FacZ is a GpsB-interacting protein that prevents aberrant division-site placement in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive pathogen responsible for antibiotic-resistant infections. To identify vulnerabilities in cell envelope biogenesis that may overcome resistance, we enriched for S. aureus transposon mutants with defects in cell surface integrity or cell division by sorting for cells that stain with propidium iodide or have increased light-scattering properties, respectively. Transposon sequencing of the sorted populations identified more than 20 previously uncharacterized factors impacting these processes. Cells inactivated for one of these proteins, factor preventing extra Z-rings (FacZ, SAOUHSC_01855), showed aberrant membrane invaginations and multiple FtsZ cytokinetic rings. These phenotypes were suppressed in mutants lacking the conserved cell-division protein GpsB, which forms an interaction hub bridging envelope biogenesis factors with the cytokinetic ring in S. aureus. FacZ was found to interact directly with GpsB in vitro and in vivo. We therefore propose that FacZ is an envelope biogenesis factor that antagonizes GpsB function to prevent aberrant division events in S. aureus.

Identification of FacZ as a division site placement factor in Staphylococcus aureus.

Staphylococcus aureus is a gram-positive pathogen responsible for life-threatening infections that are difficult to treat due to antibiotic resistance. The identification of new vulnerabilities in essential processes like cell envelope biogenesis represents a promising avenue towards the development of anti-staphylococcal therapies that overcome resistance. To this end, we performed cell sorting-based enrichments for S. aureus mutants with defects in envelope integrity and cell division. We identified many known envelope biogenesis factors as well as a large collection of new factors with roles in this process. Mutants inactivated for one of the hits, the uncharacterized SAOUHSC_01855 protein, displayed aberrant membrane invaginations and multiple FtsZ cytokinetic ring structures. This factor is broadly distributed among Firmicutes, and its inactivation in B. subtilis similarly caused division and membrane defects. We therefore renamed the protein FacZ (Firmicute-associated coordinator of Z-rings). In S. aureus, inactivation of the conserved cell division protein GpsB suppressed the division and morphological defects of facZ mutants. Additionally, FacZ and GpsB were found to interact directly in a purified system. Thus, FacZ is a novel antagonist of GpsB function with a conserved role in controlling division site placement in S. aureus and other Firmicutes.

Combining Signal Peptidase and Lipoprotein Processing Inhibitors Overcomes Ayr Resistance in Staphylococcus aureus.

Antibiotic resistance in bacterial pathogens is an ongoing public health concern. The arylomycins are a class of natural product antibiotics that target the type I signal peptidase, which carries out the terminal step in protein secretion. Here, we used transposon sequencing (Tn-Seq) to profile the effects of the optimized arylomycin derivative G0775 in Staphylococcus aureus. Our transposon libraries include both upregulation and inactivation mutants, allowing us to identify resistance mechanisms and targets for synergism. We identified several cell envelope pathways that, when inactivated, sensitize S. aureus to the arylomycin G0775. These pathways include the lipoprotein processing pathway, and we have shown that inhibitors of this pathway synergize with G0775 even though lipoprotein processing is nonessential in S. aureus. Moreover, we found that blocking this pathway completely reverses Ayr resistance, which is a major resistance mechanism to arylomycins, including G0775. Our Tn-Seq data also showed that upregulation of mprF and several other genes is protective against G0775. Because a subset of these genes was previously found in a Tn-Seq profile of the clinically important antibiotic daptomycin, we tested a set of daptomycin-nonsusceptible clinical isolates with gain-of-function mutations in mprF for susceptibility to arylomycin G0775. Despite structural and mechanistic differences between these antibiotics, we observed similar decreases in susceptibility. Taken together, our results highlight how Tn-Seq profiles that include both gene inactivation and upregulation can identify targets, antibiotic resistance mechanisms, and strategies to overcome resistance.

Rapid Inhibitor Discovery by Exploiting Synthetic Lethality.

Synthetic lethality occurs when inactivation of two genes is lethal but inactivation of either single gene is not. This phenomenon provides an opportunity for efficient compound discovery. Using differential growth screens, one can identify biologically active compounds that selectively inhibit proteins within the synthetic lethal network of any inactivated gene. Here, based purely on synthetic lethalities, we identified two compounds as the only possible inhibitors of Staphylococcus aureus lipoteichoic acid (LTA) biosynthesis from a screen of ∼230,000 compounds. Both compounds proved to inhibit the glycosyltransferase UgtP, which assembles the LTA glycolipid anchor. UgtP is required for ß-lactam resistance in methicillin-resistant S. aureus (MRSA), and the inhibitors restored sensitivity to oxacillin in a highly resistant S. aureus strain. As no other compounds were pursued as possible LTA glycolipid assembly inhibitors, this work demonstrates the extraordinary efficiency of screens that exploit synthetic lethality to discover compounds that target specified pathways. The general approach should be applicable not only to other bacteria but also to eukaryotic cells.

Defects in The First Step of Lipoprotein Maturation Underlie The Synthetic Lethality of Escherichia coli Lacking The Inner Membrane Proteins YciB And DcrB.

Nearly a quarter of the Escherichia coli genome encodes for inner membrane proteins of which approximately a third have unassigned or poorly understood function. We had previously demonstrated that the synergy between the functional roles of the inner membrane-spanning YciB and the inner membrane lipoprotein DcrB, is essential in maintaining cell envelope integrity. In yciB dcrB cells, the abundant outer membrane lipoprotein, Lpp, mislocalizes to the inner membrane where it forms toxic linkages to peptidoglycan. Here, we report that the aberrant localization of Lpp in this double mutant is due to inefficient lipid modification at the first step in lipoprotein maturation. Both Cpx and Rcs signaling systems are upregulated in response to the envelope stress. The phosphatidylglycerol-pre-prolipoprotein diacylglyceryl transferase, Lgt, catalyzes the initial step in lipoprotein maturation. Our results suggest that the attenuation in Lgt-mediated transacylation in the double mutant is not a consequence of lowered phosphatidylglycerol levels. Instead, we posit that altered membrane fluidity, perhaps due to changes in lipid homeostasis, may lead to the impairment in Lgt function. Consistent with this idea, a dcrB null is not viable when grown at low temperatures, conditions which impact membrane fluidity. Like the yciB dcrB double mutant, dcrB null-mediated toxicity can be overcome in distinct ways - by increased expression of Lgt, deletion of lpp, or removal of Lpp-peptidoglycan linkages. The last of these events leads to elevated membrane vesiculation and lipid loss, which may, in turn, impact membrane homeostasis in the double mutant.Importance A distinguishing feature of Gram-negative bacteria is their double-membraned cell envelope which presents a formidable barrier against environmental stress. In E. coli, more than a quarter of the cellular proteins reside at the inner membrane but about a third of these proteins are functionally unassigned or their function is incompletely understood. Here, we show that the synthetic lethality underlying the inactivation of two inner membrane proteins, a small integral membrane protein YciB, and a lipoprotein, DcrB, results from the attenuation of the first step of lipoprotein maturation at the inner membrane. We propose that these two inner membrane proteins YciB and DcrB play a role in membrane homeostasis in E. coli and related bacteria.

A synergistic role for two predicted inner membrane proteins of Escherichia coli in cell envelope integrity.

The bacterial cytoplasmic membrane is a principal site of protein translocation, lipid and peptidoglycan biogenesis, signal transduction, transporters and energy generating components of the respiratory chain. Although 25-30% of bacterial proteomes consist of membrane proteins, a comprehensive understanding of their influence on fundamental cellular processes is incomplete. Here, we show that YciB and DcrB, two small cytoplasmic membrane proteins of previously unknown functions, play an essential synergistic role in maintaining cell envelope integrity of Escherichia coli. Lack of both YciB and DcrB results in pleiotropic cell defects including increased levels of lipopolysaccharide, membrane vesiculation, dynamic shrinking and extension of the cytoplasmic membrane accompanied by lysis and cell death. The stalling of an abundant outer membrane lipoprotein, Lpp, at the periplasmic face of the inner membrane leads to lethal inner membrane-peptidoglycan linkages. Additionally, the periplasmic chaperone Skp contributes to yciB dcrB mutant cell death by possibly mistargeting stalled porins into the inner membrane. Consistent with the idea of a compromised envelope in the yciB dcrB mutant, multiple envelope stress response systems are induced, with Cpx signal transduction being required for growth. Taken together, our results suggest a fundamental role for YciB and DcrB in cell envelope biogenesis.

Characterization of the FtsZ C-Terminal Variable (CTV) Region in Z-Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in E. coli Cytokinesis.

Polymerization of a ring-like cytoskeletal structure, the Z-ring, at midcell is a highly conserved feature in virtually all bacteria. The Z-ring is composed of short protofilaments of the tubulin homolog FtsZ, randomly arranged and held together through lateral interactions. In vitro, lateral associations between FtsZ protofilaments are stabilized by crowding agents, high concentrations of divalent cations, or in some cases, low pH. In vivo, the last 4-10 amino acid residues at the C-terminus of FtsZ (the C-terminal variable region, CTV) have been implicated in mediating lateral associations between FtsZ protofilaments through charge shielding. Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring in vivo. Here we characterize the complementary role/s of the CTV of E. coli FtsZ and the FtsZ-ring stabilizing protein ZapD, in FtsZ assembly. We show that the net charge of the FtsZ CTV not only affects FtsZ protofilament bundling, confirming earlier observations, but likely also the length of the FtsZ protofilaments in vitro. The CTV residues also have important consequences for Z-ring assembly and interaction with ZapD in the cell. ZapD requires the FtsZ CTV region for interaction with FtsZ in vitro and for localization to midcell in vivo. Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD. Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the ß- γ-proteobacterial species.