A hemoglobin-binding outer membrane protein is involved in virulence expression by Haemophilus ducreyi in an animal model.
Haemophilus ducreyi exhibits a requirement for exogenously supplied heme for aerobic growth in vitro. Nine of ten wild-type isolates of H. ducreyi were shown to contain a readily detectable hemoglobin-binding activity. Spontaneous hemoglobin-binding-negative mutants of two of these wild-type isolates lost the ability to express an outer membrane protein with an apparent molecular mass of approximately 100 kDa. Similarly, the single wild-type isolate that lacked the ability to bind hemoglobin also appeared to lack expression of this same 100-kDa protein. A monoclonal antibody (5A9) to this 100-kDa protein was used to identify a recombinant clone which possessed an H. ducreyi chromosomal fragment containing the gene encoding the 100-kDa protein; this protein was designated hemoglobin utilization protein A (HupA). Nucleotide sequence analysis of the hupA gene revealed that the predicted protein, with a calculated molecular mass of 108 kDa, was similar to TonB-dependent outer membrane proteins of other bacteria. Increasing the concentration of heme in the growth medium resulted in decreased expression of the HupA protein. Mutant analysis was used to prove that the HupA protein was essential for the utilization by H. ducreyi of both hemoglobin and hemoglobin-haptoglobin as sources of heme in vitro. In addition, it was found that an isogenic hupA mutant was less virulent than the wild-type parent strain in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi.
Stevens
M K
MK
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235-9048, USA.
Porcella
S
S
Klesney-Tait
J
J
Lumbley
S
S
Thomas
S E
SE
Norgard
M V
MV
Radolf
J D
JD
Hansen
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002602:Chancroid / Q000209:etiology / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004195:Disease Models, Animal; D005798:Genes, Bacterial; D006191:Haemophilus ducreyi / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011485:Protein Binding; D011817:Rabbits; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014774:Virulence / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D006454:Hemoglobins; C099188:HupA protein, bacteria