Previous work in our laboratory suggested that lspA1
were under the control of different regulatory mechanisms, based on the observation that LspA1 was apparently more abundant than LspA2 in whole-cell lysates and CCS (64
), together with the fact that inactivation of lspA1
resulted in the detection of increased levels of LspA2 in whole-cell lysates and CCS (63
). Analysis of LspA1 and LspA2 expression profiles during growth in vitro in the present study indicated that LspA2 had a temporal pattern of expression, with relatively high levels being attained only in the late exponential and stationary phases (Fig. ). In contrast, LspA1 appeared to be expressed throughout the growth period (Fig. ). Taken together with our previously published findings (63
), these data supported the hypothesis that LspA1 and LspA2 are differentially regulated. In the present study, we obtained evidence strongly suggesting that the Cpx stress response system (47
) is involved in the negative regulation of the lspB-lspA2
operon. In addition, we discovered that FCS might serve as an environmental signal to upregulate a variety of virulence factors that could potentially aid in the infection process.
It has been well established that bacteria are able to sense their environment and respond to different stimuli by altering gene expression (27
). During the infection process, bacteria have been shown to adjust gene expression depending on the host, tissue, or cellular localization (for a review, see reference 10
). The fact that H. ducreyi
is an obligate human pathogen does not eliminate its need to be able to regulate gene expression, and there is ample evidence that other obligate human pathogens, including both Bordetella pertussis
) and Neisseria gonorrhoeae
), use two-component sensory transduction systems to control gene expression. Although there is some indirect evidence that heme restriction can regulate gene expression in H. ducreyi
), there have been no reports to date describing regulatory gene mutations in H. ducreyi
In the present study, we found two different mechanisms involved in controlling expression of the LspA proteins. The first involved FCS, which H. ducreyi
requires for optimal growth in vitro. During infection, H. ducreyi
is likely to be exposed to serum components at the site of ulceration as a consequence of vascular leakage. Several pathogens have been shown to alter gene expression in response to plasma or serum, including Streptococcus pyogenes
), Enterococcus faecalis
), and Yersinia pestis
). We had previously found that neither LspA protein could be detected in CCS when wild-type H. ducreyi
was grown in the absence of FCS (data not shown). In the present study, we found that LspA2 is not expressed in the absence of FCS (Fig. , panel 6), and this result was reflected by real-time RT-PCR analysis (Fig. and ). In contrast, expression of LspA1 is not affected by FCS (Fig. , panels 1 and 2). However, even though expression of LspA1 is not affected by FCS, LspA1 was not detectable in CCS derived from cultures grown in the absence of FCS (Fig. , panel 4), suggesting that a factor(s) present in FCS is necessary for the processing and/or release of this very large protein into the medium. Whether this factor acts directly or indirectly to effect this release is not known at this time.
Global transcriptional analysis of H. ducreyi
35000HP cells grown in the presence or absence of FCS indicated that, besides the upregulation of the lspB-lspA2
operon, other genes including both dsrA
and the tad
operon were also upregulated (Table ). Expression of these latter two genes or operons has been shown to be important for normal virulence of H. ducreyi
in the human challenge model (9
). In contrast, expression of the hemoglobin-binding protein HgbA (20
), which has been previously shown to be a virulence factor for H. ducreyi
), was downregulated in vitro by the presence of FCS (Table ).
Among the H. ducreyi
genes that were differentially expressed in the presence or absence of serum, it is interesting to note the dichotomy involving the ompP2A
ORFs. Expression of ompP2A
was upregulated by the presence of FCS, whereas the expression of ompP2B
was downregulated (Fig. ). Both of these genes encode porins, and H. ducreyi
35000HP expresses both OmpP2A and OmpP2B in vitro (45
). However, the majority of H. ducreyi
strains tested to date appear to express only OmpP2A and do not express OmpP2B as the result of different mutations in and near the ompP2B
ORF. Why H. ducreyi
35000HP would differentially regulate these two ORFs in the presence of serum is not immediately apparent, but this could suggest that the OmpP2A protein may function better as a porin than does OmpP2B.
Further analysis of the transcriptional data indicated that the CpxRA two-component regulatory system was downregulated in the presence of FCS. The CpxRA regulatory system is one of the four cell envelope stress systems in gram-negative bacteria (the other three being the alternative sigma factor σE
, BaeSR, and the phage shock response), which are induced by a variety of signals (for reviews, see references 16
). Although initially thought to only respond to stress in the cell envelope, the CpxRA regulatory system has also been found to affect expression of virulence factors in several pathogens, including Legionella pneumophila
), Salmonella enterica
), enteropathogenic E. coli
), and Yersinia
). The target genes controlled by the CpxRA system in the aforementioned bacteria are different but are mostly related to adhesion to and/or invasion of mammalian cells. The effect that this two-component regulatory system exerts on these target genes can also vary, in that in some cases it behaves as a positive modulator (21
), while in other instances the effect is negative (11
As described above, FCS enhanced the growth of H. ducreyi in CB (Fig. ). Under these conditions, it could be inferred that there is less stress on the H. ducreyi cell. Consequently, a reduction in transcription of the cpxRA genes might be expected, as was detected in the DNA microarray experiments in the present study. This in turn could affect the expression of genes regulated by CpxR. The fact that LspA2 was only expressed when H. ducreyi was grown in CB with FCS (Fig. ) suggested that CpxR might be involved in controlling the expression of this protein.
Inactivation of the cpxR ORF in H. ducreyi 35000HP resulted in increased expression of both LspB and LspA2 (Fig. ). EMSA findings indicated that a recombinant H. ducreyi CpxR protein bound to the lspB promoter region between nt −157 and +43. This is the region that contains the putative CpxR recognition site, thus providing additional evidence for the involvement of CpxR in the regulation of expression of LspB and LspA2. What element(s) controls of expression of LspA1 remains to be determined. It appears that LspA1 is constitutively expressed by the wild-type strain in vitro (Fig. ) and that expression of this protein is not affected by the presence of FCS (Fig. ). However, overexpression of CpxR (in the complemented cpxR mutant) resulted in a dramatic reduction in LspA1 synthesis (Fig. , lane 3). The DNA upstream from lspA1 apparently lacks a CpxR binding site consensus sequence, and the EMSA experiment indicated that this region does not bind CpxR (Fig. ). It is possible that overexpression of CpxR in the complemented cpxR deletion mutant affected another regulatory pathway(s), which in turn affected LspA1 expression.
Two previous studies focused on identifying bacterial genes transcribed during experimental H. ducreyi
infection. The first of these used RT-PCR to detect H. ducreyi
transcripts present in biopsies from pustules produced in human volunteers by H. ducreyi
). Both lspA1
transcripts were detected in these in vivo-derived samples. The second study used the selective capture of transcribed sequences to identify genes expressed in vivo and found a cDNA containing a nucleotide sequence common to both lspA1
). The fact that both lspA1
transcripts were detected in the former study indicates that both of these genes are transcribed in vivo, although these data do not address protein expression. At the very least, the presence of both lspA1
transcripts in vivo raises the possibility that H. ducreyi
synthesizes both of the encoded proteins, perhaps to optimize its ability to avoid phagocytosis.
In conclusion, the studies described here provide the first evidence for the involvement of the CpxRA system in the differential regulation of the LspA proteins. Although CpxR has been shown to be a negative regulator of LspB and LspA2 expression, it remains to be determined what gene product(s) control expression of LspA1. Similarly, the extent of involvement of the CpxRA system in controlling expression of other H. ducreyi virulence factors is not known. In addition, the identity of the factor(s) in FCS necessary for expression of LspA2 in vitro and for release of both LspA1 and LspA2 from the H. ducreyi cell surface is unknown at this time. These results and unanswered questions warrant more detailed investigation of the CpxRA two-component system in H. ducreyi.