Analysis of the Legionella longbeachae Genome and Transcriptome Uncovers Unique Strategies to Cause Legionnaires' Disease

Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species

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

Genome Sequence of the Saprophyte Leptospira biflexa Provides Insights into the Evolution of Leptospira and the Pathogenesis of Leptospirosis

Leptospira biflexa is a free-living saprophytic spirochete present in aquatic environments. We determined the genome sequence of L. biflexa, making it the first saprophytic Leptospira to be sequenced. The L. biflexa genome has 3,590 protein-coding genes distributed across three circular replicons: the major 3,604 chromosome, a smaller 278-kb replicon that also carries essential genes, and a third 74-kb replicon. Comparative sequence analysis provides evidence that L. biflexa is an excellent model for the study of Leptospira evolution; we conclude that 2052 genes (61%) represent a progenitor genome that existed before divergence of pathogenic and saprophytic Leptospira species. Comparisons of the L. biflexa genome with two pathogenic Leptospira species reveal several major findings. Nearly one-third of the L. biflexa genes are absent in pathogenic Leptospira. We suggest that once incorporated into the L. biflexa genome, laterally transferred DNA undergoes minimal rearrangement due to physical restrictions imposed by high gene density and limited presence of transposable elements. In contrast, the genomes of pathogenic Leptospira species undergo frequent rearrangements, often involving recombination between insertion sequences. Identification of genes common to the two pathogenic species, L. borgpetersenii and L. interrogans, but absent in L. biflexa, is consistent with a role for these genes in pathogenesis. Differences in environmental sensing capacities of L. biflexa, L. borgpetersenii, and L. interrogans suggest a model which postulates that loss of signal transduction functions in L. borgpetersenii has impaired its survival outside a mammalian host, whereas L. interrogans has retained environmental sensory functions that facilitate disease transmission through water

Comparative genomic and phylogeographic analysis of Mycobacterium leprae

Reductive evolution and massive pseudogene formation have shaped the 3.31-Mb genome of Mycobacterium leprae, an unculturable obligate pathogen that causes leprosy in humans. The complete genome sequence of M. leprae strain Br4923 from Brazil was obtained by conventional methods (6 x coverage), and Illumina resequencing technology was used to obtain the sequences of strains Thai53 (38 x coverage) and NHDP63 (46 x coverage) from Thailand and the United States, respectively. Whole-genome comparisons with the previously sequenced TN strain from India revealed that the four strains share 99.995% sequence identity and differ only in 215 polymorphic sites, mainly SNPs, and by 5 pseudogenes. Sixteen interrelated SNP subtypes were defined by genotyping both extant and extinct strains of M. leprae from around the world. The 16 SNP subtypes showed a strong geographical association that reflects the migration patterns of early humans and trade routes, with the Silk Road linking Europe to China having contributed to the spread of leprosy

Cross-reactivities between human IgMs and the four serotypes of dengue virus as probed with artificial homodimers of domain-III from the envelope proteins

International audienceBackgroundDengue fever is the most important vector-borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Infection by one serotype elicits long-lasting immunity to that serotype but not the other three. Subsequent infection by a different serotype is a risk factor for severe dengue. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by neutralizing antibodies. We determined the serotype specificity and cross-reactivity of human IgMs directed against ED3 by using a well-characterized collection of 90 DENV-infected and 89 DENV-uninfected human serums.MethodsThe recognitions between the four serotypes of ED3 and the serums were assayed with an IgM antibody-capture ELISA (MAC-ELISA) and artificial homodimeric antigens. The results were analyzed with Receiving Operator Characteristic (ROC) curves.ResultsThe DENV-infected serums contained IgMs that reacted with one or several ED3 serotypes. The discrimination by ED3 between serums infected by the homotypic DENV and uninfected serums varied with the serotype in the decreasing order DENV1 > DENV2 > DENV3 > DENV4. The ED3 domain of DENV1 gave the highest discrimination between DENV-infected and DENV-uninfected serums, whatever the infecting serotype, and thus behaved like a universal ED3 domain for the detection of IgMs against DENV. Some ED3 serotypes discriminated between IgMs directed against the homotypic and heterotypic DENVs. The patterns of cross-reactivities and discriminations varied with the serotype.ConclusionsThe results should help better understand the IgM immune response and protection against DENV since ED3 is widely used as an antigen in diagnostic assays and an immunogen in vaccine candidates

The folded and disordered domains of human ribosomal protein SA have both idiosyncratic and shared functions as membrane receptors

International audienceThe human RPSA [ribosomal protein SA; also known as LamR1(laminin receptor 1)] belongs to the ribosome but is also a membrane receptor for laminin, growth factors, prion, pathogens and the anticarcinogen EGCG (epigallocatechingallate). It contributes to the crossing of the blood-brain barrier by neurotropic viruses and bacteria, and is a biomarker of metastasis. RPSA includes an N-terminal domain, which is folded and homologous to the prokaryotic RPS2, and a C-terminal extension, which is intrinsically disordered and conserved in vertebrates. We used recombinant derivatives of RPSA and its N-and C-domains to quantify its interactions with ligands by in-vitro immunochemical and spectrofluorimetric methods. Both N-and C-domains bound laminin with K D (dissociation constants) of 300 nM. Heparin bound only to the N-domain and competed for binding to laminin with the negatively charged C-domain, which therefore mimicked heparin. EGCG bound only to the N-domain with a K D of 100 nM. Domain 3 of the envelope protein from yellow fever virus and serotypes-1 and-2 of dengue virus bound preferentially to the C-domain whereas that from West Nile virus bound only to the N-domain. Our quantitative in-vitro approach should help clarify the mechanisms of action of RPSA, and ultimately fight against cancer and infectious agents

Thermodynamic stability of domain III from the envelope protein of flaviviruses and its improvement by molecular design

International audienceThe Flavivirus genus includes widespread and severe human pathogens like the four serotypes of dengue virus (DENV1 to DENV4), yellow fever virus, Japanese encephalitis virus and West Nile virus. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by virus neutralizing antibodies. Its structural, antigenic and immunogenic properties have been thoroughly studied contrary to its physico-chemical properties. Here, the ED3 domains of the above pathogenic flaviviruses were produced in the periplasm of Escherichia coli. Their thermodynamic stabilities were measured and compared in experiments of unfolding equilibriums, induced with chemicals or heat and monitored through protein fluorescence. A designed ED3 domain, with the consensus sequence of DENV strains from all serotypes, was highly stable. The low stability of the ED3 domain from DENV3 was increased by three changes of residues in the protein core without affecting its reactivity towards DENV-infected human serums. Additional changes showed that the stability of ED3 varied with the DENV3 genotype. The T(m) of ED3 was higher than 69°C for all the tested viruses and reached 86°C for the consensus ED3. The latter, deprived of its disulfide bond by mutations, was predominantly unfolded at 20°C. These results will help better understand and design the properties of ED3 for its use as diagnostic, vaccine or therapeutic tools

Corynebacterium diphtheriae and Corynebacterium ulcerans : development of EUCAST methods and generation of data on which to determine breakpoints

International audienceAbstract Background Evidence-based clinical susceptibility breakpoints have been lacking for antimicrobial agents used for diphtheria. Objectives We aimed to evaluate broth microdilution and disc diffusion methods and create a dataset of MIC values and inhibition zone diameters (ZDs) from which breakpoints could be determined. Methods We included 400 recent clinical isolates equally distributed by species (Corynebacterium diphtheriae and Corynebacterium ulcerans) and by national surveillance programmes (France and Germany). Non-duplicate toxigenic and non-toxigenic isolates were chosen to enable the inclusion of a diversity of susceptibility levels for the 13 agents tested. Broth microdilution and disc diffusion, using EUCAST methodology for fastidious organisms, were used. Results The distributions of MIC and ZD values were largely in agreement among methods and countries. Breakpoints to allow categorization of WT isolates as susceptible, i.e. susceptible (S) or isusceptible, increased exposure (I) were determined for 12 agents. The data supported a breakpoint for benzylpenicillin and amoxicillin of resistant (R) &gt; 1 mg/L since WT isolates were inhibited by 1 mg/L or less. WT isolates were categorized as I (S ≤ 0.001 mg/L) for benzylpenicillin, emphasizing the need for increased exposure, and S (S ≤ 1 mg/L) for amoxicillin. Erythromycin breakpoints were set at S ≤ 0.06 mg/L and R &gt; 0.06 mg/L. The corresponding ZD breakpoints were determined for all agents except amoxicillin, for which categorization was based on benzylpenicillin results. Conclusions This work provided a large set of antimicrobial susceptibility data for C. diphtheriae and C. ulcerans, using a harmonized methodology. The dataset allowed EUCAST and experts in the diphtheria field to develop evidence-based breakpoints in January 2023