No abstract

Protective antibodies in Plasmodium falciparum malaria are only acquired after years of repeated infections. Chronic malaria exposure is associated with a large increase in atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent viral infections. Understanding the function of atypical MBCs and their relationship to classical MBCs will be critical to developing effective vaccines for malaria and other chronic infections. We show that VH gene repertoires and somatic hypermutation rates of atypical and classical MBCs are indistinguishable indicating a common developmental history. Atypical MBCs express an array of inhibitory receptors and B cell receptor (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses including proliferation, cytokine production and antibody secretion. Thus, in response to chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs becoming refractory to BCR-mediated activation and potentially interfering with the acquisition of malaria immunity.DOI: http://dx.doi.org/10.7554/eLife.07218.001

No abstract

The inability to propagate obligate intracellular pathogens under axenic (host cell-free) culture conditions imposes severe experimental constraints that have negatively impacted progress in understanding pathogen virulence and disease mechanisms. Coxiella burnetii, the causative agent of human Q (Query) fever, is an obligate intracellular bacterial pathogen that replicates exclusively in an acidified, lysosome-like vacuole. To define conditions that support C. burnetii growth, we systematically evaluated the organism's metabolic requirements using expression microarrays, genomic reconstruction, and metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (approximately 3 log 10) of C. burnetii in a 2.5% oxygen environment. Importantly, axenically grown C. burnetii were highly infectious for Vero cells and exhibited developmental forms characteristic of in vivo grown organisms. Axenic cultivation of C. burnetii will facilitate studies of the organism's pathogenesis and genetics and aid development of Q fever preventatives such as an effective subunit vaccine. Furthermore, the systematic approach used here may be broadly applicable to development of axenic media that support growth of other medically important obligate intracellular pathogens.axenic growth ͉ metabolism ͉ microaerophile ͉ obligate intracellular pathogen

Infections caused by drug-resistant bacteria are a major problem worldwide. Carbapenem-resistant Klebsiella pneumoniae, most notably isolates classified as multilocus sequence type (ST) 258, have emerged as an important cause of hospital deaths. ST258 isolates are predominantly multidrug resistant, and therefore infections caused by them are difficult to treat. It is not known why the ST258 lineage is the most prevalent cause of multidrug-resistant K. pneumoniae infections in the United States and other countries. Here we tested the hypothesis that carbapenem-resistant ST258 K. pneumoniae is a single genetic clone that has disseminated worldwide. We sequenced to closure the genomes of two ST258 clinical isolates and used these genomes as references for comparative genome sequencing of 83 additional clinical isolates recovered from patients at diverse geographic locations worldwide. Phylogenetic analysis of the SNPs in the core genome of these isolates revealed that ST258 K. pneumoniae organisms are two distinct genetic clades. This unexpected finding disproves the single-clone hypothesis. Notably, genetic differentiation between the two clades results from an ∼215-kb region of divergence that includes genes involved in capsule polysaccharide biosynthesis. The region of divergence appears to be a hotspot for DNA recombination events, and we suggest that this region has contributed to the success of ST258 K. pneumoniae. Our findings will accelerate research on novel diagnostic, therapeutic, and vaccine strategies designed to prevent and/or treat infections caused by multidrug resistant K. pneumoniae.antibiotic resistance | carbapenemase | Enterobacteriaceae | plasmid

Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group A Streptococcus (GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares Ϸ1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A2 (designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phagemediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS.superantigen ͉ streptococcal pyrogenic exotoxin ͉ comparative genomics

Deep RNA sequencing was used to simultaneously analyze vaccinia virus (VACV) and HeLa cell transcriptomes at progressive times following infection. VACV, the prototypic member of the poxvirus family, replicates in the cytoplasm and contains a double-stranded DNA genome with ≈200 closely spaced open reading frames (ORFs). The acquisition of a total of nearly 500 million short cDNA sequences allowed construction of temporal strand-specific maps of the entire VACV transcriptome at single-base resolution and analysis of over 14,000 host mRNAs. Before viral DNA replication, transcripts from 118 VACV ORFs were detected; after replication, transcripts from 93 additional ORFs were characterized. The high resolution permitted determination of the precise boundaries of many mRNAs including read-through transcripts and location of mRNA start sites and adjacent promoters. Temporal analysis revealed two clusters of early mRNAs that were synthesized in the presence of inhibitors of protein as well as DNA synthesis, indicating that they do not correspond to separate immediate-and delayed-early classes as defined for other DNA viruses. The proportion of viral RNAs reached 25-55% of the total at 4 h. This rapid change, resulting in a relative decrease of the vast majority of host mRNAs, can contribute to the profound shutdown of host protein synthesis and blunting of antiviral responses. At 2 h, however, a minority of cellular mRNAs was increased. The overrepresented functional categories of the up-regulated RNAs were NF-κB cascade, apoptosis, signal transduction, and ligand-mediated signaling, which likely represent the host response to invasion.human transcriptome | poxvirus transcriptome | RNA-seq | vaccinia virus mRNA | virus-host interaction

Acute rheumatic fever (ARF), a sequelae of group A Streptococcus (GAS) infection, is the most common cause of preventable childhood heart disease worldwide. The molecular basis of ARF and the subsequent rheumatic heart disease are poorly understood. Serotype M18 GAS strains have been associated for decades with ARF outbreaks in the U.S. As a first step toward gaining new insight into ARF pathogenesis, we sequenced the genome of strain MGAS8232, a serotype M18 organism isolated from a patient with ARF. The genome is a circular chromosome of 1,895,017 bp, and it shares 1.7 Mb of closely related genetic material with strain SF370 (a sequenced serotype M1 strain). Strain MGAS8232 has 178 ORFs absent in SF370. Phages, phage-like elements, and insertion sequences are the major sources of variation between the genomes. The genomes of strain MGAS8232 and SF370 encode many of the same proven or putative virulence factors. Importantly, strain MGAS8232 has genes encoding many additional secreted proteins involved in human-GAS interactions, including streptococcal pyrogenic exotoxin A (scarlet fever toxin) and two uncharacterized pyrogenic exotoxin homologues, all phage-associated. DNA microarray analysis of 36 serotype M18 strains from diverse localities showed that most regions of variation were phages or phage-like elements. Two epidemics of ARF occurring 12 years apart in Salt Lake City, UT, were caused by serotype M18 strains that were genetically identical, or nearly so. Our analysis provides a critical foundation for accelerated research into ARF pathogenesis and a molecular framework to study the plasticity of GAS genomes.Streptococcus pyogenes ͉ GAS ͉ complete genome sequence ͉ DNA microarray ͉ genomic diversity