Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers

Abstract

Haemophilus ducreyi causes the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans, H. ducreyi resides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. MunsonJr, E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014, http://dx.doi.org/10.1128/mBio.01081-13) suggested that H. ducreyi encounters growth conditions in human lesions resembling those found in stationary phase. However, how H. ducreyi transcriptionally responds to stress during human infection is unknown. Here, we determined the H. ducreyi transcriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that the in vivo transcriptome is distinct from those of in vitro growth. Compared to the inoculum (mid-log-phase bacteria), H. ducreyi harvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis. H. ducreyi upregulated few genes (hgbA, flp-tad, and lspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressed in vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that the in vivo transcriptome is distinct from those of in vitro growth and that adaptation to nutrient stress and anaerobiosis is crucial for H. ducreyi survival in humans.

FIG 1

Multidimensional scaling plot of H. ducreyi transcriptional profiles during in vivo and in vitro growth. The plot was generated from pairwise distances using edgeR. A PERMANOVA analysis was performed to determine if the in vivo and in vitro transcriptomes differed significantly from one another. Each symbol represents one independent sample.

FIG 2

Venn diagram showing the number of genes differentially expressed in vivo compared to those in mid-log, transition, and stationary growth phases. The up- and downregulated genes or operons are indicated by up and down arrows, respectively. The total number of genes differentially expressed is indicated in boldface outside the Venn diagram.

FIG 3

qRT-PCR validation of the RNA-Seq data. The fold change in expression was calculated by comparing the gene expression in H. ducreyi harvested from pustules to those harvested from mid-log phase. The expression levels of target genes were normalized to that of dnaE. The data represent the mean ratio of transcript expression in four biopsy specimens divided by expression from four broth cultures used for the RNA-Seq study. As the samples were not paired, no standard deviations were calculated.