ABSTRACT
Expression of the lspB-lspA2 operon encoding a virulence-related two-partner secretion system in Haemophilus ducreyi 35000HP is directly regulated by the CpxRA regulatory system (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402–3411, 2009). In the present study, we show that this secretion system is also regulated by the small nucleoid-associated protein Fis. Inactivation of the H. ducreyi fis gene resulted in a reduction in expression of both the H. ducreyi LspB and LspA2 proteins. DNA microarray experiments showed that a H. ducreyi fis deletion mutant exhibited altered expression levels of genes encoding other important H. ducreyi virulence factors, including DsrA and Flp1, suggesting a possible global role for Fis in the control of virulence in this obligate human pathogen. While the H. ducreyi Fis protein has a high degree of sequence and structural similarity to the Fis proteins of other bacteria, its temporal pattern of expression was very different from that of enterobacterial Fis proteins. The use of a lacZ-based transcriptional reporter provided evidence which indicated that the H. ducreyi Fis homolog is a positive regulator of gyrB, a gene that is negatively regulated by Fis in enteric bacteria. Taken together, the Fis protein expression data and the observed regulatory effects of Fis in H. ducreyi suggest that this small DNA binding protein has a regulatory role in H. ducreyi which may differ in substantial ways from that of other Fis proteins.
INTRODUCTION
Haemophilus ducreyi is the Gram-negative pathogen responsible for the sexually transmitted disease chancroid (1, 2). Information about the pathogenesis of this genital ulcer disease remains fairly limited, despite the fact that chancroid is endemic in some developing countries in Africa, Asia, and South America (2). Studies in sub-Saharan Africa provided evidence that chancroid can be a cofactor for human immunodeficiency virus acquisition and transmission (3, 4). In the United States, H. ducreyi infections are very rare and typically occur only in isolated cases that are commonly associated with the sex trade industry (5, 6).
Similar to the paucity of knowledge about the pathogenesis of chancroid, the specifics regarding which H. ducreyi gene products are responsible or essential for the expression of virulence are only partly defined. Nevertheless, the introduction of the human challenge model for experimental chancroid (7) in 1994 made possible the direct testing of H. ducreyi wild-type/mutant pairs in a well-controlled manner in a most appropriate system (8). In the subsequent 2 decades, a significant number of H. ducreyi mutants were found to be fully virulent, partially attenuated, or substantially deficient in virulence in this model system (8–15). Among these, a mutant lacking the ability to express both the LspA1 and LspA2 proteins was found to be very attenuated (16). These two very large H. ducreyi proteins are both secreted by the LspB outer membrane protein, with all three proteins comprising a two-partner secretion system (17). Expression of either LspA1 or LspA2 has been shown to be necessary for H. ducreyi to inhibit phagocytosis by macrophages and other phagocytic cell lines in vitro (18) via a mechanism that involves inhibition of Src family protein tyrosine kinases (19). Interestingly, the LspA proteins themselves contain multiple specific motifs that can be tyrosine phosphorylated by macrophages (20).
The genetic basis for control of virulence expression by H. ducreyi remains largely unexplored, with H. ducreyi regulatory systems having received scant attention. In fact, to date, only five reports addressed this issue at all. The first report involved mutant analysis of genes encoding proteins involved in the utilization of hemoglobin (21), whereas the second used a transcript capture method to identify a large number of H. ducreyi genes expressed in human volunteers after experimental challenge (22). Evidence for in vivo expression of both LspA1 and LspA2 was obtained in the latter study. It has also been established that the CpxRA two-component signal transduction system negatively regulates expression of the lspB-lspA2 operon as well as other open reading frames (ORFs) proven to be important in the human challenge model of chancroid (23, 24). Interestingly, deletion of the mtrC gene, encoding a protein involved in resistance to antimicrobial peptides, resulted in activation of the CpxRA regulon (25). Most recently, it was reported that inactivation of the H. ducreyi carbon storage regulator A (CsrA) gene resulted in multiple changes in gene expression involving virulent determinants (14).
A recent report indicated that the small nucleoid-associated protein Fis of Pasteurella multocida was involved in controlling the expression of several important virulence factors, including a two-partner secretion system composed of LspB_2 and PfhB_2 (26). These two P. multocida proteins have homology to the H. ducreyi LspB and LspA1/LspA2 proteins. In the present study, we investigated the potential involvement of Fis in the regulation of expression of the H. ducreyi LspB-LspA1/LspA2 system and determined that inactivation of the H. ducreyi fis gene resulted in decreased expression of both LspB and LspA2. We also show that Fis is involved in controlling the expression of other proven H. ducreyi virulence factors.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions.Bacterial strains and plasmids used in this study are listed in Table 1. H. ducreyi strains were routinely grown on chocolate agar (CA) as previously described (23). When kanamycin selection was necessary, H. ducreyi cells were grown on a GC agar-based medium (27). For broth culture, strains were incubated in a Columbia broth (CB)-based medium at 33°C in a gyratory water bath at 100 rpm, as described previously (23). Escherichia coli XL10-Gold and DH5α were used as hosts for general cloning manipulations and protein expression and were grown in Luria-Bertani medium supplemented with ampicillin (100 μg/ml), kanamycin (30 μg/ml), or chloramphenicol (30 μg/ml) when appropriate for maintenance of plasmids. Before complementation of H. ducreyi deletion mutants, plasmid constructs were transformed into and isolated from E. coli HB101.
Bacterial strains and plasmids used in this study
Tissue culture cells and media.The human foreskin fibroblast cell line Hs27 (ATCC CRL-1634) was obtained from the American Type Culture Collection (Manassas, VA). Hs27 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) (Fisher Scientific Co., Pittsburgh, PA) supplemented with 2 mM GlutaMAX (Gibco-BRL, Rockville, MD), 10 mM HEPES, 1 mM sodium pyruvate, and 10% (vol/vol) heat-inactivated fetal bovine serum at 37°C in a humidified incubator containing an atmosphere of 95% air–5% CO2.
Purification of a GST-tagged Fis fusion protein and development of a polyclonal Fis antibody.The complete fis ORF was amplified from H. ducreyi 35000HP chromosomal DNA by PCR using primers HD547 and HD548 (Table 2), which added BamHI and SmaI sites to the 5′ and 3′ ends of the fragment, respectively. The resulting amplicon was BamHI and SmaI digested and ligated into pGEX-T4-2 (GE Healthcare, Pittsburgh, PA) cut with the same enzymes to obtain pML165. E. coli XL10-Gold cells containing pML165 were cultured to mid-exponential phase (optical density at 600 nm [OD600] of ∼0.5), and after the addition of isopropyl-β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, the cultures were further incubated for 4 h. After induction, the cells were harvested by centrifugation, suspended in buffer A (50 mM Tris [pH 8.0], 1 mM EDTA, and 25% [wt/vol] sucrose) containing protease inhibitors (Sigma-Aldrich, St. Louis, MO), and disrupted by sonication. The sonicated mixture was centrifuged (15,000 × g for 30 min), and the resultant pellet was suspended in a solution containing 20 mM Tris (pH 8.0), 0.2 M NaCl, 1% (wt/vol) sodium deoxycholate, and 2 mM EGTA. After incubation at room temperature for 30 min, this mixture was centrifuged at 10,000 × g for 20 min. The resultant supernatant was applied onto glutathione beads (GE Healthcare), the beads were washed with buffer B (50 mM Tris [pH 8.0], 5 mM EDTA, 50 mM NaCl, and 5 mM β-mercaptoethanol), and the bound protein was eluted with buffer C (50 mM Tris [pH 8.0], 5 mM EDTA, 150 mM NaCl, 5 mM β-mercaptoethanol, and 10 mM glutathione). After dialysis against a solution of 50 mM Tris (pH 8.0) and 100 mM NaCl, the fusion protein was stored at −70°C, with the addition of glycerol to a final concentration of 20% (wt/vol). The use of a commercial facility for production of mouse antiserum was approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center. Polyclonal mouse antibody to this glutathione S-transferase (GST) fusion protein was produced by Rockland Immunochemicals (Boyertown, PA).
Oligonucleotide primers used in this study
Western blot analysis.Whole-cell lysate preparations (∼5 × 107 cells/lane) were resolved by SDS-PAGE in 4 to 20% (wt/vol) polyacrylamide separating gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Membranes were incubated in StartingBlock (phosphate-buffered saline [PBS]) blocking buffer (Thermo Scientific, Rockford, IL) containing 5% (vol/vol) normal goat serum for 1 h at room temperature or overnight at 4°C. The membranes were then incubated for 3 to 4 h at room temperature or overnight at 4°C in primary antibody at the appropriate dilution, followed by 1 h of incubation at room temperature in a 1:20,000 dilution of either goat anti-mouse IgG-horseradish peroxidase (HRP) or goat anti-rabbit IgG-HRP (Bio-Rad, Hercules, CA). LspA1-specific monoclonal antibody (MAb) 40A4 (28), LspA2-specific MAb 1H9 (28), mouse polyclonal LspB antibody (17), peptidoglycan-associated lipoprotein (PAL)-specific MAb 3B9 (29), mouse polyclonal DsrA-reactive antibody (30), rabbit polyclonal Flp1 antibody (31), and rabbit polyclonal CpxR-reactive antibody (23) were described previously. Western blots were developed by using Western Lightning Chemiluminescence Reagent Plus (New England Nuclear, Boston, MA).
Construction and complementation of H. ducreyi fis deletion mutants.An ∼1-kb fragment corresponding to the 5′ upstream region of the H. ducreyi fis ORF was amplified from chromosomal DNA with Ex Taq DNA polymerase (TaKaRa Bio Inc., Shiga, Japan) and primers HD524 and HD526 (Table 2). Another ∼500-bp fragment corresponding to the 3′ downstream region of the H. ducreyi fis ORF was amplified with primers HD527 and HD525. A cat cartridge from pSL33 (32), modified to contain its native promoter (23), was amplified from pML122 (Table 1) by using primers HD528 and HD529. Primer HD528 shared a 21-nucleotide (nt) complementary sequence with primer HD526 (Table 2), and primer HD529 shared a 21-nt complementary sequence with primer HD527 (Table 2). The three PCR fragments were gel purified, and equal amounts were mixed and used as the templates for overlapping extension PCR (33) with primers HD524 and HD525. The resultant ∼2.3-kb PCR product was subjected to restriction enzyme digestion with DpnI and gel purified, and 100 μg was used to electroporate H. ducreyi 35000HP, as previously described (11, 34). A fis deletion mutant (35000HPΔfis) was selected on CA plates containing chloramphenicol (1 μg/ml); nucleotide sequence analysis confirmed the nonpolar nature of this construct.
A H. ducreyi ΔcpxR Δfis mutant in which a kan cartridge was used in place of the cat cartridge in fis was also constructed. A modified kan cartridge containing its native promoter (35) was cloned into pCR2.1 to obtain pML168 (Table 1). This kan construct was amplified by using primers HD809 and HD810. Primer HD809 shared a 21-nt complementary sequence with primer HD526 (Table 2), and primer HD810 shared a 21-nt complementary sequence with primer HD527 (Table 2). This amplicon, together with the upstream and downstream sequences described above for the original fis mutant, was used as the template for overlapping extension PCR (33) with primers HD524 and HD525. The resultant amplicon was digested with DpnI and gel purified, and 100 μg was used to electroporate H. ducreyi 35000HPΔcpxR, as previously described (11, 34). Double mutant strain 35000HPΔcpxRΔfis was selected on GC plates containing kanamycin (30 μg/ml); nucleotide sequence analysis confirmed the nonpolar nature of the insertion in fis.
The wild-type fis gene from H. ducreyi together with ∼300 nt 5′ of the ATG translation start codon were amplified from chromosomal DNA by using primers HD543 and HD544 (Table 2). The amplicon was digested with XhoI and ligated into XhoI-digested pACYC177 (NEB) to obtain pML164. One hundred nanograms of pML164 DNA was used to transform 35000HPΔfis (as described above) to obtain kanamycin- and chloramphenicol-resistant strain 35000HPΔfis(pML164). Mutant strain 35000HPΔfis was also transformed with 100 ng of pACYC177 to be used as a vector-only control.
Phagocytosis assay.Opsonization of latex beads with human IgG was accomplished essentially as described previously (12), except that the antibody coating step was allowed to proceed overnight. The next day, the beads were washed three times with PBS, resuspended in 500 μl PBS, and incubated with 5 μl fluorescent Cy3 donkey anti-human antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature with gentle agitation. After several washes with PBS, the opsonized beads were resuspended in 500 μl of DMEM containing 10% fetal bovine serum (DMEM-S). On the day before the phagocytosis experiment, J774A.1 cells were plated at a density of 1.7 × 105 cells/well in chamber slides. On the next day, these monolayers were washed once with DMEM-S, and 500 μl DMEM-S was then added, followed by the addition of 200 μl of a bacterial cell suspension (OD600 = 0.25 to 0.5). After centrifugation at 200 × g for 5 min at room temperature, the slides were incubated at 33°C with 95% air–5% CO2 for 1 h prior to the addition of a 10-μl volume of opsonized beads. After the addition of the beads, the slides were subjected to centrifugation at 300 × g for 1 min, and the slides were then placed into a 37°C incubator with 90% air–10% CO2 for 5 min to allow for attachment of the beads to the phagocytes. Each chamber was then washed twice with 500 μl DMEM-S. A final 500-μl volume of DMEM-S was added to each chamber, and the slides were incubated at 37°C for 10 min to allow ingestion of the beads. Each slide was then placed on ice and washed with cold DMEM-S. Incubation with fluorescent Cy2 donkey anti-human antibody and nuclear staining with Hoechst 33342 were carried out essentially as described previously (12). Total beads (external and ingested) stained with Cy3 (red) and external beads stained with Cy2 (green) were counted and used to calculate the phagocytosis index via the following formula: total beads − external beads/total number of J774A.1 cells.
RNA isolation, DNA microarray analysis, and real-time RT-PCR.Total RNA was extracted from broth-grown H. ducreyi bacteria and used for DNA microarray analysis as previously described (23, 34). Briefly, 5 μg of total RNA extracted from 35000HP or 35000HPΔfis cells grown in CB medium to mid-exponential phase (∼8 h of growth) was used to obtain aminoallyl-cDNA, which was labeled posttranscriptionally with Cy3 or Cy5, as described previously (23, 34). Each experimental replicate was subjected to reverse labeling (i.e., a dye swap) to avoid dye bias. Differential expression was defined as a minimum of a 2-fold change in expression in the H. ducreyi fis deletion mutant relative to that in wild-type H. ducreyi 35000HP. The final results include only expression profiles that had a P value ≤0.05 after one-sample t test analysis. From the final DNA microarray result data, 24 genes were randomly selected for further confirmation of their relative transcription levels by two-step real-time reverse transcription-PCR (RT-PCR). Oligonucleotide primers used in this study are listed in Table 2, and real-time RT-PCR assays were performed as described previously (23, 34) on three independent biological replicates, using HD0084 (ldh) to normalize the amount of cDNA per sample. The fold change of each gene was calculated by using the 2−ΔΔCT method.
Bactericidal assay.Bactericidal assays were performed with normal human serum (NHS) obtained from a single healthy donor, exactly as described previously (13). In brief, we compared the survival rates in 50% NHS of plate-grown 35000HP; its isogenic fis, cpxA, and dsrA mutants; 35000HPΔfis(pACYC177); and 35000HPΔfis(pML164). Data were reported as percent survival in active NHS compared to that in heat-inactivated serum [(geometric mean CFU in active NHS/geometric mean CFU in heat-inactivated NHS) × 100]. Each experiment was repeated five times; the arithmetic mean and standard deviation (SD) of the percent survival were calculated. Comparison of the strains was performed by using a mixed-model analysis of variance (ANOVA) with experiment as the random effect. P values for pairwise comparisons were calculated by using the Tukey method of adjustment; an adjusted P value of <0.05 was considered significant for these assays.
Microcolony formation assay.Microcolony assays were performed as previously described (31). Briefly, 24-well tissue culture plates (Costar; Corning, NY) were seeded with 105 Hs27 human foreskin fibroblasts per well and incubated for 3 days until they achieved confluence. H. ducreyi cells grown overnight on CA plates were suspended in tissue culture medium to an OD600 of 0.1. Portions (5 μl) of the bacterial suspension were added in triplicate to individual wells, and the bacterial cells were centrifuged onto the confluent monolayers for 5 min at 1,000 × g at room temperature, after which the plates were incubated for 24 h at 33°C in 95% air–5% CO2. After this incubation, each well was washed three times with PBS (pH 7.4) and stained with crystal violet. Images were recorded by using an FSX100 BioImager Navigator instrument (Olympus, Center Valley, PA) at a ×14 magnification.
Construction of a LacZ-based transcriptional reporter plasmid for H. ducreyi.LacZ-based transcriptional reporter plasmid pASE222 (36) was digested by using the restriction enzyme BamHI. The resultant 4.1-kb fragment was filled in by using the Klenow fragment (New England BioLabs, Ipswich, MA) and ligated into a 3.2-kb BamHI-ScaI fragment from pACYC177 (New England BioLabs) containing the origin of replication and the kan gene; the resultant plasmid was designated pML303. A set of 600-bp fragments, including 500-bp upstream and 100-bp downstream of the translation start codon of lspB (HD_1155), dsrA (HD_0769), gyrB (HD_1643), flp1 (HD_1312), and ompP2B (HD_1435), was PCR amplified with NotI and SalI sites (see Table 2 for primer sequences) and subcloned into pGEM-T (Promega, Madison, WI). Promoter fragments were excised from pGEM-T by using NotI and SalI and individually cloned into NotI- and SalI-digested pML303, resulting in plasmids pML306, pML308, pML309, pML312, and pML314, respectively (Table 1). All constructs were sequenced prior to their introduction by electroporation into H. ducreyi strains.
LacZ-based transcriptional reporter assay.β-Galactosidase assays were performed as previously described (37), with minor adjustments. Briefly, H. ducreyi strains carrying the reporter constructs were grown overnight on CA plates. A 5-ml volume of CB medium was inoculated from the fresh CA plates, and the cultures were incubated at 33°C with gentle shaking (100 rpm). After overnight growth, cells from 1 ml of the culture were collected and resuspended in 1 ml of a modified Z-buffer (500 mM Na2HPO4, 4 M KCl, 1 M MgSO4, 1% [wt/vol] cetyltrimethylammonium bromide [CTAB], and 1% [wt/vol] sodium deoxycholate). Because H. ducreyi strains tend to autoaggregate, the protein content of the bacterial suspensions was used to standardize the assay. Briefly, 5 μl of the bacterial suspensions in modified Z-buffer was added to DC Protein Assay reagents (Bio-Rad) according to the manufacturer's protocol, the OD750 of the sample was determined by using a microplate spectrophotometer (Biotek, Winooski, VT), and the values were used to standardize the β-galactosidase assay. Prior to the addition of o-nitrophenyl-β-d-galactoside (ONPG), β-mercaptoethanol (5.4 μl/ml) was added to the bacterial suspensions. The time of ONPG addition was recorded, the cells were incubated at 37°C for 15 min, and the reactions were stopped by the addition of 0.5 ml of 1 M Na2CO3 to the mixture. The OD420 and OD550 were recorded, and Miller units were calculated as described previously (37), except that total protein content was used in place of the OD600 values.
Microarray data accession number.The raw data from these experiments were deposited at the NCBI Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE44413.
RESULTS
Construction and characterization of a H. ducreyi Fis mutant.Recent work with P. multocida (26) revealed that the small nucleoid-associated protein Fis was involved in controlling the expression of several important virulence factors, including a two-partner secretion system composed of LspB_2 and PfhB_2. This secretion system has homology to the H. ducreyi LspB-LspA1/LspA2 system, previously shown to be an important H. ducreyi virulence factor (16, 19, 38). A nonpolar fis deletion mutant (Fig. 1A) was constructed (as described in Materials and Methods) to allow determination of whether expression of the Lsp protein system in H. ducreyi involved Fis.
Characterization of the H. ducreyi fis deletion mutant. (A) Schematic representation of the fis locus in wild-type (WT) strain 35000HP and 35000HPΔfis (Δfis). (B) Growth of wild-type parent strain 35000HP and 35000HPΔfis in broth (∗∗∗∗, P < 0.001). (C) Colony size differences between 35000HP (WT) (top) and 35000HPΔfis (bottom). (D) Total cell protein profiles for 35000HP (lane 1), 35000HPΔfis (lane 2), 35000HPΔfis(pML164) (lane 3), and 35000HPΔfis(pACYC177) (lane 4), as determined by resolving proteins by SDS-PAGE and staining with Coomassie blue. Cells were sampled at the 8-h time point. The two black stars indicate bands present in the wild type and missing or markedly reduced in the mutant. The bottom panel represents Western blot analysis with a mouse polyclonal Fis antiserum. (E) Western blot analysis of whole-cell lysates from 35000HP and 35000HPΔfis probed with the Fis antiserum (top). Cells were sampled at 4, 8, and 16 h. PAL MAb 3B9 (bottom) was used to confirm equivalent loading among lanes.
Similar to results obtained with other bacteria (39, 40), deletion of fis in wild-type H. ducreyi 35000HP resulted in a significant growth defect that was apparent during mid-exponential growth (P < 0.001) (Fig. 1B). In addition to this broth growth defect, 35000HPΔfis colonies were smaller than those of the wild-type parent strain (Fig. 1C). Both the small-colony phenotype and the growth defect could be complemented in trans (data not shown) by using a wild-type copy of the H. ducreyi fis gene in the relatively low-copy-number vector pACYC177 (see Materials and Methods). The total protein profile of 35000HPΔfis (Fig. 1D, lane 2) was different from that of the wild-type parent strain (Fig. 1D, lane 1), and this difference could be eliminated when a wild-type copy of fis was introduced onto a plasmid (Fig. 1D, lane 3). Fis has been shown to be involved in several processes, including transcription, replication, and recombination, and its expression typically peaks in early exponential phase (39, 41), suggesting that Fis is most active when bacterial cells are rapidly dividing. In contrast, the Fis expression level in H. ducreyi remained relatively constant through the growth phase (Fig. 1E), suggesting that the regulatory activities of Fis in H. ducreyi might be different from those observed in other bacterial species.
Deletion of fis in H. ducreyi results in decreased expression of the LspB/LspA2 two-partner secretion system.To ascertain whether H. ducreyi Fis was involved in the regulation of expression of the LspB, LspA2, and LspA1 proteins, whole-cell lysates of wild-type, mutant, and complemented mutant strains were subjected to Western blot analysis. Deletion of the fis gene in H. ducreyi 35000HP resulted in decreased expression levels of both LspB and LspA2 (Fig. 2A, lane 2), suggesting that Fis could modulate the expression of these proteins. It should be noted here that lspB and lspA2 were previously shown to comprise a bicistronic operon in H. ducreyi (17). In contrast to LspB/LspA2, the expression level of LspA1 from an unlinked locus remained relatively unchanged in the fis deletion mutant (Fig. 2A, lane 2). Complementation of the fis mutation in trans in 35000HPΔfis(pML164) (Fig. 2A, lane 3) resulted in an increase in LspB and LspA2 expression levels to levels similar to those observed in wild-type cells (Fig. 2A, lane1).
Analysis of protein expression and phagocytosis activity in wild-type and mutant H. ducreyi strains. (A) Western blot analysis of whole-cell lysates from 35000HP (lane 1), 35000HPΔfis (lane 2), 35000HPΔfis(pML164) (lane 3), and 35000HPΔfis(pACYC177) (lane 4) probed with a LspB polyclonal antibody, LspA2 MAb 1H9, LspA1 MAb 40A4, or a CpxR polyclonal antibody. Cells were harvested at 8 h, and the same set of four whole-cell lysates was loaded onto multiple different gels, one set per primary antibody probe. PAL MAb 3B9 was used to confirm equivalent loading among lanes. It should be noted that the LspA1 and LspA2 proteins do not exhibit discrete banding patterns by Western blot analysis but instead form smears (17, 38). Fis protein expression by these same four strains is depicted in Fig. 1D. (B) Phagocytosis assay. The ability of 35000HP (column 1), 35000HPΩ12 (column 2), 35000HPΔfis (column 3), 35000HPΔfis(pML164) (column 4), and 35000HPΔfis(pACYC177) (column 5) to inhibit the phagocytic activity of murine J774A.1 macrophages, measured by the uptake of opsonized latex beads, was tested. A representative experiment is shown. The multiplicity of infection (MOI) used for each strain is listed at the top of each column. ∗∗∗, P < 0.001; ns, not significant.
The H. ducreyi LspB-LspA2/LspA1 two-partner secretion system was previously shown to be necessary for the inhibition of phagocytosis in macrophages (18); therefore, the ability of 35000HPΔfis to inhibit the uptake of opsonized secondary targets by J774A.1 macrophages was tested. In this assay, the level of phagocytosis inhibition exerted by 35000HPΔfis (Fig. 2B, column 3) was similar (P value was nonsignificant) to that of the wild-type strain (Fig. 2B, column 1), with a phagocytosis index significantly lower than that of lspA1 lspA2 double mutant strain 35000HPΩ12 (P < 0.001) (Fig. 2B, column 2). The ability of this fis mutant to effectively inhibit phagocytosis can be explained by the facts that expression of LspB, albeit reduced, was not abolished, and expression of LspA1 was not affected. Either LspA1 or LspA2 is sufficient to effectively inhibit phagocytosis (18).
H. ducreyi Fis: structural considerations.To glean information regarding the structure of the H. ducreyi Fis protein, we used its amino acid sequence to query the sequence databases (42). Nearly every protein returned from the search was annotated as a Fis homolog. Fis is a dimeric DNA binding protein that causes B-form DNA to bend, and thus, it has important functions in DNA rearrangements, replication, transcription, and other activities (43). The current Fis consensus sequence is 5′-GXXXXXXXXXXXXXC-3′, where the central portion (5 to 7 bases) is enriched for A and T (44). Many of the returned proteins are very similar to the H. ducreyi Fis protein; for example, the E. coli homologs (several strains were identified) have amino acid sequence identities to H. ducreyi Fis of about 70%. Using a hidden Markov model search method (45, 46), the Protein Data Bank (PDB) was queried for likely structural homologs. The R71L mutant of E. coli Fis (PDB accession number 1ETO) (47) was returned as the most likely, with a probability of 99.8%.
Given the high amino acid sequence identity and the high probability of a structural match, it was deemed that the use of E. coli Fis as a template for the construction of a homology model of an H. ducreyi Fis monomer was feasible. The MODELLER program (48) was used for this task; the result is shown in Fig. S1A in the supplemental material. For this model, the untemplated parts (resulting from unmodeled residues in E. coli Fis) were eliminated, as was a β-hairpin near the N terminus whose structure is known to adopt many positions with respect to the remainder of the protein (47). The H. ducreyi Fis model comprises residues L27 through G98. Overall, there is a single α-helix at the N terminus that packs against a helix-turn-helix motif that is responsible for the DNA binding activity (49). Details of E. coli Fis and other Fis structures are recapitulated in this model. For example, basic and polar residues known to contact DNA phosphates are solvent exposed in the H. ducreyi Fis model (see Fig. S1B in the supplemental material).
DNA microarray analysis of the H. ducreyi fis deletion mutant.Fis has been shown to be involved in the regulation of expression of many genes, including virulence factors, in different bacterial systems (50, 51). We used DNA microarrays to compare the global expression profile of the wild-type strain with that of the fis mutant in an attempt to determine the extent of Fis involvement in gene expression in H. ducreyi. In the absence of Fis, 9.89% of the predicted ORFs in the H. ducreyi genome were differentially expressed at least 2-fold. Of these 181 genes, 100 genes were upregulated and 81 were downregulated (P < 0.05) (see Table S1 in the supplemental material). From this list, a subset of genes was used to validate the DNA microarray data using real-time RT-PCR (correlation coefficient [R2] = 0.788) (Fig. 3). A list of the 15 most up- and downregulated genes in the absence of Fis, not including those ORFs annotated as encoding hypothetical proteins, is shown in Table 3. Among the most downregulated genes were ompP2A, ompA2, dsrA, and components of the flp operon. Both DsrA and Flp have been shown to be H. ducreyi virulence factors (52, 53). Consistent with the protein expression data presented above (Fig. 2), the level of lspA2 transcripts was reduced approximately 2-fold in this analysis (see Table S1 in the supplemental material).
Relative expression levels of selected H. ducreyi genes in 35000HPΔfis. Expression levels of 23 selected genes in 35000HPΔfis compared to wild-type 35000HP cells were measured by DNA microarrays (black bars) or real-time RT-PCR analysis (white bars), as described in Materials and Methods. These data are the means of results from three independent experiments.
Genes whose expression was most affected by the absence of Fis, as measured by DNA microarray analysisa
Effect of the fis mutation on other proven H. ducreyi virulence factors.To further validate these DNA microarray data at the protein level, whole-cell lysates of wild-type 35000HP, 35000HPΔfis, 35000HPΔfis(pML164), and 35000HPΔfis(pACYC177) were first analyzed by Western blotting for both DsrA and Flp protein expression (Fig. 4A and 5A, respectively). DsrA is responsible for serum resistance (30), and inactivation of dsrA results in attenuation of H. ducreyi in the human challenge model (52). Deletion of fis resulted in a significant reduction in DsrA levels (Fig. 4A, lane 2), which could be restored to wild-type levels by complementation in trans (Fig. 4A, lane 3). Next, the ability of a 35000HPΔfis mutant to resist killing by normal human serum (NHS) was tested in a bactericidal assay (Fig. 4B). We compared the survival rates of 35000HP, 35000HPΔfis, 35000HPΔfis(pML164), 35000HPΔfis(pACYC177), and a dsrA mutant (FX517) in 50% NHS. The resistance level of the fis mutant (Fig. 4B, column 2) was less than that of the wild type (Fig. 4B, column 1) but greater than that of the serum-sensitive dsrA mutant (Fig. 4B, column 5). Complementation of the fis mutation (Fig. 4B, column 3) resulted in serum resistance equivalent to that of the wild-type parent strain (Fig. 4B, column 1).
An H. ducreyi fis deletion mutant is sensitive to serum killing. (A) Western blot analysis of whole-cell lysates of 35000HP (lane 1), 35000HPΔfis (lane 2), 35000HPΔfis(pML164) (lane 3), and 35000HPΔfis(pACYC177) (lane 4) probed with a DsrA polyclonal antibody. Cells were harvested at 8 h. PAL MAb 3B9 was used to confirm equivalent loading among lanes. (B) Serum bactericidal activity assays. The percent survival of 35000HP (column 1), 35000HPΔfis (column 2), 35000HPΔfis(pML164) (column 3), 35000HPΔfis(pACYC177) (column 4), and dsrA mutant strain FX517 (column 5) in 50% normal human serum (NHS) was calculated as follows: (geometric mean CFU in NHS/geometric mean CFU in heat-inactivated NHS) × 100. Values represent means ± standard deviations of 5 independent experiments. All strains were compared to 35000HP in column 1. ∗∗∗∗, P < 0.0001; ∗∗, P < 0.01.
An H. ducreyi fis deletion mutant is deficient in microcolony formation. (A) Western blot analysis of the same whole-cell lysates used in Fig. 2A, probed with a Flp1 polyclonal antibody and PAL MAb 3B9. The panel depicting results obtained with MAb 3B9 is the same as that in Fig. 2A. (B) Microcolony formation assay. The ability of 35000HP, 35000HPΔfis, 35000HPΔfis(pML164), 35000HPΔfis(pACYC177), and 35000HP tadA to form microcolonies upon incubation with Hs27 human fibroblasts was tested. Cells were harvested after 16 h of growth in CB. A representative experiment is shown. Arrows indicate the positions of the microcolonies.
Previous work has shown that H. ducreyi can form microcolonies when cultured with human cells in vitro and that this phenotype requires expression of the protein products of the Flp operon (31). The DNA microarray data indicated that in the absence of Fis, the first genes in the Flp operon (i.e., flp1, flp2, and flp3) (Table 1) are all downregulated, a result which suggested that 35000HPΔfis should have a diminished ability to form microcolonies. Western blot analysis of whole-cell lysates validated our DNA microarray data and showed decreased expression levels of Flp1 in the absence of Fis (Fig. 5A, lane 2) relative to the wild-type parent strain (Fig. 5A, lane 1). This expression deficiency was corrected by complementation with the wild-type fis gene in trans (Fig. 5A, lane 3). This deficiency in Flp1 expression was also apparent in a microcolony formation assay where very few microcolonies were seen after incubation of 35000HPΔfis with Hs27 human foreskin fibroblast cells (Fig. 5B). This microcolony deficiency phenotype could be restored by complementation (Fig. 5B). As a negative control, a H. ducreyi tadA mutant (31) unable to secrete Flp proteins was used (Fig. 5B).
Fis has a positive effect on lspB expression.Using the known E. coli Fis consensus binding sequence (44) as a reference, a putative consensus binding motif was located in the lspB promoter region (see Fig. S2 in the supplemental material). Attempts to show a specific interaction of purified recombinant H. ducreyi Fis with the promoter region of lspB were unsuccessful; there was no statistical difference between the binding of Fis to the promoter region of lspB and the binding to a fragment from within the lspB ORF (data not shown). This nonspecific binding of Fis was also observed with internal fragments of 10 different ORFs selected from the DNA microarray results (data not shown). A lacZ-based transcriptional reporter (Fig. 6A) was constructed and used to test promoter activation by Fis in different H. ducreyi strain backgrounds. Although the lspB promoter in pML306 (Table 1) was not very active in the 35000HP wild-type background (Fig. 6B), in the absence of Fis (in the 35000HPΔfis mutant), promoter activity was significantly lower (Fig. 6B), which was consistent with protein expression data (Fig. 2A). The promoter regions of genes encoding other proven H. ducreyi virulence factors whose expression was shown to be affected by the absence of Fis by Western blot analysis (Fig. 4 and 5) were also introduced into this transcriptional reporter. All these promoter regions were screened for the presence of putative Fis binding motifs, and the results are shown in Fig. S2 in the supplemental material. β-Galactosidase activity assays showed reduced promoter activity for both the dsrA and flp1 reporter constructs in the 35000HPΔfis mutant (Fig. 6B), indicating a positive effect of Fis on the transcription of these genes. The promoter region of ompP2B was also tested because the DNA microarray data (Table 3) indicated that expression of this ORF was upregulated in the absence of Fis. The transcriptional reporter assay using pML314 (Table 1) corroborated these DNA microarray data and confirmed that Fis was involved in the repression of transcription of ompP2B (Fig. 6B).
Fis and CpxR are involved in the regulation of LspB expression. (A) Schematic map of the H. ducreyi LacZ-based reporter construct pML303. The location of the kanamycin gene (kan) and ori (both originally derived from pACYC177), the multicloning site (MCS), transcriptional terminators (○), and the promoterless lacZ gene (originally derived from pRS551) are indicated. The NotI and SalI sites used for directional cloning of H. ducreyi promoter regions are shown. (B) Use of a β-galactosidase assay with pML303-derived constructs to measure promoter activity. These constructs include pML306 carrying the lspB promoter region, pML308 carrying the dsrA promoter region, pML309 carrying the gyrB promoter region, pML312 carrying the flp1 promoter region, and pML314 carrying the ompP2B promoter region. The data are from a representative experiment, and error bars represent standard deviations. ∗, P = 0.0145; ∗∗, P < 0.0001. (C) Western blot analysis of whole-cell lysates from 35000HP (lane 1), 35000HPΔfis (lane 2), 35000HPΔcpxR (lane 3), and 35000HPΔcpxRΔfis (lane 4). Bacterial cells were harvested at 8 h. Blots were probed with LspB, Fis, and CpxR polyclonal antibodies and with the PAL MAb.
In other bacteria, Fis has been show to negatively regulate the gyrB gene (54, 55). To test whether the H. ducreyi Fis homologue would have the same effect on the expression of gyrB, the activity of the gyrB promoter region was tested by using this reporter. Compared to the wild-type parent strain (Fig. 6B), the activity of the gyrB promoter region in pML309 in the fis deletion mutant was significantly lower (Fig. 6B), indicating a positive effect of Fis on gyrB transcription in H. ducreyi. It is important to note that analysis of the gyrB promoter region showed the presence of two putative Fis binding motifs (see Fig. S2 in the supplemental material). These data provide further evidence that the regulatory activities of Fis in H. ducreyi might be different than those in other pathogens.
CpxR and Fis both control LspB expression.The protein expression pattern of 35000HPΔfis described above was reminiscent of that observed with a H. ducreyi recombinant strain possessing an overexpressed CpxR protein (34). In fact, when the DNA microarray results from the latter strain were compared to those obtained with the fis deletion mutant, a correlation coefficient of 0.877 was obtained. To examine the effect of a fis mutation in the absence of CpxR, double mutant strain 35000HPΔcpxRΔfis double mutant was constructed, and the levels of expression of LspB were determined by Western blotting. CpxR was previously shown to be a negative regulator of LspB expression (23), and its absence results in increased expression levels of LspB (Fig. 6C, lane 3) relative to those of the wild-type strain (Fig. 6C, lane 1). In contrast, the absence of only Fis resulted in decreased LspB expression levels (Fig. 6C, lane 2), suggesting a positive regulatory effect of Fis on LspB expression. In mutant strain 35000HPΔcpxRΔfis lacking both CpxR and Fis (Fig. 6C, lane 4), the levels of expression of LspB were higher than those in 35000HPΔfis (Fig. 6C, lane 2), lower than the LspB expression levels in 35000HPΔcpxR (Fig. 6C, lane 3), and very similar to those in the wild-type strain (Fig. 6C, lane 1). These data suggest that removal of both the positive and negative regulatory molecules (i.e., Fis and CpxR, respectively) restores LspB expression to essentially wild-type levels.
DISCUSSION
The nucleoid-associated protein Fis is one of at least 12 nucleoid-associated proteins expressed by E. coli (43). Among these proteins, perhaps the most is known about Fis functionality in the bacterial cell with respect to both physiology and virulence expression. Fis displays a high degree of sequence and function conservation among enteric pathogens (56). However, while there is a high degree of sequence and structure homology between the E. coli and H. ducreyi Fis proteins, our mutant analysis-derived data suggest that their functions might be different. In enteric bacteria like E. coli and Salmonella enterica serovar Typhimurium, Fis has been shown to have a very specific pattern of expression. Fis protein levels are highest at early stages of the growth phase as the organism responds to increased levels of nutrients, which support the increasing demands of transcription and translation as the cells grow rapidly. After this quick surge, the de novo production of Fis ends, and the amounts of Fis protein per cell decline as the bacterial cells divide rapidly, until Fis is barely detectable upon entry into stationary phase (39, 55, 57). In contrast, our data indicate that the levels of Fis protein in H. ducreyi remain relatively constant throughout the first 16 h of growth (Fig. 1E) into early stationary phase (11), suggesting that Fis might have a different role(s) in affecting gene expression in this obligate human pathogen.
In other bacterial systems, Fis indirectly regulates expression of genes through the repression of gyrB (54, 55). As the levels of GyrB decline, DNA negative supercoiling decreases, leading to a relaxed DNA state, which either allows for transcription factors to access promoter regions or prevents the binding of factors that require a specific DNA topology (55, 58, 59). Using a LacZ-based reporter construct, our data show that in H. ducreyi 35000HP, Fis has a positive effect on gyrB transcription (Fig. 6B), potentially increasing the levels of DNA supercoiling throughout the growth phase. Further work is needed to establish whether the levels of supercoiling in H. ducreyi remain constant throughout the growth phase or whether other topoisomerases are involved in this process to allow for differential gene expression during the different stages of growth.
Fis has been shown to be involved in regulation of expression of virulence factors in a number of pathogens (26, 50, 60–62). In P. multocida, a close relative of H. ducreyi, Fis has been shown to be required for expression of the two-partner secretion system composed of PfhB_2 and LspB_2 (26). This particular P. multocida system has homology to the LspB/LspA2-LspA1 two-partner secretion system of H. ducreyi. In this study, we showed that the H. ducreyi Fis homolog is involved in the regulation of expression of the lspB-lspA2 operon, which encodes the secretion component (LspB) and one of the two secreted products (LspA2) of this H. ducreyi two-partner secretion system (17). In the absence of Fis, the expression levels of LspB and LspA2 decreased significantly, while the expression level of the LspA1 protein, encoded at a different locus, remained unchanged (Fig. 2A, compare lane 1 with lane 2). The LspA proteins (LspA2 and LspA1) have been shown to be responsible for the ability of H. ducreyi to inhibit phagocytosis by macrophages in vitro (18), with either protein being sufficient to cause inhibition (18). Our new data indicate that a H. ducreyi fis mutant is able to inhibit phagocytosis at a level similar to that of the wild type (Fig. 2B, compare column 1 with column 3). It should be noted that deletion of fis reduced the expression of LspB, but it did not completely abolish it (Fig. 2A, lane 2). Consequently, enough LspB was produced to allow secretion of LspA1, the expression of which is not affected by inactivation of fis, with subsequent inhibition of phagocytosis (Fig. 2B, column 3).
While the original intent of the present study was to determine whether H. ducreyi Fis was involved in controlling expression of the LspA proteins, the wide range of both direct and indirect regulatory activities attributed to enteric Fis proteins (50, 62, 63) prompted us to widen the scope of our investigation. Using DNA microarrays to test the extent of the involvement of Fis in gene expression in H. ducreyi, we determined that deletion of fis affected expression of ∼10% of the genome. Whereas several functional categories are affected by the absence of Fis (see Fig. S3 in the supplemental material), our data also indicate that a number of genes shown to be important for virulence of H. ducreyi are also Fis dependent (Table 3 and Fig. 2, 4, and 5). These genes include those encoding the DsrA serum resistance protein (30) and the Flp1 protein involved in microcolony formation (31), in addition to the lspB-lspA2 operon. Intensive efforts to show a direct interaction of Fis with the promoter regions of dsrA, flp1, and lspB were unsuccessful due to the nonspecific binding of H. ducreyi Fis to DNA (i.e., recombinant H. ducreyi Fis readily bound to internal fragments of ORFs) (data not shown). However, the use of lacZ-based transcriptional fusions showed decreased activity of the promoters for these three ORFs in the absence of Fis (Fig. 6B), suggesting a positive influence of Fis on their expression.
We recently established that CpxR is a direct repressor of the lspB-lspA2 operon in H. ducreyi 35000HP (23, 34). In the present study, we provide evidence that Fis positively affects the expression of this same operon (Fig. 2A and 6C). In the absence of Fis, expression of the CpxR protein remained essentially constant (Fig. 2A, lane 2, and 6C, lane 2), whereas those of LspB (Fig. 2A, lane 2, and 6C, lane 2) and LspA2 (Fig. 2A, lane 2) decreased dramatically. There are at least two ways in which Fis may affect expression of this operon. Fis may alter promoter DNA topology in the presence of CpxR to allow effective transcription from the lspB-lspA2 promoter. It is also possible that the level of phosphorylation of CpxR increases in the absence of Fis, a result that would likely increase the ability of CpxR to decrease the expression level of the lspB-lspA2 operon. Consistent with the latter possibility, the DNA microarray data indicated that there was an increase in the expression level of phosphotransacetylase (Pta) (see Table S1 in the supplemental material), which was confirmed by real-time RT-PCR (data not shown). Phosphotransacetylase is involved in the synthesis of acetyl phosphate, a small phosphodonor that has been shown in E. coli to phosphorylate CpxR independent of CpxA (64). It is possible that an increase in acetyl phosphate levels in mutant strain 35000HPΔfis could increase the phosphorylation of CpxR without affecting its level of expression, resulting in a reduction in expression levels of the virulence factors negatively controlled by CpxR. In this regard, it is interesting to note that a comparison of the transcriptional profiles of mutant strains 35000HPΔfis and 35000HPΔcpxA (34), which is likely to have a highly phosphorylated CpxR (24, 34, 64), showed a correlation coefficient (R2) of 0.937.
It should be noted that deletion of both cpxR and fis resulted in levels of expression of LspB that were similar to those of the wild type (Fig. 6C, lane 4), suggesting that there may be another mechanism of positive regulation that may work in combination with the normal CpxR repression and Fis activation of this operon. In S. enterica, Fis has been shown to be involved in the control of important virulence factors, but most if not all of the Fis-dependent genes have other levels of regulation (62, 65). Similarly, in enteroaggregative E. coli, Fis is required for full expression of the Pet autotransporter toxin, where it functions in concert with C-reactive protein (CRP) (66). It is possible that these opposing actions of CpxR and Fis on the lspB-lspA2 operon are critical for the pathogenesis of H. ducreyi disease. Investigation of the relative importance and potential interaction of CpxR and Fis at the promoter region of lspB are under way.
One limitation of the present study is that it utilized 35000HP, a class I strain of H. ducreyi. Two apparently clonal populations of H. ducreyi (class I and class II) have been described (67) and confirmed by both proteomic methods (68) and additional genetic analyses (69). Whether a fis mutant of a class II strain of H. ducreyi would have a transcriptome profile different from that of 35000HPΔfis remains to be determined but would seem unlikely in view of the conservation of Fis effects among closely related bacteria (i.e., enteric organisms). Another caveat is that there are no protein expression data available to indicate how the Fis protein is regulated in the infected human host. Perhaps transcriptome sequencing (RNA-seq)-based analysis of samples from lesions formed in the human challenge model for chancroid could address this issue in the future.
ACKNOWLEDGMENTS
This study was supported by U.S. Public Health Service grant AI032011 and ARRA supplement AI032011-18S1 to E.J.H. and by U.S. Public Health Service grant AI27863 to S.M.S.
FOOTNOTES
- Received 6 June 2013.
- Returned for modification 3 August 2013.
- Accepted 15 August 2013.
- Accepted manuscript posted online 26 August 2013.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00714-13.
- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
