Precipitation of iron on the surface of Leptospira interrogans is associated with mutation of the stress response metalloprotease HtpX

Abstract

High concentrations of free metal ions in the environment can be detrimental to bacterial survival. However, bacteria utilize strategies, including the activation of stress response pathways and immobilizing chemical elements on their surface, to limit this toxicity. In this study, we characterized LA4131, the HtpX-like M48 metalloprotease from Leptospira interrogans, with a putative role in bacterial stress response and membrane homeostasis. Growth of the la4131 transposon mutant strain (L522) in 360 μM FeSO4 (10-fold the normal in vitro concentration) resulted in the production of an amorphous iron precipitate. Atomic force microscopy and transmission electron microscopy analysis of the strain demonstrated that precipitate production was associated with the generation and release of outer membrane vesicles (OMVs) from the leptospiral surface. Transcriptional studies indicated that inactivation of la4131 resulted in altered expression of a subset of metal toxicity and stress response genes. Combining these findings, this report describes OMV production in response to environmental stressors and associates OMV production with the in vitro activity of an HtpX-like metalloprotease.

Fig 1

Iron-associated phenotypic difference between the L. interrogans sv Lai la4131 mutant (L522) and the L. interrogans sv Lai (L521) parent strain after culture in EMJH lot 701688. Cell pellets were prepared from 5-ml cultures of L521 and L522 pelleted at 4,000 × g for 5 min. (A) Overlaid mass spectra generated using inductively coupled plasma mass spectrometry (ICP-MS) conducted on pelleted samples of L522 and L521. (B) The spectra in the region of 53.6 to 56.1 atomic mass units (AMU) are shown.

Fig 2

Micrographs generated using atomic force microscopy (AFM) showing the surface characteristics of L. interrogans sv Lai strain L521 (A, B, and C) and L. interrogans sv Lai la4131 mutant L522 (D, E, and F). Bacteria were grown in BD EMJH lot 8170035 supplemented with 360 μM FeSO₄. Cultures of the la4131 mutant (D) show the presence of cell-associated material. Images were acquired in tapping (A, B, D, and E) or phase-contrast (C and F) modes. Increased viscoelasticity is shown as light coloration on phase-contrast images. Cultures of the la4131 mutant showed overall increased surface viscosity. Stars (★) indicate outer membrane vesicles. Arrows (↑) indicate flagella. The fine white lines that occur in the AFM images are image-processing artifacts due to the line-by-line leveling process.

Fig 3

Transmission electron microscopy of L. interrogans strain L521 (A and B) and L. interrogans sv Lai la4131 mutant L522 (C and D) cultured in EMJH medium or EMJH medium supplemented with 360 μM FeSO₄. The bacteria were grown in BD EMJH lot 8170035 (A and C) and BD EMJH lot 8170035 supplemented with 360 μM FeSO₄ (B and D). Arrows indicate accumulated electron-dense material.