Many pathogenic and symbiotic Gram-negative bacteria employ type III secretion systems to inject ''effector'' proteins into eukaryotic host cells. These effectors manipulate signaling pathways to initiate symbiosis or disease. By using time-lapse microscopy, we have imaged delivery of the Salmonella type III effector protein SipA͞SspA into animal cells in real time. SipA delivery mostly began 10 -90 sec after docking and proceeded for 100 -600 sec until the bacterial SipA pool (6 ؎ 3 ؋ 10 3 molecules) was exhausted. Similar observations were made for the effector protein SopE. This visualization of type III secretion in real time explains the efficiency of host cell manipulation by means of this virulence system. effector protein ͉ time-lapse microscopy

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SummaryThe mammalian intestine is colonized by a dense bacterial community, called microbiota. The microbiota shields from intestinal infection (colonization resistance). Recently, we have shown that enteropathogenic Salmonella spp. can exploit inflammation to compete with the intestinal microbiota. The mechanisms explaining the enhanced pathogen growth in the inflamed intestine are elusive. Here, we analysed the function of bacterial flagella in the inflamed intestine using a mouse model for acute Salmonella Typhimurium enterocolitis. Mutations affecting flagellar assembly (Fla -) and chemotaxis (Che -) impaired the pathogen's fitness in the inflamed intestine, but not in the normal gut. This was attributable to a localized source of high-energy nutrients (e.g. galactosecontaining glyco-conjugates, mucin) released as an element of the mucosal defence. Motility allows Salmonella Typhimurium to benefit from these nutrients and utilize them for enhanced growth. Thus, nutrient availability contributes to enhanced pathogen growth in the inflamed intestine. Strategies interfering with bacterial motility or nutrient availability might offer starting points for therapeutic approaches.

The virulence of Salmonella enterica critically depends on the functions of two type III secretion systems (T3SS), with the Salmonella pathogenicity island 1 (SPI1)-encoded T3SS required for host cell invasion and the SPI2-T3SS enabling Salmonella to proliferate within host cells. A further T3SS is required for the assembly of the flagella. Most serovars of Salmonella also possess a lipopolysaccharide with a complex O-antigen (OAg) structure. The number of OAg units attached to the core polysaccharide varies between 16 and more than 100 repeats, with a trimodal distribution. This work investigated the correlation of the OAg length with the functions of the SPI1-T3SS and the SPI2-T3SS. We observed that the number of repeats of OAg units had no effect on bacterial motility. The interaction of Salmonella with epithelial cells was altered if the OAg structure was changed by mutations in regulators of OAg. Strains defective in synthesis of very long or long and very long OAg species showed increased translocation of a SPI1-T3SS effector protein and increased invasion. Invasion of a strain entirely lacking OAg was increased, but this mutant strain also showed increased adhesion. In contrast, translocation of a SPI2-T3SS effector protein and intracellular replication were not affected by modification of the OAg length. Mutant strains lacking the entire OAg or long and very long OAg were highly susceptible to complement killing. These observations indicate that the architecture of the outer membrane of Salmonella is balanced to permit sufficient T3SS function but also to confer optimal protection against antimicrobial defense mechanisms.Salmonella enterica is a remarkable pathogen with strategies for adaptation to different lifestyles in the environment as well as within various host organisms. The requirements for life within the host can vary dramatically, for example, after transition from extracellular life within the intestine to an intracellular life within a special organelle formed inside infected host cells (reviewed in reference 23). The presence of an outer membrane is an important structural feature that enables commensal as well as pathogenic bacteria to adapt to the intestine and to resist bile salts and various molecules of the host innate immune system (44). Of specific importance is the lipopolysaccharide (LPS), the major constituent of the outer leaflet of the outer membrane. LPS is composed of (i) the lipid A portion, consisting of acyl chains linked to phosphorylated N-acetylglucosamine; (ii) the inner and outer core moieties, consisting of rather conserved sugars; and (iii) a highly variable O antigen (OAg). The LPS of S. enterica serovar Typhimurium and several other serovars has special characteristics, with extreme heterogeneity in the length of the OAg repeats. LPS species with a short OAg (S-OAg) consisting of about 16 repeats of OAg units can be found. In addition, LPS species with long and very long OAg (L-OAg and VL-OAg, respectively) are present, containing about 35 and more than 100 repeat...

A genome wide RNAi screen identifies 72 host cell genes affecting S. Typhimurium entry, including actin regulators and COPI. This study implicates COPI-dependent cholesterol and sphingolipid localization as a common mechanism of infection by bacterial and viral pathogens.