Abstract
Typhoid fever remains a serious health problem worldwide and is a cause of concern especially in developing countries. Although a number of different vaccines are currently available against typhoid, each one has certain drawbacks, which has led to the development of a new generation of typhoid vaccines. In addition to Vi-polysaccharide vaccines (alone or conjugated to protein), attention has also been focused on the role of outer-membrane proteins (OMPs), particularly porins of Gram-negative bacteria, in the induction of specific immunity (Isibasi et al., 1988; Singh et al., 1999a). It is now known that in vivo expression of virulence determinants differs from expression under in vitro conditions (Smith, 1998). Therefore, there is a renewed interest in understanding the behaviour of pathogens in different host environments such as acidic pH, elevated temperature, different osmotic strengths and the presence of cationic peptides as well as the availability of ions and nutrients.
Like other enteric pathogens, Salmonella typhi requires iron for growth, proliferation and a variety of enzymic reactions. The human body, however, has an iron-withholding defence system, preventing the organism from acquiring essential iron for survival inside the host. The regular interaction of the host and the pathogen causes the latter to express certain regulons for sensing the host environment and develop survival strategies against the environmental stress. S. typhi has been reported to express iron-uptake systems, characterized by secreted siderophores, along with certain proteins on the cell's surface referred to as iron-regulated outer-membrane proteins (IROMPs).
Here, in order to assess the effect of iron availability, iron-deficient and iron-sufficient conditions were created in vitro by adding 200 µM dipyridyl and 200 µM ferrous sulphate, respectively, to nutrient broth. The iron content of the media was estimated using ferrozine by the method of Carter (1971). S. typhi (Ty2) was grown in the iron-deficient and iron-sufficient media and OMPs were extracted (Chander et al., 2004).
Fig. 1 shows the outer-membrane profile of S. typhi, indicating enhanced expression of IROMPs when under iron-deficient conditions compared with the expression under normal or iron-sufficient conditions. The expression of these proteins seems to result from a modification of the existing proteins under iron-deficient conditions rather than being from de novo synthesis. The expression of three distinct bands in the molecular mass range from 66 to 97 kDa is in agreement with an earlier study (Fernandez-Beros et al., 1989) of IROMPs from the same strain (S. typhi Ty2). However, it differs from the profile shown recently by Chibber & Bhardwaj (2004) using the same strain. The dipyridyl used in that study may not have been efficient in causing the chelation of iron present in the medium. Thus, the quantification of iron-chelation becomes essential.