AdrA as a Potential Immunomodulatory Candidate for STING-Mediated Antiviral Therapy That Required Both Type I IFN and TNF-α Production.

Several dinucleotide cyclases, including cyclic GMP-AMP synthase, and their involvement in STING-mediated immunity have been extensively studied. In this study, we tested five bacterial diguanylate cyclases from the Gram-negative bacterium Salmonella Enteritidis, identifying AdrA as the most potent inducer of a STING-mediated IFN response. AdrA wild-type (wt) or its inactive version AdrA mutant (mut) were delivered by an adenovirus (Ad) vector. Dendritic cells obtained from wt mice and infected in vitro with Ad vector containing AdrA wt, but not mut, had increased activation markers and produced large amounts of several immunostimulatory cytokines. For dendritic cells derived from STING-deficient mice, no activation was detected. The potential antiviral activity of AdrA was addressed in hepatitis B virus (HBV)-transgenic and adenovirus-associated virus (AAV)-HBV mouse models. Viremia in serum of Ad AdrA wt-treated mice was reduced significantly compared with that in Ad AdrA mut-injected mice. The viral load in the liver at sacrifice was in line with this finding. To further elucidate the molecular mechanism(s) by which AdrA confers its antiviral function, the response in mice deficient in STING or its downstream effector molecules was analyzed. wt and IFN-αR (IFNAR)-/- animals were additionally treated with anti-TNF-α (Enbrel). Interestingly, albeit less pronounced than in wt mice, in IFNAR-/- and Enbrel-treated wt mice, a reduction of serum viremia was achieved-an observation that was lost in anti-TNF-α-treated IFNAR-/- animals. No effect of AdrA wt was seen in STING-deficient animals. Thus, although STING is indispensable for the antiviral activity of AdrA, type I IFN and TNF-α are both required and act synergistically.

A DIVA vaccine strain lacking RpoS and the secondary messenger c-di-GMP for protection against salmonellosis in pigs.

Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA).

Lack of the PGA exopolysaccharide in Salmonella as an adaptive trait for survival in the host.

Many bacteria build biofilm matrices using a conserved exopolysaccharide named PGA or PNAG (poly-ß-1,6-N-acetyl-D-glucosamine). Interestingly, while E. coli and other members of the family Enterobacteriaceae encode the pgaABCD operon responsible for PGA synthesis, Salmonella lacks it. The evolutionary force driving this difference remains to be determined. Here, we report that Salmonella lost the pgaABCD operon after the divergence of Salmonella and Citrobacter clades, and previous to the diversification of the currently sequenced Salmonella strains. Reconstitution of the PGA machinery endows Salmonella with the capacity to produce PGA in a cyclic dimeric GMP (c-di-GMP) dependent manner. Outside the host, the PGA polysaccharide does not seem to provide any significant benefit to Salmonella: resistance against chlorine treatment, ultraviolet light irradiation, heavy metal stress and phage infection remained the same as in a strain producing cellulose, the main biofilm exopolysaccharide naturally produced by Salmonella. In contrast, PGA production proved to be deleterious to Salmonella survival inside the host, since it increased susceptibility to bile salts and oxidative stress, and hindered the capacity of S. Enteritidis to survive inside macrophages and to colonize extraintestinal organs, including the gallbladder. Altogether, our observations indicate that PGA is an antivirulence factor whose loss may have been a necessary event during Salmonella speciation to permit survival inside the host.

σB Inhibits Poly-N-Acetylglucosamine Exopolysaccharide Synthesis and Biofilm Formation in Staphylococcus aureus.

Staphylococcus aureus clinical strains are able to produce at least two distinct types of biofilm matrixes: biofilm matrixes made of the polysaccharide intercellular adhesin (PIA) or poly-N-acetylglucosamine (PNAG), whose synthesis is mediated by the icaADBC locus, and biofilm matrixes built of proteins (polysaccharide independent). σB is a conserved alternative sigma factor that regulates the expression of more than 100 genes in response to changes in environmental conditions. While numerous studies agree that σB is required for polysaccharide-independent biofilms, controversy persists over the role of σB in the regulation of PIA/PNAG-dependent biofilm development. Here, we show that genetically unrelated S. aureus σB-deficient strains produced stronger biofilms under both static and flow conditions and accumulated higher levels of PIA/PNAG exopolysaccharide than their corresponding wild-type strains. The increased accumulation of PIA/PNAG in the σB mutants correlated with a greater accumulation of the IcaC protein showed that it was not due to adjustments in icaADBC operon transcription and/or icaADBC mRNA stability. Overall, our results reveal that in the presence of active σB, the turnover of Ica proteins is accelerated, reducing the synthesis of PIA/PNAG exopolysaccharide and consequently the PIA/PNAG-dependent biofilm formation capacity.IMPORTANCE Due to its multifaceted lifestyle, Staphylococcus aureus needs a complex regulatory network to connect environmental signals with cellular physiology. One particular transcription factor, named σB (SigB), is involved in the general stress response and the expression of virulence factors. For many years, great confusion has existed about the role of σB in the regulation of the biofilm lifestyle in S. aureus Our study demonstrated that σB is not necessary for exopolysaccharide-dependent biofilms and, even more, that S. aureus produces stronger biofilms in the absence of σB The increased accumulation of exopolysaccharide correlates with higher stability of the proteins responsible for its synthesis. The present findings reveal an additional regulatory layer to control biofilm exopolysaccharide synthesis under stress conditions.

Evaluation of the use of sonication combined with enzymatic treatment for biofilm removal in the microbiological diagnosis of prosthetic joint infection.

Sonicating explanted prosthetic implants to physically remove biofilms is a recognized method for improving the microbiological diagnosis of prosthetic joint infection (PJI); however, chemical and enzymatic treatments have been investigated as alternative biofilm removal methods. We compared the biofilm dislodging efficacy of sonication followed by the addition of enzyme cocktails with different activity spectra in the diagnosis of PJI with that of the sonication of fluid cultures alone. Consecutive patients who underwent prosthesis explantation due to infection at our institution were prospectively enrolled for 1 year. The diagnostic procedure included the collection of five intraoperative tissue cultures, sonication of the removed devices, and conventional culture of the sonication fluid. The resulting sonication fluid was also treated with an enzyme cocktail consisting of homemade dispersin B (0.04 µg/mL) and proteinase K (Sigma; 100 µg/mL) for 45 minutes at 37°C. The resulting sonication (S) and sonication with subsequent enzymatic treatment (SE) fluids were plated for aerobic and anaerobic culture broth for 7 days (aerobic) or 14 days (anaerobic). Identification was performed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Bruker). We included 107 patients from whom a prosthetic implant had been removed, among which PJI was diagnosed in 36 (34%). The sensitivity of S alone was significantly greater than that of SE alone (82% vs 71%; P < 0.05). Four patients with PJI were positive after sonication alone but negative after sonication plus enzymatic treatment. The four microorganisms missed after the addition of the enzyme cocktail were Staphylococcus aureus, two coagulase-negative Staphylococci, and Cutibacterium acnes. In conclusion, sonication alone was more sensitive than sonication followed by enzymatic treatment. The combination of these two methods had no synergistic effect; in contrast, the results suggest that the combination of both dislodgment methods affects the viability of gram-positive microorganisms. IMPORTANCE: While the potential of sonication and enzymes as biofilm dispersal agents has been previously described, the originality of our work resides in the combination of both methods, which is hypothesized to enhance the ability to remove biofilm and, therefore, improve the microbiological diagnosis of PJI.

Coordinated cyclic-di-GMP repression of Salmonella motility through YcgR and cellulose.

Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition.

Salmonella biofilm development depends on the phosphorylation status of RcsB.

The Rcs phosphorelay pathway is a complex signaling pathway involved in the regulation of many cell surface structures in enteric bacteria. In response to environmental stimuli, the sensor histidine kinase (RcsC) autophosphorylates and then transfers the phosphate through intermediary steps to the response regulator (RcsB), which, once phosphorylated, regulates gene expression. Here, we show that Salmonella biofilm development depends on the phosphorylation status of RcsB. Thus, unphosphorylated RcsB, hitherto assumed to be inactive, is essential to activate the expression of the biofilm matrix compounds. The prevention of RcsB phosphorylation either by the disruption of the phosphorelay at the RcsC or RcsD level or by the production of a nonphosphorylatable RcsB allele induces biofilm development. On the contrary, the phosphorylation of RcsB by the constitutive activation of the Rcs pathway inhibits biofilm development, an effect that can be counteracted by the introduction of a nonphosphorylatable RcsB allele. The inhibition of biofilm development by phosphorylated RcsB is due to the repression of CsgD expression, through a mechanism dependent on the accumulation of the small noncoding RNA RprA. Our results indicate that unphosphorylated RcsB plays an active role for integrating environmental signals and, more broadly, that RcsB phosphorylation acts as a key switch between planktonic and sessile life-styles in Salmonella enterica serovar Typhimurium.

Experimental Polymorphism Survey in Intergenic Regions of the icaADBCR Locus in Staphylococcus aureus Isolates from Periprosthetic Joint Infections.

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI) characterized by bacterial biofilm formation and recalcitrance to immune-mediated clearance and antibiotics. The molecular events behind PJI infection are yet to be unraveled. In this sense, identification of polymorphisms in bacterial genomes may help to establish associations between sequence variants and the ability of S. aureus to cause PJI. Here, we report an experimental nucleotide-level survey specifically aimed at the intergenic regions (IGRs) of the icaADBCR locus, which is responsible for the synthesis of the biofilm exopolysaccharide PIA/PNAG, in a collection of strains sampled from PJI and wounds. IGRs of the icaADBCR locus were highly conserved and no PJI-specific SNPs were found. Moreover, polymorphisms in these IGRs did not significantly affect transcription of the icaADBC operon under in vitro laboratory conditions. In contrast, an SNP within the icaR coding region, resulting in a V176E change in the transcriptional repressor IcaR, led to a significant increase in icaADBC operon transcription and PIA/PNAG production and a reduction in S. aureus virulence in a Galleria mellonella infection model. In conclusion, SNPs in icaADBCR IGRs of S. aureus isolates from PJI are not associated with icaADBC expression, PIA/PNAG production and adaptation to PJI.

Functional analysis of intergenic regulatory regions of genes encoding surface adhesins in Staphylococcus aureus isolates from periprosthetic joint infections.

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI). Surface adhesins play an important role in the primary attachment to plasma proteins that coat the surface of prosthetic devices after implantation. Previous efforts to identify a genetic component of the bacterium that confers an enhanced capacity to cause PJI have focused on gene content, kmers, or single-nucleotide polymorphisms (SNPs) in coding sequences. Here, using a collection of S. aureus strains isolated from PJI and wounds, we investigated whether genetic variations in the regulatory region of genes encoding surface adhesins lead to differences in their expression levels and modulate the capacity of S. aureus to colonize implanted prosthetic devices. The data revealed that S. aureus isolates from the same clonal complex (CC) contain a specific pattern of SNPs in the regulatory region of genes encoding surface adhesins. As a consequence, each clonal lineage shows a specific profile of surface proteins expression. Co-infection experiments with representative isolates of the most prevalent CCs demonstrated that some lineages have a higher capacity to colonize implanted catheters in a murine infection model, which correlated with a greater ability to form a biofilm on coated surfaces with plasma proteins. Together, results indicate that differences in the expression level of surface adhesins may modulate the propensity of S. aureus strains to cause PJI. Given the high conservation of surface proteins among staphylococci, our work lays the framework for investigating how diversification at intergenic regulatory regions affects the capacity of S. aureus to colonize the surface of medical implants.

Role of sodium salicylate in Staphylococcus aureus quorum sensing, virulence, biofilm formation and antimicrobial susceptibility.

The widespread threat of antibiotic resistance requires new treatment options. Disrupting bacterial communication, quorum sensing (QS), has the potential to reduce pathogenesis by decreasing bacterial virulence. The aim of this study was to investigate the influence of sodium salicylate (NaSa) on Staphylococcus aureus QS, virulence production and biofilm formation. In S. aureus ATCC 25923 (agr III), with or without serum, NaSa (10 mM) downregulated the agr QS system and decreased the secretion levels of alpha-hemolysin, staphopain A and delta-hemolysin. Inhibition of agr expression caused a downregulation of delta-hemolysin, decreasing biofilm dispersal and increasing biofilm formation on polystyrene and titanium under static conditions. In contrast, NaSa did not increase biofilm biomass under flow but caused one log10 reduction in biofilm viability on polystyrene pegs, resulting in biofilms being twice as susceptible to rifampicin. A concentration-dependent effect of NaSa was further observed, where high concentrations (10 mM) decreased agr expression, while low concentrations (≤0.1 mM) increased agr expression. In S. aureus 8325-4 (agr I), a high concentration of NaSa (10 mM) decreased hla expression, and a low concentration of NaSa (≤1 mM) increased rnaIII and hla expression. The activity of NaSa on biofilm formation was dependent on agr type and material surface. Eight clinical strains isolated from prosthetic joint infection (PJI) or wound infection belonging to each of the four agr types were evaluated. The four PJI S. aureus strains did not change their biofilm phenotype with NaSa on the clinically relevant titanium surface. Half of the wound strains (agr III and IV) did not change the biofilm phenotype in the 3D collagen wound model. In addition, compared to the control, ATCC 25923 biofilms formed with 10 mM NaSa in the collagen model were more susceptible to silver. It is concluded that NaSa can inhibit QS in S. aureus, decreasing the levels of toxin production with certain modulation of biofilm formation. The effect on biofilm formation was dependent on the strain and material surface. It is suggested that the observed NaSa inhibition of bacterial communication is a potential alternative or adjuvant to traditional antibiotics.

Evaluation of a Salmonella Strain Lacking the Secondary Messenger C-di-GMP and RpoS as a Live Oral Vaccine.

Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. C-di-GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine.

Biofilm dispersion and quorum sensing.

Biofilm development and quorum sensing (QS) are closely interconnected processes. Biofilm formation is a cooperative group behaviour that involves bacterial populations living embedded in a self-produced extracellular matrix. QS is a cell-cell communication mechanism that synchronizes gene expression in response to population cell density. Intuitively, it would appear that QS might coordinate the switch to a biofilm lifestyle when the population density reaches a threshold level. However, compelling evidence obtained in different bacterial species coincides in that activation of QS occurs in the formed biofilm and activates the maturation and disassembly of the biofilm in a coordinate manner. The aim of this review is to illustrate, using four bacterial pathogens as examples, the emergent concept that QS activates the biofilm dispersion process.