The function of CozE proteins is linked to lipoteichoic acid biosynthesis in Staphylococcus aureus

ABSTRACT Coordinated membrane and cell wall synthesis is vital for maintaining cell integrity and facilitating cell division in bacteria. However, the molecular mechanisms that underpin such coordination are poorly understood. Here we uncover the pivotal roles of the staphylococcal proteins CozEa and CozEb, members of a conserved family of membrane proteins previously implicated in bacterial cell division, in the biosynthesis of lipoteichoic acids (LTA) and maintenance of membrane homeostasis in Staphylococcus aureus. We establish that there is a synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc2DAG. By contrast, in cells lacking LtaA, the flippase of Glc2DAG, the essentiality of CozE proteins was alleviated, suggesting that the function of CozE proteins is linked to the synthesis and flipping of the glycolipid anchor. CozE proteins were indeed found to modulate the flipping activity of LtaA in vitro. Furthermore, CozEb was shown to control LTA polymer length and stability. Together, these findings establish CozE proteins as novel players in membrane homeostasis and LTA biosynthesis in S. aureus. IMPORTANCE Lipoteichoic acids are major constituents of the cell wall of Gram-positive bacteria. These anionic polymers are important virulence factors and modulators of antibiotic susceptibility in the important pathogen Staphylococcus aureus. They are also critical for maintaining cell integrity and facilitating proper cell division. In this work, we discover that a family of membrane proteins named CozE is involved in the biosynthesis of lipoteichoic acids (LTAs) in S. aureus. CozE proteins have previously been shown to affect bacterial cell division, but we here show that these proteins affect LTA length and stability, as well as the flipping of glycolipids between membrane leaflets. This new mechanism of LTA control may thus have implications for the virulence and antibiotic susceptibility of S. aureus.


Fig. S2. Morphological and cell cycle analysis of single and double cozE mutants in S. aureus NCTC8325-4.
(A) Growth curves of NCTC8325-4 wild-type (MDB1), ΔcozEa (MDB2) and ΔcozEb (MDB3), as well as of a CRISPRi double knockdown strain (CRISPRi(cozEa+cozEb), MDB13) and combined knockout/knockdown strains (ΔcozEa, CRISPRi(cozEb), MDB11, and ΔcozEb, CRISPRi(cozEa), MDB12) in BHI medium at 37°C.The graphs represent averages from triplicate measurements.The CRISPRi-strains were grown with and without IPTG, as indicated.(B) Micrographs of the same strains as in (A) showing phase contrast (PC) and fluorescence microscopy of cells stained with the cell wall label VanFL and the nucleoid label DAPI.CRISPRi strains were grown in medium with IPTG to induce the CRISPRi system.White arrows point to cells with perturbed septum formation and abnormal nucleoid staining.The scale bars are 2 µm.(C) Violin plots of the cell areas (in µm 2 ) of NCTC8325-4 wild-type (1.24 ± 0.27 µm 2 ), ΔcozEa (1.19 ± 0.26 µm 2 ), ΔcozEb (1.18 ± 0.27 µm 2 ), MDB13 (1.17 ± 0.32 µm 2 ), MDB11 (1.33 ± 0.39 µm 2 ), and MDB12 (1.16 ± 0.33 µm 2 ), determined using MicrobeJ.Significant differences between the strains are indicted with asterisks (* indicates a P-value of < 0.05, ** indicates a P-value of < 0.01, and *** indicates a P-value of < 0.001, derived from a Mann-Whitney test).The number of cells analyzed for each strain is indicated in the figure.(D) Coefficient of variance (CoeV) of cell size for each strain based on the data provided in (C), showing the relative dispersion of cell area for each strain around their perspective means (CoeV = standard deviation of cell area / mean cell area).(E) Frequency of cells in each of the three cell cycle phases for NCTC8325-4 wild-type, ΔcozEa, ΔcozEb, MDB13, MDB11, and MDB12.See Fig. 1D in the main article for a schematic overview of the different phases analyzed.The distributions were obtained by manually counting the different cell cycle phases of 100-150 randomly selected VanFL stained cells from each strain.

Fig. S3. Synthetic genetic relationships between cozE genes and genes involved in LTA biosynthesis.
(A) Growth assays to identify possible genetic interactions between cozEa or cozEb and the LTA biosynthetic pathway.ugtP-ltaA or ltaS was knocked down by CRISPRi in either a NCTC8325-4 wild-type, ΔcozEa, or ΔcozEb background.The CRISPRi-strains were grown with and without IPTG, as indicated.The length of the red arrows illustrates the growth differences observed between the genetic backgrounds, wild-type, ΔcozEa, and ΔcozEb, when UgtP-LtaA or LtaS were depleted.(B) Growth assays to identify possible genetic interactions between cozEab and the LTA biosynthetic pathway.ugtP-ltaA or ltaS was knocked down along with cozEb in a ΔcozEa background.The CRISPRi-strains were grown with and without IPTG, as indicated by the colors.The red arrow points to the slight growth alleviation observed when ugtP-ltaA was knocked down together with cozEb in the ΔcozEa background (MDB25).In both (A) and (B), the graphs represent averages from triplicate measurements.For constitutive expression of sgRNA(ugtP-ltaA), amp r , cam r This work pVL2336-sgRNA(ltaS) For constitutive expression of sgRNA(ltaS), amp r , cam r This work pMAD Thermosensitive shuttle vector for allelic replacement in Gram-positive bacteria, amp r , ery r (12)   pMAD-cozEa::spc For allelic replacement of cozEa, amp r , ery r , spc r (9) pMAD-cozEb::spc For allelic replacement of cozEb, amp r , ery r , spc r (9) pMAD-cozEa::cam For allelic replacement of cozEa, amp r , ery r , cam r (9) pMAD-∆ugtP::spc For allelic replacement of ugtP, amp r , ery r , spc r

This work pMAD-cozEa-m(sf)gfp_spc
To GFP-tag cozEa in its native locus, amp r , ery r , spc r

This work pMAD-cozEb-m(sf)gfp_spc
To GFP-tag cozEb in its native locus, amp r , ery r , spc r

This work pMAD-PugtP-m(sf)gfp-ugtP_spc
To GFP-tag ugtP in a natural locus under the control of its native promotor, amp r , ery r , spc r  The restriction sites are underlined, the overhangs are bolded, and the ribosomal binding sites are italicized.b.F = forward primer, R = reverse primer, RS = restriction site, and RBS = ribosomal binding site.

Fig. S5 .
Fig. S5.Single CozE depletion in S. aureus JE2 ΔugtP and ΔltaA.Growth curves of JE2 (A) ΔugtP and (B) ΔltaA with individual depletion of CozEa or CozEb in BHI medium at 37°C.The graphs represent averages from triplicate measurements.The CRISPRi-strains were grown with and without IPTG, as indicated.(C) Additionally, growth of the JE2 ΔugtP and ΔltaA strains with single CozE depletion on solid medium.10-fold dilution series, made from noninduced overnight cultures, were spotted onto agar plates with and without IPTG, as indicated.

Fig. S10 .
Fig. S10.Subcellular localization of CozEa and CozEb.(A) The relative expression of CozEa and CozEb is indicated by the band density of CozEa-GFP and CozEb-GFP (from MK1582 and MK1584, respectively, which were incubated until they reached an OD600 of ~0.4 and subsequently normalized) in an immunoblot assay using an anti-GFP antibody.(B) The subcellular localization of CozEa and CozEb analyzed by florescent microscopy of MK1582 and MK1584, respectively.The scale bars are 2 µm.(C) The movement of the CozE proteins were analyzed by time-lapse florescent microscopy of MK1582 (with a cozEa-gfp fusion) and MK1584 (with a cozEb-gfp fusion).Images were captured every third second (x10), as indicated in the figure.White arrows highlight the membrane movement of CozEa-GFP and CozEb-GFP, as the signals pointed to in the initial images are no longer present at the same location in the membrane after 27 seconds.The dynamic spatiotemporal localization of CozEa and CozEb is further depicted in Movie S1 and S2, respectively.

Fig. S11 .
Fig. S11.Analysis of direct interaction between CozE and UgtP or LtaA.(A) Localization of GFP-UgtP in NCTC8325-4 wild-type (MDB77), as well as cells depleted of CozEa (MDB89), CozEb (MDB90), or both CozE proteins (MDB79).Phase contrast-and fluorescence images are shown.Arrows point to spots with membrane-localized GFP-UgtP.The scale bars are 2 µm.(B) Quantification of GFP-UgtP subcellular localization in the strains shown in (A).The distributions were obtained by manually counting the localization pattern of 200-250 randomly selected cells from each strain.The data presented are the averages from three independent experiments.(C) Schematic illustration of the split luciferase plasmids constructed in this work to assess pairwise protein-protein interactions in vivo in S. aureus.The proteins of interest (CozE, UgtP, and LtaA) were fused to either a large (LgBit) or small (SmBit) luciferase subunit, which upon interaction form an active luciferase enzyme that generate a bright luminescent signal.Vectors encoding CozE fused to SmBit and LgBiT, not part of a fusion protein, were used as negative controls (pAF256 vectors were used as controls in this work because they are found to have a higher occurrence of non-specific luciferase activity than pAF257 and pAF262 in S. aureus (1)).Split luciferase assays identifying potential protein-protein interactions between (D) CozEa and UgtP/LtaA and (E) CozEb and UgtP/LtaA.The average RLU/OD600 of four replicates are shown, with error bars indicating the standard deviation from the mean.Positive interactions were determined based of the negative controls, by performing a one-way ANOVA followed by Tukey's Honest Significant Difference (HSD) test (n.sindicates a P-value of > 0.05, while *** indicates a P-value of < 0.001).The assays revealed that neither CozEa nor CozEb directly interact with UgtP, while both CozE proteins interact with LtaA.

Fig. S14 .
Fig. S14.Phylogenetic distribution of CozE proteins in the Staphylococcaceae family.A maximum likelihood phylogenetic tree was constructed from a Clustal Omega sequence alignment of 56 CozE homologs from the Staphylococcaceae family, specifically Staphylococcus, Macrococcus, Jeotgalicoccus, Salinicoccus, and Nosocomiicoccus, using IQ-TREE.The phylogenetic tree was visualized and annotated using iTOL.The CozE proteins from species belonging to Staphylococcus and Macrococcus distributed into two phylogenetically separate subgroups; CozEa (marked in blue) and CozEb (marked in red).The CozE proteins from Jeotgalicoccus, Salinicoccus, and Nosocomiicoccus clustered together in another subgroup (marked in green), which is phylogenetically closer to the CozEa subgroup than the CozEb subgroup.Arrows point at the CozE proteins found in S. aureus.

Table S2 .
Plasmids used in this work.

Table S3 .
Primers used in this work.