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.

Regulation of gene expression by non-phosphorylated response regulators.

Two-component systems (TCSs) are a prominent sensory system in bacteria. A prototypical TCS comprises a membrane-bound sensor histidine kinase (HK) responsible for sensing the signal and a cytoplasmic response regulator (RR) that controls target gene expression. Signal binding activates a phosphotransfer cascade from the HK to the RR. As a result, the phosphorylated RR undergoes a conformational change that leads to activation of the response. Growing experimental evidence indicates that unphosphorylated RRs may also have regulatory functions, and thus, the classical view that the RR is only active when it is phosphorylated needs to be revisited. In this review, we highlight the most recent findings showing that RRs in the non-phosphorylated state control critical bacterial processes that range from secretion of factors to the host, antibiotic resistance, iron transport, stress response, and cell-wall metabolism to biofilm development.

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).

The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus.

RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5'UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5'UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist.

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.

Base pairing interaction between 5'- and 3'-UTRs controls icaR mRNA translation in Staphylococcus aureus.

The presence of regulatory sequences in the 3' untranslated region (3'-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 3'-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the major human pathogen Staphylococcus aureus carry 3'-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 3'-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 3'-UTRs may play in controlling mRNA expression. We showed that base pairing between the 3'-UTR and the Shine-Dalgarno (SD) region of icaR mRNA interferes with the translation initiation complex and generates a double-stranded substrate for RNase III. Deletion or substitution of the motif (UCCCCUG) within icaR 3'-UTR was sufficient to abolish this interaction and resulted in the accumulation of IcaR repressor and inhibition of biofilm development. Our findings provide a singular example of a new potential post-transcriptional regulatory mechanism to modulate bacterial gene expression through the interaction of a 3'-UTR with the 5'-UTR of the same mRNA.

Biofilm matrix exoproteins induce a protective immune response against Staphylococcus aureus biofilm infection.

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.

Bap, a biofilm matrix protein of Staphylococcus aureus prevents cellular internalization through binding to GP96 host receptor.

The biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids, plays a well-known role as a defence structure, protecting bacteria from the host immune system and antimicrobial therapy. However, little is known about its responsibility in the interaction of biofilm cells with host tissues. Staphylococcus aureus, a leading cause of biofilm-associated chronic infections, is able to develop a biofilm built on a proteinaceous Bap-mediated matrix. Here, we used the Bap protein as a model to investigate the role that components of the biofilm matrix play in the interaction of S. aureus with host cells. The results show that Bap promotes the adhesion but prevents the entry of S. aureus into epithelial cells. A broad analysis of potential interaction partners for Bap using ligand overlayer immunoblotting, immunoprecipitation with purified Bap and pull down with intact bacteria, identified a direct binding between Bap and Gp96/GRP94/Hsp90 protein. The interaction of Bap with Gp96 provokes a significant reduction in the capacity of S. aureus to invade epithelial cells by interfering with the fibronectin binding protein invasion pathway. Consistent with these results, Bap deficient bacteria displayed an enhanced capacity to invade mammary gland epithelial cells in a lactating mice mastitis model. Our observations begin to elucidate the mechanisms by which components of the biofilm matrix can facilitate the colonization of host tissues and the establishment of persistent infections.

Genome-wide antisense transcription drives mRNA processing in bacteria.

RNA deep sequencing technologies are revealing unexpected levels of complexity in bacterial transcriptomes with the discovery of abundant noncoding RNAs, antisense RNAs, long 5' and 3' untranslated regions, and alternative operon structures. Here, by applying deep RNA sequencing to both the long and short RNA fractions (<50 nucleotides) obtained from the major human pathogen Staphylococcus aureus, we have detected a collection of short RNAs that is generated genome-wide through the digestion of overlapping sense/antisense transcripts by RNase III endoribonuclease. At least 75% of sense RNAs from annotated genes are subject to this mechanism of antisense processing. Removal of RNase III activity reduces the amount of short RNAs and is accompanied by the accumulation of discrete antisense transcripts. These results suggest the production of pervasive but hidden antisense transcription used to process sense transcripts by means of creating double-stranded substrates. This process of RNase III-mediated digestion of overlapping transcripts can be observed in several evolutionarily diverse Gram-positive bacteria and is capable of providing a unique genome-wide posttranscriptional mechanism to adjust mRNA levels.

Noncontiguous operon atlas for the Staphylococcus aureus genome.

Bacteria synchronize the expression of genes with related functions by organizing genes into operons so that they are cotranscribed together in a single polycistronic messenger RNA. However, some cellular processes may benefit if the simultaneous production of the operon proteins coincides with the inhibition of the expression of an antagonist gene. To coordinate such situations, bacteria have evolved noncontiguous operons (NcOs), a subtype of operons that contain one or more genes that are transcribed in the opposite direction to the other operon genes. This structure results in overlapping transcripts whose expression is mutually repressed. The presence of NcOs cannot be predicted computationally and their identification requires a detailed knowledge of the bacterial transcriptome. In this study, we used direct RNA sequencing methodology to determine the NcOs map in the Staphylococcus aureus genome. We detected the presence of 18 NcOs in the genome of S. aureus and four in the genome of the lysogenic prophage 80α. The identified NcOs comprise genes involved in energy metabolism, metal acquisition and transport, toxin-antitoxin systems, and control of the phage life cycle. Using the menaquinone operon as a proof of concept, we show that disarrangement of the NcO architecture results in a reduction of bacterial fitness due to an increase in menaquinone levels and a decrease in the rate of oxygen consumption. Our study demonstrates the significance of NcO structures in bacterial physiology and emphasizes the importance of combining operon maps with transcriptomic data to uncover previously unnoticed functional relationships between neighbouring genes.

Beneath the water column: Uncovering microplastic pollution in the sublittoral coastal sediments of the Canary Islands, Spain.

Marine ecosystems pollution by microplastics (MPs) is a global problem of special concern. The present study examines the prevalence and distribution of MPs and cellulosic particles in sublittoral coastal sediments of the Canary Islands archipelago (Spain). At twenty-six different locations alongside seven islands, three samples were taken parallel to the shoreline between 1 and 10 m depth (n = 78). Sediment samples were primarily digested with a H2O2 solution followed by four flotations in a saturated NaCl solution. The mean concentration obtained was 3.9 ± 1.6 items/g of dry weight. A similar distribution pattern was observed across all islands concerning particles morphology, color, size and composition: mainly colorless/translucent and blue fibers (60.0%). Additionally, fragments were also found, and to a much lesser extent microbeads, films and tangled messes. MicroFourier Transform Infrared spectroscopy analysis of 12.5% of the fibers, showed that they were mainly cellulosic (54.5%) -either natural or semisynthetic- followed by polyester (22.7%) and acrylic (4.5%). The potential correlation between particle distribution in nearshore sediments and wave intensity was also explored. This work provides the first comprehensive report on the current MPs content of the seabed of the region.

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.

Staphylococcus aureus susceptibility to complestatin and corbomycin depends on the VraSR two-component system.

The overuse of antibiotics in humans and livestock has driven the emergence and spread of antimicrobial resistance and has therefore prompted research on the discovery of novel antibiotics. Complestatin (Cm) and corbomycin (Cb) are glycopeptide antibiotics with an unprecedented mechanism of action that is active even against methicillin-resistant and daptomycin-resistant Staphylococcus aureus. They bind to peptidoglycan and block the activity of peptidoglycan hydrolases required for remodeling the cell wall during growth. Bacterial signaling through two-component transduction systems (TCSs) has been associated with the development of S. aureus antimicrobial resistance. However, the role of TCSs in S. aureus susceptibility to Cm and Cb has not been previously addressed. In this study, we determined that, among all 16 S. aureus TCSs, VraSR is the only one controlling the susceptibility to Cm and Cb. Deletion of vraSR increased bacterial susceptibility to both antibiotics. Epistasis analysis with members of the vraSR regulon revealed that deletion of spdC, which encodes a membrane protein that scaffolds SagB for cleavage of peptidoglycan strands to achieve physiological length, in the vraSR mutant restored Cm and Cb susceptibility to wild-type levels. Moreover, deletion of either spdC or sagB in the wild-type strain increased resistance to both antibiotics. Further analyses revealed a significant rise in the relative amount of peptidoglycan and its total degree of cross-linkage in ΔspdC and ΔsagB mutants compared to the wild-type strain, suggesting that these changes in the cell wall provide resistance to the damaging effect of Cm and Cb. IMPORTANCE Although Staphylococcus aureus is a common colonizer of the skin and digestive tract of humans and many animals, it is also a versatile pathogen responsible for causing a wide variety and number of infections. Treatment of these infections requires the bacteria to be constantly exposed to antibiotic treatment, which facilitates the selection of antibiotic-resistant strains. The development of new antibiotics is, therefore, urgently needed. In this paper, we investigated the role of the sensory system of S. aureus in susceptibility to two new antibiotics: corbomycin and complestatin. The results shed light on the cell-wall synthesis processes that are affected by the presence of the antibiotic and the sensory system responsible for coordinating their activity.

Microplastics in snow of a high mountain national park: El Teide, Tenerife (Canary Islands, Spain).

Human activities have introduced high amounts of microplastics (MPs) into the atmosphere that can be transported long distances and be later deposited in terrestrial and aquatic ecosystems with precipitation (rain or snow). In this work, it has been assessed the presence of MPs in the snow of El Teide National Park (Tenerife, Canary Islands, Spain, 2150-3200 m above sea level) after two storm episodes (January-February 2021). The data set (63 samples) was divided into three groups: i) samples from "accessible areas" (after the first storm episode and in places with a strong previous/recent anthropogenic activity); ii) "pristine areas" (after the second storm episode, in places with no previous anthropogenic activity), and iii) "climbing areas" (after the second storm episode, in places with a soft recent anthropogenic activity). Similar pattern profiles were observed among sampling sites in terms of morphology, colour and size (predominance of blue and black microfibers of 250-750 µm length), as well as in composition (predominance of cellulosic -either natural or semisynthetic-, with a 62.7 %, polyester, 20.9 %, and acrylic, 6.3 %, microfibers); however, significant differences in MPs concentrations were found between samples collected in pristine areas (average concentration of 51 ± 72 items/L) and those obtained in places with a previous anthropogenic activity (average concentration of 167 ± 104 and 188 ± 164 items/L in "accessible areas" and "climbing areas", respectively). This study shows, for the first time, the presence of MPs in snow samples from a high altitude protected area on an insular territory and suggests that the sources of these contaminants could be atmospheric transport and local human outdoor activities.

Bacterial Colonization of Microplastics at the Beaches of an Oceanic Island, Tenerife, Canary Islands.

(1) Isolated systems, such as oceanic islands, are increasingly experiencing important problems related to microplastic debris on their beaches. The formation of microbial biofilm on the surface of microplastics present in marine environments provides potential facilities for microorganisms to survive under the biofilm. Moreover, microplastics act as a vehicle for the dispersion of pathogenic organisms, constituting a new route of exposure for humans. (2) In this study, the microbial content (FIO and Vibrio spp. and Staphylococcus aureus) of microplastics (fragments and pellets) collected from seven beaches of the oceanic island of Tenerife, in the Canary Islands (Spain), was determined. (3) Results showed that Escherichia coli was present in 57.1% of the fragments and 28.5% of the pellets studied. In the case of intestinal Enterococci, 85.7% of the fragments and 57.1% of the pellets tested positive for this parameter. Finally, 100% of the fragments and 42.8% of the pellets analyzed from the different beaches contained Vibrio spp. (4) This study shows that microplastics act as reservoirs of microorganisms that can increase the presence of bacteria indicating faecal and pathogenic contamination in bathing areas.

Three-dimensional evaluation of beaches of oceanic islands as reservoirs of plastic particles in the open ocean.

The quantification of plastic debris on beaches has been extensively used as an indicator of plastic pollution in the marine environment. However, most efforts have focused on surface layers, with few investigations looking deeper into the substrate, thus underestimating total standing stocks. Such information is crucial to improve our understanding of where plastic accumulates in the oceans. In this study, we investigated the three-dimensional distribution of plastic (>1 mm) in three sandy beaches located in oceanic islands of the North Atlantic (Azores and the Canary Islands) that are known to accumulate significant quantities of small plastic debris at the surface layer. On each beach, we collected a total of 16 sediment cores down to 1 m depth, from the high tide line up to the backshore following a stratified random sampling design spread across four different levels across the beach. Samples were taken every 10 cm down to 1 m into the sand. Our results revealed the presence of plastic items in the deepest layers with subsurface layers accounting for 84 % of the total plastic abundance and with a similar pattern in terms of size, shape, colour and composition. Furthermore, we found increasing plastic concentrations towards the upper levels of the beach, indicating longer term accumulation in the backshore. Collectively, this study suggests that the plastic items reaching sandy beaches of the Macaronesia are being incorporated into its deepest layers, acting as reservoirs of plastic in the open ocean.

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.

Deciphering the physiological blueprint of a bacterial cell: revelations of unanticipated complexity in transcriptome and proteome.

During the last few months, several pioneer genome-wide transcriptomic, proteomic and metabolomic studies have revolutionised the understanding of bacterial biological processes, leading to a picture that resembles eukaryotic complexity. Technological advances such as next-generation high-throughput sequencing and high-density oligonucleotide microarrays have allowed the determination, in several bacteria, of the entire boundaries of all expressed transcripts. Consequently, novel RNA-mediated regulatory mechanisms have been discovered including multifunctional RNAs. Moreover, resolution of bacterial proteome organisation (interactome) and global protein localisation (localizome) have unveiled an unanticipated complexity that highlights the significance of protein multifunctionality and localisation in the cell. Also, analysis of a complete bacterial metabolic network has again revealed a high fraction of multifunctional enzymes and an unexpectedly high level of metabolic responses and adaptation. Altogether, these novel approaches have permitted the deciphering of the entire physiological landscape of one of the smallest bacteria, Mycoplasma pneumoniae. Here, we summarise and discuss recent findings aimed at defining the blueprint of any prokaryote.

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella.

Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3'-5'-cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di-GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEF-domain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP-dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable.

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.