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Bacteria usually form biofilms as a defense mechanism against predation by bacterivorous nematodes. In this context, the second messenger c-di-GMP from the wild-type Pseudomonas syringae MB03 actuates the transcriptional factor FleQ03 to elicit biofilm-dependent nematicidal activity against Caenorhabditis elegans N2. P. syringae MB03 cells exhibited nematicidal activity and c-di-GMP content in P. syringae MB03 cells was increased after feeding to nematodes. Expression of a diguanylate cyclase (DGC) gene in P. syringae MB03 resulted in an increased c-di-GMP content, biofilm yield and nematicidal activity, whereas converse effects were obtained when expressing a phosphodiesterase (PDE) gene. Molecular docking and isothermal titration calorimetry assays verified the affinity activity between c-di-GMP and the FleQ03 protein. The disruption of the fleQ03 gene in P. syringae MB03, while increasing c-di-GMP content, significantly diminished both biofilm formation and nematicidal activity. Interestingly, P. syringae MB03 formed a full-body biofilm around the worms against predation, probably extending from the tail to the head, whereas it was not observed in the fleQ03 gene disrupted cells. Thus, we hypothesized that c-di-GMP incorporated FleQ03 to reinforce bacterial biofilm and biofilm-dependent pathogenicity in response to C. elegans predation, providing insights into a possible means of resisting bacterivorous nematodes by bacteria in natural ecosystems.

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BACKGROUND: Calprotectin, a calcium-binding protein, plays a crucial role in inflammation and has been associated with various inflammatory diseases, including asthma. However, its regulation and impact on steroid hyporesponsiveness, especially in severe asthma, remain poorly understood. METHODS: This study investigated the regulation of calprotectin proteins (S100A8 and S100A9) by IL-17 and its role in steroid hyporesponsiveness using in vitro and in vivo models. Calprotectin expression was assessed in primary bronchial fibroblasts from healthy controls and severe asthmatic patients, as well as in mouse models of steroid hyporesponsive lung inflammation induced by house dust mite (HDM) allergen and cyclic-di-GMP (cdiGMP) adjuvant. The effects of IL-17A stimulation on calprotectin expression and steroid response markers in bronchial epithelial and fibroblast cells were examined. Additionally, the therapeutic potential of paquinimod, a calprotectin inhibitor, in mitigating airway inflammation and restoring steroid response signatures in the mouse model was evaluated. RESULTS: The results demonstrated upregulation of calprotectin expression in asthmatic bronchial fibroblasts compared to healthy controls, as well as in refractory asthma samples compared to non-refractory asthma. IL-17 stimulation induced calprotectin expression and dysregulated glucocorticoid response signatures in lung epithelial and fibroblast cells. Treatment with paquinimod reversed IL-17-induced dysregulation of steroid signatures, indicating the involvement of calprotectin in this process. In the HDM/cdiGMP mouse model, paquinimod significantly attenuated airway inflammation and hyperresponsiveness, and restored steroid response signatures, whereas dexamethasone showed limited efficacy. Mechanistically, paquinimod inhibited MAPK/ERK and NF-κB pathways downstream of calprotectin, leading to reduced lung inflammation. CONCLUSION: These findings highlight calprotectin as a potential therapeutic target regulated by IL-17 in steroid hyporesponsive asthma. Targeting calprotectin may offer a promising approach to alleviate airway inflammation and restore steroid responsiveness in severe asthma. Further investigations are warranted to explore its therapeutic potential in clinical settings and elucidate its broader implications in steroid mechanisms of action.

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BACKGROUND: Severe bacterial infections can trigger acute lung injury (ALI) and acute respiratory distress syndrome, with bacterial pathogen-associated molecular patterns (PAMPs) exacerbating the inflammatory response, particularly in COVID-19 patients. Cyclic-di-GMP (CDG), one of the PAMPs, is synthesized by various Gram-positve and Gram-negative bacteria. Previous studies mainly focused on the inflammatory responses triggered by intracellular bacteria-released CDG. However, how extracellular CDG, which is released by bacterial autolysis or rupture, activates the inflammatory response remains unclear. METHODS: The interaction between extracellular CDG and myeloid differentiation protein 2 (MD2) was investigated using in vivo and in vitro models. MD2 blockade was achieved using specific inhibitor and genetic knockout mice. Site-directed mutagenesis, co-immunoprecipitation, SPR and Bis-ANS displacement assays were used to identify the potential binding sites of MD2 on CDG. RESULTS: Our data show that extracellular CDG directly interacts with MD2, leading to activation of the TLR4 signalling pathway and lung injury. Specific inhibitors or genetic knockout of MD2 in mice significantly alleviated CDG-induced lung injury. Moreover, isoleucine residues at positions 80 and 94, along with phenylalanine at position 121, are essential for the binding of MD2 to CDG. CONCLUSION: These results reveal that extracellular CDG induces lung injury through direct interaction with MD2 and activation of the TLR4 signalling pathway, providing valuable insights into bacteria-induced ALI mechanisms and new therapeutic approaches for the treatment of bacterial co-infection in COVID-19 patients.

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The bacterial predator Bdellovibrio bacteriovorus is considered to be obligatorily prey (host)-dependent (H-D), and thus unable to form biofilms. However, spontaneous host-independent (H-I) variants grow axenically and can form robust biofilms. A screen of 350 H-I mutants revealed that single mutations in stator genes fliL or motA were sufficient to generate flagellar motility-defective H-I strains able to adhere to surfaces but unable to develop biofilms. The variants showed large transcriptional shifts in genes related to flagella, prey-invasion, and cyclic-di-GMP (CdG), as well as large changes in CdG cellular concentration relative to the H-D parent. The introduction of the parental fliL allele resulted in a full reversion to the H-D phenotype, but we propose that specific interactions between stator proteins prevented functional complementation by fliL paralogs. In contrast, specific mutations in a pilus-associated protein (Bd0108) mutant background were necessary for biofilm formation, including secretion of extracellular DNA (eDNA), proteins, and polysaccharides matrix components. Remarkably, fliL disruption strongly reduced biofilm development. All H-I variants grew similarly without prey, showed a strain-specific reduction in predatory ability in prey suspensions, but maintained similar high efficiency in prey biofilms. Population-wide allele sequencing suggested additional routes to host independence. Thus, stator and invasion pole-dependent signaling control the H-D and the H-I biofilm-forming phenotypes, with single mutations overriding prey requirements, and enabling shifts from obligate to facultative predation, with potential consequences on community dynamics. Our findings on the facility and variety of changes leading to facultative predation also challenge the concept of Bdellovibrio and like organisms being obligate predators. IMPORTANCE: The ability of bacteria to form biofilms is a central research theme in biology, medicine, and the environment. We show that cultures of the obligate (host-dependent) "solitary" predatory bacterium Bdellovibrio bacteriovorus, which cannot replicate without prey, can use various genetic routes to spontaneously yield host-independent (H-I) variants that grow axenically (as a single species, in the absence of prey) and exhibit various surface attachment phenotypes, including biofilm formation. These routes include single mutations in flagellar stator genes that affect biofilm formation, provoke motor instability and large motility defects, and disrupt cyclic-di-GMP intracellular signaling. H-I strains also exhibit reduced predatory efficiency in suspension but high efficiency in prey biofilms. These changes override the requirements for prey, enabling a shift from obligate to facultative predation, with potential consequences on community dynamics.

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The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger cyclic di-GMP (c di-GMP) signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but five of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the four intact DGCs in a S. flexneri strain, where these four DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these four DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC pseudogenes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.

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The second messenger cyclic diguanylate monophosphate (c-di-GMP) regulates a wide range of bacterial behaviours through diverse mechanisms and binding receptors. Single-domain PilZ proteins, the most widespread and abundant known c-di-GMP receptors in bacteria, act as trans-acting adaptor proteins that enable c-di-GMP to control signalling pathways with high specificity. This study identifies a single-domain PilZ protein, XAC3402 (renamed N5MapZ), from the phytopathogen Xanthomonas citri subsp. citri (Xcc), which modulates Xcc virulence by directly interacting with the methyltransferase HemK. Through yeast two-hybrid, co-immunoprecipitation and immunofluorescent staining, we demonstrated that N5MapZ and HemK interact directly under both in vitro and in vivo conditions, with the strength of the protein-protein interaction decreasing at high c-di-GMP concentrations. This finding distinguishes N5MapZ from other characterized single-domain PilZ proteins, as it was previously known that c-di-GMP enhances the interaction between those single-domain PilZs and their protein partners. This observation is further supported by the fact that the c-di-GMP binding-defective mutant N5MapZR10A can interact with HemK to inhibit the methylation of the class 1 translation termination release factor PrfA. Additionally, we found that HemK plays an important role in Xcc pathogenesis, as the deletion of hemK leads to extensive phenotypic changes, including reduced virulence in citrus plants, decreased motility, production of extracellular enzymes and stress tolerance. Gene expression analysis has revealed that c-di-GMP and the HemK-mediated pathway regulate the expression of multiple virulence effector proteins, uncovering a novel regulatory mechanism through which c-di-GMP regulates Xcc virulence by mediating PrfA methylation via the single-domain PilZ adaptor protein N5MapZ.

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A riboswitch generally regulates the expression of its downstream genes through conformational change in its expression platform (EP) upon ligand binding. The cyclic diguanosine monophosphate (c-di-GMP) class I riboswitch Bc1 is widespread and conserved among Bacillus cereus group species. In this study, we revealed that Bc1 has a long EP with two typical ρ-independent terminator sequences 28 bp apart. The upstream terminator T1 is dominant in vitro, while downstream terminator T2 is more efficient in vivo. Through mutation analysis, we elucidated that Bc1 exerts a rare and incoherent "transcription-translation" dual regulation with T2 playing a crucial role. However, we found that Bc1 did not respond to c-di-GMP under in vitro transcription conditions, and the expressions of downstream genes did not change with fluctuation in intracellular c-di-GMP concentration. To explore this puzzle, we conducted SHAPE-MaP and confirmed the interaction of Bc1 with c-di-GMP. This shows that as c-di-GMP concentration increases, T1 unfolds but T2 remains almost intact and functional. The presence of T2 masks the effect of T1 unwinding, resulting in no response of Bc1 to c-di-GMP. The high Shannon entropy values of EP region imply the potential alternative structures of Bc1. We also found that zinc uptake regulator can specifically bind to the dual terminator coding sequence and slightly trigger the response of Bc1 to c-di-GMP. This work will shed light on the dual-regulation riboswitch and enrich our understanding of the RNA world.IMPORTANCEIn nature, riboswitches are involved in a variety of metabolic regulation, most of which preferentially regulate transcription termination or translation initiation of downstream genes in specific ways. Alternatively, the same or different riboswitches can exist in tandem to enhance regulatory effects or respond to multiple ligands. However, many putative conserved riboswitches have not yet been experimentally validated. Here, we found that the c-di-GMP riboswitch Bc1 with a long EP could form a dual terminator and exhibit non-canonical and incoherent "transcription-translation" dual regulation. Besides, zinc uptake regulator specifically bound to the coding sequence of the Bc1 EP and slightly mediated the action of Bc1. The application of SHAPE-MaP to the dual regulation mechanism of Bc1 may establish the foundation for future studies of such complex untranslated regions in other bacterial genomes.

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Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Prolonged infection allows the accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonizing upper airway environments. Here, we model this process using an experimental evolution approach. Pseudomonas aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments-sinus and lungs, under CF and non-CF conditions-selected for loss of twitching motility, increased resistance to multiple antibiotic classes, and a hyper-biofilm phenotype. These traits conferred increased airway colonization potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching, and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included the loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection.

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Bordetella bronchiseptica is a highly contagious respiratory bacterial veterinary pathogen. In this study the contribution of the transcriptional regulators BvgR, RisA, RisS, and the phosphorylation of RisA to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation were evaluated. Next Generation Sequencing (RNASeq) was used to differentiate the global gene regulation of both virulence-activated and virulence-repressed genes by each of these factors. The BvgAS system, along with BvgR, RisA, and the phosphorylation of RisA served in cyclic-di-GMP degradation. BvgR and unphosphorylated RisA were found to temporally regulate motility. Additionally, BvgR, RisA, and RisS were found to be required for biofilm formation.

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The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G-C content play a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

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In addition to comprising monomers of nucleic acids, nucleotides have signaling functions and act as second messengers in both prokaryotic and eukaryotic cells. The most common example is cyclic AMP (cAMP). Nucleotide signaling is a focus of great interest in bacteria. Cyclic di-AMP (c-di-AMP), cAMP, and cyclic di-GMP (c-di-GMP) participate in biological events such as bacterial growth, biofilm formation, sporulation, cell differentiation, motility, and virulence. Moreover, the cyclic-di-nucleotides (c-di-nucleotides) produced in pathogenic intracellular bacteria can affect eukaryotic host cells to allow for infection. On the other hand, non-cyclic nucleotide molecules pppGpp and ppGpp are alarmones involved in regulating the bacterial response to nutritional stress; they are also considered second messengers. These second messengers can potentially be used as therapeutic agents because of their immunological functions on eukaryotic cells. In this review, the role of c-di-nucleotides and cAMP as second messengers in different bacterial processes is addressed.

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Objective: This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase (DGC) in Vibrio cholerae and how its transcription is regulated by Fur and HapR. Methods: The roles of VCA0560 was investigated by utilizing various phenotypic assays, including colony morphological characterization, crystal violet staining, Cyclic di-GMP (c-di-GMP) quantification, and swimming motility assay. The regulation of the VCA0560 gene by Fur and HapR was analyzed by luminescence assay, electrophoretic mobility shift assay, and DNase I footprinting. Results: VCA0560 gene mutation did not affect biofilm formation, motility, and c-di-GMP synthesis in V. cholerae, and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity. The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator HapR. Conclusion: Overexpressed VCA0560 functions as an active DGC in V. cholerae, and its transcription is repressed by Fur and HapR.

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Pseudomonas aeruginosa grows as a biofilm under many environmental conditions, and the bacterium can disperse from biofilms via highly regulated, dynamic processes. However, physiologic triggers of biofilm dispersal remain poorly understood. Based on prior literature describing dispersal triggered by forms of starvation, we tested bacterial respiratory inhibitors for biofilm dispersal in two models resembling chronic airway infections. Our underlying hypothesis was that respiratory inhibitors could serve as a model for the downstream effects of starvation. We used two experimental conditions. In the first condition, biofilms were grown and dispersed from the surface of airway epithelial cells, and the second condition was a model where biofilms were grown on glass in cell culture media supplemented with host-relevant iron sources. In both biofilm models, the respiratory inhibitors potassium cyanide and sodium azide each triggered biofilm dispersal. We hypothesized that cyanide-induced dispersal was due to respiratory inhibition rather than signaling via an alternative mechanism, and, indeed, if respiration was supported by overexpression of cyanide-insensitive oxidase, dispersal was prevented. Dispersal required the activity of the cyclic-di-GMP regulated protease LapG, reinforcing the role of matrix degradation in dispersal. Finally, we examined the roles of individual phosphodiesterases, previously implicated in dispersal to specific triggers, and found signaling to be highly redundant. Combined deletion of the phosphodiesterases dipA, bifA, and rbdA was required to attenuate the dispersal phenotype. In summary, this work adds insight into the physiology of biofilm dispersal under environmental conditions in which bacterial respiration is abruptly limited. IMPORTANCE The bacterium Pseudomonas aeruginosa grows in biofilm communities that are very difficult to treat in human infections. Growing as a biofilm can protect bacteria from antibiotics and the immune system. Bacteria can leave a biofilm through a process called "dispersal." Dispersed bacteria seed new growth areas and are more susceptible to killing by antibiotics. The triggers for biofilm dispersal are not well understood, and if we understood dispersal better it might lead to the development of new treatments for infection. In this paper, we find that inhibiting P. aeurginosa's ability to respire (generate energy) can trigger dispersal from a biofilm grown in association with human respiratory epithelial cells in culture. The dispersal process requires a protease which is previously known to degrade the biofilm matrix. These findings give us a better understanding of how the biofilm dispersal process works so that future research can discover better ways of clearing bacteria growing in biofilms.

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The regulation of biofilm and motile states as alternate bacterial lifestyles has been studied extensively in flagellated bacteria, where the second messenger cyclic-di-GMP (cdG) plays a crucial role. However, much less is known about the mechanisms of such regulation in motile bacteria without flagella. The bacterial type IV pilus (T4P) serves as a motility apparatus that enables Myxococcus xanthus to move on solid surfaces. PilB, the T4P assembly ATPase, is, therefore, required for T4P-dependent motility in M. xanthus. Interestingly, T4P is also involved in the regulation of exopolysaccharide as the biofilm matrix material in this bacterium. A newly discovered cdG-binding domain, MshEN, is conserved in the N-terminus of PilB (PilBN) in M. xanthus and other bacteria. This suggests that cdG may bind to PilB to control the respective outputs that regulate biofilm development and T4P-powered motility. In this study, we aimed to validate M. xanthus PilB as a cdG effector protein. We performed a systematic mutational analysis of its cdG-binding domain to investigate its relationship with motility, piliation, and biofilm formation. Excluding those resulting in low levels of PilB protein, all other substitution mutations in PilBN resulted in pilB mutants with distinct and differential phenotypes in piliation and biofilm levels in M. xanthus. This suggests that the PilBN domain plays dual roles in modulating motility and biofilm levels, and these two functions of PilB can be dependent on and independent of each other in M. xanthus. IMPORTANCE The regulation of motility and biofilm by cyclic-di-GMP in flagellated bacteria has been extensively investigated. However, our knowledge regarding this regulation in motile bacteria without flagella remains limited. Here, we aimed to address this gap by investigating a non-flagellated bacterium with motility powered by bacterial type-IV pilus (T4P). Previous studies hinted at the possibility of Myxococcus xanthus PilB, the T4P assembly ATPase, serving as a cyclic-di-GMP effector involved in regulating both motility and biofilm. Our findings strongly support the hypothesis that PilB directly interacts with cyclic-di-GMP to act as a potential switch to promote biofilm formation or T4P-dependent motility. These results shed light on the bifurcation of PilB functions and its pivotal role in coordinating biofilm formation and T4P-mediated motility.

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Pectobacterium carotovorum is an important plant pathogen responsible for the destruction of crops through bacterial soft rot, which is modulated by oxygen (O2) concentration. A soluble globin coupled sensor protein, Pcc DgcO (also referred to as PccGCS) is one way through which P. carotovorum senses oxygen. DgcO contains a diguanylate cyclase output domain producing c-di-GMP. Synthesis of the bacterial second messenger c-di-GMP is increased upon oxygen binding to the sensory globin domain. This work seeks to understand regulation of function by DgcO at the transcript level. RNA sequencing and differential expression analysis revealed that the deletion of DgcO only affects transcript levels in cells grown under aerobic conditions. Differential expression analysis showed that DgcO deletion alters transcript levels for metal transporters. These results, followed by inductively coupled plasma-mass spectrometry showing decreased concentrations of six biologically relevant metals upon DgcO deletion, provide evidence that a globin coupled sensor can affect cellular metal content. These findings improve the understanding of the transcript level control of O2-dependent phenotypes in an important phytopathogen and establish a basis for further studies on c-di-GMP-dependent functions in P. carotovorum.

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Introduction: Secretory IgA (SIgA) protects the intestinal epithelium from enteric pathogens such as Salmonella enterica serovar Typhimurium (STm) through a process known as immune exclusion, where invading bacteria are aggregated via antibody cross-linking, encased in mucus, and then cleared from the intestinal tract via peristalsis. At high cell densities, the STm aggregates form a tightly packed network that is reminiscent of early bacterial biofilms. However, the underlying mechanism of how SIgA mediates this transition from a motile and invasive state to an avirulent sessile state in STm is currently unknown. Methods: In this report, we developed and validated a methodology known as the "snow globe" assay to enable real-time imaging and quantification of STm agglutination by the mouse monoclonal IgA Sal4. Results: We observed that agglutination in the snow globe assay was dose-dependent, antigen-specific, and influenced by antibody isotype. We determined that flagellar-based motility was a prerequisite for rapid onset of agglutination, even at high cell densities where cell-cell contacts are expected to be frequent. We also investigated the roles of individual cyclic-di-GMP metabolizing enzymes previously implicated in motility and biofilm formation in Sal4 IgA-mediated agglutination. Discussion: Taken together, our results demonstrate that IgA-mediated agglutination is a dynamic process influenced by bacterial motility and cell-cell collisions. We conclude that the snow globe assay is a viable platform to further decipher the molecular and genetic determinants that drive this interaction.

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The plague bacterium, Yersinia pestis, forms a biofilm-mediated blockage in the flea foregut that enhances its transmission by fleabite. Biofilm formation is positively controlled by cyclic di-GMP (c-di-GMP), which is synthesized by the diguanylate cyclases (DGC), HmsD and HmsT. While HmsD primarily promotes biofilm-mediated blockage of fleas, HmsT plays a more minor role in this process. HmsD is a component of the HmsCDE tripartite signaling system. HmsC and HmsE posttranslationally inhibit or activate HmsD, respectively. HmsT-dependent c-di-GMP levels and biofilm formation are positively regulated by the RNA-binding protein CsrA. In this study we determined whether CsrA positively regulates HmsD-dependent biofilm formation through interactions with the hmsE mRNA. Gel mobility shift assays determined that CsrA binds specifically to the hmsE transcript. RNase T1 footprint assays identified a single CsrA binding site and CsrA-induced structural changes in the hmsE leader region. Translational activation of the hmsE mRNA was confirmed in vivo using plasmid-encoded inducible translational fusion reporters and by HmsE protein expression studies. Furthermore, mutation of the CsrA binding site in the hmsE transcript significantly reduced HmsD-dependent biofilm formation. These results suggest that CsrA binding leads to structural changes in the hmsE mRNA that enhance its translation to enable increased HmsD-dependent biofilm formation. Given the requisite function of HmsD in biofilm-mediated flea blockage, this CsrA-dependent increase in HmsD activity underscores that complex and conditionally defined modulation of c-di-GMP synthesis within the flea gut is required for Y. pestis transmission. IMPORTANCE Mutations enhancing c-di-GMP biosynthesis drove the evolution of Y. pestis to flea-borne transmissibility. c-di-GMP-dependent biofilm-mediated blockage of the flea foregut enables regurgitative transmission of Y. pestis by fleabite. The Y. pestis diguanylate cyclases (DGC), HmsT and HmsD, which synthesize c-di-GMP, play significant roles in transmission. Several regulatory proteins involved in environmental sensing, as well as signal transduction and response regulation, tightly control DGC function. An example is CsrA, a global posttranscriptional regulator that modulates carbon metabolism and biofilm formation. CsrA integrates alternative carbon usage metabolism cues to activate c-di-GMP biosynthesis through HmsT. Here, we demonstrated that CsrA additionally activates hmsE translation to promote c-di-GMP biosynthesis through HmsD. This emphasizes that a highly evolved regulatory network controls c-di-GMP synthesis and Y. pestis transmission.

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Exploiting the heterologous effects of vaccines is a feasible strategy to combat different pathogens. These effects have been explained by enhanced immune responses of innate immune cells. Mycobacterium paragordonae is a rare nontuberculosis mycobacterium that has temperature-sensitive properties. Although natural killer (NK) cells exhibit heterologous immunity features, the cellular crosstalk between NK cells and dendritic cells (DCs) during live mycobacterial infection has remained elusive. We show that live but not dead M. paragordonae enhances heterologous immunity against unrelated pathogens in NK cells by IFN-ß of DCs in both mouse models and primary human immune cells. C-di-GMP from live M. paragordonae acted as a viability-associated pathogen-associated molecular pattern (Vita-PAMP), leading to STING-dependent type I IFN production in DCs via the IRE1α/XBP1s pathway. Also, increased cytosolic 2'3'-cGAMP by cGAS can induce type I IFN response in DCs by live M. paragordonae infection. We found that DC-derived IFN-ß plays a pivotal role in NK cell activation by live M. paragordonae infection, showing NK cell-mediated nonspecific protective effects against Candida albicans infection in a mouse model. Our findings indicate that the heterologous effect of live M. paragordonae vaccination is mediated by NK cells based on the crosstalk between DCs and NK cells.

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C-di-GMP is a bacterial second messenger with central role in biofilm formation. Spirochete bacteria from Leptospira genus present a wide diversity, with species of medical importance and environmental species, named as saprophytic. Leptospira form biofilms in the rat's reservoir kidneys and in the environment. Here, we performed genomic analyses to identify enzymatic and effector c-di-GMP proteins in the saprophytic biofilm-forming species Leptospira biflexa serovar Patoc. We identified 40 proteins through local alignments. Amongst them, 16 proteins are potentially functional diguanylate cyclases, phosphodiesterases, or hybrid proteins. We also identified nine effectors, including PilZ proteins. Enrichment analyses suggested that c-di-GMP interacts with cAMP signaling system, CsrA system, and flagella assembly regulation during biofilm development of L. biflexa. Finally, we identified eight proteins in the pathogen Leptospira interrogans serovar Copenhageni that share high similarity with L. biflexa c-di-GMP-related proteins. This work revealed proteins related to c-di-GMP turnover and cellular response in Leptospira and their potential roles during biofilm development.

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We previously showed that specific polyamines (PAs) present in the extracellular environment markedly affect extracellular polysaccharide (EPS) production, biofilm formation and motility in Sinorhizobium meliloti Rm8530. We hypothesized that extracellular PA signals were sensed and transduced by the NspS and MbaA proteins, respectively, which are homologs of the PA-sensing, c-di-GMP modulating NspS-MbaA proteins described in Vibrio cholerae. Here we show that the decrease in biofilm formation and EPS production in the quorum-sensing (QS)-deficient S. meliloti wild-type strain 1021 in cultures containing putrescine or spermine did not occur in a 1021 nspS mutant (1021 nspS). The transcriptional expression of nspS in strain 1021 was significantly increased in cultures containing either of these polyamines, but not by exogenous cadaverine, 1,3-diaminopropane (DAP), spermidine (Spd) or norspermidine (NSpd). Cell aggregation in liquid cultures did not differ markedly between strain 1021 and 1021 nspS in the presence or absence of PAs. The S. meliloti QS-proficient Rm8530 wild-type and nspS mutant (Rm8530 nspS) produced similar levels of biofilm under control conditions and 3.2- and 2.2-fold more biofilm, respectively, in cultures with NSpd, but these changes did not correlate with EPS production. Cells of Rm8530 nspS aggregated from two- to several-fold more than the wild-type in cultures without PAs or in those containing Spm. NSpd, Spd and DAP differently affected swimming and swarming motility in strains 1021 and Rm8530 and their respective nspS mutants. nspS transcription in strain Rm8530 was greatly reduced by exogenous Spm. Bioinformatic analysis revealed similar secondary structures and functional domains in the MbaA proteins of S. meliloti and V. cholerae, while their NspS proteins differed in some residues implicated in polyamine recognition in the latter species. NspS-MbaA homologs occur in a small subset of soil and aquatic bacterial species that commonly interact with eukaryotes. We speculate that the S. meliloti NspS-MbaA system modulates biofilm formation, EPS production and motility in response to environmental or host plant-produced PAs.

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