Influence of <i>Moraxella</i> sp. colonization on the kidney proteome of farmed gilthead sea breams (<i>Sparus aurata</i>, L.)

Background: Currently, presence of Moraxella sp. in internal organs of fish is not considered detrimental for fish farming. However, bacterial colonization of internal organs can affect fish wellness and decrease growth rate, stress resistance, and immune response. Recently, there have been reports by farmers concerning slow growth, poor feed conversion, and low average weight increase of fish farmed in offshore floating sea cages, often associated with internal organ colonization by Moraxella sp. Therefore, presence of these opportunistic bacteria deserves further investigations for elucidating incidence and impact on fish metabolism. Results: A total of 960 gilthead sea breams (Sparus aurata, L.), collected along 17 months from four offshore sea cage plants and two natural lagoons in Sardinia, were subjected to routine microbiological examination of internal organs throughout the production cycle. Thirteen subjects (1.35%) were found positive for Moraxella sp. in the kidney (7), brain (3), eye (1), spleen (1), and perivisceral fat (1). In order to investigate the influence of Moraxella sp. colonization, positive and negative kidney samples were subjected to a differential proteomics study by means of 2-D PAGE and mass spectrometry. Interestingly, Moraxella sp. infected kidneys displayed a concerted upregulation of several mitochondrial enzymes compared to negative tissues, reinforcing previous observations following lipopolysaccharide (LPS) challenge in fish. Conclusions: Presence of Moraxella sp. in farmed sea bream kidney is able to induce proteome alterations similar to those described following LPS challenge in other fish species. This study revealed that Moraxella sp. might becausing metabolic alterations in fish, and provided indications on proteins that could be investigated as markers of infection by Gram-negative bacteria within farming plants

Influence of Moraxella sp. colonization on the kidney proteome of farmed gilthead sea breams (Sparus aurata, L.)

Background: Currently, presence of Moraxella sp. in internal organs of fish is not considered detrimental for fish farming. However, bacterial colonization of internal organs can affect fish wellness and decrease growth rate, stress resistance, and immune response. Recently, there have been reports by farmers concerning slow growth, poor feed conversion, and low average weight increase of fish farmed in offshore floating sea cages, often associated with internal organ colonization by Moraxella sp. Therefore, presence of these opportunistic bacteria deserves further investigations for elucidating incidence and impact on fish metabolism. Results: A total of 960 gilthead sea breams (Sparus aurata, L.), collected along 17 months from four offshore sea cage plants and two natural lagoons in Sardinia, were subjected to routine microbiological examination of internal organs throughout the production cycle. Thirteen subjects (1.35%) were found positive for Moraxella sp. in the kidney (7), brain (3), eye (1), spleen (1), and perivisceral fat (1). In order to investigate the influence of Moraxella sp. colonization, positive and negative kidney samples were subjected to a differential proteomics study by means of 2-D PAGE and mass spectrometry. Interestingly, Moraxella sp. infected kidneys displayed a concerted upregulation of several mitochondrial enzymes compared to negative tissues, reinforcing previous observations following lipopolysaccharide (LPS) challenge in fish. Conclusions: Presence of Moraxella sp. in farmed sea bream kidney is able to induce proteome alterations similar to those described following LPS challenge in other fish species. This study revealed that Moraxella sp. might be causing metabolic alterations in fish, and provided indications on proteins that could be investigated as markers of infection by Gram-negative bacteria within farming plants.Pubblicat

Zinc uptake in Streptococcus pyogenes: characterisation of adcA

Streptococcus pyogenes (also known as Group A Streptococcus, GAS) is a capsulated Gram-positive, human-adapted pathogen. GAS strains express different virulence factors exposed on the bacterial surface or secreted outside the cell. Among the secreted virulence factors, superantigens (SAGs) are certainly the most toxic factors. A recent study demonstrates that, in vitro, the streptococcal superantigen SpeI interacts with AdcA and Lmb, the substrate binding subunits of the two zinc transporters. In particular, AdcA belongs to the high-affinity zinc transporter Adc, involved in adhesion, competence and zinc uptake. Zinc is an essential micronutrient for all living organisms but cells have to tightly control its intracellular concentration due to its toxicity. In this thesis the role of adcA in zinc uptake in GAS was studied. Three independent ΔadcA null mutants were generated in the strain MGAS5005, and their phenotype was characterised. The mutants were obtained by means of the low copy number temperature-sensitive shuttle vector pJRS233. Deletion of the adcA gene in Streptococcus pneumoniae leads to a dramatic decrease in competence. Thus, complementation using the pMU1328 plasmid was obtained by transforming the intermediate strain MGAS5005::pJRS233-ΔadcA that still contains the wild type adcA allele. The absence of a zinc transporter affects the capacity to uptake zinc from the culture medium and the mutant susceptibility to zinc starvation. The Δadca null mutants clearly displayed a higher sensitivity to zinc starvation compared to the wild type strain. The complementation of one of the mutants with the wild type gene restored the phenotype. When the ΔadcA mutant is grown in the presence of the zinc chelator TPEN, growth is rescued both by zinc or manganese. This probably means that the import of these two metals is carried out by the other zinc transporter Lmb, coded within the operon lmb-htpA. Hence, the expression of Lmb and HtpA was analysed by Western blot in different growth conditions. In wild type cells AdcA is always expressed at a high level, whereas Lmb and HtpA are highly expressed only in zinc-depleted medium or in ΔadcA mutants. This finding supports the notion that AdcA is functionally homologous to ZnuA, the major high affinity zinc transporter in many bacteria, and like ZnuA is responsible for the efficient recruitment of zinc in most conditions.Streptococcus pyogenes (detto anche Streptococco di gruppo A, GAS) è un batterio Gram-positivo capsulato, adattato ad infettare l’uomo. I ceppi di GAS esprimono diversi fattori di virulenza che possono essere esposti sulla superficie esterna o secreti fuori dalla cellula. Tra i fattori di virulenza, i Superantigeni (SAGs) sono sicuramente tra i più nocivi per l’ospite. Uno studio recente ha dimostrato che in vitro SpeI, un superantigene secreto da GAS, interagisce con AdcA a Lmb, due proteine che trasportano lo zinco. In particolare, AdcA appartiene al trasportatore ad alta affinità Adc, che è coinvolto nell’adesione, nella competenza e nel trasporto dello zinco. Questo metallo è un microelemento essenziale per tutti gli organismi viventi, ma poiché possiede una elevata tossicità, le cellule devono regolare finemente la sua concentrazione intracellulare. In questo lavoro di tesi è stato studiato il ruolo di AdcA nel trasporto dello zinco in GAS. Per questo studio sono stati generati tre mutanti indipendenti nel ceppo MGAS5005, caratterizzandone il loro fenotipo. I mutanti sono stati ottenuti mediante l’uso del vettore termosensibile pJRS233. La delezione del gene adcA in Streptococcus pneumoniae comporta una notevole diminuzione della competenza, quindi la complementazione è stata ottenuta trasformando il mutante parziale, cioè l’ “eterozigote” intermedio che contiene sia l’allele wild type che quello mutato. L’assenza del trasportatore dello zinco influisce sulla capacità di importare lo zinco dal terreno di coltura e sulla suscettibilità alla mancanza di zinco. I mutanti ΔadcA mostrano chiaramente una maggiore sensibilità alla deprivazione di zinco se comparati con il ceppo wild type. La complementazione di uno dei mutanti riporta il fenotipo del mutante a quello del ceppo wild type. Quando il mutante è cresciuto in presenza di una concentrazione inibente del chelante TPEN, la crescita è ristabilita dalla aggiunta di zinco o di manganese. Questo probabilmente significa che il trasporto di questi due metalli può avvenire tramite l’altro trasportatore Lmb, codificato all’interno dell’operone lmb-htpA. Di conseguenza, l’espressione di Lmb e HtpA è stata analizzata tramite Western blot in condizioni di crescita diverse. Nelle cellule wild type AdcA è sempre espresso ad alti livelli, invece Lmb e HtpA sono espresse ad alti livelli solo nel terreno di coltura depleto di zinco. Questo risultato avvalora il concetto che AdcA è omologo da un punto di vista funzionale a ZnuA, il maggior trasportatore ad alta affinità dello zinco in molti batteri, e come ZnuA ha il compito del reclutamento dello zinco in molte condizioni

Expression of AdcA, Lmb and HtpA during growth in zinc-depleted medium.

<p>Western blot analysis of total cell extracts from <i>S</i>. <i>pyogenes</i> MGAS5005 wild type, Δ<i>adcA</i> and Δ<i>lmb</i> null mutants grown in complete medium (THY) or in zinc-depleted medium (THY + 35 μM TPEN) containing increasing amounts of ZnCl₂. (<b>A</b>) Western blot using anti-AdcA specific antibodies. (<b>B</b>) Western blot using anti-Lmb specific antibodies. (<b>C</b>) Western blot using anti-HtpA specific antibodies.</p

Zn²⁺ Uptake in <i>Streptococcus pyogenes</i>: Characterization of <i>adcA </i>and <i>lmb</i> Null Mutants

<div><p>An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized null deletion mutants of the <i>adcA</i> and <i>lmb</i> genes of <i>Streptococcus pyogenes</i> strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in <i>Streptococcus pneumoniae</i>. Null <i>adcA</i> and <i>lmb</i> mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn²⁺ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to <i>lmb</i>, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and null mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the null <i>adcA</i> mutant, when expression of <i>lmb</i> is required to compensate for the lack of <i>adcA</i> expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.</p></div

Inhibition of growth by TPEN is rescued by the addition of Zn²⁺ ions.

<p>(<b>A</b>) Susceptibility to Zn²⁺ starvation of wild type, Δ<i>adcA</i> and Δ<i>lmb</i> null mutants grown in THY medium containing increasing concentrations of the chelating agent TPEN. (<b>B</b>) Inhibition of growth by 50 μM TPEN is rescued by the addition of either 50 μM Zn²⁺ in wild type as well as in Δ<i>adcA</i> and Δ<i>lmb</i> null mutants. Error bars represent the standard deviation.</p

Construction of <i>adcA</i> null mutants.

<p>(<b>A</b>) Schematic representation of the <i>adcA</i> locus in S. <i>pyogenes</i> MGAS5005 wild type and Δ<i>adcA</i> deletion mutants. (<b>B</b>) Map of plasmid pMU1328 used for complementation of the Δ<i>adcA</i> deletion mutants. (<b>C</b>) Western blot analysis of AdcA expression in total cell extracts of wild type, three independent Δ<i>adcA</i> null mutants and a Δ<i>adcA</i> complemented with the pMU1328-<i>adcA</i> construct using anti-AdcA specific antibodies.</p

Bacterial strains, plasmids and primers used in this study.

<p>Bacterial strains, plasmids and primers used in this study.</p

Susceptibility to Zn²⁺ starvation.

<p>Susceptibility to Zn²⁺ starvation of wild type, Δ<i>adcA</i> null mutant and complemented strains grown in THY medium containing increasing concentrations of the chelating agent N,N,N’N’-Tetrakis (2-pyridylmethyl)-1,2-ethylenediamine (TPEN). Error bars represent the standard deviation.</p