> - dGTP triphosphohydrolase; <i>deoD</i> - purine nucleoside phosphorylase; <i>gpt</i> - guanine phosphoribosyltransferase; <i>hpt</i> - hypoxanthine phosphoribosyltransferase; <i>add</i> – adenosine deaminase; <i>purR</i> – purine repressor (transcription factor controlling <i>de novo</i> synthesis of purine nucleotides) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004310#pgen.1004310-Cho1" target="_blank">[21]</a>. Green and blue – salvage and <i>de novo</i> synthesis pathways, respectively. Arrows are as follows: thin – wild-type enzyme levels; thick – elevated levels of dGTP triphosphohydrolase (<i>optA1</i> - <i>dgt</i> up-promoter) or of enzymes of the PurR regulon (<i>purR</i> deletion strain); dashed - lack of activities in a <i>gpt</i> deletion strain. Gua – guanine; Hx – hypoxanthine; Ade – adenine; dG-Rib – deoxyguanosine; IMP – inosine monophosphate; PPP_i – tripolyphosphate; dRib-1P – deoxyribose-1-phosphate; P-Rib-PP – 5′-phosphoribosyl-1-pyrophosphate (PRPP).</p

Nucleotide pool effects in Hx-starved strains.

<p>Shown are (<b>A</b>) dNTP and (<b>B</b>) NTP pools in strains grown with (grey) or without (black) hypoxanthine (Hx) at two hours after withdrawal of Hx in the indicated strains. Nucleotide amounts were normalized relative to the ATP peak as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004310#pgen.1004310-Ahluwalia1" target="_blank">[38]</a>. The amount of ATP calculated as ATP/(OD_{630 nm}×sample volume) was largely unchanged among each of the strains. CTP could not be quantitated in these experiments. Standard deviations (error bars) were calculated from three experiments. (<b>C</b>) Chromatogram showing disappearance of dGTP during Hx starvation of the <i>optA1 gpt</i> strain. The red line represents growth in presence of Hx; the blue line and green lines correspond to 2 and 4 h of Hx starvation, respectively. The small inserts show the absorption spectra of dGTP and the unknown substance (X) that appeared during the starvation and interfered with quantitation of the dGTP at later time points. (<b>D</b>) Time course for dGTP pool changes in hypoxanthine-starved <i>optA1 gpt</i> strain. Cells were grown in hypoxanthine-containing medium (+Hx). At t = 0, hypoxanthine was withdrawn, and samples were withdrawn at indicated times. The HPLC analysis protocol was modified to provide for improved resolution of the dGTP peak (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004310#s4" target="_blank">Materials and Methods</a>). The dGTP level at 15 and 30 min of starvation was below the detectable limit of the method (0.001).</p

Role of the SOS response in dGTP-starved cells.

<p>(<b>A</b>) Induction of the SOS system upon hypoxanthine (Hx) starvation. Cultures of <i>optA1</i>, <i>gpt</i>, and <i>optA1 gpt</i> strains containing a plasmid-carried SOS reporter gene (<i>umuC</i>::<i>lacZ</i>) were grown in minimal medium (with 1% casamino acids) in the absence of Hx for 2, 4, and 6 hours, and samples were processed for liquid β-galactosidase assay. (<b>B</b>) Effect of <i>recA</i> and <i>sulA</i> deletions on cell growth during dGTP starvation. Shown are the normalized viable counts (see Legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004310#pgen-1004310-g003" target="_blank">Fig. 3</a>) of the <i>optA1 gpt</i> (filled squares), <i>optA1 gpt recA</i> (open circles), and <i>optA1 gpt sulA</i> strains (open triangles) during Hx starvation initiated at time zero.</p