Deletion Of ccpA Research Articles

CcpA Regulates Staphylococcus aureus Biofilm Formation through Direct Repression of Staphylokinase Expression.

Staphylococcus aureus represents a notorious opportunistic pathogen causing various infections in biofilm nature, imposing remarkable therapeutic challenges worldwide. The catabolite control protein A (CcpA), a major regulator of carbon catabolite repression (CCR), has been recognized to modulate S. aureus biofilm formation, while the underlying mechanism remains to be fully elucidated. In this study, the reduced biofilm was firstly determined in the ccpA deletion mutant of S. aureus clinical isolate XN108 using both crystal violet staining and confocal laser scanning microscopy. RNA-seq analysis suggested that sak-encoding staphylokinase (Sak) was significantly upregulated in the mutant ∆ccpA, which was further confirmed by RT-qPCR. Consistently, the induced Sak production correlated the elevated promoter activity of sak and increased secretion in the supernatants, as demonstrated by Psak-lacZ reporter fusion expression and chromogenic detection, respectively. Notably, electrophoretic mobility shift assays showed that purified recombinant protein CcpA binds directly to the promoter region of sak, suggesting the direct negative control of sak expression by CcpA. Double isogenic deletion of ccpA and sak restored biofilm formation for mutant ∆ccpA, which could be diminished by trans-complemented sak. Furthermore, the exogenous addition of recombinant Sak inhibited biofilm formation for XN108 in a dose-dependent manner. Together, this study delineates a novel model of CcpA-controlled S. aureus biofilm through direct inhibition of sak expression, highlighting the multifaceted roles and multiple networks regulated by CcpA.

The Global Regulator CcpA of Listeria monocytogenes Confers Sensitivity to Antimicrobial Fatty Acids.

Free fatty acids (FFAs) are known to exhibit antimicrobial and anti-virulent properties against bacterial pathogens. Specific FFAs, such as lauric acid (LA; C12:0), exert both effects against the foodborne pathogen Listeria monocytogenes: at low levels, LA acts to inhibit the activity of the virulence regulator PrfA, whereas at higher levels, LA inhibits bacterial growth. Deletion of prfA is known to promote tolerance toward antimicrobial FFAs, suggesting that the response of L. monocytogenes to anti-virulent and antimicrobial FFAs could be linked. In this study, we explored the response of L. monocytogenes toward antimicrobial FFAs holding an anti-virulence activity by isolating strains that can grow at high concentrations of LA. We found that LA-tolerant isolates carry mutations in the gene encoding the global regulator CcpA. Importantly, we discovered that mutation or deletion of ccpA protect L. monocytogenes against the antimicrobial activity of FFAs, whereas the ccpA mutants remain sensitive toward FFA’s PrfA inhibitory effect. A regulatory link involving CcpA, connecting the response toward the antimicrobial and anti-virulence activities of FFAs, is therefore unlikely. To further study how deletion of ccpA promotes FFA tolerance, we performed a transcriptomic analysis of the response to LA. Our data indicated that the FFA-tolerant phenotype of the ∆ccpA strain is not induced upon LA exposure but appears to be an inherent phenotypic trait of the ccpA deletion mutation. Interestingly, we found that the bacterial surface of L. monocytogenes becomes more hydrophilic upon deletion of ccpA, and we demonstrate that CcpA plays a role in the response of L. monocytogenes to other stress conditions, including low pH and antibiotics. Altogether, our study revealed that regulatory activities of CcpA lead to an increased hydrophobicity of the bacterial surface, which may confer sensitivity of L. monocytogenes against the antimicrobial activity of FFAs. Notably, CcpA is not involved in responding to the PrfA inhibitory effect of FFAs, showing that FFA-tolerant strains can still be targeted by the anti-virulent activity of FFAs.

Regulation of CcpA on the growth and organic acid production characteristics of ruminal Streptococcus bovis at different pH

BackgroundCatabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase and pyruvate formate-lyase in Streptococcus bovis, but knowledge of its role in response to different pH is still limited. In this study, a ccpA-knockout strain of S. bovis S1 was constructed and then used to examine the effects of ccpA gene deletion on the growth and fermentation characteristics of S. bovis S1 at pH 5.5 or 6.5.ResultsThere was a significant interaction between strain and pH for the maximum specific growth rate (μmax) and growth lag period (λ), which caused a lowest μmax and a longest λ in ccpA-knockout strain at pH 5.5. Deletion of ccpA decreased the concentration and molar percentage of lactic acid, while increased those of formic acid. Strains at pH 5.5 had decreased concentrations of lactic acid and formic acid compared to pH 6.5. The significant interaction between strain and pH caused the highest production of total organic acids and acetic acid in ccpA-knockout strain at pH 6.5. The activities of α-amylase and lactate dehydrogenase decreased in ccpA-knockout strain compared to the wild-type strain, and increased at pH 5.5 compared to pH 6.5. There was a significant interaction between strain and pH for the activity of acetate kinase, which was the highest in the ccpA-knockout strain at pH 6.5. The expression of pyruvate formate-lyase and acetate kinase was higher in the ccpA-knockout strain compared to wild-type strain. The lower pH improved the relative expression of pyruvate formate-lyase, while had no effect on the relative expression of acetate kinase. The strain × pH interaction was significant for the relative expression of lactate dehydrogenase and α-amylase, both of which were highest in the wild-type strain at pH 5.5 and lowest in the ccpA-knockout strain at pH 6.5.ConclusionsOverall, low pH inhibited the growth of S. bovis S1, but did not affect the fermentation pattern. CcpA regulated S. bovis S1 growth and organic acid fermentation pattern. Moreover, there seemed to be an interaction effect between pH and ccpA deletion on regulating the growth and organic acids production of S. bovis S1.

Transcriptome Analysis Reveals Catabolite Control Protein A Regulatory Mechanisms Underlying Glucose-Excess or -Limited Conditions in a Ruminal Bacterium, Streptococcus bovis.

Ruminants may suffer from rumen acidosis when fed with high-concentrate diets due to the higher proliferation and overproduction of lactate by Streptococcus bovis. The catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase (ldh) and pyruvate formate-lyase (pfl) in S. bovis, but its role in response to different carbon concentrations remains unclear. To characterize the regulatory mechanisms of CcpA in S. bovis S1 at different levels of carbon, herein, we analyzed the transcriptomic and physiological characteristics of S. bovis S1 and its ccpA mutant strain grown in glucose-excess and glucose-limited conditions. A reduced growth rate and a shift in fermentation pattern from homofermentation to heterofermentation were observed under glucose-limited condition as compared to glucose-excess condition, in S. bovis S1. Additionally, the inactivation of ccpA significantly affected the growth and end metabolites in both conditions. For the glycolytic intermediate, fructose 1,6-bisphosphate (FBP), the concentration significantly reduced at lower glucose conditions; its concentration decreased significantly in the ccpA mutant strain. Transcriptomic results showed that about 46% of the total genes were differentially transcribed between the wild-type strain and ccpA mutant strain grown in glucose-excess conditions; while only 12% genes were differentially transcribed in glucose-limited conditions. Different glucose concentrations led to the differential expression of 38% genes in the wild-type strain, while only half of these were differentially expressed in the ccpA-knockout strain. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the substrate glucose concentration significantly affected the gene expression in histidine metabolism, nitrogen metabolism, and some carbohydrate metabolism pathways. The deletion of ccpA affected several genes involved in carbohydrate metabolism, such as glycolysis, pyruvate metabolism, fructose and mannose metabolism, as well as in fatty acid biosynthesis pathways in bacteria grown in glucose-excess conditions; this effect was attenuated under glucose-limited conditions. Overall, these findings provide new information on gene transcription and metabolic mechanisms associated with substrate glucose concentration and validate the important role of CcpA in the regulation of carbon metabolism in S. bovis S1 at differential glucose availability.

Effects of CcpA against salt stress in Lactiplantibacillus plantarum as assessed by comparative transcriptional analysis.

Lactiplantibacillus plantarum is frequently exposed to salt stress during industrial applications. Catabolite control protein (CcpA) controls the transcription of many genes, but its role in the response to salt stress remains unclear. In this study, we used transcriptome analyses to investigate differences in the logarithmic growth phases of Lactiplantibacillus plantarum ST-III and its ccpA-knockout mutant when grown with or without salt and glycine betaine (GB). The deletion of ccpA significantly affected bacterial growth under different conditions. Among the comparisons, the highest proportion of differentially expressed genes (64%) was observed in the comparison between the wild-type and ccpA mutant grown with NaCl, whereas the lowest proportion (6%) was observed in the comparison between the ccpA mutant strain cultures grown with NaCl alone or with GB together. Transcriptomic analyses showed that CcpA could regulate GB uptake, activate iron uptake, produce acetyl-CoA, and affect fatty acid composition to maintain membrane lipid homeostasis in the adaptation of high-salinity conditions. Conclusively, these results demonstrate the importance of CcpA as a master regulator of these processes in response to salt stress, and provide new insights into the complex regulatory network of lactic acid bacteria. KEY POINTS: • The absence of CcpA significantly affected growth of L. plantarum and its response to salt stress. • CcpA regulates compatible solutes absorption and ions transport to resist salt stress. • CcpA alters fatty acids composition to maintain membrane lipid homeostasis towards salt stress.

The carbon catabolite repressor CcpA mediates optimal competence development in Streptococcus oligofermentans through post-transcriptional regulation.

Natural transformation increases the genetic diversity of bacteria, but is costly and must be strictly controlled. We previously found that deletion of ccpA, a key regulator of carbon catabolite repression (CCR), reduced transformation efficiency of Streptococcus oligofermentans, the current work further investigated the regulatory mechanisms of CcpA. The competence operon comCDE is subjected to basal and autoregulatory transcription. A luciferase reporter detected a transcriptional readthrough (TRT) from the upstream tRNAArg into the comCDE operon, which was induced by L -arginine. Insertion of the Escherichia coli T1T2 terminator downstream of tRNAArg abolished TRT, and reduced the basal comCDE transcription by 77% and also the transformation efficiency. Deletion of ccpA increased tRNAArg TRT and tRNAArg -comCDE polycistronic transcript by twofold. An in vitro transcription assay determined that CcpA promoted the transcription termination of tRNAArg TRT, and RNA EMSA and SPR assays detected equal binding affinity of CcpA to both the RNA and DNA of tRNAArg . These results indicate that CcpA controls the basal comCDE transcription by post-transcriptional actions. Overexpression of comDE or its phospho-mimicking mutant comDED58E reduced transformation efficiency, indicating that excessive ComE impairs competence development. CCR-regulated competence was further confirmed by higher tRNAArg TRT but lower transformation efficiency in galactose than in glucose.

N-acetylglucosamine-Mediated Expression of nagA and nagB in Streptococcus pneumoniae.

In this study, we have explored the transcriptomic response of Streptococcus pneumoniae D39 to N-acetylglucosamine (NAG). Transcriptome comparison of S. pneumoniae D39 wild-type grown in chemically defined medium (CDM) in the presence of 0.5% NAG to that grown in the presence of 0.5% glucose revealed elevated expression of many genes/operons, including nagA, nagB, manLMN, and nanP. We have further confirmed the NAG-dependent expression of nagA, nagB, manLMN, and nanP by β-galactosidase assays. nagA, nagB and glmS are putatively regulated by a transcriptional regulator NagR. We predicted the operator site of NagR (dre site) in PnagA, PnagB, and PglmS, which was further confirmed by mutating the predicted dre site in the respective promoters (nagA, nagB, and glmS). Growth comparison of ΔnagA, ΔnagB, and ΔglmS with the D39 wild-type demonstrates that nagA and nagB are essential for S. pneumoniae D39 to grow in the presence of NAG as a sole carbon source. Furthermore, deletion of ccpA shows that CcpA has no effect on the expression of nagA, nagB, and glmS in the presence of NAG in S. pneumoniae.

Catabolite control protein a of Streptococcus suis type 2 contributes to sugar metabolism and virulence

Catabolite control protein A (CcpA) is the major transcriptional regulator in carbon catabolite repression in several Gram-positive bacteria. We attempted to characterize the role of a CcpA homologue of Streptococcus suis type 2 in sugar metabolism and virulence. Addition of glucose or sucrose to the defined medium significantly reduced the activity of raffinose-inducible α-galactosidase, cellobiose-inducible β-glucosidase, and maltose-inducible α-glucosidase of the wild-type strain by about 9, 4, and 2-3 fold, respectively. Deletion of ccpA substantially derepressed the effects of repressing sugars on α-galactosidase or β-glucosidase activity. The ccpA deletion mutant showed reduced expression of virulence genes sly and eno (P<0.05), decreased adhesion to and invasion into endothelial cells (P<0.05), and attenuated virulence to mice with significant reduction of death rate and bacterial burden in organs, as compared to the wild-type strain. Both the in vitro and in vivo defect phenotypes were reversible by ccpA complementation. Thus, this study shows that CcpA of S. suis type 2 plays an important role in carbon catabolite repression and virulence.

CcpA coordinates central metabolism and biofilm formation in Staphylococcus epidermidis

Staphylococcus epidermidis is an opportunistic bacterium whose infections often involve the formation of a biofilm on implanted biomaterials. In S. epidermidis, the exopolysaccharide facilitating bacterial adherence in a biofilm is polysaccharide intercellular adhesin (PIA), whose synthesis requires the enzymes encoded within the intercellular adhesin operon (icaADBC). In vitro, the formation of S. epidermidis biofilms is enhanced by conditions that repress tricarboxylic acid (TCA) cycle activity, such as growth in a medium containing glucose. In many Gram-positive bacteria, repression of TCA cycle genes in response to glucose is accomplished by catabolite control protein A (CcpA). CcpA is a member of the GalR-LacI repressor family that mediates carbon catabolite repression, leading us to hypothesize that catabolite control of S. epidermidis biofilm formation is indirectly regulated by CcpA-dependent repression of the TCA cycle. To test this hypothesis, ccpA deletion mutants were constructed in strain 1457 and 1457-acnA and the effects on TCA cycle activity, biofilm formation and virulence were assessed. As anticipated, deletion of ccpA derepressed TCA cycle activity and inhibited biofilm formation; however, ccpA deletion had only a modest effect on icaADBC transcription. Surprisingly, deletion of ccpA in strain 1457-acnA, a strain whose TCA cycle is inactive and where icaADBC transcription is derepressed, strongly inhibited icaADBC transcription. These observations demonstrate that CcpA is a positive effector of biofilm formation and icaADBC transcription and a repressor of TCA cycle activity.

Role of glucose and CcpA in capsule expression and virulence of Streptococcus suis

Streptococcus suis is one of the most important pathogens in pigs and is also an emerging zoonotic agent. After crossing the epithelial barrier, S. suis causes bacteraemia, resulting in meningitis, endocarditis and bronchopneumonia. Since the host environment seems to be an important regulatory component for virulence, we related expression of virulence determinants of S. suis to glucose availability during growth and to the sugar metabolism regulator catabolite control protein A (CcpA). We found that expression of the virulence-associated genes arcB, representing arcABC operon expression, cps2A, representing capsular locus expression, as well as sly, ofs, sao and epf, differed significantly between exponential and early stationary growth of a highly virulent serotype 2 strain. Deletion of ccpA altered the expression of the surface-associated virulence factors arcB, sao and eno, as well as the two currently proven virulence factors in pigs, ofs and cps2A, in early exponential growth. Global expression analysis using a cDNA expression array revealed 259 differentially expressed genes in early exponential growth, of which 141 were more highly expressed in the CcpA mutant strain 10ΔccpA and 118 were expressed to a lower extent. Interestingly, among the latter genes, 18 could be related to capsule and cell wall synthesis. Correspondingly, electron microscopy characterization of strain 10ΔccpA revealed a markedly reduced thickness of the capsule. This phenotype correlated with enhanced binding to porcine plasma proteins and a reduced resistance to killing by porcine neutrophils. Taken together, our data demonstrate that CcpA has a significant effect on the capsule synthesis and virulence properties of S. suis.

Staphylococcus aureus CcpA Affects Biofilm Formation

Biofilm formation in Staphylococcus aureus under in vitro growth conditions is generally promoted by high concentrations of sugar and/or salts. The addition of glucose to routinely used complex growth media triggered biofilm formation in S. aureus strain SA113. Deletion of ccpA, coding for the catabolite control protein A (CcpA), which regulates gene expression in response to the carbon source, abolished the capacity of SA113 to form a biofilm under static and flow conditions, while still allowing primary attachment to polystyrene surfaces. This suggested that CcpA mainly affects biofilm accumulation and intercellular aggregation. trans-Complementation of the mutant with the wild-type ccpA allele fully restored the biofilm formation. The biofilm produced by SA113 was susceptible to sodium metaperiodate, DNase I, and proteinase K treatment, indicating the presence of polysaccharide intercellular adhesin (PIA), protein factors, and extracellular DNA (eDNA). The investigation of several factors which were reported to influence biofilm formation in S. aureus (arlRS, mgrA, rbf, sarA, atl, ica, citZ, citB, and cidABC) showed that CcpA up-regulated the transcription of cidA, which was recently shown to contribute to eDNA production. Moreover, we showed that CcpA increased icaA expression and PIA production, presumably over the down-regulation of the tricarboxylic acid cycle genes citB and citZ.

Loss of penicillin tolerance by inactivating the carbon catabolite repression determinant CcpA in Streptococcus gordonii

Antibiotic tolerance is a phenomenon allowing bacteria to withstand drug-induced killing. Here, we studied a penicillin-tolerant mutant of Streptococcus gordonii (Tol1), which was shown to be deregulated in the expression of the arginine deiminase operon (arc). arc was not directly responsible for tolerance, but is controlled by the global regulator CcpA. Therefore, we sought whether CcpA might be implicated in tolerance. The ccpA gene was characterized and subsequently inactivated by PCR ligation mutagenesis in both the susceptible wild-type (WT) and Tol1. The minimal inhibitory concentration and time-kill curves for the strains were determined and the outcome of penicillin treatment in experimental endocarditis assessed. ccpA sequence and expression were similar between the WT and Tol1 strains. In killing assays, the WT lost 3.5 +/- 0.6 and 5.3 +/- 0.6 log(10) cfu/mL and Tol1 lost 0.4 +/- 0.2 and 1.4 +/- 0.9 log(10) cfu/mL after 24 and 48 h of penicillin exposure, respectively. Deletion of ccpA almost totally restored Tol1 kill susceptibility (loss of 2.5 +/- 0.7 and 4.9 +/- 0.7 log(10) cfu/mL at the same endpoints). In experimental endocarditis, penicillin treatment induced a significant reduction in vegetation bacterial densities between Tol1 (4.1 log(10) cfu/g) and Tol1DeltaccpA (2.4 log(10) cfu/g). Restitution of ccpA re-established the tolerant phenotype both in vitro and in vivo. CcpA, a global regulator of the carbon catabolite repression system, is implicated in penicillin tolerance both in vitro and in vivo. This links antibiotic survival to bacterial sugar metabolism. However, since ccpA sequence and expression were similar between the WT and Tol1 strains, other factors are probably involved in tolerance.

Staphylococcus aureus CcpA Affects Virulence Determinant Production and Antibiotic Resistance

Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue (saCOL1786) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the DeltaccpA mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of S. aureus by down-regulating RNAIII, the effector molecule of the agr locus, and altering the transcription patterns of hla, encoding alpha-hemolysin, and spa, encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant S. aureus strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant S. aureus strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased cap operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.

Complementation of a Delta ccpA mutant of Lactobacillus casei with CcpA mutants affected in the DNA- and cofactor-binding domains.

In low-G+C Gram-positive bacteria, the regulatory protein CcpA has been shown to play a major part in the so-called carbon catabolite repression (CCR) process, as well as in the induction of basic metabolic genes, for which it is considered a global regulator. A strain of Lactobacillus casei that carried a complete deletion of ccpA has been constructed and used to test the effect of CCR on N-acetylglucosaminidase activity and growth performance of a collection of seven CcpA mutations obtained by site-directed mutagenesis. The replaced amino acids were located in the DNA- and cofactor (P-Ser-HPr)-binding domains. Mutations in the DNA-binding domain lacked CCR, as found in Bacillus megaterium. However, mutations in the cofactor-binding domain of L. casei CcpA had a different phenotype to that observed in the previous studies with B. megaterium. Two of them, S80L and T307I, displayed a significant hyper-repression, an effect never reported before for CcpA. Comparison of growth capabilities provided by the different mutants and their ability to sustain CCR demonstrated that CCR, at least on the enzymic activity tested, and the growth defect caused by the CcpA mutations are unrelated features.

Contributions of XylR CcpA and cre to diauxic growth of Bacillus megaterium and to xylose isomerase expression in the presence of glucose and xylose.

Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulate xyl operon expression on diauxic growth and expression of a xylA-lacZ fusion. xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation of xylR yields a two-fold increase in expression of xylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. When xylR and cre are inactivated together a residual two-fold repression of xylA is found. Inactivation of xylR affects diauxic growth by shortening the lag phase from 70 to 40 min. In-frame deletion of ccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivated cre site in xylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously.