A novel murine model for contact lens wear reveals clandestine IL-1R dependent corneal parainflammation and susceptibility to microbial keratitis upon inoculation with Pseudomonas aeruginosa.

PURPOSE: Contact lens wear carries a risk of complications, including corneal infection. Solving these complications has been hindered by limitations of existing animal models. Here, we report development of a new murine model of contact lens wear. METHODS: C57BL/6 mice were fitted with custom-made silicone-hydrogel contact lenses with or without prior inoculation with Pseudomonas aeruginosa (PAO1-GFP). Contralateral eyes served as controls. Corneas were monitored for pathology, and examined ex vivo using high-magnification, time-lapse imaging. Fluorescent reporter mice allowed visualization of host cell membranes and immune cells. Lens-colonizing bacteria were detected by viable counts and FISH. Direct-colony PCR was used for bacterial identification. RESULTS: Without deliberate inoculation, lens-wearing corneas remained free of visible pathology, and retained a clarity similar to non-lens wearing controls. CD11c-YFP reporter mice revealed altered numbers, and distribution, of CD11c-positive cells in lens-wearing corneas after 24 h. Worn lenses showed bacterial colonization, primarily by known conjunctival or skin commensals. Corneal epithelial cells showed vacuolization during lens wear, and after 5 days, cells with phagocyte morphology appeared in the stroma that actively migrated over resident keratocytes that showed altered morphology. Immunofluorescence confirmed stromal Ly6G-positive cells after 5 days of lens wear, but not in MyD88 or IL-1R gene-knockout mice. P. aeruginosa-contaminated lenses caused infectious pathology in most mice from 1 to 13 days. CONCLUSIONS: This murine model of contact lens wear appears to faithfully mimic events occurring during human lens wear, and could be valuable for experiments, not possible in humans, that help solve the pathogenesis of lens-related complications.

The Salmonella enterica serovar Typhi type IVB self-association pili are detached from the bacterial cell by the PilV minor pilus proteins.

Salmonella enterica serovar Typhi and some strains (Vi+) of serovar Dublin use type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. Pilus-mediated self-association may be important in the pathogenesis of enteric fever. We have shown previously that the extent of DNA supercoiling controls the rate of Rci-catalyzed inversion of a DNA fragment which includes the C-terminal portions of the PilV proteins. This inversion therefore controls PilV synthesis as a high inversion rate prohibits transcription of pilV-encoding DNA. Here, we describe the manner in which PilV protein expression inhibits bacterial self-association and present data which suggest that incorporation of one or a few PilV protein molecules into a growing pilus, comprised of PilS subunits, causes the pilus to detach at the bacterial membrane. The bacteria are then unable to self-associate. We suggest that this phenomenon may be relevant to the pathogenesis of typhoid fever.

Rate of inversion of the Salmonella enterica shufflon regulates expression of invertible DNA.

Salmonella enterica serovar Typhi and some strains (Vi(+)) of serovar Dublin use type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. Pilus-mediated self-association may be important in the pathogenesis of enteric fever. We have suggested that the rate of Rci-catalyzed inversion of DNA encoding the C-terminal portions of the PilV proteins controls PilV protein synthesis. This potentially represents a novel means of transcriptional control. Here, it is initially shown that DNA inversion per se is required for inhibition of gene expression from invertible DNA. Binding, without DNA scission, of Rci to its substrate sequences on DNA cannot explain the data obtained. Next, it is shown that inversion frequencies of xylE-encoding DNA, bracketed by Rci substrate sequences, may be modulated by changes in the 19-bp consensus sequences which are essential components of Rci substrate DNA. The affinity of Rci for these sequences affects inversion frequencies, so that a greater affinity is predictive of faster inversion, and therefore less synthesis of product encoded by invertible DNA. Inversion events may inhibit transcription of DNA from external promoters. In vivo, the frequency of Rci-mediated inversion is influenced by the extent of DNA supercoiling, with increasing levels of expression of invertible genes as novobiocin inhibits DNA supercoiling and thus Rci action. This inhibition of DNA supercoiling results in increased synthesis of PilV proteins as Rci activity decreases, and, in turn, bacterial self-association (particularly in serovar Dublin) decreases.

Salmonella enterica serovar Paratyphi C carries an inactive shufflon.

Salmonella enterica serovar Typhi uses type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. This pilus-mediated event may be important in typhoid fever pathogenesis. We initially show that S. enterica serovar Paratyphi C strains harbor a pil operon very similar to that of serovar Typhi. An important difference, however, is located in the shufflon which concludes the pil operon. In serovar Typhi, the Rci recombinase acts upon two 19-bp inverted repeats to invert the terminal region of the pilV gene, thereby disrupting PilV synthesis and permitting bacterial self-association. In serovar Paratyphi C, however, the shufflon is essentially inactive because each of the Rci 19-bp substrates has acquired a single base pair insertion. A PilV protein is thus synthesized whenever the pil operon is active, and bacterial self-association therefore does not occur in serovar Paratyphi C. The data thus suggest that serovar Typhi bacterial self-association using type IVB pili may be important in the pathogenesis of epidemic enteric fever.

Salmonella enterica serovar Dublin strains which are Vi antigen-positive use type IVB pili for bacterial self-association and human intestinal cell entry.

Some strains of Salmonella enterica serovar Dublin are Vi antigen-positive. S. enterica serovar Typhi uses Type IVB pili, encoded adjacent to the viaB locus required for Vi antigen synthesis, to facilitate both eukaryotic cell attachment and bacterial self-association under conditions that favour DNA supercoiling. These pilus-mediated events may be important in typhoid fever pathogenesis. A survey of 17 isolates of S. enterica serovar Dublin showed that all strains which carried the viaB region also carried a serovar Typhi-like Type IVB pil operon, and all serovar Dublin Vi antigen-negative isolates lacked the pil operon. The pil operon was completely sequenced from one of the Vi(+) serovar Dublin strains, and was almost identical (4 nt changes; 3 aa changes, in over 10 kb) to that of serovar Typhi. A pilS mutant of one serovar Dublin strain was constructed, and shown to invade cultured human intestinal INT407 cells to an extent only 20% that of the wild-type parent. Purified prePilS protein inhibited INT407 cell entry by serovar Dublin. The wild-type serovar Dublin strain, but not the pilS mutant, self-associated. The data suggest that the serovar Dublin Type IVB pil operon may increase the human-invasiveness of serovar Dublin, compared to pil-free strains.