HmbR, a hemoglobin-binding outer membrane protein of Neisseria meningitidis, undergoes phase variation

The gram-negative diplococcus Neisseria meningitidis is a major cause of bacterial meningitis and is a significant public health problem throughout the world (7, 38). The incidence of meningococcal disease in developing countries ranges between 10 and 25 per 100,000, with periodic epidemics having attack rates of up to 1,000 per 100,000 (6, 12, 45). Approximately 2,600 cases of meningococcal disease occur annually in the United States, predominantly in children less than 2 years old (7). Since the success of the current vaccine against Haemophilus influenzae type b, N. meningitidis is the leading cause of meningitis in children in the United States (7). Despite its relatively low incidence, several facts about meningococcal disease make it a potential public health problem of major importance. Mortality from meningococcal meningitis remains, despite optimal antibiotic and supportive therapy, about 5% in children and 10 to 15% in adults (33). There have been recent reports from Europe, Canada, South Africa, and the United States of N. meningitidis isolates that show moderate resistance to penicillin (48, 51). The occurrence of a new, more virulent strain of N. meningitidis serogroup C associated with an increase in both incidence and mortality of meningococcal disease in Canada is a reminder that the evolution of N. meningitidis virulence is ongoing (49). The best preventive measure against meningococcal disease is a polyvalent polysaccharide vaccine. The vaccine is not protective against serogroup B meningococcus, however, nor can it protect children under 2 years of age (7, 14). Among new vaccine targets against N. meningitidis being explored are iron-regulated outer membrane proteins (2, 24). These proteins are attractive vaccine candidates due to their important role in virulence and surface accessibility (2, 24, 39). N. meningitidis expresses several outer membrane receptors that enable the bacterium to use human transferrin, lactoferrin, hemoglobin (Hb), heme, and haptoglobin-hemoglobin complexes (Hpt-Hb) as sources of iron (11, 13, 22, 23, 36, 42). Heme and Hb are the most abundant sources of iron in the body (29). Recently, two neisserial Hb-binding outer membrane receptors have been identified. HmbR is an iron-regulated, 89.5-kDa protein that binds Hb, extracts the heme from it, and transports the heme into the periplasm (42–44). The hmbR mutant of N. meningitidis was attenuated in an infant rat infection model, confirming the importance of Hb acquisition in meningococcal virulence (42). A second Hb-binding protein is the HpuAB bipartite receptor involved in use of Hb and Hpt-Hb complexes in N. meningitidis and Neisseria gonorrhoeae (8, 22, 23). In addition to Hb, the HpuAB receptor binds Hpt-Hb complexes and apo-Hpt (22). The HpuAB bipartite receptor is most likely the main Hb receptor of gonococci, since the hmbR gene contains a premature stop codon in all gonococcal strains tested (8, 43). Recently, the expression of the hpuAB receptor genes in N. gonorrhoeae was shown to undergo phase variation (9). A limited analysis of meningococcal clinical isolates revealed functional redundancy in their Hb utilization systems (43). In this communication, we show that this functional redundancy is a common phenomenon in clinical isolates of N. meningitidis. Moreover, we show that the expression of hmbR undergoes phase variation due to the slipped-strand mispairing (SSM) of a poly(G) tract beginning at position +1164 in the hmbR coding sequence. The rate of switching between the off and on phases of hmbR differs more than 1,000-fold between different clinical isolates. These results establish a rational explanation for the existence of functional redundancy in Hb utilization systems and support a novel hypothesis correlating the rates of phase variation and virulence.