G C A T T A C G G C A T genes Review Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection Emma Piattelli 1, Johann Peltier 1,2 and Olga Soutourina 1,3,* 1 Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Université Paris-Saclay, 91198 Gif-sur-Yvette, France; [email protected] (E.P.); [email protected] (J.P.) 2 Laboratoire Pathogenèses des Bactéries Anaérobies, Institut Pasteur, UMR CNRS 2001, Université de Paris, 75015 Paris, France 3 Institut Universitaire de France, CEDEX 05, 75231 Paris, France * Correspondence: [email protected]; Tel.: +33-169826206 Received: 10 September 2020; Accepted: 13 October 2020; Published: 16 October 2020 Abstract: The ability of pathogenic bacteria to stably infect the host depends on their capacity to respond and adapt to the host environment and on the efficiency of their defensive mechanisms. Bacterial envelope provides a physical barrier protecting against environmental threats. It also constitutes an important sensory interface where numerous sensing systems are located. Signal transduction systems include Two-Component Systems (TCSs) and alternative sigma factors. These systems are able to sense and respond to the ever-changing environment inside the host, altering the bacterial transcriptome to mitigate the impact of the stress. The regulatory networks associated with signal transduction systems comprise small regulatory RNAs (sRNAs) that can be directly involved in the expression of virulence factors. The aim of this review is to describe the importance of TCS- and alternative sigma factor-associated sRNAs in human pathogens during infection. The currently available genome-wide approaches for studies of TCS-regulated sRNAs will be discussed. The differences in the signal transduction mediated by TCSs between bacteria and higher eukaryotes and the specificity of regulatory RNAs for their targets make them appealing targets for discovery of new strategies to fight against multi-resistant bacteria. Keywords: small regulatory RNAs; TCS-associated RNAs; stress response; two-component systems; alternative sigma factors; genome-wide approaches; mixed regulatory networks; host adaptation; bacterial pathogens; quorum sensing; community behavior 1. Introduction Pathogenic and commensal bacteria that colonize mammalian digestive tract or other host locations are subjected to multiple environment fluctuations. Variations in iron and oxygen concentrations, changes in pH and temperature, competition with other bacteria for nutrient availability, host defense, and antibiotic stresses are among the parameters that can affect the life cycle of microorganisms [1,2]. Bacteria have developed different strategies to sense and respond to heterogeneous conditions that lead to the adaptation to the new environment and their survival. A well-coordinated gene expression regulation, including the control of virulence factors, underpins this adaptation [3]. Small RNAs (sRNAs) are a class of riboregulators that act generally at post-transcriptional level in all kingdoms of life [4]. Bacterial sRNAs and eukaryotic microRNAs (miRNAs) shared many features for target recognition and modulation of gene expression [5–7]. In this manuscript, we are focusing on bacterial sRNAs and their importance in the adaptation to a changing environment and in virulence control in pathogens. Bacterial sRNAs are small size, usually non-coding and heterogeneous group of molecules that regulate transcription, translation and mRNA stability through diverse mechanisms of Genes 2020, 11, 1209; doi:10.3390/genes11101209 www.mdpi.com/journal/genes Genes 2020, 11, x FOR PEER REVIEW 2 of 32 Genesheterogeneous2020, 11, 1209 group of molecules that regulate transcription, translation and mRNA stability2 of 31 through diverse mechanisms of action. sRNAs can bind proteins or effector molecules for action.riboswitches, sRNAs interact can bind with proteins foreign or DNA effector or moleculesRNA in the for case riboswitches, of clustered interact regularly with interspaced foreign DNA short or RNApalindromic in the case repeats of clustered (CRISPR) regularly RNA, or interspaced form a duplex short with palindromic mRNA target repeats via (CRISPR) base-pairing RNA, [4,8]. or form Base- a duplexpairing withsRNAs mRNA are targetimportant via base-pairing post-transcriptional [4,8]. Base-pairing regulators sRNAs that are can important either enhance post-transcriptional or repress regulatorsmRNA decay that and/or can either translation enhance by or repressbinding mRNA in most decay cases and to /theor translation 5′ untranslated by binding region in (5’ most UTR) cases of mRNA. Based on their location with respect to the target, sRNAs are differentiated between cis- and to the 50 untranslated region (5’ UTR) of mRNA. Based on their location with respect to the target, sRNAstrans-encoded, are differentiated which could between act eithercis- andin cistrans or in-encoded, trans [7,9]. which While could cis-encoded act either sRNAs in cis orbind in transthe target[7,9]. Whilewith perfectcis-encoded complementarity, sRNAs bind the trans target-encoded with perfect sRNAs complementarity, are only partiallytrans -encodedcomplementary sRNAs areto their only partiallytargets and complementary usually require to theirthe well-characterized targets and usually RNA require chaperone the well-characterized protein Hfq or RNA ProQ chaperone for their proteinaction in Hfq Gram-negative or ProQ for their bacteria action [10,11]. in Gram-negative The role of Hfq bacteria in Gram-positive [10,11]. The role bacteria of Hfq is inless Gram-positive defined and bacteriatrans-encoded is less RNAs defined may and recognizetrans-encoded their targets RNAs mayin an recognize Hfq-independent their targets manne in anr [12]. Hfq-independent Transcription mannerof most [12known]. Transcription trans-encoded of most sRNAs known istrans controlled-encoded by sRNAs transcriptional is controlled factors by transcriptional that include factors Two- thatComponent include Two-ComponentSystems (TCSs) and Systems alternative (TCSs) sigma and alternative factors. TCSs sigma allow factors. adjusting TCSs allow bacterial adjusting gene bacterialexpression gene in response expression to inenvironmental response to environmental cues. Many connections cues. Many between connections TCSs between and sRNAs TCSs have and sRNAsrecently have been recentlyunraveled been and unraveled it is becoming and it clear is becoming that they clear form that complex they form regulatory complex networks regulatory in networksbacteria (Figure in bacteria 1). Well-documented (Figure1). Well-documented examples examplesin both Gram-positive in both Gram-positive and Gram-negative and Gram-negative bacteria bacteriaillustrate illustrate this interplay this interplay between between TCSsTCSs and andsRNAs. sRNAs. For For instance instance,, porin-dependent porin-dependent membrane permeability andand quorum-sensing quorum-sensing control control of of pathogenicity pathogenicity involve involve TCS-regulated TCS-regulated sRNAs sRNAs together together with feedbackwith feedback regulatory regulatory loops. loops. Several Several review review articles articles cover cover various various aspects aspects of this of this large large subject subject [13 –[13–16]. In16]. this In this manuscript, manuscript, we arewe reviewingare reviewing the the most most recent recent advances advances in thein the characterization characterization of theof the role role of sRNAsof sRNAs related related with with virulence virulence control control in in major major human human pathogens pathogens that that areare connectedconnected withwith TCSsTCSs or stress-related sigmasigma factors in complex regulatory circuits.circuits. The main approaches for the identification identification and analysis of these sRNAs will also be discussed asas wellwell asas perspectivesperspectives forfor futurefuture applications.applications. External signal HK ATP ADP HK RR RR RR P sRNAs mRNA TCS sRNA genes sRNAs mRNA targets Hfq TCS regulon genes mRNAs RBP Proteins Niche adaptation Virulence factors Antibiotic resistance Motility Biofilm formation Phage defense Figure 1. Interplay between regulatory sRNAs and two-component systems during bacterial infection. Genes 2020, 11, 1209 3 of 31 Bacteria sense external signals through TCSs generally composed of the histidine kinase (HK) sensor and the response regulator (RR). Upon stimulation, the HK autophosphorylates on conserved histidine residues, transferring a phosphate group from the adenosine triphosphate (ATP). The phosphoryl group is then transferred to a conserved aspartate in the receiver domain of the RR. This modification activates the RR that will in turn regulate through its DNA-binding domain the expression of specific genes, activating or repressing (black arrows for activation and bar-headed lines for inhibition) their transcription. Genes regulated by phosphorylated RR encode either proteins or regulatory sRNAs. To mediate their regulatory functions, some sRNAs cooperate with chaperone proteins, such as Hfq, and interact with the mRNA target, altering the expression at post-transcriptional level. sRNAs can also sequester RNA binding proteins (RBPs). Upon detection of specific signals, proteins and sRNAs regulated by TCSs contribute to bacterial infection. Finally, TCSs often autoregulate their expression (genes in blue and orange encoding respectively the HK and the RR), altering the level