An Iron-Regulated Autolysin Remodels the Cell Wall To Facilitate Heme Acquisition in Staphylococcus lugdunensis

Abstract

Bacteria alter their cell surface in response to changing environments, including those encountered upon invasion of a host during infection. One alteration that occurs in several Gram-positive pathogens is the presentation of cell wall-anchored components of the iron-regulated surface determinant (Isd) system, which extracts heme from host hemoglobin to fulfill the bacterial requirement for iron. Staphylococcus lugdunensis, an opportunistic pathogen that causes infective endocarditis, encodes an Isd system. Unique among the known Isd systems, S. lugdunensis contains a gene encoding a putative autolysin located adjacent to the Isd operon. To elucidate the function of this putative autolysin, here named IsdP, we investigated its contribution to Isd protein localization and hemoglobin-dependent iron acquisition. S. lugdunensis IsdP was found to be iron regulated and cotranscribed with the Isd operon. IsdP is a specialized peptidoglycan hydrolase that cleaves the stem peptide and pentaglycine crossbridge of the cell wall and alters processing and anchoring of a major Isd system component, IsdC. Perturbation of IsdC localization due to isdP inactivation results in a hemoglobin utilization growth defect. These studies establish IsdP as an autolysin that functions in heme acquisition and describe a role for IsdP in cell wall reorganization to accommodate nutrient uptake systems during infection.

FIG 1

isdP is iron regulated and cotranscribed with the S. lugdunensis Isd operon. (A) Schematic of the S. lugdunensis Isd operon. SLUG_00900 is predicted to encode an energy-coupling transporter. SLUG_00910, SLUG_00920, and SLUG_00980 are predicted to encode ABC transporter components. (B) Expression of isdP (SLUG_01010) in iron-replete (TSB) or iron-depleted (TSB + DIP) conditions was determined by qRT-PCR and compared to that of the known iron-regulated genes isdC and isdG (34). (C) PCR was performed on cDNA from iron-rich (−DIP) or iron-starved S. lugdunensis (+DIP) using primers that amplify across the coding regions of isdP and isdG (arrows in panel A). (D) The PCR shown in panel C was performed using cDNA from iron-starved wild-type or ΔisdP mutant cells. RT, reverse transcriptase.

FIG 2

IsdP is a peptidoglycan hydrolase. (A) Schematic of IsdP protein domains. SEC, secretion signal; DD, disordered domain; FlgJ, flagellar muramidase domain; CHAP, amidase domain. (B) Purified IsdP and lysostaphin (lyso) were separated by 15% SDS-PAGE (left) or 15% SDS-PAGE containing lyophilized Micrococcus lysodeikticus cells (ATCC 4698; Sigma) (middle) or heat-killed, iron-starved S. lugdunensis cells (right). A zone of clearing indicates degradation. (C) IsdP was incubated with lyophilized M. lysodeikticus cells, and lysis was measured by monitoring the OD₅₇₈. Data from a representative experiment are shown. (D) An IM-MS conformational space map of a peptidoglycan treated with IsdP and lysozyme. The IM-MS plot represents data within the LC peak at 8.5 min. The peptidoglycan is annotated by the yellow box. (E) A mass spectrum selected from the area marked with the box in panel D. A theoretical isotopic distribution (dotted lines) for the proposed peptidoglycan structure (inset) is overlaid with the experimental data (solid line). Cleavage sites are indicated by arrows. Note the peak at 970.93 is the doubly charged peptidoglycan [M-CO₂ + 2H]²⁺ with cleavage at the lysine side chain/glycine bridge.

FIG 3

IsdP is required for release of IsdC. (A) Immunoblot analysis of IsdC in the supernatant of iron-starved S. lugdunensis strains. Arrows indicate the size of the ladder in kilodaltons. (B) Immunoblot analysis of the S. lugdunensis ΔisdP mutant complemented with isdP. Densitometric analysis is shown below. White bars, top IsdC-reactive band; gray bars, bottom IsdC-reactive band. (C) Immunoblot and densitometric analysis of IsdC in the supernatant of S. lugdunensis constitutively expressing isdP. (D) Immunoblot and densitometric analysis of IsdC in the supernatant of S. aureus expressing isdP. Error bars represent standard errors of the means (SEM). Asterisks denote P < 0.05 compared to wild-type S. lugdunensis pOS1 (B and C) or wild-type S. aureus (D) as calculated by Student's t test (n > 3).

FIG 4

IsdP CHAP domain is required for IsdC release. (A) Schematic of IsdP point mutants, indicated by arrows. (B) Immunoblot analysis of IsdC in the supernatant of the S. lugdunensis ΔisdP strain expressing IsdP mutants. Densitometric analysis is shown below and compared to that of the strain complemented with wild-type isdP. Error bars represent SEM. Asterisks denote a P value of <0.04 as calculated by Student's t test (n > 3).

FIG 5

IsdC, but not IsdP, must be sorted by SrtB for IsdC release. (A) Immunoblot analysis of IsdC in the cell wall or supernatant of S. lugdunensis strains. (B) Immunoblot analysis of IsdC in the cell wall or supernatant of wild-type pOS1 or the ΔisdP pOS1, ΔisdC pOS1, or ΔisdC strain expressing isdC with an altered SrtB signal. (C) Immunoblot analysis of IsdC in the cell wall or supernatant of wild-type pOS1 or the ΔisdP pOS1, ΔisdP pisdP, or ΔisdP strain expressing isdP with an altered putative SrtB motif. Densitometric analysis of supernatant immunoblots is shown below. Arrows indicate the size of the ladder in kilodaltons. White bars, top IsdC-reactive band; gray bars, bottom IsdC-reactive band. Error bars represent SEM. Statistical analyses of top IsdC-reactive bands were calculated by Student's t test (n > 3). (D) Immunoblot analysis of FLAG-tagged IsdP in cellular fractions. Densitometric analysis is shown below. White bars, protoplasts; gray bars, cell wall fraction. Error bars represent SEM.

FIG 6

IsdP alters IsdC localization in the cell wall. (A) Immunofluorescence of IsdC on the surface of S. lugdunensis cells. (B) Quantification of immunofluorescence shown in panel A relative to that of the ΔisdC mutant. Black bars, iron rich; white bars, iron poor; gray bars, iron poor plus proteinase K. (C) IsdC immunoblot and densitometric analyses of samples treated with proteinase K followed by cell wall isolation with lysostaphin. Data are presented as percent IsdC surviving proteinase K surface degradation (P→L) relative to total cell wall IsdC (L only). Gray bars, top IsdC-reactive band; white bars, bottom IsdC-reactive band. Error bars represent SEM. Statistical analysis was calculated using Student's t test, with n = 3 independent experiments. Asterisks denote P < 0.05 compared to the wild type under each condition (B) or to the wild type alone (C).

FIG 7

IsdP is required for full growth with hemoglobin as a sole iron source. (A) Representative growth assay of S. lugdunensis strains grown in iron-deplete media in the presence of hemoglobin. (B) Graphical representation of data at 48 h from three independent experiments. Error bars represent SEM. (C) Growth complementation of isdP. (D) Graphical representation of growth complementation at 24 h. P < 0.05 (*) and P < 0.0001 (**) compared to the wild type as calculated by Student's t test.

FIG 8

Model of IsdP contribution to Isd-dependent heme acquisition. Under iron-restricted conditions, IsdP is expressed with the S. lugdunensis Isd system. IsdP remodels the cell wall by cleaving the stem peptide and pentapeptide crossbridge of mature peptidoglycan, exposing amide groups to which IsdC (blue) is anchored by SrtB. A subpopulation of IsdC also is presented on the cell surface (green). Coordination of IsdP and SrtB populates the cell wall with IsdC such that surface-exposed IsdC can bind extracellular heme and IsdC spanning the peptidoglycan layer shuttles heme from the surface to the membrane.