Evasion of MAIT cell recognition by the African Salmonella Typhimurium ST313 pathovar that causes invasive disease

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal Salmonella enterica strains activate MAIT cells. However, S. Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of ribB. This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium ST313 lineage 2

A New Orbiting Deployable System for Small Satellite Observations for Ecology and Earth Observation

In this paper, we present several study cases focused on marine, oceanographic, and atmospheric environments, which would greatly benefit from the use of a deployable system for small satellite observations. As opposed to the large standard ones, small satellites have become an effective and affordable alternative access to space, owing to their lower costs, innovative design and technology, and higher revisiting times, when launched in a constellation configuration. One of the biggest challenges is created by the small satellite instrumentation working in the visible (VIS), infrared (IR), and microwave (MW) spectral ranges, for which the resolution of the acquired data depends on the physical dimension of the telescope and the antenna collecting the signal. In this respect, a deployable payload, fitting the limited size and mass imposed by the small satellite architecture, once unfolded in space, can reach performances similar to those of larger satellites. In this study, we show how ecology and Earth Observations can benefit from data acquired by small satellites, and how they can be further improved thanks to deployable payloads. We focus on DORA—Deployable Optics for Remote sensing Applications—in the VIS to TIR spectral range, and on a planned application in the MW spectral range, and we carry out a radiometric analysis to verify its performances for Earth Observation studies

Altimetry for the future: Building on 25 years of progress

In 2018 we celebrated 25 years of development of radar altimetry, and the progress achieved by this methodology in the fields of global and coastal oceanography, hydrology, geodesy and cryospheric sciences. Many symbolic major events have celebrated these developments, e.g., in Venice, Italy, the 15th (2006) and 20th (2012) years of progress and more recently, in 2018, in Ponta Delgada, Portugal, 25 Years of Progress in Radar Altimetry. On this latter occasion it was decided to collect contributions of scientists, engineers and managers involved in the worldwide altimetry community to depict the state of altimetry and propose recommendations for the altimetry of the future. This paper summarizes contributions and recommendations that were collected and provides guidance for future mission design, research activities, and sustainable operational radar altimetry data exploitation. Recommendations provided are fundamental for optimizing further scientific and operational advances of oceanographic observations by altimetry, including requirements for spatial and temporal resolution of altimetric measurements, their accuracy and continuity. There are also new challenges and new openings mentioned in the paper that are particularly crucial for observations at higher latitudes, for coastal oceanography, for cryospheric studies and for hydrology. The paper starts with a general introduction followed by a section on Earth System Science including Ocean Dynamics, Sea Level, the Coastal Ocean, Hydrology, the Cryosphere and Polar Oceans and the ‘‘Green” Ocean, extending the frontier from biogeochemistry to marine ecology. Applications are described in a subsequent section, which covers Operational Oceanography, Weather, Hurricane Wave and Wind Forecasting, Climate projection. Instruments’ development and satellite missions’ evolutions are described in a fourth section. A fifth section covers the key observations that altimeters provide and their potential complements, from other Earth observation measurements to in situ data. Section 6 identifies the data and methods and provides some accuracy and resolution requirements for the wet tropospheric correction, the orbit and other geodetic requirements, the Mean Sea Surface, Geoid and Mean Dynamic Topography, Calibration and Validation, data accuracy, data access and handling (including the DUACS system). Section 7 brings a transversal view on scales, integration, artificial intelligence, and capacity building (education and training). Section 8 reviews the programmatic issues followed by a conclusion

Altimetry for the future: building on 25 years of progress

In 2018 we celebrated 25 years of development of radar altimetry, and the progress achieved by this methodology in the fields of global and coastal oceanography, hydrology, geodesy and cryospheric sciences. Many symbolic major events have celebrated these developments, e.g., in Venice, Italy, the 15th (2006) and 20th (2012) years of progress and more recently, in 2018, in Ponta Delgada, Portugal, 25 Years of Progress in Radar Altimetry. On this latter occasion it was decided to collect contributions of scientists, engineers and managers involved in the worldwide altimetry community to depict the state of altimetry and propose recommendations for the altimetry of the future. This paper summarizes contributions and recommendations that were collected and provides guidance for future mission design, research activities, and sustainable operational radar altimetry data exploitation. Recommendations provided are fundamental for optimizing further scientific and operational advances of oceanographic observations by altimetry, including requirements for spatial and temporal resolution of altimetric measurements, their accuracy and continuity. There are also new challenges and new openings mentioned in the paper that are particularly crucial for observations at higher latitudes, for coastal oceanography, for cryospheric studies and for hydrology. The paper starts with a general introduction followed by a section on Earth System Science including Ocean Dynamics, Sea Level, the Coastal Ocean, Hydrology, the Cryosphere and Polar Oceans and the “Green” Ocean, extending the frontier from biogeochemistry to marine ecology. Applications are described in a subsequent section, which covers Operational Oceanography, Weather, Hurricane Wave and Wind Forecasting, Climate projection. Instruments’ development and satellite missions’ evolutions are described in a fourth section. A fifth section covers the key observations that altimeters provide and their potential complements, from other Earth observation measurements to in situ data. Section 6 identifies the data and methods and provides some accuracy and resolution requirements for the wet tropospheric correction, the orbit and other geodetic requirements, the Mean Sea Surface, Geoid and Mean Dynamic Topography, Calibration and Validation, data accuracy, data access and handling (including the DUACS system). Section 7 brings a transversal view on scales, integration, artificial intelligence, and capacity building (education and training). Section 8 reviews the programmatic issues followed by a conclusion

HUMAN PHAGOCYTIC CELL RESPONSE TO MYCOBACTERIUM TUBERCULOSIS ESX-5 MUTANT STRAINS

Mycobacterium tuberculosis (Mtb) is a facultative, intracellular pathogen which causes tuberculosis (TB). Among diseases with bacterial etiology TB is still the one causing most deaths globally. It is estimated that about one-third of the world's population develop a clinically silent infection and that TB is responsible for about 1.5 million deaths per year. Once inhaled, Mtb particles are readily phagocytosed, processed and presented by alveolar macrophages. Subsequently, dendritic cells (DCs) and monocyte-derived macrophages participate in the phagocytic process, playing an essential role in the initiation and maintenance of immune response against Mtb. Thus, during early stages of infection, the control of mycobacterial survival and proliferation is mainly depends on the innate immune response. Among these responses, production of pro-inflammatory cytokines plays a crucial role. Mycobacteria have evolved sophisticated mechanisms to manipulate natural biological processes of host cells to create an environment that is favourable to their survival and proliferation. One of the mechanisms used by mycobacteria to modulate the phagocyte response is their ability to manipulate the secretion of pro-inflammatory cytokines. Subversion of eukaryotic host responses by bacterial pathogens often requires specialized secretion systems that deliver effector proteins near or directly into host cells. Recently, a novel secretion pathway has been identified in mycobacteria, which has been classified as type VII secretion system. The Mtb genome harbours five gene clusters coding for type VII secretion systems, designated ESX-1 - ESX-5, that export small, highly immunogenic proteins lacking a classical N-terminal signal sequence, belonging to the Esx or WXG-100 family. Among ESX systems the ESX-5 represents the most recently evolved one. The role of the ESX-5 system in the export of selected PPE and PE proteins in M. marinum, as well as the involvement of this system in modulation of 5 cytokine responses by M. marinum-infected human macrophages are well established. By the characterization of several Mtb knock-out mutants for ESX-5 components, it has been recently demonstrated that ESX-5 plays a crucial role in host pathogen interaction also in Mtb. Here we have investigated whether the components of Mtb ESX-5 secretion system have an impact on the ability of the bacteria to modulate innate immune response of human professional phagocytes. To this aim we focused on three ESX-5 mutants: MtbeccD5ko and Mtbppe25-pe19, which inactivate the ESX-5 secretion system and show an attenuated phenotype in the SCID mouse model, and Mtbrv1794ko, whose orthologue in M. marinum was demonstrated to modulate cytokine secretion by human macrophages. First, we investigated whether the components of Mtb ESX-5 system have impact on human DC maturation and pattern of cytokine production from human macrophages and DCs. To this aim, monocyte derived dendritic cells (MoDCs) and two distinct macrophage subsets with pro-inflammatory (M1) and anti-inflammatory (M2) phenotype were stimulated with wild-type Mtb H37Rv (Mtbwt) or the ESX-5 mutant strains. We observed no difference among MoDCs infected with Mtbwt or the ESX-5 mutants both for the maturation markers analysed and the profile of cytokines secreted. Similarly, M1 and M2 infected with Mtbwt or the MtbeccD5ko and Mtbppe25-pe19 strains produced comparable amounts of the cytokines analysed. On the contrary, macrophages infected with Mtbrv1794ko produced significantly higher amounts of IL-1 and IL-18 as compared to Mtbwt or the complemented strain Mtbrv1794ko-C infected cells. This difference was abolished when the macrophages were infected with heat-killed bacteria. Both IL-1 and IL-18 are produced in inactive “pro-” form and their activation and secretion from macrophages is tightly controlled by a two-step mechanism that involves a family of cytosolic multiprotein complexes known as “inflammasomes”. Activation of inflammasome in 6 macrophages triggers caspase-1 activation, and, in turn, maturation of IL-1 and IL-18 to their active, secreted forms. Thus, to investigate the possible mechanism(s) responsible of the augmented IL-1 and IL-18 secretion by Mtbrv1794ko infected cells, in a second set of experiments, we have tried to dissect the molecular steps involved in inflammasome activation. Analyses of relative mRNA levels by quantitative RT-PCR demonstrated that both Mtbwt and Mtbrv1794ko equally activated pro-IL-1 and pro-IL-18 expression in macrophages. These data were confirmed by immunoblot analysis of pro-IL-1 protein levels in cell-lysates. In addition, a FLuorochrome Inhibitor of CAspases assay, which was used to detect active caspase-1 inside the cells, indicated higher caspase-1 activation in macrophages infected with Mtbrv1794ko compared to Mtbwt infected cells. Altogether these results suggest that the marked IL-1 and IL-18 production by Mtbrv1794ko infected macrophages may be correlated with a higher rate of processing rather than a higher production of such cytokines. Finally, we investigated whether two of the inflammasome triggering mechanisms reported to be involved in Mtb infection (i.e. the efflux of potassium and the release of lysosomial protease cathepsin B) might have a role in the observed augmented IL-1 production/secretion by Mtbrv1794ko infected macrophages. Our findings demonstrated that IL-1 and IL-18 levels were drastically reduced when KCl or the inhibitor of cathepsin B were added to the infection medium, suggesting that the release of cathepsin B from the lysosomial compartment or the decrease of intracellular [K+] may contribute to the inflammasome activation during mycobacterial infection

Ligandi batterici e recettori cellulari coinvolti nell'interazione diretta tra Mycobacterium bovis, bacillo di Calmette e Guerin, e cellule natural killer umane

Nel corso degli ultimi anni è diventato progressivamente evidente che lo sviluppo di una efficace risposta immunitaria specifica contro una varietà di agenti infettivi si determina in larga misura nelle prime fasi dell’interazione tra tali agenti ed i componenti cellulari dell’immunità innata (ad esempio macrofagi, cellule dendritiche, cellule natural killer-NK). Oltre che per la loro capacità di interferire direttamente con la moltiplicazione dei microrganismi, mediante la messa in atto di potenti funzioni effettrici, le cellule dell’immunità innata risultano, infatti, essenziali anche nel determinare il tipo e l’entità della susseguente risposta immunitaria specifica ed, in definitiva l’evoluzione più o meno favorevole di un’infezione. La prima fase della risposta immune innata prevede l’interazione di recettori espressi dagli elementi cellulari che l’agente patogeno incontra alla porta di entrata, con componenti batterici superficiali. Lo studio di tali recettori e dei corrispondenti ligandi microbici ha suscitato grande interesse negli ultimi anni, in quanto è emerso che la loro interazione rappresenta un meccanismo evolutivamente conservato in grado di allertare il sistema immunitario dell’ospite contro la presenza di un microrganismo e di indirizzare la successiva risposta specifica verso il fenotipo più adatto all’eradicazione dell’infezione. Le cellule dell’immunità innata riconoscono gli agenti infettivi tramite recettori definiti pathogen recognition receptors (PRRs) che sono codificati da geni fissi nel genoma e sono espressi, in maniera non clonale, anche su cellule appartenenti a linee differenziative diverse. Tali recettori riconoscono molecole presenti sulla superficie degli agenti infettivi, altamente conservate nell’ambito di grandi classi di microrganismi e denominate pathogen associated molecular patterns (PAMPs). Il lavoro svolto nella presente tesi rappresenta la continuazione di studi da tempo intrapresi nel laboratorio ospitante riguardanti l’interazione diretta tra cellule NK dell’uomo e Mycobacterium bovis, bacillo di Calmette e Guérin (BCG), un ceppo attenuato utilizzato in molti studi come modello per la specie virulenta M. tuberculosis, l’agente eziologico della tubercolosi nell’uomo. Tali studi hanno dimostrato che BCG è in grado di interagire direttamente con cellule NK umane e di stimolare, in assenza di cellule presentanti l’antigene, molte delle loro funzioni effettrici quali la produzione di IFN-gamma, la proliferazione e l’attività citotossica nei confronti di target cellulari sensibili. Tali funzioni effettrici vengono completamente abrogate quando i batteri e le cellule sono separati da una membrana porosa che ne impedisce il contatto e sono indotte non solo da batteri vivi, ma anche da batteri uccisi e da preparazioni antigeniche arricchite in antigeni di parete di BCG. Ciò ha suggerito che l’interazione BCG-cellule NK possa coinvolgere il riconoscimento di componenti batterici superficiali da parte di specifici recettori cellulari. E’ noto che le funzioni effettrici delle cellule NK sono il risultato di un equilibrio tra stimoli attivatori e stimoli inibitori mediati da altrettanti recettori cellulari. I recettori attivatori includono quelli appartenenti alla famiglia dei Natural Cytotoxicity Receptors (NCRs), tre proteine indicate con la sigla NKp30, NKp44 e NKp46, od alcuni membri dei Toll-Like-Receptors (TLRs), una famiglia di PRRs ampiamente caratterizzata e coinvolta nel riconoscimento di una varietà di PAMPs. Utilizzando forme solubili commerciali dei vari recettori NCRs, il laboratorio in cui è stata svolta la tesi ha dimostrato recentemente che almeno uno di essi, NKp44, è capace di legarsi alla superficie di varie specie del genere Mycobacterium, incluso BCG e la specie virulenta M. tuberculosis. Altri Autori hanno dimostrato che almeno uno dei membri della famiglia dei TLRs, il TLR2, è coinvolto nell’interazione diretta BCG-cellule NK. Scopo della presente tesi è stato quello di identificare i putativi ligandi batterici dei recettori cellulari coinvolti nell’interazione diretta tra BCG e cellule NK umane e di stabilire il ruolo di tale interazione sull’attivazione delle funzioni cellulari. Il lavoro sperimentale è stato svolto in tre fasi principali: Nella prima fase si è proceduto ad allestire un saggio immunoenzimatico con componenti micobatterici purificati. Essi includevano componenti abbondanti nella parete dei micobatteri quali il lipoarabinomannano (LAM), gli acidi micolici (AM), il peptidoglicano (PG) e l’arabinogalattano (AG) usati singolarmente ed in associazione nel micolil-arabino-galattano-peptidoglicano (mAGP) che è considerato lo scheletro fondamentale della parete di tali batteri. Nel saggio sono stati anche inclusi, come controllo, componenti parietali di batteri Gram-positivi o negativi quali, ad esempio, gli acidi teicoici od il lipopolisaccaride. I vari componenti sono stati fatti aderire a piastre da 96 pozzetti e, dopo opportuni lavaggi, sono stati incubati con forme solubili commerciali dei recettori NCR, chimera per il frammento Fc delle IgG umane. Il legame recettore/ligando(i) è stato rivelato con un anticorpo secondario marcato con perossidasi specifico per l’Fc delle IgG umane. Tali esperimenti hanno dimostrato che il recettore NKp44 in forma solubile è in grado di legare in maniera stabile e dose dipendente componenti parietali micobatterici quali AG, AM, mAGP, ma non PG o LAM. Nella seconda fase del lavoro si è proceduto ad allestire dei saggi funzionali allo scopo di valutare se i componenti parietali identificati fossero in grado di promuovere l’attivazione di cellule NK e la produzione, da parte di queste, di IFN-gamma, una citochina chiave capace di modulare l’attività di numerosi altri tipi cellulari dell’immunità innata e specifica. A tale scopo si è proceduto ad ottenere, per selezione negativa, una popolazione di cellule NK umane altamente purificate da sangue periferico di donatori sani. Tali cellule sono state stimolate in vitro con biglie di polistirene (pb) precedentemente ricoperte con lo scheletro fondamentale della parete dei micobatteri, il complesso mAGP, allo scopo di mimare la superficie batterica. I risultati ottenuti hanno dimostrato che al contrario delle biglie di controllo, quelle ricoperte con mAGP (pb-mAGP) stimolavano l’espressione su cellule NK di marker di attivazione quali il CD25 il CD69 ed anche del recettore attivatore NKp44. L’attivazione cellulare si accompagnava alla produzione di ingenti quantità di IFN-gamma, valutato nei sopranatanti di coltura dopo 3 giorni di stimolazione. Allo scopo di valutare se l’attivazione delle funzioni cellulari osservata stimolando con pb-mAGP fosse dovuta all’interazione dei componenti parietali presenti sulla superficie delle biglie con i recettori NKp44 e/o TLR2, nella terza fase del lavoro si è proceduto a ripetere gli esperimenti di stimolazione in presenza di anticorpi bloccanti anti-NKp44 ed anti-TLR2. Tali esperimenti hanno dimostrato che mentre la presenza di anti-NKp44 nella coltura non influenzava il grado di attivazione cellulare e la produzione di IFN-gamma, la presenza di anti-TLR2 riduceva tali parametri funzionali a valori paragonabili a quelli delle colture prive di stimoli. I dati ottenuti hanno dimostrato che: i) forme solubili del recettore attivatore NKp44 si legano a componenti della parete dei micobatteri quali l’arabinogalattano, gli acidi micolici ed il loro complesso che include il peptidoglicano (mAGP), ma non al peptidoglicano usato singolarmente; ii) il complesso mAGP stimola l’attivazione cellulare, induce l’espressione di NKp44 e la produzione di IFN-gamma da parte di cellule NK altamente purificate; iii) anticorpi bloccanti il TLR2, ma non NKp44 annullano l’attivazione cellulare e la produzione di IFN-gamma da parte di tali cellule NK. Nel loro insieme tali dati suggeriscono che, almeno in una prima fase, l’attivazione delle cellule NK in seguito a stimolazione diretta con mAGP non sia dovuta all’interazione di MA e AG con NKp44; tale recettore è, infatti, espresso a livelli basali molto bassi su cellule “resting” mentre viene marcatamente indotto sulle cellule attivate. Dall’altro lato, il blocco dell’attivazione cellulare osservato utilizzando anticorpi anti-TLR2 suggerisce che il TLR2 possa rappresentare il recettore primario che, interagendo con il peptidoglicano (un componente micobatterico considerato un ligando di TLR2) presente in mAGP, determini l’attivazione delle funzioni effettrici. Secondo tale ipotesi, quindi, l’interazione di TLR2 con il peptidoglicano parietale potrebbe portare ad una attivazione delle funzioni cellulari e all’induzione del recettore NKp44. Quest’ultimo recettore potrebbe, quindi, interagire con altri componenti parietali, quali l’arabinogalattano e gli acidi micolici, perpetuando e mantenendo lo stimolo attivatorio

The battle for iron in enteric infections

Iron is an essential element for almost all living organisms, but can be extremely toxic in high concentrations. All organisms must therefore employ homeostatic mechanisms to finely regulate iron uptake, usage and storage in the face of dynamic environmental conditions. The critical step in mammalian systemic iron homeostasis is the fine regulation of dietary iron absorption. However, as the gastrointestinal system is also home to >1014 bacteria, all of which engage in their own programmes of iron homeostasis, the gut represents an anatomical location where the inter-kingdom fight for iron is never-ending. Here, we explore the molecular mechanisms of, and interactions between, host and bacterial iron homeostasis in the gastrointestinal tract. We first detail how mammalian systemic and cellular iron homeostasis influences gastrointestinal iron availability. We then focus on two important human pathogens, Salmonella and Clostridia; despite their differences, they exemplify how a bacterial pathogen must navigate and exploit this web of iron homeostasis interactions to avoid host nutritional immunity and replicate successfully. We then reciprocally explore how iron availability interacts with the gastrointestinal microbiota, and the consequences of this on mammalian physiology and pathogen iron acquisition. Finally, we address how understanding the battle for iron in the gastrointestinal tract might inform clinical practice and inspire new treatments for important diseases

Multi-criteria approach for the environmental impact assessment of inland aquaculture

Trout farming, that represents the most important sector for aquaculture inland production in Italy, can cause negative effects on aquatic ecosystems. Recently, in the framework of Water Frame Directive 2000/60/EC and national law DL 152/2006, concerning the sustainable uses of water resources, multi-criteria approaches have been suggested to evaluate the impact of fish farming on aquatic ecosystems. In this study trout farms of central Italy were selected to investigate the effects of their effluents, on receiving water bodies using a multi-criteria approach based on physicochemical parameters, microbiological and macrobenthonic indicators, detected in sampling stations located upstream/downstream the trout farm. Moreover, antibiotic susceptibility against antibiotics allowed and/or forbidden by current law (D.lgs 193/56/06) was tested on E. coli strains. The results indicate variations of chemical parameters and biological indicators from upstream to downstream sites in some of the investigated farms. Antibiotic resistance of E. coli strains suggested a large use of tetracycline and a possible past use of chloramphenicol. This study represents a first contribute to the knowledge of fish farm impacts on aquatic systems in Central Italy

Multi-criteria approach for the environmental impact assessment of inland aquaculture

Trout farming, that represents the most important sector for aquaculture inland production in Italy, can cause negative effects on aquatic ecosystems. Recently, in the framework of Water Frame Directive 2000/60/EC and national law DL 152/2006, concerning the sustainable uses of water resources, multi-criteria approaches have been suggested to evaluate the impact of fish farming on aquatic ecosystems. In this study trout farms of central Italy were selected to investigate the effects of their effluents, on receiving water bodies using a multi-criteria approach based on physicochemical parameters, microbiological and macrobenthonic indicators, detected in sampling stations located upstream/downstream the trout farm. Moreover, antibiotic susceptibility against antibiotics allowed and/or forbidden by current law (D.lgs 193/56/06) was tested on E. coli strains. The results indicate variations of chemical parameters and biological indicators from upstream to downstream sites in some of the investigated farms. Antibiotic resistance of E. coli strains suggested a large use of tetracycline and a possible past use of chloramphenicol. This study represents a first contribute to the knowledge of fish farm impacts on aquatic systems in Central Italy