Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In a model of septic injury, if directly introduced into the body cavity of Drosophila, this pathogen is insensitive to the host's systemic immune response and kills flies in a day. We find that S. marcescens resistance to the Drosophila immune deficiency (imd)-mediated humoral response requires the bacterial lipopolysaccharide O-antigen. If ingested by Drosophila, bacteria cross the gut and penetrate the body cavity. During this passage, the bacteria can be observed within the cells of the intestinal epithelium. In such an oral infection model, the flies succumb to infection only after 6 days. We demonstrate that two complementary host defense mechanisms act together against such food-borne infection: an antimicrobial response in the intestine that is regulated by the imd pathway and phagocytosis by hemocytes of bacteria that have escaped into the hemolymph. Interestingly, bacteria present in the hemolymph elicit a systemic immune response only when phagocytosis is blocked. Our observations support a model wherein peptidoglycan fragments released during bacterial growth activate the imd pathway and do not back a proposed role for phagocytosis in the immune activation of the fat body. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm for deciphering intestinal pathogenesis.

As part of a study of virulence gene regulation in Streptococcus pneumoniae, we have identified a gene encoding a homologue of the staphylococcal catabolite control protein CcpA in the pneumococcal genome sequence. The pneumococcal protein, designated RegM, has significant similarity to members of the LacI/GalR family of bacterial regulatory proteins. S. pneumoniae D39 derivatives with insertion-duplication or deletion mutations in regM were significantly attenuated in virulence with respect to the wild-type strain. In defined media containing either sucrose or lactose as sole carbon sources, the in vitro growth rates of D39 and the regM mutants were essentially the same. However, in the presence of galactose the regM mutants grew significantly faster than the wild-type strain, whereas growth rates were significantly lower in the presence of glucose or maltose. These data are consistent with the involvement of regM in the catabolism of carbohydrates in S. pneumoniae. RegM was a repressor of both ␣-glucosidase and ␤-galactosidase activities in S. pneumoniae, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose. The observed attenuation in virulence was not attributable to poorer growth of the regM mutants in mouse blood ex vivo, but nevertheless, the mutants were rapidly cleared from the blood of infected mice in vivo. The regM mutation had no apparent impact on expression of several confirmed pneumococcal virulence proteins, but studies employing a lacZ transcriptional fusion construct indicated that mutation of regM resulted in a significant reduction in transcription of the capsular polysaccharide biosynthesis locus (cps). Thus, regM is the first gene outside of the cps locus to be implicated in regulation of capsular gene expression.Streptococcus pneumoniae (the pneumococcus) is a major cause of life-threatening invasive diseases such as pneumonia, meningitis, and bacteremia, as well as other less serious, but highly prevalent infections, such as otitis media and sinusitis. Invasive pneumococcal disease is almost invariably preceded by colonization of the nasopharynx, and significant proportions of the population carry S. pneumoniae asymptomatically at any one time. Progression from carriage to invasion is associated with alteration in the levels of expression of a number of virulence factors. The capacity to sense and adapt to changing host environments is likely to play a key role in this pathogenic process. Most recent works have focused on two-component signal transduction systems. Throup et al. (31) showed that 8 of the 14 two-component systems present in the S. pneumoniae genome are involved in survival in a respiratory model, but Lange et al. (16) found no evidence for involvement in systemic virulence. Although two-component systems are an important means of sensing changes in the environment, transporters associated with cytosolic regulators also play an important role. Some of the best examples of such systems are those involved in the ...

The polysaccharide capsule of Streptococcus pneumoniae and several wellcharacterized virulence proteins are known to contribute to the pathogenesis of pneumococcal disease. However, there is a paucity of data on the expression of their respective genes in vivo. In this study, the relative abundance of the mRNA transcripts of the genes encoding pneumolysin (ply), pneumococcal surface protein A (pspA), pneumococcal surface antigen A (psaA) and cholinebinding protein A (cbpA), and of the first gene of the capsular polysaccharide biosynthesis locus (cps2A), was measured in virulent type 2 pneumococci harvested from the blood of BALB/c mice at 12 h and 24 h following intraperitoneal infection. The mRNA levels were then compared, using relative quantitative RT-PCR, with those present in organisms grown in serum broth. The expression of ply was upregulated threefold at 12 h, and 10-fold at 24 h post-infection ; the expression of pspA and psaA was upregulated threefold and fivefold, respectively, at 12 h post-infection. Interestingly, the expression of pspA was 36-fold higher at 24 h post-infection whereas the expression of cps2A was upregulated approximately fourfold at 12 and 24 h post-infection. However, cbpA mRNA levels remained comparable in vivo and in vitro. When organisms were grown in whole blood or THY broth, the relative expression of these genes in the two growth media also differed markedly. This work provides direct molecular evidence that known virulence-associated genes of S. pneumoniae are differentially expressed in vivo. Data on the relative expression of these genes in different growth media also suggests that the regulation of expression of these genes is highly complex and multifactorial.Keywords : pneumococcus, pneumolysin, surface proteins, mRNA, quantitative RT-PCR INTRODUCTIONStreptococcus pneumoniae is a very important human pathogen, causing a spectrum of diseases including pneumonia, meningitis, bacteraemia and otitis media (Musher, 1992 ;Tuomanen et al., 1995). The incidence of pneumococcal disease is particularly high in children under the age of 5 years, the elderly and immunocompromised individuals (Garenne et al., 1992 ; World Health Organization, 1997 (World Health Organization, 1997). Furthermore, the alarming rate at which this organism is acquiring resistance to multiple antimicrobials has exacerbated the high morbidity and mortality resulting from pneumococcal disease (Whitney et al., 2000). The polysaccharide (PS) capsule is strongly anti-phagocytic and is a sine qua non of pneumococcal virulence (Austrian, 1981). There are 90 recognized PS serotypes ; synthesis of the serotype-specific capsular PS is encoded by distinct clusters of up to 20 tightly linked genes transcribed as single operons (Paton & Morona, 2000). Antibodies to the PS are highly protective, and the global impact of pneumococcal disease has led to the development of polyvalent PS and PS-protein conjugate A. D. Ogunniyi, P. Giammarinaro and J. C. Paton vaccine formulations. Although appreciable levels of success h...