The 3'UTR-derived sRNA RsaG coordinates redox homeostasis and metabolism adaptation in response to glucose-6-phosphate uptake in Staphylococcus aureus.

Staphylococcus aureus RsaG is a 3'-untranslated region (3'UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5'- to 3'-end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5'-end of RsaG, leading to its accumulation. RsaG together with uhpT is induced when bacteria are internalized into host cells or in the presence of mucus-secreting cells. Using MS2-affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA, and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, degrade, or repress the translation of its mRNA targets. Although RsaG is conserved only in closely related species, the uhpT 3'UTR of the ape pathogen S. simiae harbors an sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3'UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches.

A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus.

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.

RNA thermoswitches modulate Staphylococcus aureus adaptation to ambient temperatures.

Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5'UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22°C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37°C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37°C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment.

Differential evolution in 3'UTRs leads to specific gene expression in Staphylococcus.

The evolution of gene expression regulation has contributed to species differentiation. The 3' untranslated regions (3'UTRs) of mRNAs include regulatory elements that modulate gene expression; however, our knowledge of their implications in the divergence of bacterial species is currently limited. In this study, we performed genome-wide comparative analyses of mRNAs encoding orthologous proteins from the genus Staphylococcus and found that mRNA conservation was lost mostly downstream of the coding sequence (CDS), indicating the presence of high sequence diversity in the 3'UTRs of orthologous genes. Transcriptomic mapping of different staphylococcal species confirmed that 3'UTRs were also variable in length. We constructed chimeric mRNAs carrying the 3'UTR of orthologous genes and demonstrated that 3'UTR sequence variations affect protein production. This suggested that species-specific functional 3'UTRs might be specifically selected during evolution. 3'UTR variations may occur through different processes, including gene rearrangements, local nucleotide changes, and the transposition of insertion sequences. By extending the conservation analyses to specific 3'UTRs, as well as the entire set of Escherichia coli and Bacillus subtilis mRNAs, we showed that 3'UTR variability is widespread in bacteria. In summary, our work unveils an evolutionary bias within 3'UTRs that results in species-specific non-coding sequences that may contribute to bacterial diversity.

Regulation of Heterogenous LexA Expression in Staphylococcus aureus by an Antisense RNA Originating from Transcriptional Read-Through upon Natural Mispairings in the sbrB Intrinsic Terminator.

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.

Advances in bacterial transcriptome understanding: From overlapping transcription to the excludon concept.

In the last decade, the implementation of high-throughput methods for RNA profiling has uncovered that a large part of the bacterial genome is transcribed well beyond the boundaries of known genes. Therefore, the transcriptional space of a gene very often invades the space of a neighbouring gene, creating large regions of overlapping transcription. The biological significance of these findings was initially regarded with scepticism. However, mounting evidence suggests that overlapping transcription between neighbouring genes conforms to regulatory purposes and provides new strategies for coordinating bacterial gene expression. In this MicroReview, considering the discoveries made in a pioneering transcriptome analysis performed on Listeria monocytogenes as a starting point, we discuss the progress in understanding the biological meaning of overlapping transcription that has given rise to the excludon concept. We also discuss new conditional transcriptional termination events that create antisense RNAs depending on the metabolite concentrations and new genomic arrangements, known as noncontiguous operons, which contain an interspersed gene that is transcribed in the opposite direction to the rest of the operon.

One evolutionarily selected amino acid variation is sufficient to provide functional specificity in the cold shock protein paralogs of Staphylococcus aureus.

Bacterial genomes encode several families of protein paralogs. Discrimination between functional divergence and redundancy among paralogs is challenging due to their sequence conservation. Here, we investigated whether the amino acid differences present in the cold shock protein (CSP) paralogs of Staphylococcus aureus were responsible for functional specificity. Since deletion of cspA reduces the synthesis of staphyloxanthin (STX), we used it as an in vivo reporter of CSP functionality. Complementation of a ΔcspA strain with the different S. aureus CSP variants showed that only CspA could specifically restore STX production by controlling the activity of the stress-associated sigma B factor (σB ). To determine the amino acid residues responsible for CspA specificity, we created several chimeric CSPs that interchanged the amino acid differences between CspA and CspC, which shared the highest identity. We demonstrated that CspA Pro58 was responsible for the specific control of σB activity and its associated phenotypes. Interestingly, CspC gained the biological function of CspA when the E58P substitution was introduced. This study highlights how just one evolutionarily selected amino acid change may be sufficient to modify the specific functionality of CSP paralogs.

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.

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.

The Listeria transcriptional landscape from saprophytism to virulence.

The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.

A common clathrin-mediated machinery co-ordinates cell-cell adhesion and bacterial internalization.

Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E-cadherin, hijack the host adherens junction (AJ) machinery and invade non-phagocytic cells by a clathrin-dependent mechanism. Here, we investigate a potential role for clathrin in cell-cell adhesion. We observed that the initial steps of AJ formation trigger the phosphorylation of clathrin, and its transient localization at forming cell-cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of AJs. Using an InlA/E-cadherin chimera, we show that adherent cells expressing the chimera form AJs with cells expressing E-cadherin. We demonstrate that non-adherent cells expressing the InlA chimera, as bacteria, can be internalized by E-cadherin-expressing adherent cells. Together these results reveal that a common clathrin-mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes.

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.

Gut microbiota produces biofilm-associated amyloids with potential for neurodegeneration.

Age-related neurodegenerative diseases involving amyloid aggregation remain one of the biggest challenges of modern medicine. Alterations in the gastrointestinal microbiome play an active role in the aetiology of neurological disorders. Here, we dissect the amyloidogenic properties of biofilm-associated proteins (BAPs) of the gut microbiota and their implications for synucleinopathies. We demonstrate that BAPs are naturally assembled as amyloid-like fibrils in insoluble fractions isolated from the human gut microbiota. We show that BAP genes are part of the accessory genomes, revealing microbiome variability. Remarkably, the abundance of certain BAP genes in the gut microbiome is correlated with Parkinson's disease (PD) incidence. Using cultured dopaminergic neurons and Caenorhabditis elegans models, we report that BAP-derived amyloids induce α-synuclein aggregation. Our results show that the chaperone-mediated autophagy is compromised by BAP amyloids. Indeed, inoculation of BAP fibrils into the brains of wild-type mice promote key pathological features of PD. Therefore, our findings establish the use of BAP amyloids as potential targets and biomarkers of α-synucleinopathies.

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.

Bacillus thuringiensis Cyt Proteins as Enablers of Activity of Cry and Tpp Toxins against Aedes albopictus.

Aedes albopictus is a species of mosquito, originally from Southeast Asia, that belongs to the Culicidae family and the Dipteran insect order. The distribution of this vector has rapidly changed over the past decade, making most of the temperate territories in the world vulnerable to important human vector-borne diseases such as dengue, yellow fever, zika or chikungunya. Bacillus thuringiensis var. israeliensis (Bti)-based insecticides represent a realistic alternative to the most common synthetic insecticides for the control of mosquito larvae. However, several studies have revealed emerging resistances to the major Bti Crystal proteins such as Cry4Aa, Cry4Ba and Cry11Aa, making the finding of new toxins necessary to diminish the exposure to the same toxicity factors overtime. Here, we characterized the individual activity of Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa against A. albopictus and found a new protein, Cyt1A-like, that increases the activity of Cry11Aa more than 20-fold. Additionally, we demonstrated that Cyt1A-like facilitates the activity three new Bti toxins: Cry53-like, Cry56A-like and Tpp36-like. All in all, these results provide alternatives to the currently available Bti products for the control of mosquito populations and position Cyt proteins as enablers of activity for otherwise non-active crystal proteins.

In vitro modeling of polyclonal infection dynamics within the human airways by Haemophilus influenzae differential fluorescent labeling.

IMPORTANCE: Genomic diversity of nontypeable H. influenzae strains confers phenotypic heterogeneity. Multiple strains of H. influenzae can be simultaneously isolated from clinical specimens, but we lack detailed information about polyclonal infection dynamics by this pathogen. A long-term barrier to our understanding of this host-pathogen interplay is the lack of genetic tools for strain engineering and differential labeling. Here, we present a novel plasmid toolkit named pTBH (toolbox for Haemophilus), with standardized modules for fluorescent or bioluminescent labeling, adapted to H. influenzae requirements but designed to be versatile so it can be utilized in other bacterial species. We present detailed experimental and quantitative image analysis methods, together with proof-of-principle examples, and show the ample possibilities of 3D microscopy, combined with quantitative image analysis, to model H. influenzae polyclonal infection lifestyles and unravel the co-habitation and co-infection dynamics of this respiratory pathogen.

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.