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International audience; To thrive, vector-borne pathogens must survive in the vector’s gut. How these pathogens successfully exploit this environment in time and space has not been extensively characterized. Using Yersinia pestis (the plague bacillus) and its flea vector, we developed a bioluminescence-based approach and employed it to investigate the mechanisms of pathogenesis at an unprecedented level of detail. Remarkably, lipoylation of metabolic enzymes, via the biosynthesis and salvage of lipoate, increases the Y. pestis transmission rate by fleas. Interestingly, the salvage pathway’s lipoate/octanoate ligase LplA enhances the first step in lipoate biosynthesis during foregut colonization but not during midgut colonization. Lastly, Y. pestis primarily uses lipoate provided by digestive proteolysis (presumably as lipoyl peptides) rather than free lipoate in blood, which is quickly depleted by the vector. Thus, spatial and temporal factors dictate the bacterium’s lipoylation strategies during an infection, and replenishment of lipoate by digestive proteolysis in the vector might constitute an Achilles’ heel that is exploited by pathogens.

International audience; Yersinioses caused by Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are significant concerns in human and veterinary health. The link between virulence and the potent LcrV antigen has prompted the latter's selection as a major component of anti-Yersinia vaccines. Here, we report that (i) the group of Yersinia species encompassing Y. pestis and Y. pseudotuberculosis produces at least five different clades of LcrV and (ii) vaccination of mice with an LcrV-secreting Lactococcus lactis only protected against Yersinia strains producing the same LcrV clade as that of used for vaccination. By vaccinating with engineered LcrVs and challenging mice with strains producing either type of LcrV or a LcrV mutated for regions of interest, we highlight key polymorphic residues responsible for the absence of cross-protection. Our results show that an anti-LcrV-based vaccine should contain multiple LcrV clades if protection against the widest possible array of Yersinia strains is sought.

The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis. Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria., IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

International audience; Plague is a zoonosis transmitted by fleas and caused by the gram-negative bacterium Yersinia pestis. During infection, the plasmidic caf1M1A1 operon that encodes the Y. pestis F1 protein capsule is highly expressed, and anti-F1 antibodies are protective. Surprisingly, the capsule is not required for virulence after injection of cultured bacteria, even though it is an antiphagocytic factor and capsule-deficient Y. pestis strains are rarely isolated. We found that a caf-negative Y. pestis mutant was not impaired in either flea colonization or virulence in mice after intradermal inoculation of cultured bacteria. In contrast, absence of the caf operon decreased bubonic plague incidence after a flea bite. Successful development of plague in mice infected by flea bite with the caf-negative mutant required a higher number of infective bites per challenge. In addition, the mutant displayed a highly autoaggregative phenotype in infected liver and spleen. The results suggest that acquisition of the caf locus via horizontal transfer by an ancestral Y. pestis strain increased transmissibility and the potential for epidemic spread. In addition, our data support a model in which atypical caf-negative strains could emerge during climatic conditions that favor a high flea burden. Human infection with such strains would not be diagnosed by the standard clinical tests that detect F1 antibody or antigen, suggesting that more comprehensive surveillance for atypical Y. pestis strains in plague foci may be necessary. The results also highlight the importance of studying Y. pestis pathogenesis in the natural context of arthropod-borne transmission.

In adaptive immunity, Th17 lymphocytes produce the IL-17 and IL-22 cytokines that stimulate mucosal antimicrobial defenses and tissue repair. In this study, we observed that the TLR5 agonist flagellin induced swift and transient transcription of genes encoding IL-17 and IL-22 in lymphoid, gut, and lung tissues. This innate response also temporarily enhanced the expression of genes associated with the antimicrobial Th17 signature. The source of the Th17-related cytokines was identified as novel populations of CD3(neg)CD127(+) immune cells among which CD4-expressing cells resembling lymphoid tissue inducer cells. We also demonstrated that dendritic cells are essential for expression of Th17-related cytokines and so for stimulation of innate cells. These data define that TLR-induced activation of CD3(neg)CD127(+) cells and production of Th17-related cytokines may be crucial for the early defenses against pathogen invasion of host tissues.

A biological threat is an epidemic or its threat caused by a microbe or biological material of a magnitude that would overwhelm healthcare services due to the contagiousness or wide distribution of infections. A biological threat can be naturally occurring, such as the West African Ebola epidemic of 2014-2016, or the consequence of an intentional release of a microbe or toxin. The aim of this thesis was to develop and use molecular methods in order to reliably and rapidly identify potential biological threat agents. The focus was on the detection and typing of biological threat agents, whether they are naturally occurring or intentionally released. Different molecular methods were used: polymerase chain reaction (PCR) to detect and differentiate pathogenic from non-pathogenic bacterial strains, 16S ribosomal RNA (rRNA) gene sequencing to investigate polymicrobial samples, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) spacer comparison for bacterial strain typing. Cholera, a disease caused by Vibrio cholerae bacterium, is a major public health problem worldwide and a potential bioterrorism agent, according to the Centers for Disease Control and Prevention (CDC). In this thesis, an accurate PCR-based method was developed to detect V. cholerae strains: one assay for pathogenic strains and another for all V. cholerae strains. In addition, three different PCR platforms were compared. The PCR assays proved to be suitable for the reliable identification and differentiation of V. cholerae strains. The PCR platforms gave identical results, which indicate that the assays can be transferred between the platforms while maintaining sufficient sensitivity and specificity. Two 16S rRNA gene-based detection methods, using Sanger sequencing or pyrosequencing, were employed to study the presence of bacterial residues in carotid artery tissue samples and in livers of splenomegalic voles. The objectives were to observe the utility of the two methods and compare their performance. Both methods were found to be convenient approaches to detect and identify bacterial species present in different matrices and thus could be employed when investigating polymicrobial samples. In addition, the two methods gave similar results which emphasises the reliability of the methods and their results. The Yersinia genus includes three human pathogens; Y. pestis, the causal agent of plague and a potential biothreat agent, as well as Y. enterocolitica and Y. pseudotuberculosis, which commonly cause self-limiting enteritis. Due to the high level of DNA similarity between Y. pestis and Y. pseudotuberculosis, typing of Yersinia species and distinguishing them from each other has been challenging. Here, CRISPR spacers were used for typing Yersinia pseudotuberculosis complex strains. This method proved to be a promising tool, although the large diversity of different spacer sequences hindered the clustering of different strains. In addition, CRISPR data of Y. pseudotuberculosis and Y. pestis were compared to examine phylogenetic relationships, but surprising lack of shared spacers limited any further resolution from being made. In this thesis, molecular methods were developed and used to detect, identify, and type potential biological threat agents. PCR assays developed can be transferred to a field-deployable instrument and thus employed close to the patient, for example, during epidemics. PCR results were ready within a few hours, enabling a rapid and appropriate medical response. The 16S rRNA gene-based methods can be utilized in detection of biological agents, which are challenging or laborious to identify using traditional methods. The CRISPR-based sequencing method can be used for typing different strains of Y. pseudotuberculosis, if a comprehensive reference database is made available. DNA sequencing and recently next-generation sequencing have become powerful tools to identify and type biological agents. Sequencing methods can also be utilized in epidemiological investigations and source tracking. Different molecular methods have evolved recently and detection has become fast and more reliable. Rapid detection of microbes enables swift medical countermeasures, and accurate identification and typing methods facilitate the ability to distinguish a natural outbreak from an intentional release. Mikrobin tai biologisen materiaalin aiheuttamaa epidemiaa tai sen uhkaa kutsutaan biologiseksi uhkaksi, silloin kun tauti ei tartuntavaaransa vuoksi ole yhteiskunnan normaaliresurssein hoidettavissa tai kun kyseessä on laaja epidemia, jonka hallitsemiseen tavanomaiset resurssit eivät riitä. Biologinen uhka voi olla luonnollinen, kuten esimerkiksi Länsi-Afrikan laaja ebolaepidemia vuosina 2014-2016, tai tahallinen mikrobin tai toksiinin levittäminen. Väitöskirjan tavoitteena oli kehittää ja hyödyntää molekyylibiologisia menetelmiä biologisten uhka-agenssien nopeaa ja luotettavaa tunnistamista varten. Työn tarkoituksena oli tunnistaa ja tyypittää biologisia uhka-agensseja, olivat ne sitten luonnollisia ja tahallisesti levitettyjä. Työssä hyödynnettiin polymeraasiketjureaktiota (PCR) bakteerien tunnistamisessa sekä tautia aiheuttavien ja vaarattomien bakteerikantojen erottelussa toisistaan. Lisäksi käytettiin hyväksi 16S ribomaalisen RNA (rRNA) -geenin sekvensointia tutkittaessa polymikrobisia näytteitä, ja Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -geenialueiden vertailua bakteerien tyypityksessä. Kolera, Vibrio cholerae -bakteerin aiheuttama tauti, on maailmanlaajuisesti suuri kansanterveydellinen ongelma. Lisäksi Yhdysvaltain tartuntatautivirasto CDC (Centers for Disease Control and Prevention) on luokitellut V. cholerae -bakteerin yhdeksi potentiaaliseksi biouhka-agenssiksi. Työssä kehitettiin kahteen polymeraasiketjureaktioon perustuva V. cholerae -bakteerin tunnistusmenetelmä. Toisen menetelmän avulla pystytään tunnistamaan kaikki V. cholerae -kannat ja toinen havaitsee vain tautia aiheuttavat kannat. Lisäksi työssä vertailtiin kolmea eri PCR-laitetta. Työssä kehitetyt tunnistusmenetelmät osoittautuivat käyttökelpoisiksi ja luotettaviksi tunnistettaessa ja eroteltaessa V. cholerae -kantoja. Käytetyt PCR-laitteet antoivat samanlaiset tulokset, mikä mahdollisti tunnistusmenetelmän siirtämisen laitteiden välillä ilman, että sillä oli vaikutusta menetelmän herkkyyteen tai spesifisyyteen. Kahta erillistä 16S rRNA -geeniin perustuvaa menetelmää yhdistettynä Sanger- ja pyrosekvensointiin hyödynnettiin tutkittaessa bakteerijäämiä kaulavaltimokudoksesta ja myyrien maksanäytteistä. Tavoitteena oli tutkia menetelmien käytettävyyttää ja vertailla menetelmiä toisiinsa. Käytetyt menetelmät osoittautuivat soveltuviksi bakteerilajien havaitsemiseen erilaisista näytematriiseista ja menetelmiä pystytään hyödyntämään tutkittaessa polymikrobisia näytteitä. Lisäksi käytetyt kaksi eri menetelmää tunnistivat samoja bakteerisukuja samoista näytteistä, mikä lisää menetelmien ja tulosten luotettavuutta. Yersinia-bakterien sukuun kuuluu kolme ihmisille tautia aiheuttavaa lajia; Y. pestis, ruton aiheuttaja ja potentiaalinen biouhkabakteeri sekä Y. enterocolitica ja Y. pseudotuberculosis, jotka aiheuttavat suolistotulehduksia. Koska Y. pestis ja Y. pseudotuberculosis -bakteerien genomit ovat hyvin samanlaisia, kantojen tyypittäminen ja erottaminen toisistaan on haastavaa. Tässä työssä CRISPR spacer -geenialueita hyödynnettiin Yersinia-suvun kantojen tyypittämisessä. Tyypitysmenetelmä osoittautui lupaavaksi työkaluksi, vaikka toisistaan eroavien spacer-sekvenssien laaja kirjo vaikeutti kantojen ryhmittämistä ja vertailua. Lisäksi Y. pestis ja Y. pseudotuberculosis -kantojen fylogeneettistä suhdetta tutkittiin vertailemalla kantojen spacer-sekvenssejä. Yllättäen lajeilla oli hyvin vähän yhteisiä spacereita. Näin ollen tutkimus ei tuonut lisätietoa kantojen fylogeneettisistä suhteista. Väitöskirjassa hyödynnettiin molekyylibiologisia menetelmiä potentiaalisten biologisten uhka-agenssien osoittamisessa, tunnistamisessa ja tyypittämisessä. Kehitetty PCR-menetelmä pystyttiin siirtämään kenttäkelpoiselle PCR-laitteelle, mikä tekee mahdolliseksi laitteen käyttämisen lähellä potilasta esimerkiksi epidemian aikana. Tulokset olivat valmiina muutamissa tunneissa, mikä mahdollistaa nopeat lääkinnälliset toimenpiteet. Lisäksi bakteerien 16S rRNA-geenialueen sekvensointiin perustuvia menetelmiä pystytään hyödyntämään sellaisten biologisten agenssien seulonnassa ja tunnistamisessa, joiden identifiointi tavanomaisilla menetelmillä, esimerkiksi viljelemällä, olisi haastavaa tai työlästä. CRISPR-geenialueeseen perustuvaa menetelmää pystyttäisiin hyödyntämään bakteerien tyypityksessä, mikäli laaja referenssitietokanta olisi käytettävissä. DNA:n sekvensointi ja viime vuosina varsinkin uuden sukupolven sekvensointimenetelmät ovat osoittautuneet käyttökelpoisiksi työkaluiksi biologisten agenssien tunnistamisessa ja tyypittämisessä. Sekvensointimenetelmiä pystytään hyödyntämään myös epidemiologisissa tutkimuksissa ja selvitettäessä taudinaiheuttajan alkuperää. Erilaiset molekyylibiologiset menetelmät ovat kehittyneet valtavasti viimevuosina ja taudinaiheuttajien tunnistamisesta on tullut nopeaa ja luotettavaa. Nopea tunnistaminen luo perustan lääkinnällisten vastatoimien aloittamiselle. Tarkka tunnistaminen ja tyypittäminen antavat myös mahdollisuuden erottaa tahallinen levitys luonnollisesta epidemiasta.

International audience; Herein, we sought to evaluate the potential of a recombinant Lactococcus lactis strain secreting the Yersinia pseudotuberculosis low-calcium response V (LcrV) antigen for mucosal vaccination against Yersinia infections. We showed that the recombinant strain induced specific systemic and mucosal antibody and cellular immune responses after intranasal immunization and protected mice against both oral and systemic Y. pseudotuberculosis infections. This constitutes the first proof of principle for a novel anti-Yersinia mucosal vaccination strategy using recombinant lactic acid bacteria.