Impact of Acinetobacter baumannii Superoxide Dismutase on Motility, Virulence, Oxidative Stress Resistance and Susceptibility to Antibiotics

Acinetobacter baumannii is a Gram-negative bacterium appearing as an opportunistic pathogen in hospital settings. Superoxide dismutase (SOD) contributes to virulence in several pathogenic bacteria by detoxifying reactive oxygen species released in the course of host defense reactions. However, the biological role of SODs in A. baumannii has not yet been elucidated. Here, we inactivated in A. baumannii ATCC 17978 gene A1S_2343, encoding a putative SOD of the Fe-Mn type by transposon insertion, resulting in mutant ATCC 17978 sod2343::Km. The mutation was also introduced in two naturally competent A. baumannii isolates by transformation with chromosomal DNA derived from mutant ATCC 17978 sod2343::Km. We demonstrate that inactivation of sod2343 leads to significant motility defects in all three A. baumannii strains. The mutant strains were more susceptible to oxidative stress compared to their parental strains. Susceptibility to colistin and tetracycline was increased in all mutant strains while susceptibility of the mutants to gentamicin, levofloxacin and imipenem was strain-dependent. In the Galleria mellonella infection model the mutant strains were significantly attenuated. In conclusion, sod2343 plays an important role in motility, resistance to oxidative stress, susceptibility to antibiotics and virulence in A. baumannii

Characterization of the Role of Spa33 in Type 3 Secretion Regulation in Shigella flexneri

Shigella is the causative agent of shigellosis, a severe diarrheal disease that causes more than one million deaths per year, mainly in low-income settings and among children under five years of age. The rapid increase of multi-drug resistant strains and the lack of vaccine make this pathogen a global health concern. Shigella invasion of colonic epithelium and further cell to cell spread is achieved by a specialized surface nano-syringe, called type 3 secretion system (T3SS). Shigella uses its T3SS to translocate virulence proteins directly inside the host cell where they interfere with multiple host-signalling cascades. The T3SS is composed of three major parts; a cytoplasmic complex (CC), a basal body and an extracellular needle. In the absence of contact with the eukaryotic cell, tip complex (TC) proteins, IpaB and IpaD, act as a plug at the top of the needle. MxiC, a gatekeeper protein, serves as an internal plug at the base of the T3SS to prevent the premature secretion of effectors. Upon host cell contact, IpaC and IpaB form a pore in the host cell plasma membrane, through which the effector proteins are delivered directly into the host cell cytosol for bacterial entry. Pore insertion generates an activation signal, which is sensed at the tip of the needle, and then transmitted through the needle subunits, MxiH and MxiI, to the base. This signal allows the secretion of MxiC and subsequently effectors release. Nevertheless, the identity of the cytoplasmic recipient, of the activation signal, is still unknown. The primary goal of this thesis was to characterize the role of Spa33, an essential component of the T3SS cytoplasmic complex. We have first discovered that 5 fragments with different sizes were expressed from the spa33 gene namely: Spa33FL (~33 kDa), Spa33C (~12 kDa), which was previously described, Spa33CC (~7 kDa), Spa33N (~11 kDa) and Spa33X (~10 kDa). We have shown that these fragments are arising from the internal start codons (Spa33C Spa33CC) or transcriptional slippage (Spa33N). We have demonstrated that Spa33CC is essential for T3SS function. We also observed that Spa33FL, Spa33C and Spa33CC interact with each other and forms a complex. Moreover, we have found that Spa33 is implicated in the T3 secretion hierarchy, by interacting with MxiC and MxiI. In a second part of the thesis, we have demonstrated that Spa33 is phosphorylated in vivo. We have addressed the impact of Spa33 phosphorylation in the regulation of secretion hierarchy by phosphomimetic and phosphoablative mutation of 5 potential phosphorylation-sites. Our analysis has revealed that phosphomimetic mutation of some residue (Y245, T101 and T167) abolishes the T3 secretion. Moreover, phosphoablative mutations of threonine residues lead to a decrease of MxiC and/or effectors secretion, strengthening our model in which Spa33 is implicated in the regulation of T3 secretion hierarchy upon activation of T3SS. Overall, our findings provide new insights about the regulation of T3SS secretion hierarchy and identify new players involved in the regulation of T3SS.Shigella est l’agent responsable de la shigellose, une maladie diarrhéique grave qui provoque plus d’un million de décès par an, principalement dans les pays à faible revenu et chez les enfants de moins de cinq ans. L'augmentation du nombre souches multirésistantes aux antibiotiques etl’absence d’un vaccin efficace font de cet agent pathogène un problème de santé mondiale. L’invasion de l’épithélium colique par Shigella et sa dissémination dans ce dernier sont assurées par des structures spécialisées présentes à la surface bacétrienne appelées systèmes de sécrétion de type 3 (SST3). Shigella utilise le SST3 pour transloquer les protéines de virulence directement dans le cytoplasme de la cellule hôte, où elles interfèrent avec différentes cascades de signalisation cellulaire. Le T3SS est composé de trois parties principales; un complexe cytoplasmique (CC), un corps basal et une aiguille extracellulaire. En l'absence de contact avec la cellule eucaryote, deux protéines, IpaB et IpaD, localisées au sommet de l’aiguille, agissent comme un bouchon empechant la sécrétion prematurée des protéines de virulence. Lors du contact avec la cellule hôte, IpaC et IpaB forment un pore dans la membrane plasmique de la cellule hôte, à travers lequel les protéines effectrices sont délivrées directement dans le cytosol de la cellule hôte pour permettre l’entrée bactérienne. L'insertion des pores génère un signal d'activation, qui est détecté au sommet de l'aiguille, puis transmis à la base par les sous-unités de l'aiguille, MxiH et MxiI. Ce signal permet la sécrétion de MxiC, une protéine ’gatekeeper’ servant de bouchon interne à la base du T3SS. Sa sécrétion permet la libération d’une deuxième salve de protéines de virulence, appelées effecteurs. Néanmoins, la protéine recevant le signal d'activation au niveau cytoplasmique n’est, à ce jour,toujours pas identifiée. L'objectif principal de cette thèse fût de caractériser le rôle de Spa33, un composant essentiel du complexe cytoplasmique du SST3. Nous avons d’abord montré que 5 fragments de tailles différentes étaient exprimés à partir du gène spa33, à savoir :Spa33FL (~ 33 kDa), Spa33C (~ 12 kDa), décrit précédemment, Spa33CC (~ 7 kDa), Spa33N (~ 11 kDa) et Spa33X (~ 10 kDa). Nous avons montré que ces fragments provenaient de codons de démarrage alternatifs (Spa33C, Spa33CC) ou d’un glissement de transcription (Spa33N). Nous avons démontré que Spa33CC est essentiel à la fonction du SST3. Nous avons également observé que Spa33FL, Spa33C et Spa33CC interagissent et forment un complexe. De plus, nous avons montré que Spa33 est impliqué dans la hiérarchie de sécrétion, en interagissant avec MxiC et MxiI. Dans une deuxième partie de la thèse, nous avons montré que Spa33 est ?phosphorylée in vivo. Nous avons abordé l'impact de la phosphorylation de Spa33 sur la régulation de la hiérarchie de sécrétion en réalisant des mutations phospho-mimétiques et phospho-ablatives de 5 sites de phosphorylation potentiels. Notre étude a révélé que la mutation phospho-mimétique de certains résidus (Y245, T101 et T167) abolit la sécrétion de T3. De plus, les mutations phospho-ablatives des résidus thréonine entraînent une diminution de la sécrétion de MxiC et/ou des effecteurs, renforçant ainsi notre modèle dans lequel Spa33 est impliqué dans la régulation de la hiérarchie de la sécrétion lors de l'activation du SST3. En conclusion, nos résultats fournissent de nouvelles informations sur la régulation de la hiérarchie de sécrétion du SST3 et identifient de nouveaux acteurs impliqués dans cette régulation ouvrant de nouvelles perspectives dans la compréhension, et donc dans la lutte, contre une arme de virulence retrouvée chez plus de 25 pathogènes.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)info:eu-repo/semantics/nonPublishe

Multiple proteins arising from a single gene: The role of the Spa33 variants in Shigella T3SS regulation

Shigella invasion and dissemination in intestinal epithelial cells relies on a type 3 secretion system (T3SS), which mediates translocation of virulence proteins into host cells. T3SSs are composed of three major parts: an extracellular needle, a basal body, and a cytoplasmic complex. Three categories of proteins are hierarchically secreted: (a) the needle components, (b) the translocator proteins which form a pore (translocon) inside the host cell membrane and (c) the effectors interfering with the host cell signaling pathways. In the absence of host cell contact, the T3SS is maintained in an “off” state by the presence of a tip complex. Secretion is activated by host cell contact which allows the release of a gatekeeper protein called MxiC. In this work, we have investigated the role of Spa33, a component of the cytoplasmic complex, in the regulation of secretion. The spa33 gene encodes a 33-kDa protein and a smaller fragment of 12 kDa (Spa33C) which are both essential components of the cytoplasmic complex. We have shown that the spa33 gene gives rise to 5 fragments of various sizes. Among them, three are necessary for T3SS. Interestingly, we have shown that Spa33 is implicated in the regulation of secretion. Indeed, the mutation of a single residue in Spa33 induces an effector mutant phenotype, in which MxiC is sequestered. Moreover, we have shown a direct interaction between Spa33 and MxiC.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Recovery of <i>A. baumannii sod2343</i> mutant from <i>Galleria mellonella</i> larvae is minor compared to parental strain.

<p>Groups of 10 larvae were injected with 1.5×10⁶ CFU of ATCC 17978, ATCC 17978 <i>sod2343::Km</i>, and ATCC 17978 <i>sod2343::Km</i> pWH<i>sod2343</i>, respectively. Directly after injection of the 10 larvae within a group, the larvae were frozen at −80°C, homogenized and serial dilutions were plated on CHROMagar Acinetobacter to determine CFUs. Boxes indicate the 25–75% region, the median is indicated by a line in the box, the average is indicated by a small square, whiskers indicate maximum and minimum values, respectively.</p

Confirmation of <i>sod2343::Km</i> mutants by immunoblotting.

<p>Overnight cultures as indicated were diluted 1∶20, grown for another 4 hours at 37°C, then adjusted to 1 OD (600 nm) and 0.5 ml of each was centrifuged and the pellet resuspended in 50 µl of loading buffer. 10 µl of each sample was loaded on an SDS-PAGE that was subsequently electro-blotted. A polyclonal antiserum raised against GST-SOD2343 fusion protein was diluted 1∶5000 for detection. The blots shown are representative of three independent replicates.</p

Growth defects of <i>sod2343</i> mutants depend on extent of aeration.

<p>Bacterial cultures as indicated were OD-adjusted from overnight cultures and grown at 37°C in LB medium under constant shaking for 8 hours either in baffled flasks (A) or in tubes (B) and the OD (600 nm) was determined. For each strain, data obtained from three independent cultures were averaged; error bars represent plus/minus one standard deviation. Growth of all <i>sod2343</i> mutants was significantly delayed compared to their parentals when grown in baffled flasks (A) (p<0.005, Student's <i>t</i> test,two-tailed, unpaired, for the last three time points). When grown in tubes (B), only the difference between ATCC 17978 and ATCC 17978 <i>sod2343</i> was significant (p<0.05).</p

Minimal inhibitory concentrations determined by Etest.

<p>Average MIC values in [µg/ml] determined from three (MIC³) and six (MIC⁶) independent experiments, respectively; MIC value given in <b>bold</b> indicates that MIC value of mutant is significantly different from MIC value of the corresponding parental strain (p<0.05; Student's t-test, two-tailed, unpaired;</p><p>* indicates p<0.005).</p

<i>A. baumannii sod2343</i> mutants are attenuated in the <i>Galleria mellonella</i> infection model.

<p>(A) Survival of <i>Galleria</i> caterpillars injected with 3×10⁵ CFU of ATCC 17978 <i>sod2343::Km</i> (red), ATCC 17978 (green) and PBS (blue). (B) Infection with 1.5×10⁶ CFU of 07–095 and its <i>sod2343::Km</i> derivative. (C) Infection with 3×10⁵ CFU of 07–102 and its <i>sod2343::Km</i> mutant. Results represent means and standard deviations of at least three independent experiments of 16 larvae per group.</p

Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the β-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis

Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production