Heterogeneity of Salmonella-host interactions in infected host tissues

Infected host tissues have complex anatomy, diverse cell types, and dynamic inflammation. Traditional infection biology approaches largely ignore this complex host environment and its impact on pathogens, but recent single-cell technologies unravel extensively heterogeneous host-pathogen interactions in vivo. Salmonella are major model pathogens in this field due to the availability of excellent mouse disease models and facile molecular biology. The results show how Salmonella stochastically vary their virulence, exploit differential nutrient availability, experience and respond to widely varying stresses, and have disparate fates ranging from vigorous proliferation to eradication within the same host tissue. Specific Salmonella subsets drive disease progression, while others persist during antimicrobial chemotherapy. Further elucidation of the underlying mechanisms could provide a basis for improved infection control

Increased Production of Outer Membrane Vesicles by Salmonella Interferes with Complement-Mediated Innate Immune Attack

Bacterial outer membrane vesicles (OMVs) enriched with bioactive proteins, toxins, and virulence factors play a critical role in host-pathogen and microbial interactions. The two-component system PhoP-PhoQ (PhoPQ) of Salmonella enterica orchestrates the remodeling of outer membrane lipopolysaccharide (LPS) molecules and concomitantly upregulates OMV production. In this study, we document a novel use of nanoparticle tracking analysis to determine bacterial OMV size and number. Among the PhoPQ-activated genes tested,; pagC; expression had the most significant effect on the upregulation of OMV production. We provide the first evidence that PhoPQ-mediated upregulation of OMV production contributes to bacterial survival by interfering with complement activation. OMVs protected bacteria in a dose-dependent manner, and bacteria were highly susceptible to complement-mediated killing in their absence. OMVs from bacteria expressing PagC bound to complement component C3b in a dose-dependent manner and inactivated it by recruiting complement inhibitor Factor H. As we also found that Factor H binds to PagC, we propose that PagC interferes with complement-mediated killing of Salmonella in the following two steps: first by engaging Factor H, and second, through the production of PagC-enriched OMVs that divert and inactivate the complement away from the bacteria. Since PhoPQ activation occurs intracellularly, the resultant increase in PagC expression and OMV production is suggested to contribute to the local and systemic spread of Salmonella released from dying host cells that supports the infection of new cells.; IMPORTANCE; Bacterial outer membrane vesicles (OMVs) mediate critical bacterium-bacterium and host-microbial interactions that influence pathogenesis through multiple mechanisms, including the elicitation of inflammatory responses, delivery of virulence factors, and enhancement of biofilm formation. As such, there is a growing interest in understanding the underlying mechanisms of OMV production. Recent studies have revealed that OMV biogenesis is a finely tuned physiological process that requires structural organization and selective sorting of outer membrane components into the vesicles. In Salmonella, outer membrane remodeling and OMV production are tightly regulated by its PhoPQ system. In this study, we demonstrate that PhoPQ-regulated OMV production plays a significant role in defense against host innate immune attack. PhoPQ-activated PagC expression recruits the complement inhibitor Factor H and degrades the active C3 component of complement. Our results provide valuable insight into the combination of tools and environmental signals that Salmonella employs to evade complement-mediated lysis, thereby suggesting a strong evolutionary adaptation of this facultative intracellular pathogen to protect itself during its extracellular stage in the host

Type-3 Secretion System-induced pyroptosis protects Pseudomonas against cell-autonomous immunity

Inflammasome-induced pyroptosis comprises a key cell-autonomous immune process against intracellular bacteria, namely the generation of dying cell structures. These so-called pore-induced intracellular traps (PITs) entrap and weaken intracellular microbes. However, the immune importance of pyroptosis against extracellular pathogens remains unclear. Here, we report that Type-3 secretion system (T3SS)-expressing Pseudomonas aeruginosa ( P. aeruginosa ) escaped PIT immunity by inducing a NLRC4 inflammasome-dependent macrophage pyroptosis response in the extracellular environment. To the contrary, phagocytosis of Salmonella Typhimurium promoted NLRC4-dependent PIT formation and the subsequent bacterial caging. Remarkably, T3SS-deficient Pseudomonas were efficiently sequestered within PIT-dependent caging, which favored exposure to neutrophils. Conversely, both NLRC4 and caspase-11 deficient mice presented increased susceptibility to T3SS-deficient P. aeruginosa challenge, but not to T3SS-expressing P. aeruginosa. Overall, our results uncovered that P. aeruginosa uses its T3SS to overcome inflammasome-triggered pyroptosis, which is primarily effective against intracellular invaders. Importance Although innate immune components confer host protection against infections, the opportunistic bacterial pathogen Pseudomonas aeruginosa ( P. aeruginosa ) exploits the inflammatory reaction to thrive. Specifically the NLRC4 inflammasome, a crucial immune complex, triggers an Interleukin (IL)-1β and -18 deleterious host response to P. aeruginosa . Here, we provide evidence that, in addition to IL-1 cytokines, P. aeruginosa also exploits the NLRC4 inflammasome-induced pro-inflammatory cell death, namely pyroptosis, to avoid efficient uptake and killing by macrophages. Therefore, our study reveals that pyroptosis-driven immune effectiveness mainly depends on P. aeruginosa localization. This paves the way toward our comprehension of the mechanistic requirements for pyroptosis effectiveness upon microbial infections and may initiate targeted approaches in order to ameliorate the innate immune functions to infections. Graphical abstract Macrophages infected with T3SS-expressing P. aeruginosa die in a NLRC4-dependent manner, which allows bacterial escape from PIT-mediated cell-autonomous immunity and neutrophil efferocytosis. However, T3SS-deficient P. aeruginosa is detected by both NLRC4 and caspase-11 inflammasomes, which promotes bacterial trapping and subsequent efferocytosis of P. aeruginosa -containing-PITs by neutrophils

Metallome and iron homeostasie of Pseudomonas aeruginosa : a role for siderophores pyocheline and pyoverdine

Pseudomonas aeruginosa est une bactérie à Gram-négatif, pathogène et opportuniste, responsable de nombreuses et sévères infections chez l’homme. Ce microorganisme comme la plupart des organismes vivants a besoin de fer pour sa croissance ainsi que d’autres métaux biologiques comme le zinc, le cuivre, le nickel, le manganèse, le cobalt, le molybdène, le vanadium et d’autres. Afin d’acquérir le fer P. aeruginosa produit deux sidérophores majeurs, la pyoverdine (PVD), souvent considérée comme sidérophore principal, et la pyochéline (PCH). Lors de cette thèse nous avons pu démontrer les enzymes de biosynthèse de ces sidérophores adoptent une organisation spécifique aux pôles des bactéries. De plus, l’étude de la composition en métaux de P. aeruginosa dans différentes conditions de cultures a pu démontrer que la bactérie adapte sa concentration intracellulaire en métaux selon la composition du milieu extracellulaire.Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen. It is responsible for a wide range of human diseases. Iron is an essential element for this organism, like for nearly all other organisms. To a lower extent this organisms needs other metals such as zinc, copper,nickel, manganese and other for its survival. To acquire iron, P. aeruginosa secrets two major siderophores, pyoverdine (PVD) and pyochelin (PCH). During the thesis we have shown that the enzymes involved in the biosynthesis of theses siderophores adopt a specific localization at the bacterial cell poles. Furthermore, the study of the metal composition of P. aeruginosa in different growth conditions has shown that this bacterium is able to adaptits internal metal concentration to the extracellular metal availability

Efficient Dual-Negative Selection for Bacterial Genome Editing

We describe a versatile method for chromosomal gene editing based on classical consecutive single-crossovers. The method exploits rapid plasmid construction using Gibson assembly, a convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector resolution. We used this method to generate in frame deletions, insertions and point mutations in Salmonella enterica with limited hands-on time. Similar strategies allowed efficient gene editing also in Pseudomonas aeruginosa and multi-drug-resistant (MDR) Escherichia coli clinical isolates

Trojan Horse siderophore conjugates induce P. aeruginosa suicide and qualify the TonB protein as a novel antibiotic target

Rising infection rates with multidrug-resistant bacterial pathogens such as Pseudomonas combined with a shallow antibiotic pipeline urgently call for antibiotics with novel modes of action. Herein, we identify the inner membrane protein TonB, motor of active uptake in Gram negative bacteria, as a novel target in antimicrobial therapy. The interaction of the TonB box, the periplasmic N-terminal domain of ferri-siderophore transporters, with the inner membrane protein TonB is crucial for the internalization of essential bacterial metabolites. Overexpression of a TonB box-containing peptide fragment in P. aeruginosa resulted in a growth repression, even in the presence of ferric heme as an iron source. The coupling of three TonB box peptides to synthetic DOTAM and MECAM siderophores with covalent or cleavable linkers of varying length and attachment sites yielded a panel of 24 conjugates in up to 32 synthetic steps. The transporters mediating iron uptake through these conjugates were identified by molecular approaches and transporter knockout mutants to be PfeA and PirA. The conjugates 11, 13 and 17 repressed bacterial growth in P. aeruginosa strains with minimal inhibitory concentrations of 0.5, 4 and 0.1 μM, respectively. The study illustrates a variant of cellular suicide therapy where a transporter imports its own inhibitor; it also demonstrates that artificial siderophores are capable to import large cargo with molecular weights of up to 4 kDa, and suggests that TonB constitutes an attractive target for antimicrobial therapy

Microbiome diversity protects against pathogens by nutrient blocking

<p>The human gut microbiome plays an important role in resisting colonisation of the host by pathogens, but we lack the ability to predict which communities will be protective. We studied how human gut bacteria influence colonisation of two major bacterial pathogens, both <em>in vitro</em> and in gnotobiotic mice. While single species alone had negligible effects, colonisation resistance greatly increased with community diversity. Moreover, this community-level resistance rested critically upon certain species being present. We explain these ecological patterns via the collective ability of resistant communities to consume nutrients that overlap with those used by the pathogen. Further, we apply our findings to successfully predict communities that resist a novel target strain. Our work provides a reason why microbiome diversity is beneficial and suggests a route for the rational design of pathogen-resistant communities.</p><p>Funding provided by: International Human Frontier Science Program Organization<br>Crossref Funder Registry ID: https://ror.org/02ebx7v45<br>Award Number: LT000798/2020</p><p>Funding provided by: Wellcome Trust<br>Crossref Funder Registry ID: https://ror.org/029chgv08<br>Award Number: 209397/Z/17/Z</p><p>Funding provided by: European Research Council<br>Crossref Funder Registry ID: https://ror.org/0472cxd90<br>Award Number: 787932</p><p>Funding provided by: Biotechnology and Biological Sciences Research Council<br>Crossref Funder Registry ID: https://ror.org/00cwqg982<br>Award Number: Studentship</p><p>Funding provided by: Swiss National Science Foundation<br>Crossref Funder Registry ID: https://ror.org/00yjd3n13<br>Award Number: P2EZP3-199916</p><p>Funding provided by: Swiss National Science Foundation<br>Crossref Funder Registry ID: https://ror.org/00yjd3n13<br>Award Number: P500PB-210941</p><p>Various methods were used to collect data provided in this dataset including plate-reader based measurments of optical density or luminescence, flow cytometry, colony counting, and metagenomic sequencing. Raw data is provided in this dataset and allows re-plotting of figures in the manuscript.</p&gt

The pathogen Pseudomonas aeruginosa optimizes the production of the siderophore pyochelin upon environmental challenges

International audienceRepresentation of the regulation of pyochelin production, a siderophore produced by the pathogen Pseudomonas aeruginosa to access iron. This regulation involves a positive auto-regulation loop

Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development

Previous studies have suggested that antibiotic vectorization by siderophores (iron chelators produced by bacteria) considerably increases the efficacy of such drugs. The siderophore serves as a vector: when the pathogen tries to take up iron via the siderophore, it also takes up the antibiotic. Catecholates are among the most common iron-chelating compounds used in synthetic siderophore-antibiotic conjugates. Using RT-qPCR and proteomic approaches, we showed that the presence of catecholate compounds in the medium of Pseudomonas aeruginosa led to strong activation of the transcription and expression of the outer membrane transporter PfeA, the ferri-enterobactin importer. (55) Fe uptake assays on bacteria with and without PfeA expression confirmed that catechol compounds imported iron into P. aeruginosa cells via PfeA. Uptake rates were between 0.3 x 10(3) and 2 x 10(3) Fe atoms/bacterium/min according to the used catechol siderophore in iron-restricted medium, and remained as high as 0.8 x 10(3) Fe atoms/bacterium/min for enterobactin, even in iron-rich medium. RT-qPCR and proteomic approaches showed that in parallel to this switching on of PfeA expression, a repression of the expression of pyochelin (PCH) pathway genes (PCH being one of the two siderophores produced by P. aeruginosa for iron acquisition) was observed