Cytotoxicity and inflammatory potential of two Pseudomonas mosselii strains isolated from clinical samples of hospitalized patients.

BACKGROUND: The genus Pseudomonas includes a heterogeneous set of microorganisms that can be isolated from many different niches and nearly 100 different strains have been described. The best characterized bacterium is Pseudomonas aeruginosa which is the primary agent of opportunistic infection in humans, causing both acute and chronic infections. Other species like fluorescens, putida or mosselii have been sporadically isolated from hospitalized patients but their association with the pathology often remains unclear. RESULTS: This study focuses on the cytotoxicity and inflammatory potential of two strains of Pseudomonas mosselii (ATCC BAA-99 and MFY161) that were recently isolated from clinical samples of hospitalized patients. The behavior of these bacteria was compared to that of the well-known opportunistic pathogen P. aeruginosa PAO1. We found that P. mosselii ATCC BAA-99 and MFY161 are cytotoxic towards Caco-2/TC7 cells, have low invasive capacity, induce secretion of human ß-defensin 2 (HBD-2), alter the epithelial permeability of differentiated cells and damage the F-actin cytoskeleton. CONCLUSIONS: These data bring new insights into P. mosselii virulence, since this bacterium has often been neglected due to its rare occurrence in hospital.

Pseudomonas fluorescens can induce and divert the human ß-defensin-2 secretion in intestinal epithelial cells to enhance its virulence.

The effect of intestinal molecules produced by the host on the virulence of Pseudomonas fluorescens is poorly documented. In the present work, we evaluated the secretion of human ß-defensin-2 (hBD-2) by enterocytes after infection with P. fluorescens (a species previously suggested to be involved in inflammatory bowel disease) and investigated the effect of this host-defense peptide on the bacterial virulence. The results showed that P. fluorescens can induce hBD-2 production in Caco-2/TC7 cells via P38 and ERK MAPK-dependent pathways. Surprisingly, the exposure of P. fluorescens to low doses of the antimicrobial peptide was found to enhance its cytotoxic and proinflammatory effects suggesting a potential feedback mechanism in the dialog between bacteria and the host.

Full virulence of Pseudomonas aeruginosa requires OprF.

OprF is a general outer membrane porin of Pseudomonas aeruginosa, a well-known human opportunistic pathogen associated with severe hospital-acquired sepsis and chronic lung infections of cystic fibrosis patients. A multiphenotypic approach, based on the comparative study of a wild-type strain of P. aeruginosa, its isogenic oprF mutant, and an oprF-complemented strain, showed that OprF is required for P. aeruginosa virulence. The absence of OprF results in impaired adhesion to animal cells, secretion of ExoT and ExoS toxins through the type III secretion system (T3SS), and production of the quorum-sensing-dependent virulence factors pyocyanin, elastase, lectin PA-1L, and exotoxin A. Accordingly, in the oprF mutant, production of the signal molecules N-(3-oxododecanoyl)-l-homoserine lactone and N-butanoyl-l-homoserine lactone was found to be reduced and delayed, respectively. Pseudomonas quinolone signal (PQS) production was decreased, while its precursor, 4-hydroxy-2-heptylquinoline (HHQ), accumulated in the cells. Taken together, these results show the involvement of OprF in P. aeruginosa virulence, at least partly through modulation of the quorum-sensing network. This is the first study showing a link between OprF, PQS synthesis, T3SS, and virulence factor production, providing novel insights into virulence expression.

The clinical Pseudomonas fluorescens MFN1032 strain exerts a cytotoxic effect on epithelial intestinal cells and induces Interleukin-8 via the AP-1 signaling pathway.

BACKGROUND: Pseudomonas fluorescens is present in low number in the intestinal lumen and has been proposed to play a role in Crohn's disease (CD). Indeed, a highly specific antigen, I2, has been detected in CD patients and correlated to the severity of the disease. We aimed to determine whether P. fluorescens was able to adhere to human intestinal epithelial cells (IECs), induce cytotoxicity and activate a proinflammatory response. RESULTS: Behaviour of the clinical strain P. fluorescens MFN1032 was compared to that of the psychrotrophic strain P. fluorescens MF37 and the opportunistic pathogen P. aeruginosa PAO1. Both strains of P. fluorescens were found to adhere on Caco-2/TC7 and HT-29 cells. Their cytotoxicity towards these two cell lines determined by LDH release assays was dose-dependent and higher for the clinical strain MFN1032 than for MF37 but lower than P. aeruginosa PAO1. The two strains of P. fluorescens also induced IL-8 secretion by Caco-2/TC7 and HT-29 cells via the AP-1 signaling pathway whereas P. aeruginosa PAO1 potentially used the NF-kappaB pathway. CONCLUSIONS: The present work shows, for the first time, that P. fluorescens MFN1032 is able to adhere to IECs, exert cytotoxic effects and induce a proinflammatory reaction. Our results are consistent with a possible contribution of P. fluorescens in CD and could explain the presence of specific antibodies against this bacterium in the blood of patients.

Gram-negative bacterial sensors for eukaryotic signal molecules.

Ample evidence exists showing that eukaryotic signal molecules synthesized and released by the host can activate the virulence of opportunistic pathogens. The sensitivity of prokaryotes to host signal molecules requires the presence of bacterial sensors. These prokaryotic sensors, or receptors, have a double function: stereospecific recognition in a complex environment and transduction of the message in order to initiate bacterial physiological modifications. As messengers are generally unable to freely cross the bacterial membrane, they require either the presence of sensors anchored in the membrane or transporters allowing direct recognition inside the bacterial cytoplasm. Since the discovery of quorum sensing, it was established that the production of virulence factors by bacteria is tightly growth-phase regulated. It is now obvious that expression of bacterial virulence is also controlled by detection of the eukaryotic messengers released in the micro-environment as endocrine or neuro-endocrine modulators. In the presence of host physiological stress many eukaryotic factors are released and detected by Gram-negative bacteria which in return rapidly adapt their physiology. For instance, Pseudomonas aeruginosa can bind elements of the host immune system such as interferon-γ and dynorphin and then through quorum sensing circuitry enhance its virulence. Escherichia coli sensitivity to the neurohormones of the catecholamines family appears relayed by a recently identified bacterial adrenergic receptor. In the present review, we will describe the mechanisms by which various eukaryotic signal molecules produced by host may activate Gram-negative bacteria virulence. Particular attention will be paid to Pseudomonas, a genus whose representative species, P. aeruginosa, is a common opportunistic pathogen. The discussion will be particularly focused on the pivotal role played by these new types of pathogen sensors from the sensing to the transduction mechanism involved in virulence factors regulation. Finally, we will discuss the consequence of the impact of host signal molecules on commensally or opportunistic pathogens associated with different human tissue.

Pseudomonas fluorescens alters the intestinal barrier function by modulating IL-1ß expression through hematopoietic NOD2 signaling.

BACKGROUND: Ileal Crohn's disease is related to NOD2 mutations and to a gut barrier dysfunction. Pseudomonas fluorescens has also been associated with ileal Crohn's disease. The aim of this study was to determine the impact of P. fluorescens on the paracellular permeability in ileum and Peyer's patches. METHODS: To explore this question, in vivo and ex vivo experiments were performed in wild-type, Nod2, Nod2, and IL-1R mice together with in vitro analyses using the Caco-2 (epithelial) and the THP-1 (monocyte) human cell lines. RESULTS: Pseudomonas fluorescens increased the paracellular permeability of the intestinal mucosa through the secretion of IL-1ß by the immune cell populations and the activation of myosin light chain kinase in the epithelial cells. Induction of the IL-1ß pathway required the expression of Nod2 in the hematopoietic compartment, and muramyl dipeptide (a Nod2 ligand) had an inhibitory effect. CONCLUSIONS: Pseudomonas fluorescens thus alters the homeostasis of the epithelial barrier function by a mechanism similar to that previously observed for Yersinia pseudotuberculosis. This work further documents a putative role of psychrotrophic bacteria in Crohn's disease.

Growing insights into the safety of bacteriocins: the case of enterocin S37.

Very few studies have been reported on the cytotoxicity and impact of bacteriocins, and especially enterocins, upon eukaryotic cells. In order to gain more information on the safety of bacteriocins, we focused this study on enterocin S37, a bacteriocin produced by Enterococcus faecalis S37. We observed dose-dependent cytotoxicity toward undifferentiated Caco-2/TC7 cells. Moreover, no significant effect on differentiated monolayer Caco-2/TC7 and no apoptotic features were observed when cells were treated with 10 µg/ml of enterocin S37. The results obtained indicate possible safe use of enterocin S37 in the gastrointestinal tract of animals to prevent pathogen invasion and/or infection.

Pseudomonas fluorescens alters epithelial permeability and translocates across Caco-2/TC7 intestinal cells.

BACKGROUND: Pseudomonas fluorescens has long been considered as a psychrotrophic microorganism. Recently, we have shown that clinical strains of P. fluorescens (biovar 1) are able to adapt at a growth temperature of 37°C or above and induce a specific inflammatory response. Interestingly, a highly specific antigen of P. fluorescens, I2, is detected in the serum of patients with Crohn's disease but the possible role of this bacterium in the disease has not yet been explored. In the present study, we examined the ability of a psychrotrophic and a clinical strain of P. fluorescens to modulate the permeability of a Caco-2/TC7 intestinal epithelial model, reorganize the actin cytoskeleton, invade the target cells and translocate across the epithelium. The behaviour of these two strains was compared to that of the well known opportunistic pathogen P. aeruginosa PAO1. RESULTS: Both strains of P. fluorescens were found to decrease the transepithelial resistance (TER) of Caco-2/TC7 differentiated monolayers. This was associated with an increase in paracellular permeability and F-actin microfilaments rearrangements. Moreover, the invasion and translocation tests demonstrated that the two strains used in this study can invade and translocate across the differentiated Caco-2/TC7 cell monolayers. CONCLUSIONS: The present work shows for the first time, that P. fluorescens is able to alter the intestinal epithelial barrier function by disorganizing the F-actin microfilament network. Moreover, we reveal that independently of their origins, the two P. fluorescens strains can translocate across differentiated Caco-2/TC7 cell monolayers by using the transcellular pathway. These findings could, at least in part, explain the presence of the P. fluorescens specific I2 antigen in the serum of patients with Crohn's disease.