In silico selection of 197 open reading frames as putative B. anthracis vaccine candidates for in vitro screening.
Selection of B. anthracis
chromosome-derived ORFs as specific vaccine candidates was performed previously by a multistep computational screen of the B. anthracis
Ames strain draft (February 2001 version) chromosome sequence (11
). The selection procedure combined ORF determination, preliminary annotation, prediction of cellular localization, and taxonomic comparison with closely related genomes. The selection rationale was based on the following criteria: (i) putative surface exposure (anchored or secreted) of the ORF products (proteins amenable to interaction with the host immune system), (ii) lack of similarity to proteins present in nonpathogenic bacteria (removal of housekeeping genes), and (iii) putative function or sequence motifs similar to documented virulence factors of other pathogens. Together with additional filtering criteria (size and number of paralogs) and manual curation, this reductive strategy resulted in selection of 240 candidate ORFs encoding proteins with putative assigned functions and 280 hypothetical unique proteins with unknown functions having putative secretion signals and/or TM
retention segments (11
To reduce the number of candidate ORFs, we removed ORF products that are identical in B. cereus ATCC 14579, the only related genetically similar yet phenotypically distinct pathogen whose genome sequence was available at the time of the study. When there were several paralogous ORF products, a single paralog was chosen arbitrarily.
The final list consisted of 116 chromosome-derived candidate ORFs with putative functions and/or defined domains, including ORFs encoding nine S-layer homology (SLH) domain proteins (including the S-layer proteins Sap and EA1, which were used as positive controls for known chromosome-derived immunogens [60
]), 18 adhesins, lipoproteins, or transporters, 21 repeat-containing proteins, 53 membrane or secreted enzymes, and 15 other surface-anchored proteins harboring motifs characteristic of microbial virulence factors. For hypothetical ORFs with unknown functions, we selected only ORFs encoding products that were larger than 150 amino acids and contained either signal peptide or TM
In a previous study of pXO1, 11 candidate ORF products with putatively assigned functions were evaluated using similar criteria (10
). In the current study, 14 additional pXO1 ORF products with unknown functions that were unique at the time of selection (absent from the B. cereus
14579 genome) and eight pXO2-derived ORF products with putatively assigned functions were selected for further analysis. The three toxin component genes, pagA
-107), and cya
-122), were included in the study as positive controls.
In summary, an in vitro analysis was performed with 197 ORFs from the B. anthracis chromosome, as well as pXO1 and pXO2. The products of 133 of these ORFs had putative assigned functions, 59 products were hypothetical products with unknown functions, and 5 products were used as positive controls.
Considerations in evaluation of the in vitro expression and immunoreactivity of candidate ORFs.
The in vitro screening procedure for analysis of selected ORF candidates consisted of three main steps: (i) generation of the bioinformatically selected ORFs as linear PCR expression cassettes, (ii) synthesis of the corresponding protein products individually from the linear PCR amplicons by in vitro coupled T&T in the presence of [35S]methionine, and (iii) monitoring the seroreactivity of the protein products by IP of the radioactively labeled T&T reaction mixtures with polyclonal antisera to B. anthracis (Table ). A positive result was identified by development of a specific IP product, which was visualized as a discrete band at the expected molecular size. Figure shows examples of positive and negative responses.
FIG. 1. Transcription-translation and immunoreactivity profiles of selected ORFs. [35S]methionine-labeled transcription and translation products (lanes T&T) were analyzed by SDS-PAGE, followed by autoradiography. The labeled T&T products were (more ...)
All sera except R-3 (see Materials and Methods) exhibited high titers against B. anthracis toxin components, as well as against vegetative bacterial extracts representing total membrane and secreted proteins (Table ). For most of the in vitro T&T ORF products, R-1 serum exhibited higher IP titers than other sera; one example of this is the ORF BA2805 product and the S-layer protein Sap, as shown in Fig. . When R-1 results were either ambiguous or negative, other sera were used; this is shown for pXO1-130, BA3189, and BA4787 in Fig. . We found that only three of the ORF products tested reacted to the same extent both with the anti-B. anthracis sera and with the sera collected from naïve animals (NRG) (Table ); obviously, these ORFs were not considered positive ORFs.
In the absence of purified protein antigens that could be used in a quantitative immunoassay, the seroreactivities of all ORF products tested were quantified by determining IP titers of the radiolabeled T&T products. The IP titer was calculated by determining the end point of IP titration using serial serum dilutions by (i) monitoring the amount of radioactivity in the IP reaction mixture and/or (ii) visualization of the IP products by SDS-PAGE, followed by autoradiography (see Materials and Methods). We compared the IP titers to the titers determined by a standard ELISA for the PA antigen (Table ). As shown in Fig. and Table for antisera R-1 and G-1, the IP titers determined by both assays described above were in good agreement with those determined by ELISA (for R-1, 1:35,000, compared with 1:32,000 determined by the ELISA; for G-1, 1:300,000, compared with 1:500,000 determined by the ELISA).
FIG. 2. Determination of IP titers of selected T&T products. Titrations were performed with a constant volume of the T&T reaction mixture and with threefold serial dilutions of the different sera. (A) Precipitation of PA with the R-1 and G-1 antisera. (more ...)
Figure shows examples of determinations of IP titers for ORFs with different levels of T&T expression for ORFs BA5330 and BA4766 (Fig. ) or DppA and HtrA (Fig. ). The analysis of PA, DppA, and HtrA also demonstrated that the electrophoretic data were in good agreement with the data obtained from direct counting of radioactivity in the IP reaction (Fig. , lower panels).
Expression and immunoreactivity of the selected chromosomal and extrachromosomal ORF candidates.
Several parameters, including the ability to generate a T&T product, the reaction with the anti-B. anthracis antisera, and the extent of immunoreactivity, were considered when we interpreted the results of the screen. These parameters were used for evaluation of the three groups of bioinformatically selected ORFs, including (i) chromosomal ORFs with putative assigned functions, (ii) chromosomal unique ORFs with no assigned functions, and (iii) selected ORFs from the pXO1 and pXO2 plasmids.
Chromosomal ORFs with putative assigned functions.
Of the 116 chromosomal ORFs selected (for the complete list, see Table S1 in the supplemental material), 115 yielded PCR products. The only one ORF that did not generate a PCR product was a repeat-rich ORF, BA4978. Of the 115 PCR amplicons, 109 could be used for T&T product generation (see Table S1 in the supplemental material). For five of the six ORFs that did not generate T&T products (BA1094, BA1222, BA1290, BA3725, and BA4764), the failure could be explained by the length and/or extent of putative disordered or unstructured regions in the sequence. No attempt was made to manipulate these six ORFs for further analysis.
All 109 T&T products were tested with the different sera, and 43 were found to react positively with anti-B. anthracis antisera. The seropositive chromosome-encoded ORF products are listed in Table and have the following features.
Immunoreactive ORF products among the in silico-preselected annotated B. anthracis chromosomal targets
(i) Most of the seropositive ORF products harbor anchoring signals.
The peptidoglycan (cell wall [CW]) of gram-positive bacteria is a docking site for proteins, which interact with the environment (66
). In pathogens, proteins that are retained in the CW envelope are important for bacterial attachment, invasion, interaction with host proteins, and virulence (18
). Table shows the number of ORFs in the genome of B. anthracis
that have the various CW localization signals, the number of ORFs selected bioinformatically for screening, and the number of ORFs found to be seropositive. The distribution of these ORF products is described below according to their various anchoring mechanisms, domains, and motifs.
Surface localization signals harbored by the B. anthracis ORF products
Of 130 B. anthracis putative lipoproteins encoded in the genome, 12 were selected in silico for analysis, and all were immunoreactive (Table ). Ten represented ligand-binding components of ABC transporters.
(b) Sortase-mediated anchoring.
The B. anthracis genome harbors genes encoding three sortases (classes A, B, and C) and 10 substrates with diverse sorting signals. Ten candidate ORFs encoding sortase-containing signal proteins were preselected, and seven were seropositive (Table ). We noted that only 3 of the 15 preselected ORFs encoding products harboring virulence-related motifs (see Table S1 in the supplemental material) were seropositive and that all three had a sortase-anchoring signal. Thus, it appears that there is a high probability that ORFs encoding products with a sorting signal are expressed and induce an immune response following B. anthracis infection.
(c) SLH domain.
The SLH domain, a frequent gram-positive anchoring domain for CW attachment (58
), was one of the major selection criteria for candidate ORFs. Six of the nine chromosomal SLH proteins preselected for the screen were seropositive (Table ).
(d) LysM domain.
The LysM domain is recognized in a variety of enzymes involved in CW metabolism and is assumed to be necessary both for the enzymatic functions of adjacent catalytic domains and for the peptidoglycan attachment domain (39
). The LysM domain was recognized in six ORFs in the B. anthracis
genome; four were preselected for analysis, and only one was seropositive (BA3668).
The Escherichia coli
P60 domain occurs at the C terminus of a number of different bacterial and viral proteins. Several related, but distinct, catalytic activities, such as murein degradation, acyl transfer, and amide hydrolysis, occur in the NlpC/P60 superfamily (4
). The three ORFs encoding hypothetical proteins having an NlpC/P60 domain in the B. anthracis
chromosome were analyzed, and all were seropositive (Table ). Only one of these ORFs (BA5427) encodes a protein with a catalytic domain (endopeptidase LytE), while the other two (BA1952 and BA2849) encode proteins with undefined functions. BA1952 harbors an additional domain, SH3b (see below).
(f) SH3b domain.
SH3b is the bacterial homologue of SH3 (Src homology domain), a eukaryotic motif exhibited by several proteins involved in signal transduction. SH3b has been found in a number of different bacterial proteins, including endopeptidases, bacteriocins, and signaling proteins involved in cell attachment, and it may represent a putative CW targeting domain (19
). Four of the 10 B. anthracis
ORFs harboring SH3b domains in the chromosome were selected for the screen, and only one, BA1952, which also harbors an NlpC/P60 domain, was seropositive.
(g) PKD domain.
Like SH3, the PKD domain is involved in protein-protein or protein-carbohydrate interactions, which indicates possible CW exposure of the ORF products carrying it. It was first identified in PKD1, the polycystic kidney disease protein. The PKD motif occurs in the B. anthracis genome three times, all three times in ORFs encoding putative collagenases; the only ORF selected for analysis (BA3584) was seropositive.
(ii) Immunoreactivity of ORF products lacking a predicted signal peptide.
Not all of the preselected ORF products had secretion signal peptides; however, the absence of a secretion signal does not necessarily imply that a protein is not surface exposed (8
). In pathogens, secretion of some signal-less virulence factors was suggested to be mediated by alternative secretion pathways (48
), and their presence in the extracellular milieu does not necessarily reflect cell lysis.
Signal peptides were detected in 34 of the 43 seropositive ORF products (Table ). Two of the nine positive ORFs without a signal sequence, BAS5207 (LPXTG) and BA3668 (LysM), encode proteins with anchoring motifs. The other seven ORF products that do not have a signal peptide (BA3841, BA4510, BA4989, BA0309, BA0485, BA1353, and BA2805) have some characteristics that may localize them to the surface. The BA3841, BA4510, and BA4989 products have some features common to anchorless adhesins, which were first described by Chhatwal (22
) as a group of proteins from gram-positive pathogens which bind to extracellular matrix components like fibronectin and collagen. BA0309 encodes δ-1-pyrroline-5-carboxylase (RocA), which has been reported to be a component of the B. cereus
) and was recently found to be an immunodominant component of the B. anthracis
membrane proteome (23
). BA0485 is a phage lambda (B. anthracis
-specific) endolysin gene that is located adjacent to a holin gene, and BA2805 encodes another putative endolysin-like product; therefore, based on their function assignments both products are expected to be extracellular.
(iii) Immunoreactivity of ORF products containing repeat domains or motifs.
The presence of a repeated sequence domain or motif was also one of the criteria used for the in silico selection process (11
). Although proteins containing repeats are more abundant in eukaryotes than in prokaryotes, they are frequently found in pathogenic bacteria and are related to adhesion, invasion, molecular mimicry of host proteins, or antigenic variation (6
). Repeat proteins harbor either tandem repeat domains, such as ankyrin repeats, TPR-like repeats, collagen-like repeats, leucine-rich repeats, and diverse internal repeats, or repeat motifs consisting of single amino acids, periodically conserved amino acids, and oligopeptide repeats. Ten seropositive ORF products can be referred to as repeat-containing proteins (Table ). The products of six ORFs (BA0552, BA1952, BA3841, BA4510, BA4787, and BA4989) belong to the original group of preselected repeat proteins (11
). The products of the other four ORFs (BAS5205, BAS5207, BA3367, and BA4789) belong to different preselected groups. Two of the latter, the BAS5205 and BAS5207 products, are putative extracellular matrix-binding collagen adhesion proteins that have internal repeats. Similar collagen repeat-containing proteins, expressed on the surfaces of other gram-positive pathogens, are involved in attachment to host connective tissues (9
). We noted that in B. anthracis
, a collagen-like glycoprotein is a structural component of the exosporium (88
). The BA3367 ORF, encoding a sortase-anchored internal repeat protein, precedes a putative cobalt ion ABC transporter operon. The BA3367 product was recently shown to be a γ phage receptor (26
). The BA4789 product contains a near transporter (NEAT) repeat motif that is present in proteins involved in iron acquisition (see Discussion). Overall, we found that 10 of 22 (45%) expressed repeat-containing proteins reacted with the anti-B. anthracis
Chromosomal ORFs with no assigned functions.
Forty-five chromosomal ORFs to which no function could be assigned were selected for the screen (Table ). Only 31 of these 45 ORFs (70%) yielded T&T products. This yield is significantly lower than that for the annotated ORFs (109/115). It is reasonable to assume that most of the nontranslatable ORFs probably do not code for real polypeptides. Indeed, when the B. anthracis
chromosome sequence was completed (75
), 6 of the 14 ORFs not amenable to T&T expression were no longer considered ORFs.
Screened chromosomal B. anthracis ORFs with no assigned functions
The most striking observation related to the group of ORFs with no assigned functions was that only 1 of the 31 T&T products, the BA3807 product, was seropositive and exhibited a detectable IP titer (Table ). BA3807 is localized adjacent to a prophage locus and may thus have a phage-related role.
ORFs from pXO1 and pXO2.
In a previous study (10
), 12 pXO1-related ORFs were screened (including 11 novel ORFs and the PA gene as a positive control) (Table ). For 10 ORFs, a T&T product was generated, and four products were found to be seropositive: PA, pXO1
-90, and pXO1
-130. We included the following 16 additional pXO1 ORFs in our screen (Table ): the toxin genes encoding LF and EF (as positive controls) and 14 ORFs encoding hypothetical short polypeptides with unknown functions. Only nine ORFs, the two toxin genes (Fig. ) and seven ORFs with unknown functions, yielded T&T products (Fig. ). This may imply that as was observed for the chromosomal genes with unknown functions, not all the predicted pXO1 ORFs represent true genes. Finally, except for the toxin components, none of the hypothetical ORF products reacted with the sera. Thus, only 6 of 19 pXO1 ORFs tested to date which generated T&T products were seropositive, including the three toxin components (Fig. ).
B. anthracis pXO1 and pXO2 ORFs screened
FIG. 3. Transcription-translation and immunoreactivity profiles of selected pXO1 and pXO2 ORFs. (A) T&T and IP analysis of PA, LF, and EF, performed with the R-1, G-1, and R-2 antisera. (B) T&T reactions of the seven pXO1 ORFs with unknown functions (more ...)
In view of the results obtained with pXO1, we decided to restrict the pXO2 analysis to ORFs encoding products with putative assigned functions. Two of the eight annotated pXO2 ORFs selected for analysis (Table ), pXO2
-8 and pXO2
-42, reacted with the antisera (Fig. ). Both of these ORFs encode anchored CW hydrolases; the pXO2
-8 product contains an NlpC/P60 motif, and the pXO2
-42 product contains an SLH domain. The pXO2
-42 product was described previously and was found to exhibit peptidoglycan hydrolase activity (59
). It should be noted that the products of two of the three novel pXO1 positive ORFs that were identified (10
) (Fig. ), pXO1
-54 and pXO1
-90, are also SLH-anchored proteins. This observation further supports the notion that SLH anchoring is a reliable parameter for prediction of immunoreactivity, as demonstrated previously for analysis of the chromosomal genes.
Comparative immunoreactivities of seropositive ORF products.
The IP titers obtained for the various ORF products are summarized in Tables , , and . For the chromosome-derived positive ORF products (Table ), the highest scores (IP titers greater than 1,000) were detected with the following polypeptides: S-layer proteins EA1 and Sap (BA0887 and BA0885), two SLH amidases (BA0898 and BA3737), glycosyl hydrolase (BA2805), HtrA (BA3660), DppA (BA0656), a NEAT-containing protein probably involved in iron acquisition (BA4787), and a protein with an unknown function (containing SH3b and NlpC/P60 domains; BA1952). These results may be a reflection of the abundance of the proteins in vivo and/or their high immunogenicity.
As expected, for the plasmid-derived ORF products (Table ) the highest immunoreactivity was the immunoreactivity with PA. It is well established that PA is both a very potent immunogen and a major protein expressed and secreted by vegetative B. anthracis cells during infection. Of the other plasmid-derived positive immunogens (Table ), the pXO1-90 product was as immunoreactive as PA and was highly reactive with all the sera tested (Fig. ), the pXO1-130 product was also highly reactive but only with the R-2 antisera (Fig. and ), and the other pXO-derived ORF products reacted differently with different sera but were all “weakly” seropositive protein products.
Immunogenic potential of positive ORFs: DNA immunization in mice.
Once it was established that the selected ORF products could be expressed and could specifically react with relevant anti-B. anthracis
antisera, the seropositive candidate ORFs were screened for the ability to elicit a humoral response. Linear PCR amplicons coding for positive immunogens were directly cloned into a compatible plasmid vector, which allowed expression of the bacterial genes in a mammalian host following DNA vaccination (38
). A representative set (ca. 30%) of immunoreactive ORFs was selected for the analysis, and this set included antigens with both high and low IP titers. We also included two seronegative ORFs.
The IP titers of DNA-vaccinated mice are summarized in Table . For the two seronegative ORFs (pXO1-66 and pXO1-67), DNA immunization did not lead to detectable seroconversion. In contrast, almost all (10 of 12) of the seropositive ORFs selected for immunization were also capable of eliciting antibodies when they were administered individually as DNA vaccines, and all of the immunizations resulted in relatively high specific titers against the corresponding labeled T&T products. For the two seropositive ORFs that did not elicit a detectable immune response following DNA immunization (pXO1-90 and BA3807), a second round of DNA immunization was performed with full-length or truncated versions of the genes (data not shown). These manipulations did not result in any detectable positive humoral response.
Humoral responses induced by DNA immunization using selected ORFs
Since DNA immunization did not provide real added value in down-selection of the seropositive ORFs for further vaccination studies, there was no reason to extend this type of analysis, except to generate specific antibodies.
Therefore, it appears that the approach used for down-selection described here allowed us to reduce the number of ORF candidates from 197 to 52 seropositive ORFs. Each of the novel candidate ORFs still has to be evaluated in an anthrax disease animal model (guinea pig or rabbit) to determine its efficacy.