Influence of Iron-limited and Replete Continuous Culture on the Physiology and Virulence of Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae strains P9-2 (Pens) and KW2 (PenR) were grown in chemostats of nonferrous design at constant growth rate, pH and dissolved oxygen tension. Iron limitation (pmax 0.1 h-l) was imposed by omitting iron salts from the defined medium and titrating increasing concentrations of the non-metabolizable iron chelators ovotransferrin and Desferal, to progressively decrease the growth yield. Metabolic activity during iron limitation was very high, with a qGlc which was 2-or 11-fold greater than during cystine-or glucose-limited growth, respectively. More aspartate and isoleucine were metabolized during cystine-limited growth, while more glutamate, proline and serine were metabolized during glucose-or iron-limited growth. Significant concentrations of alanine or valine were excreted during cystine-or glucoselimited growth, respectively. Iron-limited growth of an initial inoculum of non-piliated, transparent colony-forming (P-0-) gonococci resulted in the selection of 100% piliated bacteria. Initial inocula of P+ 0-gonococci retained this phenotype for over 100 generations. Iron-limited gonococci were extremely virulent in the guinea-pig subcutaneous chamber model and inocula of even 12 bacteria grew rapidly and persisted. By contrast, cystine-limited (ironreplete) gonococci retained piliation but did not survive in the chambers. Transition from ironlimited to glucose-limited growth resulted in marked loss of piliation but the bacteria remained virulent. Loss of virulence did not correlate with susceptibility to killing by normal human serum, nor with changes in the content or composition of lipooligosaccharide, which contained 2.9,3.7,4.3 and 4-8 kDa moieties. Additional proteins were detectable in Sarkosyl-purified outer membranes of iron-limited gonococci but several proteins with molecular masses similar to those described in the literature for iron-restricted gonococci were detectable in cystine-or glucose-limited bacteria. I N T R O D U C T I O NA versatile physiology (Keevil et al., 1986) or rapid antigenic variation in pili (P+), proteins I1 (O+), or other outer-membrane proteins (OMPs) (Heckels, 1984) might explain the ability of Neisseria gonorrhoeae to colonize and persist at anatomically distinct sites despite the host immune response. One important environmental factor which pathogens frequently encounter in Man is iron deprivation. Iron is an essential nutrient for the majority of bacteria, particularly aerobes. Potential pathogens must therefore acquire this nutrient in competition with the host, since many body fluids contain iron-binding proteins (Weinberg, 1978 ;Griffiths, 1983 ;Brown & Williams, 1985). For example, the mucosal surfaces invaded by N. gonorrhoeae secrete t Present address: Department of Microbiology, University of Bristol, Bristol BS8 ITD, UK.$ Present address: Department of Applied Biology, UWIST, Cardiff CF1 3NU, UK.Abbreviations: KDO, 2-keto-3-deoxyoctonate (3-deoxy-~-rnanno-2-otulosonate); MIRP, major iron-regulated protein; OMP, outer-membrane protein. 0001-4838 0 19...

Isolation and characterization of a mutant of Neisseria gonorrhoeae that is defective in the uptake of iron from transferrin and haemoglobin and is avirulent in mouse subcutaneous chambers

“…Corbeil et al (1979) reported that the addition of 15% gastric mucin and haemoglobin to the inoculum increased the infectivity of gonococci for the mouse subcutaneous chamber model by more than 100-fold. Recently, Keevil et al (1989) observed that ironlimited gonococci were highly virulent in the guinea pig subcutaneous chamber model.…”

Quantitative Proteomics of the Neisseria Gonorrhoeae Cell Envelope and Membrane Vesicles for the Discovery of Potential Therapeutic Targets

“…Although the potential importance of proteins localized to the GC cell envelope and MVs has been reported previously (25,26), only two proteomic studies have been published addressing GC membrane composition (27,28). Most studies have focused on extensive characterization of factors involved in direct host cell interaction: protruding surface proteins (pili), outer membrane adhesins Opa, porins P.IA and P.IB, lipooligosaccharide, and several iron utilization proteins (3, 4, 15, 29 -32).…”

Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae

“…Thus, in establishing infection, the gonococcus must have the ability to adapt readily to a changing host environment. In this study, we investigated this topic by analyzing the protein profiles of conventionally grown, nonstressed, and nutrient-or oxygen-limited, stressed gonococci and found The existence of nutrient-specific regulation of protein synthesis in N. gonorrhoeae has been recognized previously by several investigators (12,28,32,36,62). The availability of iron, for instance, has been found to regulate the synthesis of various cytosolic and membrane proteins, including a number of proteins involved in iron acquisition, probably via the presence of iron-responsive elements adjacent to the genes involved (53).…”