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Nanoarchaeum equitansandIgnicoccus hospitalisrepresent a unique, intimate association of two archaea. Both form a stable coculture which is mandatory forN.equitansbut not for the hostI.hospitalis. Here, we investigated interactions and mutual influence between these microorganisms. Fermentation studies revealed that during exponential growth only about 25% ofI.hospitaliscells are occupied byN.equitanscells (one to three cells). The latter strongly proliferate in the stationary phase ofI.hospitalis, until 80 to 90% of theI.hospitaliscells carry around 10N.equitanscells. Furthermore, the expulsion of H2S, the major metabolic end product ofI.hospitalis, by strong gas stripping yields huge amounts of freeN.equitanscells.N.equitanshad no influence on the doubling times, final cell concentrations, and growth temperature, pH, or salt concentration ranges or optima ofI.hospitalis. However, isolation studies using optical tweezers revealed that infection withN.equitansinhibited the proliferation of individualI.hospitaliscells. This inhibition might be caused by deprivation of the host of cell components like amino acids, as demonstrated by13C-labeling studies. The strong dependence ofN.equitansonI.hospitaliswas affirmed by live-dead staining and electron microscopic analyses, which indicated a tight physiological and structural connection between the two microorganisms. No alternative hosts, including otherIgnicoccusspecies, were accepted byN.equitans. In summary, the data show a highly specialized association ofN.equitansandI.hospitaliswhich so far cannot be assigned to a classical symbiosis, commensalism, or parasitism.

Leptospirosis, caused by a pathogenic Leptospira bacteria, is the most prevalent zoonosis worldwide and in this context has been extensively investigated through a One Health framework. Diagnosis of human leptospirosis includes molecular and serological tools, with serological Microscopic Agglutination Test (MAT) still being considered as a gold standard. Mammals considered as biological reservoirs include species or populations that are able to maintain chronic infection and shed the bacteria via their urine in the environment. Leptospira bacteria are often investigated using the same diagnosis tool, serological MAT. However, MAT testing of putative animal reservoirs can lead to mis-interpretations as it can signal previous infection and not necessarily bring in robust information regarding the capacity of such sero-positive animals to maintain chronic infection. We use previously published data and present new results on introduced and endemic small mammals to show that MAT should not be used for the identification of reservoirs. By contrast, serological data are informative on the level of exposure of animals occupying a specific environment. Finally, we present a sequential methodology to investigate human leptospirosis in a One Health framework that associates molecular detection in humans and animals, together with MAT of human samples using Leptospira isolates obtained from reservoir animals occurring in the same environment.

International audience; Parasites of the genus Leishmania infect the mammalian hosts, including mice and humans and cause cutaneous or visceral leishm aniasis depending upon the parasite species transmitted by the vector sandfly. Leishmania amazonensis is one of the Leishmania species responsible for th e cutaneous form of the disease. We have inoculated with these parasites the ear dermis of mice. RNA preparations were performed from fragmented tissues using a buffer containing guanidin isothiocynate (RLT buffer, RNeasy Mini Kit, Qiagen, SAS, France) and β mercaptoethanol. Both reagents facilitate the isolation of intact RNA from tissues and the use of the RNeasy Kits present with several advantages that facilitate the isolation of pure non degraded total RNA: i) T his method allows to avoid the presence o f phenol in the RNA extraction buffer, commonly used in alternative protocols; ii) Moreover Diethylpyrocarbonate (DEPC) treatment of glassware, to avoid RNAses contamination of the samples, is not required with this protocol; iii) F inally, it is a fast pro cedure and the isolated total RNA may be concentrated in a small volume thus facilitating its use for downstream experimental procedures.

International audience; Yellow Fever (YF) remains a major public health issue in Sub-Saharan Africa and South America, despite the availability of an effective vaccine. In Africa, most YF outbreaks are reported in West Africa. However, urban outbreaks occurred in 2016 in both Angola and the Democratic Republic of Congo (DRC), and imported cases were reported in Chinese workers coming back from Africa. In Central Africa, Cameroon and the Republic of Congo host a high proportion of non-vaccinated populations increasing the risk of urban outbreaks. The main vector is Aedes aegypti and possibly, Aedes albopictus, both being anthropophilic and domestic mosquitoes. Here, we provide evidence that both Ae. aegypti and Ae. albopictus in Cameroon and the Republic of Congo are able to transmit Yellow fever virus (YFV) with higher rates of infection, dissemination, and transmission for Ae. aegypti. We conclude that the potential of both Aedes species to transmit YFV could increase the risk of urban YF transmission and urge public health authorities to intensify their efforts to control domestic vectors, and extend vaccine coverage to prevent major YFV outbreak.

International audience; Macrophages are key cells of innate immune response and serve as the first line of defense against bacteria. Transcription profiling of bacteria-infected macrophages could provide important insights on the pathogenicity and host defense mechanisms during infection. We have examined transcription profiles of bovine monocyte-derived macrophages (bMDMs) isolated from the blood of 12 animals and infected in vitro with two strains of Streptococcus agalactiae. Illumina sequencing of RNA from 36 bMDMs cultures exposed in vitro to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h and unchallenged controls was performed. Analyses of over 1,656 million high-quality paired-end sequence reads revealed 5,936 and 6,443 differentially expressed genes (p < 0.05) in bMDMs infected with ST103 and ST12, respectively, versus unchallenged controls. Moreover, 588 genes differentially expressed between bMDMs infected with ST103 versus ST12 were identified. Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST103 revealed significant enrichment for granulocyte adhesion and diapedesis, while significant enrichment for the phagosome formation pathway was found among down-regulated genes. Moreover, Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST12 showed significant enrichment for type 1/type 2 T helper cell activation, while the complement activation pathway was overrepresented in the down-regulated genes. Our study identified pathogen-induced regulation of key genes and pathways involved in the immune response of macrophages against infection but also likely involved in bacterial evasion of the host immune system. These results may contribute to better understanding of the mechanisms underlying subclinical infection such as bovine streptococcal mastitis.

International audience; Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by H2O2 leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans, a rare example of PerR lacking a structural metal-binding site. In vivo studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a perR mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion. In vitro, leptospiral PerR could bind to the perR promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA-binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress.

International audience; Immune responses are essential for the protection of the host against external dangers or infections and are normally efficient in the clearance of invading microbes. However, some intracellular pathogens have developed strategies to replicate and survive within host cells resulting in latent infection associated with strong inflammation. This excessive response can cause cell and tissue damage and lead to the release of the intracellular content, in particular the nucleotide pool, into the extracellular space. Over the last decade, new studies have implicated metabolites from the purinergic pathway in shaping the host immune response against intracellular pathogens and proved their importance in the outcome of the infection. This review aims to summarize how the immune system employs the purinergic system either to fight the pathogen, or to control collateral tissue damage. This will be achieved by focusing on the macrophage response against two intracellular pathogens, the human etiologic agent of tuberculosis, Mycobacterium tuberculosis and the protozoan parasite, Toxoplasma gondii.

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen andthat bio-selection and changes in BabA bindingproperties through mutation and recombination with babA-related genes are selected by differencesamong individuals and by changes in gastric acidity over time. These processes generate diverse H.pylori subpopulations, in which BabA's adaptive evolution contributes to H.pylori persistence and overt gastric disease.

International audience; OBJECTIVES:Resistance to fluoroquinolones (FQs) in Mycobacterium tuberculosis (Mtb) is mainly due to mutations in DNA gyrase (GyrA2B2), with the most common substitutions located at positions 90 and 94 in GyrA. Two clinical MDR Mtb (MDR-TB) strains harbouring an A90E or D94N substitution in GyrA were found to be surprisingly susceptible to FQs (ofloxacin MIC ≤2 mg/L). We studied the impact of the additional GyrA substitutions found in these strains (T80A and T80A + A90G, respectively) on FQ susceptibility.METHODS:Mutants of interest were generated by site-specific mutagenesis of GyrA alleles. WT and mutant TB DNA gyrase subunits were overexpressed in Escherichia coli and purified, and the in vitro susceptibility to FQs of their DNA supercoiling reaction was studied.RESULTS:IC50s of mutant gyrase complexes bearing GyrA D94N and A90E were 3- to 36-fold higher than WT IC50s, whereas IC50s of gyrase bearing T80A + A90G + D94N and T80A + A90E were close to the WT IC50s.CONCLUSIONS:We demonstrated that substitutions T80A and A90G restore FQ susceptibility when associated with a substitution implicated in high-level FQ resistance. Line probe assay misclassification of MDR-TB strains as pre-XDR or XDR can be corrected by sequence analysis of gyrA.

Background Rift Valley fever (RVF) is a vector-borne disease affecting ruminants and humans. Madagascar was heavily affected by RVF in 2008–2009, with evidence of a large and heterogeneous spread of the disease. The identification of at-risk environments is essential to optimize the available resources by targeting RVF surveillance in Madagascar. Herein, the objectives of our study were: (i) to identify the environmental factors and areas favorable to RVF transmission to both cattle and human and (ii) to identify human behaviors favoring human infections in Malagasy contexts. Methodology/Principal Findings First, we characterized the environments of Malagasy communes using a Multiple Factor Analysis (MFA). Then, we analyzed cattle and human serological data collected at national level using Generalized Linear Mixed Models, with the individual serological status (cattle or human) as the response, and MFA factors, as well as other potential risk factors (cattle density, human behavior) as explanatory variables. Cattle and human seroprevalence rates were positively associated to humid environments (p, Author Summary Rift Valley fever virus (RVFV) is a pathogen that causes a vector-borne tropical disease. The disease affects ruminants and humans and severely impacts the health and economy of affected countries. Madagascar was heavily affected by Rift Valley fever (RVF) in 2008–2009, with evidence of a large and heterogeneous spread of the disease. Our study aims at identifying environmental and human-related risk factors for RVFV transmission. First, we characterized Malagasy environments according to their putative influence on RVFV mosquito density and population dynamics. Then we statistically analyzed cattle and human serological data collected at a national level with the individual serological status as response, and Malagasy environments previously characterized by climatic and landscape variables as well as other potential risk factors as explanatory variables. Our results identified humid environments of the western, north-western and eastern parts of the island as risky areas. The identification of at-risk environments is essential to focus veterinary surveillance and control of RVFV.

NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden., Instituto de Biotecnologia y Biologia Molecular, Facultad de Ciencias Exactas, Facultad de Ciencias Médicas

Antimicrobials: mismatch excels when ampicillin runs low. [Nat Rev Microbiol. 2013]; International audience; Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies.

To attempt cost-effective production of US417 phytase in Bacillus subtilis, we developed an efficient system for its large-scale production in the generally recognized as safe microorganism B. subtilis 168. Hence, the phy US417 corresponding gene was cloned in the pMSP3535 vector, and for the first time for a plasmid carrying the pAMβ1 replication origin, multimeric forms of the resulting plasmid were used to transform naturally competent B. subtilis 168 cells. Subsequently, a sequential optimization strategy based on Plackett-Burman and Box-Behnken experimental designs was applied to enhance phytase production by the recombinant Bacillus. The maximum phytase activity of 47 U ml-1 was reached in the presence of 12.5 g l-1 of yeast extract and 15 g l-1 of ammonium sulphate with shaking at 300 rpm. This is 73 fold higher than the activity produced by the native US417 strain before optimization. Characterization of the produced recombinant phytase has revealed that the enzyme exhibited improved thermostability compared to the wild type PHY US417 phytase strengthening its potential for application as feed supplement. Together, our findings strongly suggest that the strategy herein developed combining heterologous expression using a cloning vector carrying the pAMβ1 replication origin and experimental designs optimization can be generalized for recombinant proteins production in Bacillus.

The use of morphological characters for the classification of cyanobacteria has often led to ambiguous strain assignment. In the past two decades, the availability of sequences, such as those of the 16S rRNA,nif,cpcandrpoC1genes, and the use of metagenomics, has steadily increased and has made the reconstruction of evolutionary relationships of some cyanobacterial groups possible in addition to improving strain assignment. Conserved indels (insertions/deletions) are present in all cyanobacterial RpoB (βsubunit of RNA polymerase) sequences presently available in public databases. These indels are located in the Rpb2_6 domain of RpoB, which is involved in DNA binding and DNA-directed RNA polymerase activity. They are variable in length (6–44 aa) and sequence, and form part of what appears to be a longer signature sequence (43–81 aa). Indeed, a number of these sequences turn out to be distinctive among several strains of a given genus and even among strains of a given species. These signature sequences can thus be used to identify cyanobacteria at a subgenus level and can be useful molecular markers to establish the taxonomic positions of cyanobacterial isolates in laboratory cultures, and/or to assess cyanobacterial biodiversity in space and time in natural ecosystems.

International audience; Enterotoxins produced by Staphylococcus aureus are responsible for staphylococcal food-poisoning outbreaks (SFPO). In France, SFPO are the second cause of food-borne diseases after Salmonella. However, very little is known about the strains involved. The objective of this study was to characterize the staphylococcal strains related to these SFPO through phenotypic and genotypic analyses. A total of 178 coagulase-positive staphylococcal isolates recovered from 31 SFPO (1981-2002) were screened through biotyping. Thirty-three strains representative of the different biotypes in each SFPO were further examined for SmaI macrorestriction-type, phage-type, resistance to various antimicrobial drugs, presence of staphylococcal enterotoxin (se) genes sea to sei, and production of enterotoxins SEA to SED. All these 33 strains were identified as S. aureus species: 27 were of human biotypes and six ovine or non-host-specific biotypes. Most (74.1%) strains reacted with group III phages. Eleven strains were resistant to at least two classes of antibiotics and among them, two were resistant to methicillin. Twenty-nine strains carried one or several of the eight se genes tested; the gene sea was most common (n=23), and often linked to sed (n=12) or seh (n=5). The novel se genes seg-i were in all cases associated with se genes sea to sed except for one strain which carried only seg and sei. Pulsed-Field Gel Electrophoresis (PFGE) of SmaI macrorestriction digests of the 33 strains discriminated 32 PFGE patterns grouped into nine biotype-specific clusters. All five strains carrying sea and seh were grouped together into the same sub-cluster. Three of the four se-gene-negative strains were in one PFGE cluster: all four should be tested for se genes not included in this study and, if negative, be further investigated for the presence of unidentified SEs.