Deciphering why Salmonella Gallinarum is less invasive in vitro than Salmonella Enteritidis

International audienceSalmonella Gallinarum and Salmonella Enteritidis are genetically closely related however associated with different pathologies. Several studies have suggested that S. Gallinarum is less invasive in vitro than S. Enteritidis. In this study we confirm that the S. Gallinarum strains tested were much less invasive than the S. Enteritidis strains tested in cells of avian or human origin. In addition, the S. Gallinarum T3SS-1-dependent ability to invade host cells was delayed by two to three hours compared to S. Enteritidis, indicating that T3SS-1-dependent entry is less efficient in S. Gallinarum than S. Enteritidis. This was neither due to a decreased transcription of T3SS-1 related genes when bacteria come into contact with cells, as transcription of hilA, invF and sipA was similar to that observed for S. Enteritidis, nor to a lack of functionality of the S. Gallinarum T3SS-1 apparatus as this apparatus was able to secrete and translocate effector proteins into host cells. In contrast, genome comparison of four S. Gallinarum and two S. Enteritidis strains revealed that all S. Gallinarum genomes displayed the same point mutations in each of the main T3SS-1 effector genes sipA, sopE, sopE2, sopD and sopA

Systemic Administration of Avian Defensin 7: Distribution, Cellular Target, and Antibacterial Potential in Mice

Defensins are natural antimicrobial peptides. The avian beta-defensin AvBD7 isolated from the chicken bone marrow possess broad antibacterial spectrum and strong resistance to proteolysis. However, its ability to fight systemic infections of major concern for public health, such as salmonellosis, is unknown. As a first approach, fluorescence labeling of AvBD7 allowed to track its systemic distribution after intraperitoneal injection in mice using whole body live imaging. It was associated to peritoneal cells and to deeper organs such as the liver. In the next step, the use of labeled AvBD7 allowed to observe its interaction with murine macrophages in culture. After incubation, it was able to penetrate inside the cells through an endocytosis-like mechanism. Furthermore, natural AvBD7 contributed to the control of intracellular multiplication of a multidrug resistant Salmonella strain, after incubation with infected macrophages. Finally, administration in a model of systemic lethal Salmonella infection in mice led to significant improvement of mouse survival, consistently with significant reduction of the liver bacterial load. In conclusion, the results reveal a hitherto unknown intracellular antibacterial effect of AvBD7 in Salmonella target cells and support AvBD7 as a candidate of interest for the treatment of infectious diseases caused by multidrug-resistant pathogenic Enterobacteriaceae

Therapeutic potential of avian beta-defensins in experimental murine salmonellosis

National audienc

Therapeutic of avian beta-defensins in experimental murine salmonellosis

National audienc

Assessment of avian beta-defensin 7 interactions with immune cells <em>in vivo</em> and <em>in vitro</em>

National audienc

Salmonella Typhimurium is able to invade and survive intracellularly independently of its T3SS1

International audienceSalmonella Typhimurium is a bacterial pathogen with an intestinal tropism, causing foodborne diseases. To infect its hosts Salmonella employs a wide range of virulence factors that allow it to actively invade, survive and multiply in a vacuole or in the cytosol. The Salmonella Pathogenicity Island 1-encoded type III secretion system (T3SS1) was the first virulence factor described for its involvement in the invasion process and intracellular fate of Salmonella in the host cell. T3SS1 effectors coordinately mimic various host-cells protein functions, to hijack the cellular machinery allowing, Salmonella invasion and the initiation of the Salmonella containing vacuole (SCV) maturation. Since, two virulence factors Rck and PagN were shown to be involved in the invasion process. Recently, out of fifteen cell lines tested we identified five, in which the Salmonella Typhimurium invasion process is independent of the three known invasion factors. Here, we investigated the intracellular fate of Salmonella Typhimurium in the murine hepatic cell line AML12. We demonstrated that both wild-type Salmonella and T3SS1-invalidated Salmonella followed a common pathway beginning by the formation of a Salmonella containing vacuole (SCV) without classical recruitment of Rho- GTPases ending to Salmonella multiplication in SCV harboring Salmonella inducing filament. These results demonstrate that Salmonella invasion can be completely independent of the T3SS-1, and suggest that other bacterial players allow SCV early maturation. This model is of major interest for establishing the protein composition of an independent T3SS-1 SCV. A better knowledge of these vacuoles will allow the identification of new bacterial and host players necessary for the intracellular survival of Salmonella

Novel method to recover Salmonella enterica cells for Tn-Seq approaches from lettuce leaves and agricultural environments using combination of sonication, filtration, and dialysis membrane

International audienceSalmonella enterica in agricultural environments has become an important concern, due to its potential transmission to humans and the associated public health risks. To identify genes contributing to Salmonella adaptation to such environments, transposon sequencing has been used in recent years. However, isolating Salmonella from atypical hosts, such as plant leaves, can pose technical challenges due to low bacterial content and the difficulty to separate an adequate number of bacteria from host tissues. In this study, we describe a modified methodology using a combination of sonication and filtration to recover S. enterica cells from lettuce leaves. We successfully recovered over a total of 3.5 × 10 6 Salmonella cells in each biological replicate from two six-week old lettuce leaves, 7 days after infiltration with a Salmonella suspension of 5 × 10 7 colony forming units (CFU)/mL. Moreover, we have developed a dialysis membrane system as an alternative method for recovering bacteria from culture medium, mimicking a natural environment. Inoculating 10 7 CFU/mL of Salmonella into the media based on plant (lettuce and tomato) leaf and diluvial sand soil, a final concentration of 10 9.5 and 10 8.5 CFU/mL was obtained, respectively. One millilitre of the bacterial suspension after 24 h incubation at 28 • C using 60 rpm agitation was pelleted, corresponding to 10 9.5 and 10 8.5 cells from leaf-or soil-based media. The recovered bacterial population, from both lettuce leaves and environment-mimicking media, can adequately cover a presumptive library density of 10 6 mutants. In conclusion, this protocol provides an effective method to recover a Salmonella transposon sequencing library from in planta and in vitro systems. We expect this novel technique to foster the study of Salmonella in atypical hosts and environments, as well as other comparable scenarios

Faecal gut microbiota composition of chicks can predict resistance to <em>Salmonella</em> Enteritidis colonization

International audienc

The T3SS-1 : not the only way for Salmonella to invade non phagocytic cells

International audienc