The Haemophilus ducreyi Fis protein is involved in controlling expression of the lspB-lspA2 operon and other virulence factors

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.

Fig 1

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.

Fig 2

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.

Fig 3

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.

Fig 4

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.

Fig 5

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.

Fig 6

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.