Expression of Peptidoglycan-Associated Lipoprotein Is Required for Virulence in the Human Model of Haemophilus ducreyi Infection

Abstract

Haemophilus ducreyi expresses a peptidoglycan-associated lipoprotein (PAL) that exhibits extensive homology to Haemophilus influenzae protein 6. We constructed an isogenic PAL mutant (35000HP-SMS4) by the use of a suicide vector that contains lacZ as a counterselectable marker. H. ducreyi 35000HP-SMS4 and its parent, 35000HP, had similar growth rates in broth and similar lipooligosaccharide profiles. 35000HP-SMS4 formed smaller, more transparent colonies than 35000HP and, unlike its parent, was hypersensitive to antibiotics. Complementation of the mutant in trans restored the parental phenotypes. To test whether expression of PAL is required for virulence, nine human volunteers were experimentally infected. Each subject was inoculated with two doses (41 to 89 CFU) of live 35000HP and one dose of heat-killed bacteria on one arm and with three doses (ranging from 28 to 800 CFU) of live 35000HP-SMS4 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent but were significantly smaller at mutant-inoculated sites than at parent-inoculated sites. The pustule formation rate was 72% (95% confidence interval [CI], 46.5 to 90.3%) at 18 parent sites and 11% (95% CI, 2.4 to 29.2%) at 27 mutant sites (P < 0.0001). The rates of recovery of H. ducreyi from surface cultures were 8% (n ‫؍‬ 130; 95% CI, 4.3 to 14.6%) for parent-inoculated sites and 0% (n ‫؍‬ 120; 95% CI, 0.0 to 2.5%) for mutant-inoculated sites (P < 0.001). H. ducreyi was recovered from six of seven biopsied parent-inoculated sites and from one of three biopsied mutant-inoculated sites. Confocal microscopy confirmed that the bacteria present in a mutant inoculation site pustule lacked a PAL-specific epitope. Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of PAL facilitates the ability of H. ducreyi to progress to the pustular stage of disease.

The Peptidoglycan-associated lipoprotein Pal contributes to the virulence of Burkholderia mallei and provides protection against lethal aerosol challenge

“…BMA2082) was present in very low abundance and only in some of the conditions tested [54]. A review of the literature indicated that Pal is produced at readily detectable levels by most organisms in which expression of the protein has been studied including E. coli [55], Haemophilus influenzae [56], Erwinia chrysanthemi [57], Pseudomonas aeruginosa [58], and Haemophilus ducreyi [59]. Hence, the pal gene appears to be more stringently regulated in B. mallei.…”

Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form

“…Comparisons of the phenotypic characteristics of the mutant and parent may, however, provide preliminary information about the role and function of AaPAL in A. actinomycetemcomitans cell physiology. We found that the mutants had decreased growth rate and filamentous cell morphology, differing partly from the pal -deficient mutant of H. ducreyi , which showed unchanged growth rate [ 14 ]. Although there were limitations in the present methods for estimating bacterial numbers, our results are consistent with previous reports demonstrating reduced growth rate of bacteria with mutations in tol-pal genes [ 13 , 57 , 58 ].…”

“…The proinflammatory and prochemotactic properties of AaPAL may contribute to producing a long-term immune challenge in persistent A. actinomycetemcomitans -associated infections. The extracellular release of PAL is also of more general interest because bacterial lipoproteins are increasingly being recognized as playing an important role in bacterial pathogenicity [ 7 , 14 , 62 ].…”

“…The PALs of several pathogens, such as Haemophilus influenzae , Escherichia coli [ 6 - 9 ], Legionella pneumophila, Bordetella pertussis , and Campylobacter jejuni [ 10 - 12 ] are proinflammatory and/or strongly immunogenic and can contribute to serum resistance of the bacterium [ 13 ]. PAL may be an important bacterial mediator in sepsis caused by Gram-negative bacteria [ 7 ] and is crucial for full expression of virulence of Haemophilus ducreyi [ 14 ].…”

Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis

“…Studies carried out on healthy individuals showed that in comparison with the wild-type strain, an H. ducreyi pal mutant caused less profound skin changes that rarely transformed into ulcerating forms and fewer bacteria were isolated from biopsy samples taken from the forming wound. This finding could be due to the decreased survival of pal mutants or a reduced ability to induce an immune response and the lower numbers of neutrophils, lymphocytes, macrophages or dendritic cells recruited to the site of infection (Fortney et al, 2000).…”