Two-component systems in Haemophilus influenzae: a regulatory role for ArcA in serum resistance

Abstract

Knockout mutations were constructed in the arcA gene of a virulent type b strain of Haemophilus influenzae, and the behavior of the resulting mutants was investigated in a number of conditions that mimicked distinct steps in the natural infection pathway. In arcA mutants, synthesis of capsule and lipooligosaccharide (LOS) and growth in synthetic media were unaltered compared to synthesis of capsule and LOS and growth in synthetic media in the wild-type H. influenzae type b parent strain. However, the virulence of the arcA mutants for BALB/c mice was significantly reduced. Upon exposure to human blood or serum, the arcA mutants showed markedly reduced survival compared with the survival of its wild-type parent. Serum resistance could be fully restored by complementation in cis with the H. influenzae arcA gene but not by complementation in cis with the homologous gene from Escherichia coli. The proteomes of wild-type and mutant bacteria were markedly different, especially under anaerobic conditions, underscoring the global regulatory role of ArcAB in H. influenzae. Evaluation of antibody titers and classical complement activities in various serum samples pointed to complement-mediated bactericidal activity as the factor that distinguishes between the arcA mutant and wild-type phenotypes. Comparative analysis of the membrane fractions of the arcA mutants and the wild-type strain revealed several ArcA-regulated proteins, some of which may be implicated in the serum hypersensitivity phenotype.

FIG. 1.

LOS profiles of the arcA mutants and the wild type on 14% PAGE gels. Lane 1, Hib⁻ strain (unencapsulated); lane 2, strain Hib1 (arcA::kan); lane 3, strain Hib2 (arcA::tet); lane 4, wild-type strain Hib; lane 5, Hib3 (arcA-gfp-tet); lane 6, strain Hib2 after exposure to serum; lane 7, wild-type strain Hib after exposure to serum.

FIG. 2.

Bactericidal activity of serum donor 9: survival of arcA mutants Hib1 (arcA::kan) and Hib2 (arcA::tet), repaired arcA mutant Hib3 (arcA*res), and the wild type (wt) in serum from donor 9, expressed in CFU per milliliter. The initial inoculum was 10⁶ CFU/ml. At 135 min, the titer of the arcA mutants fell below the threshold of detection (100 CFU/ml), while the levels of Hib3 and the wild type remained stable.

FIG. 3.

β-Galactosidase activity in the complemented E. coli arcA mutant: comparison of E. coli strains carrying plasmids for complementing the chromosomal arcA mutation in cis. The β-galactosidase (β-gal) activity of a succinate dehydrogenase (sdh)-lacZ reporter fusion was measured during anaerobic growth.

FIG. 4.

Comparison of bactericidal activities against arcA strains carrying the pJRD215 and pJRD215::arcA plasmids. The arcA mutant, the wild-type strain (wt), and the arcA mutant complemented with plasmid-coded arcA were inoculated at a concentration of 10⁷ CFU/ml into serum. After 1 h of incubation at 37°C, the numbers of CFU were determined by serial dilution (standard error, less than 5%).

FIG. 5.

Wild-type strain Hib and arcA mutant proteomes for total cell lysates after anaerobic growth. Regions with many differences are enclosed by boxes. The arrows indicate some of the major differences.

FIG. 6.

Aerobically expressed total membrane proteins. Lane 1, molecular mass standard (the molecular masses of the proteins in the standard, in kilodaltons, are indicated on the left); lane 2, strain Hib1 (arcA::kan); lane 3, strain Hib3 (arcA-gfp-tet); lane 4, strain Hib2 (arcA::tet); lane 5, wild-type strain Hib; lane 6, whole-cell lysate. Membrane proteins were prepared, as described in Materials and Methods, from cells grown aerobically in BHI medium. It was difficult to separate the cytoplasmic and outer membrane fractions, probably due to the high capsule content. Therefore, the gels represent the total membrane fraction. The most reproducible differences were the differences in two proteins, formate dehydrogenase (FD) (115 kDa) and l-lactate dehydrogenase (LLD) (42 kDa). Other small differences were observed but were not reproducible. The probable position of porin (P) is also indicated.

FIG. 7.

Anaerobically expressed membrane proteins. Total membrane proteins of anaerobically grown Hib2 (arcA::tet) (lane 1) were compared with membranes prepared from wild-type H. influenzae (lane 2). Membrane proteins were prepared, as described in Materials and Methods, from bacteria grown anaerobically in BHI medium supplemented with KNO₃ (0.5%). The gel was electrophoresed longer in order to better resolve higher-molecular-weight bands. Among the several differences observed, differences in two proteins were identified; fumarate reductase (FR) (66 kDa) was more highly expressed in ArcA⁺ strains, and l-lactate dehydrogenase (LLD) (42 kDa) was derepressed in ArcA⁻ strains. The probable position of porin (P) is also indicated.