A novel cgMLST for genomic surveillance of Yersinia enterocolitica infections in France allowed the detection and investigation of outbreaks in 2017-2021.

Enteric yersiniosis, the third most common food-borne zoonosis in Europe, is mainly caused by the pathogen Yersinia enterocolitica. In France, the yersiniosis microbiological surveillance is conducted at the Yersinia National Reference Laboratory (YNRL). Since 2017, isolates have been characterized by whole genome sequencing (WGS) followed by a 500-gene Yersinia-cgMLST. We report here the data of the WGS-based surveillance on Y. enterocolitica isolates for the 2017-2021 period. The YNRL characterized 7,642 Y. enterocolitica strains distributed in 2,497 non-pathogenic isolates from lineages 1Aa and 1Ab, and 5,145 specimens belonging to 8 pathogenic lineages. Among pathogenic isolates, lineage 4 was the most common (87.2%) followed by lineages 2/3-9b (10.6%), 2/3-5a (1.2%), 2/3-9a (0.6%), 3-3b, 3-3c, 1B, and 3-3d (0.1% per each). Importantly, we developed a routine surveillance system based on a new typing method consisting of a 1,727-genes core genome Multilocus Sequence Typing (cgMLST) specific to the species Y. enterocolitica followed by isolate clustering. Thresholds of allelic distances (AD) were determined and fixed for the clustering of isolates: AD ≤ 5 for lineages 4, 2/3-5a, and 2/3-9a, and AD ≤ 3 for lineage 2/3-9b. Clustering programs were implemented in 2019 in routine surveillance to detect genomic clusters of pathogenic isolates. In total, 419 clusters with at least 2 isolates were identified, representing 2,504 of the 3,503 isolates characterized between 2019 and 2021. Most clusters (n = 325) comprised 2 to 5 isolates. The new typing method proved to be useful for the molecular investigation of unusual grouping of cases as well as for the detection of genomic clusters in routine surveillance. IMPORTANCE: We describe here the new typing method used for molecular surveillance of Yersinia enterocolitica infections in France based on a novel core genome Multilocus Sequence Typing (cgMLST) specific to Y. enterocolitica species. This method can reliably identify the pathogenic Y. enterocolitica subspecies and compare the isolates with a high discriminatory power. Between 2017 and 2021, 5,145 pathogenic isolates belonging to 8 lineages were characterized and lineage 4 was by far the most common followed by lineage 2/3-9b. A clustering program was implemented, and detection thresholds were cross-validated by the molecular and epidemiological investigation of three unusual groups of Y. enterocolitica infections. The routine molecular surveillance system has been able to detect genomic clusters, leading to epidemiological investigations.

Promiscuous evolution of Group A Streptococcal M and M-like proteins.

Group A Streptococcus (GAS) M and M-like proteins are essential virulence factors and represent the primary epidemiological marker of this pathogen. Protein sequences encoding 1054 M, Mrp and Enn proteins, from 1668 GAS genomes, were analysed by SplitsTree4, partitioning around medoids and co-occurrence. The splits network and groups-based analysis of all M and M-like proteins revealed four large protein groupings, with multiple evolutionary histories as represented by multiple edges for most splits, leading to 'M-family-groups' (FG) of protein sequences: FG I, Mrp; FG II, M protein and Protein H; FG III, Enn; and FG IV, M protein. M and Enn proteins formed two groups with nine sub-groups and Mrp proteins formed four groups with ten sub-groups. Discrete co-occurrence of M and M-like proteins were identified suggesting that while dynamic, evolution may be constrained by a combination of functional and virulence attributes. At a granular level, four distinct family-groups of M, Enn and Mrp proteins are observable, with Mrp representing the most genetically distinct of the family-group of proteins. While M and Enn protein families generally group into three distinct family-groups, horizontal and vertical gene flow between distinct GAS strains is ongoing.

Viral origin of eukaryotic type IIA DNA topoisomerases.

Type II DNA topoisomerases of the family A (Topo IIAs) are present in all Bacteria (DNA gyrase) and eukaryotes. In eukaryotes, they play a major role in transcription, DNA replication, chromosome segregation, and modulation of chromosome architecture. The origin of eukaryotic Topo IIA remains mysterious since they are very divergent from their bacterial homologs and have no orthologs in Archaea. Interestingly, eukaryotic Topo IIAs have close homologs in viruses of the phylum Nucleocytoviricota, an expansive assemblage of large and giant viruses formerly known as the nucleocytoplasmic large DNA viruses. Topo IIAs are also encoded by some bacterioviruses of the class Caudoviricetes (tailed bacteriophages). To elucidate the origin of the eukaryotic Topo IIA, we performed in-depth phylogenetic analyses on a dataset combining viral and cellular Topo IIA homologs. Topo IIAs encoded by Bacteria and eukaryotes form two monophyletic groups nested within Topo IIA encoded by Caudoviricetes and Nucleocytoviricota, respectively. Importantly, Nucleocytoviricota remained well separated from eukaryotes after removing both Bacteria and Caudoviricetes from the data set, indicating that the separation of Nucleocytoviricota and eukaryotes is probably not due to long-branch attraction artifact. The topologies of our trees suggest that the eukaryotic Topo IIA was probably acquired from an ancestral member of the Nucleocytoviricota of the class Megaviricetes, before the emergence of the last eukaryotic common ancestor (LECA). This result further highlights a key role of these viruses in eukaryogenesis and suggests that early proto-eukaryotes used a Topo IIB instead of a Topo IIA for solving their DNA topological problems.

First Description of a Yersinia pseudotuberculosis Clonal Outbreak in France, Confirmed Using a New Core Genome Multilocus Sequence Typing Method.

Yersinia pseudotuberculosis is an enteric pathogen causing mild enteritis that can lead to mesenteric adenitis in children and septicemia in elderly patients. Most cases are sporadic, but outbreaks have already been described in different countries. We report for the first time a Y. pseudotuberculosis clonal outbreak in France, that occurred in 2020. An epidemiological investigation based on food queries pointed toward the consumption of tomatoes as the suspected source of infection. The Yersinia National Reference Laboratory (YNRL) developed a new cgMLST scheme with 1,921 genes specific to Y. pseudotuberculosis that identified the clustering of isolates associated with the outbreak and allowed to perform molecular typing in real time. In addition, this method allowed to retrospectively identify isolates belonging to this cluster from earlier in 2020. This method, which does not require specific bioinformatic skills, is now used systematically at the YNRL and proves to display an excellent discriminatory power and is available to the scientific community. IMPORTANCE We describe in here a novel core-genome MLST method that allowed to identify in real time, and for the first time in France, a Y. pseudotuberculosis clonal outbreak that took place during the summer 2020 in Corsica. Our method allows to support epidemiological and microbiological investigations to establish a link between patients infected with closely associated Y. pseudotuberculosis isolates, and to identify the potential source of infection. In addition, we made this method available for the scientific community.

A Dual Barcoding Approach to Bacterial Strain Nomenclature: Genomic Taxonomy of Klebsiella pneumoniae Strains.

Sublineages (SLs) within microbial species can differ widely in their ecology and pathogenicity, and their precise definition is important in basic research and for industrial or public health applications. Widely accepted strategies to define SLs are currently missing, which confuses communication in population biology and epidemiological surveillance. Here, we propose a broadly applicable genomic classification and nomenclature approach for bacterial strains, using the prominent public health threat Klebsiella pneumoniae as a model. Based on a 629-gene core genome multilocus sequence typing (cgMLST) scheme, we devised a dual barcoding system that combines multilevel single linkage (MLSL) clustering and life identification numbers (LINs). Phylogenetic and clustering analyses of >7,000 genome sequences captured population structure discontinuities, which were used to guide the definition of 10 infraspecific genetic dissimilarity thresholds. The widely used 7-gene multilocus sequence typing (MLST) nomenclature was mapped onto MLSL SLs (threshold: 190 allelic mismatches) and clonal group (threshold: 43) identifiers for backwards nomenclature compatibility. The taxonomy is publicly accessible through a community-curated platform (https://bigsdb.pasteur.fr/klebsiella), which also enables external users' genomic sequences identification. The proposed strain taxonomy combines two phylogenetically informative barcode systems that provide full stability (LIN codes) and nomenclatural continuity with previous nomenclature (MLSL). This species-specific dual barcoding strategy for the genomic taxonomy of microbial strains is broadly applicable and should contribute to unify global and cross-sector collaborative knowledge on the emergence and microevolution of bacterial pathogens.

Genomic Epidemiology and Strain Taxonomy of Corynebacterium diphtheriae.

Corynebacterium diphtheriae is highly transmissible and can cause large diphtheria outbreaks where vaccination coverage is insufficient. Sporadic cases or small clusters are observed in high-vaccination settings. The phylogeography and short timescale evolution of C. diphtheriae are not well understood, in part due to a lack of harmonized analytical approaches of genomic surveillance and strain tracking. We combined 1,305 genes with highly reproducible allele calls into a core genome multilocus sequence typing (cgMLST) scheme. We analyzed cgMLST gene diversity among 602 isolates from sporadic clinical cases, small clusters, or large outbreaks. We defined sublineages based on the phylogenetic structure within C. diphtheriae and strains based on the highest number of cgMLST mismatches within documented outbreaks. We performed time-scaled phylogenetic analyses of major sublineages. The cgMLST scheme showed high allele call rate in C. diphtheriae and the closely related species C. belfantii and C. rouxii. We demonstrate its utility to delineate epidemiological case clusters and outbreaks using a 25 mismatches threshold and reveal a number of cryptic transmission chains, most of which are geographically restricted to one or a few adjacent countries. Subcultures of the vaccine strain PW8 differed by up to 20 cgMLST mismatches. Phylogenetic analyses revealed a short-timescale evolutionary gain or loss of the diphtheria toxin and biovar-associated genes. We devised a genomic taxonomy of strains and deeper sublineages (defined using a 500-cgMLST-mismatch threshold), currently comprising 151 sublineages, only a few of which are geographically widespread based on current sampling. The cgMLST genotyping tool and nomenclature was made publicly accessible (https://bigsdb.pasteur.fr/diphtheria). Standardized genome-scale strain genotyping will help tracing transmission and geographic spread of C. diphtheriae. The unified genomic taxonomy of C. diphtheriae strains provides a common language for studies of ecology, evolution, and virulence heterogeneity among C. diphtheriae sublineages.

Phylogeny of the Varidnaviria Morphogenesis Module: Congruence and Incongruence With the Tree of Life and Viral Taxonomy.

Double-stranded DNA viruses of the realm Varidnaviria (formerly PRD1-adenovirus lineage) are characterized by homologous major capsid proteins (MCPs) containing one (kingdom: Helvetiavirae) or two ß-barrel domains (kingdom: Bamfordvirae) known as the jelly roll folds. Most of them also share homologous packaging ATPases (pATPases). Remarkably, Varidnaviria infect hosts from the three domains of life, suggesting that these viruses could be very ancient and share a common ancestor. Here, we analyzed the evolutionary history of Varidnaviria based on single and concatenated phylogenies of their MCPs and pATPases. We excluded Adenoviridae from our analysis as their MCPs and pATPases are too divergent. Sphaerolipoviridae, the only family in the kingdom Helvetiavirae, exhibit a complex history: their MCPs are very divergent from those of other Varidnaviria, as expected, but their pATPases groups them with Bamfordvirae. In single and concatenated trees, Bamfordvirae infecting archaea were grouped with those infecting bacteria, in contradiction with the cellular tree of life, whereas those infecting eukaryotes were organized into three monophyletic groups: the Nucleocytoviricota phylum, formerly known as the Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs), Lavidaviridae (virophages) and Polintoviruses. Although our analysis mostly supports the recent classification proposed by the International Committee on Taxonomy of Viruses (ICTV), it also raises questions, such as the validity of the Adenoviridae and Helvetiavirae ranking. Based on our phylogeny, we discuss current hypotheses on the origin and evolution of Varidnaviria and suggest new ones to reconcile the viral and cellular trees.

Phylogenetic analysis of Harmonin homology domains.

BACKGROUND: Harmonin Homogy Domains (HHD) are recently identified orphan domains of about 70 residues folded in a compact five alpha-helix bundle that proved to be versatile in terms of function, allowing for direct binding to a partner as well as regulating the affinity and specificity of adjacent domains for their own targets. Adding their small size and rather simple fold, HHDs appear as convenient modules to regulate protein-protein interactions in various biological contexts. Surprisingly, only nine HHDs have been detected in six proteins, mainly expressed in sensory neurons. RESULTS: Here, we built a profile Hidden Markov Model to screen the entire UniProtKB for new HHD-containing proteins. Every hit was manually annotated, using a clustering approach, confirming that only a few proteins contain HHDs. We report the phylogenetic coverage of each protein and build a phylogenetic tree to trace the evolution of HHDs. We suggest that a HHD ancestor is shared with Paired Amphipathic Helices (PAH) domains, a four-helix bundle partially sharing fold and functional properties. We characterized amino-acid sequences of the various HHDs using pairwise BLASTP scoring coupled with community clustering and manually assessed sequence features among each individual family. These sequence features were analyzed using reported structures as well as homology models to highlight structural motifs underlying HHDs fold. We show that functional divergence is carried out by subtle differences in sequences that automatized approaches failed to detect. CONCLUSIONS: We provide the first HHD databases, including sequences and conservation, phylogenic trees and a list of HHD variants found in the auditory system, which are available for the community. This case study highlights surprising phylogenetic properties found in orphan domains and will assist further studies of HHDs. We unveil the implication of HHDs in their various binding interfaces using conservation across families and a new protein-protein surface predictor. Finally, we discussed the functional consequences of three identified pathogenic HHD variants involved in Hoyeraal-Hreidarsson syndrome and of three newly reported pathogenic variants identified in patients suffering from Usher Syndrome.

Ongoing diphtheria outbreak in Yemen: a cross-sectional and genomic epidemiology study.

BACKGROUND: An outbreak of diphtheria, declared in Yemen in October, 2017, is ongoing. We did a cross-sectional study to investigate the epidemiological, clinical, and microbiological features of the outbreak. METHODS: Probable cases of diphtheria that were defined clinically and recorded through a weekly electronic diseases early warning system (from 2017, week 22, to 2020, week 17) were used to identify trends of the outbreak (we divided the epidemic into three time periods: May 29, 2017, to June 10, 2018; June 11, 2018, to June 3, 2019; and June 4, 2019, to April 26, 2020). We used the line list of diphtheria reports for governorate-level descriptions. Vaccination coverage was estimated using the 2017 and 2018 annual reports by the national Expanded Programme on Immunization. To confirm cases biologically, Corynebacterium diphtheriae was isolated and identified from throat swabs using standard microbiological culture and identification procedures. We assessed differences in the temporal and geographical distributions of cases, including between different age groups. For in-depth microbiological analysis, tox gene and species-specific rpoB real-time PCR, Illumina genomic sequencing, antimicrobial susceptibility analysis (disk diffusion, E-test), and the Elek diphtheria toxin production test were done on confirmed cases. We used genomic data for phylogenetic analyses and to estimate the nucleotide substitution rate. FINDINGS: The Yemen diphtheria outbreak affected almost all governorates (provinces), with 5701 probable cases and 330 deaths recorded up to April 26, 2020. We collected clinical data for 888 probable cases with throat swab samples referred for biological confirmation, and genomic data for 42 positive cases, corresponding to 43 isolates (two isolates from one culture were included due to distinct colony morphologies). The median age of patients was 12 years (range 0·2-80). The proportion of cases in children aged 0-4 years was reduced during the second time period, after a vaccination campaign, compared with the first period (19% [95% CI 18-21] in the first period vs 14% [12-15] in the second period, p<0·0001). Among 43 tested isolates, 39 (91%) produced the diphtheria toxin and two had low level (0·25 mg/L) antimicrobial resistance to penicillin. We identified six C diphtheriae phylogenetic sublineages, four of which are genetically related to isolates from Saudi Arabia, Eritrea, and Somalia. Inter-sublineage genomic variations in genes associated with antimicrobial resistance, iron acquisition, and adhesion were observed. The predominant sublineage (30 [70%] of 43 isolates) was resistant to trimethoprim and was associated with unique genomic features, more frequent neck swelling (p=0·0029) and a younger age of patients (p=0·060) compared with the other sublineages. Its evolutionary rate was estimated at 1·67 × 10-6 substitutions per site per year, placing its most recent common ancestor in 2015, and indicating silent circulation of C diphtheriae in Yemen before the outbreak was declared. INTERPRETATION: In the Yemen outbreak, C diphtheriae shows high phylogenetic, genomic, and phenotypic variation. Laboratory capacity and real-time microbiological monitoring of diphtheria outbreaks need to be scaled up to inform case management and transmission control of diphtheria. Catch-up vaccination might have provided some protection to the targeted population (children aged 0-4 years). FUNDING: National Centre of the Public Health Laboratories (Yemen), Institut Pasteur, and the French Government Investissement d'Avenir Programme. TRANSLATION: For the Arabic translation of the abstract see Supplementary Materials section.

High-Resolution Typing of Staphylococcus epidermidis Based on Core Genome Multilocus Sequence Typing To Investigate the Hospital Spread of Multidrug-Resistant Clones.

Staphylococcus epidermidis is a pathogen emerging worldwide as a leading cause of health care-associated infections. A standardized high-resolution typing method to document transmission and dissemination of multidrug-resistant S. epidermidis strains is needed. Our aim was to provide a core genome multilocus sequence typing (cgMLST) scheme for S. epidermidis to improve the international surveillance of S. epidermidis We defined a cgMLST scheme based on 699 core genes and used it to investigate the population structure of the species and the genetic relatedness of isolates recovered from infants hospitalized in several wards of a French hospital. Our results show the long-lasting endemic persistence of S. epidermidis clones within and across wards of hospitals and demonstrate the ability of our cgMLST approach to identify and track these clones. We made the scheme publicly available through the Institut Pasteur BIGSdb server (http://bigsdb.pasteur.fr/epidermidis/). This tool should enable international harmonization of the epidemiological surveillance of multidrug-resistant S. epidermidis clones. By comparing gene distribution among infection and commensal isolates, we also confirmed the association of the mecA locus with infection isolates and of the fdh gene with commensal isolates. (This study has been registered at ClinicalTrials.gov under registration no. NCT03374371.).

Diversity and evolution of B-family DNA polymerases.

B-family DNA polymerases (PolBs) represent the most common replicases. PolB enzymes that require RNA (or DNA) primed templates for DNA synthesis are found in all domains of life and many DNA viruses. Despite extensive research on PolBs, their origins and evolution remain enigmatic. Massive accumulation of new genomic and metagenomic data from diverse habitats as well as availability of new structural information prompted us to conduct a comprehensive analysis of the PolB sequences, structures, domain organizations, taxonomic distribution and co-occurrence in genomes. Based on phylogenetic analysis, we identified a new, widespread group of bacterial PolBs that are more closely related to the catalytically active N-terminal half of the eukaryotic PolEpsilon (PolEpsilonN) than to Escherichia coli Pol II. In Archaea, we characterized six new groups of PolBs. Two of them show close relationships with eukaryotic PolBs, the first one with PolEpsilonN, and the second one with PolAlpha, PolDelta and PolZeta. In addition, structure comparisons suggested common origin of the catalytically inactive C-terminal half of PolEpsilon (PolEpsilonC) and PolAlpha. Finally, in certain archaeal PolBs we discovered C-terminal Zn-binding domains closely related to those of PolAlpha and PolEpsilonC. Collectively, the obtained results allowed us to propose a scenario for the evolution of eukaryotic PolBs.

Correction: Genus-wide Leptospira core genome multilocus sequence typing for strain taxonomy and global surveillance.

[This corrects the article DOI: 10.1371/journal.pntd.0007374.].

Novel genome sequences of cell-fusing agent virus allow comparison of virus phylogeny with the genetic structure of Aedes aegypti populations.

Flaviviruses encompass not only medically relevant arthropod-borne viruses (arboviruses) but also insect-specific flaviviruses (ISFs) that are presumably maintained primarily through vertical transmission in the insect host. Interestingly, ISFs are commonly found infecting important arbovirus vectors such as the mosquito Aedes aegypti. Cell-fusing agent virus (CFAV) was the first described ISF of mosquitoes more than four decades ago. Despite evidence for widespread CFAV infections in A.aegypti populations and for CFAV potential to interfere with arbovirus transmission, little is known about CFAV evolutionary history. Here, we generated six novel CFAV genome sequences by sequencing three new virus isolates and subjecting three mosquito samples to untargeted viral metagenomics. We used these new genome sequences together with published ones to perform a global phylogenetic analysis of CFAV genetic diversity. Although there was some degree of geographical clustering among CFAV sequences, there were also notable discrepancies between geography and phylogeny. In particular, CFAV sequences from Cambodia and Thailand diverged significantly, despite confirmation that A.aegypti populations from both locations are genetically close. The apparent phylogenetic discrepancy between CFAV and its A.aegypti host in Southeast Asia indicates that other factors than host population structure shape CFAV genetic diversity.

Genus-wide Yersinia core-genome multilocus sequence typing for species identification and strain characterization.

The genus Yersinia comprises species that differ widely in their pathogenic potential and public-health significance. Yersinia pestis is responsible for plague, while Yersinia enterocolitica is a prominent enteropathogen. Strains within some species, including Y. enterocolitica, also vary in their pathogenic properties. Phenotypic identification of Yersinia species is time-consuming, labour-intensive and may lead to incorrect identifications. Here, we developed a method to automatically identify and subtype all Yersinia isolates from their genomic sequence. A phylogenetic analysis of Yersinia isolates based on a core subset of 500 shared genes clearly demarcated all existing Yersinia species and uncovered novel, yet undefined Yersinia taxa. An automated taxonomic assignment procedure was developed using species-specific thresholds based on core-genome multilocus sequence typing (cgMLST). The performance of this method was assessed on 1843 isolates prospectively collected by the French National Surveillance System and analysed in parallel using phenotypic reference methods, leading to nearly complete (1814; 98.4 %) agreement at species and infra-specific (biotype and serotype) levels. For 29 isolates, incorrect phenotypic assignments resulted from atypical biochemical characteristics or lack of phenotypic resolution. To provide an identification tool, a database of cgMLST profiles and reference taxonomic information has been made publicly accessible (https://bigsdb.pasteur.fr/yersinia). Genomic sequencing-based identification and subtyping of any Yersinia is a powerful and reliable novel approach to define the pathogenic potential of isolates of this medically important genus.

Diversification of giant and large eukaryotic dsDNA viruses predated the origin of modern eukaryotes.

Giant and large eukaryotic double-stranded DNA viruses from the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) assemblage represent a remarkably diverse and potentially ancient component of the eukaryotic virome. However, their origin(s), evolution, and potential roles in the emergence of modern eukaryotes remain subjects of intense debate. Here we present robust phylogenetic trees of NCLDVs, based on the 8 most conserved proteins responsible for virion morphogenesis and informational processes. Our results uncover the evolutionary relationships between different NCLDV families and support the existence of 2 superclades of NCLDVs, each encompassing several families. We present evidence strongly suggesting that the NCLDV core genes, which are involved in both informational processes and virion formation, were acquired vertically from a common ancestor. Among them, the largest subunits of the DNA-dependent RNA polymerase were transferred between 2 clades of NCLDVs and proto-eukaryotes, giving rise to 2 of the 3 eukaryotic DNA-dependent RNA polymerases. Our results strongly suggest that these transfers and the diversification of NCLDVs predated the emergence of modern eukaryotes, emphasizing the major role of viruses in the evolution of cellular domains.

Simulation data for the estimation of numerical constants for approximating pairwise evolutionary distances between amino acid sequences.

Estimating the number of substitution events per site that have occurred during the evolution of a pair of amino acid sequences is a common task in phylogenetics and comparative genomics that often requires quite slow maximum-likelihood procedures when taking into account explicit evolutionary models. Data presented in this article are large sets of numbers of substitution events and associated numbers of observed differences between pairs of aligned amino acid sequences that have been generated through a simulation procedure of sequence evolution under a broad range of evolutionary models. These data are available at https://zenodo.org/record/2653704 (doi:10.5281/zenodo.2653704). They are accompanied in this paper by figures showing the strong relationship between the corresponding evolutionary and uncorrected distances, as well as estimated numerical constants that determine non-linear functions that fit the simulated data. These numerical constants can be useful to quickly estimate pairwise evolutionary distances directly from uncorrected distances between aligned amino acid sequences.

Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission.

Transmission of Plasmodium falciparum involves a complex process that starts with the ingestion of gametocytes by female Anopheles mosquitoes during a blood meal. Activation of gametocytes in the mosquito midgut triggers "rounding up" followed by egress of both male and female gametes. Egress requires secretion of a perforin-like protein, PfPLP2, from intracellular vesicles to the periphery, which leads to destabilization of peripheral membranes. Male gametes also develop flagella, which assist in binding female gametes for fertilization. This process of gametogenesis, which is key to malaria transmission, involves extensive membrane remodeling as well as vesicular discharge. Phospholipase A2 enzymes (PLA2) are known to mediate membrane remodeling and vesicle secretion in diverse organisms. Here, we show that a P. falciparum patatin-like phospholipase (PfPATPL1) with PLA2 activity plays a key role in gametogenesis. Conditional deletion of the gene encoding PfPATPL1 does not affect P. falciparum blood stage growth or gametocyte development but reduces efficiency of rounding up, egress, and exflagellation of gametocytes following activation. Interestingly, deletion of the PfPATPL1 gene inhibits secretion of PfPLP2, reducing the efficiency of gamete egress. Deletion of PfPATPL1 also reduces the efficiency of oocyst formation in mosquitoes. These studies demonstrate that PfPATPL1 plays a role in gametogenesis, thereby identifying PLA2 phospholipases such as PfPATPL1 as potential targets for the development of drugs to block malaria transmission.

Genus-wide Leptospira core genome multilocus sequence typing for strain taxonomy and global surveillance.

Leptospira is a highly heterogeneous bacterial genus that can be divided into three evolutionary lineages and >300 serovars. The causative agents of leptospirosis are responsible of an emerging zoonotic disease worldwide. To advance our understanding of the biodiversity of Leptospira strains at the global level, we evaluated the performance of whole-genome sequencing (WGS) as a genus-wide strain classification and genotyping tool. Herein we propose a set of 545 highly conserved loci as a core genome MLST (cgMLST) genotyping scheme applicable to the entire Leptospira genus, including non-pathogenic species. Evaluation of cgMLST genotyping was undertaken with 509 genomes, including 327 newly sequenced genomes, from diverse species, sources and geographical locations. Phylogenetic analysis showed that cgMLST defines species, clades, subclades, clonal groups and cgMLST sequence types (cgST), with high precision and robustness to missing data. Novel Leptospira species, including a novel subclade named S2 (saprophytes 2), were identified. We defined clonal groups (CG) optimally using a single-linkage clustering threshold of 40 allelic mismatches. While some CGs such as L. interrogans CG6 (serogroup Icterohaemorrhagiae) are globally distributed, others are geographically restricted. cgMLST was congruent with classical MLST schemes, but had greatly improved resolution and broader applicability. Single nucleotide polymorphisms within single cgST groups was limited to <30 SNPs, underlining a potential role for cgMLST in epidemiological surveillance. Finally, cgMLST allowed identification of serogroups and closely related serovars. In conclusion, the proposed cgMLST strategy allows high-resolution genotyping of Leptospira isolates across the phylogenetic breadth of the genus. The unified genomic taxonomy of Leptospira strains, available publicly at http://bigsdb.pasteur.fr/leptospira, will facilitate global harmonization of Leptospira genotyping, strain emergence follow-up and novel collaborative studies of the epidemiology and evolution of this emerging pathogen.

Genomic Sequencing of Bordetella pertussis for Epidemiology and Global Surveillance of Whooping Cough.

Bordetella pertussis causes whooping cough, a highly contagious respiratory disease that is reemerging in many world regions. The spread of antigen-deficient strains may threaten acellular vaccine efficacy. Dynamics of strain transmission are poorly defined because of shortcomings in current strain genotyping methods. Our objective was to develop a whole-genome genotyping strategy with sufficient resolution for local epidemiologic questions and sufficient reproducibility to enable international comparisons of clinical isolates. We defined a core genome multilocus sequence typing scheme comprising 2,038 loci and demonstrated its congruence with whole-genome single-nucleotide polymorphism variation. Most cases of intrafamilial groups of isolates or of multiple isolates recovered from the same patient were distinguished from temporally and geographically cocirculating isolates. However, epidemiologically unrelated isolates were sometimes nearly undistinguishable. We set up a publicly accessible core genome multilocus sequence typing database to enable global comparisons of B. pertussis isolates, opening the way for internationally coordinated surveillance.