Marilyn R. Loeb

Isolation of the outer membrane of Branhamella catarrhalis

The emergence of Branhamella catarrhalis as an important human pathogen has stimulated interest in investigations of the outer membrane (OM) of the bacterium. In this study, the OM of B. catarrhalis was isolated and partially characterized. Radiolabelled cells were lysed and fractionated by isopycnic centrifugation in a continuous sucrose gradient. Five fractions were identified. Fraction A consisted of OM fragments of varying density. Fractions B and C were OM of a discrete density containing some cytoplasmic membrane. Fraction D was cytoplasmic membrane and Fraction E contained smaller less dense fragments of cytoplasmic membrane. The protein composition of the Branhamella OM is typical for that of Gram-negative bacteria in that approximately 10 to 20 proteins were present with six to eight of these proteins predominating. Having isolated and partially characterized the OM by sucrose density centrifugation, five simpler techniques for isolating OM were employed and the preparations compared to OM isolated on the gradient. Techniques that are based on differential detergent solubility of OM and cytoplasmic membrane were ineffective in isolating OM of B. catarrhalis. By contrast, techniques that involved collection of OM vesicles were successful in isolating OM of B. catarrhalis. Collection of vesicles from broth culture supernatants and EDTA-heat-induced vesicles were identified as convenient and reliable methods for isolating OM. Isolating and partially characterizing the OM of B. catarrhalis represents an initial step in a systematic study of outer membrane antigens of the bacterium.

DO PILI PLAY A ROLE IN PATHOGENICITY OF HAEMOPHILUS INFLUENZAE TYPE B

Surface components of Haemophilus influenzae type b (Hib) responsible for mucosal adherence were identified through assay of in-vitro attachment to human buccal epithelial cells (EC). Among many Hib strains from cerebrospinal fluid, blood, or the nasopharynx (NP), two isolates from NP were found to be exceptionally adherent. Adherence correlated with agglutination of human red blood cells (RBC). The adherent strains displayed pili, not previously described for Hib or other encapsulated H. influenzae. Moreover, highly adherent, piliated subpopulations of bacteria could be selected from any strain, but were lost upon subculture. Thus, the potential to express pili may be general for Hib and may aid the NP colonisation which precedes systemic invasion by this organism. Piliation was correlated with the amount of an outer membrane protein with a molecular weight of about 20,000 daltons, which had proved to be immunogenic in an 8-month-old child.

Immunoblot method for identifying surface components, determining their cross-reactivity, and investigating cell topology: Results with Haemophilus influenzae type b

Methods currently used for identifying exposed membrane components of gram-negative bacteria can give false positive results, and also do not provide information on nonprotein components such as lipopolysaccharide and polysaccharide capsules. A method, described within, has been developed to overcome these limitations. Briefly an outer membrane preparation derived from encapsulated (type b) Haemophilus influenzae was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated components were then transferred electrophoretically to nitrocellulose. The nitrocellulose was cut into vertical strips, which were then each incubated with rabbit antiserum to the whole bacterium or with the same antiserum after absorption with any of the following: the same strain of H. influenzae, a capsule-deficient mutant of that strain, other strains of H. influenzae, or other bacteria. The strips were then incubated with 125I-protein A, and the bound antibodies were detected by autoradiography. The autoradiograph of the strip exposed to unabsorbed antisera revealed the identity of those individual outer membrane components that bound antibodies. A comparison of the intensity of the various bands on this strip with those on the strips exposed to absorbed antisera was then used to identify (1) surface-exposed components, (2) those components occluded by capsule, and (3) cross-reactivity of exposed components. This method should be applicable to other cells and subcellular particles. Its major disadvantage is that it can provide false negative results.

Unexpected effects of absorbed normal rabbit serum and bovine serum albumin on survival of Haemophilus influenzae type b in the infant rat

In the course of using the infant rat model to determine the ability of various rabbit antisera to protect against challenge by Haemophilus influenzae type b we made two unexpected observations. In these experiments 4-day-old rats were inoculated s.c. on the dorsum with either rabbit serum or physiological buffers (sham serum) and then were challenged the next day with H. influenzae type b injected i.p. Bacteremia, as a marker for disease, was measured 24 h later on day 6. We observed the following. (i) Pre-immune, i.e., normal rabbit serum, containing minimal levels of antibodies to outer membrane proteins and depleted of antibodies to capsule and lipopolysaccharide, nevertheless significantly (P less than 0.01) protected the rats from challenge with H. influenzae type b when compared to a sham inoculation of buffer; (ii) In the absence of a serum inoculation on day 4 (a buffer was used as a sham serum inoculation), the levels of bacteremia obtained after inoculation with bacteria on day 5 depended upon the composition of the buffer in which the H. influenzae inoculum was suspended. Use of phosphate buffered saline (PBS) resulted in higher levels of bacteremia than PBS containing 0.5% bovine serum albumin (PBS-BSA) (P less than 0.001), i.e. the BSA apparently acted to protect the rats from H. influenzae infection. In fact the use of PBS-BSA as an inoculum buffer masked the protective effect noted above of the absorbed normal rabbit serum.

1041 EPIDEMIOLOGY OF HAEMOPHILUS INFLUENZAE DISEASE DETERMINED BY OUTER MEMBRANE PROTEIN COMPOSITION

The outer membrane protein composition of 50 disease isolates of H. influenzae, including strains of all 6 serotypes as well as untypable strains, has been determined by SDS-PAGE. Results show the existence of considerable strain variation in the mobility of some, but not all, of the proteins. For example, the 28 type b strains examined could be classified into 8 subtypes according to differences in the mobilities of 4 of the 7 major outer membrane proteins. Untypable strains showed even greater diversity. Using these differences as an epidemiological tool, we have found the following: (1) A recent outbreak of type b meningitis involving 17 Rochester area children over a 6-week period was caused by at least 5 different strains, indicating that a single “killer” strain was not responsible for the epidemic. (2) In all 7 patients examined, type b isolates from different anatomic sites in the same patient were identical, suggesting that a single bacterium initiated the disease. (3) Isolates from a patient experiencing a repeat type b infection were identical, suggesting that the same strain was responsible for reinfection. In addition to these findings we have also discovered: (1) Some outer membrane proteins are common to all strains. (2) Children recovering from systemic type b disease raise serum antibodies to outer membrane proteins. Studies are currently in progress to identify the particular immunogenic proteins.