Citrobacter rodentium infection is a mouse model for the important human diarrheal infection caused by enteropathogenic E. coli (EPEC). The pathogenesis of both species is very similar and depends on their unique ability to form intimately epithelium-adherent microcolonies, also known as “attachment/effacement” (A/E) lesions. These microcolonies must be dynamic and able to self-renew by continuous re-infection of the rapidly regenerating epithelium. It is unknown whether sustained epithelial A/E lesion pathogenesis is achieved through re-infection by planktonic bacteria from the luminal compartment or local spread of sessile bacteria without a planktonic phase. Focusing on the earliest events as C. rodentium becomes established, we show here that all colonic epithelial A/E microcolonies are clonal bacterial populations, and thus depend on local clonal growth to persist. In wild-type mice, microcolonies are established exclusively within the first 18 hours of infection. These early events shape the ongoing intestinal geography and severity of infection despite the continuous presence of phenotypically virulent luminal bacteria. Mechanistically, induced resistance to A/E lesion de-novo formation is mediated by TLR-MyD88/Trif-dependent signaling and is induced specifically by virulent C. rodentium in a virulence gene-dependent manner. Our data demonstrate that the establishment phase of C. rodentium pathogenesis in vivo is restricted to a very short window of opportunity that determines both disease geography and severity.

There is the notion that infection with a virulent intestinal pathogen induces generally stronger mucosal adaptive immunity than the exposure to an avirulent strain. Whether the associated mucosal inflammation is important or redundant for effective induction of immunity is, however, still unclear. Here we use a model of auxotrophic Salmonella infection in germ-free mice to show that live bacterial virulence factor-driven immunogenicity can be uncoupled from inflammatory pathogenicity. Although live auxotrophic Salmonella no longer causes inflammation, its mucosal virulence factors remain the main drivers of protective mucosal immunity; virulence factor-deficient, like killed, bacteria show reduced efficacy. Assessing the involvement of innate pathogen sensing mechanisms, we show MYD88/TRIF, Caspase-1/Caspase-11 inflammasome, and NOD1/NOD2 nodosome signaling to be individually redundant. In colonized animals we show that microbiota metabolite cross-feeding may recover intestinal luminal colonization but not pathogenicity. Consequent immunoglobulin A immunity and microbial niche competition synergistically protect against Salmonella wild-type infection.

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The study demonstrates that proteolytic enzymes can alter the glycoprotein composition of the surface at different tumor cell lines. This membrane modulation measured by lectin binding, is being considered as a prerequisite for the main target, namely the individualized therapeutic use of proteases for adjuvant cancer treatment. The treatment of a human bladder carcinoma cell line and the murine melanoma cell line B16 as well as its variant F1 with bromelain and ficin resulted in a reduced binding of Concanavalin A (ConA). No difference to the controls could be seen when the B16 variant F10 was further examined, whereas a human pancreas carcinoma cell line showed an elevated ConA binding. The experiments with wheat germ agglutinin (WGA) and the lectins from elder bark (SNA) and from Maackia amurensis (MAA) with specifity for N-acetylneuraminic acid also showed a strongly reduced binding after protease treatment. Finally a panel of different proteases showed similar binding effects with the exception of actinidin that revealed no detectable changes in lectin binding. Plasmin and elastase, assayed in physiological concentrations, also showed no remarkable effect. Not unexpected but still surprising, further purification of pure bromelain resulted in a loss of surface-modulating capacity. Concerning the protease concentrations a dose dependence could not be noted in the range from 0.17-2.04 U per ml. Altogether the findings support the advanced use of enzymes as an approach for the successful adjuvant cancer treatment. This is due to the fact that the metastatic potential is modulated by enzymes as the recognition signals are being altered enzymatically.