Bicarbonate enhances expression of the endocarditis and biofilm associated pilus locus, ebpR-ebpABC, in Enterococcus faecalis

<p>Abstract</p> <p>Background</p> <p>We previously identified <it>ebpR</it>, encoding a potential member of the AtxA/Mga transcriptional regulator family, and showed that it is important for transcriptional activation of the <it>Enterococcus faecalis </it><b>e</b>ndocarditis and <b>b</b>iofilm associated <b>p</b>ilus operon, <it>ebpABC</it>. Although <it>ebpR </it>is not absolutely essential for <it>ebpABC </it>expression (100-fold reduction), its deletion led to phenotypes similar to those of an <it>ebpABC </it>mutant such as absence of pili at the cell surface and, consequently, reduced biofilm formation. A non-piliated <it>ebpABC </it>mutant has been shown to be attenuated in a rat model of endocarditis and in a murine urinary tract infection model, indicating an important participation of the <it>ebpR-ebpABC </it>locus in virulence. However, there is no report relating to the environmental conditions that affect expression of the <it>ebpR-ebpABC </it>locus.</p> <p>Results</p> <p>In this study, we examined the effect of CO₂/HCO₃^-, pH, and the Fsr system on the <it>ebpR-ebpABC </it>locus expression. The presence of 5% CO₂/0.1 M HCO₃^-increased <it>ebpR-ebpABC </it>expression, while the Fsr system was confirmed to be a weak repressor of this locus. The mechanism by which the Fsr system repressed the <it>ebpR-ebpABC </it>locus expression appears independent of the effects of CO₂^-bicarbonate. Furthermore, by using an <it>ebpA</it>::<it>lacZ </it>fusion as a reporter, we showed that addition of 0.1 M sodium bicarbonate to TSBG (buffered at pH 7.5), but not the presence of 5% CO₂, induced <it>ebpA </it>expression in TSBG broth. In addition, using microarray analysis, we found 73 genes affected by the presence of sodium bicarbonate (abs(fold) > 2, <it>P </it>< 0.05), the majority of which belong to the PTS system and ABC transporter families. Finally, pilus production correlated with <it>ebpA </it>mRNA levels under the conditions tested.</p> <p>Conclusions</p> <p>This study reports that the <it>ebp </it>locus expression is enhanced by the presence of bicarbonate with a consequential increase in the number of cells producing pili. Although the molecular basis of the bicarbonate effect remains unclear, the pathway is independent of the Fsr system. In conclusion, <it>E. faecalis </it>joins the growing family of pathogens that regulates virulence gene expression in response to bicarbonate and/or CO₂.</p

Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF

A comparison of two strains of the hospital pathogen Enterococcus faecalis suggests that mediators of virulence differ between strains and that virulence does not depend on mobile gene element

Transcriptional Analysis of the Bacillus anthracis Capsule Regulators

The poly-d-glutamic acid capsule of Bacillus anthracis is essential for virulence. Control of capsule synthesis occurs at the level of transcription and involves positive regulation of the capsule biosynthetic operon capBCAD by a CO(2)/bicarbonate signal and three plasmid-borne regulators: atxA, acpA, and acpB. Although the molecular mechanism for control of cap transcription is unknown, atxA affects cap expression via positive control of acpA and acpB, two genes with partial functional similarity. Transcriptional analyses of a genetically complete strain indicate that capB expression is several hundred-fold higher during growth in 5% CO(2) compared to growth in air. atxA was expressed appreciably during growth in air and induced only 2.5-fold by CO(2). In contrast, expression of acpA and acpB was induced up to 23-fold and 59-fold, respectively, by CO(2). The 5′-end mapping of gene transcripts revealed atxA-regulated and atxA-independent apparent transcription start sites for capB, acpA, and acpB. Transcripts mapping to all atxA-regulated start sites were increased during growth in elevated CO(2). The acpA gene has one atxA-regulated and one atxA-independent start site. acpB lies downstream of capBCAD. A single atxA-independent start site maps immediately upstream of acpB. atxA-mediated control of acpB appears to occur via transcriptional read-through from atxA-dependent start sites 5′ of capB. One atxA-independent and two atxA-regulated start sites map upstream of capB. Transcription from the atxA-regulated start sites of capBCAD was reduced significantly in an acpA acpB double mutant but unaffected in mutants with deletion of only acpA or acpB, in agreement with the current model for epistatic relationships between the regulators

Planning a Phase 1 Clinical Trial: Target Product Profile for a Novel Target CAR T-cell Therapy

https://openworks.mdanderson.org/sumexp21/1013/thumbnail.jp

Comparison of OG1RF and an Isogenic fsrB Deletion Mutant by Transcriptional Analysis: the Fsr System of Enterococcus faecalis Is More than the Activator of Gelatinase and Serine Protease

The FsrABC system of Enterococcus faecalis controls the expression of gelatinase and a serine protease via a quorum-sensing mechanism, and recent studies suggest that the Fsr system may also regulate other genes important for virulence. To investigate the possibility that Fsr influences the expression of additional genes, we used transcriptional profiling, with microarrays based on the E. faecalis strain V583 sequence, to compare the E. faecalis strain OG1RF with its isogenic mutant, TX5266, an fsrB deletion mutant. We found that the presence of an intact fsrB influences expression of numerous genes throughout the growth phases tested, namely, late log to early stationary phase. In addition, the Fsr regulon is independent of the activity of the proteases, GelE and SprE, whose expression was confirmed to be activated at all three time points tested. While expression of some genes (i.e., ef1097 and ef0750 to -757, encoding hypothetical proteins) was activated in late log phase in OG1RF versus the fsrB deletion mutant, expression of ef1617 to -1634 (eut-pdu orthologues) was highly repressed by the presence of an intact Fsr at entry into stationary phase. This is the first time that Fsr has been characterized as a negative regulator. The newly recognized Fsr-regulated targets include other factors, besides gelatinase, described as important for biofilms (BopD), and genes predicted to encode the surface proteins EF0750 to -0757 and EF1097, along with proteins implicated in several metabolic pathways, indicating that the FsrABC system may be an important regulator in strain OG1RF, with both positive and negative effects

Further Characterization of the epa Gene Cluster and Epa Polysaccharides of Enterococcus faecalis▿ †

We previously identified a gene cluster, epa (for enterocococcal polysaccharide antigen), involved in polysaccharide biosynthesis of Enterococcus faecalis and showed that disruption of epaB and epaE resulted in attenuation in translocation, biofilm formation, resistance to polymorphonuclear leukocyte (PMN) killing, and virulence in a mouse peritonitis model. Using five additional mutant disruptions in the 26-kb region between orfde2 and OG1RF_0163, we defined the epa locus as the area from epaA to epaR. Disruption of epaA, epaM, and epaN, like prior disruption of epaB and epaE, resulted in alteration in Epa polysaccharide content, more round cells versus oval cells with OG1RF, decreased biofilm formation, attenuation in a mouse peritonitis model, and resistance to lysis by the phage NPV-1 (known to lyse OG1RF), while mutants disrupted in orfde2 and OG1RF_163 (the epa locus flanking genes) behaved like OG1RF in those assays. Analysis of the purified Epa polysaccharide from OG1RF revealed the presence of rhamnose, glucose, galactose, GalNAc, and GlcNAc in this polysaccharide, while carbohydrate preparation from the epaB mutant did not contain rhamnose, suggesting that one or more of the glycosyl transferases encoded by the epaBCD operon are necessary to transfer rhamnose to the polysaccharide. In conclusion, the epa genes, uniformly present in E. faecalis strains and involved in biosynthesis of polysaccharide in OG1RF, are also important for OG1RF shape determination, biofilm formation, and NPV-1 replication/lysis, as well as for E. faecalis virulence in a mouse peritonitis model

Isolation of a Minireplicon of the Virulence Plasmid pXO2 of Bacillus anthracis and Characterization of the Plasmid-Encoded RepS Replication Protein

A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5′ and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity

Diversity of the fsr-gelE Region of the Enterococcus faecalis Genome but Conservation in Strains with Partial Deletions of the fsr Operon▿ †

Most Enterococcus faecalis isolates carry gelE, but many are gelatinase nonproducers due to the lack of fsrC (EF_1820) to EF_1841 (fsrC-EF_1841; 23.9 kb in strain V583), including most of the locus encoding Fsr, which activates gelE expression. Analysis of 22 accessible E. faecalis genomes revealed the identity of the 53-amino-acid propeptide of fsrD across multiple MLSTs (multilocus sequence types), although 12 distinctly different variations were found in the EF_1814-to-EF_1902 region. Diversity was seen in fsrABC, in the region EF_1814 to EF_1902, and in a 700-kb region surrounding fsrC-EF_1841. However, analysis of five sequenced strains carrying the fsrC-EF_1841 deletion and the putative integrative conjugative element efaB5 showed almost identical single nucleotide polymorphisms (SNPs) in gelE and an identical junction sequence, despite their unrelated MLSTs, in contrast to those shown by strains without the deletion. Further analysis confirmed the conserved gelE SNPs in 6 additional strains (11 in total) with the deletion. While we were unable to detect evidence of spontaneous deletion using OG1RF and 8 other strains, we were able to engineer a deletion of the 37-kb fsrC-EF_1841 region of OG1RF without deleterious effects, and the 37-kb mutant showed changes in biofilm and chaining similar to those shown by fsr-gelE mutants. In conclusion, we describe the identity of fsrD despite high plasticity within the fsrC-EF_1841 region and the surrounding sequence. However, strains lacking the fsrC-EF_1841 region show a distinct conservation of the sequence surrounding this deletion and in gelE, suggesting that the deletion may result from horizontal transfer and recombination