The signaling peptide PapR is required for the activity of the quorum-sensor PlcRa in Bacillus thuringiensis

The transcriptional regulator PlcR, its cognate cell-cell signaling heptapeptide PapR7, and the oligopeptide permease OppABCDF, required for PapR7 import, form a quorum-sensing system that controls the expression of virulence factors in Bacillus cereus and Bacillus thuringiensis species. In B. cereus strain ATCC 14579, the transcriptional regulator PlcRa activates the expression of abrB2 gene, which encodes an AbrB-like transcriptional regulator involved in cysteine biosynthesis. PlcRa is a structural homolog of PlcR: in particular, its C-terminal TPR peptide-binding domain could be similarly arranged as in PlcR. The signaling peptide of PlcRa is not known. As PlcRa is a PlcR-like protein, the cognate PapR7 peptide (ADLPFEF) is a relevant candidate to act as a signaling peptide for PlcRa activation. Also, the putative PapRa7 peptide (CSIPYEY), encoded by the papRa gene adjacent to the plcRa gene, is a relevant candidate as addition of synthetic PapRa7 induces a dose-dependent increase of abrB2 expression. To address the issue of peptide selectivity of PlcRa, the role of PapR and PapRa peptides in PlcRa activity was investigated in B. thuringiensis 407 strain, by genetic and functional complementation analyses. A transcriptional fusion between the promoter of abrB2 and lacZ was used to monitor the PlcRa activity in various genetic backgrounds. We demonstrated that PapR was necessary and sufficient for PlcRa activity. We showed that synthetic PapRs from pherogroups II, III and IV and synthetic PapRa7 were able to trigger abrB2 expression, suggesting that PlcRa is less selective than PlcR. Lastly, the mode of binding of PlcRa was addressed using an in silico approach. Overall, we report a new role for PapR as a signaling peptide for PlcRa activity and show a functional link between PlcR and PlcRa regulons in B. thuringiensis .

Mapping Protective Regions on a Three-Dimensional Model of theMoraxella catarrhalisVaccine Antigen Oligopeptide Permease A

ABSTRACTA vaccine againstMoraxella catarrhaliswould reduce tremendous morbidity, mortality, and financial burden by preventing otitis media in children and exacerbations of chronic obstructive pulmonary disease (COPD) in adults. Oligopeptide permease A (OppA) is a candidate vaccine antigen that is (i) a nutritional virulence factor expressed on the bacterial cell surface during infection, (ii) widely conserved among strains, (iii) highly immunogenic, and (iv) a protective antigen based on its capacity to induce protective responses in immunized animals. In the present study, we show that the antibodies to OppA following vaccination mediate accelerated clearance in animals after pulmonary challenge. To identify regions of OppA that bind protective antibodies, truncated constructs of OppA were engineered and studied to map regions of OppA with surface-accessible epitopes that bind high-avidity antibodies following vaccination. Protective epitopes were located in the N and C termini of the protein. Immunization of mice with constructs corresponding to these regions (T5 and T8) induced protective responses. Studies of overlapping peptide libraries of constructs T5 and T8 with OppA immune serum identified two discrete regions on each construct. These potentially protective regions were mapped on a three-dimensional computational model of OppA, where regions with solvent-accessible amino acids were identified as three potentially protective epitopes. In all, these studies revealed two regions with three specific epitopes in OppA that induce potentially protective antibody responses following vaccination. Detection of antibodies to these regions could serve to guide vaccine formulation and as a diagnostic tool for monitoring development of protective responses during clinical trials.

Expression of the Oligopeptide Permease Operon of Moraxella catarrhalis Is Regulated by Temperature and Nutrient Availability

Moraxella catarrhaliscauses otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. Together, these two conditions contribute to enormous morbidity and mortality worldwide. The oligopeptide permease (opp) ABC transport system is a nutritional virulence factor important for the utilization of peptides. The substrate binding protein OppA, which binds peptides for uptake, is a potential vaccine antigen, but little was known about the regulation of gene expression. The fiveoppgenesoppB,oppC,oppD,oppF, andoppAare in the same open reading frame. Sequence analysis predicted two promoters, one located upstream ofoppBand one within the intergenic region betweenoppFandoppA. We have characterized the gene cluster as an operon with two functional promoters and show that cold shock at 26°C for ≤0.5 h and the presence of a peptide substrate increase gene transcript levels. Additionally, the putative promoter upstream ofoppAcontributes to the transcription ofoppAbut is not influenced by the same environmental cues as the promoter upstream ofoppB. We conclude that temperature and nutrient availability contribute to the regulation of the Opp system, which is an important nutritional virulence factor inM. catarrhalis.

Role of the Oligopeptide Permease ABC Transporter of Moraxella catarrhalis in Nutrient Acquisition and Persistence in the Respiratory Tract

ABSTRACTMoraxella catarrhalisis a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality.M. catarrhalishas a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival.M. catarrhalishas a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB,oppC,oppD,oppF, andoppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which theoppBCDFAgene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth ofM. catarrhalisis important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-typeM. catarrhaliswas observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requiresl-arginine. AnoppAknockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract.

Identification of OppA2 Linear Epitopes as Serodiagnostic Markers for Lyme Disease

ABSTRACTLaboratory diagnosis of Lyme disease is based on the serological detection of antibodies against the etiologic agentBorrelia burgdorferi. Current diagnostics are insensitive at detecting early infection, when treatment is most effective. This deficiency results from the limited number ofB. burgdorferiantigens expressed in early infection and the use of an insensitive two-tier paradigm, put in place to deal with insufficient specificity associated with the use of whole-protein antigens and/or bacterial lysates as serodiagnostic targets. Whole-protein antigens contain epitopes that are unique toB. burgdorferias well as cross-reactive epitopes found in other bacteria. One method for overcoming the limitations imposed by cross-reactive epitopes is the use of short peptides containing epitopes unique toB. burgdorferias antigen targets. This eliminates nonspecific epitopes. Using overlapping peptide libraries, we performed epitope mapping of linear epitopes in oligopeptide permease A2 (OppA2), a member of the oligopeptide permease (Opp) family of peptide transporters, expressed during earlyB. burgdorferiinfection. We identified 9 epitopes, synthesized peptides containing these epitopes, and screened those using panels of blood from patients with early Lyme disease, rheumatoid arthritis (RA), or syphilis or from healthy individuals. Two of the peptides, OppA2 (191-225) (amino acids comprising the peptide are shown in parentheses) and OppA2 (381-400), are highly conserved among the three major pathogenicBorreliaspecies responsible for most Lyme disease cases in North America and Europe. They detected antibodies in Lyme disease patient sera with sufficient sensitivity and specificity to indicate that they could have value in a serological assay for Lyme disease.