The role of pili in Bacillus cereus intraocular infection

Abstract

Bacterial endophthalmitis is a potentially blinding intraocular infection. The bacterium Bacillus cereus causes a devastating form of this disease which progresses rapidly, resulting in significant inflammation and loss of vision within a few days. The outer surface of B. cereus incites the intraocular inflammatory response, likely through interactions with innate immune receptors such as TLRs. This study analyzed the role of B. cereus pili, adhesion appendages located on the bacterial surface, in experimental endophthalmitis. To test the hypothesis that the presence of pili contributed to intraocular inflammation and virulence, we analyzed the progress of experimental endophthalmitis in mouse eyes infected with wild type B. cereus (ATCC 14579) or its isogenic pilus-deficient mutant (ΔbcpA-srtD-bcpB or ΔPil). One hundred CFU were injected into the mid-vitreous of one eye of each mouse. Infections were analyzed by quantifying intraocular bacilli and retinal function loss, and by histology from 0 to 12 h postinfection. In vitro growth and hemolytic phenotypes of the infecting strains were also compared. There was no difference in hemolytic activity (1:8 titer), motility, or in vitro growth (p > 0.05, every 2 h, 0-18 h) between wild type B. cereus and the ΔPil mutant. However, infected eyes contained greater numbers of wild type B. cereus than ΔPil during the infection course (p ≤ 0.05, 3-12 h). Eyes infected with wild type B. cereus experienced greater losses in retinal function than eyes infected with the ΔPil mutant, but the differences were not always significant. Eyes infected with ΔPil or wild type B. cereus achieved similar degrees of severe inflammation. The results indicated that the intraocular growth of pilus-deficient B. cereus may have been better controlled, leading to a trend of greater retinal function in eyes infected with the pilus-deficient strain. Although this difference was not enough to significantly alter the severity of the inflammatory response, these results suggest a potential role for pili in protecting B. cereus from clearance during the early stages of endophthalmitis, which is a newly described virulence mechanism for this organism and this infection.

Keywords: Bacillus; Endophthalmitis; Eye; Infection; Inflammation; Pili; Virulence.

Figure 1. In vitro growth of piliated and nonpiliated B. cereus

(A) B. cereus ATCC 14579 or its isogenic pilus-deficient mutant (ΔPil) were cultured in BHI for 18 h at 37°C, then subcultured to an approximate concentration of 10³ CFU/mL in fresh BHI. Aliquots were removed every two hours to quantify bacteria. Numbers of ATCC 14579 and ΔPil were statistically similar at each time point tested (P≥0.05). Both strains reached stationary phase in BHI at approximately 8 h postinfection. Values represent the mean ± SD for N=3 samples per time point. (B) B. cereus ATCC 14579 or its isogenic pilus-deficient mutant (ΔbcpA-srtD-bcpB) were spotted onto TSA + 5% sheep blood agar and incubated for 18 h at 37°C. Spreading colonies of B. cereus were observed at each inoculation point and colonies had the characteristic double zone of hemolysis. On blood agar, these strains were phenotypically indistinguishable. (C) Transmission electron microscopy (TEM) of ATCC 14579 (WT) and ΔPil. Pili are denoted by white arrows, while black arrows denote flagella or flagellar bundles. Pili were noted surrounding ATCC 14579, but not ΔPil. Magnification, 50,000X.

Figure 2. Intraocular growth of piliated and nonpiliated B. cereus

Eyes of C57BL/6J mice were infected with 100 CFU of B. cereus ATCC 14579 (WT) or its isogenic pilus-deficient mutant (ΔPil). Eyes were harvested and B. cereus were quantified. Greater numbers of WT B. cereus were recovered from infected eyes than ΔPil B. cereus at all postinfection time points (*P≤0.03). Values represent mean ± SEM of N≥6 eyes at each time point.

Figure 3. Retinal function of eyes infected with piliated or nonpiliated B. cereus

Eyes of C57BL/6J mice were infected with 100 CFU of B. cereus ATCC 14579 (WT) or its isogenic pilus-deficient mutant (ΔPil). Retinal function was assessed by electroretinography at 6, 9, and 12 h postinfection. (A) In general, retinal function declined in all infected eyes by 12 h postinfection. Although there was an observed trend of higher retained A-wave and B-wave function in eyes infected with ΔPil compared to that of eyes infected with WT at each time point, these differences were not statistically significant (P≥0.29). Values represent mean ± SEM of N≥6 eyes each time point. (B) Representative waveforms of eyes infected with WT and ΔPil B. cereus and uninfected contralateral eyes at 12 h postinfection.

Figure 4. Retinal damage and intraocular inflammation in eyes infected with piliated or nonpiliated B. cereus

Eyes of C57BL/6J mice were infected with 100 CFU of B. cereus ATCC 14579 (WT) or its isogenic pilus-deficient mutant (ΔPil). Infected globes were harvested at 3, 6, 9, and 12 h postinfection and processed for hematoxylin and eosin staining. Uninfected C57BL/6J eyes had no inflammation and were architecturally and morphologically similar. At 3 h postinfection, eyes infected with each strain had fibrin infiltrate in the anterior chamber and posterior segments. Corneas were clear, retinas were intact, and retinal layers were distinguishable at this time. B. cereus were noted in the vitreous of eyes infected with both strains. At 6 and 9 h postinfection, significant fibrin and infiltrating cells were visible in the anterior segments of eyes infected with each strain. B. cereus were noted in the mid-vitreous, as well as along the posterior capsule and near the ciliary body in eyes infected with both strains. Retinal layers in these infected eyes remained intact. At 9 h postinfection, B. cereus were also noted in the anterior chamber of eyes infected with either strain. By 12 h postinfection, all infected eyes had severe inflammation, anterior and posterior segment infiltration, and fully detached retinas. B. cereus were noted in the same areas as described for 9 h postinfection. The gross pathological changes developing in eyes infected with each strain were indistinguishable from one another. Sections are representative of three eyes per time point. B. cereus are denoted by white arrows in the higher magnification images in 4B. In 4B: Ret, retina; Vit, vitreous; AqH, aqueous humor; Ir, iris; L, lens. Magnification in A, 10×. Magnification in B, 40×.