Regulator Of Capsule Synthesis Research Articles

Key biological processes and essential genes for Proteus mirabilis biofilm development inhibition by protocatechuic acid

Proteus mirabilis is an opportunistic pathogen linked to human urinary tract infections, and is potentially present as a foodborne pathogen within poultry products, including broiler chickens. This report outlines the inhibitory impacts of protocatechuic acid (PCA) on P. mirabilis isolated from a broiler slaughterhouse in China as well as its biofilm. This investigation encompasses assays related to motility and adhesion, bacterial metabolic activity, extracellular polymer (EPS) production, and scavenging capacity. The findings demonstrated that PCA reduced biofilm formation by 61 %. Transcriptomics findings identified that PCA limited the expression of genes like PstS that promote adhesin formation, rbsA and RcsB that alter bacterial chemotaxis, lipopolysaccharide synthesis genes LpxA and EptB, and cell wall synthesis genes MurF and MrdA, and affects the Regulator of Capsule Synthesis (RCS) two-component modulation system. Weighted gene co-expression network analysis (WGCNA) was conducted to identify the core genes. Furthermore, the binding sites of PCA to cytochrome oxidases cydA and cydB, two subunits of ATP synthase atpI and atpH, and ftsZ, which regulate bacterial division, were predicted via molecular docking. Metabolome analysis determined that PCA critically influenced coenzyme A biosynthesis, nucleotide metabolism, alanine, aspartic acid, and glutamate metabolic pathways of P. mirabilis. Therefore, PCA impacts metabolism within bacteria via various pathways, limiting the levels of extracellular polymer and bacterial viability to hinder biofilm formation. Additionally, we prepared an antibacterial plastic film containing protocatechuic acid using PVA as the monomer and CNC as the reinforcing agent. We examined the mechanical and antibacterial properties of this film. When used to wrap chicken, it reduced the total number of colonies, slowed the deterioration of chicken, and maintained the freshness of chicken. In conclusion, the information outlined in this study complements our comprehension of P. mirabilis inhibition by PCA and provides clues for the reduction of foodborne infections associated with P. mirabilis.

Escherichia coli has robust regulatory mechanisms against elevated peptidoglycan cleavage by lytic transglycosylases

Peptidoglycan (PG) is an essential and conserved exoskeletal component in all bacteria that protects cells from lysis. Gram-negative bacteria such as Escherichia coli encode multiple redundant lytic transglycosylases (LTs) that engage in PG cleavage, a potentially lethal activity requiring proper regulation to prevent autolysis. To elucidate the potential effects and cellular regulatory mechanisms of elevated LT activity, we individually cloned the periplasmic domains of two membrane-bound LTs, MltA and MltB, under the control of the arabinose-inducible system for overexpression in the periplasmic space in E.coli. Interestingly, upon induction, the culture undergoes an initial period of cell lysis followed by robust growth restoration. The LT-overexpressing E.coli exhibits altered morphology with larger spherical cells, which is in line with the weakening of the PG layer due to aberrant LT activity. On the other hand, the restored cells display a similar rod shape and PG profile that is indistinguishable from the uninduced control. Quantitative proteomics analysis of the restored cells identified significant protein enrichment in the regulator of capsule synthesis (Rcs) regulon, a two-component stress response known to be specifically activated by PG damage. We showed that LT-overexpressing E.coli with an inactivated Rcs system partially impairs the growth restoration process, supporting the involvement of the Rcs system in countering aberrant PG cleavage. Furthermore, we demonstrated that the elevated LT activity specifically potentiates β-lactam antibiotics against E.coli with a defective Rcs regulon, suggesting the dual effects of augmented PG cleavage and blocked PG synthesis as a potential antimicrobial strategy.

Conformational rearrangements in the sensory RcsF/OMP complex mediate signal transduction across the bacterial cell envelope.

Timely detection and repair of envelope damage are paramount for bacterial survival. The Regulator of Capsule Synthesis (Rcs) stress response can transduce the stress signals across the multilayered gram-negative cell envelope to regulate gene expression in the cytoplasm. Previous studies defined the overall pathway, which begins with the sensory lipoprotein RcsF interacting with several outer membrane proteins (OMPs). RcsF can also interact with the periplasmic domain of the negative regulator IgaA, derepressing the downstream RcsCDB phosphorelay. However, how the RcsF/IgaA interaction is regulated at the molecular level to activate the signaling in response to stress remains poorly understood. In this study, we used a site-saturated mutant library of rcsF to carry out several independent genetic screens to interrogate the mechanism of signal transduction from RcsF to IgaA. We analyzed several distinct classes of rcsF signaling mutants, and determined the region of RcsF that is critically important for signal transduction. This region is bifunctional as it is important for RcsF interaction with both IgaA and OMPs. The mutant analysis provides strong evidence for conformational changes in the RcsF/OMP complex mediating signal transduction to IgaA, and the first direct evidence that OMPs play an important regulatory role in Rcs signaling.

206. Using Genome-Wide Association Analysis of Pneumococcal Isolates to Identify Novel Gene Variants in Central Nervous System Infection

Abstract Background Streptococcus pneumoniae is the leading cause of meningitis in children and is associated with significant morbidity and mortality. Despite conjugate pneumococcal vaccines that have otherwise been successful in reducing invasive disease caused by vaccine serotypes, the incidence of pneumococcal meningitis remains relatively unchanged. Using genome-wide association, we identified sequence elements associated with pneumococcal central nervous system (CNS) infection. Methods From 1996 to 2018, 366 clinical pneumococcal isolates were collected from pediatric patients and paired with clinical metadata. All isolates underwent whole-genome sequencing and in silico serotyping. Fifty-four clinical isolates were from children with CNS infection. Sequence element enrichment analysis was performed using elastic net and linear mixed modeling statistical methods to find genetic variants associated with CNS infection. We identified associated genes by mapping statistically associated variants to a serotype 19F reference S. pneumoniae genome. Results CNS isolates spanned 22 unique serotypes; the most common were 22F (n = 6), 7F (n = 5), 18C (n = 4), and 35B (n = 4). We identified two intergenic sequence variants and one intragenic sequence variant significantly associated with CNS infection (p < 1.48 × 10-7). The top intragenic candidate in our analysis was located within cpsC, a gene involved in regulation of capsule synthesis. The CNS infection-associated cpsC variant was found in serotypes 22F (n = 1), 18C (n = 4), and 14 (n = 2). Conclusion The diversity of pneumococcal isolates associated with CNS infection suggests the importance of genetic determinants beyond capsular serotype. We identified a number of novel microbial genetic variants significantly associated with pneumococcal CNS infection across serotypes, including a coding mutation in cpsC. Functional characterization of cpsC variants is needed to inform mechanisms contributing to pneumococcal CNS virulence. Disclosures Anne J. Blaschke, MD, PhD, BioFire Diagnostics/Biomerieux: Advisor/Consultant|BioFire Diagnostics/Biomerieux: Grant/Research Support|BioFire Diagnostics/Biomerieux: IP licensed to BioFire Diagnostics through the University of Utah and royalties received through the University of Utah related to the FilmArray|Merck: Advisor/Consultant.

Regulation of ECP fimbriae-related genes by the transcriptional regulator RcsAB in Klebsiella pneumoniae NTUH-K2044.

Klebsiella pneumoniae is one of the major pathogens causing nosocomial infections. The regulator of capsule synthesis (Rcs) system is a complex signal transduction pathway that is involved in the regulation of virulence factors of K. pneumoniae as an important transcriptional regulator. The RcsAB box-like sequence was found to be present in the promoter-proximal regions of ykgK, one of the ECP fimbriae-related genes, which suggested the expression of ECP fimbriae may be regulated by RcsAB. The ykgK gene in K. pneumoniae has 86% similarity to the ecpR gene in Escherichia coli. Nucleotide sequence alignment revealed a similar ECP fimbriae gene cluster including six genes in K. pneumoniae, which was proved to be on the same operon in this study. The electrophoretic mobility shift assay and DNase I assay, relative fluorescence expression, β-galactosidase activity, and relative gene expression of ykgK in the wild-type and mutant strains were performed to determine the transcriptional regulation mechanism of RcsAB on ECP fimbriae. The mutant ΔykgK and complementary strain ΔykgK/cΔykgK were constructed to complete theGalleria mellonella larvae infection experiment and biofilm formation assay. This study showed that RcsAB binds directly to the promoter region of the ykgK gene to positively regulate ECP fimbriae-related gene clusters, and then positively affect the biofilm formation.

High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex function

The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the β-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF's partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494L and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.

The Rcs System in Enterobacteriaceae: Envelope Stress Responses and Virulence Regulation.

The bacterial cell envelope is a protective barrier at the frontline of bacterial interaction with the environment, and its integrity is regulated by various stress response systems. The Rcs (regulator of capsule synthesis) system, a non-orthodox two-component regulatory system (TCS) found in many members of the Enterobacteriaceae family, is one of the envelope stress response pathways. The Rcs system can sense envelope damage or defects and regulate the transcriptome to counteract stress, which is particularly important for the survival and virulence of pathogenic bacteria. In this review, we summarize the roles of the Rcs system in envelope stress responses (ESRs) and virulence regulation. We discuss the environmental and intrinsic sources of envelope stress that cause activation of the Rcs system with an emphasis on the role of RcsF in detection of envelope stress and signal transduction. Finally, the different regulation mechanisms governing the Rcs system’s control of virulence in several common pathogens are introduced. This review highlights the important role of the Rcs system in the environmental adaptation of bacteria and provides a theoretical basis for the development of new strategies for control, prevention, and treatment of bacterial infections.

RcsAB and Fur Coregulate the Iron-Acquisition System via entC in Klebsiella pneumoniae NTUH-K2044 in Response to Iron Availability.

The iron acquisition system is an essential virulence factor for human infection and is under tight regulatory control in a variety of pathogens. Ferric-uptake regulator (Fur) is one of Fe2+-responsive transcription factor that maintains iron homeostasis, and the regulator of capsule synthesis (Rcs) is known to regulate exopolysaccharide biosynthesis. We speculate the Rcs may involve in iron-acquisition given the identified regulator box in the upstream of entC that participated in the biosynthesis of enterobactin. To study the coregulation by RcsAB and Fur of entC, we measured the β-galactosidase activity and relative mRNA expression of entC in WT and mutant strains. The RcsAB- and Fur-protected regions were identified by an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay. A regulatory cascade was identified with which Fur repressed rcsA expression and reduced RcsAB and entC expression. Our study demonstrated that entC was coregulated by two different transcriptional regulators, namely, RcsAB and Fur, in response to iron availability in Klebsiella pneumoniae.

Transcriptomic analysis reveals the role of RcsB in suppressing bacterial chemotaxis, flagellar assembly and infection in Yersinia enterocolitica.

Defining the Rcs (Regulator of Capsule Synthesis) regulon in Enterobacteriaceae has been the major focus of several recent studies. The overall role of the Rcs system in Yersinia enterocolitica is largely unknown. Our previous study showed that RcsB inhibits motility, biofilm formation and c-di-GMP production by negatively regulating flhDC, hmsHFRS and hmsT expression. To identify other cellular functions regulated by the RcsB, gene expression profiles of the wild type and ΔrcsB mutant were compared by RNA-Seq in this study. A total of 132 differentially expressed genes regulated by the RcsB have been identified, of which 114 were upregulated and 18 were downregulated. Further, the results of RNA sequencing were discussed with a focus on the predictive roles of RcsB in the inhibition of bacterial chemotaxis, flagellar assembly and infection. To confirm these predictions, we experimentally verified that the ΔrcsB mutant activated chemotactic behavior and flagella biosynthesis, and exhibited enhanced adhesion and invasion of Y. enterocolitica to Caco-2 cells. Although RcsB largely inhibits these physiological activities, the presence of RcsB is still of great significance for optimizing the survival of Y. enterocolitica as evidenced by our previous report that RcsB confers some level of resistance to the cationic antimicrobial peptide polymyxin B in Y. enterocolitica. Overall, the information provided in this study complements our understanding of Rcs phosphorelay in the regulation of Y. enterocolitica pathogenicity, and, simultaneously, provides clues to additional roles of the Rcs system in other members of family Enterobacteriaceae.

Distinct domains of Escherichia coli IgaA connect envelope stress sensing and down-regulation of the Rcs phosphorelay across subcellular compartments.

In enterobacteria, the Rcs system (Regulator of capsule synthesis) monitors envelope integrity and induces a stress response when damages occur in the outer membrane or in the peptidoglycan layer. Built around a two-component system, Rcs controls gene expression via a cascade of phosphoryl transfer reactions. Being particularly complex, Rcs also involves the outer membrane lipoprotein RcsF and the inner membrane essential protein IgaA (Intracellular growth attenuator). RcsF and IgaA, which are located upstream of the phosphorelay, are required for normal Rcs functioning. Here, we establish the stress-dependent formation of a complex between RcsF and the periplasmic domain of IgaA as the molecular signal triggering Rcs. Moreover, molecular dissection of IgaA reveals that its negative regulatory role on Rcs is mostly carried by its first N-terminal cytoplasmic domain. Altogether, our results support a model in which IgaA regulates Rcs activation by playing a direct role in the transfer of signals from the cell envelope to the cytoplasm. This remarkable feature further distinguishes Rcs from other envelope stress response systems.

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins.

RcsF (regulator of capsule synthesis) is an outer membrane (OM) lipoprotein that functions to sense defects such as changes in LPS. However, LPS is found in the outer leaflet, and RcsF was thought to be tethered to the inner leaflet by its lipidated N terminus, raising the question of how it monitors LPS. We show that RcsF has a transmembrane topology with the lipidated N terminus on the cell surface and the C-terminal signaling domain in the periplasm. Strikingly, the short, unstructured, charged transmembrane domain is threaded through the lumen of β-barrel OM proteins where it is protected from the hydrophobic membrane interior. We present evidence that these unusual complexes, which contain one protein inside another, are formed by the Bam complex that assembles all β-barrel proteins in the OM. The ability of the Bam complex to expose lipoproteins at the cell surface underscores the mechanistic versatility of the β-barrel assembly machine.

Modulation of the Rcs-mediated signal transfer by conformational flexibility

The Rcs (regulator of capsule synthesis) signalling complex comprises the membrane-associated hybrid sensor kinases RcsC and RcsD, the transcriptional regulator RcsB and the two co-inducers RcsA and RcsF. Acting as a global regulatory network, the Rcs phosphorelay controls multiple cellular pathways including capsule synthesis, cell division, motility, biofilm formation and virulence mechanisms. Signal-dependent communication of the individual Rcs domains showing histidine kinase, phosphoreceiver, phosphoryl transfer and DNA-binding activities is characteristic and essential for the modulation of signal transfer. We have analysed the structures of core elements of the Rcs network including the RcsC-PR (phosphoreceiver domain of RcsC) and the RcsD-HPt (histidine phosphotransfer domain of RcsD), and we have started to characterize the dynamics and recognition mechanisms of the proteins. RcsC-PR represents a typical CheY-like alpha/beta/alpha sandwich fold and it shows a large conformational flexibility near the active-site residue Asp(875). NMR analysis revealed that RcsC-PR is able to adopt preferred conformations upon Mg(2+) co-ordination, BeF(3)(-) activation, phosphate binding and RcsD-HPt recognition. In contrast, the alpha-helical structure of RcsD-HPt is conformationally stable and contains a recognition area in close vicinity to the active-site His(842) residue. Our studies indicate the importance of protein dynamics and conformational exchange for the differential response to the variety of signals perceived by complex regulatory networks.

Modification of Lipopolysaccharide with Colanic Acid (M-antigen) Repeats in Escherichia coli

Colanic acid (CA) or M-antigen is an exopolysaccharide produced by many enterobacteria, including the majority of Escherichia coli strains. Unlike other capsular polysaccharides, which have a close association with the bacterial surface, CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. Herein we show that a highly mucoid strain of E. coli K-12 ligates CA repeats to a significant proportion of lipopolysaccharide (LPS) core acceptor molecules, forming the novel LPS glycoform we call MLPS.MLPS biosynthesis is dependent upon (i) CA induction, (ii) LPS core biosynthesis, and (iii) the O-antigen ligase WaaL. Compositional analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy of a purified MLPS sample confirmed the presence of a CA repeat unit identical in carbohydrate sequence, but differing at multiple positions in anomeric configuration and linkage, from published structures of extracellular CA. The attachment point was identified as O-7 of the L-glycero-D-manno-heptose of the outer LPS core, the same position used for O-antigen ligation. When O-antigen biosynthesis was restored in the K-12 background and grown under conditions meeting the above specifications, only MLPS was observed, suggesting E. coli can reversibly change its proximal covalently linked cell surface polysaccharide coat from O-antigen to CA in response to certain environmental stimuli. The identification of MLPS has implications for potential underlying mechanisms coordinating the synthesis of various surface polysaccharides.

Transcriptional organization and regulation of the Escherichia coli K30 group 1 capsule biosynthesis (cps) gene cluster.

Escherichia coli group 1 capsules are important virulence determinants, yet little is known about the transcriptional organization or regulation of their biosynthetic (cps) operons. Transcription of the prototype serotype K30 cluster is modulated by the JUMPStart-RfaH antitermination mechanism, with the cps promoter being localized to a region immediately upstream of the JUMPStart sequence. A putative stem-loop structure located within the K30 cps cluster separates conserved genes with products that are required for surface expression of capsule from serotype-specific genes encoding enzymes for polymer repeat-unit synthesis and polymerization. This putative stem-loop structure significantly reduces transcription in a termination-probe vector and may contribute to differential expression of the cps genes. Previous work indicated that increased amounts of group 1 capsular polysaccharide synthesis resulted from the overexpression of the Rcs (regulator of capsule synthesis) proteins. However, neither overexpression of the transcriptional activator RcsB nor an rcsB::aadA chromosomal insertion altered the level of transcription measured by cps::lacZ fusions. In the group 1 strains examined, an RcsAB box was found immediately upstream of galF, a gene involved in the production of sugar nucleotide precursors. Overexpression of RcsB was found to result in a threefold increase in transcription of a galF::lacZ chromosomal fusion. Moreover, overexpression of GalF gave rise to a two- to threefold increase in cell-free as well as cell-associated capsule, without affecting cps::lacZ activity. These results indicate that transcription of the E. coli group 1 capsule cluster itself is not regulated by the Rcs system and may, in fact, be constitutive. However, the Rcs system can potentially influence levels of capsular polysaccharide production by increasing galF transcription and influencing the available pool of biosynthetic precursors.

Identification of an RcsA/RcsB Recognition Motif in the Promoters of Exopolysaccharide Biosynthetic Operons from Erwinia amylovora and Pantoea stewartii Subspeciesstewartii

The regulation of capsule synthesis (Rcs) regulatory network is responsible for the induction of exopolysaccharide biosynthesis in many enterobacterial species. We have previously shown that two transcriptional regulators, RcsA and RcsB, do bind as a heterodimer to the promoter of amsG, the first reading frame in the operon for amylovoran biosynthesis in the plant pathogenic bacterium Erwinia amylovora. We now identified a 23-base pair fragment from position -555 to -533 upstream of the translational start site of amsG as sufficient for the specific binding of the Rcs proteins. In addition, we could detect an RcsA/RcsB-binding site in a corresponding region of the promoter of cpsA, the homologous counterpart to the E. amylovora amsG gene in the operon for stewartan biosynthesis of Pantoea stewartii. The specificity and characteristic parameters of the protein-DNA interaction were analyzed by DNA retardation, protein-DNA cross-linking, and directed mutagenesis. The central core motif TRVGAAWAWTSYG of the amsG promoter was found to be most important for the specific interaction with RcsA/RcsB, as evaluated by mutational analysis and an in vitro selection approach. The wild type P. stewartii Rcs binding motif is degenerated in two positions and an up-mutation according to our consensus motif resulted in about a 5-fold increased affinity of the RcsA/RcsB proteins.

Isolation from Klebsiella and Characterization of Two rcs Genes That Activate Colanic Acid Capsular Biosynthesis in Escherichia coli

Two genes, designated rcsA (regulation of capsule synthesis) and rcsB, that had been cloned from the chromosome of Klebsiella aerogenes (K. pneumoniae) capsular serotype K21 were capable of activating expression of colanic acid capsular polysaccharide in Escherichia coli K12. The Klebsiella rcsA gene encoded a polypeptide of 23 kDa that was required for the induction of a mucoid phenotype at less than or equal to 30 degrees C but not at greater than or equal to 37 C. The Klebsiella rcsB locus encoded no apparent polypeptides and was not capable by itself of causing the overproduction of colanic acid. However, when present in the same cell with rcsA, either in cis or in trans, rcsB caused expression of mucoidy in E. coli at all growth temperatures. These findings are best explained if the Klebsiella rcsA gene product acts as a positive regulator of colanic acid biosynthesis in E. coli and that activity of this protein is in turn subject to regulation by Lon protease. The Klebsiella rcsB locus may exert its effect by preferentially binding a negative regulator of capsular biosynthesis, possibly Lon itself. DNA sequences homologous to the Klebsiella K21b rcsA and rcsB genes were found in the genomes of all other capsular serotypes of klebsiellae examined, including K2, K12, K36 and K43. However, there was no homology between such genes and the chromosome of E. coli. The ability of these rcs genes to induce a mucoid phenotype explains the apparent conjugative transfer from klebsiellae to E. coli of the ability to produce K21 or other Klebsiella capsular polysaccharides that are structurally and antigenically related to colanic acid.

Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide.

Cryptococcus neoformans is variably encapsulated in vitro, whereas in tissues it develops a large capsule. We observed that cells of a strain with thin capsules, when growing in a standard fungal culture medium, became heavily encapsulated when incubated in serum-free cell culture medium (Dulbecco's modified Eagle's medium [DME]). Capsule size was quantitated physically by measuring cell volume, and chemically by determining the content of a capsular monosaccharide, glucuronate. The CO2/HCO-3 couple stimulated capsule development, resulting in visible enlargement by 3 h after exposure to high CO2/HCO-3. The amount of capsule per cell was directly proportional to the total millimolar CO2/HCO-3 concentration between 24 and 2.4 mM at pH 7.35, but at constant PCO2 (40 torr) and varying [HCO-3], the cells were heavily encapsulated down to pH 6.8. Concentration of CO2/HCO-3 in the physiologic range increased elaboration of polysaccharide into the medium and slowed the cell generation time from 2 to 6 h. Four other first-passage clinical isolates were all heavily encapsulated in DME with CO2/HCO-3, but variably encapsulated in DME without CO2/HCO-3. Exposure of yeast to increased CO2/HCO-3 caused a marked reduction in complement-mediated phagocytosis by mouse macrophages. A stable clone was isolated which contained capsular polysaccharide, but lacked the CO2-inducible phenotype. This clone was avirulent for steroid-treated rabbits. Thus, the prevailing CO2 concentration in mammalian tissues may be one stimulus for capsular polysaccharide synthesis. This could serve as an adaptive mechanism favoring parasite survival in the host.