Identification of a haem-utilization protein (Hup) in Haemophilus influenzae

Abstract

Haemophilus influenzae has an absolute growth requirement for a porphyrin source. This growth requirement can be satisfied in vitro by haem, haemoglobin or the haemoglobin-haptoglobin, haem-haemopexin and haem-albumin complexes. A family of proteins, termed the Hgp proteins, which are essential for utilization of the haemoglobin-haptoglobin complex, has previously been identified. A strain lacking the Hgp proteins also has a residual ability to utilize haemoglobin, indicating that additional moieties contribute to haemoglobin utilization. Using a haemoglobin affinity method an approximately 105 kDa protein was isolated. Mutation of the identified gene in an Hgp null background reduced the ability of the mutant strain to utilize haemoglobin in vitro. The mutation also resulted in a reduced ability to utilize haem, haem-haemopexin, haem-albumin and haemoglobin-haptoglobin, thus identifying a general haem-utilization protein (Hup) in Haemophilus influenzae.

Lipoprotein e (P4) of Haemophilus influenzae: role in heme utilization and pathogenesis

“…Stock solutions were prepared as previously described [9,10]. Rabbit hemopexin was prepared as described previously [11].…”

“…Primers were designated eP4-1 (5′-ATACCCTGAATGAATAGG-3′) and eP4-2 (5′-AAATCGATCTTTTTAATGG-3′). PCRs were performed as previously described [11] using an annealing temperature of 51°C. Products were successfully cloned into the TA cloning vector pCR2.1-TOPO and confirmed by DNA sequencing.…”

“…A rat model was used to assess bacteremia in both 5-day old and 30-day old rats as previously described [10,11,14]. Specified pathogen free timed-pregnant Sprague-Dawley rats were received five days prior to parturition.…”

The haem–haemopexin utilization gene cluster (hxuCBA) as a virulence factor of Haemophilus influenzae

“…Four separate pairs of primers (Table 2) were designed for use in the PCR based on the Rd KW20 genomic sequence. The PCRs were performed in a 50 ml reaction as previously described (Morton et al, 2004b) except that annealing temperatures and extension times were as below. HXUCUSA and HXUCUSB were used to amplify an~1100 bp fragment upstream of hxuC (annealing temperature 56 uC, extension time 1 min); HXUC-A and HXUC-B to amplify an~2500 bp fragment encompassing the entire coding sequnce of hxuC (annealing temperature 58 uC, extension time 2 min 40 s); HXUCDSA and HXUBDSB to amplify an~2800 bp fragment including the entire coding sequence of hxuB and part of hxuA (annealing temperature 56 uC, extension time 3 min); and HXUBDSA and HXUADSB to amplify añ 3800 bp fragment including the entire coding sequence of hxuA and~1000 bp downstream of hxuA (annealing temperature 60 uC, extension time 4 min).…”

“…Haem-deplete growth was performed in BHI broth supplemented with only 10 mg b-NAD ml 21 (haemdeplete BHI; hdBHI). H. influenzae were transformed to antibiotic resistance using a modification of the static aerobic method of Gromkova et al (1989) as previously described (Morton et al, 2004b).…”