Bacterial infection as assessed by in vivo gene expression

Abstract

In vivo expression technology (IVET) has been used to identify > 100 Salmonella typhimurium genes that are specifically expressed during infection of BALB/c mice and/or murine cultured macrophages. Induction of these genes is shown to be required for survival in the animal under conditions of the IVET selection. One class of in vivo induced (ivi) genes, iviVI-A and iviVI-B, constitute an operon that resides in a region of the Salmonella genome with low G+C content and presumably has been acquired by horizontal transfer. These ivi genes encode predicted proteins that are similar to adhesins and invasins from prokaryotic and eukaryotic pathogens (Escherichia coli [tia], Plasmodium falciparum [PfEMP1]) and have coopted the PhoPQ regulatory circuitry of Salmonella virulence genes. Examination of the in vivo induction profile indicates (i) many ivi genes encode regulatory functions (e.g., phoPQ and pmrAB) that serve to enhance the sensitivity and amplitude of virulence gene expression (e.g., spvB); (ii) the biochemical function of many metabolic genes may not represent their sole contribution to virulence; (iii) the host ecology can be inferred from the biochemical functions of ivi genes; and (iv) nutrient limitation plays a dual signaling role in pathogenesis: to induce metabolic functions that complement host nutritional deficiencies and to induce virulence functions required for immediate survival and spread to subsequent host sites.

Figure 1

Selection for bacterial genes that are specifically induced during infection. (A) Random fragments of bacterial DNA (dark arrows) were cloned into an IVET vector, 5′ to a promoterless purA or cat gene (2, 3). (B) The recombinant pool was used as an inoculum for infection of BALB/c mice and/or RAW 264.7 cultured macrophages. Bacterial survival in the animal (or cultured macrophage) is dependent on in vivo selection of bacterial promoters that drive the expression of the promoterless purA or cat genes. After incubation in the animal (or cultured macrophage), bacterial fusion strains were recovered from host tissues and plated on lactose MacConkey indicator medium. The gray arrow indicates an ivi fusion-bearing strain which is Lac⁺ (ferments lactose) (•) when grown in the animal and Lac⁻ (○) when grown on laboratory medium.

Figure 2

Induction of ivi genes is required for survival in the animal under conditions of the IVET selection. BALB/c mice were infected i.g. (10⁶ cells) or i.p. (500 cells) with either an ivi or preselected Lac⁻ or Lac⁺ bacterial fusion strain. The number of bacterial cells recovered from the spleen was determined after morbidity was observed in the Lac⁺-infected controls (5 days). (A) pIVET1 (purA) i.g. and i.p. selections. (B) pIVET8 (cat) i.p. selection. The number of bacteria recovered from the spleen after i.g. (IG) or i.p. (IP) infection is indicated by dark or gray bars, respectively. The ivi fusion joint points are as follows: phoP (MT1466); iviXII (MT1733); iviVI-A; MT1461, and iviXII (MT1501).