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Introduction: Response regulators from diverse two-component systems often function as diguanylate cyclases or phosphodiesterases, thereby enabling precise regulation of intracellular c-di-GMP levels to control bacterial virulence and motility. However, the regulatory mechanisms of c-di-GMP require further elucidation. Methods: This study confirmed that ArrS and ArrR form a two-component system via structural analysis, two-hybrid, and phosphodiesterase activity detection. To evaluate the impact of ArrS/ArrR on intracellular c-di-GMP levels, biofilm detection, motility detection, fluorescence reporter plasmids, and LC-MS/MS analysis were employed. One-hybrid, EMSA, and RT-qPCR were used to demonstrate the function of ArgR on arrSR promoter. The roles of ArrS/ArrR in Aeromonas veronii were investigated using RT-qPCR, murine model, and proteomics. Results: ArrS and ArrR constituted a two-component system in Aeromonas veronii and were transcriptionally repressed by ArgR. ArrR exhibited phosphodiesterase activity, which is inhibited through phosphorylation mediated by ArrS. In Aeromonas veronii , ArrS/ArrR significantly altered the intracellular c-di-GMP levels. In a murine model, Δ arrS exhibited increased pathogenicity, leading to elevated TNF-α and IFN-γ levels in serum, and severer toxicity to spleen and kidney. These effects might be elucidated by the upregulated inflammation-associated proteins in Δ arrS. Moreover, the exonuclease RecB was also up-regulated in Δ arrS. Discussion: We elucidated the regulatory mechanism of ArrS/ArrR on intracellular c-di-GMP levels and its impact on the virulence in Aeromonas veronii , and discussed the intricate relationship between c-di-GMP metabolism and arginine metabolism. [ABSTRACT FROM AUTHOR]

Background: The two-component system (TCS) serves as a common intracellular signal transduction pathway implicated in various processes of plant development and response to abiotic stress. With regard to the important cereal crop barley, only partial information about the occurrence of TCS signaling elements in the genome and putative functions is available. Results: In this study, we identified a total of 67 non-redundant TCS genes from all subgroups of the phosphorelay in the latest barley reference genome. Functional annotation and phylogenetic characterization was combined with a comprehensive gene expression analysis of the signaling components. Expression profiles hint at potential functions in vegetative and reproductive organs and tissue types as well as diverse stress responses. Apparently, a distinct subset of TCS genes revealed a stringent grain-specificity not being expressed elsewhere in the plant. By using laser capture microdissection (LCM)-based transcript analysis of barley grain tissues, we refined expression profiles of selected TCS genes and attributed them to individual cell types within the grain. Distinct TCS elements are exclusively expressed in the different maternal and filial cell types, particularly in the endosperm transfer cell (ETC) region. These genes are deemed to be selected in the domestication process of modern cultivars. Moreover, barley plants transformed with a synthetic sensor (TCSn::GFP) showed a high and specific activity in the ETC region of grains monitoring transcriptional output of the signaling system. Conclusions: The results provide comprehensive insights into the TCS gene family in the temperate cereal crop barley and indicate implications in various agronomic traits. The dataset is valuable for future research in different aspects of plant development and will be indispensable not only for barley, but also for other crops of the Poaceae. [ABSTRACT FROM AUTHOR]

CssRS is a two-component system that plays a pivotal role in mediating the secretion stress response in Bacillus subtilis. This system upregulates the synthesis of membrane-bound HtrA family proteases that cope with misfolded proteins that accumulate within the cell envelope as a result of overexpression or heat shock. Recent studies have shown the induction of CssRS-regulated genes in response to cell envelope stress. We investigated the induction of the CssRS-regulated htrA promoter in the presence of different cell wall- and membrane-active substances and observed induction of the CssRS-controlled genes by glycopeptides (vancomycin and teicoplanin), polymyxins B and E, certain β-lactams, and detergents. Teicoplanin was shown to elicit remarkably stronger induction than vancomycin and polymyxin B. Teicoplanin and polymyxin B induced the spxO gene expression in a CssRS-dependent fashion, resulting in increased activity of Spx, a master regulator of disulfide stress in Bacillus subtilis. The CssRS signaling pathway and Spx activity were demonstrated to be involved in Bacillus subtilis resistance to teicoplanin and polymyxin B. [ABSTRACT FROM AUTHOR]

RESEARCH HIGHLIGHTSAvian pathogenic Escherichia coli (APEC) is an important zoonotic pathogen that infects avian species by colonizing the gastrointestinal, respiratory, or reproductive tracts, leading to significant economic losses to the poultry industry worldwide and threatening food security and human health. APEC has evolved the two-component signal transduction system (TCS) to adapt and respond to extracellular environmental stresses, which are produced when the host is invaded by APEC. Here, we focus on the effect of the UhpAB TCS on the pathogenicity of APEC. The results in this study showed that the UhpAB TCS contributed to the pathogenicity of APEC in a chicken infection model. The electrophoretic mobility shift assays (EMSA) confirmed that UhpAB specifically bound to the promoters of fepG, ldrD, ycgV, and ydeI, and activated their expression, measured using real-time reverse transcription PCR (real-time RT–PCR). Furthermore, the UhpAB TCS could promote biofilm formation by activating the expression of biofilm master transcriptional regulator encoding gene csgD and enhance stress tolerance by activating the expression of stress protein encoding genes uspA and bhsA, thereby assisting APEC to evade host immune responses and inflammatory responses, and increasing the pathogenicity of APEC. These findings deepen our understanding of the pathogenic mechanism in APEC and offer new perspectives for further studies on the prevention and control of APEC infection.UhpAB increases the pathogenicity of APEC.UhpAB activates the expression of virulence genes fepG, ldrD, ycgV, and ydeI.UhpAB promotes biofilm formation and enhances stress tolerance.UhpAB contributes to APEC evading attack by the host immune system.UhpAB increases the pathogenicity of APEC.UhpAB activates the expression of virulence genes fepG, ldrD, ycgV, and ydeI.UhpAB promotes biofilm formation and enhances stress tolerance.UhpAB contributes to APEC evading attack by the host immune system. [ABSTRACT FROM AUTHOR]

Abstract Background Pseudomonas aeruginosa is able to survive, grow, and cause severe infections at different sites throughout the human body owing to its ability to sense diverse signals and precisely modulate target gene expression using its abundant signaling systems. Release of zinc (Zn) and hydrogen peroxide (H2O2) within the phagocyte are two major host strategies to defend against bacterial infections. It was previously shown that the response regulator CzcR controls global gene expression including catalase genes during Zn excess, but regulatory mechanisms of catalase gene expression and the role of CzcR in H2O2 tolerance remain unclear. Results In the study, comparative transcriptome analysis comprehensively described the CzcR-dependent and -independent gene regulatory pattern in P. aeruginosa during Zn excess, which revealed the counteractive co-regulation of two key H2O2-detoxifying catalase genes katA and katB through CzcR-dependent and -independent pathways in response to Zn excess. Protein-DNA interaction assay demonstrated that CzcR negatively regulates the expression of catalase genes katA and katB by directly binding to their promoters. While interestingly, we further showed that CzcR positively regulates H2O2 tolerance by inducing the catalase activity during Zn excess. Conclusion This study reported the opposite functions of CzcR in negatively regulating the expression of catalase genes katA and katB but in positively regulating the activity of catalase and H2O2 tolerance during Zn excess in P. aeruginosa.

Abstract The two-component system (TCS) consists of histidine kinase (HK), histidine phosphate transfer protein (HP), and response regulatory factor (RR). It is one of the most crucial components of signal transduction in plants, playing a significant role in regulating plant growth, development, and responses to various abiotic stresses. Although TCS genes have been extensively identified in a variety of plants, the genome-wide recognition and examination of TCS in switchgrass remain unreported. Accordingly, this study identified a total of 87 TCS members in the genome of switchgrass, comprising 20 HK(L)s, 10 HPs, and 57 RRs. Detailed analyses were also conducted on their gene structures, conserved domains, and phylogenetic relationships. Moreover, this study analysed the gene expression profiles across diverse organs and investigated their response patterns to adverse environmental stresses. Results revealed that 87 TCS genes were distributed across 18 chromosomes, with uneven distribution. Expansion of these genes in switchgrass was achieved through both fragment and tandem duplication. PvTCS members are relatively conservative in the evolutionary process, but the gene structure varies significantly. Various cis-acting elements, varying in types and amounts, are present in the promoter region of PvTCSs, all related to plant growth, development, and abiotic stress, due to the TCS gene structure. Protein-protein interaction and microRNA prediction suggest complex interactions and transcriptional regulation among TCS members. Additionally, most TCS members are expressed in roots and stems, with some genes showing organ-specific expression at different stages of leaf and inflorescence development. Under conditions of abiotic stress such as drought, low temperature, high temperature, and salt stress, as well as exogenous abscisic acid (ABA), the expression of most TCS genes is either stimulated or inhibited. Our systematic analysis could offer insight into the characterization of the TCS genes, and further the growth of functional studies in switchgrass.

Background: Pseudomonas aeruginosa is able to survive, grow, and cause severe infections at different sites throughout the human body owing to its ability to sense diverse signals and precisely modulate target gene expression using its abundant signaling systems. Release of zinc (Zn) and hydrogen peroxide (H2O2) within the phagocyte are two major host strategies to defend against bacterial infections. It was previously shown that the response regulator CzcR controls global gene expression including catalase genes during Zn excess, but regulatory mechanisms of catalase gene expression and the role of CzcR in H2O2 tolerance remain unclear. Results: In the study, comparative transcriptome analysis comprehensively described the CzcR-dependent and -independent gene regulatory pattern in P. aeruginosa during Zn excess, which revealed the counteractive co-regulation of two key H2O2-detoxifying catalase genes katA and katB through CzcR-dependent and -independent pathways in response to Zn excess. Protein-DNA interaction assay demonstrated that CzcR negatively regulates the expression of catalase genes katA and katB by directly binding to their promoters. While interestingly, we further showed that CzcR positively regulates H2O2 tolerance by inducing the catalase activity during Zn excess. Conclusion: This study reported the opposite functions of CzcR in negatively regulating the expression of catalase genes katA and katB but in positively regulating the activity of catalase and H2O2 tolerance during Zn excess in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that infects humans by colonizing the large intestine. Upon reaching the large intestine, EHEC mediates local signal recognition and the transcriptional regulation of virulence genes to promote adherence and colonization in a highly site-specific manner. Two-component systems (TCSs) represent an important strategy used by EHEC to couple external stimuli with the regulation of gene expression, thereby allowing EHEC to rapidly adapt to changing environmental conditions. An increasing number of studies published in recent years have shown that EHEC senses a variety of host- and microbiota-derived signals present in the human intestinal tract and coordinates the expression of virulence genes via multiple TCS-mediated signal transduction pathways to initiate the disease-causing process. Here, we summarize how EHEC detects a wide range of intestinal signals and precisely regulates virulence gene expression through multiple signal transduction pathways during the initial stages of infection, with a particular emphasis on the key roles of TCSs. This review provides valuable insights into the importance of TCSs in EHEC pathogenesis, which has relevant implications for the development of antibacterial therapies against EHEC infection. [ABSTRACT FROM AUTHOR]

The management of antibiotic-resistant, bacterial biofilm infections in skin wounds poses an increasingly challenging clinical scenario. Pseudomonas aeruginosa infection is difficult to eradicate because of biofilm formation and antibiotic resistance. In this study, we identified a new benzothiazole derivative compound, SN12 (IC 50 = 43.3 nmol/L), demonstrating remarkable biofilm inhibition at nanomolar concentrations in vitro. In further activity assays and mechanistic studies, we formulated an unconventional strategy for combating P. aeruginosa -derived infections by targeting the two-component (Gac/Rsm) system. Furthermore, SN12 slowed the development of ciprofloxacin and tobramycin resistance. By using murine skin wound infection models, we observed that SN12 significantly augmented the antibacterial effects of three widely used antibiotics—tobramycin (100-fold), vancomycin (200-fold), and ciprofloxacin (1000-fold)—compared with single-dose antibiotic treatments for P. aeruginosa infection in vivo. The findings of this study suggest the potential of SN12 as a promising antibacterial synergist, highlighting the effectiveness of targeting the two-component system in treating challenging bacterial biofilm infections in humans. Novel strategy for combating P. aeruginosa -derived infections by targeting TCSs. [Display omitted] [ABSTRACT FROM AUTHOR]

The two-component system (TCS) consists of histidine kinase (HK), histidine phosphate transfer protein (HP), and response regulatory factor (RR). It is one of the most crucial components of signal transduction in plants, playing a significant role in regulating plant growth, development, and responses to various abiotic stresses. Although TCS genes have been extensively identified in a variety of plants, the genome-wide recognition and examination of TCS in switchgrass remain unreported. Accordingly, this study identified a total of 87 TCS members in the genome of switchgrass, comprising 20 HK(L)s, 10 HPs, and 57 RRs. Detailed analyses were also conducted on their gene structures, conserved domains, and phylogenetic relationships. Moreover, this study analysed the gene expression profiles across diverse organs and investigated their response patterns to adverse environmental stresses. Results revealed that 87 TCS genes were distributed across 18 chromosomes, with uneven distribution. Expansion of these genes in switchgrass was achieved through both fragment and tandem duplication. PvTCS members are relatively conservative in the evolutionary process, but the gene structure varies significantly. Various cis-acting elements, varying in types and amounts, are present in the promoter region of PvTCSs, all related to plant growth, development, and abiotic stress, due to the TCS gene structure. Protein-protein interaction and microRNA prediction suggest complex interactions and transcriptional regulation among TCS members. Additionally, most TCS members are expressed in roots and stems, with some genes showing organ-specific expression at different stages of leaf and inflorescence development. Under conditions of abiotic stress such as drought, low temperature, high temperature, and salt stress, as well as exogenous abscisic acid (ABA), the expression of most TCS genes is either stimulated or inhibited. Our systematic analysis could offer insight into the characterization of the TCS genes, and further the growth of functional studies in switchgrass. [ABSTRACT FROM AUTHOR]

Sinorhizobium meliloti senses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novel S. meliloti chemotaxis protein CheT. While CheT and the Escherichia coli phosphatase CheZ share little sequence homology, CheT is predicted to possess an α‐helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate‐sink response regulator, CheY1~P by enhancing its decay two‐fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a KD of 2.9 μM, which is 25‐fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheT mutant and cheT DXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a KD of 19 μM, and a SEC‐MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) has emerged as a global health threat, necessitating immediate actions to develop novel antimicrobial strategies and enforce strong stewardship of existing antibiotics to manage the emergence of drug-resistant strains. This issue is particularly concerning when it comes to Gram-negative bacteria, which possess an almost impenetrable outer membrane (OM) that acts as a formidable barrier to existing antimicrobial compounds. This OM is an asymmetric structure, composed of various components that confer stability, fluidity, and integrity to the bacterial cell. The maintenance and restoration of membrane integrity are regulated by envelope stress response systems (ESRs), which monitor its assembly and detect damages caused by external insults. Bacterial communities encounter a wide range of environmental niches to which they must respond and adapt for survival, sustenance, and virulence. ESRs play crucial roles in coordinating the expression of virulence factors, adaptive physiological behaviors, and antibiotic resistance determinants. Given their role in regulating bacterial cell physiology and maintaining membrane homeostasis, ESRs present promising targets for drug development. Considering numerous studies highlighting the involvement of ESRs in virulence, antibiotic resistance, and alternative resistance mechanisms in pathogens, this review aims to present these systems as potential drug targets, thereby encouraging further research in this direction. [ABSTRACT FROM AUTHOR]

ABSTRACT Group A Streptococcus (GAS) is a human-adapted pathogen responsible for a variety of diseases. The GAS M1UK lineage has contributed significantly to the recently reported increases in scarlet fever and invasive infections. However, the basis for its evolutionary success is not yet fully understood. During the transition to systemic disease, the M1 serotype is known to give rise to spontaneous mutations in the control of virulence two-component regulatory system (CovRS) that confer a fitness advantage during invasive infections. Mutations that inactivate CovS function result in the de-repression of key GAS virulence factors such as streptolysin O (SLO), a pore-forming toxin and major trigger of inflammasome/interleukin-1β-dependent inflammation. Conversely, expression of the streptococcal cysteine protease SpeB, which is required during initial stages of colonization and onset of invasive disease, is typically lost in such mutants. In this study, we identified and characterized a novel covS single nucleotide polymorphism detected in three separate invasive M1UK isolates. The resulting CovSAla318Val mutation caused a significant upregulation of SLO resulting in increased inflammasome activation in human THP-1 macrophages, indicating an enhanced inflammatory potential. Surprisingly, SpeB production was unaffected. Site-directed mutagenesis was performed to assess the impact of this mutation on virulence and global gene expression. We found that the CovSAla318Val mutation led to subtle, virulence-specific changes of the CovRS regulon compared to previously characterized covS mutations, highlighting an unappreciated level of complexity in CovRS-dependent gene regulation. Continued longitudinal surveillance is warranted to determine whether this novel covS mutation will expand in the M1UK lineage.IMPORTANCEThe M1UK lineage of GAS has contributed to a recent global upsurge in scarlet fever and invasive infections. Understanding how GAS can become more virulent is critical for infection control and identifying new treatment approaches. The two-component CovRS system, comprising the sensor kinase CovS and transcription factor CovR, is a central regulator of GAS virulence genes. In the M1 serotype, covRS mutations are associated with an invasive phenotype. Such mutations have not been fully characterized in the M1UK lineage. This study identified a novel covS mutation in invasive Australian M1UK isolates that resulted in a more nuanced virulence gene regulation compared to previously characterized covS mutations. A representative isolate displayed upregulated SLO production and triggered amplified interleukin-1β secretion in infected human macrophages, indicating an enhanced inflammatory potential. These findings underscore the need for comprehensive analyses of covRS mutants to fully elucidate their contribution to M1UK virulence and persistence.

ABSTRACT As a universal language across the bacterial kingdom, the quorum sensing signal autoinducer-2 (AI-2) can coordinate many bacterial group behaviors. However, unknown AI-2 receptors in bacteria may be more than what has been discovered so far, and there are still many unknown functions for this signal waiting to be explored. Here, we have identified a membrane-bound histidine kinase of the pathogenic bacterium Vibrio furnissii, AsrK, as a receptor that specifically detects AI-2 under low boron conditions. In contrast with another well-known AI-2 receptor LuxP that recognizes the borated form of AI-2, AsrK is found to show higher affinity with AI-2 under borate-depleted conditions, and thus boron has a negative effect on AI-2 sensing by AsrK in regulation of the biofilm and motility phenotypes. AI-2 binds to the extracytoplasmic dCache_1 domain of AsrK to inhibit its autokinase activity, thus decreasing the phosphorylation level of its cognate response regulator AsrR and activating the phosphodiesterase activity of AsrR to degrade the cellular second messenger cyclic di-GMP (c-di-GMP). AI-2 perception by the AsrK-AsrR system remarkably reduces intracellular c-di-GMP levels and enhances tolerance of V. furnissii to oxidative stress and DNA damage by upregulating the transcription of universal stress proteins including UspA1, UspA2, and UspE. Our study reveals a previously unrecognized mechanism for AI-2 detection in bacteria and also provides new insights into the important role of AI-2 in bacterial defense against oxidative stress and DNA damage.IMPORTANCEThe QS signal AI-2 is widely synthesized in bacteria and has been implicated in the regulation of numerous bacterial group behaviors. However, in contrast to the wide distribution of this signal, its receptors have only been found in a small number of bacterial species, and the underlying mechanisms for the detection of and response to AI-2 remain elusive in most bacteria. It is worth noting that the periplasmic protein LuxP is the uniquely identified receptor for AI-2 in Vibrio spp. Here, we identify a second type of AI-2 receptor, a membrane-bound histidine kinase with a periplasmic dCache_1 sensory domain, in a member of the genus Vibrio, and thus show that AI-2 enhances the defense of V. furnissii against oxidative stress and DNA damage by modulation of c-di-GMP signaling via the AsrK-AsrR two-component system. Our results reveal a previously unrecognized AI-2 sensing mechanism and expand our understanding of the physiological roles of AI-2 in bacteria.

IntroductionResponse regulators from diverse two-component systems often function as diguanylate cyclases or phosphodiesterases, thereby enabling precise regulation of intracellular c-di-GMP levels to control bacterial virulence and motility. However, the regulatory mechanisms of c-di-GMP require further elucidation.MethodsThis study confirmed that ArrS and ArrR form a two-component system via structural analysis, two-hybrid, and phosphodiesterase activity detection. To evaluate the impact of ArrS/ArrR on intracellular c-di-GMP levels, biofilm detection, motility detection, fluorescence reporter plasmids, and LC-MS/MS analysis were employed. One-hybrid, EMSA, and RT-qPCR were used to demonstrate the function of ArgR on arrSR promoter. The roles of ArrS/ArrR in Aeromonas veronii were investigated using RT-qPCR, murine model, and proteomics.ResultsArrS and ArrR constituted a two-component system in Aeromonas veronii and were transcriptionally repressed by ArgR. ArrR exhibited phosphodiesterase activity, which is inhibited through phosphorylation mediated by ArrS. In Aeromonas veronii, ArrS/ArrR significantly altered the intracellular c-di-GMP levels. In a murine model, ΔarrS exhibited increased pathogenicity, leading to elevated TNF-α and IFN-γ levels in serum, and severer toxicity to spleen and kidney. These effects might be elucidated by the upregulated inflammation-associated proteins in ΔarrS. Moreover, the exonuclease RecB was also up-regulated in ΔarrS.DiscussionWe elucidated the regulatory mechanism of ArrS/ArrR on intracellular c-di-GMP levels and its impact on the virulence in Aeromonas veronii, and discussed the intricate relationship between c-di-GMP metabolism and arginine metabolism.

Xinyan Ma,1,2,* Ziyan Wu,1,2,* Junpeng Li,1,2 Yang Yang1,2 1College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, People’s Republic of China; 2Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses, and Joint Laboratory of International Cooperation on Prevention and Control Technology of Important Animal Diseases and Zoonoses of Jiangsu Higher Education Institutions, Yangzhou, 225009, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yang Yang, Email YangyangYZU@163.comPurpose: Biofilms significantly contribute to the persistence and antibiotic resistance of Staphylococcus aureus infections. However, the regulatory mechanisms governing biofilm formation of S. aureus remain not fully elucidated. This study aimed to investigate the function of the S. aureus lineage-specific two-component system, desKR, in biofilm regulation and pathogenicity.Methods: Bioinformatic analysis was conducted to assess the prevalence of desKR across various S. aureus lineages and to examine its structural features. The impact of desKR on S. aureus pathogenicity was evaluated using in vivo mouse models, including skin abscess, bloodstream infection, and nasal colonization models. Crystal violet staining and confocal laser scanning microscopy were utilized to examine the impact of desKR on S. aureus biofilm formation. Mechanistic insights into desKR-mediated biofilm regulation were investigated by quantifying polysaccharide intercellular adhesin (PIA) production, extracellular DNA (eDNA) release, autolysis assays, and RT-qPCR.Results: The prevalence of desKR varied among different S. aureus lineages, with notably low carriage rates in ST398 and ST59 lineages. Deletion of desKR in NCTC8325 strain resulted in decreased susceptibility to β-lactam and glycopeptide antibiotics. Although desKR did not significantly affect acute pathogenicity, the ΔdesKR mutant exhibited significantly reduced nasal colonization and biofilm-forming ability. Overexpression of desKR in naturally desKR-lacking strains (ST398 and ST59) enhanced biofilm formation, suggesting a lineage-independent effect. Phenotypic assays further revealed that the ΔdesKR mutant showed reduced PIA production, decreased eDNA release, and lower autolysis rates. RT-qPCR indicated significant downregulation of icaA, icaD, icaB, and icaC genes, along with upregulation of icaR, whereas autolysis-related genes remained unchanged.Conclusion: The desKR two-component system positively regulates S. aureus biofilm formation in a lineage-independent manner, primarily by modulating PIA synthesis via the ica operon. These findings provide new insights into the molecular mechanisms of biofilm formation in S. aureus and highlight desKR as a potential target for therapeutic strategies aimed at combating biofilm-associated infections.Keywords: Staphylococcus aureus, two-component system, desKR, biofilm

Enterococcus faecalis is the main pathogen causing refractory apical periodontitis (RAP). This bacterium can tolerate harsh environments and trigger periapical immune inflammatory responses that result in persistent infection inside and outside the root canal. Adhesion to the dentin wall of root canals and the subsequent formation of biofilms significantly enhances the drug resistance and anti-erosion ability of Enterococcus faecalis, which is the key factor mediating its pathogenesis. The adhesion of Enterococcus faecalis to dentin involves non-specific adhesion and specific adhesion, and the latter is mediated by adhesion-related virulence factors, mainly including the adhesin of collagen from enterococci (Ace), extracellular surface protein (Esp), gelatinase (GelE), serine protease (SprE), endocarditis and biofilm associated pilus (Ebp) and aggregation substance (AS), which is regulated by multiple two-component systems. The two-component system Fsr can promote the expression of gelE and sprE when the cell population density increases. GelE can further reduce Ace, while the two-component system GrvRS directly downregulates ace expression in response to the serum environment. The two-component systems CroRS and WalRK may also promote and inhibit the expression of various virulence factors, including ace and gelE, thus affecting the adhesion of Enterococcus faecalis. In addition, the mechanochemical preparation and the internal environment of the root canal can also influence the adhesion of Enterococcus faecalis to dentin. Avoiding the introduction of Enterococcus faecalis and using adhesion-interfering medications during root canal treatment can effectively prevent the adhesion of Enterococcus faecalis, and a variety of activated irrigation protocols can also be effective at increasing the clearance of Enterococcus faecalis from the root canal. The design of rational drugs targeting key factors involved in and regulators of the adhesion of Enterococcus faecalis to dentin is expected to provide new ideas and strategies for root canal infection control. The present paper reviews the adhesion of Enterococcus faecalis to dentin and its influencing factors.

Tigecycline-resistant Acinetobacter baumannii (TRAB) is increasing in Thailand, complicating antibiotic treatment due to limited antibiotic options. The specific resistance mechanism behind tigecycline resistance is still unclear, necessitating further investigation. We investigated the presence of OXA-type carbapenemases, the antimicrobial susceptibility profile, the inhibitory effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on tigecycline susceptibility, the expression levels of RND-type efflux pumps and amino acid substitutions within a two-component regulatory system on 30 Thai clinical isolates. Our investigation revealed that most of (73.3%) TRAB isolates expressed at least one member of the Ade efflux pumps. The ade B was most frequently expressed (63.3%), followed by ade R (50%), ade S (43.3%), ade J (30%) and ade G (10%). Overexpression of the AdeABC was associated with increased tigecycline minimum inhibitory concentrations (MICs) and amino acid substitutions within the AdeRS. Notably, isolates harbouring simultaneous mutations in these genes exhibited an increase in the transcription level of the ade B. Our findings highlight the significant role of the AdeABC system in tigecycline resistance among Thai clinical TRAB isolates. This is supported by point mutations within the AdeRS and upregulated expression of the ade B. These results provide valuable insights for understanding resistance mechanisms and developing novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

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The two-component system (TCS) gene family is among the most important signal transduction families in plants and is involved in the regulation of various abiotic stresses, cell growth and division. To understand the role of TCS genes in mango (Mangifera indica), a comprehensive analysis of TCS gene family was carried out in mango leading to identification of 65 MiTCS genes. Phylogenetic analysis divided MiTCSs into three groups (histidine kinases, histidine-containing phosphotransfer proteins, and response regulators) and 11 subgroups. One tandem duplication and 23 pairs of segmental duplicates were found within the MiTCSs. Promoter analysis revealed that MiTCSs contain a large number of cis -elements associated with environmental stresses, hormone response, light signalling, and plant development. Gene ontology analysis showed their involvement in various biological processes and molecular functions, particularly signal transduction. Protein–protein interaction analysis showed that MiTCS proteins interacted with each other. The expression pattern in various tissues and under many stresses (drought, cold, and disease) showed that expression levels varied among various genes in different conditions. MiTCSs 3D structure predictions showed structural conservation among members of the same groups. This information can be further used to develop improved cultivars and will serve as a foundation for gaining more functional insights into the TCS gene family. We studied the genetic blueprint of mango (Mangifera indica) trees, and identified a crucial gene family for plant health and stress responses. We identified and examined 65 genes responsible for signalling pathways within mango plants. By unravelling these genetic intricacies, we pave the way for potential advancements in mango cultivation, and offer opportunities for developing better resilience against environmental challenges and the development of superior mango varieties. This article belongs to the Collection Functional Genomics for Developing Climate Resilient Crops. [ABSTRACT FROM AUTHOR]

Sensory adaptation in bacterial chemotaxis is mediated by posttranslational modifications of methyl‐accepting chemotaxis proteins (MCPs). In Escherichia coli, the adaptation proteins CheR and CheB tether to a conserved C‐terminal receptor pentapeptide. Here,we investigated the function of the pentapeptide motif (N/D)WE(E/N)F in Sinorhizobium meliloti chemotaxis. Isothermal titration calorimetry revealed stronger affinity of the pentapeptides to CheR and activated CheB relative to unmodified CheB. Strains with mutations of the conserved tryptophan in one or all four MCP pentapeptides resulted in a significant decrease or loss of chemotaxis to glycine betaine, lysine, and acetate, chemoattractants sensed by pentapeptide‐bearing McpX and pentapeptide‐lacking McpU and McpV, respectively. Importantly, we discovered that the pentapeptide mediates chemotaxis when fused to the C‐terminus of pentapeptide‐lacking chemoreceptors via a flexible linker. We propose that adaptational assistance and a threshold number of available sites enable the efficient docking of adaptation proteins to the chemosensory array. Altogether, these results demonstrate that S. meliloti effectively utilizes a pentapeptide‐dependent adaptation system with a minimal number of tethering units to assist pentapeptide‐lacking chemoreceptors and hypothesize that the higher abundance of CheR and CheB in S. meliloti compared to E. coli allows for ample recruitment of adaptation proteins to the chemosensory array. [ABSTRACT FROM AUTHOR]

Abstract Vancomycin (VAN)‐intermediate Staphylococcus aureus (VISA) is a critical cause of VAN treatment failure worldwide. Multiple genetic changes are reportedly associated with VISA formation, whereas VISA strains often present common phenotypes, such as reduced autolysis and thickened cell wall. However, how mutated genes lead to VISA common phenotypes remains unclear. Here, we show a metabolism regulatory cascade (CcpA‐GlmS), whereby mutated two‐component systems (TCSs) link to the common phenotypes of VISA. We found that ccpA deletion decreased VAN resistance in VISA strains with diverse genetic backgrounds. Metabolic alteration in VISA was associated with ccpA upregulation, which was directly controlled by TCSs WalKR and GraSR. RNA‐sequencing revealed the crucial roles of CcpA in changing the carbon flow and nitrogen flux of VISA to promote VAN resistance. A gate enzyme (GlmS) that drives carbon flow to the cell wall precursor biosynthesis was upregulated in VISA. CcpA directly controlled glmS expression. Blocking CcpA sensitized VISA strains to VAN treatment in vitro and in vivo. Overall, this work uncovers a link between the formation of VISA phenotypes and commonly mutated genes. Inhibition of CcpA‐GlmS cascade is a promising strategy to restore the therapeutic efficiency of VAN against VISA infections.

Abstract This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein–ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at − 5.81 kcal/mol, while the docking energy for meropenem was − 4.92 kcal/mol. For Bae R, imipenem had a maximum docking energy of − 4.28 kcal/mol, compared with − 4.60 kcal/mol for meropenem. The highest binding energies for Bae S–levofloxacin and Bae R–levofloxacin were − 3.60 and − 3.65 kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16 µg/mL, whereas levofloxacin-resistant strains had MIC values of 8 µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2 h under the action of 8 µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii.

The dipeptide D‐Ala‐D‐Ala is an essential component of peptidoglycan and the target of vancomycin. Most Clostridioides difficile strains possess the vanG operon responsible for the synthesis of D‐Ala‐D‐Ser, which can replace D‐Ala‐D‐Ala in peptidoglycan. The C. difficile vanG operon is regulated by a two‐component system, VanRS, but is not induced sufficiently by vancomycin to confer resistance to this antibiotic. Surprisingly, in the absence of the VanS histidine kinase (HK), the vanG operon is still induced by vancomycin and also by another antibiotic, ramoplanin, in a VanR‐dependent manner. This suggested the cross‐regulation of VanR by another HK or kinases that are activated in the presence of certain lipid II‐targeting antibiotics. We identified these HKs as CD35990 and CD22880. However, mutations in either or both HKs did not affect the regulation of the vanG operon in wild‐type cells suggesting that intact VanS prevents the cross‐activation of VanR by non‐cognate HKs. Overproduction of VanR in the absence of VanS, CD35990, and CD22880 led to high expression of the vanG operon indicating that VanR can potentially utilize at least one more phosphate donor for its activation. Candidate targets of CD35990‐ and CD22880‐mediated regulation in the presence of vancomycin or ramoplanin were identified by RNA‐Seq. [ABSTRACT FROM AUTHOR]

Enterococcal infections frequently show high levels of antibiotic resistance, including to cell envelope‐acting antibiotics like daptomycin (DAP). While we have a good understanding of the resistance mechanisms, less is known about the control of such resistance genes in enterococci. Previous work unveiled a bacitracin resistance network, comprised of the sensory ABC transporter SapAB, the two‐component system (TCS) SapRS and the resistance ABC transporter RapAB. Interestingly, components of this system have recently been implicated in DAP resistance, a role usually regulated by the TCS LiaFSR. To better understand the regulation of DAP resistance and how this relates to mutations observed in DAP‐resistant clinical isolates of enterococci, we here explored the interplay between these two regulatory pathways. Our results show that SapR regulates an additional resistance operon, dltXABCD, a known DAP resistance determinant, and show that LiaFSR regulates the expression of sapRS. This regulatory structure places SapRS‐target genes under dual control, where expression is directly controlled by SapRS, which itself is up‐regulated through LiaFSR. The network structure described here shows how Enterococcus faecalis coordinates its response to cell envelope attack and can explain why clinical DAP resistance often emerges via mutations in regulatory components. [ABSTRACT FROM AUTHOR]

BACKGROUND: Xanthomonas oryzae pv. oryzae (Xoo) is often considered one of the most destructive bacterial pathogens causing bacterial leaf blight (BLB), resulting in significant yield and cost losses in rice. In this study, a series of novel derivatives containing the isopropanolamine moiety linked to various substituted phenols and piperazines were designed, synthesized and screened. RESULTS: Antibacterial activity results showed that most compounds had good inhibitory effects on Xoo, among which compound W2 (EC50 = 2.74 μg mL−1) exhibited the most excellent inhibitory activity, and W2 also had a certain curative effect (35.89%) on rice compared to thiodiazole copper (TC) (21.57%). Scanning electron microscopy (SEM) results indicated that compound W2 could cause rupture of the Xoo cell membrane. Subsequently, proteomics and quantitative real‐time polymerase chain reaction revealed that compound W2 affected the physiological processes of Xoo and may exert antibacterial activity by targeting the two‐component system pathway. Interestingly, W2 upregulated Xoo's methyltransferase to impact on its pathogenicity. CONCLUSION: The present study offers a promising phenolic‐piperazine‐sopropanolamine compound as an innovative antibacterial strategy by specifically targeting the two‐component system pathway and inducing upregulation of methyltransferase to effectively impact Xoo's pathogenicity. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The use of probiotic lactobacilli has been proposed as a strategy to mitigate damage associated with exposure to toxic metals. Their protective effect against cationic metal ions, such as those of mercury or lead, is believed to stem from their chelating and accumulating potential. However, their retention of anionic toxic metalloids, such as inorganic arsenic, is generally low. Through the construction of mutants in phosphate transporter genes (pst) in Lactiplantibacillus plantarum and Lacticaseibacillus paracasei strains, coupled with arsenate [As(V)] uptake and toxicity assays, we determined that the incorporation of As(V), which structurally resembles phosphate, is likely facilitated by phosphate transporters. Surprisingly, inactivation in Lc. paracasei of PhoP, the transcriptional regulator of the two-component system PhoPR, a signal transducer involved in phosphate sensing, led to an increased resistance to arsenite [As(III)]. In comparison to the wild type, the phoP strain exhibited no differences in the ability to retain As(III), and there were no observed changes in the oxidation of As(III) to the less toxic As(V). These results reinforce the idea that specific transport, and not unspecific cell retention, plays a role in As(V) biosorption by lactobacilli, while they reveal an unexpected phenotype for the lack of the pleiotropic regulator PhoP. [ABSTRACT FROM AUTHOR]

Main Conclusion: OsRR26 is a cytokinin-responsive response regulator that promotes phytohormone-mediated ROS accumulation in rice roots, regulates seedling growth, spikelet fertility, awn development, represses NADPH oxidases, and negatively affects salinity tolerance. Plant two-component systems (TCS) play a pivotal role in phytohormone signaling, stress responses, and circadian rhythm. However, a significant knowledge gap exists regarding TCS in rice. In this study, we utilized a functional genomics approach to elucidate the role of OsRR26, a type-B response regulator in rice. Our results demonstrate that OsRR26 is responsive to cytokinin, ABA, and salinity stress, serving as the ortholog of Arabidopsis ARR11. OsRR26 primarily localizes to the nucleus and plays a crucial role in seedling growth, spikelet fertility, and the suppression of awn development. Exogenous application of cytokinin led to distinct patterns of reactive oxygen species (ROS) accumulation in the roots of both WT and transgenic plants (OsRR26OE and OsRR26KD), indicating the potential involvement of OsRR26 in cytokinin-mediated ROS signaling in roots. The application of exogenous ABA resulted in varied cellular compartmentalization of ROS between the WT and transgenic lines. Stress tolerance assays of these plants revealed that OsRR26 functions as a negative regulator of salinity stress tolerance across different developmental stages in rice. Physiological and biochemical analyses unveiled that the knockdown of OsRR26 enhances salinity tolerance, characterized by improved chlorophyll retention and the accumulation of soluble sugars, K+ content, and amino acids, particularly proline. [ABSTRACT FROM AUTHOR]

Background: Biofilm development by bacteria is considered to be an essential stage in the bacterial infection. Acinetobacter nosocomialis is an important nosocomial pathogen causing a variety of human infections. However, characteristics and specific determinants of biofilm development have been poorly characterized in A. nosocomialis. Objective: The aim of this study was to investigate the factors involved in the biofilm development by A. nosocomialis. Methods: Library of random transposon mutants was constructed using the Tn5 mutagenesis. The mutant strains, in which the ability of biofilm formation was significantly impaired, were screened by gentian violet staining. The roles of BfmR and BfmS were determined by constructing a bfmR and bfmS deletion mutant and analyzing the effects of bfmR and bfmS mutation on the biofilm development and motility of A. nosocomialis. Results: We identified a biofilm-defective mutant in which a transposon insertion inactivated an open reading frame encoding the BfmR in a two-component regulatory system consisting of BfmR and BfmS. The bfmR mutant revealed a significant reduction in biofilm formation and motility compared to wild-type strain. Deficiency in the biofilm formation and motility of the bfmR mutant was restored by single copy bfmR complementation. In contrast, the bfmS mutant had no effect on biofilm formation. Conclusion: A. nosocomialis has a two-component regulatory system, BfmRS. BfmR is a response regulator required for the initial attachment and maturation of biofilm during the biofilm development as well as the bacterial growth. BfmR could be a potential drug target for A. nosocomialis infection. [ABSTRACT FROM AUTHOR]

This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein–ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at − 5.81 kcal/mol, while the docking energy for meropenem was − 4.92 kcal/mol. For Bae R, imipenem had a maximum docking energy of − 4.28 kcal/mol, compared with − 4.60 kcal/mol for meropenem. The highest binding energies for Bae S–levofloxacin and Bae R–levofloxacin were − 3.60 and − 3.65 kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16 µg/mL, whereas levofloxacin-resistant strains had MIC values of 8 µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2 h under the action of 8 µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii. [ABSTRACT FROM AUTHOR]

Stenotrophomonas maltophilia is a major threat to the food industry and human health owing to its strong protease production and biofilm formation abilities. However, information regarding regulatory factors or potential mechanisms is limited. Herein, we observed that temperature differentially regulates biofilm formation and protease production, and a cAMP receptor-like protein (Clp) negatively regulates thermosensor biofilm formation, in contrast to protease synthesis. Among four c-di-GMP-related two-component systems (TCSs), promoter fusion analysis revealed that clp transcription levels were predominantly controlled by LotS/LotR, partially controlled by both RpfC/RpfG and a novel TCS Sm0738/Sm0737, with no obvious effect caused by Sm1912/Sm1911. Biofilm formation in Δclp and ΔTCSs strains suggested that LotS/LotR controlled biofilm formation in a Clp-mediated manner, whereas both RpfC/RpfG and Sm0738/Sm0737 may occur in a distinct pathway. Furthermore, enzymatic activity analysis combined with c-di-GMP level indicated that the enzymatic activity of c-di-GMP-related metabolism proteins may not be a vital contributor to changes in c-di-GMP level, thus influencing physiological functions. Our findings elucidate that the regulatory pathway of c-di-GMP-related TCSs and Clp in controlling spoilage or the formation of potentially pathogenic factors in Stenotrophomonas expand the understanding of c-di-GMP metabolism and provide clues to control risk factors of S. maltophilia in food safety. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is a major cause of nosocomial infections, and its highly adaptive nature and broad range of antibiotic resistance enable it to persist in hospital environments. A. baumannii often employs two-component systems (TCSs) to regulate adaptive responses and virulence-related traits. This study describes a previously uncharacterized TCS in the A. baumannii ATCC19606 strain, consisting of a transcriptional sensor, DJ41_1407, and its regulator, DJ41_1408, located adjacent to GacA of the GacSA TCS. Markerless mutagenesis was performed to construct DJ41_1407 and DJ41_1408 single and double mutants. DJ41_1408 was found to upregulate 49 genes and downregulate 43 genes, most of which were associated with carbon metabolism and other metabolic pathways, such as benzoate degradation. MEME analysis revealed a putative binding box for DJ41_1408, 5′TGTAAATRATTAYCAWTWAT3′. Colony size, motility, biofilm-forming ability, virulence, and antibiotic resistance of DJ41_1407 and DJ41_1408 single and double mutant strains were assessed against wild type. DJ41_1407 was found to enhance motility, while DJ41_1408 was found to upregulate biofilm-forming ability, and may also modulate antibiotic response. Both DJ41_1407 and DJ41_1408 suppressed virulence, based on results from a G. mellonella infection assay. These results showcase a novel A. baumannii TCS involved in metabolism, with effects on motility, biofilm-forming ability, virulence, and antibiotic response. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis (Pg) utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS–FimR two-component system, making the FimS sensory protein an attractive target for preventing Pg infection. However, the specific environmental signal received by FimS remains unknown. We constructed random Pg mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of fimS, and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the fimS-knockout Pg strain using the Escherichia coli–Pg conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased fimA expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of Pg, including the elucidation of structure–function relationships of proteins of interest. [ABSTRACT FROM AUTHOR]

ABSTRACT The discovery of antimicrobials with novel mechanisms of action is crucial to tackle the foreseen global health crisis due to antimicrobial resistance. Bacterial two-component signaling systems (TCSs) are attractive targets for the discovery of novel antibacterial agents. TCS-encoding genes are found in all bacterial genomes and typically consist of a sensor histidine kinase (HK) and a response regulator. Due to the conserved Bergerat fold in the ATP-binding domain of the TCS HK and the human chaperone Hsp90, there has been much interest in repurposing inhibitors of Hsp90 as antibacterial compounds. In this study, we explore the chemical space of the known Hsp90 inhibitor scaffold 3,4-diphenylpyrazole (DPP), building on previous literature to further understand their potential for HK inhibition. Six DPP analogs inhibited HK autophosphorylation in vitro and had good antimicrobial activity against Gram-positive bacteria. However, mechanistic studies showed that their antimicrobial activity was related to damage of bacterial membranes. In addition, DPP analogs were cytotoxic to human embryonic kidney cell lines and induced the cell arrest phenotype shown for other Hsp90 inhibitors. We conclude that these DPP structures can be further optimized as specific disruptors of bacterial membranes providing binding to Hsp90 and cytotoxicity are lowered. Moreover, the X-ray crystal structure of resorcinol, a substructure of the DPP derivatives, bound to the HK CheA represents a promising starting point for the fragment-based design of novel HK inhibitors.IMPORTANCEThe discovery of novel antimicrobials is of paramount importance in tackling the imminent global health crisis of antimicrobial resistance. The discovery of novel antimicrobials with novel mechanisms of actions, e.g., targeting bacterial two-component signaling systems, is crucial to bypass existing resistance mechanisms and stimulate pharmaceutical innovations. Here, we explore the possible repurposing of compounds developed in cancer research as inhibitors of two-component systems and investigate their off-target effects such as bacterial membrane disruption and toxicity. These results highlight compounds that are promising for further development of novel bacterial membrane disruptors and two-component system inhibitors.

Abstract The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.

Antimicrobial resistance (AMR) has emerged as a global health threat, necessitating immediate actions to develop novel antimicrobial strategies and enforce strong stewardship of existing antibiotics to manage the emergence of drug-resistant strains. This issue is particularly concerning when it comes to Gram-negative bacteria, which possess an almost impenetrable outer membrane (OM) that acts as a formidable barrier to existing antimicrobial compounds. This OM is an asymmetric structure, composed of various components that confer stability, fluidity, and integrity to the bacterial cell. The maintenance and restoration of membrane integrity are regulated by envelope stress response systems (ESRs), which monitor its assembly and detect damages caused by external insults. Bacterial communities encounter a wide range of environmental niches to which they must respond and adapt for survival, sustenance, and virulence. ESRs play crucial roles in coordinating the expression of virulence factors, adaptive physiological behaviors, and antibiotic resistance determinants. Given their role in regulating bacterial cell physiology and maintaining membrane homeostasis, ESRs present promising targets for drug development. Considering numerous studies highlighting the involvement of ESRs in virulence, antibiotic resistance, and alternative resistance mechanisms in pathogens, this review aims to present these systems as potential drug targets, thereby encouraging further research in this direction.

ABSTRACT Pseudomonas aeruginosa is one of the most common nosocomial pathogens worldwide, known for its virulence, drug resistance, and elaborate sensor-response network. The primary challenge encountered by pathogens during the initial stages of infection is the immune clearance arising from the host. The resident macrophages of barrier organs serve as the frontline defense against these pathogens. Central to our understanding is the mechanism by which bacteria modify their behavior to circumvent macrophage-mediated clearance, ensuring their persistence and colonization. To successfully evade macrophage-mediated phagocytosis, bacteria must possess an adaptive response mechanism. Two-component systems provide bacteria the agility to navigate diverse environmental challenges, translating external stimuli into cellular adaptive responses. Here, we report that the well-documented histidine kinase, LadS, coupled to a cognate two-component response regulator, PA0034, governs the expression of a vital adhesin called chaperone-usher pathway pilus cupA. The LadS/PA0034 system is susceptible to interference from the reactive oxygen species likely to be produced by macrophages and further lead to a poor adhesive phenotype with scantily cupA pilus, impairing the phagocytosis efficiency of macrophages during acute infection. This dynamic underscores the intriguing interplay: as macrophages deploy reactive oxygen species to combat bacterial invasion, the bacteria recalibrate their exterior to elude these defenses.IMPORTANCEThe notoriety of Pseudomonas aeruginosa is underscored by its virulence, drug resistance, and elaborate sensor-response network. Yet, the mechanisms by which P. aeruginosa maneuvers to escape phagocytosis during acute infections remain elusive. This study pinpoints a two-component response regulator, PA0034, coupled with the histidine kinase LadS, and responds to macrophage-derived reactive oxygen species. The macrophage-derived reactive oxygen species can impair the LadS/PA0034 system, resulting in reduced expression of cupA pilus in the exterior of P. aeruginosa. Since the cupA pilus is an important adhesin of P. aeruginosa, its deficiency reduces bacterial adhesion and changes their behavior to adopt a planktonic lifestyle, subsequently inhibiting the phagocytosis of macrophages by interfering with bacterial adhesion. Briefly, reactive oxygen species may act as environmental cues for the LadS/PA0034 system. Upon recognition, P. aeruginosa may transition to a poorly adhesive state, efficiently avoiding engulfment by macrophages.

ABSTRACT Strict management of intracellular heme pools, which are both toxic and beneficial, is crucial for bacterial survival during infection. The human pathogen Staphylococcus aureus uses a two-component heme sensing system (HssRS), which counteracts environmental heme toxicity by triggering expression of the efflux transporter HrtBA. The HssS heme sensor is a HisKA-type histidine kinase, characterized as a membrane-bound homodimer containing an extracellular sensor and a cytoplasmic conserved catalytic domain. To elucidate HssS heme-sensing mechanism, a structural simulation of the HssS dimer based on Alphafold2 was docked with heme. In this model, a heme-binding site is present in the HssS dimer between the membrane and extracellular domains. Heme is embedded in the membrane bilayer with its two protruding porphyrin propionates interacting with two conserved Arg94 and Arg163 that are located extracellularly. Single substitutions of these arginines and two highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic pocket limited the ability of HssS to induce HrtBA synthesis. Combination of the four substitutions abolished HssS activation. Wild-type (WT) HssS copurified with heme from Escherichia coli, whereas heme binding was strongly attenuated in the variants. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation and induce HrtBA-mediated heme extrusion from the membrane. This “gatekeeper” mechanism could limit intracellular diffusion of exogenous heme in S. aureus and may serve as a paradigm for how efflux transporters control detoxification of exogenous hydrophobic stressors.IMPORTANCEIn the host blood, pathogenic bacteria are exposed to the red pigment heme that concentrates in their lipid membranes, generating cytotoxicity. To overcome heme toxicity, Staphylococcus aureus expresses a membrane sensor protein, HssS. Activation of HssS by heme triggers a phosphotransfer mechanism leading to the expression of a heme efflux system, HrtBA. This detoxification system prevents intracellular accumulation of heme. Our structural and functional data reveal a heme-binding hydrophobic cavity in HssS within the transmembrane domains (TM) helices at the interface with the extracellular domain. This structural pocket is important for the function of HssS as a heme sensor. Our findings provide a new basis for the elucidation of pathogen-sensing mechanisms as a prerequisite to the discovery of inhibitors.

ABSTRACTIn the Staphylococcus aureus genome, a set of highly conserved two-component systems (TCSs) composed of histidine kinases (HKs) and their cognate response regulators (RRs) sense and respond to environmental stimuli, which drive the adaptation of the bacteria. This study investigates the complex interplay between TCSs in S. aureus USA300, a predominant methicillin-resistant S. aureus strain, revealing shared and unique virulence regulatory pathways and genetic variations mediating signal specificity within TCSs. Using TCS-related mutants from the Nebraska Transposon Mutant Library, we analyzed the effects of inactivated TCS HKs and RRs on the production of various virulence factors, in vitro infection abilities, and adhesion assays. We found that the TCSs’ influence on virulence determinants was not associated with their phylogenetic relationship, indicating divergent functional evolution. Using the co-crystallized structure of the DesK-DesR from Bacillus subtilis and the modeled structures of the four NarL TCSs in S. aureus, we identified interacting residues, revealing specificity determinants and conservation within the same TCS, even from different strain backgrounds. The interacting residues were highly conserved within strains but varied between species due to selection pressures and the coevolution of cognate pairs. This study unveils the complex interplay and divergent functional evolution of TCSs, highlighting their potential for future experimental exploration of phosphotransfer between cognate and non-cognate recombinant HK and RRs.IMPORTANCEGiven the widespread conservation of two-component systems (TCSs) in bacteria and their pivotal role in regulating metabolic and virulence pathways, they present a compelling target for anti-microbial agents, especially in the face of rising multi-drug-resistant infections. Harnessing TCSs therapeutically necessitates a profound understanding of their evolutionary trajectory in signal transduction, as this underlies their unique or shared virulence regulatory pathways. Such insights are critical for effectively targeting TCS components, ensuring an optimized impact on bacterial virulence, and mitigating the risk of resistance emergence via the evolution of alternative pathways. Our research offers an in-depth exploration of virulence determinants controlled by TCSs in S. aureus, shedding light on the evolving specificity determinants that orchestrate interactions between their cognate pairs.

Zinc is an important transition metal that is essential for numerous physiological processes while excessive zinc is cytotoxic. Pseudomonas aeruginosa is a ubiquitous opportunistic human pathogen equipped with an exquisite zinc homeostatic system, and the two-component system CzcS/CzcR plays a key role in zinc detox ification. Although an increasing number of studies have shown the versatility of CzcS/CzcR, its physiological functions are still not fully understood. In this study, transcriptome analysis was performed, which revealed that CzcS/CzcR is silenced in the absence of the zinc signal but modulates global gene expression when the pathogen encounters zinc excess. CzcR was demonstrated to positively regulate the copper tolerance gene ptrA and negatively regulate the pyochelin biosynthesis regulatory gene pchR through direct binding to their promoters. Remarkably, the upregulation of ptrA and downregulation of pchR were shown to rescue the impaired capacity of copper tolerance and prevent pyochelin overproduction, respectively, caused by zinc excess. This study not only advances our understanding of the regulatory spectrum of CzcS/CzcR but also provides new insights into stress adaptation mediated by two-component systems in bacteria to balance the cellular processes that are disturbed by their signals. IMPORTANCE CzcS/CzcR is a two-component system that has been found to modulate zinc homeostasis, quorum sensing, and antibiotic resistance in Pseudomonas aeruginosa. To fully understand the physiological functions of CzcS/CzcR, we performed a comparative transcriptome analysis in this study and discovered that CzcS/CzcR controls global gene expression when it is activated during zinc excess. In particular, we demonstrated that CzcS/CzcR is critical for maintaining copper tolerance and iron homeostasis, which are disrupted during zinc excess, by inducing the expression of the copper tolerance gene ptrA and repressing the pyochelin biosynthesis genes through pchR. This study revealed the global regulatory functions of CzcS/CzcR and described a new and intricate adaptive mechanism in response to zinc excess in P. aeruginosa. The findings of this study have important implications for novel anti-infective interventions by incorporating metal-based drugs. [ABSTRACT FROM AUTHOR]

Background: Previous studies have confirmed the involvement of EmaSR (ethanol metabolism a sensor/regulator) in the regulation of Acinetobacter baumannii ATCC 19606 ethanol and acetate metabolism. RNA-seq analysis further revealed that DJ41_568-571, DJ41_2796, DJ41_3218, and DJ41_3568 regulatory gene clusters potentially participate in ethanol and acetate metabolism under the control of EmaSR. Methods: This study fused the EmaSR regulon promoter segments with reporter genes and used fluorescence expression levels to determine whether EmaSR influences regulon expression in ethanol or acetate salt environments. The enzymatic function and kinetics of significantly regulated regulons were also studied. Results: The EmaSR regulons P2796 and P3218 exhibited > 2-fold increase in fluorescence expression in wild type compared to mutant strains in both ethanol and acetate environments, and PemaR demonstrated a comparable trend. Moreover, increases in DJ41_2796 concentration enhanced the conversion of acetate and succinyl-CoA into acetyl-CoA and succinate, suggesting that DJ41_2796 possesses acetate: succinyl-CoA transferase (ASCT) activity. The kcat/KM values for DJ41_2796 with potassium acetate, sodium acetate, and succinyl-CoA were 0.2131, 0.4547, and 20.4623 mM−1s−1, respectively. Conclusions: In A. baumannii, EmaSR controls genes involved in ethanol and acetate metabolism, and the EmaSR regulon DJ41_2796 was found to possess ASCT activity. [ABSTRACT FROM AUTHOR]