The Gene tia, Harbored by the Subtilase-Encoding Pathogenicity Island, Is Involved in the Ability of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing Escherichia coli Strains To Invade Monolayers of Epithelial Cells.

Locus of enterocyte effacement (LEE)-negative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are human pathogens that lack the LEE locus, a pathogenicity island (PAI) involved in the intimate adhesion of LEE-positive strains to the host gut epithelium. The mechanism used by LEE-negative STEC strains to colonize the host intestinal mucosa is still not clear. The cell invasion determinant tia, previously described in enterotoxigenic E. coli strains, has been identified in LEE-negative STEC strains that possess the subtilase-encoding pathogenicity island (SE-PAI). We evaluated the role of the gene tia, present in these LEE-negative STEC strains, in the invasion of monolayers of cultured cells. We observed that these strains were able to invade Caco-2 and HEp-2 cell monolayers and compared their invasion ability with that of a mutant strain in which the gene tia had been inactivated. Mutation of the gene tia resulted in a strong reduction of the invasive phenotype, and complementation of the tia mutation with a functional copy of the gene restored the invasion activity. Moreover, we show that the gene tia is overexpressed in bacteria actively invading cell monolayers, demonstrating that tia is involved in the ability to invade cultured monolayers of epithelial cells shown by SE-PAI-positive E. coli, including STEC, strains. However, the expression of the tia gene in the E. coli K-12 strain JM109 was not sufficient, in its own right, to confer to this strain the ability to invade cell monolayers, suggesting that at least another factor must be involved in the invasion ability displayed by the SE-PAI-positive strains.

Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and virulence.

Efflux pumps of the resistance-nodulation-cell-division (RND) family increase antibiotic resistance in many bacterial pathogens, representing candidate targets for the development of antibiotic adjuvants. RND pumps have also been proposed to contribute to bacterial infection, implying that efflux pump inhibitors (EPIs) could also act as anti-virulence drugs. Nevertheless, EPIs are usually investigated only for their properties as antibiotic adjuvants, while their potential anti-virulence activity is seldom taken into account. In this study it is shown that RND efflux pumps contribute to Pseudomonas aeruginosa PAO1 pathogenicity in an insect model of infection, and that the well-characterized EPI Phe-Arg-ß-naphthylamide (PAßN) is able to reduce in vivo virulence of the P. aeruginosa PAO1 laboratory strain, as well as of clinical isolates. The production of quorum sensing (QS) molecules and of QS-dependent virulence phenotypes is differentially affected by PAßN, depending on the strain. Transcriptomic and phenotypic analyses showed that the protection exerted by PAßN from P. aeruginosa PAO1 infection in vivo correlates with the down-regulation of key virulence genes (e.g. genes involved in iron and phosphate starvation). Since PAßN impacts P. aeruginosa virulence, anti-virulence properties of EPIs are worthy to be explored, taking into account possible strain-specificity of their activity.

The multi-output incoherent feedforward loop constituted by the transcriptional regulators LasR and RsaL confers robustness to a subset of quorum sensing genes in Pseudomonas aeruginosa.

Quorum sensing (QS) is an intercellular communication system which controls virulence-related phenotypes in the human pathogen Pseudomonas aeruginosa. LasR is the QS receptor protein which responds to the signal molecule N-(3-oxododecanoyl)homoserine lactone (3OC12-HSL) and promotes signal production by increasing the transcription of the 3OC12-HSL synthase gene, lasI. LasR also activates the expression of other genes, including rsaL, coding for the RsaL protein which acts as a transcriptional repressor of lasI. Direct gene activation and RsaL-mediated gene repression, both exerted by LasR on the expression of the output gene lasI, generate a regulatory network motif known as the type 1 incoherent feedforward loop (IFFL-1) that governs 3OC12-HSL production. In addition to lasI, RsaL directly represses a set of LasR-activated genes; hence, the IFFL-1 generated by LasR and RsaL is a multi-output IFFL-1. Here we demonstrate that the multi-output IFFL-1 constituted by LasR and RsaL confers robustness with respect to fluctuations in the levels of LasR to the phenotypes controlled by both these transcriptional regulators (e.g. 3OC12-HSL synthesis and pyocyanin production). In contrast, other virulence-related phenotypes controlled by LasR but not by RsaL (e.g. elastase and protease production) are sensitive to changes in LasR levels. Overall, the multi-output IFFL-1 generated by LasR and RsaL splits the QS regulon into two distinct sub-regulons with different robustness with respect to LasR fluctuations. This emerging regulatory property enhances the phenotypic plasticity of P. aeruginosa, thus contributing to its adaptation to changing environments.

Whole-Genome Characterization and Strain Comparison of VT2f-Producing Escherichia coli Causing Hemolytic Uremic Syndrome.

Verotoxigenic Escherichia coli infections in humans cause disease ranging from uncomplicated intestinal illnesses to bloody diarrhea and systemic sequelae, such as hemolytic uremic syndrome (HUS). Previous research indicated that pigeons may be a reservoir for a population of verotoxigenic E. coli producing the VT2f variant. We used whole-genome sequencing to characterize a set of VT2f-producing E. coli strains from human patients with diarrhea or HUS and from healthy pigeons. We describe a phage conveying the vtx2f genes and provide evidence that the strains causing milder diarrheal disease may be transmitted to humans from pigeons. The strains causing HUS could derive from VT2f phage acquisition by E. coli strains with a virulence genes asset resembling that of typical HUS-associated verotoxigenic E. coli.

Toward Repositioning Niclosamide for Antivirulence Therapy of Pseudomonas aeruginosa Lung Infections: Development of Inhalable Formulations through Nanosuspension Technology.

Inhaled antivirulence drugs are currently considered a promising therapeutic option to treat Pseudomonas aeruginosa lung infections in cystic fibrosis (CF). We have recently shown that the anthelmintic drug niclosamide (NCL) has strong quorum sensing (QS) inhibiting activity against P. aeruginosa and could be repurposed as an antivirulence drug. In this work, we developed dry powders containing NCL nanoparticles that can be reconstituted in saline solution to produce inhalable nanosuspensions. NCL nanoparticles were produced by high-pressure homogenization (HPH) using polysorbate 20 or polysorbate 80 as stabilizers. After 20 cycles of HPH, all formulations showed similar properties in the form of needle-shape nanocrystals with a hydrodynamic diameter of approximately 450 nm and a zeta potential of -20 mV. Nanosuspensions stabilized with polysorbate 80 at 10% w/w to NCL (T80_10) showed an optimal solubility profile in simulated interstitial lung fluid. T80_10 was successfully dried into mannitol-based dry powder by spray drying. Dry powder (T80_10 DP) was reconstituted in saline solution and showed optimal in vitro aerosol performance. Both T80_10 and T80_10 DP were able to inhibit P. aeruginosa QS at NCL concentrations of 2.5-10 µM. NCL, and these formulations did not significantly affect the viability of CF bronchial epithelial cells in vitro at microbiologically active concentrations (i.e., ≤10 µM). In vivo acute toxicity studies in rats confirmed no observable toxicity of the NCL T80_10 DP formulation upon intratracheal administration at a concentration 100-fold higher than the anti-QS activity concentration. These preliminary results suggest that NCL repurposed in the form of inhalable nanosuspensions has great potential for the local treatment of P. aeruginosa lung infections as in the case of CF patients.

Affecting Pseudomonas aeruginosa phenotypic plasticity by quorum sensing dysregulation hampers pathogenicity in murine chronic lung infection.

In Pseudomonas aeruginosa quorum sensing (QS) activates the production of virulence factors, playing a critical role in pathogenesis. Multiple negative regulators modulate the timing and the extent of the QS response either in the pre-quorum or post-quorum phases of growth. This regulation likely increases P. aeruginosa phenotypic plasticity and population fitness, facilitating colonization of challenging environments such as higher organisms. Accordingly, in addition to the factors required for QS signals synthesis and response, also QS regulators have been proposed as targets for anti-virulence therapies. However, while it is known that P. aeruginosa mutants impaired in QS are attenuated in their pathogenic potential, the effect of mutations causing a dysregulated timing and/or magnitude of the QS response has been poorly investigated so far in animal models of infection. In order to investigate the impact of QS dysregulation on P. aeruginosa pathogenesis in a murine model of lung infection, the QteE and RsaL proteins have been selected as representatives of negative regulators controlling P. aeruginosa QS in the pre- and post-quorum periods, respectively. Results showed that the qteE mutation does not affect P. aeruginosa lethality and ability to establish chronic infection in mice, despite causing a premature QS response and enhanced virulence factors production in test tube cultures compared to the wild type. Conversely, the post-quorum dysregulation caused by the rsaL mutation hampers the establishment of P. aeruginosa chronic lung infection in mice without affecting the mortality rate. On the whole, this study contributes to a better understanding of the impact of QS regulation on P. aeruginosa phenotypic plasticity during the infection process. Possible fallouts of these findings in the anti-virulence therapy field are also discussed.

A new transcriptional repressor of the pseudomonas aeruginosa quorum sensing receptor gene lasR.

Pseudomonas aeruginosa pathogenic potential is controlled via multiple regulatory pathways, including three quorum sensing (QS) systems. LasR is a key QS signal receptor since it acts as a global transcriptional regulator required for optimal expression of main virulence factors. P. aeruginosa modulates the QS response by integrating this cell density-dependent circuit to environmental and metabolic cues. Hence, QS also controls the adaptation to challenging environmental niches, such as infection sites. However, little is known about the molecular mechanisms connecting QS and other signalling pathways. In this work, DNA-affinity chromatography was used to identify new lasR transcriptional regulators. This approach led to the identification and functional characterization of the TetR-like transcriptional repressor PA3699. This protein was purified and shown to directly bind to the lasR promoter region in vitro. The induction of PA3699 expression in P. aeruginosa PAO1 cultures repressed lasR promoter activity and the production of LasR-dependent virulence factors, such as elastase, pyocyanin, and proteases. These findings suggest a role for PA3699 in P. aeruginosa pathogenicity. P. aeruginosa genome encodes at least 38 TetR-family proteins, and PA3699 is the eighth member of this group functionally characterized so far and the first one shown to bind the lasR promoter in vitro.